SAP-6-Val proteins and methods

Claim 2

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.

Claim 3

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

Claim 4

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

Claim 5

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

Claim 6

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.

Claim 7

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

Claim 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: ##STR6##

Claim 9

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##

Claim 10

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##

Claim 11

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##

Claim 12

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##

Claim 13

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##

Claim 14

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.

Claim 15

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

Claim 16

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

Claim 17

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

Claim 18

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

Claim 19

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.

Claim 20

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

Claim 21

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

Claim 22

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

Claim 23

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

Claim 24

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.

Claim 25

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

Claim 26

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

Claim 27

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

Claim 30

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.

Claim 31

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.

Claim 32

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%.

Claim 33

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.

Claim 34

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.

Claim 35

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.

Claim 36

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.

Claim 37

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.

Claim 38

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.

Claim 39

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.

Claim 40

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.

Claim 41

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.

Claim 42

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%.

Claim 43

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.

Claim 44

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.

Claim 45

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.

Claim 46

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.

Claim 47

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.

Claim 48

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

Claim 49

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

Claim 50

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.

Claim 51

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

Claim 52

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

Claim 53

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##

Claim 54

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##

Claim 55

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.

Claim 56

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##

Claim 57

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##

Claim 58

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.

Claim 59

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.

Claim 60

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

Claim 61

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.