JPH0129171B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a synthetic pulmonary surfactant having a surface activity similar to that of a natural pulmonary surfactant, and a therapeutic agent for respiratory distress syndrome containing the same as an active ingredient. In the alveoli of animals, physiologically active substances mainly composed of phospholipids, called pulmonary surfactants, are present. It covers the inner wall of the alveoli and has a protective effect on the alveolar epithelium, as well as an important physiological function in maintaining the respiratory function of animals. In other words, lung surfactants have a unique surface activity that changes the surface tension on the inner surface of the alveoli during exhalation and inspiration, and contributes to the stability between alveoli, resulting in anti-inflammatory properties. It is said to have a pneumolung effect. Regarding pulmonary surfactant substances, many studies have been carried out on various animals, and the full extent of these substances is being clarified. That is, it is known that this active substance contains phospholipids, neutral lipids, proteins, etc. as its components, and its main component is dipalmitoyl lecithin, which is one of the phospholipids. Recently, Fujiwara et al. added dipalmitoyl lecithin etc. to active substances extracted from bovine lungs to artificially obtain a more active lung surfactant, and used this to treat neonatal respiratory distress syndrome (IRDS) through the airway. reported that good results were obtained by supplementation therapy (Pediatrics, Vol. 32, No. 7, p. 1343). In addition, Kobayashi et al. separated the active substance from pig lung lavage fluid, prepared a lung surface active substance with Ca ⁺⁺ coexisting in order to further increase the activity, and succeeded in performing IRDS replacement therapy (Japan Interface). Journal of the Medical Society, Volume 2, No. 1
No. (1981)). In these methods, the lipid part is extracted from natural pulmonary surfactants using the Folch method, in which the organic solvent-soluble part is extracted with a mixed solvent of chloroform-methanol, etc., and foreign proteins derived from cows and pigs are extracted. Although it has been removed, the removal is not complete, and approximately 1 to 3% of foreign proteins are found to be mixed in. In addition, King et al. also recognized the existence of lung-specific proteins, which are different from normal serum proteins, among the lung surfactants obtained from nature (KingS.
Am.J.Phyoiol. 224 788-795, 1973, Fed.Proc.
332238-2241, 1974). It is known that it is a fat-soluble protein whose main component has a molecular weight of 34,000 and contains a large amount of hydrophobic amino acids. Based on these facts, as mentioned above, it is necessary to prepare a lung surfactant by extracting the organic solvent soluble part from a natural lung surfactant by Folch's method. Not only that, but lung-specific proteins were also extracted, and their contamination is considered inevitable.
Contamination with animal-derived proteins can cause antigenicity,
There is a possibility that side effects such as anaphylaxis may be induced, and these are not preferable when considering the commercialization of pulmonary surfactant substances. In order to improve the above drawbacks, CJ
Morley et al. attempted to prepare a synthetic pulmonary surfactant free of foreign proteins and applied it to respiratory distress syndrome of prematurity, with clinical success (The Lancet,
January 10, 1981). This synthetic pulmonary surfactant is
It is a powder containing dipalmitoyl phosphatidylcholine and phosphatidylglycerol in a weight ratio of 7:3. The dipalmitoyl phosphatidylcholine used in this preparation may be a synthetic product, but phosphatidylglycerol is obtained by separating phosphatidylcholine from egg yolk, converting it into a glycerol form using the enzyme phospholipase D, and purifying it. However, the fatty acid residues have a very complex composition, making it difficult to obtain a constant composition, and the disadvantage is that reproducibility is poor. Moreover, its synthesis is complicated and it is extremely difficult to supply it on an industrial scale. The present inventors have conducted intensive studies to develop a synthetic pulmonary surfactant that does not contain foreign proteins, is composed of readily available compounds, and has a specific surface activity unique to pulmonary surfactants. We have discovered that high surface activity can be obtained by using zircholine and cardiolipin, which is widely distributed in animal and plant tissues, as the basic composition, and have arrived at the present invention. That is, the present invention is a synthetic pulmonary surfactant characterized in that the main components are phospholipids phosphatidylcholine and cardiolipin, and the phosphatidylcholine accounts for 55 to 80% by weight of the total weight. The phospholipid phosphatidylcholine in the present invention is a compound represented by the following general formula []. (R ₁ and R ₂ represent fatty acid groups.) The phospholipid phosphatidylcholine may be either naturally obtained L-form or synthetically obtained DL-form. of phosphatidylcholine
It is preferable that 80% by weight or more has two linear saturated fatty acid residues having 14 to 20 carbon atoms (R ₁ and R ₂ in formula []). Examples of these include dimyristoylphosphatidylcholine, dipalmitoylphosphatidylcholine, distearylphosphatidylcholine, and dipalmitoylphosphatidylcholine is particularly preferred. Cardiolipin in the present invention is a compound represented by the following general formula []. (R′ ₁ , R ₂ , R′ ₃ , and R′ ₄ represent fatty acid groups) Cardiolipin may be derived from either natural or synthetic sources, and natural ones include cardiac muscle of animals such as cows, horses, and pigs. Extracted from organs such as lungs and kidneys.
For synthetic products, for example, the method of Van Deenen et al. (Advances in Lipid Research, P167,
Academic Press (1964)). In either case, it contains an unsaturated fatty acid (residue) group. That is, R′ ₁ , R′ ₂ in formula [],
Most of R' ₃ and R' ₄ are linear fatty acid groups having 14 to 20 carbon atoms, and preferably 30% by weight or more of them are unsaturated fatty acid groups. Unsaturated fatty acids include monoenes such as myristoleic acid, palmitoleic acid, oleic acid, and elaidic acid;
Alternatively, there are polyenes such as linoleic acid, linolenic acid, eleostearic acid, and arachidonic acid. Cardiolipin is usually commercially available in the form of sodium salt or ammonium salt, and can be obtained as these salts. The more common ones are:
It is a sodium salt, and the sodium salt was also used in the present invention. The main components of the synthetic pulmonary surfactant of the present invention are the phospholipid phosphatidylcholine and cardiolipin, with the former being 55 to 80% by weight and the latter being 55 to 80% by weight.
20-45% by weight. In addition, it may contain some phospholipids, neutral lipids such as dilinolein, cholesterol, carbohydrates, etc., as long as the surface activity is not reduced. The synthetic pulmonary surfactant of the present invention can be obtained by uniformly mixing the phospholipid phosphatidylcholine and cardiolipin. The mixing method and means are not limited in any way, but a homogeneous mixture can be easily prepared, for example, by dissolving both in a common solvent such as chloroform and then removing the solvent. The synthetic lung surfactant thus obtained is usually wax-like, and when its surface activity is directly measured by the Wilhelmy balance described below, the minimum surface tension is 10 dyne/cm or less, and the maximum surface tension is 36 to 55 dyne/cm. cm, and the stability index is 1.2 or higher, and they exhibit a specific hysteresis loop as shown in Figure 1, similar to natural pulmonary surfactants, and have high surface activity. The synthetic pulmonary surfactant of the present invention is usually in the form of a wax, so when it is actually administered as a preparation, it is used after being dispersed in an electrolyte solution such as water or physiological saline, or it is used in the form of a medically acceptable powder. Preferably, the substance is used as an excipient and administered as a powdered pulmonary surfactant. As the powder substance, a water-soluble and non-toxic powder substance is preferable, and the proportion thereof is 2 to 50 parts by weight, especially 5 parts by weight, per 1 part by weight of the synthetic pulmonary surfactant.
~20 parts by weight is suitable. Preferred powdered substances that do not reduce surface activity include amino acids, sugars, etc. Amino acids include the essential amino acids glycine, alanine, tryptophan, cystine, etc., and sugars include glycose, mannitol, sorbitol, etc. . As for the powdering method, the synthetic pulmonary surfactant and the powdered substance may be thoroughly mixed, but preferably, the active substance is dissolved in an organic solvent, and an appropriate amount of finely divided powder such as an amino acid is added to the solution, and the powder is uniformly mixed. to be dispersed.
Then, the mixture is evaporated to dryness using a rotary evaporator or the like under reduced pressure with rotational stirring, and the resulting powder is further made into a uniform fine powder to form a formulation. Another powdering method involves uniformly dispersing the active agent in water or a salt solution such as common salt, and dissolving a predetermined amount of the above-mentioned sugar or amino acid in the dispersion to prepare a uniform dispersion. Next, freeze-drying is performed and the resulting powder is further made into a uniform fine powder to form a formulation. Synthetic pulmonary surfactants are uniformly deposited on the surface of these powdered substances and used as preparations in powder form. In this way, even if it is powdered as a preparation, this surface activity does not change, and as mentioned above, it has a surface activity equivalent to that of the natural lung surfactant. Therefore, the powdered synthetic lung surfactant obtained by the present invention The agent can be administered by methods such as inhalation or spraying.
It is expected that this drug will fully demonstrate its medicinal efficacy as a treatment for respiratory distress syndrome such as IRDS. The present invention will be explained in detail with reference to Examples below. The surface activity of the pulmonary surfactant was measured by the following method. [Measurement of surface tension of synthetic lung surfactant and calculation of stability index] A Wilhelmy balance manufactured by Acoma was used for the measurement. 50ml of physiological saline was poured into this Teflon water tank, and the test sample was placed on top of it. A small amount of dry matter was gently placed on the plate and measured. In addition, when the measurement sample was a dispersion liquid, 50 ml of the dispersion liquid was placed in a Teflon water tank and measured. Then, the surface area of the liquid is increased by 40 at a rate of 0.3 cycles/min.
Cycling continuously between cm ² and 13 cm ²
-A surface area-surface tension diagram as shown in FIG. 1 was recorded using a Y recorder. The minimum surface tension (rmin) value, the maximum surface tension (rmax) value, and the area surrounded by the hysteresis loop were determined from the hysteresis loop that converged to a constant value in the 5th or 6th cycle after the start of cycling. Stability index (SI), which is an index of stability, was calculated from the following formula. SI=2(rmax−rmin)/rmax+rmin Example 1 L-α-dipalmitoylphosphatidylcholine
650 mg was dissolved in 30 ml of chloroform, and 350 mg of sodium salt of cardiolipin extracted from bovine heart was added and dissolved to form a homogeneous solution. The solution was then evaporated to dryness on a rotary evaporator under reduced pressure at room temperature and further dried under high vacuum to obtain a white waxy solid. A small amount of this material was taken and the surface activity was measured using a Wilhelmy balance. When the sample was placed on the interface, the surface tension instantly decreased, indicating good dispersion at the interface and a large hysteresis loop. The results are shown in Table 1, and showed physical properties similar to natural lung surfactant. Example 2 L-α-dipalmitoylphosphatidylcholine
1400 mg and 600 mg of cardiolipin sodium salt were dissolved in chloroform and evaporated to dryness in the same manner as in Example 1 to obtain a white waxy solid. Surface activity was measured in the same manner as in Example 1, and the results are shown in Table 1.
Furthermore, 500 mg of this product was dissolved in 50 ml of chloroform, and 5.0 g of mannitol, which had been previously pulverized into a fine powder, was uniformly dispersed in the solution. It was then rotary evaporated to dryness under reduced pressure on a rotary evaporator at room temperature and further dried under high vacuum. The obtained powder was further pulverized to obtain a uniform white free-flowing fine powder. The surface activity of this product was measured and the results are shown in Table 1. This product had very good dispersibility on the water surface, exhibited a large hysteresis loop, and no decrease in activity was observed even when powdered with mannitol. Example 3 Weight ratio of L-α-dipalmitoylphosphatidylcholine obtained in Example 2 to cardiolipin: 7:3
1.0 g of the solid was uniformly dispersed in 100 ml of water, and 20 g of mannitol was dissolved therein to prepare a uniform dispersion solution. Next, the dispersion was freeze-dried to obtain a white freeze-dried product. This is made using a small ball mill.
After grinding for 30 hours, a white uniform fine powder was obtained. The surface activity of this product is as shown in Table 1,
Further, the dispersibility was also very good. Example 4 Weight ratio of L-α-dipalmitoylphosphatidylcholine obtained in Example 2 to cardiolipin: 7:3
300 mg of the solid was dissolved in chloroform and pulverized in the same manner as in Example 2 using 3 g of tryptophan instead of mannitol. The surface activity of this product was as shown in Table 1, and no decrease in activity was observed as a result of powdering with tryptophan. Example 5 L-α-dipalmitoylphosphatidylcholine
Example 1: 650 mg, cardiolipin sodium salt 330 mg, and dilinolein 20 mg were dissolved in chloroform.
The mixture was evaporated to dryness in the same manner as above to obtain a white waxy solid. The surface activity of this product was measured and the results are shown in Table 1. Further, this product was uniformly dispersed in 50 ml of an aqueous mannitol solution having a concentration of about 20% by weight, and freeze-dried. The obtained white frozen product was powdered in a small ball mill in a cold room for 30 hours to obtain a white homogeneous derivative. The surface activity of this product was measured and the results are shown in Table 1. The dispersibility of this product on the water surface was very good. Example 6 L-α-dipalmitoylphosphatidylcholine
620 mg was dissolved in 30 ml of chloroform, and 380 mg of sodium salt of cardiolipin extracted from bovine heart was added and dissolved to form a homogeneous solution. The solution was then dried in the same manner as in Example 1 to obtain a white waxy solid. The surface activity of this product was as shown in Table 1. Next, 100 mg of this product was taken and dispersed in 50 ml of physiological saline to prepare a cloudy white dispersion. The Wilhelmy balance surface activity of the entire dispersion was as shown in Table 1. Example 7 L-α-dipalmitoylphosphatidylcholine
300 mg, cardiolipin sodium salt 190 mg and palmitic acid 10 ml were dissolved in 50 ml of chloroform,
The mixture was evaporated to dryness in the same manner as in Example 1 to obtain a white waxy solid. Next, 100 mg of this product was taken and dispersed in 50 ml of physiological saline, and the surface activity was measured in the same manner as in Example 6. The surface activity of this product was as shown in Table 1. Example 8 DL-dipalmitoylphosphatidylcholine 325
mg and 175 mg of the sodium salt of cardiolipin were dissolved in 30 ml of chloroform and evaporated to dryness in the same manner as in Example 1 to obtain a white waxy substance. Next this one 100
mg was taken and dispersed in 50 ml of a 0.9% aqueous sodium chloride solution containing 0.02% calcium chloride, and the surface activity was measured in the same manner as in Example 6. The surface activity of this product was as shown in Table 1. 【table】

[Brief explanation of drawings]

FIG. 1 shows a surface area-surface tension diagram of a synthetic pulmonary surfactant.

Claims (1)

[Scope of Claims] 1. A synthetic pulmonary surfactant comprising phospholipids phosphatidylcholine and cardiolipin as main components, and wherein the phosphatidylcholine accounts for 55 to 80% by weight of the total weight. 2. The synthetic lung surface activity according to claim 1, wherein 80% by weight or more of the phosphatidylcholine is phosphatidylcholine having two linear saturated fatty acid residues having 14 to 20 carbon atoms. agent. 3. The synthetic pulmonary surfactant according to claim 2, wherein the straight chain saturated fatty acid is palmitic acid. 4 Medically acceptable water-soluble powder for 1 part by weight of a synthetic pulmonary active agent whose main components are the phospholipids phosphatidylcholine and cardiolipin, in which the phosphatidylcholine accounts for 55 to 80% by weight of the total weight. A powdered respiratory distress syndrome treatment comprising 2 to 50 parts by weight of the substance. 5. The therapeutic agent for respiratory distress syndrome according to claim 4, wherein the water-soluble powder substance is an amino acid, a sugar, or a mixture thereof. 6. A liquid respiratory distress syndrome therapeutic agent prepared by dispersing in water or an electrolyte solution a synthetic pulmonary surfactant whose main components are phospholipids phosphatidylcholine and cardiolipin, with the phosphatidylcholine accounting for 55 to 80% by weight of the total weight. .