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.
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. ? ? ?
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