CN101233151A - Tight junction modulating peptide components for enhancing mucosal delivery of therapeutic agents - Google Patents

Abstract

Compounds and components including sequences for mucosal epithelial transport of an active agent are given. Tight junction modulating peptide components are described for use in transport and delivery. Permeability can be enhanced with reversibility. Compounds and components for enhanced delivery may be peptide or protein variants, conjugates, or other analog types and structures.

Description

Translated fromChinese

增强治疗剂的黏膜递送的紧密连接调控肽成分Tight junction modulating peptide components that enhance mucosal delivery of therapeutic agents

发明背景Background of the invention

在任何疾病或病况的治疗中基本的关心是保证将治疗剂药物安全并且有效地递送到受试者。治疗剂递送的常规途径包括静脉内注射和口服施用。然而，这些递送方法有缺点，并且因此需要备选的递送系统。A fundamental concern in the treatment of any disease or condition is ensuring safe and effective delivery of the therapeutic agent drug to the subject. Conventional routes of therapeutic agent delivery include intravenous injection and oral administration. However, these delivery methods have disadvantages, and therefore alternative delivery systems are needed.

通过注射施用药物的一个主要缺点是通常需要由培训过的人员来施用药物。另外，当通过注射施用药物时，培训过的人员处在危险中。对于自我-施用的药物，许多患者勉强或不能在正规基础上给他们自己注射。注射还与增加的感染危险有关。药物注射的其它缺点包括个体之间递送结果的可变性，以及药物作用的不可预知的强度和持续时间。A major disadvantage of administering drugs by injection is that trained personnel are often required to administer the drugs. Additionally, trained personnel are at risk when administering drugs by injection. With self-administered drugs, many patients are reluctant or unable to inject themselves on a regular basis. Injections are also associated with an increased risk of infection. Other disadvantages of drug injection include variability in delivery results between individuals, and the unpredictable intensity and duration of drug action.

尽管具有这些缺点，对于许多重要的治疗化合物，注射仍为唯一经过核准的递送方式。这些包括常规药物，以及快速发展的肽和蛋白生物治疗剂列表。这些化合物通过备选施用途径例如口服、鼻和其它黏膜途径的递送是理想的，但是可能提供更低的生物利用度。对于大分子种类，例如，肽和蛋白治疗化合物，备选施用途径可能受到易失活性和经过黏膜屏障的低吸收的限制。Despite these disadvantages, injection remains the only approved mode of delivery for many important therapeutic compounds. These include conventional drugs, as well as the rapidly growing list of peptide and protein biotherapeutics. Delivery of these compounds by alternative routes of administration such as oral, nasal and other mucosal routes is desirable, but may provide lower bioavailability. For macromolecular species, eg, peptide and protein therapeutic compounds, alternative routes of administration may be limited by facile activity and low absorption across mucosal barriers.

由于肝的第一遍代谢(hepatic first-pass metabolism)和/或在胃肠道中的降解，一些治疗剂的口服施用在作用中表现出非常低的生物利用度和极大的时间延迟。Oral administration of some therapeutic agents exhibits very low bioavailability and a significant time delay in action due to hepatic first-pass metabolism and/or degradation in the gastrointestinal tract.

治疗化合物的黏膜施用与注射和其它施用方式相比提供某些优点，例如，关于递送的便利性和速度，以及减少或消除顺应性问题和副作用。然而，生物活性药剂的黏膜递送受到黏膜屏障作用和其它因素的限制。Mucosal administration of therapeutic compounds offers certain advantages over injection and other modes of administration, for example, with respect to convenience and speed of delivery, and reduction or elimination of compliance problems and side effects. However, mucosal delivery of bioactive agents is limited by mucosal barrier effects and other factors.

上皮细胞组成黏膜屏障，并且提供外部环境与黏膜和黏膜下组织以及细胞外区室之间的重要界面。黏膜上皮细胞的一种最重要的功能是确定并且调节黏膜渗透性。在这种情形中，上皮细胞在不同生理区室之间产生选择性渗透屏障。选择渗透性是分子通过细胞质的调控转运(跨细胞途径)和细胞间空间的调节的渗透性(旁细胞途径(paracellular pathway))的结果。Epithelial cells make up the mucosal barrier and provide an important interface between the external environment and the mucosal and submucosal tissues as well as the extracellular compartment. One of the most important functions of mucosal epithelial cells is to determine and regulate mucosal permeability. In this context, epithelial cells create a selectively permeable barrier between different physiological compartments. Selective permeability is the result of regulated transport of molecules through the cytoplasm (transcellular pathway) and regulated permeability of the intercellular space (paracellular pathway).

已知上皮细胞之间的细胞间连接参与上皮细胞屏障作用的维持和调控，以及细胞-细胞黏附。上皮和内皮细胞的紧密连接(TJ)对于调控旁细胞途径的渗透性的细胞-细胞连接特别重要，并且还将细胞表面分成顶部和基底外侧区室。紧密连接在上皮细胞之间形成连续的周围细胞间接触，并且产生水、溶质和免疫细胞的旁细胞运动的调控屏障。通过限制顶部和基底外侧膜结构域之间的膜脂质交换，它们还提供构成细胞极性的第二种类型的屏障。Intercellular junctions between epithelial cells are known to be involved in the maintenance and regulation of epithelial cell barriers, as well as cell-cell adhesion. The tight junctions (TJs) of epithelial and endothelial cells are particularly important for cell-cell junctions that regulate the permeability of paracellular pathways and also divide the cell surface into apical and basolateral compartments. Tight junctions form continuous peripheral cell-cell contacts between epithelial cells and create a regulatory barrier for water, solutes, and paracellular movement of immune cells. They also provide a second type of barrier that constitutes cell polarity by limiting membrane lipid exchange between the apical and basolateral membrane domains.

认为紧密连接直接参与上皮细胞的屏障和防御功能，分别通过产生细胞间密封以生成针对溶质通过旁细胞途径扩散的一级屏障，和通过作用为顶部和基底外侧质膜结构域之间的边界以产生并且维持细胞极性而参与。紧密连接还参与白细胞到达炎性位点的移动。应答化学引诱剂，白细胞通过穿过内皮细胞而从血液移动，并且在黏膜感染的情形中，穿过发炎的上皮细胞。移动主要沿着旁细胞途径发生，并且似乎通过以高度协调和可逆的方式开放和关闭紧密连接而调控。Tight junctions are thought to be directly involved in the barrier and defense functions of epithelial cells, respectively by creating an intercellular seal to create a primary barrier against the diffusion of solutes through the paracellular pathway, and by acting as a boundary between the apical and basolateral plasma membrane domains to Involved in the generation and maintenance of cell polarity. Tight junctions are also involved in the movement of leukocytes to inflammatory sites. In response to chemoattractants, leukocytes move from the blood by crossing endothelial cells and, in the case of mucosal infections, inflamed epithelial cells. Movement occurs mainly along paracellular pathways and appears to be regulated by opening and closing tight junctions in a highly coordinated and reversible manner.

已经鉴定许多蛋白与TJs有关，包括内部和外部质膜蛋白。目前对这些蛋白的复合体结构和相互作用功能的理解还是有限的。在与上皮细胞连接相关的许多蛋白中，已经鉴定了一些种类的跨上皮膜蛋白，其可能在上皮细胞连接的生理调控中起作用。这些包括许多“连接黏附分子”(JAMs)和叫作闭合蛋白、密蛋白和连蛋白的其它TJ-相关的分子。A number of proteins have been identified to be associated with TJs, including inner and outer plasma membrane proteins. The current understanding of the complex structure and interaction function of these proteins is still limited. Among the many proteins associated with epithelial junctions, several classes of transepithelial membrane proteins have been identified that may play a role in the physiological regulation of epithelial junctions. These include a number of "junctional adhesion molecules" (JAMs) and other TJ-related molecules called occludin, claudin, and zonulin.

JAMs、闭合蛋白、和密蛋白延伸到旁细胞空间，并且特别预测这些蛋白是形成相邻上皮细胞之间的上皮屏障和通过上皮细胞层的通道的候选物。在一种模型中，提议闭合蛋白、密蛋白和JAM作为嗜同性结合配偶体相互作用，以在上皮细胞之间产生水、溶质和免疫细胞的旁细胞运动的调控的屏障。JAMs, occludins, and claudins extend into the paracellular space, and these proteins are specifically predicted to be candidates for forming epithelial barriers between adjacent epithelial cells and channels through epithelial cell layers. In one model, claudin, claudin, and JAM are proposed to interact as homophilic binding partners to create a barrier between epithelial cells for the regulation of water, solutes, and paracellular movement of immune cells.

在药物递送的情形中，药物不受递送-增强剂的帮助而透过黏膜表面的上皮细胞层的能力似乎与许多因素有关，包括分子大小、脂溶性、和离子化。通常，低于约300-1,000道尔顿的小分子经常能够透过黏膜屏障，然而，随着分子大小增加，渗透性快速减小。由于这些原因，黏膜药物施用典型地比注射施用需要更大量的药物。其它治疗化合物，包括大分子药物，通常很难通过黏膜递送。除了低的内在渗透性之外，大的大分子药物通常经受有限的扩散，以及内腔和细胞酶促降解和在黏膜位点的快速清除。因此，为了以治疗有效量递送这些更大的分子，需要细胞透性增强剂来帮助它们穿过这些黏膜表面，并且进入系统循环，在那里它们可以迅速地在目标组织上作用。In the case of drug delivery, the ability of a drug to penetrate the epithelial layer of a mucosal surface without the aid of a delivery-enhancing agent appears to be related to a number of factors, including molecular size, lipid solubility, and ionization. Generally, small molecules below about 300-1,000 Daltons are often able to penetrate mucosal barriers, however, as the size of the molecule increases, the permeability decreases rapidly. For these reasons, mucosal drug administration typically requires larger amounts of drug than injectable administration. Other therapeutic compounds, including macromolecular drugs, are often difficult to deliver across the mucosa. In addition to low intrinsic permeability, large macromolecular drugs are often subject to limited diffusion, as well as luminal and cellular enzymatic degradation and rapid clearance at mucosal sites. Therefore, in order to deliver these larger molecules in therapeutically effective amounts, cell-permeability enhancers are required to help them cross these mucosal surfaces and enter the systemic circulation where they can rapidly act on target tissues.

黏膜组织为大分子的自由扩散提供基本的屏障，而在黏膜分泌物中存在的酶促活性可以严重地限制治疗剂，特别是肽和蛋白的生物利用度。在某些黏膜位点，诸如鼻黏膜，由于快速的粘液纤毛清除，递送的蛋白和其它大分子种类的典型的停留时间有限，例如，约15-30分钟或更少。Mucosal tissues provide an essential barrier to the free diffusion of macromolecules, while the presence of enzymatic activity in mucosal secretions can severely limit the bioavailability of therapeutic agents, especially peptides and proteins. At certain mucosal sites, such as the nasal mucosa, the typical residence time of delivered proteins and other macromolecular species is limited, eg, about 15-30 minutes or less, due to rapid mucociliary clearance.

在本领域内对药物制剂和施用治疗化合物的方法存在长久的和尚未满足的需求，其中所述治疗化合物提供增强的黏膜递送，包括被靶向的组织和生理区室如在中枢神经系统中。There is a long-felt and unmet need in the art for pharmaceutical formulations and methods of administering therapeutic compounds that provide enhanced mucosal delivery, including targeted tissues and physiological compartments such as in the central nervous system.

更具体地，在本领域中，对于黏膜递送用于在哺乳动物受试者中治疗疾病和其它不利病况的治疗化合物的安全和可靠的方法与组合物存在需求。对于通过一种或多种黏膜途径提供治疗量的大分子药物的有效递送的方法和组合物存在相关的需求，所述方法和组合物作用快速，易于实施，并且具有有限的不利副作用如黏膜刺激或组织损伤。More specifically, there is a need in the art for safe and reliable methods and compositions for the mucosal delivery of therapeutic compounds for the treatment of diseases and other adverse conditions in mammalian subjects. There is a related need for methods and compositions that provide effective delivery of therapeutic amounts of macromolecular drugs via one or more mucosal routes that are fast acting, easy to implement, and have limited adverse side effects such as mucosal irritation or tissue damage.

在本领域中，还对于增强克服黏膜上皮细胞屏障机制的生物治疗化合物的黏膜递送的方法和组合物存在需求。在此之前，黏膜上皮细胞的选择渗透性被描述为治疗性大分子，包括生物活性肽和蛋白的黏膜递送的主要障碍。因此，在本领域中，对于促进生物治疗化合物黏膜递送的新方法和工具存在尚未满足的需求。特别地，在本领域中，对促进此前证明很难通过黏膜屏障递送的生物治疗化合物的黏膜递送的新方法和制剂存在需求。There is also a need in the art for methods and compositions that enhance the mucosal delivery of biotherapeutic compounds that overcome mucosal epithelial cell barrier mechanisms. Previously, the selective permeability of mucosal epithelial cells was described as a major obstacle to the mucosal delivery of therapeutic macromolecules, including bioactive peptides and proteins. Accordingly, there is an unmet need in the art for new methods and tools to facilitate the mucosal delivery of biotherapeutic compounds. In particular, there is a need in the art for new methods and formulations that facilitate the mucosal delivery of biotherapeutic compounds that have previously proven difficult to deliver across mucosal barriers.

附图简述Brief description of the drawings

图1图示PN159对PTH_1-34渗透的影响，其使用具有其它增强剂(Me-β-CD，DDPC，EDTA)的PN159。Figure 1 illustrates the effect of PN159 on the penetration of PTH_1-34 using PN159 with other enhancers (Me-β-CD, DDPC, EDTA).

图2图示PN159对PTH_1-34渗透的影响，其使用无其它增强剂的PN159。Figure 2 illustrates the effect of PN159 on the penetration of PTH_1-34 using PN159 without other enhancers.

图3图示PN159对肽YY体内渗透的影响。Figure 3 illustrates the effect of PN159 on the penetration of peptide YY in vivo.

图4图示PN159对MC-4受体激动剂渗透的影响。Figure 4 illustrates the effect of PN159 on the penetration of MC-4 receptor agonists.

图5显示25-100μM PN159对40mg/ml乳酸加兰他敏体外透过上皮细胞单层的影响。Figure 5 shows the effect of 25-100μM PN159 on the permeation of epithelial cell monolayer in vitro by 40mg/ml galantamine lactate.

图6显示TJM肽在(A)5℃，(B)25℃，和(C)40℃的化学稳定性。关于pH 4.0，pH 7.3和pH 9.0的数据分别表示为实心菱形、空心正方形和实心三角形。Figure 6 shows the chemical stability of TJM peptides at (A) 5°C, (B) 25°C, and (C) 40°C. Data for pH 4.0, pH 7.3, and pH 9.0 are represented as filled diamonds, open squares, and filled triangles, respectively.

图7图示在每种紧密连接调控肽(TJMP)存在下FITC-葡聚糖MW4000的渗透动力学。PYY制剂作用为阳性对照，和磷酸缓冲盐(PBS)为阴性对照。在用TJMP和FITC-葡聚糖MW4000处理细胞15分钟后以及处理细胞60分钟后，检测细胞渗透。图表显示渗透依赖于TJMP与上皮细胞接触的时间的长短，并且显示所检测的所有TJMPs增强FITC-葡聚糖MW4000的渗透。Figure 7 illustrates the permeation kinetics of FITC-dextran MW4000 in the presence of each tight junction modulating peptide (TJMP). The PYY formulation served as a positive control, and phosphate buffered saline (PBS) as a negative control. Cell infiltration was detected after treatment of cells with TJMP and FITC-dextran MW4000 for 15 minutes and after treatment of cells for 60 minutes. The graphs show that permeation is dependent on the length of time TJMPs are in contact with the epithelial cells and show that all TJMPs tested enhance the permeation of FITC-dextran MW4000.

图8图示在PN159和PEG-PN159处理1小时后跨上皮电阻(TER)减小。Figure 8 illustrates the decrease in transepithelial electrical resistance (TER) after 1 hour of PN159 and PEG-PN159 treatment.

图9图示在用PN159和PEG-PN159处理后FITC葡聚糖3000的渗透性增加。Figure 9 illustrates the increased permeability of FITC-dextran 3000 after treatment with PN159 and PEG-PN159.

图10图示PN159和PEG-PN159的渗透比例。Figure 10 illustrates the penetration ratio of PN159 and PEG-PN159.

图11图示PN159的peg化(pegylation)减少毒性(LDH检测)。Figure 11 shows that pegylation of PN159 reduces toxicity (LDH assay).

图12图示在鼻施用PEG化的肽PN529(PEG-PN159)后增加的平均血浆PYY3-36浓度。Figure 12 illustrates increased mean plasma PYY3-36 concentrations following nasal administration of PEGylated peptide PN529 (PEG-PN159).

图13图示在鼻施用PEG化的肽PN529(PEG-PN159)后增加的平均血浆PYY3-36浓度(对数-线性图)。Figure 13 illustrates increased mean plasma PYY3-36 concentrations following nasal administration of PEGylated peptide PN529 (PEG-PN159) (log-linear graph).

发明详述Detailed description of the invention

本发明通过提供新颖的药物组合物而满足前述需求，并且实现其它目的和优点，所述新颖的药物组合物包括最新发现的紧密连接-开放肽在哺乳动物受试者中增强生物活性药剂的黏膜递送的新颖性应用。The present invention fulfills the aforementioned needs, and achieves other objects and advantages, by providing novel pharmaceutical compositions comprising newly discovered tight junction-opening peptides that enhance the mucosal adhesion of bioactive agents in mammalian subjects. Novel applications of delivery.

本发明的一个方面是一种药物制剂，其包含生物活性剂和增强黏膜递送的有效量的紧密连接调控肽(TJMP)，所述紧密连接调控肽在哺乳动物受试者中可逆地增强生物活性剂的黏膜上皮转运。One aspect of the present invention is a pharmaceutical formulation comprising a bioactive agent and a mucosal delivery enhancing effective amount of a tight junction modulating peptide (TJMP) that reversibly enhances bioactivity in a mammalian subject mucosal epithelial transport of agents.

优选地，紧密连接调控成分包含由2-500个氨基酸残基、或2-100个氨基酸残基、或2-50个氨基酸残基组成的肽或蛋白部分。紧密连接调控肽或蛋白可以是单体或寡聚体，例如，二聚体。Preferably, the tight junction modulating component comprises a peptide or protein moiety consisting of 2-500 amino acid residues, or 2-100 amino acid residues, or 2-50 amino acid residues. A tight junction modulating peptide or protein can be a monomer or an oligomer, eg, a dimer.

紧密连接调控肽可以通过与适当领域的技术人员已知的技术一致的重组或化学合成方式生产。Tight junction modulating peptides can be produced by recombinant or chemical synthesis consistent with techniques known to those skilled in the appropriate art.

先前已经描述过能够调控上皮细胞紧密连接功能的肽(Johnson，P.H.和S.C.Quay，Expert.Opin.Drug Deliv.(关于药物递送的专家观点)2：281-98，2000)。特别地，表明一种新颖的紧密连接调控(TJM)肽，PN159，减少穿过组织屏障的跨上皮电阻(TER)，并且增加具有低细胞毒性和细胞生存能力高保持性的3,000Da MW葡聚糖的旁细胞转运。Peptides capable of modulating tight junction function in epithelial cells have been previously described (Johnson, P.H. and S.C. Quay, Expert. Opin. Drug Deliv. 2:281-98, 2000). In particular, it was shown that a novel tight junction modulating (TJM) peptide, PN159, reduces the transepithelial electrical resistance (TER) across the tissue barrier and increases the 3,000 Da MW glucan with low cytotoxicity and high retention of cell viability Paracellular transport of sugars.

在本发明的优选的实施方案中，所述TJMP选自由下列各项组成的组：In preferred embodiments of the present invention, said TJMP is selected from the group consisting of:

NH2-KLALKLALKALKAALKLA-酰胺NH2-KLALKLALKALKAALKLA-amide

NH2-GWTLNSAGYLLGKINLKALAALAKKIL-酰胺NH2-GWTLNSAGYLLGKINLKALAALAKKIL-amide

NH2-LLETLLKPFQCRICMRNFSTRQARRNHRRRHRR-酰胺NH2-LLETLLKPFQCRICMRNFSTRQARRNHRRRHRR-amide

NH2-AAVALLPAVLLALLAPRKKRRQRRRPPQ-酰胺NH2-AAVALLPAVLLALLAPRKKRRQRRRPPQ-amide

NH2-RKKRRQRRRPPQCAAVALLPAVLLALLAP-酰胺NH2-RKKRRQRRRPPQCAAVALLPAVLLALLAP-amide

NH2-RQIKIWFQNRRMKWKK-酰胺NH2-RQIKIWFQNRRMKWKK-amide

NH2-KGSKKAVTKAQKKDGKKRKRSRKESYSVYVYKVLKQ-酰胺NH2-KGSKKAVTKAQKKDGKKRKRSRKESYSVYVYKVLKQ-amide

NH2-KLWSAWPSLWSSLWKP-酰胺NH2-KLWSAWPSLWSSLWKP-amide

NH2-RRRQRRKRGGDIMGEWGNEIFGAIAGFLG-酰胺NH2-RRRQRRKRGGDIMGEWGNEIFGAIAGFLG-amide

马来酰亚胺-GLGSLLKKAGKKLKQPKSKRKV-酰胺Maleimide-GLGSLLKKAGKKLKQPKSKRKV-amide

NH2-KETWWETWWTEWSQPGRKKRRQRRRRPPQ-酰胺。NH2-KETWWETWWTEWSQPGRKKRRQRRRRPPQ-amide.

在本发明的其它优选实施方案中，所述TJMP选自由下列各项组成的组：In other preferred embodiments of the present invention, said TJMP is selected from the group consisting of:

CNGRCGGKKKLKLLLKLLCNGRCGGKKKLKLLLKLL

LRKLRKRLLRLRKLRKRLLR。LRKLRKRLLRLRKLRKRLLR.

在一个方面中，本发明描述适于跨黏膜递送的治疗性小分子、肽和蛋白的制剂，其中通过紧密连接调控肽的存在而促进跨黏膜递送，其中所述肽与水溶性聚合物缀合。优选地，所述水溶性聚合物是选自由下列各项组成的组的聚环氧烷：α-取代的聚环氧烷衍生物，烷基-封端的聚环氧乙烷，双-聚环氧乙烷，聚(原酸酯)如聚(乳-共-乙交酯)及其衍生物，聚乙二醇(PEG)均聚物及其衍生物，聚丙二醇均聚物及其衍生物，聚(环氧烷)的共聚物，以及聚(环氧烷)的嵌段共聚物或其活化的衍生物。优选地，所述聚环氧烷具有约200到约50,000的分子量。更优选地，所述聚环氧烷具有约200到约20,000的分子量。特别优选的聚环氧烷是聚乙二醇和聚环氧乙烷。In one aspect, the present invention describes formulations of therapeutic small molecules, peptides and proteins suitable for transmucosal delivery, wherein transmucosal delivery is facilitated by the presence of a tight junction modulating peptide, wherein the peptide is conjugated to a water soluble polymer . Preferably, the water-soluble polymer is a polyalkylene oxide selected from the group consisting of α-substituted polyalkylene oxide derivatives, alkyl-terminated polyethylene oxide, bis-polycyclic Ethylene oxide, poly(orthoesters) such as poly(lact-co-glycolide) and its derivatives, polyethylene glycol (PEG) homopolymer and its derivatives, polypropylene glycol homopolymer and its derivatives , copolymers of poly(alkylene oxide), and block copolymers of poly(alkylene oxide) or activated derivatives thereof. Preferably, the polyalkylene oxide has a molecular weight of from about 200 to about 50,000. More preferably, the polyalkylene oxide has a molecular weight of from about 200 to about 20,000. Particularly preferred polyalkylene oxides are polyethylene glycol and polyethylene oxide.

TJMP可以与多于一条的水溶性链缀合。在一个优选的实施方案中，所述聚(环氧烷)链是聚乙二醇(PEG)链，其可以具有约0.2和约200千道尔顿(kDa)的分子大小。TJMPs can be conjugated to more than one water soluble chain. In a preferred embodiment, the poly(alkylene oxide) chains are polyethylene glycol (PEG) chains, which can have molecular sizes between about 0.2 and about 200 kilodaltons (kDa).

水溶性聚合物可以通过间隔臂与紧密连接调控肽缀合。这种连接可以是可逆的。所述水溶性聚合物可以是线性的或可以是有支链的。The water soluble polymer can be conjugated to the tight junction modulating peptide through a spacer arm. This connection can be reversible. The water soluble polymer may be linear or may be branched.

在一个实施方案中，肽与单一聚(环氧烷)链共价连接。在一个相关的实施方案中，聚(环氧烷)具有低于2.00或低于1.20的多分散性值(Mw/Mn)。所述聚(环氧烷)链可以是有支链的或没有支链的。In one embodiment, the peptide is covalently linked to a single poly(alkylene oxide) chain. In a related embodiment, the poly(alkylene oxide) has a polydispersity value (Mw/Mn) of less than 2.00 or less than 1.20. The poly(alkylene oxide) chains may be branched or unbranched.

与水溶性聚合物诸如聚(乙二醇)(PEG)及PEG的衍生物缀合已经用作提高蛋白特别是注射剂型的半衰期的策略(Caliceti，P和F.M.Veronese，Adv.Drug Deliv.Rev.(高级药物递送综述)55：1261-77，2003)。用聚合物如PEG修饰肽和蛋白的其它潜在的益处包括化学(Diwan，M.和T.G.Park，Int.J.Pharm.(国际药学杂志)252：111-22，2003)和生化稳定性(Na，D.H.等，J.Pharm.Sci.(药学科学杂志)93：256-61，2004)以及免疫原性的减少(Yang，Z.等，Cancer Res.(癌症研究)64：6673-78，2004)。Conjugation to water-soluble polymers such as poly(ethylene glycol) (PEG) and derivatives of PEG has been used as a strategy to increase the half-life of proteins, especially in injectable dosage forms (Caliceti, P and F.M. Veronese, Adv. Drug Deliv. Rev. (Advanced Drug Delivery Reviews) 55:1261-77, 2003). Other potential benefits of modifying peptides and proteins with polymers such as PEG include chemical (Diwan, M. and T.G. Park, Int. J. Pharm. (International Journal of Pharmacy) 252:111-22, 2003) and biochemical stability (Na , D.H. et al., J.Pharm.Sci. (Journal of Pharmaceutical Sciences) 93:256-61, 2004) and the reduction of immunogenicity (Yang, Z. et al., Cancer Res. (Cancer Research) 64:6673-78, 2004 ).

缀合到蛋白上的PEG应用的最常见实例是在所述PEG链具有足够长的分子量以赋予上述作用的情形中。特别地，已经描述需要至少20kDaMW的PEG。例如，Holtsberg等(Holtsberg，F.W.等，J.Control Rel.(控释杂志)80：259-71，2002)表明，对于与PEG缀合的蛋白精氨酸脱亚氨酶(deiminase)，当PEG为20kDa或更大时，在小鼠中静脉内施用时制剂的药物代谢动力学和药效特性增加。当PEG MW低于20kDa时，几乎没有效果。在另一个实例中，在大鼠中，对肽鲑鱼降钙素的单-PEG化导致提高的鼻内生物利用度，所述提高与PEG分子量(MW)成反比(Lee，K.C.等，Calcif.Tssue Int.(国际钙化组织)73：545-9，2003，和Shin，B.S.等，Chem.Pharm.Bull.(Tokyo)(化学和药学公告(东京))52：957-60，2004)，其通过引用完全结合于此。The most common example of the use of PEG conjugated to proteins is where the PEG chains are of sufficiently long molecular weight to confer the effects described above. In particular, it has been described that PEG of at least 20 kDa MW is required. For example, Holtsberg et al. (Holtsberg, F.W. et al., J. Control Rel. (Journal of Controlled Release) 80:259-71, 2002) showed that for protein arginine deiminase (deiminase) conjugated to PEG, when PEG At 20 kDa or greater, the pharmacokinetic and pharmacodynamic properties of the formulation are increased when administered intravenously in mice. When the PEG MW is below 20kDa, there is little effect. In another example, in rats, mono-PEGylation of the peptide salmon calcitonin resulted in increased intranasal bioavailability that was inversely proportional to PEG molecular weight (MW) (Lee, K.C. et al., Calcif. Tssue Int. (International Calcification Organization) 73:545-9, 2003, and Shin, B.S. et al., Chem.Pharm.Bull. (Tokyo) (Chemical and Pharmaceutical Bulletin (Tokyo)) 52:957-60, 2004), its Fully incorporated herein by reference.

一些优选的聚(环氧烷)选择由下列各项组成的组：α-取代的聚(环氧烷)衍生物，聚(乙二醇)(PEG)均聚物及其衍生物，聚(丙二醇)(PPG)均聚物及其衍生物，聚(环氧乙烷)(PEO)聚合物及其衍生物，双-聚(环氧乙烷)及其衍生物，聚(环氧烷)的共聚物，和聚(环氧烷)的嵌段共聚物，聚(丙交酯-共-乙交酯)及其衍生物，或它们的活化衍生物。所述水溶性聚合物可以具有约200到约40000Da，优选地约200到约20000Da，或约200到10000Da，或约200到5000Da的分子量。Some preferred poly(alkylene oxide)s are selected from the group consisting of α-substituted poly(alkylene oxide) derivatives, poly(ethylene glycol) (PEG) homopolymers and derivatives thereof, poly( Propylene glycol) (PPG) homopolymer and its derivatives, poly(ethylene oxide) (PEO) polymer and its derivatives, bis-poly(ethylene oxide) and its derivatives, poly(alkylene oxide) and block copolymers of poly(alkylene oxide), poly(lactide-co-glycolide) and derivatives thereof, or activated derivatives thereof. The water soluble polymer may have a molecular weight of about 200 to about 40000 Da, preferably about 200 to about 20000 Da, or about 200 to 10000 Da, or about 200 to 5000 Da.

通常，紧密连接调控肽和PEG或其它水溶性聚合物之间的缀合物可以抵抗生理作用，包括蛋白质水解、酶作用或水解。备选地，所述缀合物可以通过生物降解作用，例如前体-药物途径而裂解。优选地，所述分子与单一聚(环氧烷)链共价连接，所述聚(环氧烷)链可以无支链或有支链。缀合的方法通常是普通技术人员所知的，例如，美国专利号5,595,732；美国专利号5,766,897；美国专利号5,985,265；美国专利号6,528,485；美国专利号6,586,398；美国专利号6,869,932；和美国专利号6,706,289。Typically, conjugates between tight junction modulating peptides and PEG or other water-soluble polymers are resistant to physiological effects, including proteolysis, enzymatic action, or hydrolysis. Alternatively, the conjugate may be cleaved by biodegradation, eg, the pro-drug pathway. Preferably, the molecule is covalently linked to a single poly(alkylene oxide) chain, which may be unbranched or branched. Methods of conjugation are generally known to those of ordinary skill, for example, U.S. Patent No. 5,595,732; U.S. Patent No. 5,766,897; U.S. Patent No. 5,985,265; U.S. Patent No. 6,528,485; .

在本发明的另一个方面中，TJMP减小穿过黏膜组织屏障的电阻。在一个优选的实施方案中，电阻的减少为应用渗透增强剂前的电阻的至少80％。在一个相关的实施方案中，TJMP增加分子穿过黏膜组织屏障的渗透性，优选地至少两倍。在另一个实施方案中，所述增加的渗透性是旁细胞的。在另一个实施方案中，所增加的渗透性由紧密连接的修饰而导致。在一个备选的实施方案中，所增加的渗透性是跨细胞的，或者是跨细胞和旁细胞的结合。In another aspect of the invention, TJMPs reduce electrical resistance across mucosal tissue barriers. In a preferred embodiment, the reduction in electrical resistance is at least 80% of the electrical resistance prior to application of the penetration enhancer. In a related embodiment, the TJMP increases the permeability of the molecule across the mucosal tissue barrier, preferably at least twofold. In another embodiment, said increased permeability is paracellular. In another embodiment, the increased permeability results from modification of tight junctions. In an alternative embodiment, the increased permeability is transcellular, or a combination of transcellular and paracellular.

在本发明的另一个方面中，黏膜组织层包括上皮细胞层。在一个优选的实施方案中，上皮细胞选自由气管细胞，支气管细胞，肺泡细胞，鼻细胞，肺细胞，胃肠细胞，表皮细胞或口腔细胞组成的组，优选鼻细胞。In another aspect of the invention, the mucosal tissue layer comprises an epithelial cell layer. In a preferred embodiment, the epithelial cells are selected from the group consisting of tracheal cells, bronchial cells, alveolar cells, nasal cells, lung cells, gastrointestinal cells, epidermal cells or oral cavity cells, preferably nasal cells.

在本发明的另一个方面中，活性试剂是肽或蛋白。所述肽或蛋白可以具有2-1000个氨基酸。在一个优选的实施方案中，所述肽或蛋白包括2-50个氨基酸。在另一个实施方案中，所述肽或蛋白是环形的。在另一个实施方案中，所述肽或蛋白通过物理或化学键合形成二聚体或更高级的寡聚体。In another aspect of the invention, the active agent is a peptide or protein. The peptide or protein may have 2-1000 amino acids. In a preferred embodiment, the peptide or protein comprises 2-50 amino acids. In another embodiment, the peptide or protein is circular. In another embodiment, the peptides or proteins form dimers or higher oligomers through physical or chemical bonding.

在一个优选的实施方案中，所述肽或蛋白选自由GLP-1，PYY_3-36，PTH_1-34和胰高血糖素样肽的抑制剂-4组成的组。在另一个实施方案中，生物活性剂是蛋白，优选地选自由β-干扰素、α-干扰素、胰岛素、促红细胞生成素、G-CSF、和GM-CSF、生长激素、以及它们的任一种的类似物组成的组。In a preferred embodiment, said peptide or protein is selected from the group consisting of GLP-1,_PYY3-36 ,_PTH1-34 and inhibitor of glucagon-like peptide-4. In another embodiment, the bioactive agent is a protein, preferably selected from the group consisting of beta-interferon, alpha-interferon, insulin, erythropoietin, G-CSF, and GM-CSF, growth hormone, and any of them A group of analogs.

本发明的渗透性肽包括PN529，其包含序列WEAALAEALAEALAEHLASQPKSKRKV(SEQ ID NO 57)。Penetrant peptides of the invention include PN529, which comprises the sequence WEAALAEALAEALAEHLASQPKSKRKV (SEQ ID NO 57).

本发明的另一个方面是将分子施用给动物的方法，其包括制备任何上述制剂，并且使得所述制剂与所述动物的黏膜表面接触。在一个优选的实施方案中，所述黏膜表面是鼻内的。Another aspect of the invention is a method of administering a molecule to an animal comprising preparing any of the above formulations, and bringing said formulation into contact with a mucosal surface of said animal. In a preferred embodiment, said mucosal surface is intranasal.

本发明的另一个方面是包含任一种上述制剂的剂型，其中所述剂型是液体，优选的是小滴形式。备选地，所述剂型还可以是固体，在施用之前在液体中重构，作为散剂施用，或者以胶囊、片剂或凝胶形式。Another aspect of the invention is a dosage form comprising any of the above formulations, wherein said dosage form is a liquid, preferably in the form of droplets. Alternatively, the dosage form may also be solid, to be reconstituted in liquid prior to administration, administered as a powder, or in capsule, tablet or gel form.

本发明的另一个方面是在哺乳动物受试者中可逆地增强生物试剂的黏膜上皮转运的分子，其具有紧密连接调控成分肽(TJMP)，TJMP类似物，TJMP或TJMP类似物的缀合物，或其复合物。Another aspect of the invention is a molecule that reversibly enhances mucosal epithelial transport of biological agents in a mammalian subject, having a tight junction modulating component peptide (TJMP), a TJMP analog, a conjugate of TJMP or a TJMP analog , or their complexes.

本发明的渗透性肽包括PN159，其具有序列NH2-KLALKLALKALKAALKLA-酰胺。本发明中包含的是本文公开的PN159的类似物，这些类似物的组合，和PN159的任何衍生物，变体，片段，模拟体或融合分子。Penetrant peptides of the invention include PN159, which has the sequence NH2-KLALKLALKALKAALKLA-amide. Included in the present invention are analogs of PN159 disclosed herein, combinations of these analogs, and any derivatives, variants, fragments, mimetibodies or fusion molecules of PN159.

所述渗透性试剂可逆增加黏膜上皮旁细胞转运，其典型地通过在受试者中黏膜上皮表面调节上皮细胞紧密连接结构和/或生理性而增加。这一作用典型地包括通过渗透性试剂而抑制相邻上皮细胞的上皮膜黏附蛋白之间的同型或异型结合。用于这种同型或异型结合阻滞的靶点蛋白可以选自各种相关的连接黏附分子(JAMs)、闭合蛋白或密蛋白。The permeation agent reversibly increases mucosal paraepithelial cell transport, which is typically increased by modulating epithelial tight junction structure and/or physiology at the mucosal epithelial surface in a subject. This action typically involves inhibition of homotypic or heterotypic binding between epithelial membrane adhesion proteins of adjacent epithelial cells by permeabilizing agents. Target proteins for this homo- or heterotypic binding block can be selected from various related junctional adhesion molecules (JAMs), occludins or claudins.

上皮细胞生物学epithelial cell biology

已经克隆编码鼠连接黏附分子-1(JAM-1)的cDNA，并且其对应一种预测的I型跨膜蛋白(包含单一跨膜结构域)，具有约32-kD的分子量(Williams，等，Molecular Immunology(分子兔疫学)36：1175-1188，1999；Gupta，等，IUBMB Life(IUBMB生命)50：51-56，2000；Ozaki，等，J.Immunol.(免疫学杂志)163：553-557，1999；Martin-Padura，等，J.Cell.Biol.(细胞生物学杂志)142：117-127，1998)。所述分子的细胞外片段包含两个Ig-样结构域，其被描述为氨基端“VH-型”和羧基端“C2-型”羧基端β-夹心折叠(Bazzoni等，Microcirculation(微循环)8：143-152，2001)。鼠JAM-1还包含两个用于N-糖基化的位点，和一个细胞质结构域。JAM-1蛋白是免疫球蛋白(Ig)超家族的一员，并且位于上皮细胞和内皮细胞的紧密连接处。超级结构研究表明JAM-1被限制在包含闭合蛋白和密蛋白的原纤维(fibrils)的膜区域。A cDNA encoding the murine junctional adhesion molecule-1 (JAM-1) has been cloned and corresponds to a predicted type I transmembrane protein (containing a single transmembrane domain) with a molecular weight of approximately 32-kD (Williams, et al. Molecular Immunology (Molecular Rabbit Epidemiology) 36: 1175-1188, 1999; Gupta, et al., IUBMB Life (IUBMB Life) 50: 51-56, 2000; Ozaki, et al., J. Immunol. (Journal of Immunology) 163: 553- 557, 1999; Martin-Padura, et al., J. Cell. Biol. 142: 117-127, 1998). The extracellular fragment of the molecule contains two Ig-like domains described as an amino-terminal "VH-type" and carboxy-terminal "C2-type" carboxy-terminal β-sandwich fold (Bazzoni et al., Microcirculation (microcirculation) 8:143-152, 2001). Murine JAM-1 also contains two sites for N-glycosylation, and a cytoplasmic domain. The JAM-1 protein is a member of the immunoglobulin (Ig) superfamily and is located at the tight junctions of epithelial and endothelial cells. Superstructural studies indicate that JAM-1 is restricted to membrane regions containing fibrils of claudin and claudin.

另一个JAM家族成员，叫作“血管内皮细胞连接-相关分子”(VE-JAM)，包含两个细胞外免疫球蛋白样结构域，一个跨膜结构域，和一个相对短的细胞质尾部。VE-JAM主要位于内皮细胞的细胞内边界(Palmeri，等，J.Biol.Chem.(生物化学杂志)275：19139-19145，2000)。VE-JAM由小静脉的内皮细胞高度表达，并且还由其它血管的内皮细胞表达。另一个报道的JAM家族成员，JMA-3，具有预测的氨基酸序列，其分别与JAM-2和JAM-1表现出36％和32％的相同性。JAM-3表现出广泛的组织表达性，在肾、脑和胎盘中有明显更高的水平。在细胞水平上，JAM-3转录物在内皮细胞内表达。已经报道JAM-3和JAM-2为结合配偶体。特别地，报道JAM-3胞外结构域结合JAM2-Fc。JAM-3蛋白在激活之后在外周血淋巴细胞上上调。(Pia Arrate，等，J.Biol.Chem.(生物化学杂志)276：45826-45832，2001)。Another JAM family member, termed "vascular endothelial cell junction-associated molecule" (VE-JAM), contains two extracellular immunoglobulin-like domains, a transmembrane domain, and a relatively short cytoplasmic tail. VE-JAM is mainly located at the intracellular border of endothelial cells (Palmeri, et al., J. Biol. Chem. 275: 19139-19145, 2000). VE-JAM is highly expressed by endothelial cells of venules and is also expressed by endothelial cells of other blood vessels. Another reported JAM family member, JMA-3, has a predicted amino acid sequence that exhibits 36% and 32% identity to JAM-2 and JAM-1, respectively. JAM-3 exhibited broad tissue expression, with significantly higher levels in kidney, brain and placenta. At the cellular level, JAM-3 transcripts are expressed in endothelial cells. JAM-3 and JAM-2 have been reported as binding partners. In particular, the JAM-3 extracellular domain was reported to bind JAM2-Fc. JAM-3 protein is upregulated on peripheral blood lymphocytes following activation. (Pia Arrate, et al., J. Biol. Chem. 276:45826-45832, 2001).

另一种提议的参与上皮细胞紧密连接调控的跨膜黏附蛋白是闭合蛋白。闭合蛋白是一种约65-kD的II型跨膜蛋白，其由4个跨膜结构域、2个胞外环和一个大的C-端胞质结构域组成(Furuse，等，J.Cell.Biol.(细胞生物学杂志)123：1777-1788(1993)；Furuse，等，J.Cell.Biol.(细胞生物学杂志)127：1617-1626，1994)。当通过免疫-冷冻断裂电镜(immuno-freezefracture electron microscopy)观察时，闭合蛋白直接集中在紧密连接原纤维内(Fujimoto，J.Cell Sci.(细胞科学杂志)108：3443-3449，1995)。Another proposed transmembrane adhesion protein involved in the regulation of tight junctions in epithelial cells is occludin. Occludin is an approximately 65-kD type II transmembrane protein consisting of 4 transmembrane domains, 2 extracellular loops and a large C-terminal cytoplasmic domain (Furuse, et al., J. Cell . Biol. 123: 1777-1788 (1993); Furuse, et al., J. Cell. Biol. 127: 1617-1626, 1994). When viewed by immuno-freezefracture electron microscopy, occludin concentrates directly within tight junction fibrils (Fujimoto, J. Cell Sci. 108:3443-3449, 1995).

另外两种紧密连接整合膜蛋白，密蛋白-1和密蛋白-2，通过从鸡肝直接生化分级分离富连接的膜而鉴定(Furuse，等，J.Cell.Biol.(细胞生物学杂志)141：1539-1550，1998)。发现密蛋白-1和密蛋白-2与闭合蛋白共同纯化，作为十二烷基硫酸钠-聚丙烯酰胺凝胶电泳上的一条大约22-kD的宽凝胶条带。从小鼠cDNA文库克隆的两个非常相关的蛋白的推定的序列预测有4个跨膜螺旋，2个短的细胞外环，和短的细胞质N-和C-端。尽管与闭合蛋白拓扑相似，它们不享有序列同源性。后来，已经克隆了6个其它密蛋白基因产物(密蛋白-3到密蛋白-8)，并且表明位于紧密连接原纤维内，如通过免疫金冷冻断裂标记所确定的那样(Morita，等，Proc.Natl.Acad.Sci.USA(美国科学院学报)96：511-516，1999)。假定在不存在闭合蛋白时保持屏障，那么密蛋白-1到密蛋白-8被视为细胞外空间的主要密封-形成元件的候选物。Two other tight junction integral membrane proteins, claudin-1 and claudin-2, were identified by direct biochemical fractionation of junction-rich membranes from chicken livers (Furuse, et al., J. Cell. Biol. 141:1539-1550, 1998). Claudin-1 and claudin-2 were found to co-purify with claudin as a broad approximately 22-kD gel band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The deduced sequences of two closely related proteins cloned from mouse cDNA libraries predicted 4 transmembrane helices, 2 short extracellular loops, and short cytoplasmic N- and C-termini. Despite being topologically similar to occludins, they do not share sequence homology. Later, six other claudin gene products (claudin-3 to claudin-8) have been cloned and shown to be located within tight junction fibrils as determined by immunogold freeze-fracture labeling (Morita, et al., Proc. . Natl. Acad. Sci. USA (Proceedings of the American Academy of Sciences) 96: 511-516, 1999). Assuming that the barrier is maintained in the absence of occludins, claudin-1 to claudin-8 are considered candidates for the major seal-forming elements of the extracellular space.

位于上皮细胞连接处的其它细胞质蛋白包括连蛋白、偶对蛋白(symplekin)、带蛋白、和7H6。连蛋白报道为与闭合蛋白的细胞质尾部结合的细胞质蛋白。代表这一蛋白家族的是″ZO-1，ZO-2，和ZO-3″。推定连蛋白是亚洲霍乱弧菌(Vibrio cholerae)来源的紧密连接毒素(ZOT)的人蛋白类似物。Other cytoplasmic proteins located at epithelial junctions include zonulin, symplekin, zona, and 7H6. Zongin is reported as a cytoplasmic protein that associates with the cytoplasmic tail of occludin. Representative of this family of proteins are "ZO-1, ZO-2, and ZO-3". Zonulin is putatively a human protein analog of tight junction toxin (ZOT) of Asian Vibrio cholerae origin.

连蛋白可能在发育、生理和病理学过程--包括组织形态发生，流体、大分子和白细胞在肠腔和间质之间的运动，和炎性/自体免疫紊乱过程中的紧密连接调控中起作用(参见，例如，Wang，等，J.Cell.Sci.(细胞科学杂志)113：4435-40，2000；Fasano，等，Lancet(柳叶刀)355：1518-9，2000；Fasano，Ann.N.Y.Acad.Sci.(纽约科学院年刊)915：214-222，2000)。在腹腔疾病的急性阶段，连蛋白表达在肠组织中增加，所述腹腔疾病是紧密连接打开并且渗透性增加的一种临床病况。在体外非人灵长类肠上皮细胞中，连蛋白诱导紧密连接解体，和随后的肠渗透性增加。Zonulin may play a role in tight junction regulation during developmental, physiological and pathological processes including tissue morphogenesis, movement of fluids, macromolecules and leukocytes between the intestinal lumen and interstitium, and inflammatory/autoimmune disorders Effect (see, for example, Wang, et al., J.Cell.Sci. (Journal of Cell Science) 113:4435-40, 2000; Fasano, et al., Lancet (Lancet) 355:1518-9, 2000; Fasano, Ann .N.Y.Acad.Sci. (Annual of the New York Academy of Sciences) 915:214-222, 2000). Zonulin expression is increased in intestinal tissue during the acute phase of celiac disease, a clinical condition in which tight junctions are opened and permeability increased. Zongin induces tight junction disassembly and subsequent increase in intestinal permeability in nonhuman primate intestinal epithelial cells in vitro.

在活性亚洲霍乱弧菌(V.cholerae)ZOT片段和人连蛋白中的氨基酸比较鉴定了在这两种蛋白N端区域内的推定的受体结合结构域。ZOT生物活性结构域增加肠渗透性，这通过与哺乳动物细胞受体相互作用，随后激活胞内信号传导，导致胞内紧密连接解体而进行。所述ZOT生物活性结构域向所述蛋白的羧基端定位，并且与由亚洲霍乱弧菌产生的预测的裂解产物一致。这一结构域与连蛋白，ZOT哺乳动物类似物，共享有推定的受体-结合基序。在ZOT活性片段和连蛋白之间的氨基酸比较，与基因定点诱变实验结合，表明对着加工的ZOT的氨基端和连蛋白的氨基端的八肽受体-结合结构域。(Di Pierro，等，J.Biol.Chem.(生物化学杂志)276：19160-19165，2001)。报道ZO-1结合肌动蛋白、AF-6、ZO-相关激酶(ZAK)、胞衬蛋白，和α-联蛋白。Amino acid comparisons in active Asiatic V. cholerae ZOT fragments and human zonulin identified putative receptor binding domains within the N-terminal regions of both proteins. The ZOT biologically active domain increases intestinal permeability by interacting with mammalian cell receptors, followed by activation of intracellular signaling leading to the disassembly of intracellular tight junctions. The ZOT bioactive domain is positioned towards the carboxy-terminus of the protein and is consistent with the predicted cleavage product produced by V. cholerae Asia. This domain shares a putative receptor-binding motif with zonulin, the mammalian analog of ZOT. Amino acid comparisons between the active fragment of ZOT and zonulin, combined with gene site-directed mutagenesis experiments, revealed an octapeptide receptor-binding domain facing the amino-terminus of processed ZOT and the amino-terminus of zonulin. (Di Pierro, et al., J. Biol. Chem. 276:19160-19165, 2001). ZO-1 is reported to bind actin, AF-6, ZO-associated kinase (ZAK), fodsin, and α-catenin.

用于本发明的渗透性肽包括天然的或合成的、治疗或预防活性的肽(包括两个或多个共价连接的氨基酸)、蛋白、肽或蛋白片段、肽或蛋白类似物、肽或蛋白模拟物、以及活性肽或蛋白的化学修饰衍生物或盐。因此，当用于本文时，术语“渗透性肽”通常意欲包括所有这些活性种类，即，肽和蛋白、肽和蛋白片段、肽和蛋白类似物、肽和蛋白模拟物、以及活性肽或蛋白的化学修饰衍生物和盐。通常，渗透性肽或蛋白是易于通过部分取代、添加或删除天然存在的或天然的(例如，野生型、天然存在的突变体、或等位基因变体)肽或蛋白序列内的氨基酸而获得的突变蛋白。另外，包括天然肽或蛋白的生物活性片段。所述突变的衍生物和片段基本上保持天然肽或蛋白的需要的生物活性。在具有碳水化合物链的肽或蛋白的情形中，通过在这些碳水化合物种类中的改变而标记的生物活性变体也包括在本发明内。Penetrant peptides useful in the present invention include natural or synthetic, therapeutically or prophylactically active peptides (comprising two or more covalently linked amino acids), proteins, peptides or protein fragments, peptides or protein analogs, peptides or Protein mimics, and chemically modified derivatives or salts of active peptides or proteins. Thus, as used herein, the term "penetrant peptide" is generally intended to include all such active species, i.e., peptides and proteins, peptide and protein fragments, peptide and protein analogs, peptide and protein mimetics, and active peptide or protein Chemically modified derivatives and salts. Typically, a penetrant peptide or protein is readily obtained by partially substituting, adding, or deleting amino acids within a naturally occurring or naturally occurring (e.g., wild type, naturally occurring mutant, or allelic variant) peptide or protein sequence mutant protein. Additionally, biologically active fragments of native peptides or proteins are included. Such mutated derivatives and fragments substantially retain the desired biological activity of the native peptide or protein. In the case of peptides or proteins with carbohydrate chains, biologically active variants labeled by alterations in these carbohydrate species are also included in the present invention.

用于本发明的方法和组合物的渗透性肽、蛋白、类似物和模拟物通常配制在药物组合物中，所述药物组合物包含黏膜递送-增强或渗透有效量的渗透性肽、蛋白、类似物或模拟物，其通过在哺乳动物受试者中调控上皮细胞连接结构和/或生理学而可逆地增强黏膜上皮旁细胞转运。Penetrant peptides, proteins, analogs and mimetics for use in the methods and compositions of the invention are typically formulated in pharmaceutical compositions comprising a mucosal delivery-enhancing or permeation effective amount of a penetrant peptide, protein, An analog or mimetic that reversibly enhances mucosal paraepithelial cell transport by modulating epithelial cell junction structure and/or physiology in a mammalian subject.

生物活性剂bioactive agent

本发明的方法和组合物针对增加广谱生物活性剂的黏膜如鼻内递送，以在哺乳动物受试者中获得治疗、预防或其它理想的生理结果。当用于本申请时，术语“生物活性剂”包括当按照本文的方法和组合物黏膜施用给哺乳动物受试者时产生生理反应的任何物质。在这种情形中，有用的生物活性剂包括在临床医学的所有主要领域应用的治疗或预防试剂，以及营养物、辅因子、酶(内源的或外源的)、抗氧化剂等等。因此，所述生物活性剂可以是水溶性的或水不溶性的，并且可以包括更高分子量的蛋白、肽、碳水化合物、糖蛋白、脂质和/或糖脂、核苷、多核苷酸和其它活性剂。The methods and compositions of the invention are directed to increasing the mucosal, eg, intranasal, delivery of a broad spectrum of bioactive agents to achieve therapeutic, prophylactic, or other desirable physiological outcomes in mammalian subjects. As used herein, the term "bioactive agent" includes any substance that produces a physiological response when administered mucosally to a mammalian subject according to the methods and compositions herein. In this context, useful bioactive agents include therapeutic or prophylactic agents used in all major fields of clinical medicine, as well as nutrients, cofactors, enzymes (endogenous or exogenous), antioxidants and the like. Thus, the bioactive agent may be water soluble or water insoluble and may include higher molecular weight proteins, peptides, carbohydrates, glycoproteins, lipids and/or glycolipids, nucleosides, polynucleotides and other active agent.

在本发明的方法和组合物中有用的药剂包括含有广谱化合物的药物和大分子治疗或预防剂，其包括小分子药物，肽，蛋白和疫苗试剂。用于本发明的代表性药剂是生物活性的，用于治疗或预防受试者中选择的疾病或病况。在这种情形中，生物活性可以确定为关于生理参数、标记或与受试者疾病或病况相关的临床症状的任何显著的(即，可检测的，令人满意地显著)作用，其通过适当的体外或体内检测系统包括实际患者、细胞培养、样品检测或可接受的动物模型进行评估。Agents useful in the methods and compositions of the invention include pharmaceuticals and macromolecular therapeutic or prophylactic agents comprising a broad spectrum of compounds, including small molecule drugs, peptides, proteins and vaccine agents. Representative agents for use in the invention are biologically active for treating or preventing a disease or condition of choice in a subject. In this context, biological activity can be determined as any significant (i.e., detectable, satisfactorily significant) effect on a physiological parameter, marker, or clinical symptom associated with a disease or condition in a subject, which is determined by appropriate Our in vitro or in vivo assay systems include actual patients, cell cultures, sample assays, or acceptable animal models for evaluation.

本发明的方法和组合物为治疗哺乳动物受试者中的疾病和其它病况提供出乎意料的益处，例如，所述益处通过提供治疗和预防化合物黏膜递送的提高的速率、持续时间、保真性或控制而调控，以到达受试者中选择的生理区室(例如，进入或穿过鼻黏膜，进入系统循环或中枢神经系统(CNS)，或者到达受试者内任何选择的靶点器官、组织、流体或细胞或细胞外区室)。The methods and compositions of the present invention provide unexpected benefits for treating diseases and other conditions in mammalian subjects, for example, by providing improved rate, duration, fidelity of mucosal delivery of therapeutic and prophylactic compounds or controlled to reach a selected physiological compartment in a subject (e.g., into or through the nasal mucosa, into the systemic circulation or central nervous system (CNS), or to any selected target organ in a subject, tissue, fluid or cell or extracellular compartment).

在各种代表性实施方案中，本发明的方法和组合物可以结合选自下列各项的一种或多种生物活性剂：In various representative embodiments, the methods and compositions of the invention may incorporate one or more bioactive agents selected from the group consisting of:

阿片样物质(opiods)或阿片样物质拮抗剂，诸如吗啡，氢吗啡酮，羟吗啡酮，洛伐啡诺(lovorphanol)，左洛啡烷，可待因，纳美芬，烯丙吗啡，纳洛酮(nalozone)，纳曲酮，丁丙诺啡，布托啡诺，和纳布啡(nalbufine)；Opioids or opioid antagonists such as morphine, hydromorphone, oxymorphone, lovorphanol, levorphanol, codeine, nalmefene, nalamorphine, nalmefene, nalozone, naltrexone, buprenorphine, butorphanol, and nalbufine;

皮质酮，诸如可的松，氢化可的松，氟氢可的松，泼尼松，泼尼松龙，甲泼尼龙，曲安西龙，地塞米松(dexamethoasone)，倍他米松(betamethoasone)，帕拉米松(paramethosone)，和氟轻松；corticosterone, such as cortisone, hydrocortisone, fludrocortisone, prednisone, prednisolone, methylprednisolone, triamcinolone, dexamethoasone, betamethoasone, paramethosone, and fluocinolone;

其它抗炎药，诸如秋水仙碱，布洛芬，吲哚美辛，和吡罗昔康；抗病毒剂诸如阿昔洛韦，利巴韦林(ribavarin)，三氟胸苷(trifluorothyridine)，Ara-A(阿糖呋喃糖腺嘌呤)，酰基鸟苷，去甲脱氧鸟嘌呤核苷(nordeoxyguanosine)，叠氮胸苷，二脱氧腺苷，和二脱氧胞苷；抗雄激素药如螺内酯；Other anti-inflammatory drugs such as colchicine, ibuprofen, indomethacin, and piroxicam; antiviral agents such as acyclovir, ribavirin, trifluorothyridine, Ara- A (arabinofuranosyladenosine), acylguanosine, nordeoxyguanosine (nordeoxyguanosine), azidothymidine, dideoxyadenosine, and dideoxycytidine; antiandrogens such as spironolactone;

雄激素，如睾酮；Androgens, such as testosterone;

雌激素，如雌二醇；Estrogens, such as estradiol;

黄体酮；Progesterone;

肌肉松弛剂，如罂粟碱；muscle relaxants, such as papaverine;

血管舒张药，如硝酸甘油，血管活性肠肽和降钙素相关的基因肽；Vasodilators, such as nitroglycerin, vasoactive intestinal peptide, and calcitonin-related gene peptide;

抗组胺剂，如赛庚啶；antihistamines, such as cyproheptadine;

具有组胺受体位点阻滞活性的试剂，如多塞平，丙米嗪，和西咪替丁；Agents with histamine receptor site blocking activity, such as doxepin, imipramine, and cimetidine;

止咳药，如右美沙芬；精神安定药如氯氮平片剂；抗心律失常药；Cough suppressants such as dextromethorphan; neuroleptics such as clozapine tablets; antiarrhythmic drugs;

抗癫痫药；antiepileptic drugs;

酶，诸如过氧化物歧化酶和神经脑啡肽酶(neuroenkephalinase)；Enzymes such as superoxide dismutase and neuroenkephalinase;

抗真菌剂，诸如两性霉素B，灰黄霉素，咪康唑，酮康唑，噻康唑，伊曲康唑，和氟康唑；Antifungal agents such as amphotericin B, griseofulvin, miconazole, ketoconazole, tioconazole, itraconazole, and fluconazole;

抗细菌剂，诸如青霉素，头孢菌素，四环素，氨基糖苷，红霉素(erythromicin)，庆大霉素，多粘菌素B；Antibacterial agents such as penicillins, cephalosporins, tetracyclines, aminoglycosides, erythromicin, gentamicin, polymyxin B;

抗癌剂，诸如5-氟尿嘧啶，博来霉素，甲氨喋呤，和羟基脲，二脱氧肌苷，氟尿苷，6-巯基嘌呤，多柔比星，柔红霉素，伊达比星(I-darubicin)，泰素和紫杉醇；Anticancer agents such as 5-fluorouracil, bleomycin, methotrexate, and hydroxyurea, dideoxyinosine, floxuridine, 6-mercaptopurine, doxorubicin, daunorubicin, idarubicin star (I-darubicin), taxol and paclitaxel;

抗氧化剂，诸如维生素E，类视色素，类胡萝卜素，辅酶Q，金属鳌合剂，和植酸；Antioxidants, such as vitamin E, retinoids, carotenoids, coenzyme Q, metal chelators, and phytic acid;

抗心律失常药，诸如奎尼丁；和Antiarrhythmic drugs, such as quinidine; and

抗高血压药如哌唑嗪，维拉帕米，硝苯地平，和地尔硫；镇痛药如对乙酰氨基酚和阿司匹林；Antihypertensives such as prazosin, verapamil, nifedipine, and diltiazem ; pain relievers such as acetaminophen and aspirin;

单克隆和多克隆抗体，包括人源化抗体及抗体片段；Monoclonal and polyclonal antibodies, including humanized antibodies and antibody fragments;

反义寡核苷酸；和antisense oligonucleotides; and

RNA，DNA和包含编码治疗肽和蛋白的基因的病毒载体。RNA, DNA and viral vectors containing genes encoding therapeutic peptides and proteins.

除了这些代表性的活性剂种类和类别，本发明的方法和组合物包括上文或本文其它地方所述或本领域另外所知的任何生理活性剂，以及任何多种活性剂的组合，其在本发明的方法和组合物中单独或组合有效用于治疗或预防哺乳动物受试者中所选的疾病或病况(参见，Physicians′DeskReference (医生桌上参考)，由利通工业有限公司(Litton Industries，Inc)的分公司医学经济学公司(Medical Economics Company)出版)。In addition to these representative classes and classes of active agents, the methods and compositions of the present invention include any physiologically active agent described above or elsewhere herein or otherwise known in the art, as well as any combination of active agents, which are in The methods and compositions of the present invention are useful, alone or in combination, for the treatment or prevention of selected diseases or conditions in a mammalian subject (see, Physicians' DeskReference (doctor's desk reference), sponsored by Litton Industries, Ltd. , Inc) division Medical Economics Company (Medical Economics Company)).

不管所用的化合物的种类，用于本发明的生物活性剂应该以充分的量存在于本发明的组合物和方法中，所述量足以为受试者提供需要的生理作用，而没有显著的、不可接受的毒性或其它不利的副作用。熟练的技术人员应该容易确定所有生物活性剂的适当的剂量水平，而无需过度实验。由于本发明的方法和组合物提供提高的生物活性剂的递送，所以可以成功地应用显著低于常规剂量水平的剂量水平。一般地，所述活性物质将以总鼻内制剂的约0.01重量％到约50重量％、通常约0.1重量％-约20重量％、并且一般约1.0重量％-5重量％或10重量％的量存在于组合物中，这取决于所用的具体的物质。Regardless of the type of compound used, the bioactive agent used in the invention should be present in the compositions and methods of the invention in an amount sufficient to provide the desired physiological effect to the subject without significant, Unacceptable toxicity or other adverse side effects. Appropriate dosage levels for all bioactive agents should be readily determined by the skilled artisan without undue experimentation. Because the methods and compositions of the present invention provide enhanced delivery of bioactive agents, dosage levels significantly lower than conventional dosage levels can be successfully employed. Generally, the active substance will be present in an amount of from about 0.01% to about 50%, usually from about 0.1% to about 20%, and typically from about 1.0% to 5% or 10% by weight of the total intranasal formulation. The amount present in the composition depends on the particular material used.

当用于本文时，术语生物活性“肽”和“蛋白”包括各种大小的多肽，并且不将本发明限于任何具体大小的氨基酸聚合物。本发明包括从小至长度为几个氨基酸的肽，到任何大小的蛋白，以及肽-肽、蛋白-蛋白融合体和蛋白-肽融合体，只要所述蛋白或肽在激发特别的生理、免疫、治疗或预防性作用或反应的情形中是有生物活性的。As used herein, the terms biologically active "peptide" and "protein" include polypeptides of various sizes and do not limit the invention to polymers of amino acids of any particular size. The present invention includes peptides as small as a few amino acids in length, to proteins of any size, and peptide-peptides, protein-protein fusions and protein-peptide fusions, as long as the protein or peptide elicits specific physiological, immune, Biologically active in the context of a therapeutic or prophylactic effect or response.

本发明提供用于提高生物活性肽和蛋白的黏膜递送的新颖的制剂和协同的施用方法。用于本发明的治疗肽和蛋白的代表性实例包括，但不限于：组织血纤维蛋白溶酶原活化因子(TPA)，表皮生长因子(EGF)，成纤维细胞生长因子(FGF-酸性或碱性)，血小板衍生生长因子(PDGF)，转化生长因子(TGF-α或β)，血管活性肠肽，肿瘤坏死因子(TNF)，下丘脑释放因子，促乳素，促甲状腺激素(TSH)，促肾上腺皮质激素(ACTH)，甲状旁腺激素(PTH)，促卵泡激素(FSF)，促性腺激素释放激素(LHRH)，内啡肽，胰高血糖素，降钙素，催产素，卡贝缩宫素，aldoetecone，脑啡肽(enkaphalins)，somatostin，生长激素，生长调节素，促性腺激素，雌激素，孕酮，睾酮，α-促黑激素，非天然存在的阿片样物质，利多卡因，酮洛芬，舒芬太尼，特布他林，氟哌利多，东莨菪碱，戈那瑞林，环吡酮，丁螺环酮，降钙素，色甘酸二钠或咪达唑仑，环孢素，赖诺普利，卡托普利，地拉普利，西咪替丁，雷尼替丁，法莫替丁，过氧化物歧化酶，天冬酰胺酶，精氨酸酶，精氨酸脱氨酶，腺苷脱氨酶核糖核酸酶，胰蛋白酶，化学胰蛋白酶(chemotrypsin)，和木瓜蛋白酶。其它有用的肽的实例包括，但不限于，铃蟾肽，P物质，血管升压素，α-球蛋白，运铁蛋白，凝血因子I，β-脂蛋白，β-球蛋白，凝血因子II，血浆铜蓝蛋白，α₂-糖蛋白，α₂-球蛋白，胎球蛋白，α₁-脂蛋白，α₁-球蛋白，白蛋白，前白蛋白，以及其他生物活性蛋白和重组蛋白产物。The present invention provides novel formulations and synergistic methods of administration for enhanced mucosal delivery of bioactive peptides and proteins. Representative examples of therapeutic peptides and proteins useful in the present invention include, but are not limited to: tissue plasminogen activator (TPA), epidermal growth factor (EGF), fibroblast growth factor (FGF-acidic or alkaline sex), platelet-derived growth factor (PDGF), transforming growth factor (TGF-α or β), vasoactive intestinal peptide, tumor necrosis factor (TNF), hypothalamic releasing factor, prolactin, thyroid-stimulating hormone (TSH), Adrenocorticotropic hormone (ACTH), parathyroid hormone (PTH), follicle-stimulating hormone (FSF), gonadotropin-releasing hormone (LHRH), endorphins, glucagon, calcitonin, oxytocin, carbe Oxytocin, aldoetecone, enkephalins (enkaphalins), somatostin, growth hormone, somatomodulin, gonadotropins, estrogens, progesterone, testosterone, alpha-melanostimulating hormone, non-naturally occurring opioids, lidoca ketoprofen, sufentanil, terbutaline, droperidol, scopolamine, gonadorelin, ciclopirox, buspirone, calcitonin, disodium cromolyn, or midazolam, Cyclosporine, Lisinopril, Captopril, Delapril, Cimetidine, Ranitidine, Famotidine, Superoxide Dismutase, Asparaginase, Arginase, Arginine deaminase, adenosine deaminase ribonuclease, trypsin, chemotrypsin, and papain. Examples of other useful peptides include, but are not limited to, bombesin, substance P, vasopressin, alpha-globulin, transferrin, coagulation factor I, beta-lipoprotein, beta-globulin, coagulation factor II , ceruloplasmin, α₂ -glycoprotein, α₂ -globulin, fetuin, α₁ -lipoprotein, α₁ -globulin, albumin, prealbumin, and other bioactive proteins and recombinant protein products .

在本发明更详细的方面中，提供用于提高特别的、生物活性肽或蛋白治疗剂的黏膜递送的方法和组合物，以治疗现存的疾病或病况(即，消除、或者减少其发生或症状的严重性)，或者在鉴定为处于所述疾病或病况的危险中的受试者中预防疾病或病况的发作。用于本发明这些方面的生物活性肽和蛋白包括，但不限于，造血药；抗感染药；抗痴呆药(antidementia)；抗病毒剂；抗肿瘤药；退热药；镇痛药；消炎药；抗溃疡药；抗过敏剂；抗抑郁药；治疗精神病的药；强心剂；抗心律失常药；血管舒张剂；抗高血压药如降血压药利尿剂；抗糖尿病药；抗凝血剂；降胆固醇药；用于骨质疏松症的治疗剂；激素；抗生素；疫苗；等。In a more detailed aspect of the invention, methods and compositions are provided for enhancing the mucosal delivery of specific, bioactive peptide or protein therapeutics to treat an existing disease or condition (i.e., eliminate, or reduce the occurrence or symptoms of) severity of the disease or condition), or preventing the onset of a disease or condition in a subject identified as being at risk of the disease or condition. Bioactive peptides and proteins useful in these aspects of the invention include, but are not limited to, hematopoietics; anti-infectives; antidementias; antivirals; ; antiulcer agents; antiallergic agents; antidepressants; psychotropic agents; cardiotonic agents; antiarrhythmic agents; vasodilators; antihypertensive agents such as hypotensive agents diuretics; Cholesterol drugs; therapeutic agents for osteoporosis; hormones; antibiotics; vaccines;

用于本发明这些方面的生物活性肽和蛋白包括，但不限于，细胞因子；肽激素；生长因子；作用在心血管系统上的因子；细胞黏附因子；作用在中枢和外周神经系统上的因子；作用在体液电解质和血液有机物质上的因子；作用在骨和骨骼生长或生理上的因子；作用在胃肠系统上的因子；作用在肾和泌尿器官上的因子；作用在结缔组织和皮肤上的因子；作用在感觉器官上的因子；作用在免疫系统上的因子；作用在呼吸系统上的因子；作用在生殖器官上的因子；和各种酶类。Bioactive peptides and proteins useful in these aspects of the invention include, but are not limited to, cytokines; peptide hormones; growth factors; factors acting on the cardiovascular system; cell adhesion factors; factors acting on the central and peripheral nervous systems; Factors acting on body fluid electrolytes and blood organic matter; factors acting on bone and skeletal growth or physiology; factors acting on the gastrointestinal system; factors acting on kidney and urinary organs; factors acting on connective tissue and skin factors acting on the sensory organs; factors acting on the immune system; factors acting on the respiratory system; factors acting on the reproductive organs; and various enzymes.

例如，可以在本发明的方法和组合物内施用的激素包括雄激素、雌激素、前列腺素、生长激素、促性腺激素、白介素、类固醇和细胞因子。For example, hormones that can be administered within the methods and compositions of the invention include androgens, estrogens, prostaglandins, growth hormones, gonadotropins, interleukins, steroids, and cytokines.

可以在本发明的方法和组合物内施用的疫苗包括细菌和病毒疫苗，诸如用于肝炎、流感、呼吸道合胞病毒(RSV)、副流感病毒(PIV)、结核病、金丝雀痘(canary pox)、水痘(chicken pox)、麻疹、流行性腮腺炎、风疹、肺炎、和人类免疫缺陷性病毒(HIV)的疫苗。Vaccines that can be administered within the methods and compositions of the invention include bacterial and viral vaccines, such as for hepatitis, influenza, respiratory syncytial virus (RSV), parainfluenza virus (PIV), tuberculosis, canary pox ), chicken pox, measles, mumps, rubella, pneumonia, and human immunodeficiency virus (HIV).

可以在本发明的方法和组合物内施用的细菌类毒素包括白喉、破伤风、假单胞菌(pseudonomas)和结核分枝杆菌(mycobactrium tuberculosis)。Bacterial toxoids that can be administered within the methods and compositions of the invention include diphtheria, tetanus, pseudomonas and mycobactrium tuberculosis.

用于本发明的特别的心血管或溶血栓试剂的实例包括水蛭素(hirugen)，hirulos和蛭素(hirudine)。Examples of specific cardiovascular or thrombolytic agents useful in the present invention include hirugen, hirulos and hirudine.

用本发明有效施用的抗体试剂包括单克隆抗体、多克隆抗体、人源化抗体、抗体片段、融合体和多聚体、以及免疫球蛋白。Antibody agents usefully administered with the present invention include monoclonal antibodies, polyclonal antibodies, humanized antibodies, antibody fragments, fusions and multimers, and immunoglobulins.

当用于本文时，术语“保守氨基酸取代”是指具有相似侧链的氨基酸残基的普通互换性。例如，通常可互换的具有脂族侧链的氨基酸组为丙氨酸、缬氨酸、亮氨酸和异亮氨酸；具有脂族-羟基侧链的氨基酸组为丝氨酸和苏氨酸；具有含有酰胺的侧链的氨基酸组为天冬酰胺和谷氨酰胺；具有芳族侧链的氨基酸组为苯丙氨酸、酪氨酸和色氨酸；具有碱性侧链的氨基酸组为赖氨酸、精氨酸和组氨酸；和具有含有硫的侧链的氨基酸组为半胱氨酸和甲硫氨酸。保守取代的实例包括非极性(疏水性)残基如异亮氨酸、缬氨酸、亮氨酸或甲硫氨酸彼此之间的取代。同样地，本发明考虑极性(亲水性)残基的取代，诸如精氨酸和赖氨酸之间的取代，谷氨酰胺和天冬酰胺之间的取代，以及苏氨酸和丝氨酸之间的取代。另外，还考虑碱性残基如赖氨酸、精氨酸或组氨酸彼此之间的取代或者酸性残基如天冬氨酸或谷氨酸彼此之间的取代。代表性的保守氨基酸取代组为：缬氨酸-亮氨酸-异亮氨酸，苯丙氨酸-酪氨酸，赖氨酸-精氨酸，丙氨酸-缬氨酸，和天冬酰胺-谷氨酰胺。As used herein, the term "conservative amino acid substitution" refers to the ordinary interchangeability of amino acid residues having similar side chains. For example, generally interchangeable groups of amino acids with aliphatic side chains are alanine, valine, leucine, and isoleucine; groups of amino acids with aliphatic-hydroxyl side chains are serine and threonine; The groups of amino acids with amide-containing side chains are asparagine and glutamine; the group of amino acids with aromatic side chains are phenylalanine, tyrosine, and tryptophan; the group of amino acids with basic side chains is lysine amino acid, arginine, and histidine; and the groups of amino acids with sulfur-containing side chains are cysteine and methionine. Examples of conservative substitutions include the substitution of non-polar (hydrophobic) residues such as isoleucine, valine, leucine or methionine for each other. Likewise, the invention contemplates substitutions of polar (hydrophilic) residues, such as between arginine and lysine, between glutamine and asparagine, and between threonine and serine. replacement between. In addition, the substitution of basic residues such as lysine, arginine or histidine for each other or acidic residues such as aspartic acid or glutamic acid for each other is also contemplated. Representative conservative amino acid substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and aspartame Amide-Glutamine.

术语生物活性肽或蛋白类似物还包括天然肽或蛋白的修饰的形式，包括20种常规氨基酸的立体异构体(例如，D-氨基酸)，或非天然的氨基酸，如α，α-双取代的氨基酸、N-烷基氨基酸、乳酸。这些和其它非常规氨基酸还可以取代或插入到本发明所用的天然肽和蛋白内。非常规氨基酸的实例包括：4-羟基脯氨酸，γ-羧基谷氨酸，ε-N，N，N-三甲基赖氨酸，ε-N-乙酰基赖氨酸，O-磷酸丝氨酸，N-乙酰基丝氨酸，N-甲酰甲硫氨酸，3-甲基组氨酸，5-羟基赖氨酸，ω-N-甲基精氨酸，以及其它的类似氨基酸和亚氨基酸(例如，4-羟基脯氨酸)。另外，生物活性肽或蛋白类似物包括碳水化合物、脂质和/或蛋白质性部分的单一或多取代、删除和/或加入，所述碳水化合物、脂质和/或蛋白质性部分作为所述肽或蛋白的结构成分天然存在或人工存在，或者与所述肽或蛋白结合或另外缔合。The term biologically active peptide or protein analog also includes modified forms of natural peptides or proteins, including stereoisomers of the 20 conventional amino acids (e.g., D-amino acids), or unnatural amino acids, such as α,α-disubstituted amino acids, N-alkyl amino acids, lactic acid. These and other unconventional amino acids may also be substituted or inserted into the native peptides and proteins used in the present invention. Examples of unconventional amino acids include: 4-hydroxyproline, γ-carboxyglutamate, ε-N,N,N-trimethyllysine, ε-N-acetyllysine, O-phosphoserine , N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, ω-N-methylarginine, and other similar amino acids and imino acids ( For example, 4-hydroxyproline). In addition, biologically active peptide or protein analogues comprise single or multiple substitutions, deletions and/or additions of carbohydrate, lipid and/or proteinaceous moieties which act as the peptide A structural component of a protein or protein that occurs naturally or artificially, or is bound or otherwise associated with the peptide or protein.

在一个方面中，通过用其它侧链取代20种遗传编码的氨基酸(或D氨基酸)的一个或多个天然存在的侧链而修饰用于本发明的肽(包括多肽)，例如，用诸如烷基、低级烷基、环4-，5-，6-，至7-元烷基、酰胺、酰胺低级烷基、酰胺二(低级烷基)、低级烷氧基、羟基、羧基及其低级酯衍生物、和具有4-，5-，6-，到7-元杂环的基团，以产生肽模拟物。例如，可以制备脯氨酸类似物，其中脯氨酸残基的环大小从5元到4、6或7元变化。环基可以是饱和的或不饱和的，并且如果是不饱和的，可以是芳香基或非芳香基的。杂环基可以包含一个或多个氮、氧、和/或硫杂原子。这样的基团的实例包括呋咱基(furazanyl)，呋喃基，咪唑烷基，咪唑基，咪唑啉基，异噻唑基，异噁唑基，吗啉基(例如，吗啉代)，噁唑基，哌嗪基(例如，1-哌嗪基)，哌啶基(例如，1-哌啶基，哌啶子基)，吡喃基，吡嗪基，吡唑烷基，吡唑啉基，吡唑基，哒嗪基，吡啶基，嘧啶基，吡咯烷基(例如，1-吡咯烷基)，吡咯啉基，吡咯基，噻二唑基(thiadiazolyl)，噻唑基，噻吩基，硫代吗啉基(例如，硫代吗啉代)，和三唑基。这些杂环基可以被取代或未被取代。在基团被取代的情形中，取代基可以是烷基、烷氧基、卤素、氧、或者取代或未取代的苯基。In one aspect, peptides (including polypeptides) for use in the invention are modified by substituting one or more of the naturally occurring side chains of the 20 genetically encoded amino acids (or D amino acids) with other side chains, e.g. group, lower alkyl, ring 4-, 5-, 6-, to 7-membered alkyl, amide, amide lower alkyl, amide di(lower alkyl), lower alkoxy, hydroxyl, carboxyl and their lower esters Derivatives, and groups with 4-, 5-, 6-, to 7-membered heterocycles to produce peptidomimetics. For example, proline analogs can be prepared in which the ring size of the proline residues varies from 5 to 4, 6 or 7 members. Cyclic groups may be saturated or unsaturated, and if unsaturated, may be aromatic or non-aromatic. A heterocyclyl group may contain one or more nitrogen, oxygen, and/or sulfur heteroatoms. Examples of such groups include furazanyl, furyl, imidazolidinyl, imidazolyl, imidazolinyl, isothiazolyl, isoxazolyl, morpholinyl (e.g., morpholino), oxazole Base, piperazinyl (for example, 1-piperazinyl), piperidinyl (for example, 1-piperidinyl, piperidino), pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl , pyrazolyl, pyridazinyl, pyridyl, pyrimidinyl, pyrrolidinyl (for example, 1-pyrrolidinyl), pyrrolinyl, pyrrolyl, thiadiazolyl (thiadiazolyl), thiazolyl, thienyl, sulfur Morpholino (eg, thiomorpholino), and triazolyl. These heterocyclic groups may be substituted or unsubstituted. Where a group is substituted, the substituent may be alkyl, alkoxy, halogen, oxygen, or substituted or unsubstituted phenyl.

肽和蛋白，以及肽和蛋白类似物和模拟物还可以与许多非蛋白聚合物如聚乙二醇、聚丙二醇或聚氧化烯(polyoxyalkenes)中的一种或多种共价结合，结合以在美国专利号4,640,835；美国专利号4,496,689；美国专利号4,301,144；美国专利号4,670,417；美国专利号4,791,192；或美国专利号4,179,337中提出的方式进行。Peptides and proteins, as well as peptide and protein analogs and mimetics, can also be covalently bound to one or more of a number of non-protein polymers such as polyethylene glycol, polypropylene glycol, or polyoxyalkenes to bind to U.S. Patent No. 4,640,835; U.S. Patent No. 4,496,689; U.S. Patent No. 4,301,144; U.S. Patent No. 4,670,417; U.S. Patent No. 4,791,192; or U.S. Patent No. 4,179,337.

本发明内的其它肽和蛋白类似物和模拟物包括糖基化变体，和与其它化学部分的共价或聚集缀合物。共价衍生物可以通过本领域公知的方法将官能度与位于氨基酸侧链中或位于N-或C-端的基团连接而制备。这些衍生物可以包括，但不限于，羧基端或含有羧基侧链的残基的脂族酯或酰胺，含有羟基的残基的O-酰基衍生物，和氨基端氨基酸或含有氨基的残基如赖氨酸或精氨酸的N-酰基衍生物。酰基选自包括C3-C18正常烷基的烷基-部分的组，由此形成烷酰基芳酰基种类。还可以应用与载体蛋白例如免疫原性部分的共价附着。Other peptide and protein analogs and mimetics within the invention include glycosylation variants, and covalent or aggregated conjugates with other chemical moieties. Covalent derivatives can be prepared by attaching functionality to groups located in the side chains of the amino acids or at the N- or C-terminus by methods well known in the art. These derivatives may include, but are not limited to, aliphatic esters or amides of carboxy-terminal or carboxyl side chain-containing residues, O-acyl derivatives of hydroxyl-containing residues, and amino-terminal amino acids or amino-containing residues such as N-acyl derivatives of lysine or arginine. Acyl is selected from the group comprising alkyl-moieties of C3-C18 normal alkyl, thereby forming the alkanoylaroyl species. Covalent attachment to carrier proteins such as immunogenic moieties may also be employed.

除了这些修饰之外，可以进行生物活性肽和蛋白的糖基化改变，例如，通过在其合成和加工过程中或在其它加工步骤中修饰肽的糖基化模式而进行。完成这一修饰的特别优选的方式为，通过将所述肽暴露于来源于正常提供这样的加工的细胞的糖基化酶，例如，哺乳动物糖基化酶而进行。还成功地应用去糖基化酶，以产生本发明内有用的修饰的肽和蛋白。还包括天然一级氨基酸序列的形式，其具有其它较小修饰，包括磷酸化的氨基酸残基，例如，磷酸酪氨酸、磷酸丝氨酸或磷酸苏氨酸，或其它部分，包括核糖基或交联试剂。In addition to these modifications, changes in the glycosylation of biologically active peptides and proteins can be made, for example, by modifying the glycosylation pattern of the peptide during its synthesis and processing or during other processing steps. A particularly preferred way of accomplishing this modification is by exposing the peptide to a glycosylase derived from a cell that normally provides such processing, eg, a mammalian glycosylase. Deglycosylation enzymes have also been successfully used to produce modified peptides and proteins useful within the invention. Also included are forms of the native primary amino acid sequence with other minor modifications, including phosphorylated amino acid residues, for example, phosphotyrosine, phosphoserine, or phosphothreonine, or other moieties, including ribosyl groups or cross-links reagent.

肽模拟物还可以具有已经通过磷酸化、磺化、生物素酰化或者加入或移除其它部分、特别是具有与磷酸脂(或盐)基相似的分子形状的那些部分而化学修饰的氨基酸残基。A peptidomimetic may also have amino acid residues that have been chemically modified by phosphorylation, sulfonation, biotinylation, or addition or removal of other moieties, particularly those having a molecular shape similar to a phospholipid (or salt) group. base.

可以环化用于本发明的活性肽，或者在所述肽的末端结合脱氨基或脱羧基残基，以致不存在末端氨基或羧基，以减少对蛋白酶的敏感性，或者以限制所述肽的构象。在本发明的肽类似物和模拟物中的C端官能团包括酰胺、酰胺低级烷基、酰胺二(低级烷基)、低等烷氧基、羟基、和羧基、和其低级酯衍生物、以及其药用盐。The active peptides used in the present invention can be cyclized, or have deaminated or decarboxylated residues incorporated at the termini of the peptides, so that no terminal amino or carboxyl groups are present, to reduce susceptibility to proteases, or to limit the activity of the peptides. Conformation. C-terminal functional groups in the peptide analogs and mimetics of the present invention include amide, amide lower alkyl, amide di(lower alkyl), lower alkoxy, hydroxyl, and carboxyl, and lower ester derivatives thereof, and its medicinal salt.

本发明提供各种添加剂、稀释剂、基质(base)和递送赋形剂，其有效控制水含量以提高蛋白稳定性。在这种意义上有效作为抗-聚集剂的这些试剂和载体物质包括，例如，各种官能度的聚合物，诸如聚乙二醇、葡聚糖、二乙基氨基乙基葡聚糖、和羧甲基纤维素，其显著地增加与其混合或者与其连接的肽和蛋白的稳定性并且减少固相聚集。在一些实例中，蛋白的活性或物理稳定性还可以通过针对肽或蛋白药物的水溶液的各种添加剂而增强。例如，可以使用添加剂，诸如多元醇(包括糖)、氨基酸，蛋白，诸如胶原蛋白和明胶，以及各种盐。The present invention provides various additives, diluents, bases and delivery vehicles that effectively control water content to enhance protein stability. Such agents and carrier materials effective as anti-aggregating agents in this sense include, for example, polymers of various functionalities such as polyethylene glycol, dextran, diethylaminoethyl dextran, and Carboxymethylcellulose, which significantly increases the stability of peptides and proteins mixed with or linked to it and reduces solid phase aggregation. In some instances, the activity or physical stability of the protein can also be enhanced by various additives to the aqueous solution of the peptide or protein drug. For example, additives such as polyols (including sugars), amino acids, proteins such as collagen and gelatin, and various salts can be used.

某些添加剂，特别是糖和其它多元醇，还给干燥的如冻干的蛋白赋予显著的物理稳定性。这些添加剂还可以用于本发明以便不但在冻干过程中，还在以干燥状态存储的过程中，防止蛋白聚集。例如，在各种条件下的固相温育过程中，蔗糖和菲可70(具有蔗糖单位的聚合物)表现出显著的抗肽或蛋白聚集的保护作用。这些添加剂还可以提高包埋在聚合物基质中的固体蛋白的稳定性。Certain additives, especially sugars and other polyols, also impart significant physical stability to dried, eg lyophilized, proteins. These additives can also be used in the present invention to prevent protein aggregation not only during lyophilization but also during storage in a dry state. For example, sucrose and Ficoll 70 (a polymer with sucrose units) exhibited significant protection against peptide or protein aggregation during solid phase incubation under various conditions. These additives can also increase the stability of solid proteins embedded in polymer matrices.

然而其它添加剂，例如蔗糖，在升高的温度在潮湿氛围中稳定蛋白抗固体-状态聚集，如可以在本发明的某些缓慢恒定释放的制剂中发生的。蛋白如明胶和胶原蛋白还作用为稳定剂或填充剂以减少在这种情形中不稳定的蛋白的变性和聚集。这些添加剂可以结合到聚合物熔化加工过程中和本发明的组合物中。例如，可以通过将含有上述各种稳定的添加剂的溶液简单冻干或者喷雾干燥而制备多肽微粒。由此可以获得未聚集的肽和蛋白在延长的时间阶段的缓慢恒定释放。Yet other additives, such as sucrose, stabilize proteins against solid-state aggregation at elevated temperatures in a humid atmosphere, as can occur in certain slow constant release formulations of the invention. Proteins such as gelatin and collagen also act as stabilizers or bulking agents to reduce denaturation and aggregation of unstable proteins in this case. These additives can be incorporated during polymer melt processing and in the compositions of the present invention. For example, polypeptide microparticles can be prepared by simply lyophilizing or spray-drying a solution containing the above-mentioned various stabilizing additives. A slow constant release of unaggregated peptides and proteins over an extended period of time can thereby be obtained.

本发明提供各种其它制备成分和方法，以及特异性的配制添加剂，其产生用于黏膜递送易于聚集的肽和蛋白的制剂，其中所述肽或蛋白稳定为基本上纯的、未聚集的形式。考虑将一定范围的成分和添加剂用于这些方法和制剂。这些抗聚集试剂的实例是连接的环糊精(CDs)二聚体，其选择性地结合多肽的疏水侧链。已经发现这些CD二聚体以显著抑制聚集的方式结合蛋白的疏水片段。关于涉及的所述CD二聚体和蛋白二者，这种抑制是选择性的。所述蛋白聚集的选择性抑制在本发明的鼻内递送方法和组合物中提供额外的益处。用于这种情形的其它试剂包括具有受到特异性地阻断肽和蛋白的聚集的接头控制的可变的几何学的CD三聚体和四聚体(Breslow等，J.Am.Chem.Soc.(美国化学协会杂志)118：11678-11681，1996；Breslow等，PNAS USA(美国国家科学院学报)94：11156-11158，1997)。The present invention provides various other preparation ingredients and methods, as well as specific formulation additives, which result in formulations for mucosal delivery of aggregation-prone peptides and proteins, wherein the peptides or proteins are stabilized in a substantially pure, non-aggregated form . A range of ingredients and additives are contemplated for use in these methods and formulations. Examples of such anti-aggregation agents are dimers of linked cyclodextrins (CDs), which selectively bind to the hydrophobic side chains of polypeptides. These CD dimers have been found to bind hydrophobic fragments of the protein in a manner that significantly inhibits aggregation. This inhibition is selective with respect to both the CD dimer and protein involved. Selective inhibition of such protein aggregation provides additional benefits in the intranasal delivery methods and compositions of the invention. Other reagents for this situation include CD trimers and tetramers with variable geometries controlled by linkers that specifically block aggregation of peptides and proteins (Breslow et al., J. Am. Chem. Soc (Journal of the American Chemical Society) 118: 11678-11681, 1996; Breslow et al., PNAS USA (Proceedings of the National Academy of Sciences of the United States of America) 94: 11156-11158, 1997).

电荷修饰和pH值控制剂和方法Charge modification and pH control agents and methods

为了改善用于提高穿过疏水黏膜屏障递送的生物活性剂(例如，大分子药物、肽或蛋白)的转运特性，本发明还提供用于所选的生物活性剂或本文所述的递送-增强剂的电荷修饰的技术和试剂。在这一点上，大分子的相关的渗透性通常与它们的分配系数相关。分子的离子化程度，其取决于所述分子的pK_a和在黏膜表面上的pH值，还影响所述分子的渗透性。用于黏膜递送的生物活性剂和渗透剂的渗透和分配可以由所述活性剂或渗透剂的电荷改变或电荷分布而促进，例如，所述电荷改变或电荷分布通过改变带电官能团，通过修饰递送活性剂的递送赋形剂或溶液的pH值，或者通过协调电荷-或pH值-改变剂与所述活性剂的施用而实现。To improve the transport properties of bioactive agents (e.g., macromolecular drugs, peptides, or proteins) for enhanced delivery across a hydrophobic mucosal barrier, the invention also provides for use in selected bioactive agents or delivery-enhancing agents described herein. Techniques and reagents for charge modification of agents. In this regard, the relative permeability of macromolecules is generally related to their partition coefficients. The degree of ionization of a molecule, which depends on the_pKa of the molecule and the pH value at the mucosal surface, also affects the permeability of the molecule. Penetration and partitioning of bioactive agents and penetrants for mucosal delivery can be facilitated by altering the charge or charge distribution of the active agent or penetrant, e.g., by altering charged functional groups, by modifying the delivery The delivery vehicle of the active agent or the pH of the solution, or by coordinating the administration of a charge- or pH-altering agent with the active agent.

防腐剂preservative

防腐剂如氯丁醇、对羟基苯甲酸甲酯、对羟基苯甲酸丙酯、苯甲酸钠(0.5％)、苯酚、甲酚、对-氯-间-甲酚、苯乙醇、苯甲醇、醋酸苯汞、硼酸苯汞、硝酸苯汞、硫柳汞、山梨酸、氯化苄乙铵或benzylkonium chloride，可以加入到本发明的制剂中，以抑制微生物生长。Preservatives such as chlorobutanol, methylparaben, propylparaben, sodium benzoate (0.5%), phenol, cresol, p-chloro-m-cresol, phenylethyl alcohol, benzyl alcohol, benzene acetate Mercury, phenylmercuric borate, phenylmercuric nitrate, thimerosal, sorbic acid, benzylethylammonium chloride or benzylkonium chloride, may be added to the formulations of the present invention to inhibit microbial growth.

pH和缓冲系统pH and Buffer Systems

pH值通常使用缓冲液如包含柠檬酸和柠檬酸盐如柠檬酸钠的系统进行调节。其它适当的缓冲系统包括醋酸与醋酸盐系统、琥珀酸与琥珀酸盐系统、苹果酸与苹果酸盐系统、和葡糖酸与葡糖酸盐系统。备选地，可以使用包含混合酸/盐系统的缓冲系统，诸如醋酸和柠檬酸钠系统，柠檬酸、醋酸钠系统，和柠檬酸、柠檬酸钠、苯甲酸钠系统。对于任何缓冲系统，可以加入其它的酸如盐酸，以及其它的碱如氢氧化钠，用于最终pH值调节。The pH is usually adjusted using buffers such as systems comprising citric acid and citrates such as sodium citrate. Other suitable buffer systems include acetic acid and acetate systems, succinic acid and succinate systems, malic acid and malate systems, and gluconic acid and gluconate systems. Alternatively, buffer systems comprising mixed acid/salt systems may be used, such as acetic acid and sodium citrate systems, citric acid, sodium acetate systems, and citric acid, sodium citrate, sodium benzoate systems. As with any buffer system, other acids such as hydrochloric acid, and other bases such as sodium hydroxide may be added for final pH adjustment.

用于调节上皮细胞连接结构和/或生理的其它试剂Other agents for modulating junctional structure and/or physiology of epithelial cells

上皮细胞紧密连接通常不能透过具有约15埃的半径的分子，除非用如本发明所提供的刺激基本连接开放的连接生理学控制剂处理。在本发明的方法和组合物中作用为二级生理学调控的有用目标的“二级”紧密连接调控成分中，ZO1-ZO2杂二聚体复合物本身表现出易于受到可以容易地并且有效地改变黏膜上皮细胞的旁细胞渗透性的外源试剂的生理学调控。一种已被充分研究的这样的试剂为来自亚洲霍乱弧菌的细菌毒素，叫作“紧密连接毒素”(ZOT)。还参见，WO 96/37196；美国专利号5,945,510；5,948,629；5,912,323；5,864,014；5,827,534；5,665,389；和5,908,825。因此，ZOT及其它调控ZO1-ZO2复合物的试剂可以与一种或多种生物活性剂组合配制或者协调施用。Epithelial cell tight junctions are generally impermeable to molecules having a radius of about 15 Angstroms unless treated with a junctional physiology control agent that stimulates substantial junctional opening as provided herein. Among the "secondary" tight junction regulatory components that serve as useful targets for secondary physiological regulation in the methods and compositions of the invention, the ZO1-ZO2 heterodimer complex itself appears susceptible to changes that can be readily and efficiently altered. Physiological regulation of paracellular permeability of mucosal epithelial cells by exogenous agents. One such agent that has been well studied is a bacterial toxin from Vibrio cholerae in Asia called "tight junction toxin" (ZOT). See also, WO 96/37196; U.S. Patent Nos. 5,945,510; 5,948,629; 5,912,323; 5,864,014; 5,827,534; Thus, ZOT and other agents that modulate the ZO1-ZO2 complex can be formulated in combination or coordinated with one or more bioactive agents.

配制和施用formulation and administration

本发明的黏膜递送制剂包含典型地与一种或多种药用载体以及任选地其它治疗成分组合在一起待施用的生物活性剂。在与所述制剂的其它成分相容并且在受试者中不激发不可接受的有害作用的意义上，所述载体必须是“药用的”。所述载体为本文在上文中所述的，或者另外为药学领域的技术人员公知的。理想地，所述制剂不应该包含已知所述生物活性剂与之一起施用不相容的物质如酶或氧化剂。所述制剂可以通过药学领域公知的任何方法进行制备。The mucosal delivery formulations of the invention comprise a bioactive agent to be administered, typically in combination with one or more pharmaceutically acceptable carriers and, optionally, other therapeutic ingredients. The carrier must be "pharmaceutically acceptable" in the sense of being compatible with the other ingredients of the formulation and not provoking unacceptable deleterious effects in the subject. Such carriers are described herein above, or otherwise known to those skilled in the pharmaceutical arts. Ideally, the formulation should not contain substances such as enzymes or oxidizing agents with which the bioactive agent is known to be administered incompatible. The formulations can be prepared by any methods known in the art of pharmacy.

本发明的组合物和方法可以通过许多黏膜施用方式施用给受试者，包通过口腔、直肠、阴道、鼻内、肺内、或透皮递送，或者通过局部递送至眼、耳、皮肤或其它黏膜表面。按照本发明的组合物通常以作为鼻或肺部喷雾剂的水溶液进行施用，并且可以通过本领域的技术人员已知的各种方法以喷雾形式分散。用于分散作为鼻喷雾剂的液体的优选系统在美国专利号4,511,069中公开。这样的制剂可以便利地通过将按照本发明的组合物溶解在水中以产生水溶液，并且使得所述溶液无菌而制备。所述制剂可以存在于多剂量容器中，例如，存在于在美国专利号4,511,069中公开的密封分配系统中。其它适当的鼻喷雾剂递送系统已在Transdermal SystemicMedication(透皮系统药物治疗)，Y.W.Chien编，Elsevier出版社，纽约，1985；和在美国专利号4,778,810中得到描述。其它的气溶胶递送形式可以包括，例如，压缩空气-、喷气式-、超声-和压电式喷雾器，其递送溶解或悬浮在药物溶剂如水、乙醇或它们的混合物中的生物活性剂。The compositions and methods of the present invention can be administered to a subject by a number of mucosal modes of administration, including oral, rectal, vaginal, intranasal, intrapulmonary, or transdermal delivery, or by topical delivery to the eye, ear, skin, or other mucosal surface. Compositions according to the invention are usually administered as aqueous solutions as nasal or pulmonary sprays and can be dispersed in spray form by various methods known to those skilled in the art. A preferred system for dispersing liquids as nasal sprays is disclosed in US Patent No. 4,511,069. Such formulations may conveniently be prepared by dissolving a composition according to the invention in water to produce an aqueous solution, and rendering said solution sterile. The formulations may be presented in multi-dose containers, for example, in the sealed dispensing system disclosed in US Patent No. 4,511,069. Other suitable nasal spray delivery systems are described in Transdermal Systemic Medication, edited by Y.W. Chien, Elsevier Press, New York, 1985; and in US Patent No. 4,778,810. Other aerosol delivery forms may include, for example, compressed air-, jet-, ultrasonic-, and piezoelectric nebulizers, which deliver the bioactive agent dissolved or suspended in a pharmaceutical solvent such as water, ethanol, or mixtures thereof.

本发明的鼻和肺部喷雾溶液典型地包含药物或待递送的药物，任选地用表面活性剂如非离子表面活性剂(例如，聚山梨酯-80)和一种或多种缓冲剂、稳定剂或张力剂(tonicifiers)配制。在本发明的一些实施方案中，所述鼻喷雾溶液还包含推进剂。所述鼻喷雾溶液的pH值任选地在约pH3.0和7.2之间，但是当需要时，调整pH值使得带电荷的大分子种类(例如，治疗蛋白或肽)以基本上未离子化的状态递送最优化。所应用的药学溶剂还可以是弱酸性的水性缓冲液(pH值3-6)。用于这些组合物的适当的缓冲剂如上文所述或者如本领域另外所知。可以添加其它成分，以提高或保持化学稳定性，所述其它成分包括防腐剂、表面活性剂、分散剂、或气体。适当的防腐剂包括，但不限于，苯酚、对羟基苯甲酸甲酯、对羟基苯甲酸酯、间-甲酚、硫柳汞、苯扎氯铵(benzylalkonimum chloride)等。适当的表面活性剂包括，但不限于，油酸、失水山梨糖醇三油酸酯、聚山梨酯、磷脂酰胆碱、磷脂酰胆碱(phosphotidyl cholines)、和各种长链甘油二酯和磷脂。适当的分散剂包括，但不限于，乙二胺四乙酸等。适当的气体包括，但不限于，氮气、氦气、含氯氟烃(CFCs)、氢氟烃(HFCs)、二氧化碳、空气等。适当的稳定剂和张力剂(tonicifying agent)包括糖和其它多元醇、氨基酸、和有机与无机盐。Nasal and pulmonary spray solutions of the invention typically comprise the drug or drug to be delivered, optionally with a surfactant such as a nonionic surfactant (e.g., polysorbate-80) and one or more buffering agents, Stabilizers or tonicifiers (tonicifiers) preparation. In some embodiments of the invention, the nasal spray solution further comprises a propellant. The pH of the nasal spray solution is optionally between about pH 3.0 and 7.2, but when necessary, the pH is adjusted such that charged macromolecular species (e.g., therapeutic proteins or peptides) are substantially unionized State delivery optimization for . The applied pharmaceutical solvent can also be weakly acidic aqueous buffer solution (pH value 3-6). Suitable buffers for these compositions are described above or otherwise known in the art. Other ingredients may be added to increase or maintain chemical stability, including preservatives, surfactants, dispersants, or gases. Suitable preservatives include, but are not limited to, phenol, methylparaben, parabens, m-cresol, thimerosal, benzylalkonimum chloride, and the like. Suitable surfactants include, but are not limited to, oleic acid, sorbitan trioleate, polysorbates, phosphatidylcholines, phosphotidyl cholines, and various long chain diglycerides and phospholipids. Suitable dispersants include, but are not limited to, ethylenediaminetetraacetic acid and the like. Suitable gases include, but are not limited to, nitrogen, helium, chlorofluorocarbons (CFCs), hydrofluorocarbons (HFCs), carbon dioxide, air, and the like. Suitable stabilizers and tonicifying agents include sugars and other polyols, amino acids, and organic and inorganic salts.

液体透皮制剂可以作为滴剂例如装置(installation)或作为小滴(喷雾)进行施用。喷雾可以通过泵、喷雾器或者通过本领域所述的其它方法而产生。对于肺部递送，用于深入肺部沉积的液体小滴表现出适于在肺部通道中沉积的最小的颗粒大小，通常约小于10μm质量中值当量空气动力学直径(mass median equivalent aerodynamic diameter，MMEAD)，通常约小于5μm MMEAD，通常约小于约2μm MMEAD。对于鼻递送，所述液体小滴颗粒大小通常约小于1000μm MMEAD，通常小于100μm MMEAD。Liquid transdermal formulations can be applied as drops, eg, an installation, or as droplets (spray). Sprays can be produced by pumps, nebulizers, or by other methods described in the art. For pulmonary delivery, liquid droplets for deep lung deposition exhibit the smallest particle size suitable for deposition in the pulmonary passage, typically less than about 10 μm mass median equivalent aerodynamic diameter (mass median equivalent aerodynamic diameter, MMEAD), usually less than about 5 μm MMEAD, usually about less than about 2 μm MMEAD. For nasal delivery, the liquid droplet particle size is typically less than about 1000 μm MMEAD, usually less than 100 μm MMEAD.

在备选实施方案中，黏膜制剂作为干粉制剂施用，其包含以适当颗粒大小或者在适宜的颗粒大小范围之内的干燥的通常是冻干形式的生物活性剂，用于鼻内递送。对于肺部递送，用于深入肺部沉积的粉末颗粒表现出适于在肺部通道中沉积的最小颗粒大小，通常约小于10μm质量中值当量空气动力学直径(MMEAD)，通常约小于5μm MMEAD，通常约小于约2μm MMEAD。对于鼻递送，所述粉末颗粒大小通常约小于1000μmMMEAD，通常小于100μm MMEAD。在这些大小范围内的鼻内可吸入粉末可以通过各种常规技术诸如喷射研磨、喷雾干燥、溶剂沉淀、超临界流体冷凝等生产。这些适当MMEAD的干粉可以通过常规干粉吸入器(DPI)施用给患者，所述常规干粉吸入器依赖患者的呼吸，当肺或鼻吸入时，以将所述散剂分散成气溶胶化的量。备选地，所述干粉可以通过空气辅助装置进行施用，所述空气辅助装置使用外部能源，以将所述散剂分散成气溶胶化的量，例如使用活塞泵进行。药物粉末颗粒可以在干燥状态配制成聚集成大颗粒(＞100um MMEAD)的颗粒，其包含适当的载体，诸如乳糖，其中当分配所述散剂时，药物颗粒和载体颗粒的聚集体被破坏。In an alternative embodiment, the mucosal formulation is administered as a dry powder formulation comprising the bioactive agent in dry, usually lyophilized form, at or within a suitable particle size range for intranasal delivery. For pulmonary delivery, powder particles for deep lung deposition exhibit a minimum particle size suitable for deposition in the lung passages, typically less than about 10 μm mass median equivalent aerodynamic diameter (MMEAD), typically less than about 5 μm MMEAD , typically less than about 2 μm MMEAD. For nasal delivery, the powder particle size is usually less than about 1000 μm MMEAD, usually less than 100 μm MMEAD. Intranasal inhalable powders within these size ranges can be produced by various conventional techniques such as jet milling, spray drying, solvent precipitation, supercritical fluid condensation and the like. Dry powders of these appropriate MMEADs can be administered to patients by conventional dry powder inhalers (DPIs) that rely on the patient's breathing, when lung or nasal inhalation, to disperse the powder into an aerosolized amount. Alternatively, the dry powder may be administered by an air-assisted device using an external energy source to disperse the powder into aerosolized quantities, for example using a piston pump. Drug powder particles can be formulated in the dry state as granules agglomerated into large particles (>100um MMEAD) containing a suitable carrier, such as lactose, wherein when the powder is dispensed, the aggregates of drug particles and carrier particles are disrupted.

干粉装置典型地需要约1mg-20mg范围的粉末质量，以产生单一的气溶胶化剂量(“喷烟”)。如果生物活性剂的需要的或理想的剂量低于这一量，那么粉状的活性剂将典型地与药物干燥填充(bulking)粉末组合，以提供需要的总散剂质量。优选的干燥填充粉末包括蔗糖、乳糖、右旋糖、甘露醇、甘氨酸、海藻糖、人血清白蛋白(HSA)、和淀粉。其它适当的干填充粉末包括纤维二糖、葡聚糖、麦芽三糖、果胶、柠檬酸钠、抗坏血酸钠、等等。Dry powder devices typically require powder masses in the range of about 1 mg-20 mg to produce a single aerosolized dose ("puff"). If the required or desired dosage of bioactive agent is below this amount, then the powdered active agent will typically be combined with a pharmaceutical dry bulking powder to provide the desired total powder mass. Preferred dry bulk powders include sucrose, lactose, dextrose, mannitol, glycine, trehalose, human serum albumin (HSA), and starch. Other suitable dry-fill powders include cellobiose, dextran, maltotriose, pectin, sodium citrate, sodium ascorbate, and the like.

为了配制用于本发明中黏膜递送的组合物，所述生物活性剂可以与各种药用添加剂、以及用于分散所述活性剂的基质或载体组合。理想的添加剂包括，但不限于，pH值控制剂，诸如精氨酸、氢氧化钠、甘氨酸、盐酸、柠檬酸等。另外，可以包含局部麻醉剂(例如，苯甲醇)、等渗剂(例如，氯化钠、甘露醇、山梨糖醇)、吸附抑制剂(例如，吐温80)、溶解性增强剂(例如，环糊精及其衍生物)、稳定剂(例如，血清白蛋白)、和还原剂(例如，谷胱甘肽)。当用于黏膜递送的组合物是液体时，如参照视为统一的0.9％(w/v)生理盐水溶液的张力进行检测时，制剂的张力典型地被调整到这样的值，即，在所述张力值，在施用位点的鼻黏膜中将不会诱导基本的、不可逆的组织损伤。通常，将溶液的张力调整到约1/3到3，或1/2到2，或3/4到1.7的值。To formulate compositions for mucosal delivery in the present invention, the bioactive agent may be combined with various pharmaceutical additives, and a matrix or carrier for dispersing the active agent. Desirable additives include, but are not limited to, pH control agents such as arginine, sodium hydroxide, glycine, hydrochloric acid, citric acid, and the like. Additionally, local anesthetics (e.g., benzyl alcohol), isotonic agents (e.g., sodium chloride, mannitol, sorbitol), adsorption inhibitors (e.g., Tween 80), solubility enhancers (e.g., cyclic dextrin and its derivatives), stabilizers (eg, serum albumin), and reducing agents (eg, glutathione). When the composition for mucosal delivery is liquid, the tonicity of the formulation is typically adjusted to a value such that, at the At the stated tension values, no substantial, irreversible tissue damage will be induced in the nasal mucosa at the site of application. Typically, the tonicity of the solution is adjusted to a value of about 1/3 to 3, or 1/2 to 2, or 3/4 to 1.7.

所述生物活性剂可以分散在基质或赋形剂中，其可以包含具有分散所述活性剂的能力的亲水性化合物和任何需要的添加剂。所述基质可以选自宽范围的适当的载体，包括但不限于，聚羧酸的共聚物或其盐，羧酸酐(例如，顺式丁烯二酸酐)，与其它单体(例如，甲基(甲)丙烯酸酯、丙烯酸等)的共聚物，亲水性乙烯基聚合物如聚乙烯乙酸酯、聚乙烯醇、聚乙烯吡咯烷酮，纤维素衍生物如羟甲基纤维素、羟丙基纤维素等，和天然聚合物如脱乙酰壳多糖、胶原蛋白、藻酸钠、明胶、透明质酸及它们的非毒性金属盐。通常，选择可生物降解的聚合物作为基质或载体，例如，聚乳酸，聚(乳酸-乙醇酸)共聚物，多羟基丁酸，聚(羟基丁酸-乙醇酸)共聚物及它们的混合物。备选地或另外地，合成的脂肪酸酯如聚甘油脂肪酸酯、脂肪酸糖酯等可以用作载体。亲水性聚合物和其它载体可以单独使用或者组合使用，并且可以通过部分结晶化、离子键合、交联等赋予所述载体提高的结构完整性。载体可以以各种形式提供，包括，流体或黏性溶液、凝胶、糊剂、散剂、微球体和薄膜，它们直接用于鼻黏膜。在这种情形中，使用选择的载体可以导致促进生物活性剂的吸收。The bioactive agent may be dispersed in a matrix or vehicle, which may contain a hydrophilic compound having the ability to disperse the active agent and any desired additives. The matrix can be selected from a wide range of suitable carriers including, but not limited to, copolymers of polycarboxylic acids or salts thereof, carboxylic anhydrides (e.g., maleic anhydride), and other monomers (e.g., methyl (a) Copolymers of acrylate, acrylic acid, etc.), hydrophilic vinyl polymers such as polyvinyl acetate, polyvinyl alcohol, polyvinylpyrrolidone, cellulose derivatives such as hydroxymethyl cellulose, hydroxypropyl cellulose and natural polymers such as chitosan, collagen, sodium alginate, gelatin, hyaluronic acid and their non-toxic metal salts. Typically, a biodegradable polymer is chosen as the matrix or carrier, for example, polylactic acid, poly(lactic-co-glycolic acid), polyhydroxybutyric acid, poly(hydroxybutyric-co-glycolic acid) and mixtures thereof. Alternatively or additionally, synthetic fatty acid esters such as polyglycerol fatty acid esters, fatty acid sugar esters, etc. can be used as carriers. Hydrophilic polymers and other supports may be used alone or in combination, and enhanced structural integrity may be imparted to the support by partial crystallization, ionic bonding, cross-linking, and the like. Carriers can be presented in a variety of forms, including, fluid or viscous solutions, gels, pastes, powders, microspheres and films, which are applied directly to the nasal mucosa. In such cases, use of the carrier of choice can result in enhanced absorption of the bioactive agent.

所述生物活性剂可以与基质或载体按照各种方法组合，并且所述活性剂的释放可以通过扩散、载体的崩解、或者水道的缔合制剂而进行。在一些情形中，所述活性剂分散在由适当的聚合物例如2-氰基丙烯酸异丁酯制成的微胶囊(微球体)或纳米胶囊(纳米球体)中(参见，例如，Michael，等，J.Pharmacy Pharmacol.(药学药理学杂志)43：1-5，1991)，并且分散在用于鼻黏膜的生物相容性分散介质中，其产生随着延长的时间缓慢恒定的递送和生物活性。The bioactive agent can be combined with a matrix or carrier in various ways, and release of the active agent can be by diffusion, disintegration of the carrier, or associative formulation of waterways. In some cases, the active agent is dispersed in microcapsules (microspheres) or nanocapsules (nanospheres) made of a suitable polymer such as isobutyl 2-cyanoacrylate (see, e.g., Michael, et al. , J.Pharmacy Pharmacol. (Journal of Pharmaceutical Pharmacology) 43: 1-5, 1991), and dispersed in a biocompatible dispersion medium for nasal mucosa, which produces slow and constant delivery and biological active.

为了进一步增强本发明的药剂的黏膜递送，包含所述活性剂的制剂还可以含有亲水性低分子量化合物作为基质或赋形剂。所述亲水性低分子量化合物提供通道介质，通过其水溶性活性剂如生理活性肽或蛋白可以通过所述基质扩散到吸收所述活性剂的机体表面。所述亲水性低分子量化合物任选地吸收来自黏膜、或施用氛围的湿气，并且溶解水溶性活性肽。所述亲水性低分子量化合物的分子量通常不超过10000，并且优选不超过3000。代表性的亲水性低分子量化合物包括多元醇化合物，诸如寡-、二-和单糖如蔗糖、甘露醇、乳糖、L-阿拉伯糖、D-赤藓糖，D-核糖、D-木糖、D-甘露糖、D-半乳糖、乳酮糖、纤维二糖、龙胆二糖(gentibiose)、甘油和聚乙二醇。在本发明内用作载体的其它亲水性低分子量化合物的实例包括N-甲基吡咯烷酮和醇(例如，寡乙烯醇、乙醇、乙二醇、丙二醇等)。这些亲水性低分子量化合物可以单独使用或者彼此组合或者与所述鼻内制剂的其它活性或无活性成分组合使用。To further enhance the mucosal delivery of the agents of the invention, formulations comprising the active agent may also contain hydrophilic low molecular weight compounds as bases or excipients. The hydrophilic low molecular weight compound provides a channel medium through which a water-soluble active agent such as a physiologically active peptide or protein can diffuse through the matrix to the surface of the body where the active agent is absorbed. The hydrophilic low molecular weight compound optionally absorbs moisture from the mucosa, or the application atmosphere, and dissolves the water-soluble active peptide. The molecular weight of the hydrophilic low molecular weight compound is usually not more than 10000, and preferably not more than 3000. Representative hydrophilic low molecular weight compounds include polyol compounds such as oligo-, di- and monosaccharides such as sucrose, mannitol, lactose, L-arabinose, D-erythrose, D-ribose, D-xylose , D-mannose, D-galactose, lactulose, cellobiose, gentibiose, glycerol and polyethylene glycol. Examples of other hydrophilic low-molecular-weight compounds used as carriers in the present invention include N-methylpyrrolidone and alcohols (eg, oligovinyl alcohol, ethanol, ethylene glycol, propylene glycol, etc.). These hydrophilic low molecular weight compounds can be used alone or in combination with each other or with other active or inactive ingredients of the intranasal preparations.

本发明的组合物可以备选地包含适当的生理条件需要的药用载体物质，诸如pH值调节剂和缓冲剂、张力调节剂、湿润剂等，例如，乙酸钠、乳酸钠、氯化钠、氯化钾、氯化钙、失水山梨糖醇单月桂酸酯、三乙醇胺油酸酯等。关于固体组合物，可以使用常规无毒性药用载体，例如，其包括药学级甘露醇、乳糖、淀粉、硬脂酸镁、糖精钠、滑石、纤维素、葡萄糖、蔗糖、碳酸镁等。The compositions of the present invention may alternatively contain pharmaceutically acceptable carrier substances required for appropriate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents, etc., for example, sodium acetate, sodium lactate, sodium chloride, chloride Potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc. For solid compositions, conventional nontoxic pharmaceutical carriers can be used, including, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talc, cellulose, glucose, sucrose, magnesium carbonate, and the like.

在本发明的某些实施方案中，所述生物活性剂在定时释放制剂中施用，例如，在包含缓慢释放聚合物的组合物中施用。所述活性剂可以用防止快速释放的载体制备，例如，控制释放的赋形剂，如聚合物、微封装的递送系统或生物黏合凝胶。所述生物活性剂在本发明的各种组合物中的延长的递送可以通过在所述组合物中包含延缓吸收的试剂例如一硬脂酸铝水凝胶和明胶而导致。In certain embodiments of the invention, the bioactive agent is administered in a timed release formulation, eg, in a composition comprising a slow release polymer. The active agent can be prepared with carriers that will prevent rapid release, eg, controlled release excipients such as polymers, microencapsulated delivery systems or bioadhesive gels. Prolonged delivery of the bioactive agent in the various compositions of the invention can be brought about by including in the compositions an agent which delays absorption, for example, aluminum stearate hydrogel and gelatin.

当用于本文时，术语“受试者”意指可以施用本发明的组合物的任何哺乳动物患者。As used herein, the term "subject" means any mammalian patient to whom a composition of the invention may be administered.

试剂盒Reagent test kit

本发明还包括含有上述药物组合物、活性成分的试剂盒、包装和多容器单元(unit)，和/或用于施用其的方式，这些试剂盒、包装和多容器单元和/或施用方式用于在哺乳动物受试者中预防和治疗疾病和其它病况。简言之，这些试剂盒包括含有配制在用于黏膜递送的药物制剂中的一种或多种生物活性剂的容器或制剂。所述生物活性剂任选地包含在填充分配容器(bulk dispensing container)或单元或多单元剂型中。例如，肺部或鼻内喷雾涂药器可以提供任选的分配方式。包装材料任选地包括指示其中包装的药剂可以黏膜应用，例如，鼻内应用以治疗或预防具体的疾病或病况的标记或说明。The present invention also includes kits, packs and multicontainer units (units) containing the above pharmaceutical compositions, active ingredients, and/or means for administering them, which kits, packs and multicontainer units and/or means for administration are For the prevention and treatment of diseases and other conditions in mammalian subjects. Briefly, these kits include a container or formulation containing one or more bioactive agents formulated in a pharmaceutical formulation for mucosal delivery. The bioactive agent is optionally contained in a bulk dispensing container or in a unit or multiple unit dosage form. For example, pulmonary or intranasal spray applicators may provide optional means of dispensing. The packaging material optionally includes indicia or instructions indicating that the pharmaceutical agent packaged therein may be applied mucosally, eg, intranasally, to treat or prevent a particular disease or condition.

提高多核苷酸递送的多肽Polypeptides that enhance polynucleotide delivery

在本发明的其它实施方案中，选择或者合理设计提高多核苷酸递送的多肽以包含两亲性氨基酸序列。例如，可以选择有用的增强多核苷酸递送的多肽，其包含形成疏水序列结构域或基序的多个非极性或疏水性氨基酸残基，与形成带电荷序列结构域或基序的多个带电荷的氨基酸残基连接，产生两亲性肽。In other embodiments of the invention, polypeptides that enhance polynucleotide delivery are selected or rationally designed to comprise amphipathic amino acid sequences. For example, useful polynucleotide delivery-enhancing polypeptides can be selected to comprise a plurality of nonpolar or hydrophobic amino acid residues forming a hydrophobic sequence domain or motif, combined with a plurality of charged sequence domains or motifs. The charged amino acid residues are linked, resulting in an amphipathic peptide.

在其它实施方案中，选择包含蛋白转导结构域或基序和融合肽结构域或基序的提高多核苷酸递送的多肽。蛋白转导结构域是能够插入并且优选地通过细胞膜转运的肽序列。融合肽是能够使得脂质膜例如质膜或围绕内体的膜不稳定的肽，其可以在低pH值被增强。代表性的融合结构域或基序在广泛多样性的病毒融合蛋白和其它蛋白例如成纤维细胞生长因子4(FGF4)中找到。In other embodiments, polynucleotide delivery-enhancing polypeptides are selected that comprise a protein transduction domain or motif and a fusion peptide domain or motif. Protein transduction domains are peptide sequences capable of insertion and preferably transport across cell membranes. Fusion peptides are peptides capable of destabilizing lipid membranes such as plasma membranes or membranes surrounding endosomes, which can be enhanced at low pH. Representative fusion domains or motifs are found in a wide diversity of viral fusion proteins and other proteins such as fibroblast growth factor 4 (FGF4).

为了合理设计本发明的提高多核苷酸递送的多肽，将蛋白转导结构域用作促进核酸通过质膜进入细胞的基序。在某些实施方案中，转运的核酸将被封装在内体中。内体的内部具有低的pH值，导致融合肽基序使得内体的膜不稳定。内体膜的不稳定和破坏允许siNA释放到细胞质中，在这里siNA可以与RISC复合物缔合并且可以导向其靶点mRNA。In order to rationally design polynucleotide delivery-enhancing polypeptides of the invention, protein transduction domains are used as motifs to facilitate entry of nucleic acids into cells through the plasma membrane. In certain embodiments, the transferred nucleic acid will be encapsulated within endosomes. The interior of the endosome has a low pH, resulting in a fusion peptide motif that destabilizes the endosome's membrane. Destabilization and disruption of the endosomal membrane allows the release of siNA into the cytoplasm where it can associate with the RISC complex and can be directed to its target mRNA.

用于任选地结合到本发明的提高多核苷酸递送的多肽中的蛋白转导结构域的实例包括：Examples of protein transduction domains for optional incorporation into polynucleotide delivery enhancing polypeptides of the invention include:

1.TAT蛋白转导结构域(PTD)(SEQ ID NO：1)KRRQRRR；1. TAT protein transduction domain (PTD) (SEQ ID NO: 1) KRRQRRR;

2.Penetratin PTD(SEQ ID NO：2)RQIKIWFQNRRMKWKK；2. Penetratin PTD (SEQ ID NO: 2) RQIKIWFQNRRMKWKK;

3.VP22 PTD(SEQ ID NO：3)DAATATRGRSAASRPTERPRAPARSASRPRRPVD；3. VP22 PTD (SEQ ID NO: 3) DAATATRGRSAASRPTERPRAPARSASRPRRPVD;

4.卡波西(Kaposi)FGF信号序列(SEQ ID NO：4)AAVALLPAVLLALLAP，和SEQ ID NO：5)AAVLLPVLLPVLLAAP；4. Kaposi (Kaposi) FGF signal sequence (SEQ ID NO: 4) AAVALLPAVLLALLAP, and SEQ ID NO: 5) AAVLLPPVLLPVLLAAP;

5.人β3整联蛋白信号序列(SEQ ID NO：6)VTVLALGALAGVGVG；5. Human β3 integrin signal sequence (SEQ ID NO: 6) VTVLALGALAGVGVG;

6.gp41融合序列(SEQ ID NO：7)GALFLGWLGAAGSTMGA；6. gp41 fusion sequence (SEQ ID NO: 7) GALFLGWLGAAGSTMGA;

7.凯门鳄(Caiman crocodylus)Ig(v)轻链(SEQ ID NO：8)MGLGLHLLVLAAALQGA；7. Caiman crocodylus (Caiman crocodylus) Ig (v) light chain (SEQ ID NO: 8) MGLGLHLLVLAAALQGA;

8.hCT-来源的肽(SEQ ID NO：9)LGTYTQDFNKFHTFPQTAIGVGAP；8. hCT-derived peptide (SEQ ID NO:9) LGTYTQDFNKFHTFPQTAIGVGAP;

9.Transportan(SEQ ID NO：10)GWTLNSAGYLLKINLKALAALAKKIL；9. Transportan (SEQ ID NO: 10) GWTLNSAGYLLKINLKALAALAKKIL;

10.Loligomer(SEQ ID NO：11)TPPKKKRKVEDPKKKK；10. Loligomer (SEQ ID NO: 11) TPPKKKRKVEDPKKKK;

11.精氨酸肽(SEQ ID NO：12)RRRRRRR；和11. Arginine peptide (SEQ ID NO: 12) RRRRRR; and

12.两亲性模式肽(SEQ ID NO：13)KLALKLALKALKAALKLA。12. The amphipathic pattern peptide (SEQ ID NO: 13) KLALKLALKALKAALKLA.

用于任选地结合到本发明的提高多核苷酸递送的多肽中的病毒融合肽融合结构域的实例包括：Examples of viral fusion peptide fusion domains for optional incorporation into polynucleotide delivery enhancing polypeptides of the invention include:

1.流感HA2(SEQ ID NO：14)GLFGAIAGFIENGWEG；1. Influenza HA2 (SEQ ID NO: 14) GLFGAIAGFIENGWEG;

2.仙台病毒(Sendai)F1(SEQ ID NO：15)FFGAVIGTIALGVATA；2. Sendai virus (Sendai) F1 (SEQ ID NO: 15) FFGAVIGTIALGVATA;

3.呼吸道合胞病毒F1(SEQ ID NO：16)FLGFLLGVGSAIASGV；3. Respiratory syncytial virus F1 (SEQ ID NO: 16) FLGFLLGVGSAIASGV;

4.HIV gp41(SEQ ID NO：17)GVFVLGFLGFLATAGS；和4. HIV gp41 (SEQ ID NO: 17) GVFVLGFLGFLATAGS; and

5.依波拉病毒(Ebola)GP2(SEQ ID NO：18)GAAIGLAWIPYFGPAA。5. Ebola virus (Ebola) GP2 (SEQ ID NO: 18) GAAIGLAWIPYFGPAA.

然而在本发明的其它实施方案中，提供提高多核苷酸递送的多肽，其结合促进本发明的方法和组合物内的多肽-siNA复合物形成和/或增强siNAs的递送的DNA-结合结构域或基序。在这种情形中代表性的DNA结合结构域包括许多“锌指”结构域，如关于DNA-结合调控蛋白和下文鉴定的其它蛋白所述(参见，例如，Simpson，等，J.Biol.Chem.(生物化学杂志)278：28011-28018，2003)。In other embodiments of the invention, however, polynucleotide delivery-enhancing polypeptides are provided that bind DNA-binding domains that facilitate polypeptide-siNA complex formation within the methods and compositions of the invention and/or enhance delivery of siNAs or motif. Representative DNA-binding domains in this context include a number of "zinc finger" domains, as described for DNA-binding regulatory proteins and other proteins identified below (see, e.g., Simpson, et al., J. Biol. Chem. . (Journal of Biochemistry) 278:28011-28018, 2003).

表1：Table 1:

不同DNA-结合蛋白的代表性锌指基序Representative zinc finger motifs of different DNA-binding proteins

^*本表显示双链DNA结合的保守锌指基序，其特征在于C-x(2，4)-C-x(12)-H-x(3)-H基序模式，其本身可以用来选择并且设计按照本发明的提高多核苷酸递送的多肽。^* This table shows a conserved zinc finger motif for double-stranded DNA binding, characterized by the Cx(2,4)-Cx(12)-Hx(3)-H motif pattern, which itself can be used to select and design according to this Inventive polypeptides that enhance polynucleotide delivery.

^**表1中关于Sp1，Sp2，Sp3，Sp4，DrosBtd，DrosSp，CeT22C8.5和Y4pB1A.4所示的序列在本文分别编号为SEQ ID NOs 19，20，21，22，23，24，25，和26。^** The sequences shown in Table 1 for Sp1, Sp2, Sp3, Sp4, DrosBtd, DrosSp, CeT22C8.5 and Y4pB1A.4 are numbered herein asSEQ ID NOs 19, 20, 21, 22, 23, 24, 25, respectively , and 26.

用于构建本发明提高多核苷酸递送的多肽的备选DNA结合结构域包括，例如，HIV Tat蛋白序列的部分(参见，实施例，如下)。Alternative DNA binding domains useful in constructing polynucleotide delivery-enhancing polypeptides of the invention include, for example, portions of the HIV Tat protein sequence (see, Examples, below).

在本文下述的本发明的代表性实施方案中，通过将任何前述结构元件、结构域或基序组合到单一多肽中，可以合理地设计并且构建提高多核苷酸递送的多肽，以有效地调控siNAs到靶点细胞的增强的递送。例如，将TAT多肽的蛋白转导结构域融合到叫作HA2的流感病毒血凝素蛋白的N-端20个氨基酸中，以产生一种代表性的本发明的提高多核苷酸递送的多肽。在本发明公开内容中提供各种其它的提高多核苷酸递送的多肽构建体，这表明本发明的概念广泛地适用于产生并且应用用于提高siNA递送的有效的提高多核苷酸递送的多肽的各种集合。In representative embodiments of the invention described below, polypeptides that enhance polynucleotide delivery can be rationally designed and constructed by combining any of the aforementioned structural elements, domains or motifs into a single polypeptide to efficiently regulate Enhanced delivery of siNAs to target cells. For example, the protein transduction domain of the TAT polypeptide was fused to the N-terminal 20 amino acids of the influenza virus hemagglutinin protein designated HA2 to generate a representative polynucleotide delivery-enhancing polypeptide of the invention. Various other polynucleotide delivery-enhancing polypeptide constructs are provided in the present disclosure, which demonstrates that the concepts of the present invention are broadly applicable to the generation and application of effective polynucleotide delivery-enhancing polypeptides for enhancing siNA delivery. Various collections.

本发明内其它代表性的提高多核苷酸递送的多肽可以选自下述肽：WWETWKPFQCRICMRNFSTRQARRNHRRRHR(SEQ ID NO：27)；GKINLKALAALAKKIL(SEQ ID NO：28)，RVIRVWFQNKRCKDKK(SEQID NO：29)，GRKKRRQRRRPPQGRKKRRQRRRPPQGRKKRRQRRRPPQ(SEQ ID NO：30)，GEQIAQLIAGYIDIILKKKKSK(SEQ ID NO：31)，聚Lys-Trp，4∶1，MW 20,000-50,000；和聚Orn-Trp，4∶1，MW 20,000-50,000。其它用于本发明的组合物和方法的提高多核苷酸递送的多肽包含所提及的(mellitin)蛋白序列的全部或部分。Other representative polynucleotide delivery-enhancing polypeptides within the present invention can be selected from the following peptides: WWETWKPFQCRICMRNFSTRQARRNHRRRHR (SEQ ID NO: 27); GKINLKALAALAKKIL (SEQ ID NO: 28), RVIRVWFQNKRCKDKK (SEQ ID NO: 29), GRKKRRQRRRPPQGRKKRRQRPKRRPPQGR (SEQ ID NO: 29) ID NO: 30), GEQIAQLIAGYIDIILKKKKSK (SEQ ID NO: 31), poly Lys-Trp, 4:1, MW 20,000-50,000; and poly Orn-Trp, 4:1, MW 20,000-50,000. Other polynucleotide delivery enhancing polypeptides useful in the compositions and methods of the invention comprise all or part of the mentioned (mellitin) protein sequences.

实施例Example

本发明通过下述实施例进行举例说明，所述实施例不限制在权利要求中描述的本发明的范围。The invention is illustrated by the following examples, which do not limit the scope of the invention described in the claims.

实施例1Example 1

黏膜递送-渗透动力学和细胞毒性Mucosal Delivery - Osmotic Kinetics and Cytotoxicity

器官型模型organotypic model

下述方法通常用于评估关于生物活性治疗剂和提高黏膜递送的有效量的渗透性肽的黏膜递送参量、动力学和副作用，所述提高黏膜递送的有效量的渗透性肽通过调控哺乳动物受试者中上皮细胞连接结构和/或生理而可逆地提高黏膜上皮旁细胞转运。The methods described below are generally used to assess mucosal delivery parameters, kinetics, and side effects for biologically active therapeutic agents and mucosal delivery-enhancing effective amounts of penetrant peptides that regulate Epithelial cell junction structure and/or physiology reversibly increased mucosal paraepithelial cell transport in subjects.

EpiAirway^TM系统由马特泰克(MatTek)公司(阿什兰，MA)开发，作为内衬于呼吸道的假复层上皮的模型。上皮细胞生长在气液界面处的以多孔膜为底部的细胞培养插入物上，这导致细胞分化成高度极化的形态。顶面有纤毛，具有微绒毛超结构，并且上皮产生粘液(通过免疫印迹已经证实了粘蛋白的存在)。插入物的直径为0.875cm，提供0.6cm²的表面积。在运输前约3周在工厂将细胞铺在插入物上。一个“试剂盒”由24个单元组成。The EpiAirway^™ system was developed by MatTek (Ashland, MA) as a model for the pseudostratified epithelium lining the airways. Epithelial cells are grown on porous membrane-bottomed cell culture inserts at the air-liquid interface, which leads to differentiation of cells into a highly polarized morphology. The apical surface is ciliated with a microvilli ultrastructure, and the epithelium produces mucus (the presence of mucin has been confirmed by immunoblotting). The insert has a diameter of 0.875 cm, providing a surface area of 0.6^cm2 . Cells were plated on the inserts at the factory approximately 3 weeks prior to shipping. A "kit" consists of 24 units.

A.当到达时，将所述单元放置在6孔微量培养板中的无菌支持物上。每个孔接受5mL专有的(proprietary)培养基。这种基于DMEM的培养基不含血清，但是补充了表皮生长因子和其它因子。一直检测所述培养基中认为用于鼻内递送的任何细胞因子或生长因子的内源水平，但是除胰岛素外不含所有细胞因子和迄今为止已研究的因子。5mL的体积恰好足以提供与单元自身底部的接触，但是允许上皮的顶面保持与空气直接接触。在这一步骤及在包括将单元转移到含有液体的孔中的所有后续步骤中都使用无菌镊子，以确保在所述单元的底部和培养基之间没有截留空气。A. Upon arrival, the units were placed on sterile supports in 6-well microplates. Each well received 5 mL of proprietary medium. This DMEM-based medium is serum-free but supplemented with epidermal growth factor and other factors. The medium was always tested for endogenous levels of any cytokine or growth factor thought to be useful for intranasal delivery, but was free of all cytokines and factors studied so far except insulin. A volume of 5 mL was just enough to provide contact with the bottom of the cell itself, but allow the top surface of the epithelium to remain in direct contact with the air. Sterile forceps were used at this step and in all subsequent steps including transferring the cells into wells containing the liquid to ensure that no air was trapped between the bottom of the cells and the medium.

B.将在它们的平板中的单元在培养箱中在空气中含有5％CO₂的氛围中在37℃保持24小时。在这一时间末，用新鲜培养基替换所述培养基，并且将所述单元放回培养箱继续保持24小时。B. Keep the cells in their plates at 37 °C for 24 h in an incubator in an atmosphere containing 5%_CO2 in air. At the end of this time, the medium was replaced with fresh medium and the unit was returned to the incubator for an additional 24 hours.

实验方案--渗透动力学Experimental Protocol - Osmotic Kinetics

A.24EpiAirway^TM单元的“试剂盒”可常规用于评估5种不同的制剂，每种制剂都应用在一式四份的孔中。每个孔用来确定渗透动力学(4个时间点)、跨上皮电阻(TER)。另一组孔用作对照，它们在确定渗透动力学过程中进行假处理，但是其他的与用于确定跨上皮细胞阻抗和存活能力的含有测试样品的单元同样处理。A. The "kit" of the 24 EpiAirway^™ unit was routinely used to evaluate 5 different formulations, each applied in quadruplicate wells. Each well was used to determine permeation kinetics (4 time points), transepithelial electrical resistance (TER). Another set of wells was used as a control, which was sham-treated during the determination of permeation kinetics, but otherwise treated identically to the cells containing the test sample for the determination of transepithelial impedance and viability.

B.在所有实验中，将待研究的黏膜递送制剂以100μL的体积应用到每个单元的顶面，所述体积足以覆盖整个顶面。留出适当体积的应用到顶面的浓度的测试制剂(通常所需要的不超过100μL)，以随后通过ELISA或其它指定的检测而确定活性物质的浓度。B. In all experiments, the mucosal delivery formulation under study was applied to the top surface of each unit in a volume of 100 μL, sufficient to cover the entire top surface. Set aside an appropriate volume of test formulation at the concentration applied to the top surface (no more than 100 [mu]L is usually required) for subsequent determination of the active substance concentration by ELISA or other designated assay.

C.将所述单元放置在无架台的6孔平板中用于本实验：每孔含有0.9mL培养基，其足以与所述单元的多孔膜底部接触，但是对所述单元不产生任何显著的向上的流体静力学压力。C. The unit was placed in a rackless 6-well plate for this experiment: each well contained 0.9 mL of medium, which was sufficient to contact the bottom of the porous membrane of the unit, but did not cause any significant effect on the unit Upward hydrostatic pressure.

D.为了将误差的潜在来源最小化并且避免任何浓度梯度的形成，在研究中的每一时间点，将所述单元从一个含有0.9mL的孔转移到另一个孔。基于将100μL体积的测试物质应用到顶面的零时刻，这些转移在下列时间点进行：15分钟，30分钟，60分钟，和120分钟。D. To minimize potential sources of error and avoid the formation of any concentration gradients, the units were transferred from one well containing 0.9 mL to the other at each time point in the study. These transfers were performed at the following time points: 15 minutes, 30 minutes, 60 minutes, and 120 minutes, based on time zero when a volume of 100 μL of test substance was applied to the top surface.

E.在时间点之间，将在它们的平板中的单元保持在37℃培养箱中。还将每孔含有0.9mL培养基的平板保持在培养箱中，以致在当将平板移出并且用无菌镊子将单元从一个孔转移到另一个孔的短时间内发生温度的最小变化。E. Keep the units in their plates in a 37°C incubator between time points. The plates containing 0.9 mL of medium per well were also kept in the incubator so that minimal changes in temperature occurred during the short time when the plates were removed and cells were transferred from one well to another with sterile forceps.

F.每个时间点完成时，将培养基从转移每个单元的孔移出，并且整分到两个管中(一个管接收700μL，和另一个管接收200μL)，以用于确定渗透的测试物质的浓度，并且在所述测试物质是细胞毒性的情形中，以用于从上皮细胞释放细胞质酶，乳酸脱氢酶。如果检测将在24小时内进行，那么将这些样品存放在冰箱中，或者将所述样品亚整分，并且在-80℃冷冻保存，直到解冻一次进行检测。避免重复的冷冻-解冻循环。F. At the completion of each time point, media was removed from the well from which each unit was transferred and aliquoted into two tubes (one tube received 700 μL, and the other tube received 200 μL) for testing to determine penetration concentration of the substance and, in the case that the test substance is cytotoxic, for the release of the cytoplasmic enzyme, lactate dehydrogenase, from the epithelial cells. If testing is to be performed within 24 hours, store these samples in the refrigerator, or sub-aliquot the samples and store frozen at -80°C until thawed once for testing. Avoid repeated freeze-thaw cycles.

G.为了将误差最小化，在开始实验之前将所有的管、平板和孔都预先标记。G. To minimize error, pre-label all tubes, plates and wells prior to starting the experiment.

H.在120分钟时间点结束时，将单元从最后含有0.9mL的孔转移到每孔含有0.3mL培养基的24孔微量培养板中。这一体积也足以接触单元底部，但是不会对所述单元施加向上的流体静力学压力。在检测跨上皮细胞阻抗之前将所述单元放回培养箱中。H. At the end of the 120 minute time point, transfer units from the last well containing 0.9 mL to a 24-well microplate containing 0.3 mL of medium per well. This volume is also sufficient to contact the bottom of the cell without exerting upward hydrostatic pressure on the cell. The unit was returned to the incubator prior to measurement of transepithelial impedance.

实验方案--跨上皮电阻Experimental Protocol - Transepithelial Resistance

A.呼吸道上皮细胞在体内和体外形成紧密连接，并且由此限制溶质穿过组织的流动。这些连接在切除的呼吸道组织中赋予几百欧姆×cm²的跨上皮电阻。在马特泰克(MatTek)EpiAirway^TM单元中，生产商声称跨上皮电阻(TER)通常在约1000欧姆×cm²。本文确定的数据表明，在渗透研究中的步骤顺序中假暴露的对照EpiAirway^TM单元的TER稍低(700-800欧姆×cm²)，但是，由于小分子的渗透与TER的倒数成比例，所以这一值仍然足够高，足以为渗透提供基本的屏障。相反，不含细胞的以多孔膜为底的单元只提供最小的跨膜电阻(约5-20欧姆×cm²)。A. Airway epithelial cells form tight junctions in vivo and in vitro and thereby restrict the flow of solutes through the tissue. These connections confer transepithelial resistances of several hundred ohms ×^cm in resected airway tissue. In the MatTek EpiAirway^™ unit, the manufacturer states that the transepithelial resistance (TER) is typically around 1000 ohms x^cm2 . The data identified here indicate that the TER of the sham-exposed control^EpiAirwayTM unit in the sequence of steps in the permeation study was slightly lower (700-800 ohm x^cm2 ), however, since the permeation of small molecules is proportional to the inverse of the TER, the This value is still high enough to provide a basic barrier to infiltration. In contrast, cell-free porous membrane-based cells offer only minimal transmembrane resistance (approximately 5-20 ohms x^cm2 ).

B.TER的准确测定需要将欧姆计的电极置于膜上和膜下的显著的表面积上，并且从所述膜到电极的距离应该可被再现地控制。由MatTek推荐并且本发明的所有实验都应用的TER确定方法使用来自WorldPrecision Instruments，Inc.(世界精密仪器公司)，萨拉索塔，FL的″EVOM″^TM上皮细胞伏特欧姆计和″ENDOHM″^TM组织电阻测量室(″ENDOHM″^TM tissue resistance measurement chamber)。B. Accurate determination of TER requires placing the electrodes of the ohmmeter over a significant surface area above and below the membrane, and the distance from the membrane to the electrodes should be reproducibly controlled. The TER determination method recommended by MatTek and applied in all experiments of the present invention uses the "EVOM"^™ epithelial cell volt-ohmmeter and "ENDOHM"^™ from WorldPrecision Instruments, Inc., Sarasota, FL. Tissue resistance measurement chamber ("ENDOHM"^TM tissue resistance measurement chamber).

C.在测定TER之前，将所述室开始充满杜尔贝科(Dulbecco’s)磷酸缓冲盐水(PBS)至少20分钟，以平衡电极。C. Initially fill the chamber with Dulbecco's phosphate buffered saline (PBS) for at least 20 minutes to equilibrate the electrodes prior to measuring TER.

D.用所述室中的1.5mL PBS和被测量的以膜为底的单元中的350μLPBS测定TER。将顶部电极调整到正好在不含细胞(但是含有350μL PBS)的一个单元的膜上部的位置，然后固定，以保证可再现地放置。不含细胞的单元的电阻典型地为5-20欧姆×cm²(″背景电阻″)。D. TER was determined with 1.5 mL of PBS in the chamber and 350 μL of PBS in the membrane-bottomed unit being measured. The top electrode was adjusted to position just above the membrane of one cell without cells (but containing 350 [mu]L PBS) and fixed to ensure reproducible placement. Cell-free cells typically have an electrical resistance of 5-20 ohms x^cm2 ("background electrical resistance").

E.一旦所述室准备好并且记录了背景电阻，那么将刚才用于渗透测定的24孔平板从培养箱中移出，并且单独放置在所述室中进行TER测定。E. Once the chamber was prepared and the background resistance was recorded, the 24-well plate just used for the permeability assay was removed from the incubator and placed in the chamber alone for the TER assay.

F.首先将每个单元转移到含有PBS的培养皿中，以确保膜底部湿润。将350μL PBS的等分试样添加到所述单元中，然后小心地吸入标记的管中以润洗顶面。然后对所述单元应用350μL PBS再次洗涤，并且将PBS吸入到同一收集管中。F. First transfer each cell to a Petri dish containing PBS to ensure that the bottom of the membrane is wet. Add an aliquot of 350 µL of PBS to the cell and carefully pipette into the labeled tube to rinse the top surface. The cells were then washed again with 350 μL of PBS, and the PBS was pipetted into the same collection tube.

G.在置于所述室(含有新鲜的1.5mL PBS等分试样)之前，将所述单元外表面轻轻印迹除去过多的PBS。在将顶部电极放置于所述室上之前将350μL PBS等分试样添加到所述单元中，并且在EVOM计上读取TER。G. Gently blot the outer surface of the unit to remove excess PBS before placing in the chamber (containing a fresh 1.5 mL aliquot of PBS). A 350 μL aliquot of PBS was added to the cell before placing the top electrode on the chamber and TER was read on the EVOM meter.

H.在ENDOHM室中读取所述单元的TER之后，移出所述单元，吸出PBS并且保存，并且将所述单元放回到每孔含有0.3mL培养基的24孔平板，在所述单元的顶面具有空气界面。H. After reading the TER of the unit in the ENDOHM chamber, remove the unit, aspirate the PBS and save, and put the unit back into the 24-well plate containing 0.3 mL of medium per well, in the The top surface has an air interface.

I.所述单元以下述顺序读取：所有假处理的对照，然后是所有制剂-处理的样品，接着是每个假处理的对照的第二次TER读数。所有的TER值报告为组织表面积的函数。I. The cells are read in the following order: all sham-treated controls, then all formulation-treated samples, followed by a second TER reading for each sham-treated control. All TER values are reported as a function of tissue surface area.

TER计算为：TER is calculated as:

TER＝(R₁-R_b)×ATER=(R₁ -R_b )×A

其中R_I是具有膜的插入物的电阻，R_b是空白插入物的电阻，并且A是膜的面积(0.6cm²)。包含增强鼻内递送增强试剂例如渗透性肽的药物制剂的作用通过穿过EpiAirway^TM细胞膜(黏膜上皮细胞层)的TER检测。渗透性肽以1.0mM的浓度应用到EpiAirway^TM细胞膜。TER值相对于对照值(对照＝约1000欧姆-cm²；标准化到100)的减小表明细胞膜电阻的减小和黏膜上皮细胞渗透性的增加。where R_I is the resistance of the insert with the membrane,_Rb is the resistance of the blank insert, and A is the area of the membrane (0.6 cm² ). The effect of drug formulations containing enhanced intranasal delivery enhancing agents such as penetrative peptides is detected by TER across EpiAirway^™ cell membranes (mucosal epithelial cell layer). The penetrating peptide was applied to the EpiAirway^™ cell membrane at a concentration of 1.0 mM. A decrease in TER values relative to control values (control = about 1000 ohm-^cm2 ; normalized to 100) indicates a decrease in cell membrane resistance and an increase in permeability of mucosal epithelial cells.

实验方案--LDH检测Experimental protocol--LDH detection

通过使用CytoTox 96细胞毒性检测试剂盒(普洛麦格(Promega)公司，麦迪逊，威斯康星州)检测来自细胞的乳酸脱氢酶(LDH)的损失而测定细胞死亡量。将50微升样品上样到96孔检测平板上。新鲜的不含细胞的培养基用作空白。向每个孔中加入50μl底物溶液，并且将平板在黑暗中在室温温育30分钟。温育后，向每个孔中加入50μl终止溶液，并且将所述平板在光学密度平板读数仪上在490nm读数。The amount of cell death was determined by detecting the loss of lactate dehydrogenase (LDH) from cells using the CytoTox 96 Cytotoxicity Assay Kit (Promega, Madison, Wisconsin).Load 50 microliters of sample onto a 96-well assay plate. Fresh medium without cells was used as blank. 50 μl of substrate solution was added to each well and the plate was incubated for 30 minutes at room temperature in the dark. After incubation, 50 μl of stop solution was added to each well and the plate was read at 490 nm on an optical density plate reader.

实验方案--EIA方法Experimental protocol--EIA method

EIA试剂盒(p/n S-1178(EIAH6101)购自半岛实验室公司(PeninsulaLaboratories Inc.BACHEM分部，圣卡洛斯，加利福尼亚，800-922-1516)。17×120mm聚丙烯圆锥管(p/n 352097，法欧肯(Falcon)，富兰克林湖(Franklin Lakes)，新泽西)用于所有样品制备。8种标准物用于PTH定量。其余的检测步骤与试剂盒插入物相同。EIA kit (p/n S-1178 (EIAH6101) was purchased from Peninsula Laboratories Inc. BACHEM Division, San Carlos, California, 800-922-1516). 17 × 120mm polypropylene conical tube (p/n n 352097, Falcon, Franklin Lakes, NJ) was used for all sample preparation. Eight standards were used for PTH quantification. The remaining detection steps were the same as for the kit insert.

实施例2Example 2

通过PN159增强上皮渗透作用Enhanced epithelial penetration by PN159

本发明下述实施例表明，本发明的渗透作用增强肽，以PN159为例，增强肽治疗药物包括PTH和肽YY的黏膜渗透。由PN159表明的本发明的肽的这种渗透增强活性，可能等价于，或者大于通过使用一种或多种小分子渗透增强剂而获得的上皮渗透增强。The following examples of the present invention show that the penetration-enhancing peptides of the present invention, taking PN159 as an example, can enhance the mucosal penetration of peptide therapeutic drugs including PTH and peptide YY. This penetration enhancing activity of the peptides of the invention, as demonstrated by PN159, may be equivalent to, or greater than, the epithelial penetration enhancement obtained by using one or more small molecule penetration enhancers.

肽YY_3-36(PYY _3-36)是一种已经成为许多临床试验的主题的34个氨基酸的肽。这种生物活性肽的黏膜递送可以在包含小分子渗透增强剂的制剂中得到增强。因此，本研究评估以PN159为例的本发明的渗透增强肽是否可以代替小分子渗透增强剂促进肽YY的黏膜递送的作用。这些研究包括体外评估PN159减小跨上皮电阻(TEER)和增加标记物质的渗透的作用，以及证明与所述体外结果一致的相关的体内研究。Peptide YY_3-36 (PYY_3-36 ) is a 34 amino acid peptide that has been the subject of many clinical trials. Mucosal delivery of such bioactive peptides can be enhanced in formulations containing small molecule penetration enhancers. Therefore, this study evaluates whether the penetration-enhancing peptides of the present invention, PN159 as an example, can replace small-molecule penetration enhancers to promote the mucosal delivery of peptide YY. These studies included an in vitro assessment of the effect of PN159 on reducing transepithelial electrical resistance (TEER) and increasing penetration of labeled substances, as well as related in vivo studies demonstrating consistency with the in vitro results.

在本实施例中，描述PN159与PTH的组合。PTH可以是全长肽(1-84)、或片段如(1-34)。所述制剂还可以是PTH、渗透性肽、和一种或多种其它渗透增强剂的组合。所述制剂还可以包含缓冲剂、张力剂、pH值调节剂、和肽/蛋白稳定剂如氨基酸、糖或多元醇、聚合物、和盐。In this example, the combination of PN159 and PTH is described. PTH can be a full length peptide (1-84), or a fragment such as (1-34). The formulation may also be a combination of PTH, a penetrating peptide, and one or more other penetration enhancers. The formulations may also contain buffers, tonicity agents, pH adjusters, and peptide/protein stabilizers such as amino acids, sugars or polyols, polymers, and salts.

设计本研究以评估PN159自身或与其它渗透增强剂组合对PTH渗透的作用。评估的PN159浓度为25，50，和100μM。其它渗透增强剂为45mg/ml M-β-CD，1mg/ml DDPC，和1mg/ml EDTA。山梨醇用作张力剂(146-190mM)以将制剂的摩尔渗透压浓度(osmolarity)调节到220mOsm/kg。将制剂pH值固定在4.5。在本实施例中选择PTH作为模型肽。2mg/ml PTH与PN159与或不与其它渗透增强剂组合。使用体外上皮组织模型检测所述组合，以通过LDH检测而监测PTH渗透、跨上皮电阻(TER)，和所述制剂的细胞毒性。This study was designed to evaluate the effect of PN159 on PTH penetration by itself or in combination with other penetration enhancers. PN159 concentrations evaluated were 25, 50, and 100 μM. Other penetration enhancers are 45mg/ml M-β-CD, 1mg/ml DDPC, and 1mg/ml EDTA. Sorbitol was used as a tonicity agent (146-190 mM) to adjust the osmolarity of the formulation to 220 mOsm/kg. The formulation pH was fixed at 4.5. In this example, PTH was chosen as the model peptide. 2 mg/ml PTH in combination with PN159 with or without other penetration enhancers. The combinations were tested using an in vitro epithelial tissue model to monitor PTH penetration, transepithelial electrical resistance (TER), and cytotoxicity of the formulations by LDH assay.

跨上皮电阻transepithelial resistance

来自本研究的TER检测的结果表明大于80％的TER减少由PN159引起。用增加的PN159浓度观察到更高的TER减少。应用到顶面的培养基没有减小TER，而triton X处理的组如预计那样表现出显著的TER减少。The results from the TER assay in this study indicated that greater than 80% of the TER reduction was caused by PN159. Higher TER reductions were observed with increasing PN159 concentrations. Medium applied to the apex did not reduce TER, whereas the triton X treated group exhibited a significant reduction in TER as expected.

细胞毒性Cytotoxicity

本研究关于LDH的数据表明，当用25-100μM PN159处理细胞时，没有观察到显著的细胞毒性。应用到顶面的培养基没有表现出细胞毒性，而Triton X处理的组如预计那样表现出显著的细胞毒性。The data from this study on LDH indicated that no significant cytotoxicity was observed when cells were treated with 25-100 μM PN159. Medium applied to the top surface showed no cytotoxicity, while the Triton X-treated group showed significant cytotoxicity as expected.

渗透penetration

关于有和无其它增强剂的PN159的PTH_1-34渗透数据分别在图1和2中显示。在存在PN159时观察到PTH渗透的显著增加。在25，50，和100μM PN159之间没有观察到％渗透的显著的不同。PN159对PTH渗透的作用与45/1/1mg/ml M-β-CD/DDPC/EDTA的作用相当。使用45/1/1mg/mlM-b-CD/DDPC/EDTA和PN159组合观察到PTH渗透的额外的增加。The PTH_1-34 permeation data for PN159 with and without other enhancers are shown in Figures 1 and 2, respectively. A significant increase in PTH permeation was observed in the presence of PN159. No significant difference in % penetration was observed between 25, 50, and 100 [mu]M PN159. The effect of PN159 on PTH penetration was comparable to that of 45/1/1 mg/ml M-β-CD/DDPC/EDTA. An additional increase in PTH penetration was observed with the 45/1/1 mg/ml M-b-CD/DDPC/EDTA and PN159 combination.

实施例3Example 3

PN159对肽激素治疗剂的体内渗透增强作用等于或超过小分子渗透增强In vivo penetration enhancement of peptide hormone therapeutics by PN159 equals or exceeds small molecule penetration enhancement剂的增强作用Enhancement of the agent

将20只3-6月龄并且重2.1-3.0kg的雄性新西兰白兔随机分成5个处理组，每组4只动物。将测试动物以15μl/kg并且通过吸管鼻内给药。下表5表示5种不同的给药组的组合物。Twenty male New Zealand white rabbits aged 3-6 months and weighing 2.1-3.0 kg were randomly divided into 5 treatment groups of 4 animals each. Test animals were dosed intranasally at 15 μl/kg and via pipette. Table 5 below shows the composition of 5 different dosing groups.

对于给药组1(见表2)，使用包含小分子渗透增强剂的PYY临床制剂。在这些研究中的小分子增强剂包括甲基-β环糊精、二癸酰基磷脂酰胆碱(DDPC)、和/或EDTA。给药组2接受溶解在磷酸缓冲的盐水(PBS)中的PYY。对于给药组3-5，将不同浓度的PN159加到给药组2中，以致给药组3-5中的每一组由PYY，PN159和PBS组成。For dosing group 1 (see Table 2), a clinical formulation of PYY containing a small molecule penetration enhancer was used. Small molecule enhancers in these studies included methyl-beta cyclodextrin, didecanoylphosphatidylcholine (DDPC), and/or EDTA.Dosing group 2 received PYY dissolved in phosphate buffered saline (PBS). For administration groups 3-5, different concentrations of PN159 were added toadministration group 2 so that each of administration groups 3-5 consisted of PYY, PN159 and PBS.

表2Table 2

组Group动物animal渗透增强剂penetration enhancer  给药浓度(mg/ml)Dosing concentration (mg/ml)  给药体积(ml/kg)Dosing volume (ml/kg)  PYY剂量(μg/kg)PYY dose (μg/kg)  1 1  4M4M  小分子渗透增强剂Small molecule penetration enhancer  13.6713.67  0.0150.015  205205  2 2  4M4M无none  13.6713.67  0.0150.015  205205  33  4M4M  25μM PN15925μM PN159  13.6713.67  0.0150.015  205205  44  4M4M  50μM PN15950μM PN159  13.6713.67  0.0150.015  205205  55  4M4M  100μM PN159100μM PN159  13.6713.67  0.0150.015  205205

通过直接在耳缘静脉进行直接静脉穿刺而收集连续的血液样品(每份约2ml)，收集到含有EDTA作为抗凝血剂的血液收集管中。在给药后0，2.5，5，10，15，30，45，60，和120分钟收集血液样品。收集血液之后，管子轻轻摇动几次，以抗凝血，然后加入50μl牛胰蛋白酶抑制剂溶液。在大约4℃以约1,600xg离心血液15分钟，并且血浆样品分配到一式两份等分试样中，并且在大约-70℃冷冻保存。Serial blood samples (approximately 2 ml each) were collected by direct venipuncture in the marginal ear vein into blood collection tubes containing EDTA as an anticoagulant. Blood samples were collected at 0, 2.5, 5, 10, 15, 30, 45, 60, and 120 minutes after dosing. After blood was collected, the tubes were shaken gently several times to anticoagulate, and then 50 [mu]l bovine trypsin inhibitor solution was added. Blood was centrifuged at approximately 1,600 xg for 15 minutes at approximately 4°C, and plasma samples were distributed into duplicate aliquots and stored frozen at approximately -70°C.

平均在一个处理组中的所有4只动物，检测下述PYY的血浆浓度(表3)：Averaged over all 4 animals in one treatment group, the plasma concentrations of the following PYY were determined (Table 3):

表3table 3

  组1Group 1  组2Group 2  组3Group 3  组4Group 4  组5Group 5  时间，分钟time, minutes  小分子渗透增强剂Small molecule penetration enhancer  无渗透增强剂No penetrationenhancer  25μMPN15925μMPN159  50μMPN15950μMPN159100μM PN159100μM PN159  00  183.825183.825  257.3257.3  228.675228.675  424.4424.4  294.225294.225  2.52.5  1280.71280.7  242.8242.8  526.375526.375  749.975749.975  1748.2251748.225  55  1449.4251449.425  273.675273.675  1430.151430.15  1293.41293.4  3088.23088.2

  1010  8251.88251.8  372.05372.05  6521.76521.7  12517.212517.2  14486.614486.6  1515  13731.213731.2  398.225398.225  12563.07512563.075  34455.334455.3  20882.72520882.725  3030  19537.5519537.55  476.475476.475  15222.615222.6  35294.37535294.375  25470.47525470.475  4545  13036.07513036.075  340.7340.7  9081.1259081.125  21582.22521582.225  16499.5516499.55  6060  7080.8757080.875  283.825283.825  4843.154843.15  9461.9259461.925  10676.62510676.625  120120  1671.91671.9  192.575192.575  1224.21224.2  2337.7752337.775  1891.2751891.275

从上述数据计算的药物代谢动力学数据显示在下表4中：Pharmacokinetic data calculated from the above data are shown in Table 4 below:

表4Table 4

与组2(无增强剂)制剂比较，确定下述相对增强比例(表5)：Compared with the Group 2 (no enhancer) formulation, the following relative enhancement ratios were determined (Table 5):

表5table 5

  组 Group  制剂preparation  相对CmaxRelative to Cmax 相对AUClastRelative toAUClast  1 1  小分子渗透增强剂Small molecule penetrationenhancer  38x38x 27x27x  33  PN159，25μmPN159,25μm  30x30x 21x21x  44  PN159，50μmPN159,50μm  79x79x 46x46x  55  PN159，100μmPN159, 100μm  54x54x 38x38x

前述数据图表显示在图3中，并且表明，与小分子渗透增强剂比较，以PN159为例的本发明的渗透性肽能够在体内将人激素肽治疗剂的鼻内渗透增强到相等的或更大的程度。在50μM浓度观察到所述肽的最大作用。100μM浓度导致稍低的渗透，尽管二者都导致比小分子渗透增强剂更高的渗透性。The aforementioned data graph is shown in Figure 3 and shows that, compared with small molecule penetration enhancers, the penetrating peptides of the present invention, exemplified by PN159, can enhance the intranasal penetration of human hormone peptide therapeutics to equal or greater levels in vivo. to a great extent. The maximum effect of the peptide was observed at a concentration of 50 μM. The 100 μΜ concentration resulted in somewhat lower penetration, although both resulted in higher permeability than the small molecule penetration enhancers.

实施例4Example 4

PN159对寡肽治疗剂的渗透增强作用Penetration-enhancing effect of PN159 on oligopeptide therapeutics

本实施例表明本发明的一种代表性肽，PN159增强一种环五肽，黑皮质素4受体激动剂(MC-4RA)，一种用于哺乳动物细胞受体的模式寡肽激动剂的上皮渗透的功效。在这一实施例中，描述一种或多种渗透性肽与MC-4RA的组合。在这种情形中有用的制剂可以包括寡肽治疗剂、渗透性肽和一种或多种其它渗透增强剂的组合。所述制剂还可以含有缓冲剂、张力剂、pH值调节剂、和肽/蛋白稳定剂如氨基酸、糖或多元醇、聚合物、和盐。This example demonstrates that a representative peptide of the invention, PN159, enhances a cyclic pentapeptide,melanocortin 4 receptor agonist (MC-4RA), a model oligopeptide agonist for mammalian cell receptors Efficacy of epithelial penetration. In this example, the combination of one or more penetrant peptides with MC-4RA is described. Formulations useful in this context may include a combination of an oligopeptide therapeutic, a penetrating peptide, and one or more other penetration enhancers. The formulations may also contain buffers, tonicity agents, pH adjusters, and peptide/protein stabilizers such as amino acids, sugars or polyols, polymers, and salts.

在本研究中评估PN159对MC-4RA渗透的作用。MC-4RA是一种具有约1,100Da分子量的甲烷磺酸盐，其调控MC-4受体的活性。评估的PN159浓度为5，25，50，和100μM。45mg/ml M-β-CD用作所有制剂的稳定剂，以获得10mg/ml的肽浓度。评估PN159自身或与EDTA(1，2.5，5，或10mg/ml)组合的作用。制剂的pH值固定在4，并且摩尔渗透压浓度为220mOsm/kg。The effect of PN159 on MC-4RA penetration was assessed in this study. MC-4RA is a methanesulfonate salt with a molecular weight of approximately 1,100 Da that modulates the activity of the MC-4 receptor. PN159 concentrations evaluated were 5, 25, 50, and 100 μM. 45 mg/ml M-β-CD was used as a stabilizer for all formulations to obtain a peptide concentration of 10 mg/ml. The effect of PN159 by itself or in combination with EDTA (1, 2.5, 5, or 10 mg/ml) was assessed. The pH of the formulation was fixed at 4 and the osmolarity was 220 mOsm/kg.

HPLC方法HPLC method

通过RP-HPLC分析MC-4RA在基底外侧部培养基中的浓度，所述RP-HPLC使用C18RP层析，流速1mL/分钟，和柱温25℃。The concentration of MC-4RA in the basolateral lateral medium was analyzed by RP-HPLC using C18RP chromatography at a flow rate of 1 mL/min and a column temperature of 25°C.

溶剂A：在水中0.1％的TFA；溶剂B：在ACN中0.1％的TFASolvent A: 0.1% TFA in water; Solvent B: 0.1% TFA in ACN

注射体积：50μLInjection volume: 50 μL

检测：220nmDetection: 220nm

运行时间：15分钟Run time: 15 minutes

MC-4RA与5，25，50，和100μM PN159组合，并且在pH值4和摩尔渗透压浓度～220mOsm/kg。使用体外上皮组织模型检测所述组合，以通过MTT和LDH检测而监测PTH渗透、跨上皮电阻(TER)、和所述制剂的细胞毒性。MC-4RA was combined with 5, 25, 50, and 100 μM PN159 atpH 4 and osmolarity ~220 mOsm/kg. The combinations were tested using an in vitro epithelial tissue model to monitor PTH penetration, transepithelial electrical resistance (TER), and cytotoxicity of the formulations by MTT and LDH assays.

MC-4RA渗透的研究结果显示在图4中。这些研究表明，除了增强肽激素治疗剂的黏膜渗透之外，PN159还显著增强寡肽治疗剂的上皮渗透。The results of the MC-4RA penetration study are shown in Figure 4. These studies demonstrated that, in addition to enhancing the mucosal penetration of peptide hormone therapeutics, PN159 also significantly enhanced the epithelial penetration of oligopeptide therapeutics.

实施例5Example 5

PN159对小分子药物的渗透增强作用Penetration Enhancement Effect of PN159 on Small Molecule Drugs

本实施例表明本发明的一种代表性肽，PN159增强以乙酰胆碱酯酶(ACE)抑制剂加兰他敏为例的小分子药物的上皮渗透的功效。在本实施例中，描述一种或多种渗透性肽与小分子药物的组合。在这种情形中有用的制剂可以包括小分子药物、渗透性肽、和一种或多种其他渗透增强剂的组合。所述制剂还可以包含缓冲剂、张力剂、pH值调节剂、稳定剂和/或防腐剂。This example demonstrates the efficacy of a representative peptide of the invention, PN159, to enhance epithelial penetration of small molecule drugs exemplified by the acetylcholinesterase (ACE) inhibitor galantamine. In this example, the combination of one or more penetrant peptides with a small molecule drug is described. Formulations useful in this context may include a combination of a small molecule drug, a penetrating peptide, and one or more other penetration enhancers. The formulations may also contain buffers, tonicity agents, pH adjusters, stabilizers and/or preservatives.

本发明将加兰他敏与PN159组合，以增强加兰他敏穿过鼻黏膜的渗透。由于加兰他敏是一种可以独立地透过鼻上皮膜的小分子，所以这种药物渗透的增加是出乎意料的。因此，通过加入增强肽渗透的赋形剂而介导的加兰他敏通过上皮渗透的显著增强作用是令人惊讶的，这是基于通常没有预计到所述赋形剂显著增加加兰他敏穿过上皮组织层的渗透。因此，本发明将通过增加它们的生物利用度而促进加兰他敏及其它小分子药物的鼻递送。The present invention combines galantamine with PN159 to enhance the penetration of galantamine through the nasal mucosa. Since galantamine is a small molecule that can independently penetrate nasal epithelial membranes, this increase in drug penetration was unexpected. Thus, the significant enhancement of galantamine penetration through the epithelium mediated by the addition of peptide penetration-enhancing excipients was surprising, based on the fact that such excipients are not generally expected to significantly increase galantamine Penetration through epithelial tissue layers. Thus, the present invention will facilitate nasal delivery of galantamine and other small molecule drugs by increasing their bioavailability.

在本研究中，40mg/ml乳酸盐形式的加兰他敏与25，50，和100μMPN159在溶液中、pH 5.0和摩尔渗透压浓度～270mOsm组合。使用体外上皮组织模型检测所述组合，以通过上文所述的LDH和MTT检测而监测加兰他敏渗透、跨上皮电阻(TER)、和所述制剂的细胞毒性。加兰他敏的渗透检测通过标准HPLC分析进行，如下。In this study, 40 mg/ml of galantamine in lactate form was combined with 25, 50, and 100 μM PN159 in solution at pH 5.0 and osmolarity ~270 mOsm. The combinations were tested using an in vitro epithelial tissue model to monitor galantamine penetration, transepithelial electrical resistance (TER), and cytotoxicity of the formulations by the LDH and MTT assays described above. Penetration detection of galantamine was performed by standard HPLC analysis, as follows.

HPLC分析HPLC analysis

应用无梯度LC(Waters Alliance(沃特斯联合))方法和UV检测，确定制剂中和在基底外侧培养基(渗透样品)中的加兰他敏浓度。The concentration of galantamine in the formulation and in the basolateral medium (infiltrated samples) was determined using a gradient-free LC (Waters Alliance) method with UV detection.

柱：沃特斯对称柱(Waters Symmetry Shield)，C18，5μm，25×0.46cmColumn: Waters Symmetry Shield, C18, 5μm, 25×0.46cm

流动相：在50mM甲酸铵中的5％ACN，pH 3.0Mobile phase: 5% ACN in 50 mM ammonium formate, pH 3.0

流速：1ml/分钟Flow rate: 1ml/min

柱温：30℃Column temperature: 30°C

校准曲线：0-400μg/ml溴化加兰他敏Calibration curve: 0-400μg/ml galantamine bromide

检测：在285nm的紫外线(UV)Detection: Ultraviolet (UV) at 285nm

基于前述研究，PN159提高小分子的跨黏膜递送。加兰他敏被选作模型低分子量药物，并且认为这一分子的结果是用于其它小分子药物的渗透性肽活性的预期。为了评估这种情形中的渗透性活性，将40mg/ml乳酸盐形式的加兰他敏与25，50，和100μM PN159在溶液中、pH 5.0和摩尔渗透压浓度～270mOsm组合。使用体外上皮组织模型检测所述组合，以通过LDH和MTT检测而监测加兰他敏渗透、跨上皮电阻(TER)、和所述制剂的细胞毒性。Based on the previous studies, PN159 enhances the transmucosal delivery of small molecules. Galantamine was chosen as a model low molecular weight drug, and the results for this molecule were considered to be expected for the penetrating peptide activity of other small molecule drugs. To assess osmotic activity in this case, galantamine in lactate form at 40 mg/ml was combined with 25, 50, and 100 μM PN159 in solution at pH 5.0 and osmolarity ~270 mOsm. The combinations were tested using an in vitro epithelial tissue model to monitor galantamine penetration, transepithelial electrical resistance (TER), and cytotoxicity of the formulations by LDH and MTT assays.

在所述体外组织模型中，加入PN159导致药物穿过细胞屏障的渗透的显著增加。具体地，在40mg/ml加兰他敏的P_app中存在2.5-3.5倍的增加。(图5)In the in vitro tissue model, addition of PN159 resulted in a significant increase in drug penetration across cellular barriers. Specifically, there was a 2.5-3.5 fold increase in P_app at 40 mg/ml galantamine. (Figure 5)

在加兰他敏存在下，PN159减少TER，正如实施例II中所述。In the presence of galantamine, PN159 reduced TER, as described in Example II.

在所有检测浓度的乳酸加兰他敏和PN159存在下，细胞存活能力保持高(＞80％)。相反，如通过LDH检测的，在存在PN159和乳酸加兰他敏时，细胞毒性低。这些检测都表明PN159对上皮膜没有毒性。Cell viability remained high (>80%) in the presence of galantamine lactate and PN159 at all concentrations tested. In contrast, cytotoxicity was low in the presence of PN159 and galantamine lactate as detected by LDH. These tests all indicated that PN159 had no toxicity to epithelial membranes.

总结上述结果，本文已经表明PN159令人惊讶地增加作为模型低分子量药物的加兰他敏的上皮渗透。将PN159加入到加兰他敏溶液中显著增强加兰他敏穿过上皮细胞单层的渗透。证据表明PN159暂时减少穿过上皮膜的TER，而没有损坏膜中的细胞，如通过高细胞存活能力和低细胞毒性所测定那样。因此，PN159是一种在体内增强加兰他敏及其它小分子药物的生物利用度的代表性肽，其通过在本文中使用体外模型证明的相同的机制而增强。进一步预测到PN159还在更高的浓度增强加兰他敏的渗透。Summarizing the above results, it has been shown herein that PN159 surprisingly increases the epithelial penetration of galantamine, a model low molecular weight drug. Addition of PN159 to galantamine solution significantly enhanced the penetration of galantamine across epithelial cell monolayers. Evidence indicates that PN159 temporarily reduces TER across epithelial membranes without damaging cells in the membrane, as determined by high cell viability and low cytotoxicity. Thus, PN159 is a representative peptide that enhances the bioavailability of galantamine and other small molecule drugs in vivo by the same mechanism demonstrated here using an in vitro model. It is further predicted that PN159 also enhances the penetration of galantamine at higher concentrations.

化学稳定性chemical stability

在治疗相关的储存条件下确定PN159的化学稳定性。应用表示稳定性的HPLC方法。溶液(50mM)保存在不同的pH值(4.0，7.3，和9.0)和温度(5℃，25℃，35℃，40℃，和50℃)条件下。在pH值4的样品含有10mM柠檬酸缓冲液。在pH值7.3和9.0的样品含有10mM磷酸缓冲液。典型的储存稳定性数据(包括阿伦尼乌斯作图法(Arrhenius plot))显示在图6中。可以看出，在低温度和pH值PN159在化学上是最稳定的。例如，在5℃和pH 4.0或pH7.3，对于6个月的储存，存在基本上100％的PN159恢复。当保存温度升高到25℃时，6个月后，对于在pH 4或pH 7的样品分别存在7％和26％的天然PN159损失。在pH 9和/或升高的温度，例如，40-50℃，跟着发生PN159的快速消耗。4.0-7.3的pH值范围和冷冻到环境温度的温度范围对于鼻内制剂是最相关的。因此，这些数据支持PN159可以在与IN制剂相关的储存条件下保持化学完整性。在药物相对时间渗透的速率中存在显著的增加。这些数据用来计算渗透性常数(P_app)，显示在表6中。The chemical stability of PN159 was determined under therapeutically relevant storage conditions. An HPLC method indicating stability was applied. Solutions (50 mM) were kept at different pH values (4.0, 7.3, and 9.0) and temperatures (5°C, 25°C, 35°C, 40°C, and 50°C). Samples atpH 4 contained 10 mM citrate buffer. Samples at pH 7.3 and 9.0 contained 10 mM phosphate buffer. Typical storage stability data (including Arrhenius plot) are shown in FIG. 6 . It can be seen that PN159 is chemically the most stable at low temperature and pH. For example, at 5°C and pH 4.0 or pH 7.3, there was essentially 100% recovery of PN159 for 6 months of storage. When the storage temperature was increased to 25°C, there was a loss of native PN159 of 7% and 26% after 6 months for the samples atpH 4 or pH 7, respectively. AtpH 9 and/or elevated temperature, eg, 40-50°C, rapid consumption of PN159 ensues. A pH range of 4.0-7.3 and a temperature range of freezing to ambient temperature are most relevant for intranasal formulations. Thus, these data support that PN159 can maintain chemical integrity under storage conditions associated with IN formulations. There was a significant increase in the rate of drug permeation versus time. These data were used to calculate permeability constants (P_app ), shown in Table 6.

表6Table 6

使用体外组织模型检测的P_appP_app detected using an in vitro tissue model

^apH为5.0。^a pH is 5.0.

在不存在PN159时，加兰他敏的P_app为约2.1×10^-6cm/s。当存在25，50和100mM PN159时，P_app分另为5.1×10^-6，6.2×10^-6，和7.2×10^-6cm/s。因此，在这种模式低分子量药物的P_app中，PN159提供2.4-到3.4-倍的增加。In the absence of PN159, the P_a p of galantamine is about 2.1×10^-6 cm/s. In the presence of 25, 50 and 100 mM PN159, P_app was 5.1×10^-6 , 6.2×10^-6 , and 7.2×10^-6 cm/s, respectively. Thus, PN159 provided a 2.4- to 3.4-fold increase in the P_app of low molecular weight drugs in this model.

已经确定PN159用于低分子量化合物跨黏膜制剂的用途，重要地是辨别这些观察是否能够外推到更大的分子，例如治疗性肽和蛋白。为了这一目的，在不存在和存在25，50，和100mM PN159时，对作为模式治疗肽的鲑鱼降钙素进行体外组织研究。当不存在PN159时，对于降钙素的P_app为约1×10^-7cm/s，低于加兰他敏的P_app约一个数量级，推测这是由于分子量的不同导致的。当存在PN159时，数据表明降钙素渗透的显著增加，与只有降钙素的情形相比，在P_app中达到23-到47-倍增加(表6)。Having established the use of PN159 for transmucosal formulation of low molecular weight compounds, it will be important to discern whether these observations can be extrapolated to larger molecules such as therapeutic peptides and proteins. For this purpose, in vitro tissue studies were performed on salmon calcitonin as a model therapeutic peptide in the absence and presence of 25, 50, and 100 mM PN159. In the absence of PN159, the P_app for calcitonin was about 1×10^-7 cm/s, about an order of magnitude lower than that for galantamine, presumably due_to the difference in molecular weight. When PN159 was present, the data indicated a significant increase in calcitonin penetration, reaching a 23- to 47-fold increase in P_app compared to calcitonin alone (Table 6).

为了研究这些发现的普遍性，在不存在和存在PN159时，在所述体外模型中检验两种其它的肽，即人甲状旁腺激素1-34(PTH_1-34)和人肽YY3-36(PYY_3-36)(P_app数据显示在表6中)。当不存在PN159时，这两种肽的P_app与降钙素的P_app一致。在PTH_1-34的情形中，PN159的存在在P_app中提供约3-5倍的增加。当在PN159存在下配制PYY_3-36时，Papp增加约12-到17-倍。这些数据证实我们的发现--PN159具有增强跨黏膜药物递送的用途--的普遍性。To investigate the generality of these findings, two other peptides, human parathyroid hormone 1-34 (PTH_1-34 ) and human peptide YY3-36, were tested in the in vitro model in the absence and presence of PN159 (PYY_3-36 ) (P_app data are shown in Table 6). When PN159 is absent, the P_app of these two peptides is consistent with_that of calcitonin. In the case of PTH_1-34 , the presence of PN159 provided about a 3-5 fold increase in P_app . Papp increased approximately 12- to 17-fold when PYY_3-36 was formulated in the presence of PN159. These data confirm the generality of our findings that PN159 has utility in enhancing transmucosal drug delivery.

实施例6Example 6

PN159的D-氨基酸形式D-amino acid form of PN159

合成并且纯化在表7中列出的D-氨基酸取代的PN159肽，并且使用在上述实施例中所述的方法检测它们增强TER和渗透性的能力。The D-amino acid substituted PN159 peptides listed in Table 7 were synthesized and purified, and tested for their ability to enhance TER and permeability using the methods described in the above Examples.

表7Table 7

D-氨基酸取代D-amino acid substitution

PN407表现出较小但是统计学显著的渗透性提高。所有的D和反逆反(retro inverso)形式的PN159都表现出减小的TER恢复，这表明可以用于体内递送的更长的TER减小作用。随机D取代(PN434)可以在TER减小和渗透性增强二者上引起无效活性。PN407 exhibited a small but statistically significant increase in permeability. All D and retro inverso forms of PN159 exhibited reduced TER recovery, suggesting a longer TER reduction that could be used for in vivo delivery. A random D substitution (PN434) could cause ineffective activity in both TER reduction and permeability enhancement.

实施例7Example 7

PN159长度变化PN159 length variation

合成并且纯化在表8中列出的具有长度变化的PN159肽，并且使用在上述实施例中所述的方法检测它们增强TER和渗透性的能力。PN159 peptides listed in Table 8 with varying lengths were synthesized and purified, and tested for their ability to enhance TER and permeability using the methods described in the Examples above.

表8Table 8

不同大小different size

肽peptide序列sequence描述describe  TER(x)/TER(159)+/-SEM^＊TER(x)/TER(159)+/-SEM^*  Perm(x)/Perm(159)+/-SEM^*Perm(x)/Perm(159)+/-SEM^*PN159PN159NH2-KLALKLALKALKAALKLA-酰胺NH2-KLALKLALKALKAALKLA-amide  模式肽model peptide  1.00+/-0.141.00+/-0.14  1.00+/-0.131.00+/-0.13PN417PN417NH2-KLALKLALKALKAA-酰胺(SEQ ID NO：39)NH2-KLALKLALKALKAA-amide (SEQ ID NO: 39)  缩短至14个氨基酸shortened to 14 amino acids0.19+/0.010.19+/0.010.04+/-0.010.04+/-0.01PN418PN418NH2-KLALKLALKALKAALK-酰胺(SEQ ID NO：40)NH2-KLALKLALKALKAALK-amide (SEQ ID NO: 40)  缩短至16个氨基酸shortened to 16 amino acids1.05+/-0.051.05+/-0.050.64+/-0.080.64+/-0.08PN419PN419NH2-KLALKLALKALKAALKLALK-酰胺(SEQ ID NO：41)NH2-KLALKLALKALKAALKLALK-amide (SEQ ID NO: 41)  延长至20个氨基酸Extended to 20 amino acids1.23+/-0.011.23+/-0.010.74+/-0.130.74+/-0.13PN420PN420NH2-KLALKLALKALKAALKLALKLA-酰胺(SEQ ID NO：42)NH2-KLALKLALKALKAALKLALKLA-amide (SEQ ID NO: 42)  延长至22个氨基酸Extended to 22 amino acids0.77+/-0.050.77+/-0.050.24+/-0.050.24+/-0.05PN421PN421  NH2-KLALKLALKALKAALKLALKLALK-酰胺(SEQ ID NO：43)NH2-KLALKLALKALKAALKLALKLALK-amide (SEQ ID NO: 43)  延长至24个氨基酸Extended to 24 amino acids0.74+/-0.110.74+/-0.110.17+/-0.060.17+/-0.06PN422PN422  NH2-KLALKLALKALKAALKLALKLALKAL-酰胺(SEQ ID NO：44)NH2-KLALKLALKALKAALKLALKLALKAL-amide (SEQ ID NO: 44)  延长至26个氨基酸Extended to 26 amino acids0.47+/-0.070.47+/-0.070.07+/-0.010.07+/-0.01

^*来自多次重复的平均值^* Average from multiple replicates

结果表明，PN159的长度对于其TER减少和增强的渗透性活性很重要。将PN159延长到20个氨基酸增加TER减少作用但是减小渗透性作用。TER恢复更缓慢。将PN159缩短到16个氨基酸对TER减少没有作用，但是减小渗透性作用。将PN159缩短到14个氨基酸显著减小渗透性，这表明PN159的长度对渗透性非常重要。与渗透性作用相反，PN159长度对TER减少的作用更缓和。The results indicated that the length of PN159 is important for its TER reduction and enhanced permeability activity. Extending PN159 to 20 amino acids increases the TER-reducing effect but reduces the permeability effect. TER recovers more slowly. Shortening PN159 to 16 amino acids had no effect on TER reduction, but reduced the permeability effect. Shortening PN159 to 14 amino acids significantly reduced permeability, suggesting that the length of PN159 is important for permeability. In contrast to the effect of permeability, the effect of PN159 length on TER reduction was more moderate.

实施例8Example 8

PN159中的色氨酸和精氨酸取代Tryptophan and arginine substitutions in PN159

合成并且纯化在表9中列出的具有氨基酸取代的PN159肽，并且使用在上述实施例中所述的方法检测它们增强TER和渗透性的能力。PN159 peptides with amino acid substitutions listed in Table 9 were synthesized and purified, and tested for their ability to enhance TER and permeability using the methods described in the above Examples.

表9Table 9

氨基酸取代amino acid substitution

结果表明，精氨酸胍头部基团(headgroup)比赖氨酸和组氨酸更有效。色氨酸是水-膜界面上的优选氨基酸。PN407对渗透性表现出较小但是统计学显著的提高。精氨酸取代赖氨酸显著减小渗透性，但是对TER减少有很少影响，这表明赖氨酸的重要性是渗透性。在氨基酸10用天冬酰胺单一取代丙氨酸消除了渗透性，这表明α螺旋对PN159活性的重要性。The results showed that the arginine guanidine headgroup was more effective than lysine and histidine. Tryptophan is the preferred amino acid at the water-membrane interface. PN407 showed a small but statistically significant increase in permeability. Substitution of arginine for lysine significantly reduced permeability but had little effect on TER reduction, suggesting the importance of lysine for permeability. A single substitution of alanine with asparagine atamino acid 10 abolished permeability, suggesting the importance of the alpha helix for PN159 activity.

实施例9Example 9

PN159中的疏水性变化Hydrophobic changes in PN159

合成并且纯化在表10中列出的具有氨基酸取代的PN159肽，并且使用在上述实施例中所述的方法检测它们增强TER和渗透性的能力。PN159 peptides with amino acid substitutions listed in Table 10 were synthesized and purified, and tested for their ability to enhance TER and permeability using the methods described in the above Examples.

表10Table 10

疏水面Hydrophobic surface

  肽peptide  序列sequence 描述 describe  TER(x)/TER(159)+/-SEM^*TER(x)/TER(159)+/-SEM^*  Perm(x)/Perm(159)+/-SEM^*Perm(x)/Perm(159)+/-SEM^*PN159PN159  NH2-KLALKLALKALKAALKLA-酰胺NH2-KLALKLALKALKAALKLA-amide模式肽model peptide1.00+/-0.141.00+/-0.141.00+/-0.131.00+/-0.13PN424PN424  NH2-KALKLKAALALLAKLKLA-酰胺(SEQ ID NO：53)NH2-KALKLKAALALLAKLKLA-amide (SEQ ID NO: 53)非两亲性的non-amphipathic0.59+/-0.070.59+/-0.070.20+/-0.040.20+/-0.04PN441PN441  NH2-KLAAALLKKAKKLAAALL-酰胺(SEQ ID NO：54)NH2-KLAAALLKKAKKLAAALL-amide (SEQ ID NO: 54)200°疏水面200° hydrophobic surface0.54+/-0.040.54+/-0.040.35+/-0.040.35+/-0.04PN442PN442  NH2-KALAALLKKAAKLLAALK-酰胺(SEQ ID NO：55)NH2-KALAALLKKAAKLLAALK-amide (SEQ ID NO: 55)180°面180° face0.93+/-0.030.93+/-0.030.81+/-0.030.81+/-0.03PN444PN444  NH2-KALAALLKKLAKLLAALK-酰胺(SEQ ID NO：56)NH2-KALAALLKKLAKLLAALK-amide (SEQ ID NO: 56)180°面180° face0.82+/-0.050.82+/-0.050.41+/-0.080.41+/-0.08

^*来自多次重复的平均值^* Average from multiple replicates

PN159具有280°的疏水面。结果表明，疏水面的减小可以引起PN159活性的减小。PN159的两亲性对于它的活性也是重要的。PN159 has a 280° hydrophobic face. The results showed that the reduction of the hydrophobic surface can lead to the reduction of PN159 activity. The amphipathic nature of PN159 is also important for its activity.

体外方法和方案In vitro methods and protocols

测定每一TJMP的跨上皮电阻(TER)、TER恢复、细胞毒性(LDH)和样品渗透(EIA)。用于每种检测的细胞培养条件和方案在下文中详细阐释。Transepithelial electrical resistance (TER), TER recovery, cytotoxicity (LDH) and sample penetration (EIA) were determined for each TJMP. Cell culture conditions and protocols used for each assay are explained in detail below.

实施例10Example 10

体外方法和方案In vitro methods and protocols

紧密连接调控肽或TJMPs是这样的肽，即，所述肽能够以在上皮细胞之间产生开孔的作用损害(compromising)紧密连接的完整性并且因此减少上皮的屏障功能。可以通过检测样品透过人鼻上皮组织模型系统的电阻和程度水平，而体外检测紧密连接完整性的状态。电阻的减少和增强的渗透表明，紧密连接已被损害，并且已在上皮细胞之间产生开孔。有效地，将诱导可检测的叫作(TER)减少的穿过组织膜电阻的减少，并且促进小分子通过组织膜的增强的渗透的肽分类为TJMPs。另外，还评估关于TJMPs的细胞毒性水平，以确定这些肽是否能够在药物穿过黏膜表面的递送例如鼻内(IN)药物递送中作用为紧密连接调控肽。Tight junction modulating peptides or TJMPs are peptides that are capable of compromising the integrity of tight junctions by creating openings between epithelial cells and thus reducing the barrier function of the epithelium. The status of tight junction integrity can be tested in vitro by measuring the resistance and extent of sample penetration through a human nasal epithelial tissue model system. A decrease in electrical resistance and increased penetration indicates that tight junctions have been compromised and pores have been created between epithelial cells. Effectively, peptides that induce a detectable reduction in resistance across tissue membranes, termed (TER) reduction, and promote enhanced penetration of small molecules through tissue membranes, are classified as TJMPs. In addition, the level of cytotoxicity with respect to TJMPs was also assessed to determine whether these peptides could function as tight junction modulating peptides in the delivery of drugs across mucosal surfaces, such as intranasal (IN) drug delivery.

在本实施例中描述用于筛选本发明代表性的肽(参考实施例25的表23)的检测。这些检测包括跨上皮电阻(TER)、细胞毒性(LDH)、和样品渗透。本实施例还描述所用的试剂和细胞培养条件。The assay used to screen representative peptides of the invention (see Table 23 of Example 25) is described in this example. These assays include transepithelial electrical resistance (TER), cytotoxicity (LDH), and sample penetration. This example also describes the reagents and cell culture conditions used.

表11举例说明用于后续实施例中的样品试剂。Table 11 illustrates sample reagents used in subsequent examples.

表11Table 11

样品试剂sample reagent

  试剂Reagent  等级level  生产商 manufacturer  城市，州city, state  Lot#Lot#  MWMW  1X DPBS++1X DPBS++  TCTC  基麦(Gibco)/英杰(Invitrogen)^TMGibco/Invitrogen^TM  卡尔斯巴德，加利福尼亚州Carlsbad, CA  12130611213061  无菌、无核酸酶的水Sterile, nuclease-free water  艾姆比恩(Ambion)^TMAmbion^TM  奥斯汀，德克萨斯州Austin, Texas  065P053618A065P053618A  荧光葡聚糖Fluorescent dextran  分子探针/英杰(Invitrogen)^TMMolecular Probes/Invitrogen^TM  卡尔斯巴德，加利福尼亚州Carlsbad, CA  111105111105  30003000  空气-100培养基^TMAir-100^MediumTM  TCTC  马特泰克(MatTek)^TMMatTek^™  阿什兰，MAAshland, MA  1111056511110565  空气-196插入物^TMAir-196^InsertTM  马特泰克(MatTek)^TMMatTek^™  阿什兰，MAAshland, MA  71187118  细胞毒性96检测^TMCytotoxicity 96^AssayTM  普洛麦格(Promega)^TMPromega^™  麦迪逊，威斯康星州Madison, Wisconsin  210634210634

TC＝组织培养TC = tissue culture

细胞培养cell culture

EpiAirway^TM系统由马特泰克(MatTek)公司(阿什兰，MA)开发，作为内衬于呼吸道的假复层上皮的模型。上皮细胞生长在气液界面处的以多孔膜为底的细胞培养插入物上，这导致细胞分化成高度极化的形态。顶面有纤毛，具有微绒毛超结构，上皮产生粘液(通过免疫印迹已经证实了粘蛋白的存在)。在运输前约3周在工厂将细胞铺在插入物上。The EpiAirway^™ system was developed by MatTek (Ashland, MA) as a model for the pseudostratified epithelium lining the airways. Epithelial cells are grown on porous membrane-based cell culture inserts at the air-liquid interface, which leads to differentiation of cells into a highly polarized morphology. The apical surface is ciliated, with a microvilli ultrastructure, and the epithelium produces mucus (the presence of mucin has been confirmed by immunoblotting). Cells were plated on the inserts at the factory approximately 3 weeks prior to shipping.

在实验开始前那天收到EpiAirway^TM培养膜。它们在不含酚红和不含氢化可的松的杜尔贝科(Dulbecco’s)改良的Eagle培养基(DMEM)中运输。细胞有纤毛并且是假复层的，在包括聚碳酸酯过滤系统的密斯博(Millipore)多筛(Multiscreen)Caco-296-孔检测系统上生长至汇合。当收到时，插入物系统未开封在4℃保存，和/或在使用前在每孔250μl基本培养基(不含酚红和不含氢化可的松的杜尔贝科改良的Eagle培养基(DMEM))中在37℃/5％CO2培养24小时。EpiAirway^™ culture membranes were received the day before the start of the experiment. They were shipped in Dulbecco's Modified Eagle's Medium (DMEM) without phenol red and without hydrocortisone. Cells were ciliated and pseudostratified and grown to confluence on a Millipore Multiscreen Caco-296-well assay system including a polycarbonate filter system. When received, store the insert system unopened at 4°C, and/or in 250 µl per well of minimal medium (Dulbecco's Modified Eagle's Medium without phenol red and without hydrocortisone) before use (DMEM)) at 37°C/5% CO2 for 24 hours.

这种模型系统用来评估TJMPs调控TEER、影响细胞毒性和提高上皮细胞单层的渗透的功效。This model system was used to evaluate the efficacy of TJMPs to regulate TEER, affect cytotoxicity, and enhance permeability of epithelial cell monolayers.

马特泰克(MatTek)公司(阿什兰，MA)的细胞系是正常的、来源于人的气管/支气管上皮细胞(EpiAirway^TM组织模型)的来源。提供细胞为插入物，其在包括透明的亲水性特氟隆(PTFE)的密斯博(Millipore)小室(Milicell)-CM滤器上生长至汇合。当收到时，在使用之前将所述膜在1ml基本培养基(不含酚红和不含氢化可的松的杜尔贝科改良的Eagle培养基(DMEM))中在37℃/5％CO2培养24-48小时。给插入物在恢复的每一天提供营养。The MatTek (Ashland, MA) cell line is a source of normal, human-derived tracheal/bronchial epithelial cells (EpiAirway^™ tissue model). Cells were provided as inserts grown to confluency on Millipore-CM filters comprising clear hydrophilic Teflon (PTFE). When received, the membrane was incubated in 1 ml of minimal medium (Dulbecco's Modified Eagle's Medium (DMEM) without phenol red and without hydrocortisone) at 37°C/5% before use. CO2 culture for 24-48 hours. Provide nourishment to the insert each day as it recovers.

将Madin-Darbey犬肾细胞(MDCK)、人肠上皮细胞(Caco-2)和人支气管上皮细胞(16HBE14o-)的细胞接种在来自密斯博的多筛(Multi-Screen)Caco-296-孔插入物上。这些细胞作为单层并且在与EpiAirway上皮细胞相似的条件下生长。Cells of Madin-Darbey canine kidney cells (MDCK), human intestinal epithelial cells (Caco-2) and human bronchial epithelial cells (16HBE14o-) were seeded in Multi-Screen Caco-296-well inserts from Misab things. These cells were grown as a monolayer and were grown under conditions similar to EpiAirway epithelial cells.

肽合成peptide synthesis

使用诺瓦生物化学(NovaBiochem)TGR树脂以50微摩尔的规模在Rainin Symphony合成仪上进行肽合成。通过用20％的在DMF中的哌啶处理两次，10分钟而进行去保护作用。去保护后，将树脂用10mL含有5％HOBt的DMF洗涤一次(30s)，并且用10mL DMF洗涤4次(30s)。通过将5倍过量的在DMF中的Fmoc氨基酸递送到反应容器中然后递送等体积的激活剂溶液，所述激活剂溶液含有6.25倍过量的N-甲基吗啉和5倍过量的HCTU，而进行偶联。在合成过程中应用40分钟的偶联时间。在第一次偶联反应后，在开始第二次偶联步骤之前，将树脂用10mL DMF洗涤两次(30s)。对于PEG化的肽，当完成肽合成时，去除N端Fmoc基团，并且人工向反应容器中加入2当量的在DMF中的O-(N-Fmoc-2-氨基乙基)-O′-(2-羧基乙基)-十一亚乙基二醇。尽管以人工方式，但是将2当量的激活剂溶液递送到反应容器中，并且允许偶联过夜进行。通常，获得大于97％的偶联效率，并且任何未反应的肽通过乙酸酐封端。Peptide synthesis was performed on a Rainin Symphony synthesizer at a 50 micromolar scale using NovaBiochem TGR resin. Deprotection was performed by treating twice for 10 minutes with 20% piperidine in DMF. After deprotection, the resin was washed once (30 s) with 10 mL of DMF containing 5% HOBt and 4 times (30 s) with 10 mL of DMF. By delivering a 5-fold excess of Fmoc amino acids in DMF into the reaction vessel and then delivering an equal volume of an activator solution containing a 6.25-fold excess of N-methylmorpholine and a 5-fold excess of HCTU, and Perform coupling. A coupling time of 40 min was applied during the synthesis. After the first coupling reaction, the resin was washed twice (30 s) with 10 mL DMF before starting the second coupling step. For PEGylated peptides, when the peptide synthesis is complete, the N-terminal Fmoc group is removed and 2 equivalents of O-(N-Fmoc-2-aminoethyl)-O'- in DMF are manually added to the reaction vessel. (2-Carboxyethyl)-undecaethylene glycol. Although manually, 2 equivalents of the activator solution were delivered into the reaction vessel and the coupling was allowed to proceed overnight. Typically, coupling efficiencies greater than 97% were obtained, and any unreacted peptide was capped by acetic anhydride.

通过递送10mL含有2.5％TIS、2.5％水的TFA然后轻轻氮搅动3小时，在单个反应容器中进行裂解。将裂解溶液自动收集到圆锥管中，合并在一起，并且通过在减压下蒸发而减小体积。将得到的溶液用过量的冰冷的醚研磨，过滤并且用冰冷的醚彻底洗涤。干燥后，粗肽吸收在密斯博(Millipore)水中，并且冻干到干燥。Lysis was performed in a single reaction vessel by delivering 10 mL of TFA containing 2.5% TIS, 2.5% water followed by gentle nitrogen agitation for 3 hours. Lysis solutions were automatically collected into conical tubes, pooled together, and reduced in volume by evaporation under reduced pressure. The resulting solution was triturated with excess ice-cold ether, filtered and washed thoroughly with ice-cold ether. After drying, the crude peptide was taken up in Millipore water and lyophilized to dryness.

FITC (荧光素-5-异硫氰酸酯)-葡聚糖渗透检测FITC (fluorescein-5-isothiocyanate)-dextran permeation assay

使用分子量3000的FITC标记的葡聚糖(FD3)来评估个体TJMP对上皮细胞单层渗透的作用。将组织插入物平板转移到含有200μl DPBS++作为基本培养基的96-孔接收平板上。每个组织培养插入物的顶面用20μl单一检测制剂样品(关于检测制剂的详情参考实施例25的表24)在37℃在黑暗中在振荡器(～100rpm)上温育1个小时。在1个小时的温育时间后，从每个组织培养插入物采集在下面的基本培养基样品，并且暂时在室温下保存在暗处，直到通过荧光光谱学量化FD3水平。关于FD3测量，将150μl基本培养基样品转移到黑的、清楚的底部的96-孔平板中。使用来自博奥泰克仪器(Biotek Instrument)的FLx800荧光平板读数仪测量在485/20激发后在528/20的荧光发射。FITC-dextran (FD3) with a molecular weight of 3000 was used to assess the effect of individual TJMPs on the penetration of epithelial cell monolayers. Transfer the tissue insert plate to a 96-well receiver plate containing 200 μl DPBS++ as minimal medium. The top surface of each tissue culture insert was incubated with 20 μl of a single assay formulation sample (see Table 24 of Example 25 for details on assay formulation) at 37° C. in the dark on a shaker (˜100 rpm) for 1 hour. Following an incubation time of 1 hour, samples of the underlying minimal medium were taken from each tissue culture insert and temporarily kept in the dark at room temperature until quantification of FD3 levels by fluorescence spectroscopy. For FD3 measurements, 150 μl minimal media samples were transferred to black, clear bottom 96-well plates. Fluorescence emission at 528/20 after excitation at 485/20 was measured using a FLx800 fluorescence plate reader from Biotek Instruments.

渗透计算为：Penetration is calculated as:

定义的关于渗透的式术语：Defined formula terms for penetration:

Cb：基底外侧部浓度Cb: basolateral concentration

Ca：顶部浓度Ca: top concentration

Vb：基底外侧部体积Vb: basolateral lateral volume

Va：顶部体积Va: top volume

SA：滤器表面积SA: filter surface area

dt：实耗时间dt: elapsed time

每个组织插入物置于含有1ml马特泰克(MatTek)基本培养基的单个孔中。在插入物的顶面，按照研究设计将应用25μl检测制剂，并且将样品放置在振荡器(～100rpm)上在37℃1.5小时。将FITC-标记的葡聚糖溶液顶部加入到插入物中，并且在温育时间后从基底外侧培养基进行荧光测量。FITC-葡聚糖的浓度表示为应用到细胞上的起始物质的百分数。具有分子量4000(MW4000)的FITC标记的葡聚糖用来评估关于个体TJMP渗透的负荷(cargo)大小限制。注意，可以获得各种大小的FITC标记的葡聚糖，以进行大小限制性研究。Each tissue insert was placed in a single well containing 1 ml of MatTek minimal medium. On the top side of the insert, 25 μl of detection formulation will be applied according to the study design, and the samples will be placed on a shaker (~100 rpm) for 1.5 hours at 37°C. FITC-labeled dextran solution was added topically to the inserts and fluorescence measurements were taken from the basolateral medium after the incubation time. The concentration of FITC-dextran is expressed as a percentage of the starting material applied to the cells. FITC-labeled dextran with a molecular weight of 4000 (MW4000) was used to assess the cargo size limitation on penetration of individual TJMPs. Note that FITC-dextran is available in various sizes for size-restricted studies.

跨上皮电阻(TER)和TER恢复Transepithelial resistance (TER) and TER recovery

使用与具有电极导线的EVOM上皮细胞伏特欧姆表(世界精密仪器(World Precision Instruments)，萨拉索塔，FL)连接的Endohm-12组织电阻测量室，完成TER检测。在检验校准前，关闭电源，将电极和组织培养空白插入物在马特泰克(MatTek)培养基中平衡至少20分钟。用Endohm组织室中的1.5ml培养基和空白插入物中的300μl培养基测量背景电阻。这样调整顶部电极，以致其正好接近，但不接触插入物膜顶面。空白插入物的背景电阻应该为约5-20欧姆。对于每次TER测定，将300μl马特泰克(MatTek)培养基加入到插入物中，然后置于在Endohm室中。所有的TER值报道为组织表面积的函数。TER assays were accomplished using an Endohm-12 tissue resistance measurement chamber connected to an EVOM epithelial cell volt-ohmmeter (World Precision Instruments, Sarasota, FL) with electrode leads. Power was turned off and electrodes and tissue culture blank inserts were equilibrated in MatTek medium for at least 20 minutes before checking calibration. Background resistance was measured with 1.5 ml medium in the Endohm tissue chamber and 300 μl medium in the blank insert. The top electrode was adjusted so that it was just close to, but not touching, the top surface of the insert membrane. The background resistance of the blank insert should be about 5-20 ohms. For each TER assay, 300 [mu]l of MatTek medium was added to the insert and then placed in the Endohm chamber. All TER values are reported as a function of tissue surface area.

TER计算为：TER is calculated as:

TER＝(R_I-R_b)×ATER＝(R_I -R_b )×A

其中R_I是具有膜的插入物的电阻，R_b是空白插入物的电阻，并且A是膜的面积(0.6cm²)。TER值相对于对照值(对照＝约1000欧姆-cm²；标准化到100)的减小表明细胞膜电阻的减小和黏膜上皮细胞渗透性的增加。where R_I is the resistance of the insert with the membrane,_Rb is the resistance of the blank insert, and A is the area of the membrane (0.6 cm² ). A decrease in TER values relative to control values (control = about 1000 ohm-^cm2 ; normalized to 100) indicates a decrease in cell membrane resistance and an increase in permeability of mucosal epithelial cells.

对于TER恢复，在处理后第1，3，5，和21小时测量TER’s。TER百分数计算为：For TER recovery, TER's were measured at 1, 3, 5, and 21 hours after treatment. The TER percentage is calculated as:

％TER＝(TER T_处理后/TER T₀)/(TER T_处理后/TER T₀对于对照培养基)。% TER = (_{after TER T treatment} /TER T₀ )/(_{after TER T treatment} /TER T₀ for control medium).

在一些实施方案中，使用具有电极导线的REMS自动取样器(世界精密仪器(World Precision Instruments)，萨拉索塔，FL)进行TER测量。在检验校准前，关闭电源，将电极和组织培养空白插入物在马特泰克(MatTek)Air-100TM培养基中平衡至少20分钟。通过多次测量空白插入物平板，已经确定插入物系统的背景电阻，并且将同一值用于在该平台上的每次检测。在将插入物与检测制剂温育前测量零时刻TER(TER0)。这样调整顶部电极，以致其正好接近，但不接触插入物膜的顶面。空白插入物的背景电阻应该为约5-20欧姆。对于每次TER测定，将100μl马特泰克(MatTek)Air-100TM培养基加入到插入物中，并且将250μl加入到基底孔中，然后置于Endohm室中。所有的TER值报道为组织表面积的函数。电阻表示为欧姆*cm2和初始TER值的百分数。In some embodiments, TER measurements are performed using a REMS autosampler (World Precision Instruments, Sarasota, FL) with electrode leads. Power was turned off and electrodes and tissue culture blank inserts were equilibrated in MatTek Air-100™ medium for at least 20 minutes prior to verification of calibration. The background resistance of the insert system has been determined by measuring blank insert plates multiple times, and the same value is used for each assay on this platform. Time zero TER (TER0) was measured before incubating the insert with the detection formulation. The top electrode was adjusted so that it was just close to, but not touching, the top surface of the insert membrane. The background resistance of the blank insert should be about 5-20 ohms. For each TER assay, 100 μl of MatTek Air-100™ medium was added to the inserts and 250 μl was added to the base wells and then placed in the Endohm chamber. All TER values are reported as a function of tissue surface area. Resistance is expressed as ohms*cm2 and as a percentage of the initial TER value.

TER值计算为：The TER value is calculated as:

标准电阻，Ohm*cm²＝(TERt-空白)*0.12Standard resistance, Ohm*cm² =(TERt-blank)*0.12

定义的关于TER计算的式术语：Defined formula terms for TER calculation:

TER0：在零时刻的TER测量TER0: TER measurement at time zero

TERt：在检测制剂温育后在时刻t进行的TER测量TERt: TER measurement at time t after incubation of assay preparation

空白：背景电阻测量Blank: background resistance measurement

TER值相对于对照值的减小表明细胞膜电阻的减小和黏膜上皮细胞渗透性的增加。A decrease in TER values relative to control values indicates a decrease in cell membrane resistance and an increase in permeability of mucosal epithelial cells.

细胞毒性(LDH测定)Cytotoxicity (LDH assay)

通过使用CytoTox 96细胞毒性测定试剂盒(普洛麦格(Promega)公司，麦迪逊，威斯康星州)测量来自细胞的乳酸脱氢酶(LDH)的损失而测定细胞死亡量。将50微升样品上样到96孔测定平板上。新鲜的不含细胞的培养基用作空白。向每个孔中加入50μl底物溶液，并且将平板在黑暗中在室温温育30分钟。温育后，向每个孔中加入50μl终止溶液，并且将所述平板在光学密度平板读数仪上在490nm读数。释放到基底外侧培养基中的LDH测量表示样品的相对细胞毒性。将具有0.3％辛基酚聚(乙二醇醚)x(TritonX-100)对照插入物作为100％裂解，允许LDH值表示为总裂解的百分数。Cell death was determined by measuring the loss of lactate dehydrogenase (LDH) from the cells using the CytoTox 96 Cytotoxicity Assay Kit (Promega, Madison, Wisconsin). 50 microliters of samples were loaded onto 96-well assay plates. Fresh medium without cells was used as blank. 50 μl of substrate solution was added to each well and the plate was incubated for 30 minutes at room temperature in the dark. After incubation, 50 μl of stop solution was added to each well and the plate was read at 490 nm on an optical density plate reader. The measurement of LDH released into the basolateral medium indicates the relative cytotoxicity of the samples. Control inserts with 0.3% octylphenol poly(glycol ether) x (TritonX-100) were taken as 100% cleavage, allowing LDH values to be expressed as a percentage of total lysis.

备选地，可以使用WST-1测定测量细胞毒性。WST-1测定基于线粒体代谢活性测量细胞存活能力。在肽处理、洗涤和在处理后10分钟TER测量后，将细胞单层的顶面用WST-1试剂(罗氏(Roche))在37℃温育4个小时。使用微量平板读数仪在OD 450nm测量顶部细胞上清液。％值＝样品_OD 450/培养基对照_OD 450·。Alternatively, cytotoxicity can be measured using the WST-1 assay. The WST-1 assay measures cell viability based on mitochondrial metabolic activity. After peptide treatment, washing andTER measurement 10 minutes after treatment, the top surface of the cell monolayer was incubated with WST-1 reagent (Roche) for 4 hours at 37°C. The top cell supernatant was measured at OD 450nm using a microplate reader. % value = sample_{OD 450} /medium control_{OD 450} ·.

在一些实施方案中，通过使用CytoTox 96细胞毒性检测试剂盒(普洛麦格(Promega)公司，麦迪逊，威斯康星州)检测乳酸脱氢酶(LDH)从细胞向顶部培养基中的释放而测定细胞死亡量。稀释在磷酸缓冲的盐水(PBS)中的1％辛基酚聚(乙二醇醚)x(Triton X-100^TM)在培养的细胞中引起100％的裂解，并且在本文作为LDH检测的阳性对照。在用检测制剂(关于检测制剂的详情参考实施例25的表24)温育1个小时的时间后，用培养基将每个插入物的总液体体积达到200μl的终体积。然后，通过用设定为100μl体积的多通道移液管吹吸4次，而将顶部培养基混合。混合后，将来自每个插入物顶面的100μl样品转移到新的96-孔平板中。将顶部培养基样品用平板密封物密封，并且保存在室温用于在同一天进行分析，或者在4℃保存过夜用于第二天分析。为了检测LDH水平，将100μl顶部培养基样品中的5μl在新的96-孔平板中稀释在45μl DPBS中。将新鲜的不含细胞的培养基用作空白。向每个孔中加入50μl底物溶液，并且将其在室温远离直接的光线温育30分钟。30分钟温育后，向每个孔中加入50μl终止溶液。使用来自博奥泰克仪器(Biotek Instruments)的uQuant吸收平板读数仪检测在490nm的光密度(OD)。释放到顶部培养基中的LDH检测表示样品的相对细胞毒性。通过从测定的个体检测制剂的吸光度减去测定的PBS对照的吸光度(LDH释放的基底水平)，然后将所述值除以对于1％Triton X-100^TM阳性对照所测量的吸光度，乘以100，而计算每种检测制剂的细胞毒性百分数。In some embodiments, it is determined by detecting the release of lactate dehydrogenase (LDH) from the cells into the apical medium using the CytoTox 96 Cytotoxicity Assay Kit (Promega, Madison, WI). amount of cell death. 1% octylphenol poly(glycol ether) x (Triton X-100^™ ) diluted in phosphate-buffered saline (PBS) caused 100% lysis in cultured cells and was tested here as positive for LDH control. After a period of 1 hour of incubation with the detection formulation (see Table 24 of Example 25 for details on the detection formulation), the total liquid volume of each insert was brought to a final volume of 200 μl with culture medium. The top medium was then mixed by pipetting 4 times with a multichannel pipette set to a volume of 100 μl. After mixing, 100 μl of sample from the top surface of each insert was transferred to a new 96-well plate. Top media samples were sealed with plate sealers and stored at room temperature for analysis the same day, or overnight at 4°C for analysis the next day. To measure LDH levels, 5 μl of a 100 μl top media sample was diluted in 45 μl DPBS in a new 96-well plate. Fresh medium without cells was used as blank. 50 μl of substrate solution was added to each well and incubated for 30 minutes at room temperature away from direct light. After the 30 min incubation, 50 μl of stop solution was added to each well. Optical density (OD) at 490 nm was measured using a uQuant absorbance plate reader from Biotek Instruments. Detection of LDH released into the top medium indicates the relative cytotoxicity of the samples. By subtracting the measured absorbance of the PBS control (basal level of LDH release) from the measured absorbance of the individual test preparations, then dividing the value by the measured absorbance for the 1% Triton X-100^™ positive control, multiplying by 100 , and the percentage cytotoxicity of each test preparation was calculated.

用于计算细胞毒性百分数的式如下：The formula used to calculate percent cytotoxicity is as follows:

摩尔渗透压浓度Osmolarity

通过来自高级仪器公司(Advanced Instruments Inc.)(诺伍德，马萨诸塞州)的模型20200检测样品。Samples were tested by a Model 20200 from Advanced Instruments Inc. (Norwood, MA).

实施例11Example 11

在体外调控上皮紧密连接并且增强上皮细胞层渗透的肽Peptides that regulate epithelial tight junctions and enhance epithelial cell layer penetration in vitro

表12显示在体外调控紧密连接蛋白并且增强上皮细胞层渗透的11种肽的氨基酸序列，其通过TER检测和渗透动力学而测定。为了这些实施例的目的，由于它们相似的活性，所以选择PN27代表PN27和PN28。Table 12 shows the amino acid sequences of 11 peptides that modulate tight junction proteins and enhance epithelial cell layer penetration in vitro, as determined by TER assay and penetration kinetics. For the purposes of these examples, PN27 was chosen to represent PN27 and PN28 due to their similar activities.

表12Table 12

  肽peptide氨基酸序列amino acid sequence  PN159PN159NH2-KLALKLALKALKAALKLA-酰胺NH2-KLALKLALKALKAALKLA-amide  PN161PN161NH2-GWTLNSAGYLLGKINLKALAALAKKIL-酰胺(SEQ ID NO：63)NH2-GWTLNSAGYLLGKINLKALAALAKKIL-amide (SEQ ID NO: 63)  PN202PN202NH2-LLETLLKPFQCRICMRNFSTRQARRNHRRRHRR-酰胺(SEQ ID NO：64)NH2-LLETLLKPFQCRICMRNFSTRQARRNHRRRHRR-amide (SEQ ID NO: 64)  PN27PN27NH2-AAVALLPAVLLALLAPRKKRRQRRRPPQ-酰胺(SEQ ID NO：65)NH2-AAVALLPAVLLALLAPRKKRRQRRRPPQ-amide (SEQ ID NO: 65)

  PN28PN28  NH2-RKKRRQRRRPPQCAAVALLPAVLLALLAP-酰胺(SEQ ID NO：66)NH2-RKKRRQRRRPPQCAAVALLPAVLLALLAP-amide (SEQ ID NO: 66)  PN58PN58  NH2-RQIKIWFQMRRMKWKK-酰胺(SEQ ID NO：67)NH2-RQIKIWFQMRRMKWKK-amide (SEQ ID NO: 67)  PN73PN73  NH2-KGSKKAVTKAQKKDGKKRKRSRKESYSVYVYKVLKQ-酰胺(SEQ ID NO：68)NH2-KGSKKAVTKAQKKDGKKRKRSRKESYSVYVYKVLKQ-amide (SEQ ID NO: 68)  PN228PN228  NH2-KLWSAWPSLWSSLWKP-酰胺(SEQ ID NO：69)NH2-KLWSAWPSLWSSLWKP-amide (SEQ ID NO: 69)  PN250PN250  NH2-RRRQRRKRGGDIMGEWGNEIFGAIAGFLG-酰胺(SEQ ID NO：70)NH2-RRRQRRKRGGDIMGEWGNEIFGAIAGFLG-amide (SEQ ID NO: 70)  PN283PN283  马来酰亚胺-GLGSLLKKAGKKLKQPKSKRKV-酰胺(SEQ ID NO：71)Maleimide-GLGSLLKKAGKKLKQPKSKRKV-amide (SEQ ID NO: 71)  PN183PN183  NH2-KETWWETWWTEWSQPGRKKRRQRRRRPPQ-酰胺(SEQ ID NO：72)NH2-KETWWETWWTEWSQPGRKKRRQRRRRPPQ-amide (SEQ ID NO: 72)

实施例12Example 12

紧密连接调控肽减小TERTight junction regulatory peptides reduce TER

本实施例评估各种肽在体外调控上皮细胞单层中的紧密连接蛋白的作用，其通过TER减少而检测。对于每种TJMP从在EpiAirway上皮细胞中进行的实验获得的TER数据的总结显示在表13中。在所述表中突出的方格代表对于TJMP在检测浓度范围内观察到的最高TER减少。This example evaluates the effect of various peptides in modulating tight junction proteins in epithelial cell monolayers in vitro, as detected by TER reduction. A summary of TER data obtained from experiments performed in EpiAirway epithelial cells for each TJMP is shown in Table 13. Highlighted squares in the table represent the highest TER reduction observed for TJMP over the concentration range tested.

表13Table 13

  肽peptide  1000μM1000μM  500μM500μM  250μM250μM  125μM125μM  100μM100μM  50μM50μM  25μM25μM  10μM10μM  2.5μM2.5μM 1μM1μM  PN159PN15994％94%89％89%79％79% 54％54%  PN161PN161  84％84%  73％73%  43％43%  11％11%  PN202PN202  95％95%  95％95%  57％57%  3％3%  PN27PN27  94％94%  91％91%  81％81%  PN283PN283  92％92%  86％86%  39％39%  PN250PN250  84％84%  79％79%  58％58%  17％17%  3％3%  PN228PN228  82％82%  9％ 9%  PN73PN73  83％83%  38％38%  8％ 8%  PN58PN58  88％88%  64％64%  -6％-6%  PN183PN183  55％55%  41％41%  25％25%

PN159，PN202，PN27，和PN283减少TER超过90％，而PN161，PN250，PN228，PN73，和PN58减少TER达到82％-88％。PN28未显示，但是它功能与Pn27相等。最后PN183具有55％的TER减少。这些数据表明所有检测的TJMPs能够在体外损害上皮细胞紧密连接。PN159, PN202, PN27, and PN283 reduced TER by more than 90%, while PN161, PN250, PN228, PN73, and PN58 reduced TER by 82%-88%. PN28 is not shown, but it is functionally equivalent to Pn27. Finally PN183 had a 55% reduction in TER. These data demonstrate that all tested TJMPs are capable of impairing epithelial tight junctions in vitro.

另外，进行TER恢复分析以确定用TJMPs处理后EpitAirway上皮细胞层恢复的速率。令人吃惊地，结果表明，在所有检测的TJMPs中，PN250，PN202，和PN161具有最快的恢复时间。这些数据表明TJMPs对上皮细胞层的作用本质上是短暂的。Additionally, TER recovery assays were performed to determine the rate of EpitAirway epithelial cell layer recovery after treatment with TJMPs. Surprisingly, the results showed that PN250, PN202, and PN161 had the fastest recovery times among all tested TJMPs. These data suggest that the effects of TJMPs on the epithelial cell layer are transient in nature.

实施例13Example 13

紧密连接调控肽的体外渗透动力学In vitro permeation kinetics of tight junction-modulating peptides

在本实施例中，检测TJMPs调控EpiAirway上皮细胞渗透的作用。下表14显示以渗透百分数表示的每种TJMP的渗透动力学总结。在所述表中突出的方格代表对于该TJMP在检测浓度范围内观察到的最大程度的渗透。In this example, the role of TJMPs in regulating epithelial cell penetration of the EpiAirway was examined. Table 14 below shows a summary of the permeation kinetics for each TJMP expressed as percent permeation. Highlighted squares in the table represent the maximum degree of penetration observed for that TJMP over the concentration range tested.

表14Table 14

  肽peptide  1000μM1000μM  500μM500μM  250μM250μM  125μM125μM  100μM100μM  50μM50μM  25μM25μM  10μM10μM  2.5μM2.5μM  1μM1μM  PN159PN159  12.5％12.5%6.5％6.5%1.9％1.9%0.6％0.6%  PN161PN161  7.1％7.1%  2.9％2.9%  1.6％1.6%  0.3％0.3%  PN202PN2025.9％5.9%2.8％2.8%  1.5％1.5%0.2％0.2%  PN27PN27  8.4％8.4%  7.3％7.3%  7.7％7.7%  PN283PN283  5.2％5.2%  3.9％3.9%  0.7％0.7%  PN250PN250  4.2％4.2%  3.3％3.3%  1.7％1.7%  0.5％0.5%  0.3％0.3%  PN228PN228  1.7％1.7%  0.2％0.2%  PN73PN730.8％0.8%0.2％0.2%  0.1％0.1%  PN58PN586.3％6.3%4.5％4.5%0.9％0.9%0.3％0.3%  0.3％0.3%  PN183PN183  0.6％0.6%  0.5％0.5%  0.2％0.2%

这些数据表明，所有检测的TJMPs能够在体外增强上皮细胞单层的渗透。通常，渗透性程度与肽减少TER的能力相关。These data demonstrate that all tested TJMPs are capable of enhancing the penetration of epithelial cell monolayers in vitro. In general, the degree of permeability correlates with the ability of the peptide to reduce TER.

实施例14Example 14

紧密连接调控肽不会引起显著的细胞毒性Tight junction regulatory peptides do not cause significant cytotoxicity

本实施例评估在暴露于TJMPs之后对上皮细胞的细胞毒性作用。在用每种肽处理15分钟和60分钟之后进行LDH检测。在所有的情形中，在15分钟处理后几乎没有观察到LDH释放。在60分钟处理后，在所检测的肽中细胞毒性水平不同，但是在可接受的水平之内，这表明所有检测的肽都不会引起显著的细胞损伤。This example evaluates cytotoxic effects on epithelial cells following exposure to TJMPs. LDH assays were performed after 15 and 60 minutes of treatment with each peptide. In all cases, little LDH release was observed after 15 minutes of treatment. After 60 minutes of treatment, levels of cytotoxicity varied among the peptides tested, but were within acceptable levels, indicating that none of the peptides tested caused significant cell damage.

实施例15Example 15

在所有检测的上皮细胞类型中，紧密连接调控肽减少TER是一致的Tight junction regulatory peptides reduce TER consistently across all epithelial cell types examined

为了确定在EpitAirway上皮细胞培养系统中观察到的TER结果是否代表其他上皮细胞类型，将MDCK，Caco-2，和16HBE14o-细胞用TJMps处理，并且检测TER。在所有情形中，使用这些细胞类型观察到的TER结果与使用EpiAirway上皮细胞观察到的TER结果一致，这表明这些TJMPs具有减少所有上皮细胞类型中的TER的能力。To determine whether the TER results observed in the EpitAirway epithelial cell culture system were representative of other epithelial cell types, MDCK, Caco-2, and 16HBE14o- cells were treated with TJMps and TER was assayed. In all cases, the TER results observed with these cell types were consistent with those observed with EpiAirway epithelial cells, suggesting that these TJMPs have the ability to reduce TER in all epithelial cell types.

实施例16Example 16

基于表现排列紧密连接调控肽Ranking tight junction regulatory peptides based on expression

排列9种TJMPs，并且按照在表15中显示的它们的渗透性水平、TER值、TER恢复速率和细胞毒性，而将其分成4种不同的表现等级。表15中不包括PN183和PN28。下表总结在用所述肽处理EpiAirway上皮细胞15分钟后和用所述肽处理EpitAirway上皮细胞60分钟后，每种TJMPs的最佳浓度(即，与最高渗透性水平相关的最大程度的TER减少并且不表现出显著细胞毒性)和相对应的渗透百分数。另外，对每种肽显示关于15分钟和60分钟处理的LDH值(细胞毒性)。还显示TER恢复。TER恢复速率与斜率值直接相关(即，更大的斜率值与更快的TER恢复相关)。The 9 TJMPs were ranked and classified into 4 different performance classes according to their permeability level, TER value, TER recovery rate and cytotoxicity shown in Table 15. PN183 and PN28 are not included in Table 15. The table below summarizes the optimal concentration of each TJMP (i.e., the greatest reduction in TER associated with the highest permeability level) after 15 minutes of treatment of EpiAirway epithelial cells with the peptide and after 60 minutes of treatment of EpitAirway epithelial cells with the peptide. and does not exhibit significant cytotoxicity) and the corresponding percent penetration. In addition, LDH values (cytotoxicity) for 15-minute and 60-minute treatments are shown for each peptide. Also shows TER recovery. The rate of TER recovery is directly related to the slope value (ie, larger slope values are associated with faster TER recovery).

表15Table 15

实施例17Example 17

紧密连接调控肽增强FITC-葡聚糖MW4000穿过上皮细胞单层的渗透Tight junction regulatory peptides enhance the penetration of FITC-dextran MW4000 across epithelial cell monolayers

在本实施例中，进行研究以确定存在每种TJMP时FITC-葡聚糖MW4000的渗透动力学。这一实验评估渗透是否取决于肽与上皮细胞单层一起温育的时间，以及渗透是否是负荷大小依赖性的。在用TJMP和FITC-葡聚糖MW4000处理细胞15分钟后以及处理细胞60分钟后检测细胞渗透(图7)。PYY制剂用作阳性对照，并且磷酸缓冲盐水(PBS)用作阴性对照。在表现出与最高渗透性水平相关的最大TER减少程度并且不表现显著的细胞毒性的浓度检测所述肽。In this example, a study was performed to determine the permeation kinetics of FITC-dextran MW4000 in the presence of each TJMP. This experiment assesses whether permeation depends on the time the peptide is incubated with the epithelial cell monolayer and whether permeation is load size dependent. Cell infiltration was detected after treatment of cells with TJMP and FITC-dextran MW4000 for 15 minutes and after treatment of cells for 60 minutes (Figure 7). PYY formulation was used as positive control and phosphate buffered saline (PBS) was used as negative control. The peptides were tested at concentrations that exhibited the greatest degree of TER reduction associated with the highest permeability levels and did not exhibit significant cytotoxicity.

对于同一种TJMP，60分钟的处理表现出比15分钟的处理显著更高的渗透程度。令人吃惊地，PN161，PN127，和PN228表现出与PN159相等的渗透水平(大约7.5％)。在与细胞温育60分钟后，TJMPs PN250，PN283，PN202，PN58获得大约5％的渗透，所述渗透恰好没有达到通过PN161，PN127，PN228和PN159获得的渗透。这些数据表明，所有检测的TJMPs都能够增强FITC-葡聚糖MW4000的渗透，并且这种增强作用取决于肽与上皮细胞层接触时间的长短。For the same TJMP, the 60 min treatment showed a significantly higher degree of infiltration than the 15 min treatment. Surprisingly, PN161, PN127, and PN228 exhibited the same level of penetration as PN159 (approximately 7.5%). After 60 min of incubation with the cells, TJMPs PN250, PN283, PN202, PN58 achieved about 5% infiltration which just missed that obtained by PN161, PN127, PN228 and PN159. These data indicate that all tested TJMPs were able to enhance the penetration of FITC-dextran MW4000 and that this enhancement was dependent on the length of time the peptide was in contact with the epithelial cell layer.

上述实验证明，所检测的TJMPs能够在体外增强上皮细胞单层的渗透。The experiments described above demonstrate that the tested TJMPs are able to enhance the penetration of epithelial cell monolayers in vitro.

实施例18Example 18

紧密连接调控肽在体外增强渗透性与在体内观察到的增强的渗透高度相                            关Enhanced permeability of tight junction modulating peptides in vitrocorrelates highly with enhanced permeability observed in vivo

进行线性回归分析，以确定在体外EpiAirway上皮细胞模型系统中观察到的TJMP渗透动力学是否与对于同一种TJMP观察到的体内药物代谢动力学数据相关。为了确定体外渗透数据是否作为体内效果的良好的指示，将从使用PYY和TJMPs进行的体内药物代谢动力学研究获得的曲线下面积-最终(last)值(AUC-last)针对使用PYY和TJMPs进行的体外上皮细胞单层渗透研究绘图。体外渗透表示为百分数，而AUC-last(AUC-最终)表示为分钟*pg/ml。关于10种不同的TJMPs的体外和体内研究进行绘图，并且进行线性回归。得到0.82的R²值(82％相关性)，这表明对于来源于体内的AUC值和体外观察的渗透性百分数存在高度的相关性。令人惊讶地，当排除检测间(inter-assay)可变性时，得到0.996的R²值(基本上100％)，这表明在体外渗透性和体内效果之间存在直接的相关性。因此，体外渗透可以用来预测体内效果。Linear regression analysis was performed to determine whether the TJMP permeation kinetics observed in the EpiAirway epithelial cell model system in vitro correlated with the in vivo pharmacokinetic data observed for the same TJMP. To determine whether the in vitro permeation data serve as a good indicator of in vivo efficacy, the area under the curve-last value (AUC-last) obtained from the in vivo pharmacokinetic studies using PYY and TJMPs was compared against the use of PYY and TJMPs Drawing of in vitro epithelial cell monolayer permeation studies. In vitro permeation is expressed as a percentage, while AUC-last (AUC-last) is expressed as min*pg/ml. In vitro and in vivo studies for 10 different TJMPs were plotted and linear regression was performed. An^R2 value of 0.82 (82% correlation) was obtained, indicating a high correlation between the AUC values derived in vivo and the percent permeability observed in vitro. Surprisingly, when inter-assay variability was excluded, an^R2 value of 0.996 (essentially 100%) was obtained, indicating a direct correlation between in vitro permeability and in vivo effect. Therefore, in vitro permeation can be used to predict in vivo effects.

实施例19Example 19

TJMP对肽激素治疗剂的体内渗透增强作用等于或者超过小分子渗透增强In vivo penetration enhancement of peptide hormone therapeutics by TJMP equals or exceeds small molecule penetration enhancement剂的增强作用Enhancement of the agent

将20只3-6月龄并且重2.1-3.0kg的雄性新西兰白兔随机分成5个处理组，每组4只动物。将测试动物以15μl/kg并且通过吸管鼻内给药。下表19表示5种不同的给药组的组成。Twenty male New Zealand white rabbits aged 3-6 months and weighing 2.1-3.0 kg were randomly divided into 5 treatment groups of 4 animals each. Test animals were dosed intranasally at 15 μl/kg and via pipette. Table 19 below shows the composition of 5 different dosing groups.

对于给药组1(见表16)，使用包含小分子渗透增强剂的PYY临床制剂。在这些研究中的小分子增强剂包括甲基-β-环糊精、二癸酰基磷脂酰胆碱(DDPC)、和/或EDTA。给药组2接受溶解在磷酸缓冲盐水(PBS)中的PYY。对于给药组3-5，将不同浓度的PN159加到给药组2中，以致给药组3-5中的每一组由PYY，PN159和PBS组成For dosing group 1 (see Table 16), a clinical formulation of PYY containing a small molecule penetration enhancer was used. Small molecule enhancers in these studies included methyl-β-cyclodextrin, didecanoylphosphatidylcholine (DDPC), and/or EDTA.Dosing group 2 received PYY dissolved in phosphate buffered saline (PBS). For administration groups 3-5, different concentrations of PN159 were added toadministration group 2, so that each of administration groups 3-5 consisted of PYY, PN159 and PBS

表16Table 16

组Group动物animal渗透增强剂penetration enhancer  给药浓度(mg/ml)Dosing concentration (mg/ml)  给药体积(ml/kg)Dosing volume (ml/kg)  PYY剂量(μg/kg)PYY dose (μg/kg)  1 1  4M4M  小分子渗透增强剂Small molecule penetration enhancer  13.6713.67  0.0150.015  205205  2 2  4M4M  无 none  13.6713.67  0.0150.015  205205

  33  4M4M  25μMPN15925μMPN159  13.6713.67  0.0150.015  205205  44  4M4M  50μM PN15950μM PN159  13.6713.67  0.0150.015  205205  55  4M4M  100μM PN159100μM PN159  13.6713.67  0.0150.015  205205

通过直接在耳缘静脉进行直接静脉穿刺而收集连续的血液样品(每份约2ml)，收集到含有EDTA作为抗凝血剂的血液收集管中。在给药后0，2.5，5，10，15，30，45，60，和120分钟收集血液样品。收集血液之后，管子轻轻摇动几次，以抗凝血，然后加入50μl牛胰蛋白酶抑制剂溶液。在大约4℃以约1,600xg离心血液15分钟，并且将血浆样品分配到一式两份等分试样中，并且在大约-70℃冷冻保存。Serial blood samples (approximately 2 ml each) were collected by direct venipuncture in the marginal ear vein into blood collection tubes containing EDTA as an anticoagulant. Blood samples were collected at 0, 2.5, 5, 10, 15, 30, 45, 60, and 120 minutes after dosing. After blood was collected, the tubes were shaken gently several times to anticoagulate, and then 50 [mu]l bovine trypsin inhibitor solution was added. Blood was centrifuged at approximately 1,600 xg for 15 minutes at approximately 4°C, and plasma samples were divided into duplicate aliquots and stored frozen at approximately -70°C.

平均在一个处理组中的所有4只动物，检测下述PYY的血浆浓度(表17)：Averaged over all 4 animals in one treatment group, the plasma concentrations of the following PYY were determined (Table 17):

表17Table 17

  组1Group 1  组2Group 2  组3Group 3  组4Group 4  组5Group 5  时间，分钟time, minutes  小分子渗透增强剂Small molecule penetration enhancer  无渗透增强剂Nopenetration enhancer25μM PN15925 μMPN15950μM PN15950μM PN159100μM PN159100μM PN159  00  183.825183.825  257.3257.3  228.675228.675  424.4424.4  294.225294.225  2.52.5  1280.71280.7  242.8242.8  526.375526.375  749.975749.975  1748.2251748.225  55  1449.4251449.425  273.675273.675  1430.151430.15  1293.41293.4  3088.23088.2  1010  8251.88251.8  372.05372.05  6521.76521.7  12517.212517.2  14486.614486.6  1515  13731.213731.2  398.225398.225  12563.07512563.075  34455.334455.3  20882.72520882.725  3030  19537.5519537.55  476.475476.475  15222.615222.6  35294.37535294.375  25470.47525470.475  4545  13036.07513036.075  340.7340.7  9081.1259081.125  21582.22521582.225  16499.5516499.55  6060  7080.8757080.875  283.825283.825  4843.154843.15  9461.9259461.925  10676.62510676.625  120120  1671.91671.9  192.575192.575  1224.21224.2  2337.7752337.775  1891.2751891.275

从上述数据计算的药物代谢动力学数据显示在下表18中：Pharmacokinetic data calculated from the above data are shown in Table 18 below:

表18Table 18

与组2(无增强剂)制剂比较，确定下述相对增强比例(表19)：Compared with the Group 2 (no enhancer) formulation, the following relative enhancement ratios were determined (Table 19):

表19Table 19

  组 Group  制剂preparation  相对CmaxRelative to Cmax  相对AUC lastRelative AUC last  1 1  小分子渗透增强剂Small molecule penetrationenhancer  38x38x  27x27x  33  PN159，25μmPN159,25μm  30x30x  21x21x  44  PN159，50μmPN159,50μm  79x79x  46x46x  55  PN159，100μmPN159, 100μm  54x54x  38x38x

前述数据表明，与小分子渗透增强剂比较，TJMP在体内将人激素肽治疗剂的鼻内渗透增强到相等的或更大的程度。在50μM浓度观察到所述肽的最大作用。100μM浓度导致稍低的渗透，尽管二者都导致比小分子渗透增强剂更高的渗透性。The foregoing data indicate that TJMPs enhance intranasal penetration of human hormone peptide therapeutics to an equal or greater extent in vivo than small molecule penetration enhancers. The maximum effect of the peptide was observed at a concentration of 50 μM. The 100 μΜ concentration resulted in somewhat lower penetration, although both resulted in higher permeability than the small molecule penetration enhancers.

实施例20Example 20

TJMP对寡肽治疗剂的渗透增强作用Penetration enhancement of oligopeptide therapeutics by TJMP

本实施例表明本发明的一种代表性肽，PN159增强一种环五肽，黑皮质素-4受体激动剂(MC-4RA)，一种用于哺乳动物细胞受体的模式寡肽激动剂，的上皮细胞渗透的功效。在这一实施例中，描述一种或多种渗透性肽与MC-4RA的组合。在这种情形中有用的制剂包括寡肽治疗剂、渗透性肽和一种或多种其它渗透增强剂的组合。所述制剂还可以含有缓冲剂、张力剂、pH值调节剂、和肽/蛋白稳定剂如氨基酸、糖或多元醇、聚合物、和盐。This example demonstrates that a representative peptide of the invention, PN159, enhances agonism of a cyclic pentapeptide, melanocortin-4 receptor agonist (MC-4RA), a model oligopeptide for mammalian cell receptors agent, the efficacy of epithelial cell penetration. In this example, the combination of one or more penetrant peptides with MC-4RA is described. Useful formulations in this context include a combination of an oligopeptide therapeutic, a penetrating peptide, and one or more other penetration enhancers. The formulations may also contain buffers, tonicity agents, pH adjusters, and peptide/protein stabilizers such as amino acids, sugars or polyols, polymers, and salts.

在本研究中评估PN159对MC-4RA渗透的作用。MC-4RA是一种具有约1,100Da分子量的甲烷磺酸盐，其调控MC-4受体的活性。评估的PN159浓度为5，25，50，和100μM。45mg/ml M-β-CD用作所有制剂的增溶剂，以获得10mg/ml的肽浓度。评估PN159自身或与EDTA(1，2.5，5，或10mg/ml)组合的作用。制剂的pH值固定在4，并且摩尔渗透压浓度为220mOsm/kg。The effect of PN159 on MC-4RA penetration was assessed in this study. MC-4RA is a methanesulfonate salt with a molecular weight of approximately 1,100 Da that modulates the activity of the MC-4 receptor. PN159 concentrations evaluated were 5, 25, 50, and 100 μM. 45 mg/ml M-β-CD was used as a solubilizer for all formulations to obtain a peptide concentration of 10 mg/ml. The effect of PN159 by itself or in combination with EDTA (1, 2.5, 5, or 10 mg/ml) was assessed. The pH of the formulation was fixed at 4 and the osmolarity was 220 mOsm/kg.

HPLC方法HPLC method

通过RP-HPLC分析MC-4RA在基底外侧部培养基中的浓度，所述RP-HPLC使用C18RP层析，流速1mL/分钟，和柱温25℃。The concentration of MC-4RA in the basolateral lateral medium was analyzed by RP-HPLC using C18RP chromatography at a flow rate of 1 mL/min and a column temperature of 25°C.

溶剂A：在水中0.1％的TFA；溶剂B：在ACN中0.1％的TFASolvent A: 0.1% TFA in water; Solvent B: 0.1% TFA in ACN

注射体积：50μLInjection volume: 50 μL

检测：220nmDetection: 220nm

运行时间：15分钟Run time: 15 minutes

MC-4RA与5，25，50，和100μM PN159组合，并且在pH值4和摩尔渗透压浓度～220mOsm/kg。使用体外上皮组织模型检测所述组合，以通过MTT和LDH检测而监测PTH渗透、跨上皮电阻(TER)、和所述制剂的细胞毒性。MC-4RA was combined with 5, 25, 50, and 100 μM PN159 atpH 4 and osmolarity ~220 mOsm/kg. The combinations were tested using an in vitro epithelial tissue model to monitor PTH penetration, transepithelial electrical resistance (TER), and cytotoxicity of the formulations by MTT and LDH assays.

MC-4RA渗透的研究结果表明，该TJMP，除了增强肽激素治疗剂的黏膜渗透之外，还显著增强寡肽治疗剂的上皮渗透。The results of the MC-4RA penetration study showed that the TJMP, in addition to enhancing the mucosal penetration of peptide hormone therapeutics, also significantly enhanced the epithelial penetration of oligopeptide therapeutics.

实施例21Example 21

TJMP对小分子药物的渗透增强作用Penetration enhancement of small molecule drugs by TJMP

本实施例表明本发明的一种代表性肽，PN159增强以乙酰胆碱酯酶(ACE)抑制剂加兰他敏为例的小分子药物的上皮渗透的功效。在本实施例中，描述一种或多种渗透性肽与小分子药物的组合。在这种情形中有用的制剂可以包括小分子药物、渗透性肽、和一种或多种其它渗透增强剂的组合。所述制剂还可以包含缓冲剂、张力剂、pH值调节剂、稳定剂和/或防腐剂。This example demonstrates the efficacy of a representative peptide of the invention, PN159, to enhance epithelial penetration of small molecule drugs exemplified by the acetylcholinesterase (ACE) inhibitor galantamine. In this example, the combination of one or more penetrant peptides with a small molecule drug is described. Formulations useful in this context may include a combination of a small molecule drug, a penetrating peptide, and one or more other penetration enhancers. The formulations may also contain buffers, tonicity agents, pH adjusters, stabilizers and/or preservatives.

本发明将加兰他敏与PN159组合，以增强加兰他敏穿过鼻黏膜的渗透。由于加兰他敏是一种可以独立地透过鼻上皮膜的小分子，所以这种药物渗透的增加是出乎意料的。因此，通过加入增强肽渗透性的赋形剂而介导的加兰他敏通过上皮渗透的显著增强作用是令人惊讶的，这是基于通常没有预计到所述赋形剂显著增加加兰他敏穿过上皮组织层的渗透。因此，本发明将通过增加它们的生物利用度而促进加兰他敏及其它小分子药物的鼻递送。The present invention combines galantamine with PN159 to enhance the penetration of galantamine through the nasal mucosa. Since galantamine is a small molecule that can independently penetrate nasal epithelial membranes, this increase in drug penetration was unexpected. Thus, the significant enhancement of galantamine penetration through the epithelium mediated by the addition of peptide permeability-enhancing excipients was surprising, based on the fact that such excipients are not generally expected to significantly increase galantamine penetration. Penetration of sensitizers across epithelial tissue layers. Thus, the present invention will facilitate nasal delivery of galantamine and other small molecule drugs by increasing their bioavailability.

在本研究中，40mg/ml乳酸盐形式的加兰他敏与25，50，和100μMPN159在溶液，pH 5.0和摩尔渗透压浓度～270mOsm中组合。使用体外上皮组织模型检测所述组合，以通过上文所述的LDH和MTT检测而监测加兰他敏渗透、跨上皮电阻(TER)、和所述制剂的细胞毒性。加兰他敏的渗透检测通过标准HPLC分析进行，如下。In this study, 40 mg/ml of galantamine in lactate form was combined with 25, 50, and 100 μM PN159 in solution, pH 5.0 and osmolarity ~270 mOsm. The combinations were tested using an in vitro epithelial tissue model to monitor galantamine penetration, transepithelial electrical resistance (TER), and cytotoxicity of the formulations by the LDH and MTT assays described above. Penetration detection of galantamine was performed by standard HPLC analysis, as follows.

HPLC分析HPLC analysis

应用无梯度LC(Waters Alliance(沃特斯联合))方法和UV检测，确定制剂中和在基底外侧培养基(渗透样品)中的加兰他敏浓度。The concentration of galantamine in the formulation and in the basolateral medium (infiltrated samples) was determined using a gradient-free LC (Waters Alliance) method with UV detection.

柱：沃特斯对称柱(Waters Symmetry Shield)，C 18，5um，25×0.46cmColumn: Waters Symmetry Shield, C 18, 5um, 25×0.46cm

流动相：在50mM甲酸铵中的5％ACN，pH 3.0Mobile phase: 5% ACN in 50 mM ammonium formate, pH 3.0

流速：1ml/分钟Flow rate: 1ml/min

柱温：30℃Column temperature: 30°C

校准曲线：0-400μg/ml溴化加兰他敏Calibration curve: 0-400μg/ml galantamine bromide

检测：在285nm的紫外线(UV)Detection: Ultraviolet (UV) at 285nm

基于前述研究，PN159提高小分子的跨黏膜递送。选择加兰他敏作为模型低分子量药物，并且认为这一分子的结果是用于其它小分子药物的渗透性肽活性的预测。为了评估这种情形中的渗透性活性，将40mg/ml乳酸盐形式的加兰他敏与25，50，和100μM PN159在溶液，pH 5.0和摩尔渗透压浓度～270mOsm中组合。使用体外上皮组织模型检测所述组合，以通过LDH和MTT检测而监测加兰他敏渗透、跨上皮电阻(TER)、和所述制剂的细胞毒性。Based on the previous studies, PN159 enhances the transmucosal delivery of small molecules. Galantamine was chosen as a model low molecular weight drug and results for this molecule were considered to be useful for prediction of penetrating peptide activity for other small molecule drugs. To assess osmotic activity in this case, galantamine in the lactate form at 40 mg/ml was combined with 25, 50, and 100 μM PN159 in solution, pH 5.0 and osmolarity ~270 mOsm. The combinations were tested using an in vitro epithelial tissue model to monitor galantamine penetration, transepithelial electrical resistance (TER), and cytotoxicity of the formulations by LDH and MTT assays.

在所述体外组织模型中，加入PN159导致药物穿过细胞屏障的渗透的显著增加。具体地，在40mg/ml加兰他敏的P_app中存在2.5-3.5倍的增加。In the in vitro tissue model, addition of PN159 resulted in a significant increase in drug penetration across cellular barriers. Specifically, there was a 2.5-3.5 fold increase in P_app at 40 mg/ml galantamine.

在加兰他敏存在下，PN159减少TER，正如前述实施例中所述。In the presence of galantamine, PN159 reduced TER, as described in the previous examples.

在所有检测浓度的乳酸加兰他敏和PN159存在下，细胞存活能力保持较高(＞80％)。相反，如通过LDH所检测，在存在PN159和乳酸加兰他敏时，细胞毒性较低。这些检测都表明PN159对上皮膜没有毒性。Cell viability remained high (>80%) in the presence of galantamine lactate and PN159 at all concentrations tested. In contrast, cytotoxicity was lower in the presence of PN159 and galantamine lactate as detected by LDH. These tests all indicated that PN159 had no toxicity to epithelial membranes.

在不存在PN159时，加兰他敏的P_app为约2.1×10^-6cm/s。当存在25，50和100mM PN159时，P_app分别为5.1×10^-6，6.2×10^-6，和7.2×10^-6cm/s。因此，在这种模型低分子量药物的P_app中，PN159提供2.4-至3.4-倍的增加。In the absence of PN159, the P_app for galantamine is about 2.1 x 10^-6 cm/s. P_app was 5.1×10^-6 , 6.2×10^-6 , and 7.2×10^-6 cm/s in the presence of 25, 50 and 100 mM PN159, respectively. Thus, PN159 provided a 2.4- to 3.4-fold increase in the P_app of this model low molecular weight drug.

TJMP令人惊讶地增加作为模型低分子量药物的加兰他敏的上皮渗透。将PN159加入到加兰他敏溶液中显著增强加兰他敏穿过上皮细胞单层的渗透。证据表明PN159暂时减少穿过上皮膜的TER，而没有损坏膜中的细胞，如通过高细胞存活能力和低细胞毒性所测定那样。TJMP在体内增强加兰他敏及其它小分子药物的生物利用度，其通过在本文中使用体外模型证明的相同的机制而增强。进一步预测到TJMP还在更高的浓度增强加兰他敏的渗透。TJMP surprisingly increases the epithelial penetration of galantamine as a model low molecular weight drug. Addition of PN159 to galantamine solution significantly enhanced the penetration of galantamine across epithelial cell monolayers. Evidence indicates that PN159 temporarily reduces TER across epithelial membranes without damaging cells in the membrane, as determined by high cell viability and low cytotoxicity. TJMPs enhance the bioavailability of galantamine and other small molecule drugs in vivo by the same mechanism demonstrated here using an in vitro model. It is further predicted that TJMP also enhances the penetration of galantamine at higher concentrations.

实施例22Example 22

TJMP对蛋白的渗透增强作用Penetration-enhancing effect of TJMP on proteins

已经确定PN159用于低分子量化合物的跨黏膜制剂的用途，重要地是辨别这些观察是否能够外推到更大的分子，例如治疗性肽和蛋白。为了这一目的，在不存在和存在25，50，和100mM PN159时，对作为模式治疗肽的鲑鱼降钙素进行体外组织研究。当不存在PN159时，对于降钙素的P_app为约1×10^-7cm/s，低于加兰他敏的P_app约一个数量级，推测这是由于分子量的不同。当存在PN159时，数据表明降钙素渗透的显著增加，与只有降钙素的情形相比，在P_app中达到23-到47-倍增加(表20)。Having established the use of PN159 for transmucosal formulations of low molecular weight compounds, it will be important to discern whether these observations can be extrapolated to larger molecules such as therapeutic peptides and proteins. For this purpose, in vitro tissue studies were performed on salmon calcitonin as a model therapeutic peptide in the absence and presence of 25, 50, and 100 mM PN159. In the absence of_PN159 , the P_app for calcitonin was about 1 x 10^-7 cm/s, about an order of magnitude lower than that for galantamine, presumably due to the difference in molecular weight. When PN159 was present, the data showed a significant increase in calcitonin penetration, reaching a 23- to 47-fold increase in P_app compared to calcitonin alone (Table 20).

表20Table 20

使用体外组织模型检测的P_appP_app detected using an in vitro tissue model

^apH为5.0。^a pH is 5.0.

为了研究这些发现的普遍性，在不存在和存在PN159时，在所述体外模型中检验两种其它的肽，即人甲状旁腺激素1-34(PTH_1-34)和人肽YY3-36(PYY_3-36)(P_app数据显示在表20中)。当不存在PN159时，这两种肽的P_app与降钙素的P_app一致。在PTH_1-34的情形中，PN159的存在在P_app中提供约3-5倍的增加。当在PN159存在下配制PYY_3-36时，Papp增加约12-到17-倍。这些数据证实我们的发现，即TJMP增强小分子和蛋白的跨黏膜药物递送的普遍性。To investigate the generality of these findings, two other peptides, human parathyroid hormone 1-34 (PTH_1-34 ) and human peptide YY3-36, were tested in the in vitro model in the absence and presence of PN159 (PYY_3-36 ) (P_app data are shown in Table 20). When PN159 is absent, the P_app of these two peptides is consistent with_that of calcitonin. In the case of PTH_1-34 , the presence of PN159 provided about a 3-5 fold increase in P_app . Papp increased approximately 12- to 17-fold when PYY_3-36 was formulated in the presence of PN159. These data confirm our finding that TJMPs enhance the generality of transmucosal drug delivery of small molecules and proteins.

实施例23Example 23

TJMP的化学稳定性Chemical stability of TJMP

在治疗相关的储存条件下确定PN159的化学稳定性。应用表示稳定性的HPLC方法。溶液(50mM)保存在不同的pH值(4.0，7.3，和9.0)和温度(5℃，25℃，35℃，40℃，和50℃)条件下。在pH值4的样品含有10mM柠檬酸盐缓冲液。在pH值7.3和9.0的样品含有10mM磷酸盐缓冲液。储存稳定性结果(包括阿伦尼乌斯作图法(Arrhenius plot))表明，在低温度和pH值PN159在化学上是最稳定的。例如，在5℃和pH 4.0或pH7.3，对于6个月的储存，存在基本上100％的PN159恢复。当保存温度升高到25℃时，6个月后，对于在pH 4或pH 7的样品分别存在7％和26％的天然PN159损失。在pH 9和/或升高的温度，例如，40-50℃，跟着发生PN159的快速消耗。4.0-7.3的pH值范围和冷冻到环境温度的温度范围对于鼻内制剂是最相关的。因此，这些数据支持TJMP可以在与IN制剂相关的储存条件下保持化学完整性。The chemical stability of PN159 was determined under therapeutically relevant storage conditions. An HPLC method indicating stability was applied. Solutions (50 mM) were kept at different pH values (4.0, 7.3, and 9.0) and temperatures (5°C, 25°C, 35°C, 40°C, and 50°C). Samples atpH 4 contained 10 mM citrate buffer. Samples at pH 7.3 and 9.0 contained 10 mM phosphate buffer. Storage stability results (including Arrhenius plot) indicated that PN159 was chemically most stable at low temperature and pH. For example, at 5°C and pH 4.0 or pH 7.3, there was essentially 100% recovery of PN159 for 6 months of storage. When the storage temperature was increased to 25°C, after 6 months, there was 7% and 26% loss of native PN159 for the samples atpH 4 or pH 7, respectively. AtpH 9 and/or elevated temperature, e.g., 40-50°C, rapid consumption of PN159 ensues. A pH range of 4.0-7.3 and a temperature range of freezing to ambient temperature are most relevant for intranasal formulations. Thus, these data support that TJMPs can maintain chemical integrity under storage conditions associated with IN formulations.

实施例24Example 24

在兔中通过鼻内施用在体内评估紧密连接调控肽In vivo assessment of tight junction-modulating peptides by intranasal administration in rabbits

在兔中进行药物代谢动力学(PK)研究，以评估通过鼻内(IN)递送施用的肽YY(PYY)与各种紧密连接调控肽(TJMPs)的血浆药物代谢动力学特征。Pharmacokinetic (PK) studies were performed in rabbits to evaluate the plasma pharmacokinetic profile of peptide YY (PYY) administered by intranasal (IN) delivery with various tight junction modulating peptides (TJMPs).

动物模型animal model

在本研究中，新西兰白兔(Hra：(NZW)SPF)用作测试受试者，以通过鼻内施用和静脉内输注而评估MC-4RA的血浆药物代谢动力学。动物的处理按照在USDA动物福利法(USDA Animal Welfare Act)中列出的规章(9CFR第1、2和3部分)和实验动物饲养管理和使用指南(Guide for theCare and Use of Laboratory Animals，(ILAR出版，1996，国家科学院印刷))中指定的条件进行。In this study, New Zealand white rabbits (Hra: (NZW)SPF) were used as test subjects to evaluate the plasma pharmacokinetics of MC-4RA by intranasal administration and intravenous infusion. Animals were handled in accordance with the regulations listed in the USDA Animal Welfare Act (9CFR Parts 1, 2 and 3) and the Guide for the Care and Use of Laboratory Animals (ILAR Published, 1996, Printed by the National Academy of Sciences)) under the conditions specified.

由于从施用给兔的药物获得的药物代谢动力学曲线非常近似于相同的药物在人中的PK曲线，所以选择兔作为本研究的动物受试者。Rabbits were chosen as animal subjects for this study because the pharmacokinetic profile obtained from a drug administered to rabbits closely approximates the PK profile of the same drug in humans.

给药施用Administration

关于9种检测的TJMPs的实验设计和给药方案总结在表21中。所有的实验组都通过鼻内(IN)施用给予同单个TJMP或磷酸缓冲盐水(PBS：阴性对照)组合的205μg/kg PYY(3-36)。使用自动移液器和一次性塑料吸头，将每种制剂一次施用到左鼻孔中。将动物头部向后翘起，并且在动物吸入时施用所述剂量，以便允许毛细管作用将溶液吸入鼻孔。在IN施用后，控制动物头部在向后翘起的位置约15秒，以防止施用的剂量的任何损失。在这一步骤过程中，采取极度的小心，以避免由与鼻内黏膜接触导致的任何潜在的组织损伤。The experimental design and dosing regimens for the nine tested TJMPs are summarized in Table 21. All experimental groups received 205 μg/kg PYY(3-36) in combination with TJMP alone or phosphate buffered saline (PBS: negative control) by intranasal (IN) administration. Each formulation was administered once into the left nostril using an automatic pipette and disposable plastic tips. The head of the animal is tilted back and the dose is administered as the animal inhales to allow capillary action to draw the solution into the nostrils. Following IN administration, the animal was held head-back in the tilted position for approximately 15 seconds to prevent any loss of the administered dose. During this step, extreme care is taken to avoid any potential tissue damage caused by contact with the endonasal mucosa.

表21Table 21

  组 Group  动物数量number of animals  途径way  紧密连接调控剂(浓度)Tight junction modulator (concentration)  PYY3(μg/kg)PYY3 (μg/kg)  1 1  5M5M  鼻内intranasal  PBSPBS  205205  2 2  5M5M  鼻内intranasal  PN159(50μM)PN159 (50μM)  205205  33  5M5M  鼻内intranasal  PN161(100μM)PN161 (100μM)  205205  44  5M5M  鼻内intranasal  PN202(100μM)PN202 (100μM)  205205  55  5M5M  鼻内intranasal  PN27(250μM)PN27 (250μM)  205205  66  5M5M  鼻内intranasal  PN58(500μM)PN58 (500μM)  205205  77  5M5M  鼻内intranasal  PN73(500μM)PN73 (500μM)  205205  8 8  5M5M  鼻内intranasal  PN228(500μM)PN228 (500μM)  205205  9 9  5M5M  鼻内intranasal  PN183(1000μM)PN183 (1000μM)  205205  1010  5M5M  鼻内intranasal  PN556(1000μM)PN556 (1000μM)  205205

PN556具有与PN283相同的一级序列，但是在肽的N端没有马来酰亚胺修饰。PN556 has the same primary sequence as PN283, but without the maleimide modification at the N-terminus of the peptide.

血液和血浆样品收集Blood and Plasma Sample Collection

通过IN给药施用后，通过直接在耳缘静脉进行静脉穿刺而从每只动物收集连续的血液样品。在给药前，给药后5，10，15，20，30，45，60，90，120和180分钟收集血液样品。样品收集到含有EDTA二钾作为抗凝血剂的管中。管子在离心之前冷却。所有的样品在收集1小时之内进行离心。收集血浆，并且转移到预先标记的塑料小瓶中，在干冰/丙酮浴中冷冻，然后在进行药物代谢动力学分析之前保存在大约-70℃。Following administration by IN dosing, serial blood samples were collected from each animal by venipuncture directly in the marginal ear vein. Blood samples were collected before dosing, 5, 10, 15, 20, 30, 45, 60, 90, 120 and 180 minutes after dosing. Samples were collected into tubes containing dipotassium EDTA as an anticoagulant. Tubes were cooled before centrifugation. All samples were centrifuged within 1 hour of collection. Plasma was collected and transferred into pre-labeled plastic vials, frozen in a dry ice/acetone bath, and stored at approximately -70°C until pharmacokinetic analysis.

在每次血液采样时刻进行临床观察，并且恰好在5分钟之前和鼻内给药后1小时对在IN施用检测组中的所有动物的两个鼻孔进行检查。Clinical observations were made at the time of each blood sampling and both nostrils of all animals in the IN administered test group were examined just 5 minutes before and 1 hour after intranasal administration.

分析方法Analytical method

通过应用ELISA，分析来自所有研究组中每只动物的样品的PYY(3-36)水平。将给药之前和给药后的检测样品在HPLC上运行进行质量控制。血浆等分试样(0.1mL)是在加入生物分析内标后用乙腈沉淀的蛋白。将上清液用氮气干燥，在HPLC缓冲液中重构，并且然后注射到HPLC系统中。通过阳离子电喷雾离子化串联三极四极质谱仪(positive ion electrosprayionization tandem triple quadrupole mass spectrometer)检测流出物。PK数据通过WinNonlin(法萨特公司(Pharsight Corp.)，芒廷维尤(MountainView))进行分析。Samples from each animal in all study groups were analyzed for PYY(3-36) levels by application of ELISA. Pre-dose and post-dose test samples were run on HPLC for quality control. Plasma aliquots (0.1 mL) were protein precipitated with acetonitrile after addition of bioassay internal standards. The supernatant was dried with nitrogen, reconstituted in HPLC buffer, and then injected into the HPLC system. The effluent was monitored by a positive ion electrosprayionization tandem triple quadrupole mass spectrometer. PK data were analyzed by WinNonlin (Pharsight Corp., Mountain View).

结果result

关于每个检测组的平均血浆PK参数总结在表22中。在施用任何制剂后，没有观察到不利的临床迹象。通过IN施用制剂的动物的两个鼻孔的鼻内检查后显示没有任何发红，没有肿胀。PK研究评估C_max(最大观察浓度)，T_max(最大浓度的时刻)和AUC(曲线下面积)末端和无穷大极限(inf)。按照它们的体内渗透性水平排列8种TJMPs，并且分成4种不同的表现等级，等级I包含具有最大体内渗透性水平的TJMPs，并且每个后续等级包含具有逐渐减小的体内渗透性水平的TJMPs。The mean plasma PK parameters for each test group are summarized in Table 22. No adverse clinical signs were observed after administration of any formulation. Intranasal examination of both nostrils of animals administered the formulation by IN showed no redness, no swelling. The PK study evaluates C_max (maximum observed concentration), T_max (moment of maximum concentration) and AUC (area under the curve) terminal and limit of infinity (inf). The 8 TJMPs are arranged according to their levels of in vivo permeability and are grouped into 4 different performance classes, with class I containing TJMPs with the greatest level of in vivo permeability and each subsequent class containing TJMPs with decreasing levels of in vivo permeability .

表22Table 22

这些数据表明对于PN161和PN27二者观察到的体内渗透性与PN159相当；并且其余的TJMPs，在所检测的浓度，获得低于PN159的体内渗透性水平。These data indicate that the in vivo permeability observed for both PN161 and PN27 was comparable to PN159; and that the remaining TJMPs, at the concentrations tested, achieved lower levels of in vivo permeability than PN159.

实施例25Example 25

在体外增强上皮细胞层渗透的紧密连接调控肽Tight junction regulatory peptides that enhance epithelial cell layer penetration in vitro

本实施例描述筛选本发明的代表性肽PN679和PN745(表23所示)以及每种肽的检测制剂(表24所示)，以确定每种肽关于上皮细胞单层渗透增强作用的有效浓度范围。This example describes the screening of representative peptides PN679 and PN745 of the invention (shown in Table 23) and test formulations of each peptide (shown in Table 24) to determine the effective concentration of each peptide for the permeation enhancement effect of epithelial cell monolayers scope.

表23Table 23

紧密连接调控肽tight junction regulatory peptide

肽#Peptide#氨基酸序列amino acid sequence分子量molecular weight批号batch number  纯度(％)Purity (%)PN679PN679  CNGRCGGKKKLKLLLKLL(SEQ ID NO：32)CNGRCGGKKKLKLLLKLL (SEQ ID NO: 32)1984.781984.7805-1882-75805-1882-75894.0194.01PN745PN745  LRKLRKRLLRLRKLRKRLLR-酰胺(SEQ ID NO：33)LRKLRKRLLRLRKLRKRLLR-amide (SEQ ID NO: 33)2684.532684.5305-1882-76105-1882-76199.2999.29

下表24描述含有本发明的代表性肽(表24中的“活性剂”栏)的个别检测制剂，和作为阳性和阴性检测制剂对照的检测制剂，它们通过TER、LDH(细胞毒性)和样品渗透增强作用检测而进行检验。每种肽在25μM，100μM，250μM，500μM和1000μM浓度进行检测。此处的PN159(检测制剂#11)作为TJMP阳性对照，并且先前已经表现出在25μM有效减小TER和增强样品渗透的能力。1％的Triton X-100^TM(检测制剂#14)作为细胞毒性(LDH)检测和TER减小检测二者的阳性对照。“特别的加料(specialsauce)”(SS)在此处作为小分子渗透增强剂。DPBS++作为阴性对照。除了具有pH 5的目的pH值的检测制剂#12外，每种检测制剂具有300μl的终体积，和pH 7的目的pH值。1％的Triton X-100^TM(检测制剂#14)作为细胞毒性(LDH)检测的阳性对照。Table 24 below describes individual test formulations containing representative peptides of the invention (column "Active" in Table 24), and test formulations as controls for positive and negative test formulations, which passed TER, LDH (cytotoxicity), and sample Tested for penetration enhancement. Each peptide was tested at 25 μM, 100 μM, 250 μM, 500 μM and 1000 μM concentrations. PN159 (assay formulation #11) here serves as a TJMP positive control and has previously been shown to effectively reduce TER and enhance sample penetration at 25 μΜ. 1% Triton X-100^™ (Assay Preparation #14) served as a positive control for both the cytotoxicity (LDH) assay and the TER reduction assay. "Special sauce" (SS) is used herein as a small molecule penetration enhancer. DPBS++ served as a negative control. Each test formulation had a final volume of 300 μl, and a target pH of pH 7, except test formulation #12, which had a target pH ofpH 5. 1% Triton X-100^™ (Assay Preparation #14) was used as a positive control for the cytotoxicity (LDH) assay.

在300μl总体积的每种检测制剂中，只将20μl样品应用到来源于人的气管/支气管上皮细胞(EpiAirway^TM组织模型系统)，以评估每种检测制剂对TER、LDH和样品渗透的作用。In a total volume of 300 μl of each assay formulation, only 20 μl of sample was applied to human-derived tracheal/bronchial epithelial cells (EpiAirway^™ tissue model system) to assess the effect of each assay formulation on TER, LDH and sample penetration.

表24Table 24

检测制剂Test preparation

SS＝″特别的加料″SS = "Special Addition"

实施例26Example 26

PN679和PN745在体外调控紧密连接蛋白PN679 and PN745 regulate tight junction proteins in vitro

本实施例表明，代表性肽PN679和PN745以剂量依赖型方式有效减小TER并且显著增强样品的渗透，而没有引起显著的细胞毒性，这表明这些肽是有效的TJMPs。表25总结了关于在实施例25的表24中描述的检测制剂的TER、LDH和样品渗透(FD3)数据。关于PN679的检测制剂#1和关于PN745的检测制剂#6检测两次。关于TER、LDH和样品渗透的其它检测结果显示在括号中。This example shows that representative peptides PN679 and PN745 effectively reduce TER and significantly enhance the penetration of samples in a dose-dependent manner without causing significant cytotoxicity, suggesting that these peptides are potent TJMPs. Table 25 summarizes the TER, LDH and sample penetration (FD3) data for the test formulations described in Table 24 of Example 25.Test formulation #1 on PN679 andtest formulation #6 on PN745 were tested twice. Additional assay results for TER, LDH, and sample permeation are shown in parentheses.

表25Table 25

TER、LDH和样品渗透增强作用数据的总结Summary of TER, LDH and sample permeation enhancement data

SS＝″特别的加料″SS = "Special Addition"

包含100μM，250μM，500μM和1000μM本发明的代表性肽PN679(检测制剂#1，#2，#3和#4)或PN745(检测制剂#6，#7，#8和#9)的任一种的检测制剂将TER减小到与″特别的加料″相等的程度，并且显著低于确定的TJMP对照物PN159的程度。如预计那样，DPBS++阴性对照没有显著地减小TER。这两种肽减小TER的能力与它们增强FD3分子的渗透的能力高度相关。100μM剂量的PN679(检测制剂#4)和PN745(检测制剂#9)都表现出与PN159TJMP相似的渗透百分数，但是具有较低的细胞毒性(较低的％LDH释放)。更高浓度的任一种肽导致增加的FD3渗透水平，所述水平超过PN159的水平，但是也增加LDH的释放水平，这表明增加的细胞毒性。如预计那样，DPBS++对照没有诱导可检测到的LDH释放。基于所观察到的TER减少、样品渗透和细胞毒性(LDH释放)，100μM剂量的任一种代表性肽PN679和PN745似乎用于进一步分析这两种TJMPs最理想。Any of the representative peptides PN679 (detection preparations #1, #2, #3 and #4) or PN745 (detection preparations #6, #7, #8 and #9) comprising 100 μM, 250 μM, 500 μM and 1000 μM All test formulations reduced TER to an extent equal to that of "special addition" and significantly less than that of the established TJMP control PN159. The DPBS++ negative control did not significantly reduce TER, as expected. The ability of these two peptides to reduce TER is highly correlated with their ability to enhance the penetration of FD3 molecules. Both PN679 (test formulation #4) and PN745 (test formulation #9) at a dose of 100 μM showed similar percent penetration to PN159TJMP, but were less cytotoxic (lower % LDH release). Higher concentrations of either peptide resulted in increased levels of FD3 infiltration over that of PN159, but also increased levels of LDH release, indicating increased cytotoxicity. As expected, the DPBS++ control did not induce detectable LDH release. Based on the observed reduction in TER, sample penetration and cytotoxicity (LDH release), a dose of 100 [mu]M of either of the representative peptides PN679 and PN745 seemed optimal for further analysis of these two TJMPs.

前述数据表明出乎意料的发现，即，代表性的肽PN679和PN745在体外减小人上皮细胞单层的TER，并且增强小分子渗透，而没有显著的毒性。这些数据表明，这些紧密连接调控肽(TMJP)是用于穿过黏膜表面的药物递送例如鼻内(IN)药物递送的极好的候选物。The foregoing data demonstrate the unexpected finding that representative peptides PN679 and PN745 reduce TER in human epithelial cell monolayers in vitro and enhance small molecule penetration without significant toxicity. These data suggest that these tight junction modulating peptides (TMJPs) are excellent candidates for drug delivery across mucosal surfaces, such as intranasal (IN) drug delivery.

实施例27Example 27

紧密连接调控肽在体外增强渗透与在体内观察到的增强的渗透高度相关Enhanced penetration of tight junction modulating peptides in vitro correlates highly with enhanced penetration observed in vivo

进行线性回归分析，以确定在体外EpitAirway上皮细胞模型系统中观察到的TJMP渗透动力学是否与对于同一种TJMP观察到的体内药物代谢动力学数据相关。为了确定体外渗透数据是否作为体内效果的良好的指示，将从使用PYY和TJMPs进行的体内药物代谢动力学研究获得的曲线下面积-最终(last)值(AUC-last)针对使用PYY和TJMPs进行的体外上皮细胞单层渗透研究绘图。体外渗透表示为百分数，而AUC-last(AUC-最终)表示为分钟*pg/ml。关于10种不同的TJMPs的体外和体内研究进行绘图，并且进行线性回归。得到0.82的R²值(82％相关性)，这表明对于来源于体内的AUC值和体外观察的渗透性百分数存在高度的相关性。令人惊讶地，当排除检测间(inter-assay)可变性时，得到0.996的R²值(基本上100％)，这表明在体外渗透性和体内效果之间存在直接的相关性。因此，体外渗透可以用来预测体内效果。Linear regression analysis was performed to determine whether the TJMP permeation kinetics observed in the EpitAirway epithelial cell model system in vitro correlated with the in vivo pharmacokinetic data observed for the same TJMP. To determine whether the in vitro permeation data serve as a good indicator of in vivo efficacy, the area under the curve-last value (AUC-last) obtained from the in vivo pharmacokinetic studies using PYY and TJMPs was compared against the use of PYY and TJMPs Drawing of in vitro epithelial cell monolayer permeation studies. In vitro permeation is expressed as a percentage, while AUC-last (AUC-last) is expressed as min*pg/ml. In vitro and in vivo studies for 10 different TJMPs were plotted and linear regression was performed. An^R2 value of 0.82 (82% correlation) was obtained, indicating a high correlation between the AUC values derived in vivo and the percent permeability observed in vitro. Surprisingly, when inter-assay variability was excluded, an^R2 value of 0.996 (essentially 100%) was obtained, indicating a direct correlation between in vitro permeability and in vivo effect. Therefore, in vitro permeation can be used to predict in vivo effects.

实施例28Example 28

TJMP对肽激素治疗剂的体内渗透增强作用等于或者超过小分子渗透增强In vivo penetration enhancement of peptide hormone therapeutics by TJMP equals or exceeds small molecule penetration enhancement剂的增强作用Enhancement of the agent

将20只3-6月龄并且重2.1-3.0kg的雄性新西兰白兔随机分成5个处理组，每组4只动物。将测试动物以15μl/kg并且通过吸管鼻内给药。下表26表示5种不同的给药组的组成。Twenty male New Zealand white rabbits aged 3-6 months and weighing 2.1-3.0 kg were randomly divided into 5 treatment groups of 4 animals each. Test animals were dosed intranasally at 15 μl/kg and via pipette. Table 26 below shows the composition of 5 different dosing groups.

对于给药组1(见表26)，使用包含小分子渗透增强剂的PYY临床制剂。在这些研究中的小分子增强剂包括甲基-β-环糊精、二癸酰基磷脂酰胆碱(DDPC)、和/或EDTA。给药组2接受溶解在磷酸缓冲盐水(PBS)中的PYY。对于给药组3-5，将不同浓度的PN159加到给药组2中，以致给药组3-5中的每一组由PYY，PN159和PBS组成。For dosing group 1 (see Table 26), a clinical formulation of PYY containing a small molecule penetration enhancer was used. Small molecule enhancers in these studies included methyl-β-cyclodextrin, didecanoylphosphatidylcholine (DDPC), and/or EDTA.Dosing group 2 received PYY dissolved in phosphate buffered saline (PBS). For administration groups 3-5, different concentrations of PN159 were added toadministration group 2 so that each of administration groups 3-5 consisted of PYY, PN159 and PBS.

表26Table 26

给药组G

组Group动物animal渗透增强剂penetration enhancer  给药浓度(mg/ml)Dosing concentration (mg/ml)  给药体积(ml/kg)Dosing volume (ml/kg)  PYY剂量(μg/kg)PYY dose (μg/kg)  1 1  4M4M  小分子渗透增强剂Small molecule penetration enhancer  13.6713.67  0.0150.015  205205  2 2  4M4M  无 none  13.6713.67  0.0150.015  205205  33  4M4M  25μMPN15925μMPN159  13.6713.67  0.0150.015  205205  44  4M4M  50μMPN15950μMPN159  13.6713.67  0.0150.015  205205  55  4M4M  100μM PN159100μM PN159  13.6713.67  0.0150.015  205205

通过直接在耳缘静脉进行静脉穿刺而收集连续的血液样品(每份约2ml)，收集到含有EDTA作为抗凝血剂的血液收集管中。在给药后0，2.5，5，10，15，30，45，60，和120分钟收集血液样品。收集血液之后，管子轻轻摇动几次，以抗凝血，然后加入50μl牛胰蛋白酶抑制剂溶液。在大约4℃以约1,600xg离心血液15分钟，并且将血浆样品分配到一式两份等分试样中，并且在大约-70℃冷冻保存。Serial blood samples (approximately 2 ml each) were collected by venipuncture directly in the marginal ear vein into blood collection tubes containing EDTA as an anticoagulant. Blood samples were collected at 0, 2.5, 5, 10, 15, 30, 45, 60, and 120 minutes after dosing. After blood was collected, the tubes were shaken gently several times to anticoagulate, and then 50 [mu]l bovine trypsin inhibitor solution was added. Blood was centrifuged at approximately 1,600 xg for 15 minutes at approximately 4°C, and plasma samples were divided into duplicate aliquots and stored frozen at approximately -70°C.

平均在一个处理组中的所有4只动物，检测下述PYY的血浆浓度(表27)：Averaged over all 4 animals in one treatment group, the plasma concentrations of the following PYY were determined (Table 27):

表27Table 27

检测组PYY血浆浓度的总结Summary of PYY Plasma Concentrations by Test Groups

  组1Group 1  组2Group 2  组3Group 3  组4Group 4  组5Group 5  时间，分钟time, minutes  小分子渗透增强剂Small molecule penetration enhancer  无渗透增强剂Nopenetration enhancer25μM PN15925 μMPN15950μM PN15950μM PN159100μM PN159100μM PN159  00  183.825183.825  257.3257.3  228.675228.675  424.4424.4  294.225294.225  2.52.5  1280.71280.7  242.8242.8  526.375526.375  749.975749.975  1748.2251748.225  55  1449.4251449.425  273.675273.675  1430.151430.15  1293.41293.4  3088.23088.2  1010  8251.88251.8  372.05372.05  6521.76521.7  12517.212517.2  14486.614486.6

  1515  13731.213731.2  398.225398.225  12563.07512563.075  34455.334455.3  20882.72520882.725  3030  19537.5519537.55  476.475476.475  15222.615222.6  35294.37535294.375  25470.47525470.475  4545  13036.07513036.075  340.7340.7  9081.1259081.125  21582.22521582.225  16499.5516499.55  6060  7080.8757080.875  283.825283.825  4843.154843.15  9461.9259461.925  10676.62510676.625  120120  1671.91671.9  192.575192.575  1224.21224.2  2337.7752337.775  1891.2751891.275

从上述数据计算的药物代谢动力学数据显示在下表28中：Pharmacokinetic data calculated from the above data are shown in Table 28 below:

表28Table 28

药物代谢动力学数据总结Pharmacokinetic Data Summary

与组2(无增强剂)制剂比较，确定下述相对增强比例(表29)：Compared with the Group 2 (no enhancer) formulation, the following relative enhancement ratios were determined (Table 29):

表29Table 29

相对增强比例relative enhancement ratio

  组 Group  制剂preparation  相对CmaxRelative to Cmax  相对AUC lastRelative AUC last  1 1  小分子渗透增强剂Small molecule penetrationenhancer  38x38x  27x27x  33  PN159，25μmPN159,25μm  30x30x  21x21x  44  PN159，50μmPN159,50μm  79x79x  46x46x  55  PN159，100μmPN159, 100μm  54x54x  38x38x

前述数据表明，与小分子渗透增强剂比较，TJMP在体内将人激素肽治疗剂的鼻内渗透增强到相等的或更大的程度。在50μM浓度观察到所述肽的最大作用。100μM浓度导致稍低的渗透，尽管二者都导致比小分子渗透增强剂更高的渗透。The foregoing data indicate that TJMPs enhance intranasal penetration of human hormone peptide therapeutics to an equal or greater extent in vivo than small molecule penetration enhancers. The maximum effect of the peptide was observed at a concentration of 50 μM. The 100 μΜ concentration resulted in somewhat lower penetration, although both resulted in higher penetration than the small molecule penetration enhancers.

实施例29Example 29

TJMP对寡肽治疗剂的渗透增强作用Penetration enhancement of oligopeptide therapeutics by TJMP

本实施例表明本发明的一种代表性肽，PN159增强一种环五肽，黑皮质素-4受体激动剂(MC-4RA)，一种用于哺乳动物细胞受体的模式寡肽激动剂的上皮渗透的功效。在这一实施例中，描述一种或多种渗透性肽与MC-4RA的组合。在这种情形中有用的制剂可以包括寡肽治疗剂、渗透性肽和一种或多种其它渗透增强剂的组合。所述制剂还可以含有缓冲剂、张力剂、pH值调节剂、和肽/蛋白稳定剂如氨基酸、糖或多元醇、聚合物、和盐。This example demonstrates that a representative peptide of the invention, PN159, enhances agonism of a cyclic pentapeptide, melanocortin-4 receptor agonist (MC-4RA), a model oligopeptide for mammalian cell receptors Efficacy of epithelial penetration of agents. In this example, the combination of one or more penetrant peptides with MC-4RA is described. Formulations useful in this context may include a combination of an oligopeptide therapeutic, a penetrating peptide, and one or more other penetration enhancers. The formulations may also contain buffers, tonicity agents, pH adjusters, and peptide/protein stabilizers such as amino acids, sugars or polyols, polymers, and salts.

在本研究中评估PN159对MC-4RA渗透的作用。MC-4RA是一种具有约1,100Da分子量的甲烷磺酸盐，其调控MC-4受体的活性。评估的PN159浓度为5，25，50，和100μM。45mg/ml M-β-CD用作所有制剂的增溶剂，以获得10mg/ml的肽浓度。评估PN159自身或与EDTA(1，2.5，5，或10mg/ml)组合的作用。制剂的pH值固定在4，并且摩尔渗透压浓度为220mOsm/kg。The effect of PN159 on MC-4RA penetration was assessed in this study. MC-4RA is a methanesulfonate salt with a molecular weight of approximately 1,100 Da that modulates the activity of the MC-4 receptor. PN159 concentrations evaluated were 5, 25, 50, and 100 μM. 45 mg/ml M-β-CD was used as a solubilizer for all formulations to obtain a peptide concentration of 10 mg/ml. The effect of PN159 by itself or in combination with EDTA (1, 2.5, 5, or 10 mg/ml) was assessed. The pH of the formulation was fixed at 4 and the osmolarity was 220 mOsm/kg.

HPLC方法HPLC method

通过RP-HPLC分析MC-4RA在基底外侧部培养基中的浓度，所述RP-HPLC使用C18RP层析，流速1mL/分钟，和柱温25℃。The concentration of MC-4RA in the basolateral lateral medium was analyzed by RP-HPLC using C18RP chromatography at a flow rate of 1 mL/min and a column temperature of 25°C.

溶剂A：在水中0.1％的TFA；溶剂B：在ACN中0.1％的TFASolvent A: 0.1% TFA in water; Solvent B: 0.1% TFA in ACN

注射体积：50μLInjection volume: 50 μL

检测：220nmDetection: 220nm

运行时间：15分钟Run time: 15 minutes

MC-4RA与5，25，50，和100μM PN159组合，并且在pH值4和摩尔渗透压浓度～220mOsm/kg。使用体外上皮组织模型检测所述组合，以通过MTT和LDH检测而监测PTH渗透、跨上皮电阻(TER)、和所述制剂的细胞毒性。MC-4RA was combined with 5, 25, 50, and 100 μM PN159 atpH 4 and osmolarity ~220 mOsm/kg. The combinations were tested using an in vitro epithelial tissue model to monitor PTH penetration, transepithelial electrical resistance (TER), and cytotoxicity of the formulations by MTT and LDH assays.

MC-4RA渗透的研究结果表明，除了增强肽激素治疗剂的黏膜渗透之外，TJMP还显著增强寡肽治疗剂的上皮渗透。The results of the MC-4RA penetration study showed that, in addition to enhancing the mucosal penetration of peptide hormone therapeutics, TJMP also significantly enhanced the epithelial penetration of oligopeptide therapeutics.

实施例30Example 30

TJMP对小分子药物的渗透增强作用Penetration enhancement of small molecule drugs by TJMP

本实施例表明本发明的一种代表性肽，PN159增强以乙酰胆碱酯酶(ACE)抑制剂加兰他敏为例的小分子药物的上皮渗透的功效。在本实施例中，描述一种或多种渗透性肽与小分子药物的组合。在这种情形中有用的制剂可以包括小分子药物、渗透性肽、和一种或多种其它渗透增强剂的组合。所述制剂还可以包含缓冲剂、张力剂、pH值调节剂、稳定剂和/或防腐剂。This example demonstrates the efficacy of a representative peptide of the invention, PN159, to enhance epithelial penetration of small molecule drugs exemplified by the acetylcholinesterase (ACE) inhibitor galantamine. In this example, the combination of one or more penetrant peptides with a small molecule drug is described. Formulations useful in this context may include a combination of a small molecule drug, a penetrating peptide, and one or more other penetration enhancers. The formulations may also contain buffers, tonicity agents, pH adjusters, stabilizers and/or preservatives.

本发明将加兰他敏与PN159组合，以增强加兰他敏穿过鼻黏膜的渗透。由于加兰他敏是一种可以独立地透过鼻上皮膜的小分子，所以这种药物渗透的增加是出乎意料的。因此，通过加入增强肽渗透的赋形剂而介导的加兰他敏通过上皮渗透的显著增强作用是令人惊讶的，这是基于通常没有预计到所述赋形剂显著增加加兰他敏穿过上皮组织层的渗透。因此，本发明将通过增加它们的生物利用度而促进加兰他敏及其它小分子药物的鼻递送。The present invention combines galantamine with PN159 to enhance the penetration of galantamine through the nasal mucosa. Since galantamine is a small molecule that can independently penetrate nasal epithelial membranes, this increase in drug penetration was unexpected. Thus, the significant enhancement of galantamine penetration through the epithelium mediated by the addition of peptide penetration-enhancing excipients was surprising, based on the fact that such excipients are not generally expected to significantly increase galantamine Penetration through epithelial tissue layers. Thus, the present invention will facilitate nasal delivery of galantamine and other small molecule drugs by increasing their bioavailability.

在本研究中，40mg/ml乳酸盐形式的加兰他敏与25，50，和100μMPN159在溶液，pH 5.0和摩尔渗透压浓度～270mOsm中组合。使用体外上皮组织模型检测所述组合，以通过上文所述的LDH和MTT检测而监测加兰他敏渗透、跨上皮电阻(TER)、和所述制剂的细胞毒性。加兰他敏的渗透检测通过标准HPLC分析进行，如下。In this study, 40 mg/ml of galantamine in lactate form was combined with 25, 50, and 100 μM PN159 in solution, pH 5.0 and osmolarity ~270 mOsm. The combinations were tested using an in vitro epithelial tissue model to monitor galantamine penetration, transepithelial electrical resistance (TER), and cytotoxicity of the formulations by the LDH and MTT assays described above. Penetration detection of galantamine was performed by standard HPLC analysis, as follows.

HPLC分析HPLC analysis

应用无梯度LC(Waters Alliance(沃特斯联合))方法和UV检测，确定制剂中和在基底外侧培养基(渗透样品)中的加兰他敏浓度。The concentration of galantamine in the formulation and in the basolateral medium (infiltrated samples) was determined using a gradient-free LC (Waters Alliance) method with UV detection.

柱：沃特斯对称柱(Waters Symmetry Shield)，C18，5um，25×0.46cmColumn: Waters Symmetry Shield, C18, 5um, 25×0.46cm

流动相：在50mM甲酸铵中的5％ACN，pH 3.0Mobile phase: 5% ACN in 50 mM ammonium formate, pH 3.0

流速：1ml/分钟Flow rate: 1ml/min

柱温：30℃Column temperature: 30°C

校准曲线：0-400μg/ml溴化加兰他敏Calibration curve: 0-400μg/ml galantamine bromide

检测：在285nm的紫外线(UV)Detection: Ultraviolet (UV) at 285nm

基于前述研究，PN159提高小分子的跨黏膜递送。选择加兰他敏作为模型低分子量药物，并且认为这一分子的结果是用于其它小分子药物的渗透性肽活性的预测。为了评估这种情形中的渗透性活性，将40mg/ml乳酸盐形式的加兰他敏与25，50，和100μM PN159在溶液中、pH 5.0和摩尔渗透压浓度～270mOsm组合。使用体外上皮组织模型检测所述组合，以通过LDH和MTT检测而监测加兰他敏渗透、跨上皮电阻(TER)、和所述制剂的细胞毒性。Based on the previous studies, PN159 enhances the transmucosal delivery of small molecules. Galantamine was chosen as a model low molecular weight drug and results for this molecule were considered to be useful for prediction of penetrating peptide activity for other small molecule drugs. To assess osmotic activity in this case, galantamine in lactate form at 40 mg/ml was combined with 25, 50, and 100 μM PN159 in solution at pH 5.0 and osmolarity ~270 mOsm. The combinations were tested using an in vitro epithelial tissue model to monitor galantamine penetration, transepithelial electrical resistance (TER), and cytotoxicity of the formulations by LDH and MTT assays.

在所述体外组织模型中，加入PN159导致药物穿过细胞屏障的渗透的显著增加。具体地，在40mg/ml加兰他敏的P_app中存在2.5-3.5倍的增加。In the in vitro tissue model, addition of PN159 resulted in a significant increase in drug penetration across cellular barriers. Specifically, there was a 2.5-3.5 fold increase in P_app at 40 mg/ml galantamine.

在加兰他敏存在下，PN159减少TER，正如前述实施例中所述。In the presence of galantamine, PN159 reduced TER, as described in the previous examples.

在所有检测浓度的乳酸加兰他敏和PN159存在下，细胞存活能力保持较高(＞80％)。相反，当通过LDH检测时，在存在PN159和乳酸加兰他敏时，细胞毒性低。这些检测都表明PN159对上皮膜没有毒性。Cell viability remained high (>80%) in the presence of galantamine lactate and PN159 at all concentrations tested. In contrast, cytotoxicity was low in the presence of PN159 and galantamine lactate when detected by LDH. These tests all indicated that PN159 had no toxicity to epithelial membranes.

在不存在PN159时，加兰他敏的P_app为约2.1×10^-6cm/s。当存在25，50和100mM PN159时，P_app分别为5.1×10^-6，6.2×10^-6，和7.2×10^-6cm/s。因此，在这种模型低分子量药物的P_app中，PN159提供2.4-至3.4-倍的增加。In the absence of PN159, the P_app for galantamine is about 2.1 x 10^-6 cm/s. P_app was 5.1×10^-6 , 6.2×10^-6 , and 7.2×10^-6 cm/s in the presence of 25, 50 and 100 mM PN159, respectively. Thus, PN159 provided a 2.4- to 3.4-fold increase in the P_app of this model low molecular weight drug.

TJMP令人惊讶地增加作为模型低分子量药物的加兰他敏的上皮渗透。将PN159加入到加兰他敏溶液中显著增强加兰他敏穿过上皮细胞单层的渗透。证据表明PN159暂时减少穿过上皮膜的TER，而没有损坏膜中的细胞，如通过高细胞存活能力和低细胞毒性所测定那样。TJMP在体内增强加兰他敏及其它小分子药物的生物利用度，其通过在本文中使用体外模型证明的相同的机制而增强。进一步预测到TJMP还在更高的浓度增强加兰他敏的渗透。TJMP surprisingly increases the epithelial penetration of galantamine as a model low molecular weight drug. Addition of PN159 to galantamine solution significantly enhanced the penetration of galantamine across epithelial cell monolayers. Evidence indicates that PN159 temporarily reduces TER across epithelial membranes without damaging cells in the membrane, as determined by high cell viability and low cytotoxicity. TJMPs enhance the bioavailability of galantamine and other small molecule drugs in vivo by the same mechanism demonstrated here using an in vitro model. It is further predicted that TJMP also enhances the penetration of galantamine at higher concentrations.

实施例31Example 31

TJMP对蛋白的渗透增强作用Penetration-enhancing effect of TJMP on proteins

已经确定PN159用于低分子量化合物的跨黏膜制剂的用途，重要地是辨别这些观察是否能够外推到更大的分子，例如治疗性肽和蛋白。为了这一目的，在不存在和存在25，50，和100mM PN159时，对作为模式治疗肽的鲑鱼降钙素进行体外组织研究。当不存在PN159时，对于降钙素的P_app为约1×10^-7cm/s，低于加兰他敏的P_app约一个数量级，推测这是由于分子量的不同所导致的。当存在PN159时，数据表明降钙素渗透的显著增加，与只有降钙素的情形相比，在P_app中达到23-到47-倍增加(表30)。Having established the use of PN159 for transmucosal formulations of low molecular weight compounds, it will be important to discern whether these observations can be extrapolated to larger molecules such as therapeutic peptides and proteins. For this purpose, in vitro tissue studies were performed on salmon calcitonin as a model therapeutic peptide in the absence and presence of 25, 50, and 100 mM PN159. In the absence of PN159, the P_app for calcitonin was about 1×10^-7 cm/s, about an order of magnitude lower than that of galantamine, presumably due_to the difference in molecular weight. When PN159 was present, the data showed a significant increase in calcitonin penetration, reaching a 23- to 47-fold increase in P_app compared to calcitonin alone (Table 30).

表30Table 30

使用体外组织模型检测的PappPapp detected using in vitro tissue models

^apH值为5.0^a pH 5.0

为了研究这些发现的普遍性，在不存在和存在PN159时，在所述体外模型中检验两种其它的肽，即人甲状旁腺激素1-34(PTH_1-34)和人肽YY3-36(PYY_3-36)(P_app数据显示在表30中)。当不存在PN159时，这两种肽的P_app与降钙素的P_app一致。在PTH_1-34的情形中，PN159的存在在P_app中提供约3-5倍的增加。当在PN159存在下配制PYY_3-36时，Papp增加约12-到17-倍。这些数据证实我们的发现，即TJMP增强小分子和蛋白的跨黏膜药物递送的普遍性。To investigate the generality of these findings, two other peptides, human parathyroid hormone 1-34 (PTH_1-34 ) and human peptide YY3-36, were tested in the in vitro model in the absence and presence of PN159 (PYY_3-36 ) (P_app data are shown in Table 30). When PN159 is absent, the P_app of these two peptides is consistent with_that of calcitonin. In the case of PTH_1-34 , the presence of PN159 provided about a 3-5 fold increase in P_app . Papp increased approximately 12- to 17-fold when PYY_3-36 was formulated in the presence of PN159. These data confirm our finding that TJMPs enhance the generality of transmucosal drug delivery of small molecules and proteins.

实施例32Example 32

TJMP的化学稳定性Chemical stability of TJMP

在治疗相关的储存条件下确定PN159的化学稳定性。应用表示稳定性的HPLC方法。溶液(50mM)保存在不同的pH值(4.0，7.3，和9.0)和温度(5℃，25℃，35℃，40℃，和50℃)条件下。在pH值4的样品含有10mM柠檬酸盐缓冲液。在pH值7.3和9.0的样品含有10mM磷酸缓冲液。储存稳定性结果(包括阿伦尼乌斯作图法(Arrhenius plot))表明，在低温度和pH值PN159在化学上是最稳定的。例如，在5℃和pH 4.0或pH7.3，对于6个月的储存，存在基本上100％的PN159恢复。当保存温度升高到25℃时，6个月后，对于在pH 4或pH 7的样品分别存在7％和26％的天然PN159损失。在pH 9和/或升高的温度，例如，40-50℃，跟着发生PN159的快速消耗。4.0-7.3的pH值范围和冷冻到环境温度的温度范围对于鼻内制剂是最相关的。因此，这些数据支持TJMP可以在与IN制剂相关的储存条件下保持化学完整性。The chemical stability of PN159 was determined under therapeutically relevant storage conditions. An HPLC method indicating stability was applied. Solutions (50 mM) were kept at different pH values (4.0, 7.3, and 9.0) and temperatures (5°C, 25°C, 35°C, 40°C, and 50°C). Samples atpH 4 contained 10 mM citrate buffer. Samples at pH 7.3 and 9.0 contained 10 mM phosphate buffer. Storage stability results (including Arrhenius plot) indicated that PN159 was chemically most stable at low temperature and pH. For example, at 5°C and pH 4.0 or pH 7.3, there was essentially 100% recovery of PN159 for 6 months of storage. When the storage temperature was increased to 25°C, after 6 months, there was 7% and 26% loss of native PN159 for the samples atpH 4 or pH 7, respectively. AtpH 9 and/or elevated temperature, e.g., 40-50°C, rapid consumption of PN159 ensues. A pH range of 4.0-7.3 and a temperature range of freezing to ambient temperature are most relevant for intranasal formulations. Thus, these data support that TJMPs can maintain chemical integrity under storage conditions associated with IN formulations.

实施例33Example 33

在兔中通过鼻内施用在体内评估紧密连接调控肽In vivo assessment of tight junction-modulating peptides by intranasal administration in rabbits

在兔中进行药物代谢动力学(PK)研究，以评估通过鼻内(IN)递送施用的肽YY(PYY)与各种紧密连接调控肽(TJMPs)的血浆药物代谢动力学特征。Pharmacokinetic (PK) studies were performed in rabbits to evaluate the plasma pharmacokinetic profile of peptide YY (PYY) administered by intranasal (IN) delivery with various tight junction modulating peptides (TJMPs).

动物模型animal model

在本研究中，新西兰白兔(Hra：(NZW)SPF)用作测试受试者，以通过鼻内施用和静脉内输注而评估MC-4RA的血浆药物代谢动力学。动物的处理按照在USDA动物福利法(USDAAnimal Welfare Act)中列出的规章(9CFR第1、2和3部分)和实验动物饲养管理和使用指南(ILAR出版，1996，国家科学院印刷)中指定的条件进行。In this study, New Zealand white rabbits (Hra: (NZW)SPF) were used as test subjects to evaluate the plasma pharmacokinetics of MC-4RA by intranasal administration and intravenous infusion. Animals were handled in accordance with the regulations set forth in the USDA Animal Welfare Act (USDA Animal Welfare Act) (9CFR Parts 1, 2, and 3) and the Guidelines for the Care and Use of Laboratory Animals (published by ILAR, 1996, printed by the National Academy of Sciences) conditions.

由于从施用给兔的药物获得的代谢动力学曲线非常近似于相同的药物在人中的PK曲线，所以选择兔作为本研究的动物受试者。Rabbits were chosen as animal subjects for this study because the metabolic kinetic profile obtained from a drug administered to rabbits closely approximates the PK profile of the same drug in humans.

给药施用Administration

关于9种检测的TJMPs的实验设计和给药方案总结在表31中。所有的实验组都通过鼻内(IN)施用给予同单个TJMP或磷酸缓冲盐水(PBS：阴性对照)组合的205μg/kg PYY(3-36)。使用自动移液器和一次性塑料吸头，将每种制剂一次施用到左鼻孔中。将动物头部向后翘起，并且在动物吸入时施用所述剂量，以便允许毛细管作用将溶液吸入鼻孔。在IN施用后，控制动物头部在向后翘起的位置约15秒，以防止施用的剂量的任何损失。在这一步骤过程中，采取极度的小心，以避免由与鼻内黏膜接触导致的任何潜在的组织损伤。The experimental design and dosing regimens for the nine tested TJMPs are summarized in Table 31. All experimental groups received 205 μg/kg PYY(3-36) in combination with TJMP alone or phosphate buffered saline (PBS: negative control) by intranasal (IN) administration. Each formulation was administered once into the left nostril using an automatic pipette and disposable plastic tips. The head of the animal is tilted back and the dose is administered as the animal inhales to allow capillary action to draw the solution into the nostrils. Following IN administration, the animal was held head-back in the tilted position for approximately 15 seconds to prevent any loss of the administered dose. During this step, extreme care is taken to avoid any potential tissue damage caused by contact with the endonasal mucosa.

表31Table 31

检测组总结Summary of the testing team

  组 Group  动物数量number of animals  途径way  紧密连接调控剂(浓度)Tight junction modulator (concentration)  PYY3(μg/kg)PYY3 (μg/kg)  1 1  5M5M  鼻内intranasal  PBSPBS  205205  2 2  5M5M  鼻内intranasal  PN159(50μM)PN159 (50μM)  205205  33  5M5M  鼻内intranasal  PN161(100μM)PN161 (100μM)  205205  44  5M5M  鼻内intranasal  PN202(100μM)PN202 (100μM)  205205  55  5M5M  鼻内intranasal  PN27(250μM)PN27 (250μM)  205205  66  5M5M  鼻内intranasal  PN58(500μM)PN58 (500μM)  205205  77  5M5M  鼻内intranasal  PN73(500μM)PN73 (500μM)  205205  8 8  5M5M  鼻内intranasal  PN228(500μM)PN228 (500μM)  205205  9 9  5M5M  鼻内intranasal  PN183(1000μM)PN183 (1000μM)  205205  1010  5M5M  鼻内intranasal  PN556(1000μM)PN556 (1000μM)  205205

PN556具有与PN283相同的一级序列，但是在肽的N端没有马来酰亚胺修饰。PN556 has the same primary sequence as PN283, but without the maleimide modification at the N-terminus of the peptide.

血液和血浆样品收集Blood and Plasma Sample Collection

通过IN给药施用后，通过直接在耳缘静脉进行静脉穿刺而从每只动物收集连续的血液样品。在给药前，给药后5，10，15，20，30，45，60，90，120和180分钟收集血液样品。样品收集到含有EDTA二钾作为抗凝血剂的管中。管子在离心之前冷却。所有的样品在收集1小时之内进行离心。收集血浆，并且转移到预先标记的塑料小瓶中，在干冰/丙酮浴中冷冻，然后在进行药物代谢动力学分析之前保存在大约-70℃。Following administration by IN dosing, serial blood samples were collected from each animal by venipuncture directly in the marginal ear vein. Blood samples were collected before dosing, 5, 10, 15, 20, 30, 45, 60, 90, 120 and 180 minutes after dosing. Samples were collected into tubes containing dipotassium EDTA as an anticoagulant. Tubes were cooled before centrifugation. All samples were centrifuged within 1 hour of collection. Plasma was collected and transferred into pre-labeled plastic vials, frozen in a dry ice/acetone bath, and stored at approximately -70°C until pharmacokinetic analysis.

在每次血液采样时刻进行临床观察，并且恰好在5分钟之前和鼻内给药后1小时对在IN施用检测组中的所有动物的两个鼻孔进行检查。Clinical observations were made at the time of each blood sampling and both nostrils of all animals in the IN administered test group were examined just 5 minutes before and 1 hour after intranasal administration.

分析方法Analytical method

通过应用ELISA，分析来自所有研究组中每只动物的样品的PYY(3-36)水平。将给药之前和给药后的检测样品在HPLC上运行进行质量控制。血浆等分试样(0.1mL)是在加入生物分析内标后用乙腈沉淀的蛋白。将上清液用氮气干燥，在HPLC缓冲液中重构，并且然后注射到HPLC系统中。通过阳离子电喷雾离子化串联三极四极质谱仪检测流出物。PK数据通过WinNonlin(法萨特公司(Pharsight Corp.)，芒廷维尤(Mountain View))进行分析。Samples from each animal in all study groups were analyzed for PYY(3-36) levels by application of ELISA. Pre-dose and post-dose test samples were run on HPLC for quality control. Plasma aliquots (0.1 mL) were protein precipitated with acetonitrile after addition of bioassay internal standards. The supernatant was dried with nitrogen, reconstituted in HPLC buffer, and then injected into the HPLC system. The effluent was monitored by cationic electrospray ionization tandem triple quadrupole mass spectrometer. PK data were analyzed by WinNonlin (Pharsight Corp., Mountain View).

结果result

将关于每个检测组的平均血浆PK参数总结在表32中。在施用任何制剂后，没有观察到不利的临床迹象。通过IN施用制剂的动物的两个鼻孔的鼻内检查后显示没有任何发红，也没有肿胀。PK研究评估C_max(最大观察浓度)，T_max(最大浓度的时刻)和AUC(曲线下面积)末端和无穷大极限(inf)。按照它们的体内渗透性水平排列8种TJMPs，并且分成4种不同的表现等级，等级I包含具有最大体内渗透性水平的TJMPs，并且每个后续等级包含具有逐渐减小的体内渗透性水平的TJMPs。The mean plasma PK parameters for each test group are summarized in Table 32. No adverse clinical signs were observed after administration of any formulation. Intranasal examination of both nostrils of animals administered the formulation by IN did not reveal any redness nor swelling. The PK study evaluates C_max (maximum observed concentration), T_max (moment of maximum concentration) and AUC (area under the curve) terminal and limit of infinity (inf). The 8 TJMPs are arranged according to their levels of in vivo permeability and are grouped into 4 different performance classes, with class I containing TJMPs with the greatest level of in vivo permeability and each subsequent class containing TJMPs with decreasing levels of in vivo permeability .

表32Table 32

药物代谢动力学数据总结Pharmacokinetic Data Summary

  组 Group  体内等级排列In vivo ranking  T_1/2T_1/2  T_max(分钟)T_max (minutes)  C_max(pg/mL)C_max (pg/mL)  AUClast(分钟＊pg/mL)AUClast (min*pg/mL)  AUCinf(分钟＊pg/mL)AUCinf(min*pg/mL)  PBSPBS  86.086.0  22.022.0  806806  4.5×10⁴4.5×10⁴  6.81×10⁴6.81×10⁴  PN159PN159  II  30.230.2  17.017.0  3020030200  1.52×10⁶1.52×10⁶  1.55×10⁶1.55×10⁶  PN161PN161  II  34.334.3  24.024.0  3210032100  1.62×10⁶1.62×10⁶  1.65×10⁶1.65×10⁶  PN27PN27  II  29.929.9  33.033.0  2930029300  1.67×10⁶1.67×10⁶  1.71×10⁶1.71×10⁶  PN228PN228  IIII  30.430.4  31.031.0  2120021200  1.06×10⁶1.06×10⁶  1.08×10⁶1.08×10⁶  PN202PN202  IIII  34.134.1  32.032.0  1270012700  7.35×10⁵7.35×10⁵  7.63×10⁵7.63×10⁵  PN58PN58  IIIIII  29.529.5  43.043.0  1280012800  8.3×10⁵8.3×10⁵  8.71×10⁵8.71×10⁵  PN73PN73  IVIV  53.853.8  37.037.0  82208220  3.46×10⁵3.46×10⁵  3.55×10⁵3.55×10⁵  PN183PN183  IVIV  33.733.7  22.022.0  54505450  2.58×10⁵2.58×10⁵  2.75×10⁵2.75×10⁵  PN556PN556  IVIV  51.251.2  22.022.0  46204620  2.47×10⁵2.47×10⁵  2.80×10⁵2.80×10⁵

实施例34Example 34

纯化purification

已经合成下述PEG化的PN159肽(表33)：The following PEGylated PN159 peptides have been synthesized (Table 33):

表33Table 33

合成的PEG化的PN0159肽列表List of Synthetic PEGylated PN0159 Peptides

  PN526PN526  (SEQ.ID NO 58)PEG1-KLALKLALKALKAALKLA-酰胺(SEQ.ID NO 58) PEG1-KLALKLALKALKAALKLA-amide  PN537PN537  (SEQ.ID NO 59)PEG(5000Da)-KLALKLALKALKAALKLA-酰胺(SEQ.ID NO 59)PEG(5000Da)-KLALKLALKALKAALKLA-amide  PN570PN570  (SEQ.ID NO 60)NH2-KLALKLALKALKAALKLA-PEG 1-酰胺(SEQ.ID NO 60)NH2-KLALKLALKALKAALKLA-PEG 1-amide  PN571PN571  (SEQ.ID NO 61)PEG1-KLALKLALKALKAALKLA-PEG1-酰胺(SEQ.ID NO 61) PEG1-KLALKLALKALKAALKLA-PEG1-amide  PN572PN572  (SEQ.ID NO 62)PEG3-KLALKLALKALKAALKLA-酰胺(SEQ.ID NO 62) PEG3-KLALKLALKALKAALKLA-amide

将150mg量的粗肽吸收在15mL含有0.1％TFA和3mL乙酸的水中。在搅拌和超声处理后，将混合物转移到1.5mL Eppendorf管中，并且在13000rpm离心。收集上清液，并且通过Millex GV 0.22um注射滤器过滤。将这种溶液通过5mL注射环以5mL/分钟的流速上样到Zorbax 300SBC18柱(21.2mm ID×250mm，7um颗粒大小)上。通过运行0.2％B/分钟的线性AB梯度完成纯化，其中溶剂A是在水中的0.1％TFA，和溶剂B是在乙腈中的0.1％TFA。在这些条件下，肽在15-17％B的范围洗脱下来。A 150 mg amount of crude peptide was taken up in 15 mL of water containing 0.1% TFA and 3 mL of acetic acid. After stirring and sonication, the mixture was transferred to a 1.5 mL Eppendorf tube and centrifuged at 13000 rpm. The supernatant was collected and filtered through a Millex GV 0.22um syringe filter. This solution was loaded onto a Zorbax 300SBC18 column (21.2mm ID x 250mm, 7um particle size) through a 5mL injection loop at a flow rate of 5mL/min. Purification was accomplished by running a linear AB gradient of 0.2% B/min, where solvent A was 0.1% TFA in water, and solvent B was 0.1% TFA in acetonitrile. Under these conditions, peptides eluted in the range of 15-17%B.

实施例35Example 35

细胞cell

EpiAirway^TM细胞(以96孔形式(Air-196-HTS)或单独的24孔插入物(Air-100))，一种人气管/支气管组织模型，购自马特泰克(MatTek)公司(阿什兰，MA)，以筛选紧密连接调控肽(TJMPs)，筛选基于它们对跨上皮电阻(TER)和渗透性的作用而进行。培养组织来源于单一供体，并且对于HIV、乙型肝炎、丙型肝炎、支原体、细菌、酵母菌和真菌筛选是为阴性。^EpiAirwayTM cells (in 96-well format (Air-196-HTS) or individual 24-well inserts (Air-100)), a human trachea/bronchial tissue model, were purchased from MatTek (Ash Lan, MA) to screen for tight junction modulating peptides (TJMPs) based on their effects on transepithelial electrical resistance (TER) and permeability. Cultured tissue was derived from a single donor and screened negative for HIV, hepatitis B, hepatitis C, mycoplasma, bacteria, yeast, and fungi.

EpiAirway组织在补充琼脂糖凝胶的培养基上冷冻运输。EpiAirway组织用生产商提供的培养基在37℃复苏24小时。用于EpiAirway模型的完全培养基(Epi-CM)含有DMEM、EFG和其它因子、庆大霉素(5μg/ml)、两性霉素B(0.25μg/ml)以及作为pH指示剂的酚红。EpiAirway tissues are shipped frozen on media supplemented with agarose gel. EpiAirway tissues were recovered for 24 hours at 37°C in the medium provided by the manufacturer. Complete medium (Epi-CM) for the EpiAirway model contained DMEM, EFG and other factors, gentamicin (5 μg/ml), amphotericin B (0.25 μg/ml) and phenol red as a pH indicator.

实施例36Example 36

确定TERDetermine TER

使用自动化的组织电阻系统(REMS)(世界精密仪器(World PrecessionInstrument)(WPI)有限公司，(萨拉索塔，佛罗里达州)对Air-196-HTS进行TER检测。为了监测在96孔HTS形式中的TER，在组织培养箱(hood)中使用Endhom-Multi(STX)，以防止污染。当过夜复苏插入物时，将100μl培养基用于顶面，250μl培养基用于基底室。背景TER用空白插入物(密斯博(Millipore))检测，并且从组织插入物中减去。通过将所述插入物倒转在纸巾上而将培养基倾出。然后，将插入物在纸巾上轻轻敲打，以确保最大限度去除顶部培养基。对于其它的TER检测时间点，在处理后立即将插入物用150μl Epi-CM轻轻润洗三次，并且在TER检测之前完全排尽。Air-196-HTS was tested for TER using an automated Tissue Resistance System (REMS) (World Precession Instruments (WPI) Inc., (Sarasota, FL). For monitoring in a 96-well HTS format For TER, use Endhom-Multi (STX) in a tissue culture incubator (hood) to prevent contamination. When resuscitating inserts overnight, use 100 μl medium for the top surface and 250 μl medium for the basal chamber. Background TER is used Blank inserts (Millipore) were detected and subtracted from tissue inserts. Media was decanted by inverting the inserts on paper towels. Inserts were then tapped gently on paper towels, To ensure maximum media removal on top. For other TER assay time points, inserts were gently rinsed three times with 150 μl Epi-CM immediately after treatment and drained completely prior to TER assay.

结果(图8)表明，在单层上皮细胞上检测的本发明的紧密连接调控肽PN159和PN159的PEG化形式都具有强的、可逆的增强上皮渗透性的作用。使用二者观察到的作用以可预测的方式发生。此外，结果表明，PEG-159比单独的PN159显著地增强离子渗透性(减小TER)。PEG-PN159和159之间在TER中的最大差别在50μM PEG-PN159。The results ( FIG. 8 ) indicated that both the tight junction modulating peptide PN159 of the present invention and the PEGylated form of PN159 tested on monolayer epithelial cells had a strong and reversible effect of enhancing epithelial permeability. The effects observed with both occurred in a predictable manner. Furthermore, the results indicated that PEG-159 significantly enhanced ion permeability (reduced TER) over PN159 alone. The largest difference in TER between PEG-PN159 and 159 was at 50 μM PEG-PN159.

实施例37Example 37

渗透性检测Penetration testing

将荧光素异硫氰酸酯(FITC)标记的糊精(MW 3,000)以0.1-1mg/ml添加到处理混合物中。将处理混合物加到顶壁的侧面，并且将平板在轨道振荡器(新布伦斯维克科学，埃迪逊，新泽西州(New Brunswick Scientific，Edison，NJ))中以100rpm在37℃温育指定的时间。在温育末，将一式三份200μl的基底培养基转移到黑壁荧光读取平板上。通过微量培养板荧光读取仪FLx800(博奥泰克(BIO-TEK)仪器有限公司，Winooski，VT)检测在470nm波长的荧光强度。标准物的连续稀释用来获得标准曲线和计算浓度。渗透性以两种方式检测，作为供体质量(顶部室)的比例或者作为受体质量(基底室)的比例，以百分数表示。Fluorescein isothiocyanate (FITC)-labeled dextrin (MW 3,000) was added to the treatment mixture at 0.1-1 mg/ml. The treatment mixture was added to the side of the top wall and the plate was incubated in an orbital shaker (New Brunswick Scientific, Edison, NJ) at 100 rpm at 37°C for the indicated time. At the end of the incubation,triplicate 200 μl of basal medium were transferred to black-walled fluorescence reading plates. Fluorescence intensity at a wavelength of 470 nm was detected by a microplate fluorescence reader FLx800 (BIO-TEK Instruments Co., Ltd., Winooski, VT). Serial dilutions of standards were used to obtain standard curves and calculate concentrations. Permeability was measured in two ways, expressed as a percentage of the donor mass (apical compartment) or as a fraction of the acceptor mass (basal compartment).

在存在本发明的PN159和PEG-PN159时，观察到PTH渗透的显著增加(图9)。使用二者观察到的作用在10μM和100μM之间有些浓度依赖性。此外，结果表明PEG-PN159比PN159显著增强分子渗透性。A significant increase in PTH penetration was observed in the presence of PN159 and PEG-PN159 of the present invention (Figure 9). The effects observed with both were somewhat concentration dependent between 10 μM and 100 μM. In addition, the results showed that PEG-PN159 significantly enhanced molecular permeability than PN159.

当PEG-PN159的渗透性增加与PN159比较时(在图10中作为两个值之间的比例绘图)，渗透增加的最大差别是在50μM浓度。When the permeability increase of PEG-PN159 was compared to PN159 (plotted in Figure 10 as a ratio between the two values), the greatest difference in permeability increase was at the 50 μΜ concentration.

实施例38Example 38

细胞毒性检测Cytotoxicity Assay

LDH检测用来评估所述处理的细胞毒性。LDH水平通过CytoTox96无放射性细胞毒性检测(普洛麦格(Promega)，麦迪逊，威斯康星州)按照生产商的方法确定。对于基底侧LDH水平，将一式三份50μl的基底培养基用来确定LDH水平。对于顶部LDH水平，通过向顶室中加入150μl Epi-CM，而去除150μl的稀释的顶部样品，将培养基通过上下吹吸而混合，并且去除150μl培养基并稀释2倍(最终8倍稀释)，用于以50μl的一式三份检测。总LDH水平通过在终浓度0.9％的Triton-X100中裂解细胞而确定。每种样品中的LDH表示为Triton-X100细胞裂解的百分数。结果(图11)表明，PEG-PN159具有比PN159更低的细胞毒性。LDH assay was used to assess the cytotoxicity of the treatments. LDH levels were determined by CytoTox96 radioactive cytotoxicity assay (Promega, Madison, WI) according to the manufacturer's protocol. For basolateral LDH levels,triplicate 50 μl of basal medium was used to determine LDH levels. For top LDH levels, 150 μl of diluted top sample was removed by adding 150 μl of Epi-CM to the top chamber, medium was mixed by pipetting up and down, and 150 μl of medium was removed and diluted 2-fold (final 8-fold dilution) , for triplicate assays in 50 μl. Total LDH levels were determined by lysing cells in a final concentration of 0.9% Triton-X100. LDH in each sample is expressed as a percentage of Triton-X100 cell lysis. The results ( FIG. 11 ) indicated that PEG-PN159 had lower cytotoxicity than PN159.

实施例39Example 39

兔中的药物代谢动力学数据Pharmacokinetic data in rabbits

将25只雄性新西兰白兔，大约3月龄，用于本研究。兔接受单一的鼻内施用，一个鼻孔一剂量的紧密连接(TJ)肽和PYY3-36组，这使用自动移液器和一次性塑料吸头进行。兔按照在表34中显示的TH肽和对照组给药。TJ肽(PN407，PN408，PN526(PEG-PN159)，和PN159)都在具有钙和镁的0.75x DPBS中。阴性对照是只含有钙和镁的0.75x DPBS(PBS)。不含在柠檬酸盐缓冲液中包含TJ肽的DDPC、EDTA和MbCD的阳性PYY3-36对照制剂用于比较(PDF)。Twenty-five male New Zealand White rabbits, approximately 3 months old, were used in this study. Rabbits received a single intranasal administration, one nostril dose of tight junction (TJ) peptide and PYY3-36 groups, using an automatic pipette and disposable plastic tips. Rabbits were dosed according to the TH peptides shown in Table 34 and the control group. TJ peptides (PN407, PN408, PN526 (PEG-PN159), and PN159) were all in 0.75x DPBS with calcium and magnesium. The negative control is 0.75x DPBS (PBS) containing only calcium and magnesium. Positive PYY3-36 control formulations without DDPC, EDTA and MbCD containing TJ peptide in citrate buffer were used for comparison (PDF).

当递送所述剂量时，将动物的头部稍微向后翘起。给药后，控制动物头部在向后翘起的位置约15秒。通过直接在耳缘静脉进行静脉穿刺而收集连续的血液样品(每份约1.5mL)，收集到含有EDTA作为抗凝血剂的血液收集管中。在鼻内组给药后0(给药前)，5，10，15，30，45，60，120和240分钟收集血液样品。收集之后，管子轻轻倒转几次，以抗凝血，然后向收集管中加入50μl牛胰蛋白酶抑制剂，并且轻轻但是彻底混合。在大约4℃以约1,600xg离心15分钟之前，将混合的样品放置在冷冻包装上。将血浆样品分成一式两份等分试样(每份约0.35mL)，并且然后保存在大约-70℃。The animal's head was tilted slightly back when the dose was delivered. After dosing, the animal's head was held in the tilted-back position for about 15 seconds. Serial blood samples (approximately 1.5 mL each) were collected by venipuncture directly in the marginal ear vein into blood collection tubes containing EDTA as an anticoagulant. Blood samples were collected at 0 (pre-dose), 5, 10, 15, 30, 45, 60, 120 and 240 minutes after intranasal group dosing. After collection, the tubes were gently inverted several times to anticoagulate, then 50 [mu]l bovine trypsin inhibitor was added to the collection tube and mixed gently but thoroughly. The pooled samples were placed on freezer packs prior to centrifugation at approximately 1,600 xg for 15 minutes at approximately 4°C. Plasma samples were divided into duplicate aliquots (approximately 0.35 mL each) and then stored at approximately -70°C.

表34Table 34

用于兔药物代谢动力学研究的给药组Dosing groups for rabbit pharmacokinetic studies

使用商购ELISA试剂盒(″活性总肽YY(PYY)ELISA″，目录号.DSL-10-33600，诊断系统实验有限公司(Diagnostic Systems Laboratories，Inc.)，韦伯斯特(Webster)，德克萨斯州)进行在兔血浆中的PYY3-36生物分析检测。所述检测是一种酶促扩增的“一步”夹心型免疫检测。在所述检测中，将校正物、对照和未知样品在已经用另一种抗-PYY抗体包被的微量滴定孔中用抗-PYY抗体温育。温育和洗涤后，孔用发光底物，四甲基联苯胺温育。然后加入酸性终止溶液，并且通过在450和620nm的双波长吸光度检测而确定底物的酶促转换程度。测定的吸光度与存在的PYY的浓度成比例。A commercially available ELISA kit ("Active Total Peptide YY (PYY) ELISA", Cat. No. DSL-10-33600, Diagnostic Systems Laboratories, Inc., Webster, Decker Sax) for bioanalytical detection of PYY3-36 in rabbit plasma. The assay is an enzymatically amplified "one-step" sandwich immunoassay. In the assay, calibrators, controls, and unknown samples are incubated with an anti-PYY antibody in microtiter wells that have been coated with another anti-PYY antibody. After incubation and washing, the wells are incubated with the luminescent substrate, tetramethylbenzidine. An acidic stop solution was then added, and the extent of enzymatic turnover of the substrate was determined by dual wavelength absorbance detection at 450 and 620 nm. The measured absorbance is proportional to the concentration of PYY present.

5参数的逻辑数据简化方法(five-parameter logistic data reductionmethod)用于校正结果，以产生关于每种检测的校正曲线。校正曲线用于根据它们的吸光度结果而内插入未知样品的PYY浓度值。试剂盒成分用于所有的检测步骤，除了下述例外以外：PYY_3-36参照物质用于产生校正物和对照；校正物和对照用洗脱(strip)(C18固相萃取柱)合并的兔血浆作为稀释剂而制备；并且如果需要，将未知样品稀释在洗脱合并的兔血浆中。将在这一试剂盒中的抗体组合优化，以检测完整的人PYY_1-36，并且与小鼠PYY_1-36和人PYY_3-36充分交叉反应。A five-parameter logistic data reduction method was used to calibrate the results to generate calibration curves for each assay. The calibration curve was used to interpolate PYY concentration values for unknown samples from their absorbance results. Kit components were used for all assay steps, with the following exceptions: PYY_3-36 reference material was used to generate calibrators and controls; calibrators and controls were pooled with a strip (C18 SPE column) Plasma was prepared as diluent; and if necessary, unknown samples were diluted in eluted pooled rabbit plasma. The combination of antibodies in this kit was optimized to detect intact human PYY_1-36 and to substantially cross-react with mouse PYY_1-36 and human PYY_3-36 .

关于对照(PBS和PDF)和TJ肽(PN159，PN407，PN408，和PN526)制剂的平均药物代谢动力学(PK)数据和标准偏差(SD)显示在表35中。关于每种紧密连接调控剂和对照的相对生物利用度(％BA)显示在表36中。关于药物代谢动力学变量的变化系数百分数显示在表37中。Mean pharmacokinetic (PK) data and standard deviation (SD) for control (PBS and PDF) and TJ peptide (PN159, PN407, PN408, and PN526) formulations are shown in Table 35. The relative bioavailability (% BA) for each tight junction modulator and control is shown in Table 36. The percentage coefficients of variation for the pharmacokinetic variables are shown in Table 37.

表35Table 35

关于在兔中的PYY_3-36的平均PK参数和标准偏差(SD)Mean PK parameters and standard deviation (SD) for PYY_3-36 in rabbits

制剂preparationT_max(分钟)T_max (minutes)  C_max(pg/mL)C_max (pg/mL)  AUC_last(分钟*pg/mL)AUC_last (min*pg/mL)  AUC_inf(分钟*pg/mL)AUC_inf (min*pg/mL)  t1/2(分钟)t1/2(minute)  Kel (1/分钟)Kel (1/minute)  PBSPBS  33.7533.75  2646.252646.25  118438.13118438.13  147625.18147625.18  83.1283.12  0.0090.009  SDSD  18.8718.87  1381.061381.06  23611.8623611.86  42331.6842331.68  22.5322.53  0.0030.003  PDFPDF  30.0030.00  19004.4019004.40  1289219.501289219.50  1319034.731319034.73  38.5638.56  0.0190.019  SDSD  10.6110.61  8174.328174.32  589127.80589127.80  612688.59612688.59  11.1211.12  0.0050.005  PN159PN159  27.0027.00  18346.6018346.60  973038.80973038.80  985572.89985572.89  34.4334.43  0.0210.021  SDSD  19.5619.56  9671.729671.72  549668.76549668.76  546060.77546060.77  7.207.20  0.0050.005  PN407PN407  21.0021.00  13980.2013980.20  725950.50725950.50  753080.86753080.86  47.4647.46  0.0160.016  SDSD  8.228.22  7124.997124.99  388368.38388368.38  397975.49397975.49  14.5114.51  0.0040.004  PN408PN408  15.0015.00  15420.0015420.00  721601.50721601.50  758951.24758951.24  44.2344.23  0.0160.016  SDSD  0.000.00  7644.407644.40  361013.89361013.89  360247.20360247.20  8.238.23  0.0030.003  PN526PN526  27.0027.00  36066.2036066.20  1786973.501786973.50  1819888.301819888.30  41.0441.04  0.0180.018  SDSD  6.716.71  22447.1322447.13  1065867.601065867.60  1084222.741084222.74  9.669.66  0.0050.005

表36Table 36

紧密连接调控剂的％生物利用度% Bioavailability of Tight Junction Modulators

制剂preparation  AUC_last(分钟*pg/mL)AUC_last (min*pg/mL)％F%F  PBSPBS  118438.13118438.13  9.199.19  PDFPDF  1289219.501289219.50  PN159PN159  973038.80973038.80  75.4875.48  PN407PN407  725950.50725950.50  56.3156.31  PN408PN408  721601.50721601.50  55.9755.97  PN526PN526  1786973.501786973.50  138.61138.61

表37Table 37

关于药物代谢动力学参数的变化％系数% Coefficients of Variation for Pharmacokinetic Parameters

制剂preparation  T_max(分钟)T_max (minutes)  C_max(pg/mL)C_max (pg/mL)  AUC_last(分钟*pg/mL)AUC_last (min*pg/mL)  AUC_inf(分钟*pg/mL)AUC_inf (min*pg/mL)  PBSPBS  55.955.9  52.252.2  19.919.9  28.728.7  PDFPDF  35.435.4  43.043.0  45.745.7  46.446.4  PN159PN159  72.472.4  52.752.7  56.556.5  55.455.4  PN407PN407  39.139.1  51.051.0  53.553.5  52.852.8  PN408PN408  0.00.0  49.649.6  50.050.0  47.547.5  PN526PN526  24.824.8  62.262.2  59.659.6  59.659.6

量化的下限(LLOQ)认为是15.8pg/mL。将任何＜NUMBER的原始数据值设为7.9pg/mL用来分析。鼻施用后平均PYY_3-36血浆浓度在图12中以线性图显示，在图13中以对数-线性图显示。施用鼻剂量的动物的平均PYY_3-36血清浓度表示所有组在给药后15-34分钟之间的峰值浓度(T_max)。对于以205μg/kg的剂量水平的鼻PBS；PDF；PN159；PN407；PN408和PN526的平均C_max分别为2,646.25；19,004.40；18,346.60；13,980.20；15,420.00和36,066.20pg/mL。对于鼻PBS；PDF；PN159；PN407；PN408和PN526的平均AUC_last分别为118,438.13；1,289,219.50；973,038.80；725,950.50；721,601.50和1,786,973.50分钟*pg/mL。对于鼻PBS；PDF；PN159；PN407；PN408和PN526的平均AUC_inf分别为147,625.18；1,319,034.73；985,572.89；753,080.86；758,951.24和1,819,888.30分钟*pg/mL。对于所有鼻制剂t1/2为约35-48分钟；然而，PBS为83分钟。参见表35关于包括标准偏差的所有药物代谢动力学参数的完整列表。对于PN159，PN407，PN408和PN526，基于关于紧密连接调控剂相对PDF制剂的AUC_last的％BA分别为75，56，56和139％。与PDF相比，PBS的％生物利用度只为9％。还比较了变异系数(表37)。当在制剂之间比较药物代谢动力学参数C_max，和AUC时，所有的紧密连接调控剂具有相似的变异。使用一种方式方差分析模型分析所有5种制剂组之间的药物代谢动力学变化，并且发现关于C_max，AUC_last和AUC_infPBS制剂显著低于PN526。(T_max：p＝0.27；C_max：p＝0.009；AUC_last：p＝0.008；AUC_inf：p＝0.0097)。The lower limit of quantification (LLOQ) was considered to be 15.8 pg/mL. Any raw data value < NUMBER was set at 7.9 pg/mL for analysis. Mean PYY_3-36 plasma concentrations after nasal administration are shown as a linear graph in FIG. 12 and as a log-linear graph in FIG. 13 . Mean PYY_3-36 serum concentrations in animals administered nasal doses represent peak concentrations (T_max ) for all groups between 15-34 minutes post-dose. The mean_Cmax for nasal PBS; PDF; PN159; PN407; PN408 and PN526 at a dose level of 205 μg/kg were 2,646.25; 19,004.40; 18,346.60; 13,980.20; The average AUC_last for nasal PBS; PDF; PN159; PN407; PN408 and PN526 were 118,438.13; 1,289,219.50; 973,038.80; 725,950.50; The mean AUC_inf for nasal PBS; PDF; PN159; PN407; PN408 and PN526 were 147,625.18; 1,319,034.73; 985,572.89; 753,080.86; 758,951.24 and 1,819,888.30 min*pg/mL, respectively. t1/2 was approximately 35-48 minutes for all nasal formulations; however, 83 minutes for PBS. See Table 35 for a complete list of all pharmacokinetic parameters including standard deviation. The %BA based on AUC_last for tight junction modulators versus PDF formulations was 75, 56, 56 and 139% for PN159, PN407, PN408 and PN526, respectively. Compared to PDF, the % bioavailability of PBS was only 9%. Coefficients of variation were also compared (Table 37). When comparing the pharmacokinetic parameters C_max , and AUC between the formulations, all tight junction modulators had similar variability. Pharmacokinetic variation between all 5 formulation groups was analyzed using a one way ANOVA model, and the PBS formulation was found to be significantly lower with respect to_Cmax ,_AUClast and_AUCinf than PN526. (T_max : p=0.27; C_max : p=0.009; AUC_last : p=0.008; AUC_inf : p=0.0097).

比较C_max，PEG化的紧密连接调控剂PN526高于PDF 1.9倍，并且分别高于PBS、PN407和PN40813.6、2.6和2.3倍。比较AUC_last，PEG化的紧密连接调控剂PN526高于PDF 1.4倍，并且分别高于PBS、PN407和PN40815.1、2.5和2.5倍。除了PBS在80分钟以外，对于所有组t1/2约40分钟。Comparing C_max , the PEGylated tight junction modulator PN526 was 1.9-fold higher than PDF, and 3.6, 2.6 and 2.3-fold higher than PBS, PN407 and PN408, respectively. Compared with AUC_last , the PEGylated tight junction regulator PN526 was 1.4 times higher than PDF, and 5.1, 2.5 and 2.5 times higher than PBS, PN407 and PN408, respectively. t1/2 was approximately 40 minutes for all groups except PBS at 80 minutes.

当比较药物代谢动力学参数，C_max和AUC时，在PN526和PBS制剂之间存在显著的差异；然而，在紧密连接调控剂之间不存在显著性。When comparing pharmacokinetic parameters,_Cmax and AUC, there were significant differences between PN526 and PBS formulations; however, no significance existed between tight junction modulators.

与所有其它紧密连接调控剂相比，使用PN526生物利用度增加，并且与PBS对照制剂相比，药物代谢动力学参数是统计学显著性的。这些数据表明，PEG化的肽制剂，PN526，具有增加的％BA，其高于没有PEG化的肽的制剂，PN159，PN407，PN408，和PBS。此外，PN526的％BA也高于没有PEG化的肽的阳性对照PDF。Bioavailability was increased with PN526 compared to all other tight junction modulators and pharmacokinetic parameters were statistically significant compared to the PBS control formulation. These data indicate that the PEGylated peptide formulation, PN526, had an increased % BA that was higher than the formulations without PEGylated peptide, PN159, PN407, PN408, and PBS. In addition, the %BA of PN526 was also higher than the positive control PDF without PEGylated peptide.

本文给出的实施例只是用于举例说明的目的，并且不意欲限制本发明如权利要求所述的范围。尽管在本文中应用具体的术语和值，但是所述术语和值应该理解为是代表性的，并且不限制本发明的范围。The examples given herein are for illustrative purposes only and are not intended to limit the scope of the invention as claimed. Although specific terms and values are employed herein, the terms and values should be understood to be representative and not to limit the scope of the invention.

出于所有目的，在本公开内容中引用的所有出版物和参考文献通过引用完全结合于此。All publications and references cited in this disclosure are hereby fully incorporated by reference for all purposes.

权利要求书(按照条约第19条的修改)Claims (as amended under Article 19 of the Treaty)

1. 一种含有肽的化合物或其药用盐，其具有在哺乳动物黏膜中通过调控所述黏膜的渗透性而增强活性剂的黏膜上皮转运的活性，其中所述肽具有小于10千道尔顿的分子量，并且其中所述肽包含延长一个或多个氨基酸残基的SEQ ID NO：34的氨基酸序列。1. A peptide-containing compound or a pharmaceutically acceptable salt thereof, which has the activity of enhancing the mucosal epithelial transport of active agents in mammalian mucosa by regulating the permeability of said mucosa, wherein said peptide has an activity of less than 10 kdal The molecular weight of the peptide, and wherein said peptide comprises the amino acid sequence of SEQ ID NO: 34 extended by one or more amino acid residues.

2. 权利要求1的化合物，其中所述肽包括选自由SEQ ID NOS：41-43组成的组的氨基酸序列。2. The compound ofclaim 1, wherein said peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOS: 41-43.

3. 一种含有肽的化合物或其药用盐，其具有在哺乳动物黏膜中通过调控所述黏膜的渗透性而增强活性剂的黏膜上皮转运的活性，其中所述肽具有小于10千道尔顿的分子量，并且包含SEQ ID NO：35的氨基酸序列。3. A peptide-containing compound or a pharmaceutically acceptable salt thereof, which has the activity of enhancing the mucosal epithelial transport of active agents in mammalian mucosa by regulating the permeability of said mucosa, wherein said peptide has an activity of less than 10 kdal The molecular weight of Dun, and comprise the aminoacid sequence of SEQ ID NO:35.

4. 权利要求3的化合物，其中所述肽选自由SEQ ID NO：35组成的组。4. The compound ofclaim 3, wherein said peptide is selected from the group consisting of SEQ ID NO:35.

5. 一种含有肽的化合物或其药用盐，其具有在哺乳动物黏膜中通过调控所述黏膜的渗透性而增强活性剂的黏膜上皮转运的活性，其中所述肽具有小于10千道尔顿的分子量，并且包含SEQ ID NO：38的氨基酸序列。5. A peptide-containing compound or a pharmaceutically acceptable salt thereof, which has the activity of enhancing the mucosal epithelial transport of an active agent in mammalian mucosa by regulating the permeability of said mucosa, wherein said peptide has an activity of less than 10 kdal The molecular weight of Dun, and comprise the aminoacid sequence of SEQ ID NO:38.

6. 权利要求5的化合物，其中所述肽选自由SEQ ID NO：38组成的组。6. The compound ofclaim 5, wherein said peptide is selected from the group consisting of SEQ ID NO:38.

7. 一种含有肽的化合物或其药用盐，其具有在哺乳动物黏膜中通过调控所述黏膜的渗透性而增强活性剂的黏膜上皮转运的活性，其中所述肽具有小于10千道尔顿的分子量，并且包含富含至少60％的赖氨酸、亮氨酸和/或丙氨酸的SEQ ID NO：34的氨基酸序列。7. A peptide-containing compound or a pharmaceutically acceptable salt thereof, which has the activity of enhancing the mucosal epithelial transport of active agents in mammalian mucosa by regulating the permeability of said mucosa, wherein said peptide has an activity of less than 10 kdal and comprising an amino acid sequence of SEQ ID NO: 34 enriched in at least 60% lysine, leucine and/or alanine.

8. 权利要求7的化合物，其中所述肽包括选自由SEQ ID NOS：32，33，36和50组成的组的氨基酸序列。8. The compound of claim 7, wherein said peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOS: 32, 33, 36 and 50.

9. 权利要求1-8中任一项的化合物，其中在黏膜中所述渗透性增强，而保持细胞存活能力。9. The compound according to any one of claims 1-8, wherein in the mucosa said permeability is enhanced while maintaining cell viability.

10. 权利要求1-8中任一项的化合物，其中所述化合物与水溶性链共价连接。10. The compound of any one of claims 1-8, wherein the compound is covalently linked to a water soluble chain.

11. 权利要求10的化合物，其中所述化合物是聚(环氧烷)链。11. The compound ofclaim 10, wherein said compound is a poly(alkylene oxide) chain.

12. 权利要求11的化合物，其中所述聚(环氧烷)链是有支链的或无支链的。12. The compound of claim 11, wherein the poly(alkylene oxide) chain is branched or unbranched.

13. 权利要求12的化合物，其中所述聚(环氧烷)链是聚乙二醇(PEG)链。13. The compound of claim 12, wherein the poly(alkylene oxide) chain is a polyethylene glycol (PEG) chain.

14. 权利要求13的化合物，其中所述PEG具有约0.2和约200千道尔顿(kDa)之间的分子大小。14. The compound of claim 13, wherein the PEG has a molecular size between about 0.2 and about 200 kilodaltons (kDa).

15. 权利要求13的化合物，其中所述PEG具有低于40kDa的大小，优选地其中所述PEG具有低于20kDa的大小，更优选地其中所述PEG具有低于10kDa的大小。15. The compound of claim 13, wherein said PEG has a size below 40 kDa, preferably wherein said PEG has a size below 20 kDa, more preferably wherein said PEG has a size below 10 kDa.

16. 权利要求13的化合物，其中所述PEG具有低于5kDa的大小，优选地其中所述PEG具有低于2kDa的大小。16. The compound of claim 13, wherein said PEG has a size below 5kDa, preferably wherein said PEG has a size below 2kDa.

17. 权利要求13的化合物，其中所述聚(环氧烷)具有小于2.00的多分散性值(Mw/Mn)。17. The compound of claim 13, wherein the poly(alkylene oxide) has a polydispersity value (Mw/Mn) of less than 2.00.

18. 权利要求13的化合物，其中所述聚(环氧烷)具有小于1.20的多分散性值(Mw/Mn).18. The compound of claim 13, wherein the poly(alkylene oxide) has a polydispersity value (Mw/Mn) of less than 1.20.

19. 一种药物制剂，其包含增强黏膜上皮转运有效量的权利要求1-18中任一项的化合物或包含所述SEQ ID NO：34的氨基酸序列的化合物，和治疗有效量的活性剂。19. A pharmaceutical preparation comprising the compound according to any one of claims 1-18 or a compound comprising the amino acid sequence of SEQ ID NO: 34 in an effective dose for enhancing mucosal epithelial transport, and an active agent in a therapeutically effective dose.

20. 权利要求19的制剂，其中所述制剂减小穿过黏膜组织屏障的电阻。20. The formulation of claim 19, wherein the formulation reduces the resistance across the mucosal tissue barrier.

21. 权利要求20的制剂，其中所述电阻的减少为至少80％。21. The formulation ofclaim 20, wherein the reduction in electrical resistance is at least 80%.

22. 权利要求21的制剂，其中所述制剂相对于不包含权利要求1-33中任一项的化合物或者含有SEQ ID NO：34的氨基酸序列的化合物的类似的制剂增加所述活性剂穿过黏膜组织屏障的渗透性。22. The formulation of claim 21, wherein said formulation increases the passage of said active agent relative to a similar formulation not comprising a compound of any one of claims 1-33 or a compound comprising the amino acid sequence of SEQ ID NO:34 Permeability of the mucosal tissue barrier.

23. 权利要求22的制剂，其中渗透性的增加为至少两倍。23. The formulation of claim 22, wherein the increase in permeability is at least two-fold.

24. 权利要求22的制剂，其中所述渗透性是旁细胞的。24. The formulation of claim 22, wherein the permeability is paracellular.

25. 权利要求22的制剂，其中所述增加的渗透性由调控紧密连接导致。25. The formulation of claim 22, wherein said increased permeability results from modulation of tight junctions.

26. 权利要求22的制剂，其中所述渗透性是跨细胞的或者是跨细胞和旁细胞的混合。26. The formulation of claim 22, wherein the permeability is transcellular or a mixture of transcellular and paracellular.

27. 权利要求22的制剂，其中所述黏膜组织屏障是上皮细胞层。27. The formulation of claim 22, wherein the mucosal tissue barrier is an epithelial cell layer.

28. 权利要求27的制剂，其中所述上皮细胞选自由气管细胞，支气管细胞，肺泡细胞，鼻细胞，肺细胞，胃肠细胞，表皮细胞和口腔细胞组成的组。28. The formulation of claim 27, wherein the epithelial cells are selected from the group consisting of tracheal cells, bronchial cells, alveolar cells, nasal cells, lung cells, gastrointestinal cells, epidermal cells, and oral cavity cells.

29. 权利要求28的制剂，其中所述上皮细胞是鼻细胞。29. The formulation of claim 28, wherein the epithelial cells are nasal cells.

30. 权利要求19的制剂，其中所述活性剂是肽、蛋白或核酸。30. The formulation of claim 19, wherein the active agent is a peptide, protein or nucleic acid.

31. 权利要求30的制剂，其中所述肽或蛋白包括2-1000个氨基酸。31. The formulation ofclaim 30, wherein the peptide or protein comprises 2-1000 amino acids.

32. 权利要求30的制剂，其中所述肽或蛋白包括2-50个氨基酸。32. The formulation ofclaim 30, wherein the peptide or protein comprises 2-50 amino acids.

33. 权利要求30的制剂，其中所述肽或蛋白是环形的。33. The formulation ofclaim 30, wherein the peptide or protein is cyclic.

34. 权利要求30的制剂，其中所述肽或蛋白是二聚体或寡聚体。34. The formulation ofclaim 30, wherein the peptide or protein is a dimer or oligomer.

35. 权利要求30的制剂，其中所述肽或蛋白选自由GLP-1、PYY3-36、PTH1-34和胰高血糖素样肽的抑制剂-4组成的组。35. The formulation ofclaim 30, wherein the peptide or protein is selected from the group consisting of GLP-1, PYY3-36, PTH1-34, and inhibitor of glucagon-like peptide-4.

36. 权利要求30的制剂，其中所述蛋白选自由β-干扰素、α-干扰素、胰岛素、促红细胞生成素、G-CSF、GM-CSF、生长激素、以及它们的类似物组成的组。36. The formulation ofclaim 30, wherein the protein is selected from the group consisting of beta-interferon, alpha-interferon, insulin, erythropoietin, G-CSF, GM-CSF, growth hormone, and their analogs .

37. 包含权利要求19-36中任一项的制剂的剂型，其中所述剂型是液体。37. A dosage form comprising the formulation of any one of claims 19-36, wherein said dosage form is a liquid.

38. 权利要求37的剂型，其中所述液体是小滴形式。38. The dosage form of claim 37, wherein the liquid is in the form of droplets.

39. 权利要求37的剂型，其中所述液体是气溶胶形式。39. The dosage form of claim 37, wherein the liquid is in aerosol form.

40. 包含权利要求19-36中任一项的制剂的剂型，其中所述剂型是固体。40. A dosage form comprising the formulation of any one of claims 19-36, wherein said dosage form is a solid.

41. 权利要求40的剂型，其中所述固体在施用之前在液体中重构。41. The dosage form ofclaim 40, wherein the solid is reconstituted in a liquid prior to administration.

42. 权利要求40的剂型，其中所述固体作为散剂施用。42. The dosage form ofclaim 40, wherein the solid is administered as a powder.

43. 权利要求40的剂型，其中所述固体是胶囊、片剂或凝胶形式。43. The dosage form ofclaim 40, wherein the solid is in the form of a capsule, tablet or gel.

44. 一种给动物施用分子的方法，其包括提供如权利要求19-36中任一项的制剂，并且将所述制剂与所述动物的黏膜表面接触。44. A method of administering a molecule to an animal comprising providing the formulation of any one of claims 19-36, and contacting the formulation with a mucosal surface of the animal.

45. 权利要求44的方法，其中所述黏膜表面是鼻内。45. The method of claim 44, wherein the mucosal surface is intranasal.

46. 一种在哺乳动物中增加鼻内施用的活性剂的生物利用度的方法，其包括提供如权利要求19-36中任一项的制剂，并且将所述制剂施用给所述哺乳动物。46. A method for increasing the bioavailability of an intranasally administered active agent in a mammal, comprising providing a formulation according to any one of claims 19-36, and administering said formulation to said mammal.

47. 权利要求1-18中任一项的化合物或者权利要求19-36中任一项的制剂或者权利要求37-43中任一项的剂型，其中所述活性剂是siRNA。47. The compound of any one of claims 1-18 or the formulation of any one of claims 19-36 or the dosage form of any one of claims 37-43, wherein the active agent is siRNA.

48. 权利要求1-18中任一项的化合物或者权利要求19-36中任一项的制剂或者权利要求37-43中任一项的剂型，其中所述活性剂是dsDNA。48. The compound of any one of claims 1-18 or the formulation of any one of claims 19-36 or the dosage form of any one of claims 37-43, wherein the active agent is dsDNA.

49. 权利要求1-18中任一项的化合物或者权利要求19-36中任一项的制剂或者权利要求37-43中任一项的剂型，其中所述活性剂是造血药；抗感染药；抗痴呆药；抗病毒剂，抗肿瘤药，退热药，镇痛药，消炎药，抗溃疡药，抗过敏剂，抗抑郁药，治疗精神病的药，强心剂，抗心律失常药，血管舒张剂，抗高血压药，降血压药利尿剂，抗糖尿病药，抗凝血剂，降胆固醇药，用于骨质疏松症的治疗剂，激素，抗生素，或疫苗。49. The compound of any one of claims 1-18 or the formulation of any one of claims 19-36 or the dosage form of any one of claims 37-43, wherein the active agent is a hematopoietic drug; an anti-infective Antidementia Drugs; Antiviral Agents, Antineoplastic Agents, Antipyretics, Analgesics, Antiinflammatory Agents, Antiulcer Agents, Antiallergic Agents, Antidepressants, Psychotropic Agents, Cardiotonic Agents, Antiarrhythmic Agents, Vasodilation antihypertensive drugs, blood pressure lowering drugs diuretics, antidiabetic drugs, anticoagulants, cholesterol lowering drugs, therapeutic agents for osteoporosis, hormones, antibiotics, or vaccines.

50. 权利要求1-18中任一项的化合物或者权利要求19-36中任一项的制剂或者权利要求37-43中任一项的剂型，其中所述活性剂是细胞因子，肽激素，生长因子，心血管因子，细胞粘附因子，中枢或外周神经系统因子，体液电解质因子，血液有机物质，骨生长因子，胃肠系统因子，肾因子，结缔组织因子，感觉器官因子，免疫系统因子，呼吸系统因子，或生殖器官因子。50. The compound of any one of claims 1-18 or the formulation of any one of claims 19-36 or the dosage form of any one of claims 37-43, wherein the active agent is a cytokine, a peptide hormone, Growth factors, cardiovascular factors, cell adhesion factors, central or peripheral nervous system factors, humoral electrolyte factors, blood organic matter, bone growth factors, gastrointestinal system factors, renal factors, connective tissue factors, sensory organ factors, immune system factors , respiratory system factors, or reproductive organ factors.

51. 权利要求1-18中任一项的化合物或者权利要求19-36中任一项的制剂或者权利要求37-43中任一项的剂型，其中所述活性剂是雄激素、雌激素、前列腺素、生长激素、促性腺激素、白介素、类固醇或细胞因子。51. The compound of any one of claims 1-18 or the formulation of any one of claims 19-36 or the dosage form of any one of claims 37-43, wherein the active agent is an androgen, an estrogen, Prostaglandins, growth hormones, gonadotropins, interleukins, steroids, or cytokines.

52. 权利要求1-18中任一项的化合物或者权利要求19-36中任一项的制剂或者权利要求37-43中任一项的剂型，其中所述活性剂是用于肝炎、流感、呼吸道合胞病毒(RSV)、副流感病毒(PIV)、结核病、金丝雀痘、水痘、麻疹、流行性腮腺炎、风疹、肺炎、或人类免疫缺陷性病毒(HIV)的疫苗。52. The compound of any one of claims 1-18 or the formulation of any one of claims 19-36 or the dosage form of any one of claims 37-43, wherein the active agent is for hepatitis, influenza, Vaccines for respiratory syncytial virus (RSV), parainfluenza virus (PIV), tuberculosis, canarypox, chickenpox, measles, mumps, rubella, pneumonia, or human immunodeficiency virus (HIV).

53. 权利要求1-18中任一项的化合物或者权利要求19-36中任一项的制剂或者权利要求37-43中任一项的剂型，其中所述活性剂是白喉、破伤风、假单胞菌(pseudomonas)或结核分枝杆菌(mycobactriumtuberculosis)的细菌类毒素。53. The compound of any one of claims 1-18 or the formulation of any one of claims 19-36 or the dosage form of any one of claims 37-43, wherein the active agent is diphtheria, tetanus, Bacterial toxoids of pseudomonas or mycobacterium tuberculosis.

54. 权利要求1-18中任一项的化合物或者权利要求19-36中任一项的制剂或者权利要求37-43中任一项的剂型，其中所述活性剂是水蛭素，hirulos或蛭素。54. The compound of any one of claims 1-18 or the formulation of any one of claims 19-36 or the dosage form of any one of claims 37-43, wherein the active agent is hirudin, hirulos or leeches white.

55. 权利要求1-18中任一项的化合物或者权利要求19-36中任一项的制剂或者权利要求37-43中任一项的剂型，其中所述活性剂是单克隆抗体、多克隆抗体、人源化抗体、抗体片段、或免疫球蛋白。55. The compound of any one of claims 1-18 or the formulation of any one of claims 19-36 or the dosage form of any one of claims 37-43, wherein the active agent is a monoclonal antibody, polyclonal Antibody, humanized antibody, antibody fragment, or immunoglobulin.

56. 权利要求1-18中任一项的化合物或者权利要求19-36中任一项的制剂或者权利要求37-43中任一项的剂型，其中所述活性剂是吗啡，氢吗啡酮，羟吗啡酮，洛伐啡诺，左洛啡烷，可待因，纳美芬，烯丙吗啡，纳洛酮，纳曲酮，丁丙诺啡，布托啡诺，或纳布啡。56. The compound of any one of claims 1-18 or the formulation of any one of claims 19-36 or the dosage form of any one of claims 37-43, wherein the active agent is morphine, hydromorphone, Oxymorphone, lovaphanol, levolorphanol, codeine, nalmefene, nalamorphine, naloxone, naltrexone, buprenorphine, butorphanol, or nalbuphine.

57. 权利要求1-18中任一项的化合物或者权利要求19-36中任一项的制剂或者权利要求37-43中任一项的剂型，其中所述活性剂是可的松，氢化可的松，氟氢可的松，泼尼松，泼尼松龙，甲泼尼龙，曲安西龙，地塞米松，倍他米松，帕拉米松，或氟轻松。57. The compound of any one of claims 1-18 or the formulation of any one of claims 19-36 or the dosage form of any one of claims 37-43, wherein the active agent is cortisone, hydrocortisone Dexamethasone, fludrocortisone, prednisone, prednisolone, methylprednisolone, triamcinolone, dexamethasone, betamethasone, paramethasone, or fluocinolone.

58. 权利要求1-18中任一项的化合物或者权利要求19-36中任一项的制剂或者权利要求37-43中任一项的剂型，其中所述活性剂是秋水仙碱，对乙酰氨基酚，阿司匹林，布洛芬，酮洛芬，吲哚美辛，萘普生，美洛昔康，或吡罗昔康。58. The compound of any one of claims 1-18 or the formulation of any one of claims 19-36 or the dosage form of any one of claims 37-43, wherein the active agent is colchicine, paraacetyl Aminophenols, aspirin, ibuprofen, ketoprofen, indomethacin, naproxen, meloxicam, or piroxicam.

59. 权利要求1-18中任一项的化合物或者权利要求19-36中任一项的制剂或者权利要求37-43中任一项的剂型，其中所述活性剂是阿昔洛韦，利巴韦林，三氟胸苷，Ara-A(阿糖呋喃糖腺嘌呤)，酰基鸟苷，去甲脱氧鸟嘌呤核苷，叠氮胸苷，二脱氧腺苷，或二脱氧胞苷。59. The compound of any one of claims 1-18 or the formulation of any one of claims 19-36 or the dosage form of any one of claims 37-43, wherein the active agent is acyclovir, Bavirin, trifluorothymidine, Ara-A (arabinofuranosyladenosine), acylguanosine, nordeoxyguanosine, azidothymidine, dideoxyadenosine, or dideoxycytidine.

60. 权利要求1-18中任一项的化合物或者权利要求19-36中任一项的制剂或者权利要求37-43中任一项的剂型，其中所述活性剂是螺内酯，睾酮，雌二醇，黄体酮，促性腺激素，雌激素或孕酮。60. The compound of any one of claims 1-18 or the formulation of any one of claims 19-36 or the dosage form of any one of claims 37-43, wherein the active agent is spironolactone, testosterone, estradiol Alcohol, progesterone, gonadotropins, estrogen, or progesterone.

61. 权利要求1-18中任一项的化合物或者权利要求19-36中任一项的制剂或者权利要求37-43中任一项的剂型，其中所述活性剂是罂粟碱，硝酸甘油，血管活性肠肽，降钙素相关的基因肽，赛庚啶，多塞平，丙米嗪，西咪替丁，右美沙芬，氯氮平片剂，过氧化物歧化酶，神经脑啡肽酶，两性霉素B，灰黄霉素，咪康唑，酮康唑，噻康唑，伊曲康唑，氟康唑，头孢菌素，四环素，氨基糖苷，红霉素，庆大霉素，多粘菌素B，5-氟尿嘧啶，博来霉素，甲氨喋呤，和羟基脲，二脱氧肌苷，氟尿苷，6-巯基嘌呤，多柔比星，柔红霉素，伊达比星，泰素，紫杉醇，维生素E，奎尼丁，哌唑嗪，维拉帕米，硝苯地平，或地尔硫

61. The compound of any one of claims 1-18 or the formulation of any one of claims 19-36 or the dosage form of any one of claims 37-43, wherein the active agent is papaverine, nitroglycerin, Vasoactive intestinal peptide, calcitonin-related gene peptide, cyproheptadine, doxepin, imipramine, cimetidine, dextromethorphan, clozapine tablet, superoxide dismutase, neuroenkephalin Enzymes, amphotericin B, griseofulvin, miconazole, ketoconazole, tioconazole, itraconazole, fluconazole, cephalosporins, tetracyclines, aminoglycosides, erythromycin, gentamicin , polymyxin B, 5-fluorouracil, bleomycin, methotrexate, and hydroxyurea, dideoxyinosine, floxuridine, 6-mercaptopurine, doxorubicin, daunorubicin, i Darubicin, taxol, paclitaxel, vitamin E, quinidine, prazosin, verapamil, nifedipine, or diltiazem

62. 权利要求1-18中任一项的化合物或者权利要求19-36中任一项的制剂或者权利要求37-43中任一项的剂型，其中所述活性剂是组织血纤维蛋白溶酶原活化因子(TPA)，表皮生长因子(EGF)，成纤维细胞生长因子(FGF-酸性或碱性)，血小板衍生生长因子(PDGF)，转化生长因子(TGF-α或β)，血管活性肠肽，肿瘤坏死因子(TNF)，下丘脑释放因子，促乳素，促甲状腺激素(TSH)，促肾上腺皮质激素(ACTH)，甲状旁腺激素(PTH)，促卵泡激素(FSF)，促性腺激素释放激素(LHRH)，内啡肽，胰高血糖素，降钙素，催产素，卡贝缩宫素，aldoetecone，脑啡肽，somatostin，生长激素，生长调节素，α-促黑激素，利多卡因，舒芬太尼，特布他林，氟哌利多，东莨菪碱，戈那瑞林，环吡酮，丁螺环酮，降钙素，色甘酸二钠或咪达唑仑，环孢素，赖诺普利，卡托普利，地拉普利，雷尼替丁，法莫替丁，过氧化物歧化酶，天冬酰胺酶，精氨酸酶，精氨酸脱氨酶，腺苷脱氨酶核糖核酸酶，胰蛋白酶，化学胰蛋白酶，木瓜蛋白酶，铃蟾肽，P物质，血管升压素，α-球蛋白，运铁蛋白，凝血因子I，β-脂蛋白，β-球蛋白，凝血因子II，血浆铜蓝蛋白，α2-糖蛋白，α2-球蛋白，胎球蛋白，α1-脂蛋白，α1-球蛋白，白蛋白，或前白蛋白。62. The compound of any one of claims 1-18 or the formulation of any one of claims 19-36 or the dosage form of any one of claims 37-43, wherein the active agent is tissue plasmin Proactivator activator (TPA), epidermal growth factor (EGF), fibroblast growth factor (FGF-acidic or basic), platelet-derived growth factor (PDGF), transforming growth factor (TGF-alpha or beta), vasoactive intestinal Peptides, Tumor Necrosis Factor (TNF), Hypothalamic Release Factor, Prolactin, Thyroid Stimulating Hormone (TSH), Adrenocorticotropic Hormone (ACTH), Parathyroid Hormone (PTH), Follicle Stimulating Hormone (FSF), Gonadotropins Hormone-releasing hormone (LHRH), endorphins, glucagon, calcitonin, oxytocin, carbetocin, aldoetecone, enkephalin, somatostin, growth hormone, somatomodulin, alpha-melanostimulating hormone, Lidocaine, sufentanil, terbutaline, droperidol, scopolamine, gonadorelin, ciclopirox, buspirone, calcitonin, cromolyn disodium or midazolam, cyclosporine Lisinopril, Captopril, Delapril, Ranitidine, Famotidine, Superoxide Dismutase, Asparaginase, Arginase, Arginine Deaminase, Adenosine deaminase ribonuclease, trypsin, chemical trypsin, papain, bombesin, substance P, vasopressin, alpha-globulin, transferrin, coagulation factor I, beta-lipoprotein, beta - globulin, coagulation factor II, ceruloplasmin, α2-glycoprotein, α2-globulin, fetuin, α1-lipoprotein, α1-globulin, albumin, or prealbumin.

63. 一种药物产品，其包括含有权利要求1-18中任一项的化合物或者权利要求19-36中任一项的制剂或者权利要求37-43中任一项的剂型的溶液，和用于黏膜、鼻内、或肺部喷雾的调节器。63. A pharmaceutical product comprising a solution containing the compound of any one of claims 1-18 or the formulation of any one of claims 19-36 or the dosage form of any one of claims 37-43, and Regulators for mucous membrane, intranasal, or pulmonary sprays.

Claims (78)

Translated fromChinese

1.一种含有肽的化合物，其具有在哺乳动物黏膜中通过调控黏膜的渗透性而增强活性剂的黏膜上皮转运的活性，或其药用盐。CLAIMS 1. A peptide-containing compound having an activity of enhancing mucosal epithelial transport of an active agent in mammalian mucosa by regulating the permeability of the mucosa, or a pharmaceutically acceptable salt thereof.2.权利要求1的化合物，其中所述肽具有小于10千道尔顿的分子量。2. The compound of claim 1, wherein said peptide has a molecular weight of less than 10 kilodaltons.3.权利要求1的化合物，其中所述肽是紧密连接调控肽。3. The compound of claim 1, wherein said peptide is a tight junction modulating peptide.4.权利要求1的化合物，其中所述化合物含有蛋白转导结构域、DNA-结合结构域或融合结构域。4. The compound of claim 1, wherein said compound contains a protein transduction domain, a DNA-binding domain, or a fusion domain.5.权利要求1的化合物，其中所述化合物含有锌指结构域。5. The compound of claim 1, wherein said compound contains a zinc finger domain.6.权利要求1的化合物，其中所述化合物含有至少60％的赖氨酸、亮氨酸和/或丙氨酸残基。6. The compound of claim 1, wherein said compound contains at least 60% lysine, leucine and/or alanine residues.7.权利要求1的化合物，其中所述化合物形成α-螺旋。7. The compound of claim 1, wherein said compound forms an [alpha]-helix.8.权利要求1的化合物，其中所述渗透性是体外或体内的。8. The compound of claim 1, wherein said permeability is in vitro or in vivo.9.权利要求1的化合物，其中所述渗透性可逆增强。9. The compound of claim 1, wherein said permeability is reversibly enhanced.10.权利要求1的化合物，其中所述渗透性是基本上小于约90分钟的时间周期。10. The compound of claim 1, wherein said permeability is for a period of time substantially less than about 90 minutes.11.权利要求1的化合物，其中所述渗透性基本上在少于约60分钟的时间周期内被增强。11. The compound of claim 1, wherein said permeability is substantially enhanced for a period of time of less than about 60 minutes.12.权利要求1的化合物，其中在所述黏膜中所述渗透性被增强，而没有诱导基本的细胞溶解。12. The compound of claim 1, wherein said permeability is enhanced in said mucosa without inducing substantial cell lysis.13.权利要求1的化合物，其中在所述黏膜中所述渗透性被增强，而保持细胞存活能力。13. The compound of claim 1, wherein said permeability is enhanced in said mucosa while maintaining cell viability.14.权利要求1的化合物，其中所述渗透性通过增加黏膜脂质流动性而增强。14. The compound of claim 1, wherein said permeability is enhanced by increasing mucosal lipid fluidity.15.权利要求1的化合物，其中所述化合物是肽PN159。15. The compound of claim 1, wherein said compound is peptide PN159.16.权利要求1的化合物，其中所述化合物是PN159的类似物、缀合物、衍生物、变体、片段、模拟物、融合分子或复合物。16. The compound of claim 1, wherein said compound is an analog, conjugate, derivative, variant, fragment, mimetic, fusion molecule or complex of PN159.17.权利要求1的化合物，其中所述肽选自由SEQ.ID NOs 1-72组成的组。17. The compound of claim 1, wherein said peptide is selected from the group consisting of SEQ.ID NOs 1-72.18.权利要求1的化合物，其中所述肽选自由SEQ.ID NOs 32-35，38，46-49，53，和55组成的组。18. The compound of claim 1, wherein said peptide is selected from the group consisting of SEQ.ID NOs 32-35, 38, 46-49, 53, and 55.19.权利要求1-18中任一项的化合物，其中所述化合物与水溶性链共价连接。19. The compound of any one of claims 1-18, wherein the compound is covalently linked to a water soluble chain.20.权利要求19的化合物，其中所述化合物具有小于约300千道尔顿的分子量。20. The compound of claim 19, wherein said compound has a molecular weight of less than about 300 kilodaltons.21.权利要求19的化合物，其中所述化合物具有小于约200千道尔顿的分子量。21. The compound of claim 19, wherein said compound has a molecular weight of less than about 200 kilodaltons.22.权利要求1-21中任一项的化合物，其中所述化合物与聚(环氧烷)链共价连接。22. The compound of any one of claims 1-21, wherein the compound is covalently attached to a poly(alkylene oxide) chain.23.权利要求22的化合物，其中所述聚(环氧烷)链是有支链的或无支链的。23. The compound of claim 22, wherein the poly(alkylene oxide) chain is branched or unbranched.24.权利要求22的化合物，其中所述聚(环氧烷)链是聚乙二醇(PEG)链。24. The compound of claim 22, wherein the poly(alkylene oxide) chain is a polyethylene glycol (PEG) chain.25.权利要求24的化合物，其中所述PEG具有约0.2和约200千道尔顿(kDa)之间的分子大小。25. The compound of claim 24, wherein the PEG has a molecular size between about 0.2 and about 200 kilodaltons (kDa).26.权利要求24的化合物，其中所述PEG具有低于40kDa的大小。26. The compound of claim 24, wherein said PEG has a size below 40 kDa.27.权利要求24的化合物，其中所述PEG具有低于20kDa的大小。27. The compound of claim 24, wherein said PEG has a size below 20 kDa.28.权利要求24的化合物，其中所述PEG具有低于10kDa的大小。28. The compound of claim 24, wherein said PEG has a size below 10 kDa.29.权利要求24的化合物，其中所述PEG具有低于5kDa的大小。29. The compound of claim 24, wherein said PEG has a size below 5 kDa.30.权利要求24的化合物，其中所述PEG具有低于2kDa的大小。30. The compound of claim 24, wherein said PEG has a size below 2 kDa.31.权利要求22的化合物，其中所述聚(环氧烷)具有小于2.00的多分散性值(Mw/Mn)。31. The compound of claim 22, wherein the poly(alkylene oxide) has a polydispersity value (Mw/Mn) of less than 2.00.32.权利要求22的化合物，其中所述聚(环氧烷)具有小于1.20的多分散性值(Mw/Mn)。32. The compound of claim 22, wherein the poly(alkylene oxide) has a polydispersity value (Mw/Mn) of less than 1.20.33.权利要求22的化合物，其中所述化合物和聚(环氧烷)通过间隔部分缀合。33. The compound of claim 22, wherein the compound and poly(alkylene oxide) are conjugated through a spacer moiety.34.一种药物制剂，其包含增强黏膜上皮转运有效量的权利要求1-33中任一项的化合物和治疗有效量的活性剂。34. A pharmaceutical formulation comprising a mucosal epithelial transport enhancing effective amount of a compound according to any one of claims 1-33 and a therapeutically effective amount of an active agent.35.权利要求34的制剂，其中所述制剂减小穿过黏膜组织屏障的电阻。35. The formulation of claim 34, wherein the formulation reduces electrical resistance across the mucosal tissue barrier.36.权利要求35的制剂，其中所述电阻的减少为至少80％。36. The formulation of claim 35, wherein the reduction in electrical resistance is at least 80%.37.权利要求34的制剂，其中所述制剂相对于不包含权利要求1-33中任一项的化合物的类似的制剂增加所述活性剂穿过黏膜组织屏障的渗透性。37. The formulation of claim 34, wherein said formulation increases the permeability of said active agent across the mucosal tissue barrier relative to a similar formulation not comprising the compound of any one of claims 1-33.38.权利要求37的制剂，其中渗透性的增加为至少两倍。38. The formulation of claim 37, wherein the increase in permeability is at least two-fold.39.权利要求37的制剂，其中所述渗透性是旁细胞的。39. The formulation of claim 37, wherein said permeability is paracellular.40.权利要求37的制剂，其中所述增加的渗透性由调控紧密连接导致。40. The formulation of claim 37, wherein said increased permeability results from modulation of tight junctions.41.权利要求37的制剂，其中所述渗透性是跨细胞的或者是跨细胞和旁细胞的混合。41. The formulation of claim 37, wherein said permeability is transcellular or a mixture of transcellular and paracellular.42.权利要求37的制剂，其中所述黏膜组织屏障是上皮细胞层。42. The formulation of claim 37, wherein the mucosal tissue barrier is an epithelial cell layer.43.权利要求42的制剂，其中所述上皮细胞选自由气管细胞，支气管细胞，肺泡细胞，鼻细胞，肺细胞，胃肠细胞，表皮细胞和口腔细胞组成的组。43. The formulation of claim 42, wherein said epithelial cells are selected from the group consisting of tracheal cells, bronchial cells, alveolar cells, nasal cells, lung cells, gastrointestinal cells, epidermal cells, and oral cavity cells.44.权利要求42的制剂，其中所述上皮细胞是鼻细胞。44. The formulation of claim 42, wherein said epithelial cells are nasal cells.45.权利要求34的制剂，其中所述活性剂是肽、蛋白或核酸。45. The formulation of claim 34, wherein the active agent is a peptide, protein or nucleic acid.46.权利要求45的制剂，其中所述肽或蛋白包括2-1000个氨基酸。46. The formulation of claim 45, wherein said peptide or protein comprises 2-1000 amino acids.47.权利要求45的制剂，其中所述肽或蛋包括2-50个氨基酸。47. The formulation of claim 45, wherein the peptide or egg comprises 2-50 amino acids.48.权利要求45的制剂，其中所述肽或蛋白是环形的。48. The formulation of claim 45, wherein the peptide or protein is circular.49.权利要求45的制剂，其中所述肽或蛋白是二聚体或寡聚体。49. The formulation of claim 45, wherein the peptide or protein is a dimer or oligomer.50.权利要求45的制剂，其中所述肽或蛋白选自由GLP-1、PYY3-36、PTH1-34和胰高血糖素样肽的抑制剂-4组成的组。50. The formulation of claim 45, wherein said peptide or protein is selected from the group consisting of GLP-1, PYY3-36, PTH1-34, and inhibitor of glucagon-like peptide-4.51.权利要求45的制剂，其中所述蛋白选自由β-干扰素、α-干扰素、胰岛素、促红细胞生成素、G-CSF、GM-CSF、生长激素、以及它们的类似物组成的组。51. The formulation of claim 45, wherein said protein is selected from the group consisting of beta-interferon, alpha-interferon, insulin, erythropoietin, G-CSF, GM-CSF, growth hormone, and their analogs .52.包含权利要求34-51中任一项的制剂的剂型，其中所述剂型是液体。52. A dosage form comprising the formulation of any one of claims 34-51, wherein said dosage form is a liquid.53.权利要求52的剂型，其中所述液体是小滴形式。53. The dosage form of claim 52, wherein said liquid is in the form of droplets.54.权利要求52的剂型，其中所述液体是气溶胶形式。54. The dosage form of claim 52, wherein said liquid is in the form of an aerosol.55.包含权利要求34-51中任一项的制剂的剂型，其中所述剂型是固体。55. A dosage form comprising the formulation of any one of claims 34-51, wherein said dosage form is a solid.56.权利要求55的剂型，其中所述固体在施用之前在液体中重构。56. The dosage form of claim 55, wherein the solid is reconstituted in a liquid prior to administration.57.权利要求55的剂型，其中所述固体作为散剂施用。57. The dosage form of claim 55, wherein said solid is administered as a powder.58.权利要求55的剂型，其中所述固体是胶囊、片剂或凝胶形式。58. The dosage form of claim 55, wherein said solid is in the form of a capsule, tablet or gel.59.一种给动物施用分子的方法，其包括提供如权利要求34-58中任一项的制剂，并且将所述制剂与所述动物的黏膜表面接触。59. A method of administering a molecule to an animal comprising providing a formulation according to any one of claims 34-58, and contacting said formulation with a mucosal surface of said animal.60.权利要求59的方法，其中所述黏膜表面是鼻内。60. The method of claim 59, wherein said mucosal surface is intranasal.61.一种在哺乳动物中增加鼻内施用的活性剂的生物利用度的方法，其包括提供如权利要求34-58中任一项的制剂，并且将所述制剂施用给所述哺乳动物。61. A method of increasing the bioavailability of an intranasally administered active agent in a mammal comprising providing a formulation according to any one of claims 34-58, and administering said formulation to said mammal.62.权利要求1的化合物，其中所述活性剂是siRNA。62. The compound of claim 1, wherein the active agent is siRNA.63.权利要求1的化合物，其中所述活性剂是dsDNA。63. The compound of claim 1, wherein the active agent is dsDNA.64.权利要求1的化合物，其中所述活性剂是造血药；抗感染药；抗痴呆药；抗病毒剂，抗肿瘤药，退热药，镇痛药，消炎药，抗溃疡药，抗过敏剂，抗抑郁药，治疗精神病的药，强心剂，抗心律失常药，血管舒张剂，抗高血压药，降血压药利尿剂，抗糖尿病药，  抗凝血剂，降胆固醇药，用于骨质疏松症的治疗剂，激素，抗生素，或疫苗。64. The compound of claim 1, wherein said active agent is a hematopoietic drug; an anti-infective drug; an anti-dementia drug; an anti-viral agent, an antineoplastic drug, an antipyretic, an analgesic, an anti-inflammatory drug, an anti-ulcer drug, an anti-allergic drug antidepressants, psychotropic drugs, cardiotonic agents, antiarrhythmic drugs, vasodilators, antihypertensive drugs, hypotensive drugs diuretics, antidiabetic drugs, anticoagulants, cholesterol lowering drugs, for bone Treatments for osteoporosis, hormones, antibiotics, or vaccines.65.权利要求1的化合物，其中所述活性剂是细胞因子，肽激素，生长因子，心血管因子，细胞粘附因子，中枢或外周神经系统因子，体液电解质因子，血液有机物质，骨生长因子，胃肠系统因子，肾因子，结缔组织因子，感觉器官因子，免疫系统因子，呼吸系统因子，或生殖器官因子。65. The compound of claim 1, wherein the active agent is a cytokine, a peptide hormone, a growth factor, a cardiovascular factor, a cell adhesion factor, a central or peripheral nervous system factor, a humoral electrolyte factor, a blood organic substance, a bone growth factor , gastrointestinal system factor, kidney factor, connective tissue factor, sensory organ factor, immune system factor, respiratory system factor, or reproductive organ factor.66.权利要求1的化合物，其中所述活性剂是雄激素、雌激素、前列腺素、生长激素、促性腺激素、白介素、类固醇或细胞因子。66. The compound of claim 1, wherein the active agent is an androgen, an estrogen, a prostaglandin, a growth hormone, a gonadotropin, an interleukin, a steroid, or a cytokine.67.权利要求1的化合物，其中所述活性剂是用于肝炎、流感、呼吸道合胞病毒(RSV)、副流感病毒(PIV)、结核病、金丝雀痘、水痘、  麻疹、流行性腮腺炎、风疹、肺炎、或人类免疫缺陷性病毒(HIV)的疫苗。67. The compound of claim 1, wherein said active agent is for hepatitis, influenza, respiratory syncytial virus (RSV), parainfluenza virus (PIV), tuberculosis, canary pox, chickenpox, measles, mumps , rubella, pneumonia, or human immunodeficiency virus (HIV).68.权利要求1的化合物，其中所述活性剂是白喉、破伤风、假单胞菌(pseudomonas)或结核分枝杆菌(mycobactrium tuberculosis)的细菌类毒素。68. The compound of claim 1, wherein the active agent is a bacterial toxoid of diphtheria, tetanus, pseudomonas or mycobactrium tuberculosis.69.权利要求1的化合物，其中所述活性剂是水蛭素，hirulos或蛭素。69. The compound of claim 1, wherein said active agent is hirudin, hirulos or hirudin.70.权利要求1的化合物，其中所述活性剂是单克隆抗体、多克隆抗体、人源化抗体、抗体片段、或免疫球蛋白。70. The compound of claim 1, wherein the active agent is a monoclonal antibody, polyclonal antibody, humanized antibody, antibody fragment, or immunoglobulin.71.权利要求1的化合物，其中所述活性剂是吗啡，氢吗啡酮，羟吗啡酮，洛伐啡诺(lovorphanol)，左洛啡烷，可待因，纳美芬，烯丙吗啡，纳洛酮，纳曲酮，丁丙诺啡，布托啡诺，或纳布啡。71. The compound of claim 1, wherein said active agent is morphine, hydromorphone, oxymorphone, lovorphanol (lovorphanol), levolorphan, codeine, nalmefene, nalamorphine, nalmefene, Loxone, naltrexone, buprenorphine, butorphanol, or nalbuphine.72.权利要求1的化合物，其中所述活性剂是可的松，氢化可的松，氟氢可的松，泼尼松，泼尼松龙，甲泼尼龙，曲安西龙，地塞米松，倍他米松，帕拉米松，或氟轻松。72. The compound of claim 1, wherein said active agent is cortisone, hydrocortisone, fludrocortisone, prednisone, prednisolone, methylprednisolone, triamcinolone, dexamethasone, Betamethasone, paramethasone, or fluocinolone.73.权利要求1的化合物，其中所述活性剂是秋水仙碱，对乙酰氨基酚，阿司匹林，布洛芬，酮洛芬，吲哚美辛，萘普生，美洛昔康，或吡罗昔康。73. The compound of claim 1, wherein the active agent is colchicine, acetaminophen, aspirin, ibuprofen, ketoprofen, indomethacin, naproxen, meloxicam, or piroxicam .74.权利要求1的化合物，其中所述活性剂是阿昔洛韦，利巴韦林，三氟胸苷，Ara-A(阿糖呋喃糖腺嘌呤)，酰基鸟苷，去甲脱氧鸟嘌呤核苷，叠氮胸苷，二脱氧腺苷，或二脱氧胞苷。74. The compound of claim 1, wherein the active agent is acyclovir, ribavirin, trifluorothymidine, Ara-A (arabinofuranosyladenosine), acylguanosine, nordeoxyguanine nucleoside, zidovudine, dideoxyadenosine, or dideoxycytidine.75.权利要求1的化合物，其中所述活性剂是螺内酯，睾酮，雌二醇，黄体酮，促性腺激素，雌激素或孕酮。75. The compound of claim 1, wherein the active agent is spironolactone, testosterone, estradiol, progesterone, gonadotropin, estrogen or progesterone.76.权利要求1的化合物，其中所述活性剂是罂粟碱，硝酸甘油，血管活性肠肽，降钙素相关的基因肽，赛庚啶，多塞平，丙米嗪，西咪替丁，右美沙芬，氯氮平片剂，过氧化物歧化酶，神经脑啡肽酶，两性霉素B，灰黄霉素，咪康唑，酮康唑，噻康唑，伊曲康唑，氟康唑，头孢菌素，四环素，氨基糖苷，红霉素，庆大霉素，多粘菌素B，5-氟尿嘧啶，博来霉素，甲氨喋呤，和羟基脲，二脱氧肌苷，氟尿苷，6-巯基嘌呤，多柔比星，柔红霉素，伊达比星，泰素，紫杉醇，维生素E，奎尼丁，哌唑嗪，维拉帕米，硝苯地平，或地尔硫76. The compound of claim 1, wherein said active agent is papaverine, nitroglycerin, vasoactive intestinal peptide, calcitonin-related gene peptide, cyproheptadine, doxepin, imipramine, cimetidine, Dextromethorphan, Clozapine Tablets, Superoxide Dismutase, Neprilysin, Amphotericin B, Griseofulvin, Miconazole, Ketoconazole, Tioconazole, Itraconazole, Fluoride Conazole, cephalosporins, tetracyclines, aminoglycosides, erythromycin, gentamicin, polymyxin B, 5-fluorouracil, bleomycin, methotrexate, and hydroxyurea, dideoxyinosine, Floxuridine, 6-mercaptopurine, doxorubicin, daunorubicin, idarubicin, taxol, paclitaxel, vitamin E, quinidine, prazosin, verapamil, nifedipine, or diltiazem77.权利要求1的化合物，其中所述活性剂是组织血纤维蛋白溶酶原活化因子(TPA)，表皮生长因子(EGF)，成纤维细胞生长因子(FGF-酸性或碱性)，血小板衍生生长因子(PDGF)，转化生长因子(TGF-α或β)，血管活性肠肽，肿瘤坏死因子(TNF)，下丘脑释放因子，促乳素，促甲状腺激素(TSH)，促肾上腺皮质激素(ACTH)，甲状旁腺激素(PTH)，促卵泡激素(FSF)，促性腺激素释放激素(LHRH)，内啡肽，胰高血糖素，降钙素，催产素，卡贝缩宫素，aldoetecone，脑啡肽，somatostin，生长激素，生长调节素，α-促黑激素，利多卡因，舒芬太尼，特布他林，氟哌利多，东莨菪碱，戈那瑞林，环吡酮，丁螺环酮，降钙素，色甘酸二钠或咪达唑仑，环孢素，赖诺普利，卡托普利，地拉普利，雷尼替丁，法莫替丁，过氧化物歧化酶，天冬酰胺酶，精氨酸酶，精氨酸脱氨酶，腺苷脱氨酶核糖核酸酶，胰蛋白酶，化学胰蛋白酶，木瓜蛋白酶，铃蟾肽，P物质，血管升压素，α-球蛋白，运铁蛋白，凝血因子I，β-脂蛋白，β-球蛋白，凝血因子II，血浆铜蓝蛋白，α2-糖蛋白，α2-球蛋白，胎球蛋白，α1-脂蛋白，α1-球蛋白，白蛋白，或前白蛋白。77. The compound of claim 1, wherein said active agent is tissue plasminogen activator (TPA), epidermal growth factor (EGF), fibroblast growth factor (FGF-acidic or basic), platelet-derived growth factor (PDGF), transforming growth factor (TGF-α or β), vasoactive intestinal peptide, tumor necrosis factor (TNF), hypothalamic releasing factor, prolactin, thyroid-stimulating hormone (TSH), corticotropin ( ACTH), parathyroid hormone (PTH), follicle-stimulating hormone (FSF), gonadotropin-releasing hormone (LHRH), endorphins, glucagon, calcitonin, oxytocin, carbetocin, aldoetecone , enkephalin, somatostin, growth hormone, somatomodulin, α-melanostimulating hormone, lidocaine, sufentanil, terbutaline, droperidol, scopolamine, gonadorelin, ciclopirox, butyl spirone, calcitonin, disodium cromoglycate or midazolam, cyclosporine, lisinopril, captopril, delapril, ranitidine, famotidine, peroxide Dismutase, asparaginase, arginase, arginine deaminase, adenosine deaminase ribonuclease, trypsin, chemical trypsin, papain, bombesin, substance P, vasopressin , α-globulin, transferrin, coagulation factor I, β-lipoprotein, β-globulin, coagulation factor II, ceruloplasmin, α2-glycoprotein, α2-globulin, fetuin, α1-lipid protein, α1-globulin, albumin, or prealbumin.78.一种药物产品，其包括含有权利要求1的化合物的溶液，和用于黏膜、鼻内、或肺部喷雾的调节器。78. A pharmaceutical product comprising a solution comprising a compound of claim 1, and a regulator for mucosal, intranasal, or pulmonary spray.

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