WO1993021225A1

WO1993021225A1 - Synthetic peptide, pulmonary surfactant containing the same, and remedy for respiratory distress syndrome

Info

Publication number: WO1993021225A1
Application number: PCT/JP1993/000492
Authority: WO
Inventors: Tsunetomo Takei; Toshimitsu Aiba; Kaoru Sakai; Tetsuro Fujiwara
Original assignee: Tokyo Tanabe Company Limited
Priority date: 1992-04-17
Filing date: 1993-04-16
Publication date: 1993-10-28
Also published as: JP3376582B2; AU3905693A; JPH05294996A

Abstract

A peptide represented by the sequence (I), a pulmonary surfactant comprising the peptide and a lipid mixture, and a remedy for respiratory distress syndrome containing the surfactant as the active ingredient. In sequence (I) Xaa means the absence of any member or represents Cys, Ser or Ala; Xbb represents Cys, Ser or Ala; Xcc represents His or Asn; Xdd represents Leu or Ile; Xee represents Val or Ile; Xff represents Ile, Leu or Val; and Xgg means the absence of any member or represents Leu. The peptide is easy to isolate and purify, can be mass produced in a short time and has a potent surface activity when mixed with a lipid mixture. The pulmonary surfactant has a good suspending activity and a potent surface activity, thus being useful as a remedy for respiratory distress syndrome.

Description

Description Synthetic peptides, pulmonary surfactants containing them, and therapeutic agents for respiratory distress group

The present invention relates to synthetic peptides. More specifically, the present invention relates to a synthetic peptide having a strong surface activity by being combined with a lipid mixture, a pulmonary surfactant comprising the synthetic peptide and a lipid mixture, and a therapeutic agent for respiratory distress syndrome containing the pulmonary surfactant as an active ingredient. . Background technology,

Respiratory distress syndrome is a disease that causes severe respiratory distress as a result of the collapse of the alveoli due to a lack of pulmonary surfactant.

In recent years, replacement therapy has been developed to administer pulmonary surfactant from the outside via the airway to respiratory distress syndrome, and has achieved remarkable therapeutic effects. Pulmonary surfactants to be supplemented include substances consisting of phospholipids, neutral lipids, total cholesterol and carbohydrates and a small amount of proteins present in lung tissue of mammals (Japanese Patent Publication No. 61-92925), In addition to the components, a substance containing fatty acids (hereinafter referred to as “S-TA”: Japanese Patent Publication No. 61-92924), a substance separated from pig lung lavage fluid and added with Ca (Japanese Journal of the Japanese Society of Surface Medicine, Vol. 12, No. 1, p. 1, 1989), corin phosphoglycerides, acidic phospholipids, fatty acids, and lipoproteins derived from animal lungs at specific ratios Substances (Japanese Patent Publication No. 3-78.371), substances composed of dipalmitoylphosphatidylcholine and fatty alcohol No. 3, 4 3 2 5 2).

Some of the present inventors previously separated a lipoprotein from an animal-derived lung surfactant, and added the lipoprotein to a lipid mixture because the lipoprotein is an essential component for exhibiting lung surface activity. As a result, the surfactant exerts an excellent surface tension lowering effect, shortens the surface wavefront diffusion effect of the surfactant, and exerts a low surface tension on the flat mouth, thereby securing sufficient alveolar cavity volume, etc., resulting in respiratory distress syndrome. It has been discovered that it can be used to treat (Japanese Patent Publication No. 3-788371).

In recent years, as apoproteins specific to mammalian lung surfactant, lipophilic surfactant apoprotein A and surfactant apoprotein D, and hydrophobic surfactant apoprotein B (hereinafter referred to as “SP-B”) and surfactant apoprotein C have been used. (Hereinafter referred to as “SP-C”) [A review on the structure and function of apoproteins (Toyoaki Akino, Yoshio Kuroki, Respiration and Circulation, Vol. 38, No. 18, No. 7 22 pages, 1990; Ed. Yasuda, K. et al., “Biosurfactant_ [, Chapter 2 Biochemistry of Surfactants—Surfactants and Apoproteins” Page 31, 1990, Science Co., Ltd. Forum)]

SP-C derived from human lung is an apoprotein with extremely strong hydrophobicity, which is composed of 35 amino acid residues and whose N-terminal amino acid is phenylalanine and rich in hydrophobic amino acids such as amino acids. SP-C isolated from the lungs of pigs, pigs, rats, etc. also consists of 34 to 35 amino acids, and although the amino acid sequence at the N-terminal varies depending on the animal species, it is not Are extremely high in homology. Human SP — amino acid sequence of c

Phe Gly lie Pro Cys Cys Pro Val His Leu Lys Arg Leu Leu lie Val

1 5 10 15

Val Val Val Val Val Leu lie Val Val Val lie Val Gly Ala Leu Leu

20 25 30

Met Gly Leu

35

In Japanese Patent Application Laid-Open No. Hei 3-5002095 and Japanese Unexamined Patent Publication No. Hei 3-90033, SP-B or SP-C promotes adsorption and diffusion of lung surfactant to the gas-liquid interface. To improve the surface activity of pulmonary surfatatantes.

Therapeutic agent for respiratory distress syndrome, which contains a pulmonary surfactant consisting of SP-C and a lipid mixture as an active ingredient, is extremely effective, but SP-C is extremely hydrophobic and difficult to isolate and purify. Due to the fact that it is contained in living organisms in extremely small amounts, it has not been put to practical use. Japanese Patent Application Laid-Open No. 3-52095 states that a mixture of a synthetic peptide containing the SP-C partial structure shown below and a lipid is effective for the treatment of respiratory distress syndrome Have been. The publication also describes a peptide having 32 amino acid residues having the following sequence as a minimum unit exhibiting high surface activity. In addition, a comparison of the surface activity of this minimal unit of peptide with synthetic peptides having shorter amino acid residues indicates that the loss of surface activity is not due to the loss of specific residues, but to the polypeptide. They conclude that this is due to a decrease in peptide length.

Cys Cys Pro Val His Leu Lys Arg Leu Leu lie Val Val Val Val Val 1 5 10 15 Val Leu lie Val Val Val lie Val Gly Ala Leu Leu Met Gly Leu His

20 25 30 However, in general, the production of synthetic peptides requires that the longer the amino acid sequence becomes, the more frequently immature peptides are produced during synthesis, which makes isolation and purification difficult, and that production requires a long time. It is said that there are drawbacks such as the necessity and difficulty in mass synthesis.

By the way, the pulmonary surfactant preparation is often provided as a dry powder preparation which is converted into a physiological saline suspension wave at the time of use from the viewpoint of quality conservation.

However, pulmonary surfactant (hereinafter referred to as “S-35”) preparations in which a synthetic peptide having the amino acid sequence of SP-C is mixed with a lipid mixture composed of choline phosphoglyceride, acidic phospholipid, and fatty acids, are: Since the cysteine residue present in the peptide forms a disulfide bond, the dispersibility in physiological saline is extremely poor due to factors such as high peptide agglutinability and strong hydrophobicity of the lung surfactant itself. It was difficult to make the suspension uniform enough to be used as such.

In addition, as a method for improving the suspendability of a pulmonary surfactant preparation, a method of adding a suspending agent such as mannitol (Japanese Patent Application Laid-Open No. Sho 60-34905) and a method in which the temporary freezing temperature during drying is reduced to 1: 1. Although a freezing method performed at temperatures of up to 11 ° C. has been proposed (Japanese Patent Application Laid-Open No. Sho 63-107718), the operation is complicated, and it is hoped that a more convenient method for producing a drug product will be developed. Disclosure of the Invention

In view of the above findings, the inventors of the present invention have conducted intensive studies on a synthetic peptide having a strong surface activity by being combined with a lipid mixture, and as a result, a peptide represented by the following specific sequence (hereinafter referred to as “the synthetic peptide of the present invention”) The pulmonary surfatatant containing) as an active ingredient was produced by a conventional freeze-drying method performed at 120 ° C or lower without the addition of a suspending agent, even when S-35, corin phosphoglycerol was used. Only lipid mixtures consisting of acidic phospholipids and fatty acids It has better uniform suspension than S-TA, and has the same strong surface activity as S-35 or S-TA. Knowing this, they completed the present invention.

Xaa Xbb Pro Val Xcc Xdd Lys Arg Leu Leu Xee Val Val Val Val Val 1 5 10 15

Val Leu Xff Val Val Val He Val Gly Ala Leu Xgg

20 25

(In the sequence, Xaa is absent or represents Cys, Ser or Ala, Xbb represents Cys, Ser or Ala, Xcc represents His or Asn, Xdd represents Leu or Ile, Xee is Val or l ie, Xff represents I le, Leu or Val, and Xgg does not exist or represents Leu.)

According to the present invention, it is a synthetic peptide which is easy to isolate and purify due to the low frequency of production of immature peptides during production, and can be produced in large quantities in a short time. A synthetic peptide having good suspension properties and strong surface activity by being blended, a lung surfactant comprising a mixture of the synthetic peptide and lipid, and a treatment for respiratory distress syndrome containing the lung surfactant as an effective component An agent is provided.

The synthetic peptide of the present invention can be produced by a chemical or genetic engineering technique, but a chemical production method is preferred in terms of isolation and purification.

Examples of chemical production methods include “Peptide synthesis (by Nobuo Izumiya, Maruzen Co., Ltd., 1979)”, “Biochemistry—Lecture, Volume 1, Protein Chemistry IV, Chemical Modification and Peptides” Synthesis-(Shunpei Sakakibara, Tokyo Chemical Doujin Co., Ltd., 1973) "," Semi-chemical Chemistry Laboratory, Vol. 2, Protein Chemistry (below), Peptide Synthesis (Kimura Terutoshi, Tokyo Chemical) Dojin Co., Ltd., 1987), "Solid phase peptide synthesis, a practical approach;, Senoreton (E. Atherton) and Shieno De Shepherd, RC Sheppard, 25-: I89, Oxford University Press, Oxford, 1989) "and Kenichi, Akagi et aL (Chem. Pharm. Bull., Vol. 37, Vol. 1) No. 0, 2661-26464, 1989) "or" The Peptides "(Gross, E. and Meinenhofe, J., Barany (Barany, G.) and Merrifield, R., Vol. 2, pp. 1-284, Academic Press, New York, 19 & 0)]] , Azide method, acid chloride method, acid anhydride method, mixed acid anhydride method, DCC method, active ester method (ρ-ditrophenyl ester method, P-hydroxysuccinic acid imid ester method, etc.), carboimidazole It can be manufactured by a liquid phase synthesis method such as a redox method, a redox method, a DCC-activation method, or a solid synthesis method.

Of these, solid phase synthesis is preferred, and synthesis can be performed by an automatic synthesizer. Examples of the automatic synthesizer include a 431A peptide synthesizer (trademark; manufactured by Applied Biosystems) or a peptide synthesizer model 9900E (trademark; manufactured by Beckman).

A pulmonary surfactant (hereinafter referred to as “surfactant of the present invention”) can be produced by blending choline phosphoglyceride, acidic phospholipid and fatty acids as a lipid mixture with the synthetic peptide of the present invention. The compounding ratio is 0.1 to 5.0% (W / W) for synthetic peptide and 50.6 to 85.0 for choline phosphoglyceride, based on the weight ratio of these components to the total dry weight of the final product. % (W / W) 適当 It is appropriate to set acidic phospholipids to 4.5 to 37.6% (W / W) and fatty acids to 4.6 to 24.6% (WZW). It is.

Choline phosphodalicerides that can be used in the surfactants of the present invention include 1,2-dipalmitoyl glyceride— (3.)-Phosphocholine (also known as dipalmitoyl phosphatidyl choline) and 1,2-distearoyl glyceride. Lysero (3)-phosphocorin, 1-palmitoyl 2- 2-stearoyl glycerol (3)-phosphocholine or 1-stearoyl-2-palmitoyl glycerol (3)-1,2 such as phosphocorin-diacylglycerose-(3) —Phosphocholine, 1-hexadecyl—2—palmitoylglycerol (3) —phosphocholine or 1-year-old kutadecyl-2—palmitoylglycerol (3) —1-alkyl, such as phosphocorin-2—angluglycerol (3) — 1,2-Dialkylglycerol (3) -phosphocholine such as phosphocorin or 1,2-dihexadecylglycerol (3) -phosphocholine is suitable. These compounds have optical isomers based on the carbon at the 2-position of the glycerol residue, and any of the D-form, L-form and DL-form can be used in the surfactant of the present invention. In addition to this, in addition to the above-mentioned single-part corin phosphoglyceride, corynephosphoglyceride may be an acyl group having 12 to 24 carbon atoms, preferably two saturated acyl groups. , 2-Diacylglycerose- (3) -A mixture of two or more phosphocholins, and a mixture of the mixture and the above-mentioned single product can also be used.

Examples of acidic phospholipids include 1,2-diacyl-sn-glycerol (3) monophosphoric acid (also known as L-α-phosphatidic acid), 1,2-diacyl-sη-glycerol— (3) -phospho-serine (Also known as phosphatidylserine), 1,2-diasyl-sη-glycerol— (3) —phospho-sη-glycerol (also known as phosphatidylglycerol) or 1,2-diasyl-sη-glycerol (3) —phospho (1) — L-1 my 0—Inositol (also known as phosphatidylinositol) is suitable. In these compounds, the 1-position and the 2-position may be substituted with the same or different kinds of acyl groups, respectively. Here, the carbon number of the acyl group is preferably from 12 to 24. Fatty acids include free fatty acids, alkali metal salts of fatty acids, and fatty acids. Suitable are & alkyl esters, fatty acid glycerin esters, fatty acid amides, or mixtures of two or more of these, and furthermore, fatty alcohols or aliphatic amines.

In the present specification, "on fatty acids" is meant to include the fatty alcohols and aliphatic amines referred to herein.

Myristic acid, palmitic acid or stearic acid is suitable as a free fatty acid. Lumitic acid !? Good.

Preferably, sodium palmitate is used as the metal salt of fatty acid, ethyl palmitate is used as fatty acid alkyl ester, monopalmitin is used as fatty acid glycerin ester, and palmitic acid amide is used as fatty acid amide.

Hexadecyl alcohol is preferred as the fatty alcohol, and hexadecylamine is preferred as the aliphatic amine.

The above-mentioned choline phosphoglycerides, acidic phospholipids and fatty acids may be any of products isolated from animals and plants, semi-synthetic products or chemically synthesized products, and commercially available products thereof can be used.

The surfactant of the present invention is obtained by drying a mixed solution of the synthetic peptide solution of the present invention and the above-mentioned lipid mixture or compound solution under reduced pressure, suspending the obtained residue using a suitable suspending solvent, and then freeze-drying. It can be manufactured by a method. Examples of the solvent used for preparing the synthetic peptide solution of the present invention include formic acid, trifluoroacetic acid (TFA), trifluoroethanol, dimethyl sulfoxide (DMS0), chloroform / methanol, and chloroform.

Examples of the solvent used for preparing the lipid mixture solution include chloroform, chloroform / methanol [2: 1 to 5: 1. (V / V)] —. Examples of the suspending solvent include water or a mixed solution of water and ethanol [4: 1 to 20: 1 (V / V)], and a mixed solution of water and ethanol is preferable. The suspension is carried out at 30 to 60 ° C., preferably 40 to 50 ° C., for 5 to 60 minutes, preferably for 15 to 30 minutes.

In the surfactant of the present invention, it is inevitable that a trace amount of water remains in the production method, but it is preferable to dry the surfactant until the remaining weight ratio becomes 5.0% (W / W) or less based on the total weight. If it is dried to such an extent, residual ethanol cannot be detected when a water-ethanol mixture is used.

In addition, the surfactant dry powder preparation of the present invention can be prepared in a variable-speed mixer or an ultrasonic generator at an appropriate physiological concentration of a monovalent or divalent metal salt, for example, 0.9% sodium chloride or 1.5%. It can be used by uniformly suspending and dispersing it using mM calcium chloride or a physiological buffer containing them.

Next, the surface activity, suspendability and pharmacological properties of the surfactant of the present invention thus produced will be described in detail.

[Surface activity]

①Surface tension lowering action

The surface tension lowering effect was measured according to the method of Tanaka et al. (Journal of the Surface Science Society of Japan, Vol. 13, No. 2, No. 87, p. 87, 1982).

The surfactant of the present invention is dropped onto physiological saline (surface area: 54.0 cm ² ) so that the surfactant of the present invention is 1.0 to 2.0 / g per cm ² , and the surface area is reduced. 54. The surface tension during compression and expansion in the range of 0 to 2 1.6 cm ² over 2 to 5 minutes was measured at 37 ° C with a Wilhelmy surface tension measurement device (manufactured by Kyowa Interface Science Co., Ltd.). Measured continuously. The surface tension reducing effect of the surfactant of the present invention is as follows.

7 ~ 34.5 dyne cm, minimum surface tension is 1.4 ~ 8.9 dyne Z cm, which was found to lower the surface tension of physiological saline. The surface tension lowering effect of SF-3 measured in the same manner was a maximum surface tension of 25.8 to 50.3 dyne / cm and a minimum awakening power of 1.0 to 13.5 dyne / cm. o

The initial surface tension of saline at 37 ° C was 70.5 dyne

/ cm m rare o

② Gas-liquid diffusion

0.8 to 1.5 g of the surfactant suspension of the present invention per 1 cm ^{2 of} surface area was dropped on the surface of physiological saline ice, and the surface tension immediately after the dropping was measured over time by a vertical plate method. The measurement temperature was 37 ° C.

The arrival time refers to the time required for the surface tension to reach a constant value immediately after dropping of the sample, and the equilibrium surface tension refers to the value at that time.

The surfactant of the present invention formed a film on the gas-liquid surface in a short time of 3 to 65 seconds, and reduced the surface tension to 27.9 to 34.8 dyneZcm.

In the gas-liquid surface diffusion effect of SF-3 measured in the same manner, the surface tension after 120 seconds passed was 38.1-52.9 dyneZcm.

③ Gas-liquid surface adsorption

A suspension of physiological saline at 37 ° C containing 0.2 to 1.0 mg of the surfactant of the present invention per liter was prepared, and the suspension of the surfactant of the present invention to the saline gas-liquid surface was prepared. Was measured.

The adsorption rate was measured according to the method of King et al. (American Journal of Physiology, Vol. 223, Vol. 7, pp. 15, 1972).

In other words, the suspension was poured into the bottom of a 5 cm diameter water tank containing physiological saline, and the mixture was stirred gently with a magnetic stirrer. The speed was determined. The surfactant of the present invention reduced the surface tension to a range of 28.:! To 39.5 dyne cm after 30 to 120 seconds had elapsed after the stirring was stopped, and then showed a constant value.

This indicates that the surfactant of the present invention in the suspension state floated and adsorbed on the gas-liquid surface in 30 to 120 seconds, and formed a film having strong surface activity. The SF-3 measured in the same manner showed a constant value in the surface tension range of 42.2 to 58.3 dyne Zcm, and the required time was 150 seconds or more.

This indicates that the gas-liquid surface adsorption effect of SF-3 is weaker than that of the surfactant of the present invention, and that the surfactant of the present invention has a strong surface adsorption promoting power.

[Suspension]

The suspension test of the lung surfactant was performed according to the method disclosed in Japanese Patent Application Laid-Open No. Sho 63-107718.

That is, the suspendability was determined based on the dispersion ratio at predetermined time intervals after the start of the suspension and the maximum dispersed particle diameter after 2 minutes from the start of the suspension.

To test the dispersibility, 60 mg of lung surfactant was dispensed into a 20 ml vial, 2 ml of physiological saline was injected, and the vial was placed on an Iwaki KM Shiaker V-S shaker (Iwaki Co., Ltd.). Attach and shake at 270 strokes / minute.After 30 seconds of shaking, disperse each sample every minute from 1 minute to 4 minutes, and every 2 minutes from 4 minutes to 10 minutes. The condition was visually observed from outside the container through a loop.

The determination of the suspension state was carried out by two persons, 10 samples each time at each time, and no judgment was made as to whether or not the suspension was any small lump in the container, and the formulation was uniformly dispersed in physiological saline This was done by determining whether a white, slightly viscous suspension had formed. The dispersion ratio was calculated as a percentage of the total number of samples (10 tubes) in which each individual completed the suspension at each time, and the average value was shown by the two individuals. The maximum dispersed particle size was determined by dispensing 6 mg of lung surfactant into a 20 ml vial of each sample, injecting 2 ml of physiological saline, and shaking continuously for 2 minutes under the same shaking conditions as described above. The largest particle was found by using a microscope and its diameter was determined by measuring with a vernier caliper.

Most of the surfactants of the present invention were suspended within 2 minutes in most cases, and had a maximum particle diameter of 0.9 mm or less, and showed good suspendability.

[Pharmacological properties]

①Acute toxicity

The acute toxicity of the surfactant of the present invention was tested using 5-week-old male ICR mice and Dister rats. Oral LD _{5 in} mice. And intraperitoneal LD ₅ . Is 2.5-10.0 / ¾: and 1.5-5.O gZkg, and those on the rat are 1.5-5.0 11 and 1.5-2.5 g Z kg.

② Subacute toxicity

The surfactant of the present invention was intraperitoneally administered to mature Wistar rats for 3 months 0 to 600 mgZkg for 1 month, but no change in body weight or abnormality in macroscopic and histological observation of major organs was observed. .

③ Alveolar cavity volume maintenance

A rabbit immature fetus with a gestation period of 27 days produces little pulmonary surfactant and is in a pulmonary surfactant-deficient state, and is therefore a model animal for neonatal respiratory distress syndrome.

Using 5 rabbit fetuses with a gestation period of 27, the alveolar cavity volume (hereinafter, referred to as lung volume) was measured at 37 ° C under increasing and decreasing airway pressure.

The measurement was continuously performed 5 minutes after the surfactant of the present invention was intratracheally administered using a water manometer connected to the trachea by incising the neck of the fetus. 2-channel independent drive cylinder with tracheal pressure connected to the trachea The pressure was increased to 30 cm water pressure using a dipump No. 940 (manufactured by Harvard, USA) to expand the alveoli. Next, the airway pressure was reduced to 0 cm water pressure, the alveoli were contracted, and the lung volume at each water pressure was measured. Lung volume was expressed in milliliters per kilogram of body weight (m1Zkg).

The administration of the surfactant of the present invention has a concentration of 1.0 to 6.0% (W /

V) was performed by a method of injecting 0.05 to 0.5 ml of a physiological saline suspension prepared directly into the airway.

The greater the lung capacity at 5 cm water pressure during decompression, which indicates functional residual capacity, the higher the pulmonary surfactant activity.

As a control, physiological saline was injected instead of the surfactant suspension of the present invention. In the control group, the lung capacity (5 cm water pressure) of the immature rabbit at gestational age 27 was 1-5 m 1 / kg, and the alveoli were hardly expanded.

Also, gestational age 30 fetuses with normal levels of pulmonary surfactant have a lung volume (5 cm water pressure) of 39-53 m 1 / kg and alveolar dilation. Shows that it is possible to breathe normally. When one SF-3 was administered, the lung volume (5 cm water pressure) of the immature fetus was 15 to 25 m 1 / kg and the alveolar expansion was insufficient.

The lung capacity (5 cm water pressure) of the immature fetus to which the surfactant of the present invention was administered was 35 to 53 m 1 Zkg, indicating that the surfactant of the present invention improved the lung capacity of the immature fetus to a normal level.

As described above, the present synthetic peptide has an action of strongly activating the surface activity of the lipid mixture, and the present surfactant comprising the present synthetic peptide and the lipid mixture exhibits surface activity, suspendability and Based on its pharmacological properties, it is an effective remedy for respiratory distress syndrome.

The remedy for respiratory distress syndrome provided by the present invention is a single dose of 50 to 100 mg for children and 500 to 500 mg for adults. Contains the surfactant of the present invention. This dose is suspended in water, physiological saline, or a physiologically acceptable buffer, and adjusted to a concentration of 1.0 to 10% by weight (WZV). Use by injecting or spraying 1 to 10 times into the respiratory tract 48 hours immediately after. In addition, they can be inhaled directly as powder without being suspended. Dosage, use and frequency may be adjusted as appropriate for the patient's condition and combination therapy.

The therapeutic agent of the present invention may contain, if necessary, pharmaceutical additives such as stabilizers, preservatives, isotonic agents, buffers, suspending agents, or pharmaceuticals such as bronchodilators, antiallergic agents, anticancer agents, and antibacterial agents. It can be contained.

The dosage form is suitably a liquid preparation or a powder preparation to be used in suspension at the time of use. The therapeutic agent of the present invention is filled in a sealed container such as a vial or ampoule and stored as a sterile preparation.

Hereinafter, the present invention will be described with reference to examples.

[Production of peptides]

(Example 1)

The peptide described in SEQ ID NO: 1 (hereinafter, referred to as “peptide A”) was converted to “Solid phase peptide synthesis” by E. Atherton and RC Sheppard. a practical approach) ”p. 25-189, 1989, Oxford University Press, Oxford] and KenicM, Akagi et al. [Chem. Pharm. Bull., 37 (10), p. 9 89)], the solid phase was synthesized using a multi-peptide solid-phase synthesis system “Koksan” (trade name; manufactured by Kokusaikagaku Co., Ltd.).

As the first resin, N-α-9-fluorenylmethyloxycarbo-2-leucine (Fmoc-Leu) was added to [4-1 (hydroxymethyl) Phenoxymethyl-copoly (styrene 1% divinylbenzene)] N- «-9-Fluorenylmethyloxycarbocarbone-to-resin bonded to resin-0-Resin (Fmoc-Leu-0- Resin) 0.2 g of 0.5 mm was used. After swelling the resin with N, N-dimethylformamide (DMF) for 20 minutes, the resin was washed four times with DMF. A 20% piperidine-DMF solution was added and shaken to perform deprotection. This operation was repeated three times to completely perform this deprotection. Then, the resin was washed three times with DMF, three times with N-methyl-2-pyrrolidone, and three times with DMF to remove excess piberidine in the resin. At this time, the presence or absence of peridine was confirmed using pH test paper. ―

Then add 9 ml of DMF, 5 mmo of Fmoc-Leu O.5 mmo1, 0.5 mm of N-hydroxybenzotriazole and 0.5 mm of N, N'-diisopropylcarboimide and add 90 mm of 90 The mixture was shaken for a minute to carry out a condensation reaction. The resin was then washed four times with DMF to remove excess reagent. Confirmation of this condensation reaction was performed by Kaiser test by the ninhydrin method.

Thus, according to the synthesis plan, the amino acid was sequentially extended in the N-terminal direction on the resin to synthesize a peptide 0-resin in which the N-terminal and the functional group were completely protected. The condensation reaction during the introduction of Arg, Lys, His. Pro, and Cys was performed twice in 120 minutes. Then, add 20% piperidine-DMF solution to the protected peptide-10-resin to deprotect the N-terminal Fmoc protecting group, and then treat the peptide10-resin 6 times with DMF and 6 times with methanol. It was washed and dried under reduced pressure. While stirring the dried peptide 0-resin (10 Omg) under ice cooling, m-cresol (0.2 ml), 1,2-ethanedithiol (0.5 ml), thioanisol ( 1.2 ml), TFA (7.5 ml) and trimethylsilyl bromide (1.4 ml). After that, the mixture was stirred for 12 minutes under ice-cooling, and the peptide was cut out from the resin together with the deprotection of the functional side chain, and filtered with a glass filter (G3). The filtrate was concentrated under reduced pressure to about 5 ml with an evaporator, and a peptide was precipitated by adding getyl ether. The peptide precipitate was collected by filtration with a glass filter (G3), washed five times with getyl ether, and dried under reduced pressure to obtain 55 mg of crude peptide A.

The amino group at the N-terminus of all amino acids was protected with an Fmoc group, and the functional side chains were protected with the following groups.

A rg--M tr; (4-methoxy-2,3,6-trimethylbenzenesulfonyl).

Lys-Boc; (t-butyloxycarbonyl).

Cys-Trt; (trityl).

H I s -T r t; (trityl).

About 7 mg of this crude peptide was dissolved in 0.6 ml of TFA. Further, formaldehyde-methanol (CZM) [2: 1, (VXV)] was added to the solution to make a final volume of 3.0 ml. The sample was purified on a Sephadex LH-60 column (ø2.5 cm x 90 cm) equilibrated with a CZM mixed solvent [2: 1, (VZV)], and peptide A was collected.

The presence of the peptide in the eluate Φ was monitored by 245 nm (spectrophotometer; Model 870-UV of JASCO Corporation) and a differential refractometer (Shimadzu Corporation; Model R ID-6A).

(Example 2)

The peptide described in SEQ ID NO: 2 (peptide B) was referred to as “The Peptides [Gross, E.) and Meinenhofe (J.), Barany, G. and Merrif (Merrif). ierd, R), Vol. 2, pp. 1-284, Academic Press, New York, 1980)]] According to the method described in (1), synthesis was carried out on a solid-phase synthesis method on fuyunyl acetamidomethyl (PAM) resin.

The leucine at the C-terminal amino acid residue is designated as t-butyloxycarboxy-l-isocyanate (Boc-Leu), which is bound to the PAM resin via an oxymethylphenylacetamide bond. I let it. After binding the C-terminal, B 0 c—L eu—PAM resin (0.70 mol / g, 0.35 g) is used as a reaction vessel in a peptide synthesizer (Model 990E, manufactured by Beckman). Moved to The protected amino acid was extended in the N-terminal direction on the resin by a preformed symmetric anhydride method to synthesize a completely protected peptide 10-resin. However, when condensing arginine, N, N-dicyclohexylcarpoimide (DCC) / hydroxybenzotriazole [Kony et al., Chem. Ber., 103, 788-798 (1970) ] To double force. The amino group at the N-terminus of all amino acids was protected with a Boc group, and the functional side chains were protected with the following groups.

A r g -T os; (tosyl).

L y s— 2 C L Z; (2-chloro benzyloxycarbonyl).

Cys-4MeBzl; (4-methylbenzyl).

H s -T 0 s; (Tosyl).

Confirmation of this condensation reaction was carried out by Kaiser test by the Ninh Ndolin method.

The completely protected peptide 0—resin (155 mg) was swollen in methylene chloride for 5 minutes. The N—a—B0c protecting group was deprotected using TFA containing 1% (v / v) indole and 0.1% (v / v) ethanedithiol. The deprotected peptide-0-resin was then treated with p-cresol (1 ml), p-thiocresol (0.2 g) and DMSO (Lm1) in anhydrous hydrogen fluoride (HF) ( 1 1 m 1) at 0 ° C for 60 minutes Upon treatment, the peptide was excised from the resin.

HF and DMS 0 were distilled off at 0 ° C. under vacuum. The cut out peptide and resin were washed three times with 15 ml of cold getyl ether, and then the free peptide was extracted by washing three times with 10 ml of cold TFA washing solution. The extract was filtered immediately and added to ice-cold water (12 Om1-150 ml) to precipitate crude peptide B. Then, the crude peptide B was centrifuged at 1,000 X g, 0 ° C for 30 minutes and collected as a precipitate. The precipitate was washed with getyl ether (15 ml). This washing step was further repeated using getyl ether, ethyl acetate and distilled water to obtain 83 mg of peptide B.

The obtained peptide B was dissolved in a 50% aqueous solution of DMS and purified by high-performance liquid chromatography using a Bondasphere and C8-300 column.

As an eluent, a 50% aqueous solution of acetonitrile containing 1% TFA was used for elution for 5 minutes. The eluate was then eluted with a linear concentration gradient of 80% acetonitrile aqueous solution containing 0.1% TFA for 30 minutes. The presence of the peptide in the eluate was determined at 245 nm (spectrophotometer; JASCO Corporation). It was monitored with a model company model 870-UV) and a differential refractometer (Shimadzu Corporation Model RID-6A).

(Example 3)

The peptide of SEQ ID NO: 3 (peptide C) was prepared in the same manner as in (Example 1).

-(Example 4)

The peptide of SEQ ID NO: 4 (peptide D) was prepared in the same manner as in (Example 1). (Example 5)

The peptide of SEQ ID NO: 5 (peptide E) was prepared in the same manner as in (Example 1).

(Example 6)

The peptide of SEQ ID NO: 6 (peptide F) was prepared in the same manner as in (Example 1).

(Example 7)

The peptide of SEQ ID NO: 7 (peptide G) was prepared in the same manner as in (Example 1).

(Example 8)

The peptide of SEQ ID NO: 8 (peptide H) was prepared in the same manner as in (Example 1).

(Example 9)

The peptide of SEQ ID NO: 9 (peptide I) was prepared in the same manner as in (Example 1).

(Example 10)

The peptide of SEQ ID NO: 10 (peptide J) was prepared in the same manner as in (Example 1).

[Amino acid composition of peptide]

The synthetic peptide of the present invention was prepared using 1,2N hydrochloric acid ZTFA [2: 1 (V / V)] containing 5% (v / v) funinol under vacuum at 150 ° C for 2, 4, 6, 1 After acid hydrolysis for 2, 24, 48 and 72 hours to remove the acid, the hydrolyzed products were analyzed by Shimadzu Amino Acid Automatic Analysis System (LC-19A). In the hydrolysis for 2 to 72 hours, the amino acid value showing higher recovery was adopted, and the amino acid composition value was calculated.

[Molecular weight of peptide] The molecular weight of the synthetic peptide of the present invention was measured by the fast atom bombardment method (FABMS). A JMS-S102A type (manufactured by JEOL Ltd.) was used as a mass spectrometer, and a cesium gun (10 KeV) was used as an ion source.

The results of amino acid composition and mass analysis of the obtained peptide are shown in [Table 1].

Table 1 Amino acid composition value and molecular weight of the synthetic peptide of the present invention

(The values in the table indicate the amino acid composition values when Gly is set to 1.0.) (Production of the surfactant of the present invention)

The surfactant of the present invention is prepared by using the synthetic peptide and 1,2-dipalmitoylglycerol (3) -phosphocholine, 1,2-diasyl-sn-glycerol- (3) -phospho-sn-glycerol and palmitic acid as lipid components. The three components of the acid were prepared by mixing.

(Example 11)

Aseptically treated 2-divalmitoyl glycerol (3) -phosphocholine 660 mg, I, 2-diacyl s II-glycerol (3) -phospho sn-glycerol (14 to 24 carbon atoms in the acyl group: Sigma) 22 Orag and l-Omg of balmitic acid are dissolved at room temperature in a mixed solution of form-methanol in mouth and [2: 1 (VZV)] 100 Hi I, and 12 mg of peptide A is dissolved in 5 ml of TFAO. Was dissolved. These solutions were mixed and evaporated to dryness under reduced pressure. The obtained residue was suspended in 100 ml of a mixed solution of water and ethanol [9: 1 (VV)] at 40 ° C. for 5 minutes. This suspension was added for 150. The mixture was frozen with C and dried at a vacuum of 85 to 100 iHg for 36 hours to obtain Surfactant ¾22 mg as a white powder.

No ethanol remained in this powder, and the content of each component relative to the total weight of the surfactant was 64.6% (w / w) for 1,2-dipalmitoylglycerol (3) -phosphocorrin. 1,2-diacyl-sn-glycerol (3) -phospho-sn-glycerol is 21.5% (w / w), palmitic acid is 9.8% (/ w), peptide A is 1.2% (w / w) w / w) and water 2.9% (/ w).

The actions of the obtained surfactant were as follows.

Surface tension lowering action:

Maximum surface tension: 32.8 dyn e / cm

Minimum surface tension: 2.2 dyne / cm Gas-liquid interface diffusion:

Arrival time: 40 seconds, equilibrium surface tension: 29.8 dyne / cm

Arrival time: 60 seconds, equilibrium surface tension: 31.9 dyne eZcm Alveolar cavity volume maintenance action:

Lung capacity (5 cm water pressure); 51 m1 / kg

(Example 1 2)

1,2-Dipalmitoyl glycerol— (3) —Phosphocholine 204 mg, 1,2-Diacyl-s η-glycerol (3) —Phosphos η-glycerol (Chain 14 to 24 carbon atoms: Sigma) 6 3. Omg and palmitic acid 27. Omg was dissolved in a mixture of form-methanol (2: 1 (V / V)) 30 Om1 and 2.8 mg of peptide B was added to 3 ml of TFAO. Dissolved. These solutions were mixed and evaporated to dryness under reduced pressure. The obtained residue was suspended in 100 ml of a water-ethanol mixture [9: 1 (V / V)] at 45 ° C for 20 minutes. The suspension was frozen at −60 ° C. and dried at a vacuum of 60 to 110 ° Hg for 40 hours to obtain 301.9 mg of a white powdered surfactant.

No ethanol remained in the powder, and the content of each component relative to the total weight of the surfactant was 1,7.6-dipalmitoylglycerol- (3) -phosphocorrin was 67.6% (w / w). 1,2-Diacyl-sn-glycerol (3) -phospho-sn-glycerol 20.9% (w / w), palmitic acid 8.9% (w / w), peptide B 0.9% (w / w) and water 1.7% (w / w).

The actions of the obtained surfactant were as follows.

Surface tension lowering action:

Maximum surface tension: 34.5 dyne / cm Minimum surface tension; 6.7 dyne / cm

Gas-liquid interface diffusion:

Arrival time: 60 seconds Equilibrium surface tension: 31.2 dyne / cm Gas-liquid surface adsorption:

Arrival time: 1 20 seconds Equilibrium surface tension; 34.7 dyne / cm alveolar volume maintenance:

Lung capacity (5 cm water pressure) 49 m 1 / kg- (Example 13)

A surfactant was produced in the same manner as in (Example 12) except that peptide C was used instead of peptide B.

No ethanol remained in this powder, and the content of each component relative to the total weight of surfatatant was as follows: l _r 2-dipalmitoyl glycerol (3) 67.0% (w / w) , 1,2-diacyl-sn-glycerol (3) -phospho-sn-glycerol is 20.7% (w / w), palmitic acid is 8.9% (wZw), and peptide C is 0.9% (w / w) w / w) and water 2.5% iw / w).

The actions of the obtained surfactant were as follows.

Surface tension lowering action:

Maximum surface tension; 29.7 dyn e / cm

Minimum surface tension; 2.3 dyn e / cm

Gas-liquid interface diffusion:

Arrival time: 30 seconds Equilibrium surface tension: 27.9 dyne / cm Air wave surface adsorption:

Arrival time: 30 seconds Flat mouth surface tension; 2 &. L dyn eZcm Alveolar cavity volume maintenance action:

Lung capacity (5 cm water pressure) 53 m1 / kg- (Example 14)

Surfactants were produced in the same manner as in (Example 12) except that peptide D was used instead of peptide B.

No ethanol remained in this powder, and the content of each component relative to the total weight of the surfactant was 1,8.5-dipalmitoylglycerol (3) -phosphocorrin was 68.5% (w / w). 1,2—diacyl sn—glycerol (3) —phospho sn—glycerol 21.2% (w / w), palmitic acid 9.1% (w / w), peptide D 0.9% (w / w) and water 0.3% (w / w).

The actions of the obtained surfactant were as follows.

Surface tension lowering action:

Maximum surface tension: 30.8 dyne / cm

Minimum surface tension; 5.4 dyne / cm

Gas-liquid interface diffusion:

Arrival time: 45 seconds Equilibrium surface tension; 28.3 dvn / cm Gas / liquid surface adsorption:

Arrival time: 40 seconds Equilibrium surface tension: 30.7 dyne / cm alveolar cavity volume maintenance action:

Lung capacity (5 cm water pressure) 49 m1 / kg

(Example 15)

A surfactant was produced in the same manner as in (Example 12) except that peptide E was used instead of peptide B.

No ethanol remained in this powder, and the content of each component relative to the total weight of the surfactant was 1,2-dipalmitoylglycerol (3) -phosphocorrin was 67.8% (w / w), 1, 2-diacyl- sn-grease mouth-(3)-phospho- sn-glycerol is 20.9% (w / w), And palmitic acid were 9.0% (w / w), peptide E was 0.9% (/) and water was 1.4% (w / w).

The actions of the obtained surfactant were as follows.

Surface tension lowering action:

Maximum surface tension; 30.3 dyne / cm

Minimum surface tension; 1. dyn eX cm

Gas-liquid interface diffusion:

Arrival time: 60 seconds Equilibrium surface tension: 29.8 dyne / cm

Arrival time; 90 seconds Equilibrium surface tension; 32 dyne / cm alveolar volume maintenance:

Lung capacity (5 cm water pressure) 4 7 m 1 / kg

(Example 16)

Surfactants were produced in the same manner as in (Example 12) except that peptide F was used instead of peptide B.

No ethanol remained in this powder, and the content of each component relative to the total weight of the surfactant was 1,2-dipalmitoylglycerol (3) -phosphocholine was 68.5% (ww) .1,2. —Diacyl mono sn—Glycerol (3) —Phospho sn—Glycerol is 21.1% (w / w), and Balmitic acid is 9.1% (w / w), peptide F is 0.9% (/ w) and water was 0.4% (wZw).

The actions of the obtained surfactant were as follows.

Surface tension lowering action:

Maximum surface tension; 34.1 dyn e / cm

Minimum surface tension: 8.9 dyne / cm diffusion at gas-liquid interface: Arrival time: 65 seconds, equilibrium surface tension: 34.8 dyne / cm Gas-liquid adsorption:

Arrival time: 115 seconds, equilibrium surface tension; 39.5 dyne / cm alveolar cavity volume maintenance action:

Lung volume (5 cm water pressure); 39 ml / kg

(Example 17)

1,2-dipalmitoyl glycerol— (3) —phosphocholin 210 mg, 1,2-diacyl-sη-glycerol (3) —phospho-sη—glyceric ester (carbon number of the acyl group: 14 to 2 90 mg of Omg and 33.Omg of palmitic acid were dissolved in a mixture of form-methanol [3: 1 (V / V)] 40 Om1 and 3.Omg of peptide D was dissolved. Dissolved in 5 ml of TFAO. These solutions were mixed and evaporated to dryness under reduced pressure. The obtained residue was suspended in 120 ml of a water-ethanol mixture [9: 1 (V / V)] at 50 ° C for 15 minutes. This suspension was frozen in one 6 O ^e C was dried 2 8 hours at a vacuum degree 5 0~ 1 0 0 H g, was 3 3 7. 9 mg of Safakutan bets white powder.

No ethanol remained in this powder, and the content of each component relative to the total-weight of the surfactant was 1,2-dipalmitoylglycerol (3)-62.1% (w / w) of phosphocorrin , 1,2-diacyl-sn-glycerol (3) -phospho-sn-glycerol is 26.6% (w / w), palmitic acid is 9.8% (w / w), and peptide G is 0. 9% (w / w) and water 0.6% (w / w).

The actions of the obtained surfactant were as follows.

Surface tension lowering action:

Maximum surface tension: 29.6 dyn eZcm-Minimum surface tension: 1.8 dyne / cm Gas-liquid interface diffusion:

Arrival time: 45 seconds Equilibrium surface tension: 29.1 dyne / cm Gas-liquid surface adsorption:

Arrival time: 80 seconds Flat street surface tension: 3 2.3 dynec m Alveolar cavity volume maintenance action:

Lung capacity (5 cm water pressure) 48 m I / kg

(Example 18)

A surfactant was produced in the same manner as in (Example 17) except that peptide H was used instead of peptide G.

No ethanol remained in this powder, and the content of each component relative to the total weight of the surfatatant was 1,2-dipalmitoylglycerol— (3) 1-phosphocholine was 61.9% (/ w), 1 , 2-diacyl-sn-glycerol (3)-phospho-sn-glycerol 26.5% (w / w), palmitic acid 9.7% Cw / w), peptide H 0.9. % (w / w) and water 0.9% (w / w).

The actions of the obtained surfactant were as follows.

Surface tension lowering effect:

Maximum table ¾ tension 3 2.4 dyn e / cm

Minimum surface tension 1.3 dyne / cm

Gas-liquid interface diffusion

Arrival time: 6 seconds Equilibrium surface tension; 33.1 dyne / cm Gas-liquid surface adsorption:

Arrival time: 90 seconds Equilibrium surface tension: 34.6 dyne xcm Alveolar cavity volume maintenance:

Lung capacity (5 cm water pressure) 41 ni1 / kg

(Example 19) Surfactant was produced in the same manner as in (Example 17) except that peptide I was used instead of peptide G.

No ethanol remained in this powder, and the content of each component relative to the total weight of the surfactant was 61.6% (w / w) for 1,2-dipalmitoylglycerol (3) -phosphocholine. 1, 2-diacyl-sn-glycerol (3)-phospho-sn-glycerol is 26.4% (w / w), palmitic acid is 9.7% of w / w), and peptide I is 0.9. % (wZw) and water 1.4% (w / w).

The actions of the obtained surfactant were as follows.

Surface tension lowering action:

Maximum table tension; 32.3 dyne / cm

Minimum surface tension; 5. e d y n e / cm

Gas-liquid interface diffusion:

Arrival time: 55 seconds Equilibrium surface tension: 2 9. Q dyne / cm Gas-liquid surface adsorption:

Arrival time; 70 seconds Equilibrium surface tension; 31.7 dyne / cm alveolar cavity volume maintenance action:

Lung capacity (5 cm water pressure) 43 m1 / kg

(Example 20)

Surfactant was produced in the same manner as in (Example 17) except that peptide J was used instead of peptide G.

No ethanol remained in this powder, and the content of each component relative to the total weight of the surfactant was 1,1.5-dipalmitoyl glycerol (3) -monophosphoricone was 61.5% (w / w), 1,2—Diacyl sn—Glycerol (3) —Phospho sn—Glycerol 26.4% (w / w), Palmitic acid 9.7% (w / w), Peptide J 0.9 % (w / ) And water 1.6% (/).

The actions of the obtained surfactant were as follows.

Maximum force 3 3 9 d y n e no c m

Minimum surface tension 4 / dyn e / cm

Gas-liquid interface diffusion

Arrival time: 65 seconds Equilibrium surface tension: 3 2. 9 dyn e / cm Gas-liquid surface adsorption:

Arrival time; 1 15 seconds Equilibrium surface tension; 34.2 dyne / cm Alveolar cavity volume maintenance action:

Lung capacity (5 cm water pressure) 40 m1 / kg

The results of the suspension test of the present invention Safakutan preparative shown in Table 2 ₍

Table 2 Suspension properties of the surfactant of the present invention

Claims

Sequence list SEQ ID NO: 1

Array length: 27

Sequence type: amino acid

Topology: linear

Sequence type: peptide

Array

Cys Pro Val His Leu Lys Arg Leu Leu lie Val Val Val Yal Val Val 1 5 10 15

Leu lie Val Val Val He Val Gl Ala Leu Leu

20 25

SEQ ID NO: 2

Array length: 27

Sequence type: amino acid

Topology: linear

Sequence type: peptide

Array

Cys Cys Pro Val His Leu Lys Arg Leu Leu lie Val Val Val Val Val

1 5 10 15

Val Leu He Yal Val Val He Val Gly Ala Leu

20 25 SEQ ID NO: 3

Array length: 2 7

Sequence type: amino acid

Topology: linear

Sequence type: Peptide

Array

Cys Pro Val Asn lie Lys Arg Leu Leu lie Val Val Val Val Val Val

1 5 10 15

Leu Leu Val Val Val lie Val Gly Ala Leu Leu

20 25 SEQ ID NO: 4

Sequence length: 27-Sequence type: amino acid

Topology: linear

Sequence type: peptide

Array

Cys Cys Pro Val Asn lie Lys Arg Leu Leu lie Val Val Val Val Val 1 5 10 15 Val Leu Leu Val Val Val lie Val Gly Ala Leu

20 1 25 SEQ ID NO: 5

Array length: 2 7

Sequence type: amino acid

Topology: linear Sequence type: Peptide

Array

Cys Pro Val Asn Leu Lys Arg Le Leu Val Val Val Val Val Val Val,

1 5 10 15

Leu Val Val Val Val lie Val Gly Ala Leu Leu

20 25

SEQ ID NO: 6

Array length: 2 7

Sequence type: amino acid

Topology: linear

Sequence type: Peptide

Array

Cys Cys Pro Yal Asn Leu Lys Arg Leu Leu Val Val Val Val Yal Val

1 5 10 15

Val Leu Yal Val Val Val lie Val Gly Ala Leu

20 25

SEQ ID NO: r 7

Array length: 2 7

Sequence type: amino acid

Topology: linear

Sequence type: peptide

Array Ser Pro Val His Leu Lys Arg Leu Leu lie Val Val Val Val Val Val

1 5 10 15

Leu lie Val Val Val lie Val Gly Ala Leu Leu

20 25 SEQ ID NO: 8

Array length: 2 7

Sequence type: amino acid

Topology: linear

Sequence type: Peptide

Array

Ser Ser Pro Val His Leu Lys Arg Leu Leu lie Val Val Val Val Val

1 5 10 15

Val Leu lie Val Val Val lie Val Gly Ala Leu

20 25 SEQ ID NO: 9

Array length: 27

Sequence type: amino acid

Topology: linear

Sequence type: peptide

Array

Ala Pro Val His Leu Lys Arg Leu Leu lie Val Val Val Val Val Val 1 5 10 15 Leu lie Val Val Val lie Val Gly Ala Leu Leu

20 25 SEQ ID NO: 10

Sequence length: 27 <Sequence type: Amino acid

Topology: linear

Sequence type: peptide

Array

Ala Ala Pro Val His Leu Lys Arg Leu Leu He Val Val Val Val Val

1 5 10 15

Val Leu lie Val Val Val lie Val Gly Ala Leu

20 25