JPH05294996A - Synthetic peptides, lung surfactant containing the same and therapeutic agents for respiration distress syndrome - Google Patents

Abstract

PURPOSE:To provide a synthetic peptide which has a specific amino acid sequence, can readily be isolated and purified and produced in a large amount in a shortened time, and develops surface activating action in combination with a lipid mixture, thus can be used in a therapeutic agent for respiration distress syndrome as a pulmonary surfactant. CONSTITUTION:In the solid-phase peptide synthesis, 4--(hydroxymethyl) phenoxymethyl-copoly (styrene-1%-divinylbenzene) resin is used a support and an amino-protected amino acid is linked to the support followed by protection, then amino acid sequence is extended on the resin in the N-terminus direction according to the peptide synthesis plan to obtain a resin-O-peptide whose N- terminus and functional groups are completely protected. The protected peptide- O-resin is treated with trifluoroacetic acid or the like to effect deprotection and elimination from the resin whereby the synthetic peptide of the formula (Xaa is absent or Cys; Xbb is His; Xcc is Leu; Xdd is Val; Xee is Ile; Xbb is absent or Leu).

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to synthetic peptides. More specifically, the present invention relates to a synthetic peptide having a strong surface activity by blending 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.

[0002]

2. Description of the Related Art Respiratory distress syndrome is a disease that causes serious respiratory distress as a result of collapse of alveoli due to deficiency of pulmonary surfactant.

In recent years, a replacement therapy for externally administering respiratory surfactant to the respiratory distress syndrome has been developed, and has a remarkable therapeutic effect.

As the lung surfactant to be supplemented,
Phospholipids, neutral lipids present in mammalian lung tissue,
A substance consisting of total cholesterol and carbohydrates and a trace amount of protein (Japanese Patent Publication No. 61-9925), a substance containing a fatty acid in addition to the above components (hereinafter referred to as S-TA: Japanese Patent Publication No. 61-9924), pig. Substances derived from lung lavage fluid with Ca ⁺⁺ added (Journal of the Japanese Society for Interfacial Medicine, Vol. 12, No. 1, page 1, 1980), choline phosphoglycerides, acidic phospholipids, fatty acids and animal lungs Known in the art (Japanese Patent Publication No. 3-78371), a substance comprising dipalmitoylphosphatidylcholine and a fatty alcohol (Japanese Patent Publication No. 3-43252), and the like are known.

Some of the inventors of the present invention isolated lipoprotein from lung surfactant of animal origin first, and the lipoprotein is an indispensable component because it exhibits lung surface activity. By incorporating it into a lipid mixture, By exhibiting the excellent surface tension lowering effect of surfactant, shortening surfactant gas-liquid surface diffusion action, and exhibiting low equilibrium surface tension, it is possible to secure sufficient alveolar cavity volume, etc., and it can be used for treatment of respiratory distress syndrome. Discovered (3-7837
No. 1).

In recent years, hydrophilic surfactant apoprotein A and surfactant apoprotein D as hydrophobic apoproteins specific to mammalian lung surfactant, and hydrophobic surfactant apoprotein B (hereinafter referred to as SP-B).
Say. ) And surfactant apoprotein C (hereinafter,
It is called SP-C. ) Were confirmed [A review of the structure and function of apoproteins (Toyoaki Akino, Yoshio Kuroki, Respiration and circulation, Vol. 38, No. 18, p. 722, 1990;
Hiroki Yasuda et al., "Biosurfactant", Chapter 2
Biochemistry of Surfactants-Surfactants and Apoproteins, p. 131, 1990, Science Forum Co., Ltd.)].

[0007] Human lung-derived SP-C is a highly hydrophobic apoprotein consisting of 35 amino acid residues, having N-terminal amino acid phenylalanine and rich in hydrophobic amino acids such as valine. Further, SP-C isolated from the lungs of bovine, porcine, rat, etc. is also composed of amino acid residues 34 to 35,
The terminal amino acid sequence differs depending on the animal species, but the homology with human is extremely high.

Japanese Laid-Open Patent Publication No. 3-502095, Japanese Patent Laid-Open No.
JP-A-90033 describes that SP-B or SP-C promotes adsorption and diffusion of lung surfactant to the air-liquid interface, and improves the surface activity of lung surfactant.

[0009]

Pulmonary surfactant containing SP-C as an active ingredient is extremely effective as a therapeutic agent for respiratory distress syndrome, but SP-C
It has not been put to practical use because it is extremely hydrophobic and is difficult to isolate and purify, and it is contained in an extremely small amount in the living body.

Further, Japanese Patent Publication No. 3-502095 describes that a mixture of a synthetic peptide having a partial structure of SP-C and a lipid is effective for treating respiratory distress syndrome.

In general, however, in the production of synthetic peptides, the longer the amino acid sequence, the higher the frequency of production of immature peptides during synthesis, which makes isolation and purification difficult, and the long production time. It is said that there are drawbacks such as the need for the preparation and the difficulty of large-scale synthesis.

By the way, from the viewpoint of quality preservation, lung surfactant preparations are often provided as dry powder preparations which are made into physiological saline suspensions at the time of use.

However, a pulmonary surfactant (hereinafter referred to as S-35) preparation in which a synthetic peptide having the amino acid sequence of SP-C is mixed with a lipid mixture consisting of choline phosphoglyceride, acidic phospholipid and fatty acid is contained in the peptide. Due to factors such as high peptide aggregation due to existing cysteine residues forming disulfide bonds and strong hydrophobicity of lung surfactant itself, dispersibility in physiological saline is extremely poor and it can be used as a formulation. It was difficult to make a uniform suspension.

As a method for improving the suspension property of the pulmonary surfactant preparation, a method of adding a suspending agent such as mannitol (Japanese Patent Laid-Open No. 34905/1985) and a temporary freezing temperature of -1 to -10 ° C during freeze-drying. Freezing Method (Japanese Patent Application Laid-Open No. 63-1
No. 0718) has been proposed, but the operation is complicated, and the development of a simpler preparation manufacturing method has been desired.

[0015]

In view of the above findings, the inventors of the present invention have earnestly studied synthetic peptides having a strong surface-active action by blending with a lipid mixture, and as a result, surprisingly, the N-terminal Even when the synthetic peptide of the present invention having a cysteine residue in S is produced by the usual freeze-drying method performed at −20 ° C. or lower without adding a suspending agent, S-3
5, compared to synthetic lung surfactant (hereinafter referred to as SF-3) or S-TA consisting only of a lipid mixture consisting of choline phosphoglyceride, acidic phospholipids and fatty acids,
The inventors have completed the present invention by discovering that they have a good uniform suspension property and have a strong surface active action equivalent to that of S-35 or S-TA.

According to the present invention, the peptide represented by the following specific sequence (hereinafter referred to as the synthetic peptide of the present invention), that is, the immature peptide at the time of production has a low production frequency, so that isolation and purification are easy, A synthetic peptide that can be produced in a large amount in a short time, has good suspension properties when mixed with a lipid mixture, and has a strong surface-active action, from the synthetic peptide and lipid mixture There is provided a pulmonary surfactant and a therapeutic agent for respiratory distress syndrome containing the pulmonary surfactant as an active ingredient.

[0017]

[Chemical 2]

The synthetic peptide of the present invention can be produced by a chemical or genetic engineering method, but a chemical production method is preferable from the viewpoint of isolation and purification.

As the chemical production method, "peptide synthesis (Nobuo Izumiya et al., Maruzen Co., Ltd., 1975)", "Biochemistry Experiment Course, Volume 1, Chemistry IV-Chemical Modification of Proteins-Peptide Synthesis- ( Shunpei Sakakibara, Tokyo Kagaku Doujin Co., Ltd., 1973) "," Seikagaku Chemistry Laboratory Course, Volume 2, Protein Chemistry (2) Peptide Synthesis (Akitoshi Kimura, Tokyo Kagaku Dojin Co., Ltd., 1987) " , `` Solid phase pepitde synthesis (Solid phase pepitde syn
thesis-a practical approrch) (Aselton (E.At
herton) and Shepard (RC Sheppard), 25-1
89, Oxford University Press, Oxford, 1989.
) ”And“ Kenichi, Akagi et al. (Chem., Pharm.,
Bull., Vol. 37, No. 10, pp. 2661-2664,
1989) "or" The Peptides [Gross, E. and Meinenhofe, J.].
Edited by Barany, G. and Merrifield
riffierd, R), Vol. 2, pp. 1-284, Academic Press, New York, 1980)] ", for example, azide method, acid chloride method, acid anhydride method, mixed acid anhydride. Method, DCC method, active ester method (p-nitrophenyl ester method, p-hydroxysuccinimide ester method, etc.), carboimidazole method, redox method, DCC-activation method, etc. It can be manufactured by a method or the like.

Of these, the solid phase synthesis method is preferable, and it is possible to synthesize by an automatic synthesizer. Examples of the automatic synthesizer include 431A peptide synthesizer (manufactured by Applied Biosystems) or peptide synthesizer model 990E (manufactured by Beckman).

By incorporating choline phosphoglyceride, acidic phospholipids and fatty acids into the synthetic peptide of the present invention as a lipid mixture, lung surfactant (hereinafter referred to as the surfactant of the present invention) can be produced.

The mixing ratio is such that the weight ratio of these components to the total dry weight of the final product is 0.1 to 0.1% for the synthetic peptide.
5.0% (W / W), 5 for choline phosphoglyceride
0.6-85.0% (W / W), acidic phospholipid is 4.5-3
7.6% (W / W), fatty acids 4.6 to 24.6% (W / W)
It is appropriate to set so that

Examples of choline phosphoglycerides that can be used in the surfactant of the present invention include 1,2-dipalmitoylglycero- (3) -phosphocholine (also known as dipalmitoylphosphatidylcholine) and 1,2-distearoylglycero-
(3) -phosphocholine, 1-palmitoyl-2-stearoylglycero- (3) -phosphocholine or 1-stearoyl-2
-Palmitoylglycero- (3) -phosphocholine and other 1,2-diacylglycero- (3) -phosphocholine, 1-hexadecyl-2
-Palmitoyl glycero- (3) -phosphocholine or 1-
Octadecyl-2-palmitoylglycero- (3) -phosphocholine and other 1-alkyl-2-acylglycero- (3) -phosphocholine or 1,2-dihexadecylglycero- (3) -phosphocholine and other 1,2-dialkyl Glycero- (3) -phosphocholine is suitable. These compounds have optical isomers based on the carbon at the 2-position of the glycerol residue, but in the surfactant of the present invention, any of D-form, L-form or DL-form can be used. In addition to this, as choline phosphoglyceride, 1,2-diacylglycero- (3 having an acyl group having 12 to 24 carbon atoms, preferably two saturated acyl groups, in addition to the choline phosphoglyceride consisting of the above-mentioned single product ) -A mixture of two or more kinds of phosphocholine, and a mixture of the mixture and the above-mentioned single product can also be used.

As the acidic phospholipid, 1,2-diacyl-sn-
Glycero- (3) -phosphoric acid (also known as L-α-phosphatidic acid)
, 1,2-diacyl-sn-glycero- (3) -phospho-L-serine
(Also known as phosphatidylserine), 1,2-diacyl-sn-glycero- (3) -phospho-sn-glycerol (also known as phosphatidylglycerol) or 1,2-diacyl-sn-glycero- (3)-
Phospho- (1) -L-myo-inositol (also known as phosphatidylinositol) is suitable. In these compounds, the 1-position and 2-position may be substituted with the same kind or different kinds of acyl groups, respectively. Here, the carbon number of the acyl group is preferably 12 to 24.

Suitable fatty acids are free fatty acids, alkali metal salts of fatty acids, fatty acid alkyl esters, fatty acid glycerin esters or fatty acid amides or mixtures of two or more thereof, and further fatty alcohols or aliphatic amines.

In the present specification, the term "fatty acids" is meant to include fatty alcohols and aliphatic amines as used herein.

Suitable free fatty acids are myristic acid, palmitic acid or stearic acid, with palmitic acid being preferred.

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

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

The above-mentioned choline phosphoglyceride, acidic phospholipids and fatty acids may be 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 a method in which a mixed solution of the synthetic peptide solution of the present invention and the above lipid mixture solution is dried under reduced pressure, the resulting residue is suspended in a suitable suspending solvent, and then freeze-dried. Can be manufactured by.

Examples of the solvent used for preparing the synthetic peptide solution of the present invention include formic acid and trifluoroacetic acid (T
FA), trifluoroethanol, dimethyl sulfoxide (DMSO), chloroform / methanol or chloroform.

Examples of the solvent used for preparing the lipid mixture solution include chloroform and chloroform / methanol [2: 1 to 5: 1 (V / V)].

Examples of the suspending solvent include water and a water-ethanol mixed solution [4: 1 to 20: 1 (V / V)].
Ethanol mixtures are preferred. The suspension is carried out at 30 to 60 ° C, preferably 40 to 50 ° C for 5 to 60 minutes, preferably 1
Do this for 5 to 30 minutes.

Although a slight amount of water remains inevitable in the surfactant of the present invention in the manufacturing method, it is desirable to dry it until the remaining weight ratio becomes 5.0% (W / W) or less based on the total weight. .. When dried to such an extent, residual ethanol becomes undetectable when a water-ethanol mixture is used.

Further, the surfactant dry powder preparation of the present invention contains a monovalent or divalent metal salt having a suitable physiological concentration, for example, 0.
It can be used by uniformly suspending and dispersing it using 9% sodium chloride or 1.5 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.

(1) Surface activity Surface tension lowering action; measurement of the surface tension lowering method is carried out by the method of Tanaka et al. (Journal of the Japanese Society for Interfacial Medicine, Volume 13, No. 2, No. 8).
P. 7, 1982).

[0039] The present invention surfactant suspension saline; on (surface area 54.0cm ^2), 1cm per ² present invention surfactant is added dropwise so that 1.0～2.0Myug, the said surface area 54. ^{2 in} the range of 0 to 21.6 cm 2.
The surface tension when compressed / expanded for 5 minutes was continuously measured at 37 ° C. by a Wilhelmy surface tension measuring device (manufactured by Kyowa Interface Science Co., Ltd.). The surface tension reducing action of the surfactant of the present invention has a maximum surface tension of 29.7.
~ 34.5dyne / cm, minimum surface tension is 1.4 ~ 8.9dy
It was ne / cm 2 and was found to reduce the surface tension of saline.

The surface tension lowering effect of SF-3 measured in the same manner is that the maximum surface tension is 25.8 to 50.3 dyne / cm.
The minimum surface tension was 1.0 to 13.5 dyne / cm.

The initial surface tension of physiological saline at 37 ° C. was 70.5 dyne / cm 2.

Gas-liquid surface diffusion action: on the liquid surface of physiological saline,
0.8 to 1.5 μg of the surfactant suspension of the present invention was dropped per 1 cm ^{2 of the} surface area, and the surface tension immediately after the dropping was measured with the vertical plate method with time. The measurement temperature was 37 ° C.

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

The surfactant of the present invention forms a film on the gas-liquid surface in a short time of 30 to 65 seconds and has a surface tension of 27.9.
It was reduced to ˜34.8 dyne / cm 2.

The gas-liquid surface diffusion effect of SF-3 measured in the same manner was that the surface tension after 120 seconds was 38.1 to 52.
It was 9 dyne / cm.

Gas-liquid surface adsorption: 0.2 to 1 ml
A 37 ° C. physiological saline suspension containing 1.0 mg of the surfactant of the present invention was prepared, and the adsorption rate of the suspended surfactant of the present invention on the liquid-vapor surface of the physiological saline was measured.

The adsorption rate was measured by the method of King et al.
can Journal of Physlology, 223rd, 715th
P., 1972).

That is, the suspension was poured into the bottom of a Teflon water tank having a diameter of 5 cm and containing physiological saline, and the mixture was slowly stirred with a magnetic stirrer, and the adsorption rate was obtained from the fluctuation value of the surface tension after the stirring was stopped. It was

The surface tension of the surfactant of the present invention is 28.1 to 30 seconds after the stirring is stopped.
It was lowered to the range of 39.5 dyne / cm, and then showed a constant value.

This indicates that the surfactant of the present invention in a suspended state floated and adsorbed on the gas-liquid surface in 30 to 120 seconds to form a film having a strong surface activity.

SF-3 measured in the same manner has a constant surface tension in the range of 42.2 to 58.3 dyne / cm,
The required time was 150 seconds or more.

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

(2) Suspension property A suspension property test of pulmonary surfactant was conducted according to Japanese Patent Laid-Open No. 63-10.
It carried out according to the method of 718 gazette.

That is, according to the dispersion rate at a predetermined time after the start of suspension and the maximum dispersed particle diameter at 2 minutes after the start of suspension,
The suspension property was determined.

To test the dispersion ratio, 60 mg of lung surfactant was dispensed into a 20 ml vial, 2 ml of physiological saline was injected, and the vial was attached to an Iwaki KM shaker VS type shaker (Iwaki Co., Ltd.). 270 strokes /
Shake for 30 minutes, and after 30 seconds of shaking, visually observe the dispersion state of each sample every minute from 1 minute to 4 minutes, and every 2 minutes from 4 minutes to 10 minutes through a magnifying glass from outside the container. I went by.

The determination of the suspended state was made for each sample 1 at each time.
It was carried out by two people, 0 bottles each, and it was judged whether they were suspended or not. No small particles were found in the container, and the preparation was uniformly dispersed in physiological saline to form a white, slightly viscous suspension. I went with or without.

The dispersion rate was calculated as a percentage of the total number (10) of the samples in which suspension was completed by each person at each time, and expressed as an average value by the two persons.

The maximum dispersed particle size was obtained by aliquoting each sample into 60 ml of lung surfactant in a 20 ml vial.
ml, and shake continuously for 2 minutes under the same shaking conditions as above to find the largest particles in suspension using a microscope,
It was determined by measuring the diameter with a caliper.

Most of the surfactants of the present invention were suspended within 2 minutes, and the maximum particle size was 0.9.
It was less than mm, and the suspension property was good.

(3) Pharmacological properties Acute toxicity: The acute toxicity of the surfactant of the present invention was tested using 5-week-old male ICR mice and Wistar rats. Oral LD ₅₀ and intraperitoneal LD in mice
₅₀ is 2.5-10.0 g / kg and 1.5-5.0 g / kg, those in rats are 1.5-5.0 g / kg and 1.
It was 5-2.5 g / kg.

Subacute toxicity: 300-600 mg / kg daily
Each week, the surfactant of the present invention was intraperitoneally administered to Wistar adult rats, but no change in body weight and no abnormality in macroscopic or histological observation of major organs were observed.

Alveolar space capacity-maintaining activity: An immature rabbit fetus with a gestational period of 27 days produces almost no lung surfactant and is deficient in lung surfactant, and is therefore considered as a model animal for neonatal respiratory distress syndrome.

The alveolar cavity volume (hereinafter referred to as the lung volume) was measured at 37 ° C. under the change of the airway pressure using 5 rabbit fetuses having a gestational period of 27 days.

The measurement was carried out continuously by using a water manometer incised in the neck of the fetus and connected to the trachea from 5 minutes after the surfactant of the present invention was administered transtracheally. 2 channel independent drive syringe pump No. Using 940 (manufactured by Harvard, USA), the pressure was increased to 30 cm to expand the alveoli. Next, the airway pressure was reduced to 0 cm water pressure to contract the alveoli, and the lung volume at each water pressure was measured. Lung volume was expressed in milliliters per kg body weight (ml / kg).

The surfactant of the present invention is administered by directly injecting 0.05 to 0.5 ml of a physiological saline suspension prepared to have a concentration of 1.0 to 6.0% (W / V) into the respiratory tract. I went the way.

The larger the lung volume of 5 cm water pressure at the time of decompression showing the functional residual capacity, the higher the lung surfactant activity.

As a control, physiological saline was administered instead of the surfactant suspension of the present invention. In the control group, the lung volume (5 cm water pressure) of immature rabbits with a gestational age of 27 was 1 to 5 ml.
At / kg, the alveoli were barely dilated.

Further, a 30-day-old gestational fetus having a normal level of lung surfactant had a lung volume (5 cm water pressure) of 39 to 53 ml / kg, and the alveoli were sufficiently expanded,
Indicates that normal breathing is possible.

When SF-3 was administered, the lung volume of the immature fetus (5 cm water pressure) was 15 to 25 ml / kg, and the alveolar expansion was insufficient.

The lung volume (5 cm water pressure) of immature fetuses to which the surfactant of the present invention was administered was 39 to 53 ml / kg, and it was confirmed that the surfactant of the present invention improves the lung volume of immature fetuses 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 surfactant of the present invention comprising the present synthetic peptide and the lipid mixture exhibits the surface activity, suspension property and pharmacology. It can be said that it is an effective therapeutic agent for respiratory distress syndrome due to its unique properties.

The therapeutic agent for respiratory distress syndrome provided by the present invention has a single dose of 50 to 1000 for children.
mg, for adults, 500 to 5000 mg of the surfactant of the present invention is contained. This dose is suspended in water, physiological saline, physiologically acceptable buffer, etc., and the concentration is 1.0 to
The concentration is adjusted to 10.0% (W / V), and this is used by injecting or spraying 1 to 10 times into the respiratory tract immediately after the onset of respiratory disorder for 48 hours. In addition, it can be directly inhaled as a powder without being suspended. The dose, method of use and frequency may be appropriately changed depending on the symptoms of the patient and the combination therapy.

The therapeutic agent of the present invention may optionally contain stabilizers, preservatives, isotonic agents, buffers, suspending agents and other pharmaceutical additives or bronchodilators, antiallergic agents, anticancer agents, antibacterial agents, etc. The medicines of can be included.

As the dosage form, a liquid agent or a powder agent used by suspending at the time of use is suitable. The therapeutic agent of the present invention is filled in a sealed container such as a vial bottle or an ampoule bottle and stored as a sterile preparation.

The present invention will be described below with reference to examples.

[0076]

【Example】

[Production of peptide] In the following examples, the molecular weight of the synthesized peptide 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.

[Example 1] The peptide described in [SEQ ID NO: 1] (hereinafter referred to as "peptide A") was converted to E.Athe.
rton) and Shepard (RC Sheppard) in Solid phase pepitde s.
ynthesis-a practical approrch) '' (p.25-189,19
89, Oxford University Press, Oxford and Kenichi, A
kagi et al. Chem., Pharm., Bull., 37 (10), p.2661 ~ 266
According to the method described in 4 (1989), solid phase synthesis was carried out by a multi-peptide solid phase synthesis system "Cock-san" (trade name; manufactured by Kokusan Kagaku Co., Ltd.).

As a starting resin, N-α-9-fluorenylmethyloxycarbonyl-leucine (Fmoc-Leu) was bound to 4- (hydroxymethyl) phenoxymethyl-copoly (styrene 1% divinylbenzene) resin. 0.20 mmol / 0.5 g of N-α-9-fluorenylmethyloxycarbonyl-leucine-O-resin (Fmoc-Leu-O-resin) was used. The resin was swollen with N, N-dimethylformamide (DMF) for 20 minutes and then washed with DMF four times.
A 20% piperidine-DMF solution was added and shaken for deprotection. This operation was repeated 3 times in order to complete this deprotection. Then, to remove excess piperidine in the resin, 3 times with DMF, 3 times with N-methyl-2-pyrrolidone,
Further, it was washed with DMF three times. At this time, the presence or absence of piperidine was confirmed using pH test paper.

Then, DMF 6 ml, Fmoc-Leu 0.5 mmo
l, N-hydroxybenzotriazole 0.5 mmol and N,
Add 0.5 mmol of N'-diisopropylcarbodiimide 9
The mixture was shaken for 0 minutes to carry out a condensation reaction. The resin was then washed 4 times with DMF to remove excess reagent. This condensation reaction was confirmed by Kaiser test by the ninhydrin method.

Thus, according to the synthetic scheme, amino acids were sequentially extended on the resin in the N-terminal direction to synthesize a peptide-O-resin in which the N-terminal and functional groups were completely protected.

Arg, Lys, His, Pro, Cys, Ph
The condensation reaction at the time of introducing e was performed twice for 120 minutes.

Then, the protected peptide-O-resin was
A 0% piperidine-DMF solution was added to deprotect the Fmoc protecting group at the N-terminus, and this peptide-O-resin was added with DMF
Once, washed 6 times with methanol, and dried under reduced pressure. While stirring the dried peptide-O-resin (100 mg) under ice cooling, m-cresol (0.2 ml), 1,2-ethanedithiol (0.5 ml), thioanisole (1.2 ml), TF.
A (7.5 ml) and trimethylsilyl bromide (1.
(4 ml) was added, and the mixture was stirred for 120 minutes under ice-cooling, the peptide was cleaved from the resin along with deprotection of the functional side chain, and filtered through a glass filter (G3). The filtrate was concentrated under reduced pressure to about 5 ml with an evaporator, and diethyl ether was added to precipitate the peptide. The peptide precipitate was collected by filtration with a glass filter (G3), washed with diethyl ether 5 times, and dried under reduced pressure to remove peptide A 5
Obtained 5 mg.

The N-terminal amino groups of all amino acids were protected with Fmoc groups, and the functional side chains were protected with the following groups.

Arg-Mtr; (4-methoxy-2,3,6-
Trimethylbenzenesulfonyl). Lys-Boc; (t-butyloxycarbonyl). Cys-Trt; (Trityl). His-Trt; (Trityl). About 7 mg of this peptide was dissolved in 0.6 ml TFA. Furthermore, chloroform-methanol (C / M) was added to the same solution.
2: 1 (V / V) was added to a final volume of 3.0 ml. The same sample was purified by a Sephadex LH-60 column (φ2.5 cm × 90 cm) equilibrated with a C / M mixed solvent 2: 1, (V / V), and pure peptide A was collected.

The presence of peptide in the eluate was 245 nm.
(Spectrophotometer; JASCO Corporation Model 870-UV)
And differential refractometer (Shimadzu Corporation; Model RID-
6A).

FABMS (M + H ⁺ ); 2878.0
(Calculated molecular weight; 2876.7).

[Example 2] The peptide (peptide B) described in [SEQ ID NO: 2] was designated as "The Peptides".
(Gross, E. and Meinenhofe,
J.), Barany, G. and Merrifield (M)
errifierd, R), Vol. 2, pp. 1-284, Academic Press, New York, 1980)] by solid phase synthesis on phenylacetamidomethyl (PAM) resin.

Leucine at the C-terminal amino acid residue was changed to t-butyloxycarbonyl-leucine (Boc-Leu), which was attached to the PAM resin via an oxymethylphenylacetamide bond. Boc-Leu-PAM resin (0.70
mol / g, 0.35g) to a peptide synthesizer (model 99
OE, manufactured by Beckman). Peptide completely protected by extending a protected amino acid in the N-terminal direction on the resin by a preformed symmetrical anhydride method
O-resin was synthesized. However, when condensing arginine, N, N-dicyclohexylcarbodiimide (DCC) /
Hydroxybenzotriazole [Cony et al., Chem. Ber.,
103,788-798 (1970)] was used for double coupling.

The N-terminal amino groups of all amino acids were protected with Boc groups, and the functional side chains were protected with the following groups.

Arg-Tos; (Tosyl). Lys-2CLZ; (2-chlorobenzyloxycarbonyl). Cys-4MeBzl; (4-methylbenzyl). His-Tos; (Tosyl). This condensation reaction was confirmed by Kaiser test by the ninhydrin method.

Fully protected peptide-O-resin (15
5 mg) was swollen in methylene chloride for 5 minutes. N-α-Boc
The protecting groups were deprotected with TFA containing 1% (v / v) indole and 0.1% (v / v) ethanedithiol. The deprotected peptide-O-resin was then added to p-cresol (1 ml), p-thiocresol (0.2 g) and DM.
Anhydrous hydrogen fluoride (HF) (11 ml) added with SO (1 ml)
ml) for 60 minutes at 0 ° C. to cleave the peptide from the resin.

HF and DMSO were distilled off under vacuum at 0 ° C. The excised peptide and resin were washed 3 times with 15 ml cold diethyl ether and then the free peptide was extracted 3 times with a 10 ml wash of cold TFA. The extract was immediately filtered and cooled with ice-cold water (120 ml ~
150 ml) to precipitate the crude peptide. This crude peptide was then added to 1000 xg at 0 ° C for 30
It was centrifuged for a minute and collected as a precipitate. The precipitate was washed with diethyl ether (15 ml). This washing step was repeated using diethyl ether, ethyl acetate and distilled water to obtain 83 mg of peptide B.

This crude peptide was dissolved in a 50% DMSO aqueous solution and purified by reverse phase high performance liquid chromatography using a μ-bonder sphere and C8-300Å column to collect pure peptide B.

The eluent contains 5% of 0.1% TFA.
Elution was performed with 0% aqueous acetonitrile for 5 minutes. Then, elution was performed for 30 minutes with a linear concentration gradient using the same eluent and an 80% acetonitrile aqueous solution containing 0.1% TFA.

The presence of peptide in the eluate was 245 nm
(Spectrophotometer; JASCO Corporation Model 870-UV)
And differential refractometer (Model RID-Shimadzu Corporation)
6A).

FABMS (M + H ⁺ ); 2867.5
(Calculated molecular weight; 2866.7).

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

FABMS (M + H ⁺ ); 2854.6
(Calculated molecular weight; 2853.6).

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

FABMS (M + H ⁺ ); 2844.3
(Calculated molecular weight; 2843.6).

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

FABMS (M + H ⁺ ); 2826.8
(Calculated value of molecular weight; 2285.6).

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

FABMS (M + H ⁺ ); 2816.7
(Calculated molecular weight; 2815.6).

The synthetic peptide of the present invention was treated with 12N hydrochloric acid / TFA [2: 1 (V / V)] containing 5% (v / v) phenol under vacuum at 150 ° C. for 2, 4, 6, 12, After acid hydrolysis for 24, 48 and 72 hours to remove the acid, the hydrolysis product was analyzed by Shimadzu amino acid automatic analysis system (LC-9A). In the hydrolysis for 2 to 72 hours, the amino acid value showing a higher recovery rate was adopted and the amino acid composition value was calculated. The results are shown in [Table 1].

[0106]

[Table 1]

[Production of Surfactant of the Present Invention] The surfactant of the present invention is used as the synthetic peptide and a lipid component.
1,2-dipalmitoyl glycero- (3) -phosphocholine, 1,2-
It was prepared by mixing diacyl-sn-glycero- (3) -phospho-sn-glycerol and three components of palmitic acid.

Example 7 Aseptically treated 1,2-dipalmitoylglycero- (3) -phosphocholine 660 mg, 1,2-diacyl-sn-glycero- (3) -phospho-sn-glycerol (carbon of acyl group) (14 to 24) (manufactured by Sigma) (220 mg) and palmitic acid (100 mg) were dissolved in chloroform-methanol mixed solution (2: 1 (V / V)) (1000 ml) at room temperature, and peptide A (12 mg) was dissolved in TFA (0.5 ml). These solutions were mixed and dried under reduced pressure. The obtained residue was mixed with water-ethanol mixture at 40 ° C for 15 minutes [9: 1 (V / V)].
Suspended in 100 ml. The suspension was frozen at -50 ° C and dried at a vacuum degree of 85 to 100 µHg for 36 hours to obtain 1022 mg of surfactant as a white powder.

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

Each action of the obtained surfactant was as follows.

Surface tension lowering action: maximum surface tension; 32.8 dyne / cm, minimum surface tension; 2.2 dyne / cm.

Diffusion action at gas-liquid interface: arrival time; 40 seconds, equilibrium surface tension 29.8 dyne / c
m.

Gas-liquid surface adsorption action: arrival time; 60 seconds, equilibrium surface tension; 31.9 dyne / c
m.

Alveolar volume maintenance effect: Lung volume (5 cm water pressure); 51 ml / kg.

Example 8 1,2-Dipalmitoyl glycero
-(3)-Phosphocholine 204 mg, 1,2-diacyl-sn-glycero- (3) -phospho-sn-glycerol (acyl group having 1 carbon atom
4 to 24 pieces: manufactured by Sigma) 63.0 mg and palmitic acid 27.0 mg are mixed with a chloroform-methanol mixture [2: 1.
(V / V)] 300 ml, and 2.8 mg of peptide B was dissolved in 0.3 ml of TFA. These solutions were mixed and dried under reduced pressure. The obtained residue was suspended in 100 ml of a water-ethanol mixed solution [9: 1 (V / V)] at 45 ° C for 20 minutes. This suspension was frozen at -60 ° C to obtain a vacuum degree of 60-
After drying at 110 μHg for 40 hours, 301.9 mg of a white powder of surfactant was obtained.

No ethanol remained in this powder, and the content of each component was 67.6% (w / w) for 1,2-dipalmitoylglycero- (3) -phosphocholine with respect to the total weight of the surfactant. , 1,2-diacyl-sn-glycero- (3) -phospho-sn-glycerol was 20.9% (w / w), palmitic acid was 8.9% (w / w), and peptide B was 0. 9% (w / w)
And water was 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.

Diffusion action at gas-liquid interface: arrival time; 60 seconds, equilibrium surface tension; 31.2 dyne / c
m.

Gas-liquid surface adsorption action: arrival time; 120 seconds, equilibrium surface tension; 34.7 dyne / c
m.

Alveolar volume maintenance effect: Lung volume (5 cm water pressure); 49 ml / kg.

Example 9 A surfactant was produced in the same manner as in Example 8 except that peptide C was used instead of peptide B.

No residual ethanol was observed in this powder, and the content of each component relative to the total weight of the surfactant was 67.0% (w / w) for 1,2-dipalmitoylglycero- (3) -phosphocholine. , 1,2-diacyl-sn-glycero- (3) -phospho-sn-glycerol was 20.7% (w / w), palmitic acid was 8.9% (w / w), and peptide C was 0. 9% (w /
w) and water 2.5% (w / w).

The actions of the obtained surfactant were as follows.

Surface tension lowering action: maximum surface tension; 29.7 dyne / cm 2, minimum surface tension; 2.3 dyne / cm 2.

Air-liquid interface diffusion action: arrival time; 30 seconds, equilibrium surface tension; 27.9 dyne / c
m.

Gas-liquid surface adsorption action: arrival time; 30 seconds, equilibrium surface tension; 28.1 dyne / c
m.

Alveolar volume maintenance effect: Lung volume (5 cm water pressure); 53 ml / kg.

[Example 10] A surfactant was produced in the same manner as in [Example 8] except that peptide D was used in place of peptide B.

No ethanol remained in this powder, and the content of each component was 68.5% (w / w) for 1,2-dipalmitoylglycero- (3) -phosphocholine with respect to the total weight of the surfactant. , 1,2-diacyl-sn-glycero- (3) -phospho-sn-glycerol was 21.2% (w / w), palmitic acid was 9.1% (w / w), and peptide D was 0.1%. 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.

Air-liquid interface diffusion action: Arrival time: 45 seconds, equilibrium surface tension; 28.3 dyne / c
m.

Gas-liquid surface adsorption action: arrival time; 40 seconds, equilibrium surface tension; 30.7 dyne / c
m.

Alveolar volume maintenance effect: Lung volume (5 cm water pressure); 49 ml / kg.

[Example 11] A surfactant was produced in the same manner as in [Example 8] except that peptide E was used instead of peptide B.

No ethanol remained in this powder, and the content of each component was 67.8% (w / w) for 1,2-dipalmitoylglycero- (3) -phosphocholine with respect to the total weight of the surfactant. , 1,2-diacyl-sn-glycero- (3) -phospho-sn-glycerol was 20.9% (w / w), palmitic acid was 9.0% (w / w), and peptide E was 0. 9% (w /
w) and water 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; 1.4 dyne / cm.

Air-liquid interface diffusion action: arrival time; 60 seconds, equilibrium surface tension; 29.8 dyne / c
m.

Gas-liquid surface adsorption action: arrival time; 90 seconds, equilibrium surface tension; 32.1 dyne / c
m.

Alveolar volume maintenance effect: Lung volume (5 cm water pressure); 47 ml / kg.

Example 12 A surfactant was produced in the same manner as in Example 8 except that peptide F was used instead of peptide B.

No ethanol remained in this powder, and the content of each component was 68.5% (w / w) for 1,2-dipalmitoylglycero- (3) -phosphocholine with respect to the total weight of the surfactant. , 1,2-diacyl-sn-glycero- (3) -phospho-sn-glycerol was 21.1% (w / w), palmitic acid was 9.1% (w / w), and peptide F was 0. 9% (w /
w) and water 0.4% (w / w).

The actions of the obtained surfactant were as follows.

Surface tension lowering action: maximum surface tension; 34.1 dyne / cm, minimum surface tension; 8.9 dyne / cm.

Air-liquid interface diffusion action: arrival time; 65 seconds, equilibrium surface tension; 34.8 dyne / c
m.

Gas-liquid surface adsorption action: arrival time; 115 seconds, equilibrium surface tension; 39.5 dyne / c
m.

Alveolar volume maintenance effect: Lung volume (5 cm water pressure); 39 ml / kg.

[0150]

[Table 2]

The results of the suspension test of the surfactant of the present invention are shown in [Table 2].

[0152]

[Sequence list]

SEQ ID NO: 1 Sequence length: 27 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Sequence Cys Pro Val His Leu Lys Arg Leu Leu
Ile Val Val Val Val Val 15
10 15 Leu Ile Val Val Val Ile Val Gly Ala
Leu Leu 20 25

SEQ ID NO: 2 Sequence Length: 27 Sequence Type: Amino Acid Topology: Linear Sequence Type: Peptide Sequence Cys Cys Pro Val His Leu Lys Arg Leu Leu Ile Val Val Val Val Val 1 5 10 15 Val Leu Ile Val Val Val Ile Val Gly Ala Leu 20 25

SEQ ID NO: 3 Sequence Length: 27 Sequence Type: Amino Acid Topology: Linear Sequence Type: Peptide Sequence Cys Pro Val Asn Ile Lys Arg Leu Leu Ile Val Val Val Val Val Val 1 5 10 15 Leu Leu Val Val Val Ile Val Gly Ala Leu Leu 20 25

SEQ ID NO: 4 Sequence Length: 27 Sequence Type: Amino Acid Topology: Linear Sequence Type: Peptide Sequence Cys Cys Pro Val Asn Ile Lys Arg Leu Leu Ile Val Val Val Val Val 1 5 10 15 Val Leu Leu Val Val Val Ile Val Gly Ala Leu 20 25

SEQ ID NO: 5 Sequence length: 27 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Sequence Cys Pro Val Asn Leu Lys Arg Leu Leu Val Val Val Val Val Val Val 1 5 10 15 Leu Val Val Val Val Ile Val Gly Ala Leu Leu 20 25

SEQ ID NO: 6 Sequence length: 27 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Sequence Cys Cys Pro Val Asn Leu Lys Arg Leu Leu Val Val Val Val Val Val 1 5 10 15 Val Leu Val Val Val Val Ile Val Gly Ala Leu 20 25

Continuation of the front page (51) Int.Cl. ⁵ Identification number Office reference number FI technical display location A61K 37/02 ACD 8314-4C 37/22 8314-4C // C07K 99:00

Claims (4)

[Claims] 1. The following specific sequence: The peptide represented by. 2. A lung surfactant comprising a peptide having a specific sequence, choline phosphoglyceride, acidic phospholipid and fatty acid. 3. A therapeutic agent for respiratory distress syndrome, which comprises, as an active ingredient, a pulmonary surfactant containing a peptide having a specific sequence, choline phosphoglyceride, acidic phospholipid and fatty acid. 4. The peptide according to claim 1, 2 or 3, wherein the peptide having the specific sequence is any one of SEQ ID NOS: 1-6.