Production of a Lung Surfactant SP-B Mimic

肺表面活性剂 SP-B 模拟物的生产

• 批准号: 7688621
• 负责人: Gary Fujii
• 金额: $ 62.54万
• 依托单位: MOLECULAR EXPRESS, INC.
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-05-01 至 2011-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7688621
• 关键词: Acute; Acute Lung Injury; Acute respiratory failure; Adult Respiratory Distress Syndrome; Air; Amino Acids; Animal Model; Animals; Anti-Inflammatory Agents; Antibiotics; Asthma; Behavior; Biological Assay; Bronchoalveolar Lavage; C-terminal; Cell Culture Techniques; Charge; Chemical Surfactants; Chemicals; Classification; Cleaved cell; Clinical; Development; Dialysis procedure; Digestion; Disulfides; Drug Formulations; Ensure; Exhibits; Future; Genetic Materials; Grant; High Pressure Liquid Chromatography; In Situ; In Vitro; Infasurf; Inflammation; Inflammatory; Lead; Length; Lipids; Liposomes; Lung; Lung diseases; LytA enzyme; Mass Spectrum Analysis; Measures; Methods; Modeling; Molecular; Monitor; Newborn Respiratory Distress Syndrome; Oryctolagus cuniculus; Patients; Peptides; Pharmaceutical Preparations; Phase; Phase I Clinical Trials; Phospholipase; Physiological; Preparation; Process; Production; Property; Proteins; Pulmonary Surfactant-Associated Protein B; Pulmonary Surfactant-Associated Proteins; Pulmonary Surfactants; Quality Control; Rattus; Research; Resistance; Running; Saposins; Small Business Innovation Research Grant; Solvents; Spectroscopy, Fourier Transform Infrared; Stress Tests; Structure; Surface; Surface Properties; Survanta; System; Temperature; Therapeutic; Tissues; Toxic effect; Viscosity; base; clinically significant; crosslink; cytotoxic; design; gene therapy; improved; in vitro testing; in vivo; inhibitor/antagonist; interfacial; lung injury; novel; oxidation; peptide B; public health relevance; solid state; surfactant; synthetic peptide; three dimensional structure

DESCRIPTION (provided by applicant): Surfactant Protein B (SP-B) is crucial in the function of native lung surfactant, and is equally important for activity in clinical exogenous surfactants as well. Based on the known three-dimensional structures of proteins in the "Saposin" class, early SP-B models predicted that the disulfide cross-linked, N- and C-terminal domains fold as functionally essential charged amphipathic helices. For the current Phase II SBIR research, we have designed and chemically synthesized a 41 residue Super Mini-B (S-MB) peptide construct that emulates many of the in vitro and in vivo structural and functional properties of the 79 residue native protein. This S-MB peptide has activity exceeding that of the promising 34 amino acid Mini-B peptide studied in our completed Phase I SBIR and initial Phase II proposal (April, 2007). In the current application, S-MB peptide is combined with synthetic lipids to generate two complementary fully-synthetic exogenous lung surfactants (Minisurf(tm) and Minisurf-R(tm)) that have extremely high activity and commercial potential. Two types of synthetic lipids are utilized to allow the production of these S-MB surfactants: (i) a mix of synthetic lipids (L) modeled after those in native lung surfactant; and (ii) novel phospholipase-resistant lipids (RL) synthesized to have enhanced surface properties as components in therapeutic surfactants plus reduced degradation in inflammatory lung injuries where lytic enzymes are present in increased concentrations in pulmonary tissue. In the current grant, we develop commercially viable production and quality-control methods for S-MB peptide and active synthetic L-SMB (Minisurf(tm)) and RL-S-MB (Minisurf-R(tm)) surfactants for treating acute respiratory failure in the Neonatal Respiratory Distress Syndrome (NRDS), clinical Acute Lung Injury (ALI), and the Acute Respiratory Distress Syndrome (ARDS). Minisurf(tm) and Minisurf-R(tm) surfactants can also potentially be used in novel pulmonary delivery systems for antibiotics, anti-inflammatory agents, and genetic material. This Phase II SBIR proposal comprises Aims leading to the following key Milestones: 1) to define and establish process development activities required to produce commercially-viable quantities of purified active S-MB and; 2) to demonstrate that S-MB peptide can be reproducibly formulated and quality-controlled in final Minisurf(tm) and Minisurf-R(tm) formulations that have significant potential advantages in production economy, surface activity, inhibition resistance, and physiological activity compared to current animal-derived and synthetic surfactant drug products while exhibiting minimal toxicity. PUBLIC HEALTH RELEVANCE: This SBIR research will lead to the development and production of novel fully synthetic exogenous surfactants having significant advantages in production economy, activity, and inhibition resistance for future clinical use in treating prevalent and severe respiratory diseases (NRDS, ALI, and ARDS). This SBIR Phase II application will synthesize, quality-control, and define the high activity of synthetic lung surfactants (Minisurf(tm) and Minisurf-R(tm)) based on the novel synthetic peptide Super Mini-B (S-MB), a 41 residue construct incorporating key functional domains of full-length lung surfactant protein (SP)-B. This S-MB peptide has been proven in preliminary studies to be even more active than the Mini-B (MB) peptide used in our successful Phase I studies. The synthetic S-MB surfactants of this Phase II proposal are designed to have significant activity in the therapy of the neonatal respiratory distress syndrome (NRDS), clinical acute lung injury (ALI), and the acute respiratory distress syndrome (ARDS). Minisurf(tm) formulations contain the S-MB peptide combined with synthetic lipids (L) reflecting those in native surfactant, and Minisurf-R(tm) formulations contain S-MB combined with novel phospholipase-resistant lipids (RL) with molecular analogy to native lipids but designed to have improved surface behavior and structural resistance to degradation during inflammatory lung injury. Minisurf(tm) and Minisurf-R(tm) final products can both be used in NRDS and ALI/ARDS, with Minisurf- R(tm) having particular potential advantages for patients with lung injury involving high degrees of inflammation. This Phase II grant will chemically synthesize and quality-control S-BM peptide in Aim 1, and will optimize final Minisurf(tm) and Minisurf-R(tm) surfactants in Aim 2 by assessing surface activity and pulmonary efficacy in animal models relevant for NRDS and ALI/ARDS. In addition to their use in surfactant therapy for clinically significant and prevalent lung diseases, Minisurf(tm) and Minisurf-R(tm) also could be utilized in new liposomal delivery systems for pulmonary antibiotics, anti-inflammatory agents, asthma drugs, or gene therapy material. The present proposal, however, focuses primarily on the synthesis and quality-control of S-MB, and the formulation of Minisurf(tm) and Minisurf-R(tm) products with optimal surface activity and efficacy in animal models of NRDS and ALI/ARDS. Preliminary studies have documented the production of S-MB peptide by solid-state chemical methods analogous to those in our successful Phase I studies with MB. These methods are extended in Aim 1 to include the use of a Symphony Multiple Peptide Synthesizer that we have recently documented can prepare preparative scale (gram) amounts of S-MB per run. Aim I will apply and refine our preliminary Symphony Synthesizer methods to maximize S-MB yield, and will develop a commercially-relevant set of quality-control methods based on those used successfully with MB and in our preliminary syntheses of S-MB. Crude linear SMB from the Symphony Synthesizer will be cleaved, deprotected, precipitated, lyophilized, reduced and purified by preparative reverse phase (RP)-HPLC. The mass of purified, reduced S-MB will be verified by mass spectroscopy (MS), and the peptide will then be air-oxidized in structure-promoting solvent and re-purified by RP-HPLC and dialysis. Complete oxidation of purified S-MB will be confirmed by enzymatic digestion and MS analysis, and folding into the desired disulfide-stabilized helix-hairpin will be quality-controlled with Fourier transform infrared (FTIR) spectroscopy. Final folded, oxidized S-MB will be lyophilized and stored for formulation with lipids in Minisurf(tm) and Minisurf-R(tm) formulations in Aim 2. Batches of final S-MB will also be evaluated in preliminary stability and stress testing. Aim 2 focuses on formulating optimal final Minisurf(tm) and Minisurf-R(tm) surfactants containing S-MB peptide combined with L or RL lipids, and evaluating and quality-controlling their surface activity and their pulmonary activity in animal lung models relevant for NRDS and ALI/ARDS. Surface activity will be measured in vitro using the pulsating and captive bubble surfactometers, both of which have been shown to be applicable for assessing functionally-relevant interfacial behaviors in native and synthetic lung surfactants. Preliminary studies detailed in the grant have already identified formulations of both Minisurf(tm) and Minisurf-R(tm) that have extremely high surface and physiological activity, but systematic assessments in Aim 2 will allow us to optimize final product compositions. Aim 2 will also use chemical and spectroscopic methods to ensure the quality of surfactant formulations, measure surfactant preparation viscosity, and define any possible cytotoxic or hemolytic effects in cell culture assays. Batches of Minisurf(tm) and Minisurf-R(tm) will also be monitored under different conditions (temperature, pH) in preliminary stability and stress testing. The most promising Minisurf(tm) and Minisurf-R(tm) formulations from in vitro testing will be evaluated for pulmonary activity in situ using an FDA accepted excised rat lung model in the presence and absence of inhibitor substances relevant for lung injury, and in vivo activity studies will be performed in ventilated lung-lavaged rabbits. These animal assessments are directly related to NRDS and ALI/ARDS, and will determine the physiological efficacy of Minisurf(tm) and Minisurf-R(tm) compared to current/pending commercial surfactants (e.g., Surfaxin(r), Infasurf(r), Survanta(r)). Results already obtained for initial Minisurf(tm) and Minisurf-R(tm) formulations show that they have very favorable surface and physiological activity compared to currently available animal and synthetic clinical surfactants as detailed in the body of this Phase II proposal.

描述（由申请人提供）：表面活性剂蛋白 B (SP-B) 对于天然肺表面活性剂的功能至关重要，并且对于临床外源性表面活性剂的活性也同样重要。基于“Saposin”类蛋白质的已知三维结构，早期的 SP-B 模型预测二硫键交联的 N 端和 C 端结构域折叠为功能上必需的带电两亲性螺旋。对于当前的 II 期 SBIR 研究，我们设计并化学合成了 41 残基 Super Mini-B (S-MB) 肽构建体，该构建体模拟了 79 残基天然蛋白的许多体外和体内结构和功能特性。这种 S-MB 肽的活性超过了我们已完成的 I 期 SBIR 和初始 II 期提案（2007 年 4 月）中研究的有前景的 34 个氨基酸 Mini-B 肽。在目前的应用中，S-MB肽与合成脂质结合生成两种互补的全合成外源性肺表面活性剂（Minisurf(tm)和Minisurf-R(tm)），它们具有极高的活性和商业潜力。使用两种类型的合成脂质来生产这些 S-MB 表面活性剂：(i) 模仿天然肺表面活性剂的合成脂质 (L) 的混合物； (ii) 合成的新型抗磷脂酶脂质（RL）作为治疗性表面活性剂的成分具有增强的表面特性，并且减少了炎症性肺损伤的降解，其中肺组织中溶解酶的浓度增加。在目前的资助中，我们开发了商业上可行的 S-MB 肽和活性合成 L-SMB (Minisurf(tm)) 和 RL-S-MB (Minisurf-R(tm)) 表面活性剂的生产和质量控制方法，用于治疗新生儿呼吸窘迫综合征 (NRDS)、临床急性肺损伤 (ALI) 和急性呼吸窘迫中的急性呼吸衰竭 综合症（ARDS）。 Minisurf(tm) 和 Minisurf-R(tm) 表面活性剂还可用于抗生素、抗炎剂和遗传物质的新型肺部输送系统。该第二阶段 SBIR 提案包括实现以下关键里程碑的目标： 1) 定义和建立生产商业上可行数量的纯化活性 S-MB 所需的工艺开发活动； 2) 证明 S-MB 肽可以在最终的 Minisurf(tm) 和 Minisurf-R(tm) 制剂中进行可重复配制和质量控制，与目前的动物源性和合成表面活性剂药品相比，这些制剂在生产经济性、表面活性、抑制抗性和生理活性方面具有显着的潜在优势，同时表现出最小的毒性。公共健康相关性：这项 SBIR 研究将导致新型全合成外源表面活性剂的开发和生产，这些表面活性剂在生产经济性、活性和抗抑制性方面具有显着优势，可用于未来临床治疗流行和严重呼吸系统疾病（NRDS、ALI 和 ARDS）。 该 SBIR II 期应用将基于新型合成肽 Super Mini-B (S-MB) 合成、质量控制和定义高活性的合成肺表面活性剂（Minisurf(tm) 和 Minisurf-R(tm)），Super Mini-B 是一种包含全长肺表面活性剂蛋白 (SP)-B 关键功能域的 41 个残基结构。这种 S-MB 肽已在初步研究中被证明比我们成功的 I 期研究中使用的 Mini-B (MB) 肽更具活性。该 II 期提案的合成 S-MB 表面活性剂旨在在治疗新生儿呼吸窘迫综合征 (NRDS)、临床急性肺损伤 (ALI) 和急性呼吸窘迫综合征 (ARDS) 方面具有显着活性。 Minisurf(tm) 配方含有与合成脂质 (L) 结合的 S-MB 肽，反映了天然表面活性剂中的脂质，而 Minisurf-R(tm) 配方含有 S-MB 与新型抗磷脂酶脂质 (RL) 组合，其分子类似于天然脂质，但旨在改善炎症性肺损伤期间的表面行为和结构抗降解性。 Minisurf(tm) 和 Minisurf-R(tm) 最终产品均可用于 NRDS 和 ALI/ARDS，其中 Minisurf-R(tm) 对于涉及高度炎症的肺损伤患者具有特殊的潜在优势。该第二阶段资助将在目标 1 中化学合成和质量控制 S-BM 肽，并将通过评估与 NRDS 和 ALI/ARDS 相关的动物模型中的表面活性和肺功效来优化目标 2 中最终的 Minisurf(tm) 和 Minisurf-R(tm) 表面活性剂。除了用于临床上显着且普遍的肺部疾病的表面活性剂治疗外，Minisurf(tm) 和 Minisurf-R(tm) 还可用于肺部抗生素、抗炎剂、哮喘药物或基因治疗材料的新型脂质体递送系统。然而，目前的提案主要侧重于 S-MB 的合成和质量控制，以及在 NRDS 和 ALI/ARDS 动物模型中具有最佳表面活性和功效的 Minisurf(tm) 和 Minisurf-R(tm) 产品的配方。 初步研究已经记录了通过固态化学方法生产 S-MB 肽，类似于我们成功的第一阶段 MB 研究中的方法。这些方法在目标 1 中进行了扩展，包括使用我们最近记录的 Symphony 多肽合成器，该合成器可以每次运行制备制备规模（克）量的 S-MB。目标 我将应用和完善我们的初步 Symphony Synthesizer 方法，以最大限度地提高 S-MB 产量，并将基于成功用于 MB 和 S-MB 初步合成的方法，开发一套商业相关的质量控制方法。来自 Symphony 合成器的粗线性 SMB 将通过制备型反相 (RP)-HPLC 进行裂解、脱保护、沉淀、冻干、还原和纯化。纯化、还原的 S-MB 的质量将通过质谱 (MS) 进行验证，然后肽将在结构促进溶剂中进行空气氧化，并通过 RP-HPLC 和透析重新纯化。纯化的 S-MB 的完全氧化将通过酶消化和 MS 分析来确认，并且折叠成所需的二硫键稳定的螺旋发夹将通过傅里叶变换红外 (FTIR) 光谱进行质量控制。最终折叠的氧化 S-MB 将被冻干并储存，以便与目标 2 中的 Minisurf(tm) 和 Minisurf-R(tm) 配方中的脂质一起配制。最终 S-MB 的批次也将在初步稳定性和压力测试中进行评估。 目标 2 重点是配制含有 S-MB 肽与 L 或 RL 脂质组合的最佳最终 Minisurf(tm) 和 Minisurf-R(tm) 表面活性剂，并在与 NRDS 和 ALI/ARDS 相关的动物肺模型中评估和质量控制其表面活性和肺活性。将使用脉动和捕获气泡表面活性剂在体外测量表面活性，这两种表面活性剂已被证明适用于评估天然和合成肺表面活性剂中功能相关的界面行为。资助中详细的初步研究已经确定了 Minisurf(tm) 和 Minisurf-R(tm) 的配方具有极高的表面和生理活性，但 Aim 2 中的系统评估将使我们能够优化最终产品的成分。目标 2 还将使用化学和光谱方法来确保表面活性剂配方的质量，测量表面活性剂制剂的粘度，并确定细胞培养测定中任何可能的细胞毒性或溶血效应。 Minisurf(tm) 和 Minisurf-R(tm) 批次也将在不同条件（温度、pH）下进行初步稳定性和压力测试。体外测试中最有前途的 Minisurf(tm) 和 Minisurf-R(tm) 制剂将使用 FDA 接受的切除大鼠肺模型在存在和不存在与肺损伤相关的抑制剂物质的情况下原位评估肺活性，并且体内活性研究将在通气肺灌洗的兔子中进行。这些动物评估与 NRDS 和 ALI/ARDS 直接相关，并将确定 Minisurf(tm) 和 Minisurf-R(tm) 与当前/待定商业表面活性剂（例如 Surfaxin(r)、Infasurf(r)、Survanta(r)）相比的生理功效。初始 Minisurf(tm) 和 Minisurf-R(tm) 配方已获得的结果表明，与目前可用的动物和合成临床表面活性剂相比，它们具有非常有利的表面和生理活性，如本 II 期提案正文中详述。