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WO1987006943A1 - Proteine associee a un agent tensio-actif hydrophobe pulmonaire d'un poids moleculaire de 6000 daltons, et ses multimeres - Google Patents

Proteine associee a un agent tensio-actif hydrophobe pulmonaire d'un poids moleculaire de 6000 daltons, et ses multimeres Download PDF

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Publication number
WO1987006943A1
WO1987006943A1 PCT/US1986/002258 US8602258W WO8706943A1 WO 1987006943 A1 WO1987006943 A1 WO 1987006943A1 US 8602258 W US8602258 W US 8602258W WO 8706943 A1 WO8706943 A1 WO 8706943A1
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Prior art keywords
sap
val
leu
pro
surfactant
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PCT/US1986/002258
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English (en)
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Jeffrey A. Whitsett
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Children's Hospital Medical Center
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Application filed by Children's Hospital Medical Center filed Critical Children's Hospital Medical Center
Priority to EP19870907365 priority Critical patent/EP0290516A4/fr
Priority to PCT/US1987/002536 priority patent/WO1988003170A1/fr
Publication of WO1987006943A1 publication Critical patent/WO1987006943A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6884—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids from lung
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/785—Alveolar surfactant peptides; Pulmonary surfactant peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans

Definitions

  • Hyaline membrane disease is a common disorder of premature infants and is related to diffuse atelectesls, hypoxia and resultant respiratory impairment. More particularly, HMD relates to the lack of vital pulmonary materials necessary for reducing surface tension in the airways of the alveoli. As a result, the alveoli or terminal respiratory sacs of patients suffering from HMD normally collapse. And, because the surface tension at the gas-liquid interface in HMD patients is elevated, their alveoli or terminal respiratory sacs are very difficult to reinflate. Consequently, HMD may be associated with significant morbidity and mortality, especially in premature infants.
  • Natural pulmonary surfactant material is a complex material composed primarily of phospholipids and surfactant-associated proteins or apolipoproteins.
  • the phospholipids mainly phosphatidycholine (PC), disaturated phosphatidycholine (DSPC) and phosphatidylglycerol (PG), are of paramount importance for the physiological role of natural pulmonary surfactant material in reducing surface tension in the alveoli.
  • Phospholipids, of which DSPC is the principal component are synthesized in the endoplasmic reticulu of Type II epithelial cells, packaged into lamellar bodies, then secreted into the alveolar space by an exocytotic process.
  • Several of the phospholipids are apparently not catabolized and resynthesized, but rather it is presently believed that they are reutilized primarily as intact molecules and constitute the major components of the naturally existing pulmonary surfactant material.
  • Surfactant-associated proteins or apolipoproteins include both serum and lung specific proteins.
  • This protein, herein called SAP-35 is synthesized from Mr»28-30,000 dalton translation products which undergo glycosylation, hydroxylation of proline residues and sulfhydryl-dependent cross-linking to form large oligomers which can be detected in the airway.
  • Proteolytic fragments of SAP-35 have been identified in protein preparations isolated from lavage of patients with alveolar proteinosis and from other mammalian surfactants migrating as proteins of small molecular weight by Whitsett et al, Characteristics of Human Surfactant-Associated Glycoprotein(s) A, Pediatr Res 19:501-508, 1985. While the glycoprotein SAP-35 binds phospholipids and may confer the structural organization of tubular myelin to surfactant lipids, it remains unclear whether SAP-35 is required for the biophysical activity of surfactants. See King et al, Metabolism of the Apoproteins in Pulmonary Surfactant, J Appl Physio1 42:483-491, 1977.
  • Schilling, et al describe a human 35,000 dalton protein and nonhuman 10,000 dalton proteins. It was disclosed that both classes of proteins were needed to confer full biophysical activity to surfactant phospholipids. Also, the complete DNA sequence encoding the 35,000 dalton protein and the NH 2 -terminal amino acid sequences for the 10,000 dalton nonhuman proteins were disclosed. However, the 10,000 dalton proteins were unfortunately not purified for use alone or in combination with the 35,000 dalton protein for biophysical assays with phospholipids. Instead, it appears that Schilling et al used a 35,000 dalton protein derived by recombinant DNA methods in combination with naturally-occurring nonhuman 10,000 dalton proteins.
  • Schilling, et al disclose a preliminary (81 bp) partial sequence for a human cDNA clone conjectured to be the 10,000 dalton protein, which was chosen based on its homology to a canine cDNA clone.
  • the present invention alleviates and overcomes the above problems and shortcomings of the present state of the art. This was accomplished through the discovery of novel, isolated, substantially pure, hydrophobic surfactant-associated protein of 6,000 dalton simple molecular weight which, when isolated from animal tissue, comprises two hydrophobic proteins of molecular weights of 6,000 daltons each and may also comprise larger multimers thereof (hereinafter "SAP-6").
  • SAP-6 when combined with lipids, has significant pulmonary biophysical surfactant activity that can be utilized to effectively treat and prevent HMD and other syndromes associated with lack or insufficient amounts of natural pulmonary surfactant material.
  • SAP-6 is lung specific, its pulmonary biophysical surfactant activity is believed not to be species specific. Therefore, SAP-6 can be purified from animal tissue, specifically pulmonary tissue or amniotic fluid, extracted from a variety of animals, such as dog, cow, human, pig, rabbit, rat and the like, or made by recombinant DNA methods or direct peptide synthesis. The concentration of SAP-6 in pulmonary tissue and lavage is probably greater than that found in amniotic fluid.
  • SAP-6 is believed to be present in substantially smaller or undetectable concentrations or completely absent. It is believed that when SAP-6 is combined with lipids, it enhances the surfactant properties of the lipids imparting to the combination significant pulmonary biophysical surfactant activity. As a result of this remarkable property, such a combination is highly useful for replacing or supplementing natural pulmonary surfactant material and for reducing or maintaining normal surface tension in the lungs, especially in the lungs of patients suffering from HMD and other syndromes associated with the lack or insufficient amounts of natural pulmonary surfactant material.
  • hydrophobic surfactant-associated protein and "SAP-6" are used interchangeably herein, and that whenever referenced herein, they are meant to include any small hydrophobic surfactant-associated protein that has surfactant-like activity, that has a simple molecular weight of about 6,000 daltons determined in polyacrylamide gel containing sodium dodecyl sulfate and which is substantially resistant to protease enzymes, endoglycosidase F and collagenase.
  • the present invention further resides in a method of separating SAP-6 from animal tissue which involves separating the animal tissue into a particulate fraction and a liquid fraction, and extracting from the liquid fraction the SAP-6 in a substantially pure state.
  • the methods of this invention are further concerned with separating SAP-6 from the larger, novel hydrophobic multimers thereof. As already mentioned above, this can be accomplished by, for instance, gel electrophoresis migration or other suitable techniques.
  • the present invention still further contemplates novel medicaments, preparations and methods employed to treat animals, including human infants, suffering from HMD and other syndromes related to the lack or insufficient amounts of natural pulmonary surfactant material.
  • novel antibodies and antisera directed against SAP-6 are also contemplated by the present invention. Accordingly, it can be appreciated that the present invention provides a solution to the art that has long sought to understand natural pulmonary surfactant material and effective means to treat or prevent HMD and other syndromes associated with the lack or insufficient amounts of natural pulmonary surfactant material.
  • Fig. 1 SAP-6 from bovine.
  • SAP-6 was purified and delipidated as described herein and applied to 10-20% sodium dodecyl sulfate- polyacrylamide gel electrophoresis gels in the absence of beta-mercaptoethanol.
  • Lane B represents approximately 2 micrograms of protein detected by silver staining.
  • SAP-6 from bovine, Lane B, migrated at Mr 6,000, 14,000, 20,000 and 26,000.
  • SAP-6 from human, canine and bovine SAP-6 was purified and delipidated as described herein and applied to 10-20% sodium dodecyl sulfate-polyacryl mide gel electrophoresis gels in the presence of beta-mercaptoethanol. Each lane represents approximately 2 micrograms of each protein detected by silver staining.
  • SAP-6 isolated from animal tissue, specifically from pulmonary tissue and amniotic fluid or made by recombinant DNA methods or direct peptide synthesis.
  • SAP-6 when isolated from animal tissue, comprises two small hydrophobic surfactant-associated proteins with simple molecular weights of about 6,000 daltons each (hereinafter referred to as "SAP-6 monomers"), and may also comprise larger, hydrophobic protein multimers which may be covalent or noncovalent aggregates of SAP-6 monomers having molecular weights of about 14,000, about 20,000 and about 26,000 daltons based on size estimation using SDS-PAGE gel electrophoresis (hereinafter referred to as "SAP-6 multimers").
  • SAP-6 multimers may exist but in smaller undetectable concentrations.
  • "simple molecular weight” refers to the molecular mass of what is thought to be the smallest polypeptide chain after sulfhydryl reduction which serves as a repeating building block for a SAP-6 multimer.
  • a gel with about 3-27% polyacrylamide, in particular about 15% polyacrylamide (PAGE), and containing about 2% of sodium dodecyl sulfate (SDS) can be used to separate and determine the molecular weights of SAP-6 monomers and multimers in SDS-PAGE gel.
  • PAGE polyacrylamide
  • SDS sodium dodecyl sulfate
  • low molecular weight protein markers such as trypsin inhibitor (6,200), lysozyme (14,000), beta- lactalbumin (18,400), alpha-chymotrypsin (25,700) and ovalbumin (43,000) can be used. These can be obtained from BRL Inc., of Bethesda, MD.
  • hydrophobic refers to solubility in non-polar solvents, such as 3:1 ether/ethanol, chloroform, chloroform/methanol in various ratios, such as 3:1, and having an abundance of hydrophobic non-charged a ino acids.
  • surfacta ⁇ f-associated protein refers to proteins associated with binding to or co-purifying with the phospholipid components of mammalian surfactants during centrifugation in isotonic solution.
  • SAP-6 can be further characterized as being substantially resistant to protease enzymes (trypsin, chymotrypsin and staph V-8), endoglycosidase F, and collagenase. It has been discovered that SAP-6 is not degraded nor is its size heterogeneity significantly altered by these enzymes.
  • SAP-6 is localized in pulmonary type II cells and lung surfactant in a variety of mammalian species, as well as in human amniotic fluid near term gestation.
  • purified samples are hydrolyzed by two different methods.
  • the samples are hydrolyzed in constantly boiling 5.7N HC1 containing 0.3% phenol and 0.1% beta- mercaptoethanol at 110°C under vacuum for about 24 and 48 hours.
  • these same samples are hydrolyzed in 12N HCl/trifluoroacetic acid (2:1) containing 0.3% phenol at 150°C under vacuum for 2 hours, 6 hours, 24 hours and 48 hours.
  • the cysteine composition of these samples was determined separately as cysteic acid after performic acid oxidation.
  • Table I illustrates the amino acid compositions that have been determined for bovine and canine SAP-6. It should be understood to those skilled in the art that since the amino acid compositions have been determined using a Beckman 6300 analyzer, the amino acid residue compositions per protein molecule are only estimates based upon this analytical technique.
  • SAP-6 comprises two 6,000 molecular weight proteins or SAP-6 monomers.
  • each protein will be separately referred to as SAP-6-Val and SAP-6-Phe.
  • the SAP-6 samples are prepared by Ed an sequence analysis by first dialyzing the samples against water. The samples are taken up in methanol and applied to a glass fiber filter. The analysis is performed on an Applied BioSystems model 470A Gas Phase Protein Sequencer. Analysis of the resulting phenylthiohydantoins (PTH) is accomplished by high pressure liquid chromatography (HPLC) at 50°C on an Altex reversed-phase PTH-C,g column. A binary buffer system consisting of ammonium acetate buffered acetonitrile, pH of 4.5, is used.
  • the chromatography procedure is conducted utilizing buffer A which contains about 10% acetonitrile and buffer B which contains about 90% acetonitrile.
  • the initial chromatography conditions are 70% buffer A and 30% buffer B with a flowrate of 1.0 ml per minute.
  • concentration of buffer B is linearly increased to 50% over a 3 minute period and held at this level for 8 minutes.
  • concentration of Buffer B then is reduced to 30% over one minute.
  • re- equilibration which takes approximately 10 minutes, another PTH sample is injected.
  • SAP-6 comprises two 6,000 dalton hydrophobic surfactant-associated proteins. With respect to these SAP-6 monomers, the following has been observed. First, the NH 2 -terminal amino acid residue sequences of SAP-6-Val for canine, bovine and human, although similar to one another as indicated above, appear to be substantially different from the NH -terminal amino acid residue sequences of SAP-6-Phe for all three species. Similarly the NH 2 ⁇ terminal amino acid residue sequences of SAP-6-Phe for all three species are similar to one another but substantially different from the SAP-6-Val sequences.
  • SAP-6 monomers are possibly bonded together via sulfhydryl bonds in view of their migration patterns in the absence and presence of beta-mercaptoethanol , as illustrated in
  • SAP-6 can be purified from, for example, animal tissue and fluids, cells, cultivated cells, suitable pulmonary surfactant replacement preparations available, such as CLSE or Surfactant-TA, and the like.
  • animal tissue as used herein is meant in a broad sense to encompass all appropriate sources for SAP-6, including but not limited to animal tissue and fluid, cells, cultivated cells, pulmonary surfactant replacement preparations, such as CLSE and Surfactant-TA, and the like. It is preferred, however, to isolate SAP-6 from animal pulmonary tissue or amniotic fluid, and more preferably human pulmonary tissue. Solutions containing SAP-6 are obtained by extraction of animal tissue in which SAP-6 occurs.
  • the trachea preferably should be cannulated and washed repeatedly with several volumes of an iced, buffered solution, such as 0.9% NaCl (pH 7.0-7.4), to remove surfactant from the alveoli.
  • the wash is centrifuged at low speed, for example, 1,000 x g for ten minutes, and should be repeated if necessary to remove contaminating blood and white cells or macrophages. All procedures preferably should be performed at about 2-4°C. This surfactant material should then be centrifuged at high speed from the alveoli wash to sediment particulate materials at about 10,000 x g for about 30 minutes or longer.
  • This pellet preferably should be resuspended and washed by repeated centrifugation and sonicated or freeze thawed, several times if necessary, in a suitable buffer, such as the one above, to uniformly resuspend the materials.
  • the resulting white surfactant is pelleted by centrifugation, the aqueous material is removed and is extracted with about 10-100 volumes of approximately 2:1 ether/ethanol, 3:1 chloroform/methanol or other suitable organic solvents at about -30°C to produce a supernatant containing SAP-6. Extractions should proceed overnight. The material should then be centrifuged again at about 10,000 x g for about 20 minutes or longer.
  • the resulting liquid fraction will contain lipid and include the SAP-6 of this invention.
  • the liquid fraction thereafter can be dried (concentrated) with, for instance, N 2 stream, resuspended in an organic solvent, such as chloroform or other suitable organic solvents, and applied to a silicic acid column or LH-20 column, approximately 50cm x 2.5cm in size which is pre-equilibrated with chloroform or other suitable organic solvents at room temperature.
  • the column is then eluted with, for example, 50 to 200 mis of a mixture of chloroform and alcohol, such as methanol or the like, increasing the alcohol concentration from 0% to about 100% in stepwise or gradient fashion.
  • SAP-6 is eluted in the solvents containing alcohol having concentrations of greater than about 40% up to about 100% and more particularly about 40% to about 80%.
  • SAP-6 lipids present in the SAP-6 will be separated from the proteins at this stage. It should be appreciated to those skilled in the art that the silicic acid column can also be eluted with any other suitable solvents capable of extracting therefrom the SAP-6 of this invention.
  • An example of isolating SAP-6 is disclosed in the following example:
  • Surfactant associated proteins are isolated from lung lavage material which is obtained from adult dogs and cows after sacrifice. Proteins are also isolated from human surfactant which are obtained from human cadavers after autopsy or from lung lavage of consenting adults. In animal studies, the trachea is cannulated and the lung is lavaged three times with several volumes of iced 0.9% NaCl, 50 mM Na 2 H 0 4 , and 5 mM EDTA, pH 7.2. Cells and debris are removed by centrifugation at 80 x g for about 10 minutes (twice) and a particular fraction is collected by centrifugation at 40,000 x g for about 30 minutes at 4°C.
  • the pelleted material is then resuspended in the above buffer containing 1 mM phenylmethylsulfonylfluoride and sonicated for 10 seconds with a Branson sonifier.
  • Surfactant is pelleted by repeated centrifugation at about 40,000 x g for about 30 minutes at 4°C.
  • the surfactant pellet is further processed by extraction in about 3:1 ether/ethanol or chloroform/ methanol at about -30°C for about 16 hours.
  • Ether/ ethanol or chloroform/methanol extracts containing SAP-6 are evaporated to near dryness with N 2 gas and are redissolved in chloroform.
  • the redissolved residue in chloroform is applied to a BioSil-HA column (silicic column dimensioned 2.5 cm by 40 cm that is obtained from Bio-Rad, Richmond, California) and is equilibrated in chloroform.
  • Lipid-protein fractions are recovered by stepwise elution with chloroform-methanol mixtures (200 ml each) with successive increases in concentration of methanol (10% intervals) in chloroform.
  • the SAP-6 proteins are eluted between 3:2 and 1:4 chloroform/ methanol solvent elution or about 40% to about 80% methanol in chloroform.
  • SAP-6 Fractions containing the SAP-6 of this invention now can be assessed by, for example, fluorescamine assay and by gel electrophoresis migration followed by staining with silver stain or Coomassie brilliant blue staining.
  • the SAP-6 isolated at this point is considered to be substantially pure and homogeneous.
  • substantially pure it is meant herein that the SAP-6 proteins are substantially free of contaminants, such as cells, cellular debris, DNA, RNA, and major surfactant glycoproteins or fragments thereof. It should be appreciated, however, that some lipids still remain in the now substantially pure SAP-6. In the most preferred form, the substantially pure SAP-6 contains on the order of about 10% or less of contaminants.
  • the fractions containing the eluted SAP-6 preferably are pooled, evaporated to near dryness and subjected to repeated dialysis in, for example, a cellulose dialysis bag with about 100-500 volumes of about 2:1 of chloroform/ methanol or other suitable mixtures or acidified chloroform/methanol mixtures acidified with HC1 or other appropriate acids at about room temperature. This procedure ⁇ should proceed until the SAP-6 is virtually free of contaminating lipids.
  • This substantially purified SAP-6 can again be assessed by fluorescamine assay and gel electrophoresis migration.
  • SAP-6 of this invention has useful "surfactant-like activity".
  • surfactant-like activity refers to the ability to interact with phospholipids or other lipids to reduce surface tension spread on a surface and/or reduce surface tension at an air/liquid interface.
  • SAP-6 i.e., SAP-6 monomers, or the monomers in combination with multimers thereof, are mixed with lipids, their surfactant-like activity is believed to impart to the mixture significant pulmonary biophysical surfactant activity.
  • Such a mixture with this activity is highly useful for replacing or supplementing natural pulmonary surfactant material for reducing or maintaining normal surface tension in lungs, especially in the lungs of animals suffering from HMD and other syndromes associated with the lack or insufficient amounts of natural pulmonary surfactant material.
  • isolated SAP-6 can be stored in chloroform under nitrogen or air at about -30°C prior to reconstitution with lipids. Preparations may also be made with alcohols.
  • Those lipids, including neutral or other lipids as well as phospholipids, that may be employed can be derived from a number of suitable phospholipids, such as phosphatidylcholine, disaturated phosphatidylcholine, phosphatidylglycerol, dipalmitoylphosphatidylcholine, phosphatidylinositol and mixtures thereof, cholesterol or cholesterol esters and mixtures thereof, and the like.
  • purified SAP-6 can be mixed in an amount of about 0.1 to about 2.0% with synthetic phospholipids, such as about 65% dipalmitoylphosphatidylcholine, about 20% egg-phosphatidylcholine, about 7.5% of soy-phospha ⁇ tidylinositol, and about 7.5% egg-phosphatidylglycerol.
  • synthetic phospholipids such as about 65% dipalmitoylphosphatidylcholine, about 20% egg-phosphatidylcholine, about 7.5% of soy-phospha ⁇ tidylinositol, and about 7.5% egg-phosphatidylglycerol.
  • the mixture may further contain a physiological buffer comprising 0.9% sodium chloride with or without about 0.5 to about 1.5 mM calcium chloride or any other suitable pharmaceutically accepted carrier.
  • the material should be sonicated or vortexed and tested for surface tension lowering capacity and adsorption on a Wilhelmy balance or other surface tension measuring devices.
  • Adsorption studies can be conducted by the methodology disclosed by Notter et al, Chem. Phys . Lipids 33: 67-80, 1983 and Ross et al, Phospholipid Binding and Biophysical Activity of Pulmonary Surfactant-Associated Protein SAP-35 and Its Non-Collagenous C-Terminal Domains, In press, 1986 in which they are incorporated herein by reference in their entirety.
  • a phospholipid mixture containing about 65% dipalmitoylphosphatidylcholine (DDPC), about 20% egg- phosphatidylcholine (PC), about 7.5% egg-phosphatidyl ⁇ glycerol (PG) and about 7.5% soy-phosphatidylinositol (PI) is combined with delipidated SAP-6 and tested in a Wilhelmy balance and nitrogen bubble surfactometer.
  • the SAP-6 phospholipid mixture is absorbed rapidly, lowers surface tension by about 30-47 dynes/cm and is found to be more active than the phospholipid mixture or SAP-35, the major surfactant-associated glycoprotein, mixed with the same phospholipid tested in identical fashion. See Tables IV-VI. The methods employed to generate Tables IV-VI can be found in detail in Notter et al and Ross et al referenced hereinabove.
  • *SM is a synthetic lipid mixture composed of DPPC:egg-PC: egg-PG:soy-PI 65:20:7.5:7.5.
  • lipid-surfactant represents optimally active surfactants; ovalbumin is used as a non-specific protein control.
  • the lipid to protein ration is (wt/wt) 99/1 to 98/2.
  • lipid-surfactant represents active lung surfactant preparation against which to compare the lipid-protein combined mixtures of cases 3-5 and 7-10.
  • mixtures containing SAP-6 and naturally occurring or synthetic phospholipids can be instilled intratracheally, for instance, in liquid form or as an aerosol spray in doses of about 0.1% to about 2.0% SAP-6 by weight, with about 20 to about 100 mg phospholipid per kilogram for treatment or prevention of HMD and other related disorders.
  • SAP-6 antibodies or antisera Another utility for SAP-6 is in the preparation of SAP-6 antibodies or antisera. These antibodies or antisera can then be used in immunochemical methods for detecting, determining or purifying SAP-6.
  • SAP-6 antibodies and antisera can be used for Immunoblot and ELISA analysis for immunological detection and quantification or other immunological assays of SAP-6.
  • SAP-6 antibodies and antisera which can be used for this purpose can be obtained as follows: Albino rabbits are immunized with repeated injections of delipidated SAP-6 and Freund's complete adjuvant. Usable antisera should be obtainable after four injections with approximately 100 micrograms of the delipidated SAP-6 per injection.
  • the SAP-6 used for injection should contain SAP-6 monomers and a smaller amount of the SAP-6 multimers assessed by silver staining analysis after one dimensional SDS-PAGE.
  • Polyclonal antibodies may be isolated from the antisera by affinity chromatography.
  • lymphocytes from mice immunized with isolated SAP-6 may be fused with mouse myeloma cells in the presence of, for example, polyethylene glycol, generally according to the technique of Kohler and Milstein, 256:495-497 (1975) which is incorporated herein by reference in it entirety.
  • the surviving hybrids would then be screened by selective cloning to obtain only cells which produce the anti-SAP-6 which, when separated from contaminating myeloma antibody by immuno-absorption, may be employed in immunoassay techniques for detection of SAP-6 in, for instance, a tissue sample.
  • such antibodies can bind to or precipitate SAP-6 from a mixture containing SAP-6, increase the sedimentation rate of SAP-6, can modify elution characteristics of SAP-6 on gel filtration, and can identify or quantify SAP-6 from a sample.
  • SAP-6 The detection and determination of SAP-6 by immunological methods is of diagnostic importance. In normal situations, since SAP-6 occurs in some tissues of the human body, for example, lung tissue, lung lavage and amniotic fluid, SAP-6 can be detected. In illnesses associated with SAP-6, the proteins will not appear or will appear in altered concentrations in the serum, tissue, such as the lungs, or other body fluids. The detection or lack of detection of these proteins can be useful for diagnostic determination of an illness or for monitoring the course of a disease and for closely controlling the therapy of such an illness. SAP-6 has been detected in human amniotic fluid near term gestation by immunoblot analysis using anti-SAP-6 antibodies.

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Abstract

Nouvelle protéine isolée associée à un agent tensio-actif hydrophobe sensiblement pur d'un poids moléculaire simple de 6000 daltons (SAP-6). Lorsque SAP-6 est combinée avec des lipides, sa caractéristique semblable à celle d'un agent tensio-actif confère à la combinaison une activité biophysique pulmonaire importante. Une telle combinaison a pour résultat une adsorption accrue des lipides avec des propriétés similaires à celles du matériau tensio-actif pulmonaire naturel. SAP-6 en combinaison avec des lipides est très utile pour remplacer ou compléter un matériau tensio-actif pulmonaire naturel dans le but de réduire ou maintenir une tension en surface normale dans les poumons, en particulier dans les poumons de patients souffrant de la maladie des membranes hyalines (HMD), ou d'autres syndromes associés à la déficience ou insuffisance de quantités d'un matériau tensio-actif pulmonaire naturel. Une combinaison de SAP-6 et de lipides peut être administrée sous la forme d'un atomiseur aérosol ou dans un sérum physiologique normal aqueux avec ou sans chlorure de calcium pour traiter ou prévenir HMD ainsi que d'autres états de déficience au niveau des matériaux tensio-actifs. Sont également décrits des procédés d'isolement de SAP-6 dans des tissus animaux.
PCT/US1986/002258 1986-05-06 1986-10-24 Proteine associee a un agent tensio-actif hydrophobe pulmonaire d'un poids moleculaire de 6000 daltons, et ses multimeres WO1987006943A1 (fr)

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EP19870907365 EP0290516A4 (fr) 1986-10-24 1987-10-02 Proteines associees a des agents tensioactifs hydrophobes pulmonaires.
PCT/US1987/002536 WO1988003170A1 (fr) 1986-10-24 1987-10-02 Proteines associees a des agents tensioactifs hydrophobes pulmonaires

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Cited By (18)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1989000167A1 (fr) * 1987-07-01 1989-01-12 Kabigen Ab Proteines, compositions de proteines et leurs utilisations
EP0335133A3 (fr) * 1988-03-31 1990-05-16 Abbott Laboratories Délivrance d'un médicament à l'aide d'un tensioactif pulmonaire pour faciliter l'absorption
EP0368823A3 (en) * 1988-10-18 1990-05-23 Kabigen Ab Biologically active lipoprotein and its use
US5024995A (en) * 1987-04-08 1991-06-18 Chiesi Farmaceutici S.P.A. Natural pulmonary surfactant, method of preparation and pharamceutical compositions
EP0458167A1 (fr) * 1990-05-21 1991-11-27 Abbott Laboratories Conjugés acide gras- tensioactif pulmonaire
US5169761A (en) * 1984-12-11 1992-12-08 California Biotechnology Inc. Dna encoding and expression systems for alveolar surfactant proteins
US5258496A (en) * 1990-07-10 1993-11-02 Scios Nova Inc. Isolation and purification of lung surfactant protein
EP0593094A3 (fr) * 1988-01-06 1994-11-02 Scripps Clinic Res Protéine de tensio-actif pulmonaire et polypeptide s'y rapportant.
US5430020A (en) * 1984-12-11 1995-07-04 Scios Nova Inc. Recombinant alveolar surfactant protein
US5683982A (en) * 1991-11-04 1997-11-04 Merrell Pharmaceuticals Inc. Synthetic lung surfactant having antioxidant properties
US5840527A (en) * 1984-12-11 1998-11-24 Byk Gulden Lomberg Chemische Fabrik Gmbh Recombinant alveolar surfactant protein
US5958902A (en) * 1997-04-16 1999-09-28 Wisconsin Alumni Research Foundation Method and composition for treating sleep apnea
US6613734B2 (en) 1988-01-06 2003-09-02 The Scripps Research Institute Peptides-containing liposomal surfactants
US6982075B2 (en) 2001-10-11 2006-01-03 Altana Pharma Ag Use of pulmonary surfactant
US7053176B1 (en) 1999-09-16 2006-05-30 Altana Pharma Ag Combination of C1-INH and lung surfactant for the treatment of respiratory disorders
US7951781B2 (en) 2006-11-02 2011-05-31 University Of Iowa Research Foundation Methods and compositions related to PLUNC surfactant polypeptides
US8236750B2 (en) 2004-08-06 2012-08-07 Nycomed Gmbh Composition comprising a pulmonary surfactant and a TNF-derived peptide
US9289388B2 (en) 2008-12-10 2016-03-22 Paka Pulmonary Pharmaceuticals, Inc. Methods and compositions for delivery of medicaments to the lungs

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US4933280A (en) * 1984-12-11 1990-06-12 California Biotechnology Inc. Recombinant DNA sequence encoding Alveolar Surfactant Protein
JPH05294996A (ja) * 1992-04-17 1993-11-09 Tokyo Tanabe Co Ltd 合成ペプチド、それを含有する肺サーファクタント及び呼吸窮迫症候群治療剤

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Cited By (23)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5430020A (en) * 1984-12-11 1995-07-04 Scios Nova Inc. Recombinant alveolar surfactant protein
US5840527A (en) * 1984-12-11 1998-11-24 Byk Gulden Lomberg Chemische Fabrik Gmbh Recombinant alveolar surfactant protein
US5169761A (en) * 1984-12-11 1992-12-08 California Biotechnology Inc. Dna encoding and expression systems for alveolar surfactant proteins
US5385840A (en) * 1984-12-11 1995-01-31 Scios Nova Inc. DNA encoding analogs of human alveolar surfactant protein SP-5
US5024995A (en) * 1987-04-08 1991-06-18 Chiesi Farmaceutici S.P.A. Natural pulmonary surfactant, method of preparation and pharamceutical compositions
WO1989000167A1 (fr) * 1987-07-01 1989-01-12 Kabigen Ab Proteines, compositions de proteines et leurs utilisations
US6613734B2 (en) 1988-01-06 2003-09-02 The Scripps Research Institute Peptides-containing liposomal surfactants
EP0593094A3 (fr) * 1988-01-06 1994-11-02 Scripps Clinic Res Protéine de tensio-actif pulmonaire et polypeptide s'y rapportant.
EP0335133A3 (fr) * 1988-03-31 1990-05-16 Abbott Laboratories Délivrance d'un médicament à l'aide d'un tensioactif pulmonaire pour faciliter l'absorption
EP0368823A3 (en) * 1988-10-18 1990-05-23 Kabigen Ab Biologically active lipoprotein and its use
EP0458167A1 (fr) * 1990-05-21 1991-11-27 Abbott Laboratories Conjugés acide gras- tensioactif pulmonaire
US5700777A (en) * 1990-05-21 1997-12-23 Abbott Laboratories Fatty acid - pulmonary surfactant conjugates
US6022955A (en) * 1990-05-21 2000-02-08 Abbott Laboratories Fatty acid -- pulmonary surfactant conjugates
US5258496A (en) * 1990-07-10 1993-11-02 Scios Nova Inc. Isolation and purification of lung surfactant protein
US5403915A (en) * 1990-07-10 1995-04-04 Scios Nova Inc. Isolation and purification of lung surfactant protein
US5683982A (en) * 1991-11-04 1997-11-04 Merrell Pharmaceuticals Inc. Synthetic lung surfactant having antioxidant properties
US5958902A (en) * 1997-04-16 1999-09-28 Wisconsin Alumni Research Foundation Method and composition for treating sleep apnea
US6329352B1 (en) 1997-04-16 2001-12-11 Wisconsin Alumni Research Foundation Method and composition for treating sleep apnea
US7053176B1 (en) 1999-09-16 2006-05-30 Altana Pharma Ag Combination of C1-INH and lung surfactant for the treatment of respiratory disorders
US6982075B2 (en) 2001-10-11 2006-01-03 Altana Pharma Ag Use of pulmonary surfactant
US8236750B2 (en) 2004-08-06 2012-08-07 Nycomed Gmbh Composition comprising a pulmonary surfactant and a TNF-derived peptide
US7951781B2 (en) 2006-11-02 2011-05-31 University Of Iowa Research Foundation Methods and compositions related to PLUNC surfactant polypeptides
US9289388B2 (en) 2008-12-10 2016-03-22 Paka Pulmonary Pharmaceuticals, Inc. Methods and compositions for delivery of medicaments to the lungs

Also Published As

Publication number Publication date
EP0273916A1 (fr) 1988-07-13
JPS63503222A (ja) 1988-11-24
EP0273916A4 (fr) 1989-11-30

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