US5387746A
Expired / Lapsed
US
⏱️ 0.0 years remaining
Pulmonary hydrophobic surfactant-associated protein of 6,000 daltons molecular weight and multimers thereof
Assignee: Takeda GmbH
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Filing: 1992-11-12
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Granted: 1995-02-07
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Expires: 2012-11-12
Abstract
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.
Inventors
- 👤 Jeffrey A. Whitsett
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-23 04:03
CPC Classifications
G; G01; G01N; G01N33/00; G01N33/48; G01N33/50; G01N33/68; G01N33/6884; A; A61; A61K; A61K38/00; A61K38/04; C; C07; C07K; C07K14/00; C07K14/435; C07K14/785; C07K16/00; C07K16/18; Y; Y10; Y10S; Y10S530/00; Y10S530/827; Y10S530/848
Claims Analysis
Total Claims: 51
Claim 1
Independent Claim
91 words
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.
Claim 2
Dependent Claim
91 words
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.
Claim 3
Dependent Claim
18 words
A SAP-6-Val recombinant protein of claim 1, said recombinant protein having at least about 11 valine amino acids.
Claim 4
Dependent Claim
19 words
A recombinant protein of claim 1 said recombinant protein having a sequence which corresponds to a human SAP-6-Val protein.
Claim 5
Dependent Claim
20 words
A SAP-6-Val recombinant protein of claim 1 said recombinant protein having a sequence which corresponds to a bovine SAP-6-Val protein.
Claim 6
Dependent Claim
28 words
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.
Claim 7
Dependent Claim
35 words
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##
Claim 8
Dependent Claim
50 words
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##
Claim 9
Dependent Claim
50 words
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##
Claim 10
Dependent Claim
67 words
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.
Claim 11
Dependent Claim
15 words
A medicament of claim 9, said medicament further including multimers of the SAP-6-Val recombinant protein.
Claim 12
Dependent Claim
22 words
A medicament of claim 8, said phospholipid being selected from the group consisting of a synthetic phospholipid and a naturally occurring phospholipid.
Claim 13
Dependent Claim
23 words
A medicament of claim 11, said phospholipid being selected from the group consisting of phosphatidylcholine, disaturated phosphatidylcholine, phosphotidylglycerol, dipalmitoylphosphatidylcholine, phophatidylinositol and mixtures thereof.
Claim 14
Dependent Claim
37 words
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.
Claim 15
Dependent Claim
15 words
A preparation of claim 13, said preparation further including multimers of the SAP-6-Val recombinant protein.
Claim 16
Dependent Claim
22 words
A preparation of claim 13, said phospholipid being selected from the group consisting of a synthetic phospholipid and a naturally occurring phospholipid.
Claim 17
Dependent Claim
23 words
A preparation of claim 15, said phospholipid being selected from the group consisting of phosphatidylcholine, disaturated phosphatidylcholine, phosphatidylglycerol, dipalmitoylphosphatidylcholine, phosphatidylinositol and mixtures thereof.
Claim 18
Dependent Claim
52 words
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.
Claim 19
Dependent Claim
21 words
A method of claim 17 said administration step comprises administering the effective dose as a liquid or as an aerosol spray.
Claim 20
Dependent Claim
16 words
A method of claim 17, the effective dose further including multimers of the SAP-6-Val recombinant protein.
Claim 21
Dependent Claim
49 words
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.
Claim 22
Dependent Claim
49 words
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.
Claim 23
Dependent Claim
62 words
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.
Claim 24
Dependent Claim
22 words
A medicament of claim 21, said phospholipids being selected from the group consisting of synthetic phospholipids, naturally occurring phospholipids and mixtures thereof.
Claim 25
Dependent Claim
23 words
A medicament of claim 22, said phospholipids being selected from the group consisting of phosphatidylcholine, disaturated phosphatidylcholine, phosphotidylglycerol, dipalmitoylphosphatidylcholine, phosphatidylinositols and mixtures thereof.
Claim 26
Dependent Claim
42 words
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.
Claim 27
Dependent Claim
22 words
A preparation of claim 24, said phospholipids being selected from the group consisting of synthetic phospholipids, naturally occurring phospholipids and mixtures thereof.
Claim 28
Dependent Claim
23 words
A preparation of claim 25, said phospholipids being selected from the group consisting of phosphatidylcholine, disaturated phosphatidylcholine, phosphatidylglycerol, dipalmitoylphosphatidylcholine, phosphatidylinositol and mixtures thereof.
Claim 29
Dependent Claim
87 words
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.
Claim 30
Dependent Claim
87 words
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.
Claim 31
Dependent Claim
26 words
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.
Claim 32
Dependent Claim
17 words
An isolated lung specific hydrophobic surfactant-associated protein of claim 27 wherein said protein is a synthetic protein.
Claim 33
Dependent Claim
62 words
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.
Claim 34
Dependent Claim
40 words
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.
Claim 35
Dependent Claim
53 words
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.
Claim 36
Dependent Claim
160 words
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.
Claim 37
Dependent Claim
160 words
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.
Claim 38
Dependent Claim
20 words
A substantially pure SAP-6 protein composition of claim 33, said SAP-6-Val protein having at least about 11 valine amino acids.
Claim 39
Dependent Claim
17 words
A substantially pure SAP-6 protein composition of claim 33, said SAP-6-Val protein being a human SAP-6-Val protein.
Claim 40
Dependent Claim
17 words
A substantially pure SAP-6 protein composition of claim 33, said SAP-6-Val protein being a synthetic SAP-6Val protein.
Claim 41
Dependent Claim
24 words
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##
Claim 42
Dependent Claim
24 words
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##
Claim 43
Dependent Claim
25 words
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##
Claim 44
Dependent Claim
66 words
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.
Claim 45
Dependent Claim
39 words
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.
Claim 46
Independent Claim
348 words
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.
Claim 47
Independent Claim
348 words
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.
Claim 48
Dependent Claim
59 words
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.
Claim 49
Dependent Claim
58 words
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.
Claim 50
Dependent Claim
33 words
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%.
Claim 51
Dependent Claim
22 words
A method of claim 42, said isolating step comprising: evaporating the second eluting solvent to generate the substantially pure SAP-6 protein composition.
References Cited (Backward Citations)
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