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Systems and methods for characterizing surfactant protein d (sp-d) oligomers

A surfactant, SP-D technology, applied in biochemical equipment and methods, alveolar/pulmonary surfactant peptides, chemical instruments and methods, etc., can solve problems such as reducing potential efficacy

Pending Publication Date: 2021-01-12
AIRWAY THERAPEUTICS INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, recombinant SP-D can have variable oligomerization states and / or inactive aggregated forms, thus reducing the potential efficacy of such formulations

Method used

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  • Systems and methods for characterizing surfactant protein d (sp-d) oligomers
  • Systems and methods for characterizing surfactant protein d (sp-d) oligomers
  • Systems and methods for characterizing surfactant protein d (sp-d) oligomers

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] Atomic force microscopy analysis of embodiment 1-rhSP-D

[0061] Oligomeric species of recombinant human SP-D (rhSP-D) were characterized by atomic force microscopy (AFM). See, eg, Arroyo R, et al. (2017) Biophysical Journal 112(3):503a, which is incorporated by reference in its entirety. A solution containing 0.85 ng / μL rhSP-D in dilution buffer (200 mM NaCl, 20 mM Tris (pH 7.4), 1 mM EDTA) was placed on a freshly cut mica substrate. Samples were probed with tips from Nanotec (Nanotec Electrónica, Madrid, Spain) and PointProbePlus, type PPP-NCH (Nanosensors, Atomic force microscopy imaging of Switzerland). The image size for quantitative studies was 1 μm x 1 μm with 512 pixels and was acquired at a rate of 1 line / s. General planar subtraction is performed on the original image, and flattening is performed with background subtraction and artifact line removal.

[0062] Oligomeric species identified in the acquired images included trimers, hexamers, dodecamers, an...

Embodiment 2

[0066] AF4-MALLS analysis of embodiment 2-rhSP-D

[0067] The distribution of different oligomeric species of rhSP-D was determined by AF4-MALLS analysis. For AF4-MALLS analysis, by AF4 system (Eclipse Dual Tec, Wyatt Technology Corp., Santa Barbara, CA), then by UV (Ultimate 3000 variable wavelength detector, Dionex Corporation, Sunnyvale, CA) and MALS analysis (Dawn Heleos II detector, Wyatt Technology Corp., Santa Barbara, CA) to separate rhSP-D samples. A Dionex Ultimate 3000 HPLC system (Dionex Corporation, Sunnyvale, CA) was used to inject the samples and deliver the mobile phase to the AF4 system. The AF4 configuration used short channels with 350 μm thick spacers (Wyatt Technology Corp., Santa Barbara, CA). Data analysis and calculations were performed using Chromeleon (Dionex Corporation, Sunnyvale, CA) and ASTRA (Wyatt Technology Corp., Santa Barbara, CA) software. The results of the AF4-MALLS analysis included a fraction profile with several peaks.

[0068] AS...

Embodiment -3

[0092] Example-3 Activity of rhSP-D in TLR4 assay

[0093] Toll-like receptors (TLRs) function in both the innate and adaptive immune systems, and SP-D has the ability to modulate signaling through TLRs such as Toll-like receptor 2 (TLR2) and Toll-like receptor 4 (TLR4) conduction activity. See, eg, Haagsman HP et al., (2008) Neonatology 93:288-294; Yamazoe M. et al., (2008) J. Biol Chem. 283:35878-35888; and Vieira F. et al., (2017) Ann Anat 211 :184-201, each of which is incorporated by reference in its entirety. TLR4 activity can also modulate the severity of conditions such as bronchopulmonary dysplasia (BPD) (Malash AH et al., (2016) Gene 592:23-28, which is incorporated by reference in its entirety). Therefore, the activity of rhSP-D to modulate TLR4 activity was measured as an indication of the effect of rhSP-D on the host immune response. The activity of reconstituted formulations containing rhSP-D was tested in the LPS-TLR4 assay. In the LPS-TLR4 assay, reconsti...

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Abstract

Some embodiments of the methods and compositions provided herein include identifying and / or quantifying oligomeric species of surfactant protein-D (SP-D). Some embodiments include performing an asymmetric flow field-flow fractionation with multi-angle laser light scattering (AF4-MALLS) analysis on the SP-D.

Description

[0001] Cross References to Related Applications [0002] This application claims the U.S. provisional application titled "METHODS FOR CHARACTERIZING SURFACTANT PROTEIN D (SP-D) OLIGOMERS," filed March 29, 2018. Priority to Application Serial No. 62 / 650138, the US Provisional Application is hereby incorporated by reference in its entirety. [0003] About the sequence listing [0004] This application is filed with a sequence listing in electronic format. The sequence listing is provided as a file named AIRWY013WOSEQLIST created on March 20, 2019, which is approximately 7Kb in size. The information of the sequence listing in electronic format is incorporated herein by reference in its entirety. [0005] field of invention [0006] Some embodiments of the methods and compositions provided herein include identifying and / or quantifying oligomeric species of Surfactant Protein-D (SP-D). Some embodiments include performing asymmetric flow field flow separation and multi-angle lase...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K38/17C07K14/785B03C1/30
CPCC07K14/785A61K38/395G01N33/68G01N21/53G01N30/0005G01N15/0205G16C20/30G01N2021/4711C12Q1/025G01N33/15G01N33/5041G01N33/94G01N2500/10
Inventor 简·苏珊·罗森鲍姆马克·康奈尔·曼宁瑞恩·R·曼宁
Owner AIRWAY THERAPEUTICS INC
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