Production of a Lung Surfactant SP-B Mimic

描述（由申请人提供）：表面活性剂蛋白B （SP-B）对天然肺表面活性剂的功能至关重要，对临床外源性表面活性剂的活性也同样重要。基于已知的“皂苷”类蛋白质的三维结构，早期的SP-B模型预测二硫交联、N端和c端结构域折叠为功能必需的带电两亲螺旋。在目前的II期SBIR研究中，我们设计并化学合成了41个残基的Super Mini-B （S-MB）肽结构，模拟了79个残基天然蛋白的许多体外和体内结构和功能特性。该S-MB肽的活性超过了我们完成的I期SBIR和初始II期提案（2007年4月）中研究的有前途的34个氨基酸的Mini-B肽。在目前的应用中，S-MB肽与合成脂质结合产生两种互补的全合成外源性肺表面活性剂（Minisurf（tm）和Minisurf- r (tm)），具有极高的活性和商业潜力。两种类型的合成脂质被用来生产这些S-MB表面活性剂：(i)合成脂质的混合物(L)模仿天然肺表面活性剂；（ii）合成的新型磷脂酶抗性脂质（RL）具有增强的表面特性，作为治疗性表面活性剂的成分，并且在肺组织中溶解酶浓度增加的炎症性肺损伤中减少降解。在目前的资助下，我们开发了S-MB肽和活性合成L-SMB （Minisurf(tm)）和RL-S-MB （Minisurf- r (tm)）表面活性剂的商业上可行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）和急性呼吸窘迫综合征（ARDS）的急性呼吸衰竭。Minisurf（tm）和Minisurf- r （tm）表面活性剂也可能用于抗生素、抗炎药和遗传物质的新型肺部递送系统。该II期SBIR提案包括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）制剂中可重复配制并进行质量控制，与目前的动物源性和合成表面活性剂药物相比，这些制剂在生产经济性、表面活性、抗抑制性和生理活性方面具有显著的潜在优势，同时毒性最小。公共卫生相关性：这项SBIR研究将导致新型全合成外源性表面活性剂的开发和生产，这些外源性表面活性剂在生产经济、活性和抗抑制方面具有显著优势，可用于未来临床治疗流行和严重呼吸系统疾病（NRDS、ALI和ARDS）。

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.