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WO1989000167A1 - Proteines, compositions de proteines et leurs utilisations - Google Patents

Proteines, compositions de proteines et leurs utilisations Download PDF

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Publication number
WO1989000167A1
WO1989000167A1 PCT/SE1988/000361 SE8800361W WO8900167A1 WO 1989000167 A1 WO1989000167 A1 WO 1989000167A1 SE 8800361 W SE8800361 W SE 8800361W WO 8900167 A1 WO8900167 A1 WO 8900167A1
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WO
WIPO (PCT)
Prior art keywords
leu
val
pro
protein
gly
Prior art date
Application number
PCT/SE1988/000361
Other languages
English (en)
Inventor
Tore Curstedt
Bengt Robertsson
Hans Jörnvall
Original Assignee
Kabigen Ab
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from SE8702724A external-priority patent/SE8702724D0/xx
Priority claimed from SE8703661A external-priority patent/SE8703661D0/xx
Application filed by Kabigen Ab filed Critical Kabigen Ab
Publication of WO1989000167A1 publication Critical patent/WO1989000167A1/fr

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Classifications

    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof

Definitions

  • the present invention relates to proteins and protein compositions useful as components of pulmonary surfactants, i.e. for providing for normal respiration in mammals including man.
  • Pulmonary surfactant which is a phospholipid-prot ein complex, is essential for normal respiration by reducing surface tension at the air-liquid interface of the alveoli (1).
  • Different surfactant-specific proteins have been detected.
  • One group comprises comparatively large glycoproteins with molecular weights varying between 28 and 36 kDa, depending on the degree of glycosylation (2,3).
  • This protein is soluble in water and the primary structure of the forms from canine and human lung has been determined (4-6). In the presence of calcium ions this protein apparently participates in the formation of surface-active tubular myelin from secreted lamellar bodies (7) and increases the rate of adsorption of surfactant phospholipids (8).
  • this protein probably is functional for the endogenous surfactant, synthesized in the alveolar epithelial type II cells, it does not seem to be essential for the physiological activity of exogenous surfactant preparations designed for replacement therapy (9-11).
  • a second group of surfactant-specific proteins constitutes forms with low-molecular weights ( ⁇ 14 kDa) (2, 9, 12-20). These proteins are very hydrophobic and are composed of different proteins which may be soluble (9) or insoluble (2,16,17) in ether/ethanol. Both proteins require organic solvents for solubilization and purification, and are heterogenous by multiple start positions in the N-terminal regions (truncated forms). Recombination of either of these proteins with synthetic phospholipids yields a surfactant preparation with physical and biological properties which in many respects are similar to those of natural pulmonary surfactant.
  • the two low-molecular weight proteins have unrelated structures and sizes; the smaller form is not a fragment of the larger. Recently, cDNA segments of the longer form (21) have been described from dog, but exact structures, sizes or further properties have not been defined.
  • the present invention concerns lypophilic low-molecular weight apoproteins of mammalian origin.
  • K and L are selected from lie, Gly, Val, and wherein preferably K is lle and L is Gly or both are Val.
  • a preferred protein comprising said amino acid sequence has the following structure: 1 5 10 15
  • a protein comprising the amino acid sequence: 10 Phe-Pro-Ile-Pro-Leu-Pro-Phe-Cys-Trp-Leu-Cys-Arg-Thr-Leu-Ile-
  • protein compositions comprising protein I in combination with protein III and, alternatively, protein II in combination with protein III.
  • the present invention also involves pharmaceutical compositions comprising in combination a protein or protein composition as defined above and a phospholipid type of material.
  • the protein is a minor component, and a preferred weight range of the contents of the composition of the protein is about 0.5 to about 10% by weight thereof. It is particularly preferred that the protein constitutes about 1 to 5% by weight of the composition as a whole.
  • phospholipid material there may be mentioned phospholipids based on palmitic acid.
  • the composition of the invention may also contain other additives, such as pharmaceutically acceptable carriers or diluents, stabilising agents, and other conventionally used pharmaceutically acceptable additives.
  • the proteins according to the present invention have been found to contribute significantly to pulmonary surfactant activity. Accordingly, the proteins and compositions of the invention are particularly useful as components of pulmc nary surfactants. Furthermore, the invention includes a method for facilitating respiration in mammals including man, such method comprising administering an effective amount of a protein or composition according to the invention to the respiratory tract of a patient in need of treatment for respiratory disorders. By such administration it is possible to significantly reduce surface tension at the air-liquid interface of the patient's alveoli. The administration can take place directly into trachea or bronchi, but can also take place through the oral cavity by using an aerosol spray of a conventional type.
  • Fig. 1 shows the complete amino acid sequence of porcine surfactant fraction 1
  • Fig. 2 shows the complete amino acid sequence of porcine surfactant fraction 2
  • Fig. 3 shows the complete amino acid sequence of human surfactant fraction 2
  • Fig. 4 shows diagrams on surface activity of artificial surfactant preparations using the pulsating bubble technique
  • Fig. 5 shows the tidal volumes as a function of time intervals and insufflation pressures at varying therapeutic regimens including treatment according to the invention and in controls;
  • Fig. 6 shows representative tidal volumes in untreated and treated animals.
  • the two types of surfactant low-molecular weight protein are isolated and separated by Sephadex LH-60 chromatography: both are biologically active, structurally unrelated, and show no evidence of product relationships.
  • the larger protein containing 79 residues constitutes fraction 1 from the Sephadex LH-60 chromatography step, and is now named low- -molecular weight surfactant protein type 1.
  • the smaller protein containing 35 residues constitutes fraction 2, and is named low-molecular weight surfactant protein type 2.
  • Type 1 surfactant protein has been purified from pig lung and type 2 surfactant protein has been isolated from pig lung and human bronchoalveolar lavage or amniotic fluid.
  • Minced pig lungs were washed with saline and the mixture was filtered. Cells and debris were removed by centrifugation at 1000xg for 15 min at 20°C and the supernatant was centrifuged at 3000xg for 2 hrs at 4°C. The pellets were then extracted with chlorof orm/methanol 2:1 (v/v) and the lipid extract obtained was separated by reverse phase chromatography on a column of Lipidex-5000 (Packard Instruments Co., Downers Grove, IL) in a system of ethylene chloride/methanol 1:4 (v/v) (22). The phospholipid fraction was used for isolation of proteins.
  • Lipidex-5000 Packard Instruments Co., Downers Grove, IL
  • the proteins were also separated by HPLC on a 5 ⁇ m column (25 cm x 4.4 mm) of Nucleosil C 1 8 in methanol, containing 0.1% trif luoroacetic acid.
  • the proteins, applied in 10 ⁇ lchloroform/methanol 1:2 (v/v) were detected at 214 nm.
  • the two peaks obtained corresponded to those from the Sephadex LH-60 column.
  • EHXAMPLE 2 Isolation of human low molecular weight surfactant protein. Bronchoalveolar lavage
  • Bronchoalveolar lavage (BAD on humans was carried out with a flexible bronchoscope under local anesthesia. The bronchoscope was wedged in a middle lobe bronchus and sterile saline solution at 37°C was instilled in aliquot ⁇ of 50 ml.
  • the total volume instilled varied between 200 and 300 ml.
  • the fluid was gently suctioned back after each instillation and collected in a siliconized bottle kept on ice. Immediately after completion of the lavate the bottle was transported to the laboratory.
  • the recovered BAL fluid was strained through a double layer of Dacron nets and the volume was measured. It was centrifuged at 400 g at 4°C for 5 min and the supernatant was stored at -20°C until further analyzed. Amniotic fluid.
  • Human amniotic fluid obtained from full term pregnancies at Caesarian sections and vaginal deliveries, was filtered through a net and the volume was measured and the material was stored at -20°C until further analyzed. Isolation of hydrophobic proteins from bronchoalveolar and amniotic fluids.
  • the proteins were reduced with dithioerythrit ⁇ l (about 30 nmol/nmol peptide; 37°C; 2h) and carboxymethylated by addition of neutralized iodo ( 14 C) acetate (120 nmol/nmol peptide; 37°C; 2h) in 8 M urea, 0.4 M Tris/HCXl, 2mM EDTA.
  • Excess reagents were removed by exclusion chromatography on Sephadex LH-60 (40x1.1 cm) in chloroform/methanol 1:1 (v/v) containing 5% 0.1 M HCl.
  • SDS/polyacrylamide gel electrophoresis (using a 10% gel containing 8 M urea) (24).
  • Molecular weight markers were purchased from BDH Chemicals Ltd (England) and consisted of horse- -heart myoglobin, cleaved by cyanogen bromide. Phosphorus was analyzed according to Bartlett (25).
  • the total amount of fatty acids was determined by boiling the fractions for 10 mm in 10% boron trifluoride in methanol (25).
  • the unesterified fatty acids were determined by reaction of the fractions with diazomethane.
  • the amount of different fatty acids was analyzed by gas-liquid chromatography using a 25 m fused silica capillary column coated with OV-1.
  • the protein fractions were reduced by treatment with dithiothreitol (30 nmol/nmol polypeptide) at 37°C for 2 h, under nitrogen.
  • the reduced samples were then 14 C-carboxymethylated by treatment with neutralized lodo ( 14 C)-acetic acid (120 nmol/nmol polypeptide; 37°C; 2 h) and purified by exclusion chromatography on Sephadex LH-60.
  • Samples for sequence analysis of the 14 C-carboxymethylated protein were removed after solubilization in chloroform/methanol. Samples for cleavages with pepsin were dissolved in 100% formic acid, diluted to 5% formic acid, and then submitted to the enzyme (1:30, enzyme to substrate ratio;
  • Gas-phase sequencer analysis was performed by degradation in an Applied Biosystems 470 A instrument and phenylthiohydantoin detection by reverse-phase high performance liquid chromatography using a Hewlett Packard 1090 instrument (27).
  • Samples for liquid-phase sequencer analysis in a Beck- man 890C instrument were applied to glycine-precycled Polybrene (28), and analyzed by a similar high performance liquid chromatography system.
  • Total compositions were obtained by hydrolysis with 6 M HCl/0.5% phenol at 100°C for 24 h in vacuum. Hydrazinolysis was performed with anhydrous hydrazine in 100°C for 6 h in evacuated tubes (29).
  • DPPC 1.2-Dipalmitoyl-sn-glycero-3-phosphocholin e
  • POPC 1-palm ⁇ toyl-2-oleoyl-sn-glycero-3-phosphochol me
  • DPPG 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol
  • the surface properties of the protein-based artificial surfactants were analyzed with a pulsating bubble instrument (Surfactometer International, Toronto, Canada) (31).
  • the surfactant preparations were suspended at a phospholipid concentration of 10 mg/ml, and the pressure gradient across the bubble wall was recorded at 37°C during 50% cyclic surface compression at the rate of 40/min. Surface tension was assessed at maximal and minimal bubble sizes during the 5th cycle and after 1 and 5 min of pulsation.
  • the animals were anesthetized with intraperitoneal sodium pentobarbital (0.1 ml; 6 mg/ml), paralysed with intraperitoneal pancuronium bromide (0.1 ml; 0.2 mg/ml), kept in body plethysmographs at 37°C, and subjected to pressure-controlled artificial ventilation with 100% oxygen, a frequency of 40 breaths/min, and 50% inspiration time.
  • the insufflation pressure was first set at 35 cm H 2 O for 1 min, then lowered to 25 cm H 2 O for 15 min, 20 cm H 2 O for 5 min, 15 cm H 2 O for 5 min, and then again raised to 25 cm H 2 O for 5 min (11,17).
  • Tidal volumes (V T ) were determined with a pneumothachygraph connected to each body plethysmograph.
  • tidal volumes were recorded in control animals and in animals treated with the same artificial surfactant preparations at a concentration of 80 mg phospholipids per ml.
  • the tracings were obtained at an insufflation pressure of 25 cm H 2 O after 30 mm of ventilation.
  • Fig.6 Representative tracings are illustrated in Fig.6, showing signifiicant pulmonary surfactant activities for preparations containing the proteins of the invention as compared to phospholipids only and to controls.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Immunology (AREA)
  • Genetics & Genomics (AREA)
  • Biochemistry (AREA)
  • Zoology (AREA)
  • Toxicology (AREA)
  • Pulmonology (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biophysics (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

Protéine comprenant la séquence d'acides aminés (I) dans laquelle K et L sont sélectionnés à partir de Ile, Gly, Val et dans laquelle K représente de préférence Ile et L est Gly, ou l'un et l'autre représentent Val. On décrit également des compositions de protéines et des compositions pharmaceutiques, une telle protéine ou une composition destinée à être utilisée comme constituant d'un tensio-actif pulmonaire, et un procédé facilitant la respiration des mammifères y compris l'homme.
PCT/SE1988/000361 1987-07-01 1988-06-29 Proteines, compositions de proteines et leurs utilisations WO1989000167A1 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
SE8702724-9 1987-07-01
SE8702724A SE8702724D0 (sv) 1987-07-01 1987-07-01 Proteins and protein compositions and their use
SE8703661-2 1987-09-22
SE8703661A SE8703661D0 (sv) 1987-09-22 1987-09-22 Surfactant-protein ii

Publications (1)

Publication Number Publication Date
WO1989000167A1 true WO1989000167A1 (fr) 1989-01-12

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WO (1) WO1989000167A1 (fr)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0413957A2 (fr) * 1989-08-22 1991-02-27 Abbott Laboratories Fragments de la protéine de tensio-actif pulmonaire
US5683982A (en) * 1991-11-04 1997-11-04 Merrell Pharmaceuticals Inc. Synthetic lung surfactant having antioxidant properties
WO1997041155A3 (fr) * 1996-04-26 1998-02-19 Ony Inc Compositions et procedes servant a isoler les proteines hydrophobes sp-b et sp-c du surfactant pulmonaire
US6020307A (en) * 1997-04-25 2000-02-01 Ony, Inc. Compositions and methods for isolating lung surfactant hydrophobic proteins SP-B and SP-C

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
SE8803713D0 (sv) * 1988-10-18 1988-10-18 Kabigen Ab Biologically active lipoprotein and its use

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1987006943A1 (fr) * 1986-05-06 1987-11-19 Children's Hospital Medical Center Proteine associee a un agent tensio-actif hydrophobe pulmonaire d'un poids moleculaire de 6000 daltons, et ses multimeres
WO1988003170A1 (fr) * 1986-10-24 1988-05-05 Whitsett Jeffrey A Proteines associees a des agents tensioactifs hydrophobes pulmonaires
WO1988004324A1 (fr) * 1986-12-08 1988-06-16 Abbott Laboratories Proteines associees a des tensioactifs hydrophobes pulmonaires

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1987006943A1 (fr) * 1986-05-06 1987-11-19 Children's Hospital Medical Center Proteine associee a un agent tensio-actif hydrophobe pulmonaire d'un poids moleculaire de 6000 daltons, et ses multimeres
WO1988003170A1 (fr) * 1986-10-24 1988-05-05 Whitsett Jeffrey A Proteines associees a des agents tensioactifs hydrophobes pulmonaires
WO1988004324A1 (fr) * 1986-12-08 1988-06-16 Abbott Laboratories Proteines associees a des tensioactifs hydrophobes pulmonaires

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
PROC. NATL. ACAD. SCI. USA, Vol. 84, pp. 7915-7919, November 1987, RHONDA G. WARR et al., "Low molecular weight human pulmonary surfactant protein (SP5): Isolation, characterization, and eDNA and amino acid sequences". *

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0413957A2 (fr) * 1989-08-22 1991-02-27 Abbott Laboratories Fragments de la protéine de tensio-actif pulmonaire
EP0413957A3 (en) * 1989-08-22 1991-10-16 Abbott Laboratories Pulmonary surfactant protein fragments
US5238920A (en) * 1989-08-22 1993-08-24 Abbott Laboratories Pulmonary surfactant protein fragments
US5302581A (en) * 1989-08-22 1994-04-12 Abbott Laboratories Pulmonary surfactant protein fragments
US5683982A (en) * 1991-11-04 1997-11-04 Merrell Pharmaceuticals Inc. Synthetic lung surfactant having antioxidant properties
WO1997041155A3 (fr) * 1996-04-26 1998-02-19 Ony Inc Compositions et procedes servant a isoler les proteines hydrophobes sp-b et sp-c du surfactant pulmonaire
US6020307A (en) * 1997-04-25 2000-02-01 Ony, Inc. Compositions and methods for isolating lung surfactant hydrophobic proteins SP-B and SP-C

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AU1996388A (en) 1989-01-30

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