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Last Updated: March 28, 2024

Claims for Patent: 5,013,720


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Summary for Patent: 5,013,720
Title: SAP-6-Val proteins and methods
Abstract:A novel hydrophobic surfactant-associated protein mixture, i.e., a SAP-6 proteins, has been isolated from pulmonary animal tissue. A small, novel pulmonary hydrophobic surfactant-associated SAP-6-Val protein having a molecular weight of about 6,000 daltons as determined by SDS-PAGE and about 3,500-4,000 daltons as determined by tricine-SDS-PAGE has been further isolated from the SAP-6 protein mixture. The amino acid residue compositions of the SAP-6-Val protein for human and bovine have been determined and disclosed. When a SAP-6-Val protein is combined with phospholipids, it enhances the surfactant-like activity of the phospholipids in lungs of animals and, therefore, uniquely imparts to the mixture significant pulmonary biophysical activity. Such a mixture results in enhanced adsorption of the phospholipids with properties similar to that of natural pulmonary surfactant material. SAP-6-Val proteins in combination with phospholipids is highly useful for replacing or supplementing natural pulmonary surfactant material for reducing or maintaining normal surface tension in lungs, and especially in lungs of patients suffering from hyaline membrane disease, HMD, or other syndromes associated with the lack or insufficient amounts of natural pulmonary surfactant material. A mixture of a SAP-6-Val protein and phospholipids may be administered as an aerosol spray or in aqueous normal saline with or without calcium chloride for treating or preventing HMD and other surfactant deficiency states. Also disclosed are methods of isolating the noncanine SAP-6-Val proteins from the SAP-6 proteins isolated from animal tissue.
Inventor(s): Whitsett; Jeffrey A. (Cincinnati, OH)
Assignee: Abbott Laboratories (N/A)
Application Number:07/504,691
Patent Claims:1. An isolated lung specific hydrophobic SAP-6-Val protein, said protein comprises a plurality of valine amino acid residues having a first stretch of about six adjacent valine amino acid residues and a second stretch of about three adjacent valine amino acid residues separated from the first stretch by about two hydrophobic amino acid residues, said protein enhances surfactant-like activity of phospholipids in lungs of an animal, has a molecular weight of about 3,500-4,000 daltons as determined by tricine-SDS-PAGE and a molecular weight of about 6,000 daltons as determined by SDS-PAGE, and is substantially resistant to protease, endoglycosidase F and collagenase enzymes.

2. An isolated SAP-6-Val protein of claim 1, said protein having at least about 11 valine amino acids.

3. An isolated SAP-6-Val protein of claim 1, said protein being a human SAP-6-Val protein.

4. An isolated SAP-6-Val protein of claim 1, said protein being a bovine SAP-6-Val protein.

5. An isolated SAP-6-Val protein of claim 1, said protein being selected from a class consisting of canine, porcine, rabbit and rat SAP-6-Val proteins.

6. A SAP-6-Val protein of claim 1 said protein being a synthetically produced SAP-6-Val protein.

7. An isolated lung specific hydrophobic surfactant-associated human protein having a molecular weight of about 3,500-4,000 daltons as determined by tricine-SDS-PAGE, said human protein enhancing surfactant-like activity of phospholipids in lungs of an animal and comprising the following amino acid sequence: ##STR6##

8. An isolated lung specific hydrophobic surfactant-associated human protein having a molecular weight of about 3,500-4,000 daltons as determined by tricine-SDS-PAGE, said human protein enhancing surfactant-like activity of phospholipids in lungs of an animal and comprising the following amino acid sequence: ##STR7##

9. An isolated lung specific hydrophobic surfactant-associated bovine protein having a molecular weight of about 3,500-4,000 daltons as determined by tricine-SDS-PAGE, said bovine protein enhancing surfactant-like activity of phospholipids in lungs of an animal and comprising the following amino acid sequence: ##STR8##

10. A medicament for reducing or maintaining normal pulmonary surface tension in the alveoli of an animal comprising an isolated SAP-6-Val protein as recited in claim 1, a phospholipid and a pharmaceutically acceptable carrier, said SAP-6-Val protein and said phospholipid being present in an effective amount to reduce or maintain normal pulmonary surface tension in the alveoli of the animal.

11. A medicament of claim 10, said medicament further including multimers of the SAP-6-Val protein.

12. A medicament of claim 10, said phospholipid being selected from the group consisting of a synthetic phospholipid and a naturally occurring phospholipid.

13. A medicament of claim 12, said phospholipid being selected from the group consisting of phosphatidylcholine, disaturated phosphatidylcholine, phosphotidylglycerol, dipalmitoylphosphatidylcholine, phosphatidylinositol and mixtures thereof.

14. A medicament of claim 10, said SAP-6-Val protein being a synthetically produced SAP-6-Val protein.

15. A preparation for replacing pulmonary surfactant material in lungs of an animal comprising an isolated SAP-6-Val protein as recited in claim 1 and a phospholipid, both of which are present in said preparation in an effective amount.

16. A preparation of claim 15, said preparation further including multimers of the SAP-6-Val protein.

17. A preparation of claim 15, said SAP-6-Val protein being a synthetically produced protein.

18. A preparation of claim 15, said phospholipid being selected from the group consisting of a synthetic phospholipid and a naturally occurring phospholipid.

19. A preparation of claim 18, said phospholipid being selected from the group consisting of phosphatidylcholine, disaturated phosphatidylcholine, phosphatidylglycerol, dipalmitoylphosphatidylcholine, phosphatidylinositol and mixtures thereof.

20. A method of treating or preventing hyaline membrane disease or other syndromes associated with insufficient pulmonary surfactant material in lungs of an animal comprising:

administering to the animal an effective dose comprising an isolated SAP-6-Val protein as recited in claim 1 and a phospholipid to reduce or maintain normal pulmonary surface tension.

21. A method of claim 20, said administration step comprises administering the effective dose as a liquid or as an aerosol spray.

22. A method of claim 20, the effective dose further including multimers of SAP-6-Val protein.

23. A method of claim 20, the SAP-6-Val protein being a synthetically produced SAP-6-Val protein.

24. A method of isolating from animal pulmonary or amniotic tissue a lung specific hydrophobic surfactant-associated SAP-6-Val protein for enhancing surfactant-like activity of phospholipids in lungs of an animal, the SAP-6-Val protein having a molecular weight of about 6,000 daltons as determined in a polyacrylamide gel containing sodium dodecyl sulfate and being substantially resistant to protease, endoglycosidase F, and collagenase enzymes, said method comprises the steps of:

separating from the animal pulmonary or amniotic tissue a cell fraction and a liquid supernatant;

separating from the liquid supernatant a particulate fraction containing the SAP-6-Val protein;

separating from the particulate fraction with an organic solvent a liquid fraction which contains the SAP-6-Val protein;

concentrating the liquid fraction to generate a concentrate which contains the SAP-6-Val protein;

dispersing the concentrate in a second organic solvent to form a dispersion;

applying the dispersion to a separating column;

eluting the separating column with a first eluting solvent comprising an alcohol and chloroform wherein the concentration of alcohol is less than about 40%;

eluting the separating column with a second eluting solvent comprising an alcohol and chloroform wherein the concentration of alcohol in the second eluting solvent ranges from about 40% up to about 100% to isolate in the second eluting solvent SAP-6-Val protein; and

dialyzing the second eluting solvent to isolate the SAP-6-Val protein therefrom in substantially pure form.

25. A method of claim 24, said first eluting step comprising:

eluting the separating column in stepwise increments with selected solvents in series wherein the selected solvents comprise an alcohol and chloroform wherein the alcohol concentration in each selected solvent of the series is continuously increased from 0% up to less than about 40% with each of the stepwise increments.

26. A method of claim 24, said second eluting step comprising:

eluting the separating column in stepwise increments with selected solvents in series wherein the selected solvents comprise an alcohol and chloroform wherein the alcohol concentration in each selected solvent of the series is continuously increased from about 40% up to about 100% with each of the stepwise increments.

27. A method of claim 26, the SAP-6-Val protein being eluted during said second eluting step in the selected solvents having an alcohol concentration of about 40% up to about 80%.

28. A method of claim 24, said dialyzing step comprising:

evaporating the second eluting solvent to generate a concentrated fraction containing the SAP-6-Val protein;

resuspending the concentrated fraction in a solvent; and

purifying the resuspended fraction to remove therefrom contaminating lipids and proteins to produce the substantially pure SAP-6-Val protein.

29. A substantially pure lung specific hydrophobic surfactant-associated SAP-6-Val protein isolated from animal pulmonary or amniotic tissue having a hydrophobic region of amino acids which includes a first stretch of about 6 adjacent valine amino acids and a second stretch of about 3 adjacent valine amino acids separated from the first stretch by about 2 hydrophobic amino acid residues for enhancing surfactant-like activity of phospholipids in lungs of an animal, said SAP-6-Val protein further having a molecular weight of about 6,000 as daltons determined in a polyacrylamide gel containing sodium dodecyl sulfate and a molecular weight of about 3,500-4,000 daltons as determined by tricine-SDS-PAGE and being substantially resistant to protease, endoglycosidase F, and collagenase enzymes, said SAP-6-Val protein being produced by a process comprising the steps of:

separating from the animal pulmonary or amniotic tissue a particulate fraction which contains said SAP-6-Val protein;

obtaining from the particulate fraction with an organic solvent a liquid fraction which contains said SAP-6-Val protein;

concentrating the liquid fraction, thereby obtaining a surfactant material which contains said SAP-6-Val protein;

dispersing the concentrated surfactant material in chloroform to form a dispersion;

applying the chloroform dispersion to a separating column;

eluting the separating column with a first eluting solvent comprising an alcohol and chloroform wherein the concentration of alcohol is less than about 40%;

eluting the separating column with a second eluting solvent comprising an alcohol and chloroform wherein the concentration of alcohol in the second eluting solvent ranges from about 40% up to about 100% to isolate in the second solvent said SAP-6-Val protein; and

dialyzing the second eluting solvent to isolate said SAP-6-Val protein therefrom in substantially pure form.

30. A substantially pure lung specific hydrophobic surfactant-associated SAP-6-Val protein produced by the process of claim 29, said dialyzing step comprising:

evaporating the second eluting solvent to generate a concentrated fraction containing the SAP-6-Val protein;

resuspending the concentrated fraction in a solvent; and

purifying the resuspended fraction to remove therefrom contaminating lipids and proteins to produce the substantially pure SAP-6-Val protein.

31. A substantially pure lung specific hydrophobic surfactant-associated SAP-6-Val protein produced by the process of claim 29, the animal pulmonary or amniotic tissue being selected from a class consisting of canine, porcine, rabbit and rat pulmonary or amniotic tissue.

32. A substantially pure lung specific hydrophobic surfactant-associated SAP-6-Val protein produced by the process of claim 29, the animal pulmonary or amniotic tissue being bovine pulmonary or amniotic tissue.

33. A substantially pure lung specific hydrophobic surfactant-associated SAP-6-Val protein produced by the process of claim 29, the animal pulmonary or amniotic tissue being human pulmonary or amniotic tissue.

34. A substantially pure lung specific hydrophobic surfactant-associated SAP-6-Val protein produced by the process of claim 29, said first eluting step comprising:

eluting the separating column in stepwise increments with selected solvent in series wherein the selected solvents comprise an alcohol and chloroform wherein the alcohol concentration in each selected solvent of the series is continuously increased from 0% to less than about 40% with each of the stepwise increments.

35. A substantially pure lung specific surfactant-associated SAP-6-Val protein produced by the process of claim 29, said second eluting step comprising:

eluting the separating column in stepwise increments with selected solvents in series wherein the selected solvents comprise an alcohol and chloroform wherein the alcohol concentration in each selected solvent of the series is continuously increased from about 40% up to less than about 100% with each of the stepwise increments.

36. A substantially pure lung specific hydrophobic surfactant-associated SAP-6-Val protein produced by the process of claim 29, said SAP-6-Val protein being eluted during said second eluting step in the selected solvents having an alcohol concentration of about 40% up to about 80%.

37. A substantially pure lung specific hydrophobic surfactant-associated human SAP-6-Val protein isolated from human pulmonary or amniotic tissue having a hydrophobic region of amino acids which includes a first stretch of about 6 adjacent valine residues and a second stretch of about 3 valine residues separated from the first stretch by about 2 hydrophobic amino acid residues for enhancing surfactant-like activity of phospholipids in lungs of an animal, said protein further having a simple molecular weight of about 6,000 daltons as determined in a polyacrylamide gel containing sodium dodecyl sulfate and a molecular weight of about 3,500-4,000 daltons as determined by tricine-SDS-PAGE and being substantially resistant to protease, endoglycosidase F, and collagenase enzymes, said human SAP-6-Val protein being produced by a process comprising the steps of:

separating from the human pulmonary or amniotic tissue a cell fraction and a liquid supernatant;

separating from the liquid supernatant a particulate fraction containing said human SAP-6-Val protein;

separating from the particulate fraction with chloroform a liquid fraction which contains said human SAP-6-Val protein;

concentrating the liquid fraction to generate a concentrate which contains said human SAP-6-Val protein;

dispersing the concentrate in chloroform to form a dispersion;

applying the chloroform dispersion to a separating column;

eluting the separating column with a first eluting solvent comprising an alcohol and chloroform wherein the alcohol concentration in the selected solvent is less than about 40%;

eluting the separating column in stepwise increments with selected solvents in series wherein the selected solvents comprise an alcohol and chloroform wherein the alcohol concentration in each selected solvent of the series is continuously increased from about 40% up to about 80% with each of the stepwise increments to isolate in these selected elution solvents of the series said human SAP-6-Val protein; and

dialyzing the selected eluting solvents of the series containing from about 40% up to about 80% alcohol to isolate said human SAP-6-Val protein therefrom in substantially pure form.

38. An isolated hydrophobic surfactant-associated SAP-6-Val protein, said SAP-6-Val protein enhancing surfactant-like activity of phospholipids in lungs of an animal, having a simple molecular weight of about 6,000 daltons determined in a polyacrylamide gel containing sodium dodecyl sulfate and being substantially resistant to protease, endoglycosidase F and collagenase enzymes.

39. A medicament for reducing or maintaining normal pulmonary surface tension in the alveoli of an animal comprising a mixture of an isolated human SAP-6-Val protein as recited in claim 1, phospholipids and a pharmaceutically acceptable carrier, said SAP-6-Val protein and said phospholipids being present in an effective amount to reduce or maintain normal pulmonary surface tension in the alveoli of the animal.

40. A medicament of claim 39, said phospholipids being selected from the group consisting of synthetic phospholipids, naturally occurring phospholipids and mixtures thereof.

41. A medicament of claim 40, said phospholipids being selected from the group consisting of phosphatidylcholine, disaturated phosphatidylcholine, phosphotidylglycerol, dipalmitoylphosphatidycholine, phosphatidylinositols and mixtures thereof.

42. A preparation for replacing pulmonary surfactant material in lungs of an animal comprising a mixture of an isolated SAP-6-Val protein as recited in claim 1 and phospholipids, both said SAP-6-Val

proteins and phospholipids being present in said preparation in an effective amount.

43. A preparation of claim 42, said phospholipids being selected from the group consisting of synthetic phospholipids, naturally occurring phospholipids and mixtures thereof.

44. A preparation of claim 43, said phospholipids being selected from the group consisting of phosphatidycholine, disaturated phosphatidylcholine, phosphatidyglycerol, dipalmitoylphosphatidycholine, phosphatidylinositol and mixtures thereof.

45. An isolated, human hydrophobic surfactant-associated protein, said human protein enhancing surfactant-like activity of phospholipids in lungs of an animal and having the following sequence: ##STR9##

46. A substantially pure lung specific hydrophobic surfactant-associated human SAP-6-Val protein produced by the process of claim 37, said dialyzing step comprising:

evaporating the selected eluting solvents to generate a concentrated fraction containing the human SAP-6-Val protein;

resuspending the concentrated fraction in a solvent; and

purifying the resuspended fraction to remove therefrom contaminating lipids and proteins to produce the substantially pure human SAP-6-Val protein.

47. An isolated lung specific hydrophobic surfactant-associated canine protein having a molecular weight of about 6,000 daltons as determined by SDS-PAGE and a molecular weight of about 3,500-4,000 daltons as determined by tricine-SDS-PAGE, said canine protein enhancing surfactant-like activity of phospholipids in lungs of an animal and comprising the following amino acid sequence: ##STR10##

48. A medicament of claim 10, said isolated SAP-6-Val protein being selected from a class consisting of isolated bovine, canine, porcine, rabbit and rat SAP-6-Val proteins.

49. A preparation of claim 15, said isolated SAP-6-Val protein being selected from a class consisting of isolated bovine, canine, procine, rabbit and rat SAP-6-Val proteins.

50. A method of claim 20, the isolated SAP-6-Val protein being an isolated human SAP-6-Val protein.

51. A method of claim 20, the isolated SAP-6-Val protein being selected from a class consisting of isolated bovine, canine, procine, rabbit and rat SAP-6-Val proteins.

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