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Last Updated: September 22, 2021

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Claims for Patent: 5,387,746

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Summary for Patent: 5,387,746
Title: Pulmonary hydrophobic surfactant-associated protein of 6,000 daltons molecular weight and multimers thereof
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: Scios Nova Inc. (Mountain View, CA)
Application Number:07/975,202
Patent Claims:1. A lung specific hydrophobic SAP-6-Val recombinant protein, said recombinant 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 of about three adjacent valine amino acid residues separated from the first stretch by about two hydrophobic amino acid residues, said recombinant protein enhances surfactant-like activity of phospholipids in lungs of an animal, and is substantially resistant to protease, endoglycosidase F and collagenase enzymes.

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

3. A recombinant protein of claim 1 said recombinant protein having a sequence which corresponds to a human SAP-6-Val protein.

4. A SAP-6-Val recombinant protein of claim 1 said recombinant protein having a sequence which corresponds to a bovine SAP-6-Val protein.

5. A SAP-6-Val recombinant protein of claim 1 said recombinant protein being selected from a class of recombinant proteins having sequences corresponding to porcine, rabbit and rat SAP-6-Val proteins.

6. A lung specific hydrophobic surfactant-associated recombinant protein of claim 1, said recombinant protein enhancing surfactant-like activity of phospholipids in lungs of an animal and comprising the following amino acid sequence which corresponds to human: ##STR7##

7. A lung specific hydrophobic surfactant-associated recombinant protein of claim 1, said recombinant protein having a molecular weight of about 3,500-4,000 daltons as determined by tricine-SDS-PAGE, said recombinant protein enhancing surfactant-like activity of phospholipids in lungs of an animal and comprising the following amino acid sequence which corresponds to human: ##STR8##

8. A lung specific hydrophobic surfactant-associated recombinant protein of claim 1, said recombinant protein having a molecular weight of about 3,500-4,000 daltons as determined by tricine-SDS-PAGE, said recombinant protein enhancing surfactant-like activity of phospholipids in lungs of an animal and comprising the following amino acid sequence which corresponds to bovine: ##STR9##

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

10. A medicament of claim 9, said medicament further including multimers of the SAP-6-Val recombinant protein.

11. A medicament of claim 8, said phospholipid being selected from the group consisting of a synthetic phospholipid and a naturally occurring phospholipid.

12. A medicament of claim 11, said phospholipid being selected from the group consisting of phosphatidylcholine, disaturated phosphatidylcholine, phosphotidylglycerol, dipalmitoylphosphatidylcholine, phophatidylinositol and mixtures thereof.

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

14. A preparation of claim 13, said preparation further including multimers of the SAP-6-Val recombinant protein.

15. A preparation of claim 13, said phospholipid being selected from the group consisting of a synthetic phospholipid and a naturally occurring phospholipid.

16. A preparation of claim 15, said phospholipid being selected from the group consisting of phosphatidylcholine, disaturated phosphatidylcholine, phosphatidylglycerol, dipalmitoylphosphatidylcholine, phosphatidylinositol and mixtures thereof.

17. 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 a SAP-6-Val recombinant protein as recited in claim 1 and a phospholipid to reduct or maintain normal pulmonary surface tension.

18. A method of claim 17 said administration step comprises administering the effective dose as a liquid or as an aerosol spray.

19. A method of claim 17, the effective dose further including multimers of the SAP-6-Val recombinant protein.

20. A hydrophobic surfactant-associated SAP-6-Val recombinant protein, said SAP-6-Val recombinant 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.

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

22. A medicament of claim 21, said phospholipids being selected from the group consisting of synthetic phospholipids, naturally occurring phospholipids and mixtures thereof.

23. A medicament of claim 22, said phospholipids being selected from the group consisting of phosphatidylcholine, disaturated phosphatidylcholine, phosphotidylglycerol, dipalmitoylphosphatidylcholine, phosphatidylinositols and mixtures thereof.

24. A preparation for replacing pulmonary surfactant material in lungs of an animal comprising a mixtures of a SAP-6-Val recombinant 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.

25. A preparation of claim 24, said phospholipids being selected from the group consisting of synthetic phospholipids, naturally occurring phospholipids and mixtures thereof.

26. A preparation of claim 25, said phospholipids being selected from the group consisting of phosphatidylcholine, disaturated phosphatidylcholine, phosphatidylglycerol, dipalmitoylphosphatidylcholine, phosphatidylinositol and mixtures thereof.

27. An isolated lung specific hydrophobic surfactant-associated protein for enhancing surfactant-like activity of phospholipids in lungs of an animal, said protein having a polyvaline domain of valine amino acid residures, said polyvaline domain containing 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 being of a sufficient size for enhancing the surfactant-like activity of phospholipids in lungs of an animal.

28. An isolated lung specific hydrophobic surfactant-associated protein of claim 27, said hydrophobic amino acid residues being selected from a group consisting of leucine, isoleucine and valine.

29. An isolated lung specific hydrophobic surfactant-associated protein of claim 27 wherein said protein is a synthetic protein.

30. A medicament for reducing or maintaining normal pulmonary surface tension in the alveoli of an animal comprising

an isolated lung specific hydrophobic surfactant-associated protein as recited in claim 27, a phospholipid and a pharmaceutically acceptable carrier, said protein and said phospholipid each being present in an effective amount to reduce or maintain normal pulmonary surface tension in the alveoli of the animal.

31. A preparation for replacing pulmonary surfactant material in lungs of an animal comprising an isolated lung specific hydrophobic surfactant-associated protein as recited in claim 27 and a phospholipid, both of which are present in said preparation in an effective amount.

32. 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 a substantially isolated lung specific hydrophobic surfactant-associated protein as recited in claim 27 to reduce or maintain normal pulmonary surface tension.

33. A substantially pure lung specific hydrophobic surfactant-associated SAP-6 protein composition for enhancing surfactant-like activity of phospholipids in lungs of animals, said SAP-6 protein composition consisting essentially of:

a) a lung specific hydrophobic surfactant-associated SAP-6-Val protein, said SAP-6-Val protein having a polyvaline domain of valine amino acid residues, said polyvaline domain containing 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 SAP-6-Val protein having 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 being substantially resistant to protease, endoglycosidase F and collagenase enzymes; and

b) a second lung specific hydrophobic surfactant-associated SAP-6 protein, said second SAP-6 protein having a molecular weight of about 6,000 daltons as determined by SDS-PAGE and being resistant to protease, endoglycosidase F and collagenase enzymes.

34. A substantially pure SAP-6 protein composition of claim 33, said SAP-6-Val protein having at least about 11 valine amino acids.

35. A substantially pure SAP-6 protein composition of claim 33, said SAP-6-Val protein being a human SAP-6-Val protein.

36. A substantially pure SAP-6 protein composition of claim 33, said SAP-6-Val protein being a synthetic SAP-6Val protein.

37. A substantially pure SAP-6 protein composition of claim 33, said SAP-6-Val protein being a human SAP-6-Val protein comprising the following amino acid sequence: ##STR10##

38. A substantially pure SAP-6 protein composition of claim 33, said SAP-6-Val protein being a human SAP-6-Val protein comprising the following amino acid sequence: ##STR11##

39. A substantially pure SAP-6 protein composition of claim 33, said second SAP-6 protein being a human SAP-6 protein comprising the following amino acid sequence: ##STR12##

40. A medicament for reducing or maintaining normal pulmonary surface tension in the alveoli of an animal comprising

a substantially pure SAP-6 protein composition as recited in claim 33, a phospholipid and a pharmaceutically acceptable carrier, said SAP-6 protein composition and said phospholipid each being present in said medicament in an effective amount to reduce or maintain normal pulmonary surface tension in the alveoli of the animal.

41. A preparation for replacing pulmonary surfactant material in lungs of an animal comprising a substantially pure SAP-6 protein composition as recited in claim 33 and a phospholipid, both of which are present in said preparation in an effective amount.

42. A method of isolating from animal pulmonary or amniotic tissue a substantially pure lung specific hydrophobic surfactant-associated SAP-6 protein composition for enhancing surfactant-like activity of phospholipids in lungs of an animal, the substantially pure SAP-6 protein composition consists essentially of a) a lung specific hydrophobic surfactant-associated SAP-6-Val protein, said SAP-6-Val protein having a polyvaline domain of valine amino acid residues, said polyvaline domain including a first stretch of about six adjacent valine amino 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, the SAP-6-Val protein having 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 being substantially resistant to protease, endoglycosidase F, and collagenase enzymes, and b) a second lung specific hydrophobic surfactant-associated SAP-6 protein, the second SAP-6 protein having a molecular weight of about 6,000 daltons as determined by SDS-PAGE and being substantially resistant to protease, endoglycosidase F, and collagenase enzymes, said method comprising 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 substantially pure SAP-6 protein composition;

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

concentrating the liquid fraction to generate a concentrate which contains the substantially pure SAP-6 protein composition;

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 the substantially pure SAP-6 protein composition; and

isolating the SAP-6 protein composition from the second eluting solvent in substantially pure form.

43. A method of claim 42, 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.

44. A method of claim 42, 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.

45. A method of claim 44, the substantially pure SAP-6 protein composition being eluted during said second eluting step in the selected solvents having an alcohol concentration of about 40% up to about 80%.

46. A method of claim 42, said isolating step comprising:

evaporating the second eluting solvent to generate the substantially pure SAP-6 protein composition.

Details for Patent 5,387,746

Applicant Tradename Biologic Ingredient Dosage Form BLA Number Approval Date Patent No. Assignee Estimated Patent Expiration Status Orphan Source
Smith And Nephew SANTYL collagenase OINTMENT;TOPICAL 101995 001 1965-06-04 ⤷  Free Forever Trial Scios Nova Inc. (Mountain View, CA) 2006-05-06 RX search
>Applicant >Tradename >Biologic Ingredient >Dosage Form >BLA >Number >Approval Date >Patent No. >Assignee >Estimated Patent Expiration >Status >Orphan >Source

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