DILUTE SURFACTANT OR ISOLATED SURFACTANT PROTEIN SOLUTION FOR THE REDUCTION OF SURFACE TENSION IN THE LUNG

Abstract

In permeability lung edema, cardiogenic lung edema or neonatal respiratory distress, there is heterogeneous liquid distribution throughout the lungs. The excess alveolar liquid reduces gas exchange. Mechanical ventilation is used to improve gas exchange. In the presence of heterogeneous liquid distribution, there are surface tension-dependent stress concentrations in septa separating aerated from flooded alveoli. Mechanical ventilation, by inflating the lung above normal volumes, thus increasing surface tension above normal, exacerbates the stress concentrations and consequently injures, or exacerbates pre-existing injury of, the alveolar-capillary barrier. Any means of lowering surface tension should lessen ventilation injury of the lung. In the present invention, dilute exogenous surfactant solution or surfactant protein C solution interacts with albumin to lower surface tension, likely through effective promotion of surfactant lipid adsorption. Dilute surfactant or SP-C solution could be administered via either the trachea or the vasculature. Either solution could be delivered in the absence or presence of albumin or alternative facilitating solute, to lower surface tension and lessen ventilation injury of the heterogeneously flooded lung.

Claims

1. A method of reducing ventilation injury to a patient whose lung has regions with heterogeneous alveolar flooding by alveolar liquid, said method comprising the step of (i) delivering to the patient a solution comprising a predetermined amount of a surfactant protein C, wherein said predetermined amount of said surfactant protein C is selected to provide a concentration of said surfactant protein C in the alveolar liquid in a range of from 0.000001 weight/volume percent to 1 weight/volume percent after the performance of step (i), and wherein a negatively-charged solute is present in the alveolar liquid in a concentration which is in a range of from greater than 2 weight/volume percent to less than 12 weight/volume percent after the performance of step (i), said weight/volume percents of said surfactant protein C and said negatively-charged solute being based on the total volume of the alveolar liquid after the performance of step (i).

2. The method of claim 1, wherein said negatively-charged solute comprises at least one chemical selected from the group consisting of albumin, fibrinogen and negatively-charged dextran.

3. The method of claim 1, wherein said surfactant protein C is natural, recombinant, or synthetic.

4. The method of claim 3, wherein said surfactant protein C is synthetic and selected from sSP-Css-ion lock, sSP-Css-ion sSP-Cff-leuc, and combinations thereof.

5. The method of claim 1, wherein said concentration of said surfactant protein Cin the alveolar liquid after the performance of step (i) is in a range of from 0.0005 weight/volume percent to 1 weight/volume percent.

6. The method of claim 1, wherein said concentration of said surfactant protein C the alveolar liquid after the performance of step (i) is in a range of from 0.00001 weight/volume percent to 1 weight/volume percent.

7. The method of claim 1, wherein said concentration of said surfactant protein C in the alveolar liquid after the performance of step (i) is in a range of from 0.00001 weight/volume percent to 0.1 weightivolunie percent.

8. The method of claim 1, wherein said negatively-charged solute is present in the alveolar liquid in a concentration which is in a range of from 2.1 weight/volume percent to 11 weight/volume percent after the performance of step (i).

9. The method of claim 1, wherein said surfactant protein C is derived from an animal.

10. The method of claim 1 wherein said solution comprises a surfactant containing said surfactant protein C.

11. The method of claim 1, wherein said solution further comprises lipids.

12. The method of claim 1, wherein said alveolar liquid is contained in a patient's alveoli, said alveolar liquid having a surface tension which is lowered by the performance of step (i) such that the lowered surface tension of the alveolar liquid lessens ventilation-induced overdistension injury of the patient's lung.

13. The method of claim 12, wherein the lowered surface tension of the alveolar liquid reduces stress concentrations in the patient's lung.

14. The method of claim 13, wherein the patient's alveoli are flooded to substantially the same extent.

15. The method of claim 13, wherein the patient's alveoli are heterogeneously flooded, whereby the lowered surface tension of the alveolar liquid lessens ventilation-induced overdistension injury of intervening septa located between aerated and flooded alveoli.

16. The method of claim 1, wherein step (i) comprises the step of administering said solution comprising said surfactant protein C to a trachea or bronchus of the patient having the lung.

17. The method of claim 1, wherein some of said negatively-charged solute is already present in the alveolar liquid and some of said negatively-charged solute is delivered as part of said solution.

18. The method of claim 1, wherein said concentration of said surfactant protein C in the alveolar liquid after the performance of step (i) is in a range of from 0.00001 weight/volume % to 0.1 weight/volume percent.

19. The method of claim 1, wherein said concentration of said surfactant protein C in the alveolar liquid after the performance of step (i) is in a range of from 0.0005 weight/volume percent to 0.05 weight/volume percent.

20. The method of claim 1, wherein said concentration of said surfactant protein C in the alveolar liquid after the performance of step (i) is in a range of from 0.00001 weight/volume percent to 0.05 weight/volume percent.

21. The method of claim 1, wherein said concentration of said surfactant protein C in the alveolar liquid after the performance of step (i) is in a range of from 0.0001 weight/volume percent to 0.05 weight/volume percent.

22. The method of claim 1, wherein said concentration of said surfactant protein C in the alveolar liquid after the performance of step (i) is in a range of from 0.005 weight/volume percent to 0.05 weight/volume percent.

23. The method of claim 1, wherein said concentration of said surfactant protein C in the alveolar liquid after the performance of step (i) is in a range of from 0.0025 weight/volume percent to 0.05 weight/volume percent.

24. The method of claim 1, wherein said concentration of said surfactant protein C in the alveolar liquid after the performance of step (i) is in a range of from 0.00001 weight/volume percent to 0.01 weight/volume percent.

25. The method of claim 1, wherein said concentration of said surfactant protein C in the alveolar liquid after the performance of step (i) is in a range of from 0.0005 weight/volume percent to 0.01 weight/volume percent.

26. The method of claim 1, wherein said concentration of said surfactant protein C in the alveolar liquid after the performance of step (i) is in a range of from 0.0001 weight/volume percent to 0.01 weight/volume percent.

27. The method of claim 1, wherein said concentration of said surfactant protein C in the alveolar liquid after the performance of step (i) is in a range of from 0.005 weight/volume percent to 0.01 weight/volume percent.

28. The method of claim 1, wherein said concentration of said surfactant protein C in the alveolar liquid after the performance of step (i) is in a range of from 0.0025 weight/volume percent to 0.01 weight/volume percent.

29. The method of claim 1, wherein said concentration of said surfactant protein C in the alveolar liquid after the performance of step (i) is in a range of from 0.0005 weight/volume percent to 0.1 weight/volume percent.

30. The method of claim 1, wherein said concentration of said surfactant protein C in the alveolar liquid after the performance of step (i) is in a range of from 0.0001 weight/volume percent to 0.1 weight/volume percent.

31. The method of claim 1, wherein said concentration of said surfactant protein C in the alveolar liquid after the performance of step (i) is in a range of from 0.005 weight/volume percent to 0.1 weight/volume percent.

32. The method of claim 1, wherein said concentration of said surfactant protein C in the alveolar liquid after the performance of step (i) is in a range of from 0.0025 weight/volume percent to 0.1 weight/volume percent.

33. The method of claim 1, wherein step (i) is performed by injecting said solution into a circulatory system of a patient having the lung.

34. A method of reducing ventilation injury to a patient whose lung has regions with heterogeneous alveolar flooding by alveolar liquid which contains a negatively-charged solute, said method comprising the step of (i) delivering to the patient a solution comprising a predetermined amount of a surfactant protein C and no additional negatively-charged solute, wherein said predetermined amount of said surfactant protein C is selected to provide a concentration of said surfactant protein C in the alveolar liquid in a range of from 0.000001 weight/volume percent to 1 weight/volume percent after the performance of step (i), and wherein the negatively-charged solute is present in the alveolar liquid in a concentration which is in a range of from greater than 2 weight/volume percent to less than 12 weight/volume percent after the performance of step (i), said weight/volume percents of said surfactant protein C and said negatively-charged solute being based on the total volume of the alveolar liquid after the performance of step (i).

35. The method of claim 34, wherein said negatively-charged solute comprises at least one chemical selected from the group consisting of albumin, fibrinogen and negatively-charged dextran.

36. The method of claim 34, wherein said surfactant protein C is natural, recombinant, or synthetic.

37. The method of claim 36, wherein said surfactant protein C is synthetic and selected from sSP-Css-ion lock, sSP-Css-ion lock B sSP-Cff-leuc, and combinations thereof.

38. The method of claim 34, wherein said concentration of said surfactant protein C in the alveolar liquid after the performance of step (i) is in a range of from 0.0005 weight/volume percent to 1 weight/volume percent.

39. The method of claim 34, wherein said concentration of said surfactant protein C in the alveolar liquid after the performance of step (i) is in a range of from 0.00001 weight/volume percent to 1 weight/volume percent.

40. The method of claim 34, wherein said concentration of said surfactant protein C in the alveolar liquid after the performance of step (i) is in a range of from 0.00001 weight/volume percent to 0.1 weight/volume percent.

41. The method of claim 34, wherein said negatively-charged solute is present in the alveolar liquid in a concentration which is in a range of from 2.1 weight/volume percent to 11 weight/volume percent after the performance of step (i).

42. The method of claim 34, wherein said surfactant protein C is derived from an animal.

43. The method of claim 34, wherein said solution comprises a surfactant containing said surfactant protein C.

44. The method of claim 34, wherein said solution further comprises lipids.

45. The method of claim 34, wherein said alveolar liquid is contained in a patient's alveoli, said alveolar liquid having a surface tension which is lowered by the performance of step (i) such that the lowered surface tension of the alveolar liquid lessens ventilation-induced overdistension injury of the patient's lung.

46. The method of claim 45, wherein the lowered surface tension of the alveolar liquid reduces stress concentrations in the patient's lung.

47. The method of claim 46, wherein the patient's alveoli are flooded to substantially the same extent.

48. The method of claim 46, wherein the patient's alveoli are heterogeneously flooded, whereby the lowered surface tension of the alveolar liquid lessens ventilation-induced overdistension injury of intervening septa located between aerated and flooded alveoli

49. The method of claim 34, wherein step (i) comprises the step of administering said solution comprising said surfactant protein C to a trachea or bronchus of the patient having the king.

50. The method of claim 34, wherein said concentration of said surfactant protein C in the alveolar liquid after the performance of step (i) is in a range of from 0.00001 weight/volume % to 0.1 weight/volume percent.

51. The method of claim 34, wherein said concentration of said surfactant protein C in the alveolar liquid after the performance of step (i) is in a range of from 0.0005 weight/volume percent to 0.05 weight/volume percent.

52. The method of claim 34, wherein said concentration of said surfactant protein C in the alveolar liquid after the performance of step (i) is in a range of from 0.00001 weight/volume percent to 0.05 weight/volume percent.

53. The method of claim 34, wherein said concentration of said surfactant protein C in the alveolar liquid after the performance of step (i) is in a range of from 0.0001 weight/volume percent to 0.05 weight/volume percent.

54. The method of claim 34, wherein said concentration of said surfactant protein C in the alveolar liquid after the performance of step (i) is in a range of from 0.005 weight/volume percent to 0.05 weight/volume percent.

55. The method of claim 34, wherein said concentration of said surfactant protein C in the alveolar liquid after the performance of step (i) is in a range of from 0.0025 weight/volume percent to 0.05 weight/volume percent.

56. The method of claim 34, wherein said concentration of said surfactant protein C in the alveolar liquid after the performance of step (i) is in a range of from 0.00001 weight/volume percent to 0.01 weight/volume percent.

57. The method of claim 34, wherein said concentration of said surfactant protein C in the alveolar liquid after the performance of step (i) is in a range of from 0.0005 weight/volume percent to 0.01 weight/volume percent.

58. The method of claim 34, wherein said concentration of said surfactant protein C in the alveolar liquid after the performance of step (i) is in a range of from 0.0001 weight/volume percent to 0.01 weight/volume percent.

59. The method of claim 34, wherein said concentration of said surfactant protein C in the alveolar liquid after the performance of step (i) is in a range of from 0.005 weight/volume percent to 0.01 weight/volume percent.

60. The method of claim 34, wherein said concentration of said surfactant protein C in the alveolar liquid after the performance of step (i) is in a range of from 0.0025 weight/volume percent to 0.01 weight/volume percent.

61. The method of claim 34, wherein said concentration of said surfactant protein C in the alveolar liquid after the performance of step (i) is in a range of from 0.0005 weight/volume percent to 0.1 weight/volume percent.

62. The method of claim 34, wherein said concentration of said surfactant protein C in the alveolar liquid after the performance of step (i) is in a range of from 0.0001 weight/volume percent to 0.1 weight/volume percent.

63. The method of claim 34, wherein said concentration of said surfactant protein C in the alveolar liquid after the performance of step (i) is in a range of from 0.005 weight/volume percent to 0.1 weight/volume percent.

64. The method of claim 34, wherein said concentration of said surfactant protein C in the alveolar liquid after the performance of step (i) is in a range of from 0.0025 weight/volume percent to 0.1 weight/volume percent.

65. The method of claim 34, wherein step (i) is performed by injecting said solution into a circulatory system of a patient having the lung.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0028] For a more complete understanding of the present invention, reference is made to the following detailed description of an exemplary embodiment considered in conjunction with the accompanying drawings, in which:

[0029] FIG. 1A. Injury Score for Flooding Solutions in the Adult Rat Lung. In 275 control, aerated regions, instilled solutions are 0-5% albumin, 5% 70 kD dextran, 10 M NaOH or 5% 70 kD dextran plus 10 M NaOH, all in normal saline, without (n=24) or with (n=24) 1% SURVANTA. These solutions have surface tensions spanning the full range tested. In flooded regions, instilled liquid is normal saline with additives as specified (n=4/group). When SURVANTA is included, its concentration is 1%. Statistics: data shown as meanstandard deviation. All groups with flooding differ from control, aerated groups (p<0.01, statistics not shown on graph); *p<0.01 vs. no SURVANTA for same flooding liquid; #p<0.01 vs. 3% albumin plus SURVANTA and p<0.05 vs. 5% fibrinogen plus SURVANTA.

[0030] FIG. 1 B. Surface Tension for Flooding Solutions in the Adult Rat Lung. Surface tension in alveoli flooded with normal saline plus 31 M fluorescein and additives, as specified (n4/group). SURVANTA concentration is 0.9%. Statistics: *p<0.05 vs. no SURVANTA for same flooding liquid; #p<0.01 vs. no SURVANTA for same flooding liquid.

[0031] FIGS. 1C and 1D. Injury Score and Surface Tension for Flooding Solutions with Albumin Concentrations at the High End of the Effective Range in the

[0032] Adult Rat Lung. Flooding solution is normal saline plus solutes as specified and, in surface tension-determination experiments, 31 M fluorescein. SURVANTA concentrations are 1% for ventilation injury experiments and 0.9% for surface tension determination experiments. Control groups combine data for two albumin solutionsin the absence of SURVANTAwhose albumin concentrations bracket those of the solutions tested with SURVANTA and between which there is no difference in injury score or surface tension. Statistics: *p<0.05 vs. control group without SURVANTA; #p<0.01 vs. control group without SURVANTA.

[0033] FIG. 1E. Injury Score Data Plotted vs. Surface Tension Data. Open symbol is average of data groups between which neither injury score nor surface tension differ. R.sup.2=0.65.

[0034] FIG. 2A. Injury Score for Flooding Solutions Containing Specific Surfactant Components in the Presence of Albumin. Solutes are at about the same concentrations as present in 1% SURVANTA solution. The SP-B and SP-C used are isolated from pulmonary alveolar proteinosis patients; the SP-C, thus, is likely a mixture of fully-, partially- and non-palmitoylated peptide. Two forms of synthetic SP-C are used. One is sSP-Cff-leuc. The other is sSP-Css-ion lock-B. Only SP-C and sSP-C lower injury score, thus lower surface tension. Base solution for all groups is normal saline with 5% albumin. Due to pre-dissolution of certain solutes, at high concentration, in non-aqueous solvents, the final DPPC solution contains 2% methanol; the final SP-B and SP-C solutions contain 1.6% chloroform and 0.8% methanol; and the final sSP-Cff-leuc solution contains 1.3% chloroform and 1.3% ethanol. The sSP-Css-ion lock-B is dissolved directly in 5% albumin solution in normal saline, without additional solvents. n=1 or 2/group. From these results it appears that albumin facilitation of dilute SURVANTA solution is attributable to albumin-SP-C interaction.

[0035] FIG. 2B. Injury Score for Flooding Solutions Containing SP-C, With and Without Albumin and DPPC. In normal saline without albumin, SP-C fom pulmonary alveolar proteinosis patients loses its ability to lower injury score, thus lower surface tension. Inclusion of DPPC does not restore the ability of SP-C to lower injury score, thus surface tension, in the absence of albumin. Base solution for all groups is normal saline. Due to pre-dissolution of certain solutes, at high concentration, in non-aqueous solvents, the final SP-C solutions, with or without albumin, contain 1.6% chloroform and 0.8% methanol; and the final SP-C plus DPPC solution contains 1.6% chloroform and 2.8% methanol. n =1 or 2/group. From these results it appears that isolated SP-C is surface active only when facilitated by albumin.

[0036] FIG. 3. Opening Pressure for Tracheal Instillation Solutions in the Immature Fetal Rat Lung. Solution (4-5 l), with solutes as specified, is instilled in the trachea of the fluid-filled immature (embryonic day 18 or 19) fetal rat lung. The pressure required to inflate the fetal rat lung for the first time is proportional to surface tension. Base solution is normal saline excepting that base solution is Ringer's solution for 1% SURVANTA plus 5% albumin. The solution of 0.0005% SP-C plus 5% albumin additionally includes 1.6% chloroform and 0.8% methanol. The sSP-Css-ion lock used is not biotinylated and is dissolved directly in normal saline without additional solvents. n=1 or 2/group.

[0037] FIG. 4. Surface Tension for Solutions In Vitro. Surface tension of normal saline drops (3 l) containing 31 M fluorescein and additional solutes as specified. The SP-C solution additionally contains 1.6% chloroform and 0.8% methanol. The fourth, sixth and seventh bars show that, in the presence of 5% albumin, surface tension decreases with increasing SURVANTA concentration. The third, fourth and fifth bars demonstrate that there is an optimal albumin concentration of 5% for the facilitation of 1% SURVANTA. n=2 or 3/group.

DETAILED DESCRIPTION OF THE EXEMPLARY EMBODIMENT

[0038] The present invention overcomes the disadvantages and shortcomings discussed above. Four alternative methods are used to assess the surface activity of test solutions:

[0039] 1. direct surface tension determination in surface alveoli of the isolated adult rat lung, which contains native lung surfactant, following alveolar instillation of a test solution;

[0040] 2. degree of ventilation induced injury in a region of the isolated, perfused adult rat lung in which surface alveoli have been flooded, in a heterogeneous fashion, with a test solution;

[0041] 3. initial inflation pressure of the immature fetal rat lung, which contains a reduced quantity of native surfactant compared with the adult lung, following tracheal instillation of a test solution; and 4. surface tension determination in a normal saline drop containing known quantities of additional solutes and no lipids other than any that are added to it.

[0042] The concentrations of SURVANTA discussed below are in volume percent (vol %) based on the total volume of the liquid in which the SURVANTA is dispersed. The concentrations of SP-C, whether natural, recombinant or synthetic, discussed below are in weight/volume percent (w/v %) based on the weight (in grams) of SP-C dispersed and the volume (in tenths-of-liters) of liquid in which the SP-C is dispersed.

[0043] The concentrations of the negatively charged solute (e.g., albumin, fibrinogen or negatively charged 70 kD dextrin) discussed below are also in weight/volume percent (w/v %) based on the weight (in grams) of the negatively charged solute dispersed and the volume (in tenths-of-liters) of liquid in which the negatively charged solute is dispersed.

[0044] In practice, with human patients having an edematous lung and receiving mechanical ventilation, the volume of liquid of concern in the body (i.e., the volume of liquid in which either SP-C or a negatively charged solute, or both, is to be dispersed) is understood to be the sum of the edema liquid and blood plasma. Persons of ordinary skill in the art will be familiar with this principle and capable of calculating an estimated volume for a particular patient in need of receiving treatment, as well as calculating the therapeutically effective amount of SP-C or negatively charged solute necessary which will provide the concentrations discussed below.

[0045] The following discussion is intended to provide guidance without limiting the methods described and contemplated herein. Functional residual capacity (FRC), which is the air volume in the lung at the end of expiration, averages 2.3 liters (L) in humans. In pulmonary edema, permeability of the lung capillaries is elevated such that solutes, such as SP-C and a negatively charged solute, can pass between the alveolar edema liquid and the blood plasma. Therefore, the plasma volume, which averages 3 L, should be included in the calculation of the volume of concern. As recognized by persons of ordinary skill in the art, a particular human patient's weight and hematocrit values will aid in estimating the plasma volume.

[0046] Assuming that, in a human patient with pulmonary edema, somewhere between 5 and 80% of FRC were flooded with liquid, then the total volume of edema liquid would be 0.12-1.8 L (based on the average 2.3 L mentioned above) and the total volume of edema liquid plus blood plasma would be 3.1-4.8 L. This would be the total volume of the liquid of concern upon which to base further calculations of the range of amounts of SP-C and the negatively charged solute that would be required to be therapeutically effective at providing the concentrations discussed below. More particularly, therapeutically effective amounts of SP-C and the negatively charged solute are those amounts that, directly or indirectly, minimize mechanical ventilation injury to an edematous lung by reducing the surface tension of alveolar liquid so that stress concentrations and alveolar flooding heterogeneity are reduced. As surprisingly discovered and described herein, therapeutically effective amounts of SP-C and the negatively charged solute are those amounts that provide the concentrations of these substances in the volume of liquid of concern as discussed hereinbelow because those concentrations of SP-C and the negatively charged solute reduce the surface tension of alveolar liquid.

[0047] From the results of testing based on the four methods described in the background above, it has been found that:

[0048] 1. A dilute solution of 1 vol % SURVANTA in normal saline is not surface 405 active, but that solutions of 1-5 vol % SURVANTA are surface active when facilitated by 5% albumin solution. Further, based on the test results reported in FIG. 2, it is the SP-C in SURVANTA that interacts with albumin; solutions of various forms of natural and synthetic SP-C at concentrations comparable to that in 1 vol % SURVANTA are surface active in the adult or fetal rat lung, but only when facilitated by albumin solution. A solution of SP-C plus albumin tends to lose its surface activity in vitro in the absence of lipids, however, suggesting the SP-C and albumin, together, may reduce surface tension by promoting the adsorption of surfactant lipids.

[0049] Dilute surfactant solution or SP-C solution, where the SP-C may be natural, recombinant or synthetic, could be administered intratracheally in some embodiments. With sufficient albumin present in the alveolar liquid, dilute surfactant or SP-C solution could simply be administered in buffer (normal saline, Ringer's solution, physiologic saline solution, or equivalent). Without sufficient albumin present, a facilitating negatively charged solute (e.g., albumin, fibrinogen, negatively charged dextran or alternative negatively charged solute) could be added to the administered composition. The surfactant in the dilute surfactant solution may be SURVANTA or another surfactant isolated from an animal that comprises SP-C. Suitable recombinant or synthetic SP-C are reasonably believed to include any of those identified in U.S. Patent Application Publication No. 2015/0125515, which has already been mentioned and incorporated herein by reference above.

[0050] Dilute surfactant solution or SP-C solution, where the SP-C may be natural, recombinant or synthetic, could alternatively be administered intravascularly, in the absence or presence of exogenous albumin or of an alternative negatively charged facilitating solute. Again, the surfactant in the dilute surfactant solution may be SURVANTA or another surfactant isolated from an animal that comprises SP-C.

[0051] 2. Albumin or alternative negatively charged solutes (e.g., fibrinogen, negatively charged 70 kD dextran) facilitate the surface activity of dilute SURVANTA solution containing SP-C. The surface activity of the dilute SURVANTA solution in normal saline was assessed by three of the four surface tension determination methods discussed in the background above.

[0052] Albumin concentrations of 3-11 w/v % facilitate the surface activity of 1 vol % SURVANTA solution in the adult rat lung. Alternatively, 5 w/v % fibrinogen or 5 w/v % negatively charged 70 kD dextran (negative charge imparted by inclusion of 10 M NaOH) also facilitate 1 vol % SURVANTA. In contrast, 5 w/v % neutral 70 kD dextran does not facilitate 1 vol % SURVANTA. Thus osmotic pressure is not sufficient to facilitate 1 vol % SURVANTA; a negatively charged solute is required. Control experiments have shown 10 M NaOH alone, without dextran, has no effect on surface tension or lung injury in the absence or presence of SURVANTA (see FIGS. 1A and 1B).

[0053] In vitro, albumin is likewise required to facilitate the surface activity of SURVANTA. However, the albumin concentration range that facilitates 1 vol % SURVANTA does not extend to 10 vol %. Further, as shown by the data in FIG. 4, dose-response experiments demonstrate that, in conjunction with 5 w/v % albumin, 5 vol % SURVANTA lowers surface tension more than 1 vol % SURVANTA.

[0054] 3. Surfactant protein C is the SURVANTA component that interacts with 450 albumin to lower surface tension. SURVANTA contains 2.5% total phospholipids, which includes 1.1-1.6% DPPC, and <0.1% of SP-B and SP-C combined, with a concentration of SP-C that is up to 15 times that of SP-B. Thus 1% SURVANTA solution contains 0.025% total phospholipids, 0.01% DPPC, <0.001% SP-C and <<0.001% SP-B. The surface activity of solutions containing DPPC, SP-B and SP-C, in concentrations comparable to those in 1 vol % SURVANTA solution, was assessed by ventilation injury assay (method #2). In conjunction with 5 w/v % albumin solution, only solutions with SP-C or sSP-C lower injury score, thus surface tension. Surfactant protein C alone, without albumin, is not surface active. Neither is the combination of SP-C and DPPC, in the absence of albumin, surface active.

[0055] 4. The combination of SP-C and albumin lowers surface tension in the presence of at least a low concentration of surfactant lipids. The combination of SP-C, or sSP-C, and albumin lowers surface tension in the adult rat lung with normal levels of native surfactant and in the immature fetal rat lung with reduced surfactant levels. The combination of SP-C and albumin is likewise surface active in in vitro tests of dilute 1-5 vol % SURVANTA containing only 0.03-0.13 w/v % total phospholipids (compared with 2.5 w/v % in undiluted SURVANTA). Thus the combination of SP-C and albumin is surface active in the presence of low lipid concentrations. However, the combination of SP-C and albumin in the absence of any lipids demonstrates only low surface activity in vitro. The combination of SP-C and albumin appears to be highly effective at promoting 470 lipid adsorption, but to require the presence of at least a low lipid level for surface activity.

[0056] By extension of the above findings, it is expected that a concentration of from greater than about 2 w/v % to less than about 12 w/v % of a negatively charged solute (e.g., albumin, fibrinogen, and negatively charged 70 kD dextran), will facilitate the surface activity of a concentration of at least 0.01 vol % SURVANTA or other surfactants isolated from animals; or of from about 0.000001 w/v % to about 1 w/v % SP-C, whether natural, recombinant or synthetic, in the presence of at least low levels of surfactant lipids, the relative proportions of which might be the same as or different from that in natural lung surfactant. This effect is reasonably expected regardless of whether the negatively charged solute is included in the delivered composition or already present, for example, in edema liquid or blood plasma. The recombinant or synthetic SP-C peptides that could be used include sSP-Css ion lock; sSP-Css ion lock-B; sSP-Css-ion lock with biotin tags(s) in alternative locations; sSP-Cff-leuc; biotinylated sSP-Cff-leuc; or alternative variations of natural human or animal SP-C.

[0057] In some embodiments, the therapeutically effective concentration of SP-C (natural, recombinant or synthetic) in the liquid of concern may be, for example without limitation, from about 0.00001 w/v % to about 1 w/v %, or from about 0.0005 w/v % to about 1 w/v %, or from about 0.0001 w/v % to about 1 w/v %, or from about 0.005 to about 1 w/v %, or from about 0.0025 w/v % to about 1 w/v %, or from about 0.00001 w/v % to about 0.05 w/v %, or from about 0.0005 w/v % to about 0.05 w/v %, or from about 0.0001 w/v % to about 0.05 w/v %, or from about 0.005 to about 0.05 w/v %, or from about 0.0025 w/v % to about 0.05 w/v %, 0.00001 w/v % to about 0.01 w/v %, or from about 0.0005 w/v % to about 0.01 w/v %, or from about 0.0001 w/v % to about 0.01 w/v %, or from about 0.005 to about 0.01 w/v %, or from about 0.0025 w/v % to about 0.01 495 w/v %, or from about 0.00001 w/v % to about 0.1 w/v %, or from about 0.0005 w/v % to about 0.1 w/v %, or from about 0.0001 w/v % to about 0.1 w/v %, or from about 0.005 to about 0.1 w/v %, or from about 0.0025 w/v % to about 0.1 w/v %,

[0058] In some embodiments, the therapeutically effective concentration of the negatively charged solute in the liquid of concern may be, for example without limitation, from about 2.1 w/v % to about 11.9 w/v %, or from about 2.5 w/v % to about 11.9 w/v %, or from about 3 w/v % to about 11.9 w/v %, or from about 4 or from about 3 w/v % to about 11.9 w/v %, or from about 5 w/v % to about 11.9 w/v %, or from about 6 w/v % to about 11.9 w/v %, or from about 2.1 w/v % to about 11.5 w/v %, or from about 2.1 w/v % to about 11 w/v %, or from about 2.1 w/v % to about 10 w/v %, or from about 2.1 w/v % to about 9 w/v %, or from about 2.5 w/v % to about 11.5 w/v %.

TABLE-US-00001 TABLE1 SEQUENCELISTING <110> StevensInst.ofTech. Perlman,Carrie <120> DILUTESURFACTANTORISOLATEDSURFACTANT PROTEINSOLUTIONFORTHEREDUCTIONOFSURFACE TENSIONINTHELUNG <150> US15/194,096 <151> 2016Jun.27 <130> 101995.043102 <160> 2 <170> PatentInversion3.5 <210> 1 <211> 34 <212> PRT <213> ArtificialSequence <220> <223> sSP-Css-ionlock <400> 1 GlyIleProSerSerProValHisLeuLysArgLeu 1510 LeuIleValValValValValGluLeuIleValLys 1520 ValIleValGlyAlaLeuLeuMetGlyLeu 2530 <210> 2 <211> 34 <212> PRT <213> ArtificialSequence <220> <223> sSP-Cff-leuc <400> 2 GlyIleProPhePheProValHisLeuLysArgLeu 1510 LysLeuLeuLeuLeuLeuLeuLeuLeuIleLeuLeu 1520 LeuIleLeuGlyAlaLeuLeuMetGlyLeu 2530