KR101784651B1 - Synthetic Pulmonary Surfactant Composition Comprising Analogues of Surfactant Protein B and Surfactant Protein C - Google Patents

Abstract

The present invention relates to a synthetic pulmonary surfactant composition suitable for the prevention or treatment of respiratory distress syndrome (RDS). The pulmonary surfactant composition of the present invention not only has lung interface activity superior to that of existing animal-derived pulmonary surfactants, but also can be economically mass produced by constructing a large scale synthesis system.

Description

Synthetic Pulmonary Surfactant Composition Comprising Analogues of Surfactant Protein B and Surfactant Protein C}

The present invention relates to a synthetic pulmonary surfactant composition suitable for the prevention or treatment of respiratory distress syndrome (RDS).

The human lung consists of a number of small air sacs called alveoli, in which gas exchange takes place between the blood and the pulmonary space. In healthy individuals, this gas exchange is mediated by the presence of a protein-containing surfactant complex that prevents lung destruction at the end of exhalation. The lung surfactant complex consists mainly of lipids and contains a small amount of various proteins. If these complexes are not maintained at an appropriate level, lung dysfunction is caused, and in this case, a representative disease that can occur is respiratory distress syndrome (RDS). This is a disease that causes respiratory failure due to insufficient production of pulmonary surfactant (PS). It is the most common disease in newborns, especially premature babies, and is one of the very important diseases with high mortality rate. Intrabronchial administration of pulmonary surfactants for respiratory distress syndrome in newborns has been proven effective through clinical trials, and is currently established as the most reliable and universal treatment.

The major components of lung surfactants are phospholipids dipalmitoyl phosphatidyl choline (DPPC), phosphatidyl glycerol (PG), other phospholipids, triglycerides, etc., lung surfactant protein (SP) SP-A, B, C, It consists of D, etc. In addition, the commercially used artificial pulmonary surfactant product line can be divided into two broad categories.(i) First, it is a pulmonary surfactant formulation obtained by separating a surfactant protein from the lungs of an animal (cow or pig) and mixing it with the remaining phospholipids. It is called'natural lung surfactant' or'animal-derived lung surfactant', and most products belong to this. (ii) Second, there is an artificial'synthetic lung surfactant' manufactured by artificially synthesizing a part of a surfactant protein.

Since most animal-derived pulmonary surfactants currently clinically applied contain animal surfactant proteins, 1) animal antigenicity, 2) potential risk of infectious diseases, 3) sona There are problems such as ethical issues that require slaughtering animals such as pigs in large quantities, and 4) high manufacturing costs. Therefore, the need for the development of next-generation artificial synthetic surfactants for neonates is raised (Kim YD. new synthetic surfactants for neonates. J Korean Soc Neonatol. 2012;19:184-194., Robertson B, Johansson J, Curstedt T. Synthetic surfactants to treat neonatal lung disease.Mol Med Today 2000; 6:119-124.).

^{Exosurf ®, a} first-generation synthetic lung surfactant developed in the 1990s, did not contain surfactant proteins and consisted of phospholipids only, but its production was discontinued due to poor clinical utility and is currently not used in clinical practice. Since then, animal-derived lung surfactant products with excellent effects have been dominated around the world, and among them, research on the development of formulations containing surfactant proteins continues.

Surfactant proteins can be classified into SP-A, B, C, and D. Among them, SP-A and SP-D are water-soluble and are involved in the production and secretion of pulmonary surfactants, so the development of artificial pulmonary surfactants It is effective not to include SP-B and/or SP-C, which are mainly involved in the physical properties of the surface of the waste surfactant, not included in the component.

However, it is impossible to synthesize the amino acid structures of human SP-B and SP-C into exactly the same structure. This is because SP-B has a large molecular weight and a complex structure, especially because it is a double sulfide bond, which has limitations in its synthesis, and SP-C is a single protein and has very poor stability when synthesized (Kallberg Y, Gustafsson M, Persson B, Thyberg J, Johansson J. Prediction of amyloid fibril-forming proteins.J Biol Chem. American Society for Biochemistry and Molecular Biology; 2001 Apr 20;276(16):12945-50.).

On the other hand, since the pulmonary surfactant composition should be administered to low-weight premature infants, it should exhibit a low viscosity in order to allow a concentrated suspension formulation in an aqueous medium apart from excellent surfactant. However, pulmonary surfactants including polypeptides and phospholipids are complex mixtures whose properties such as viscosity significantly change depending on the composition of the phospholipid mixture as well as the specific phospholipid/polypeptide combination. Therefore, when two or more polypeptides are included, their combination affects the rheological properties of the composition, and their content is also one of the factors to be considered.

Under this background, the present inventors have made intensive efforts to create a synthetic pulmonary surfactant that has superior surfactant activity than conventional animal-derived pulmonary surfactants, and as a result, some sequences of SP-B and/or similar sequences of SP-C were used as pulmonary surfactants. When prepared by including in, the present invention was completed by confirming that it has an effect superior to that of a conventional waste surfactant.

One object of the present invention is a pulmonary surfactant comprising a partial polypeptide of the surfactant protein SP-B consisting of the sequence represented by SEQ ID NO: 1 and an analog polypeptide of the surfactant protein SP-C consisting of the sequence represented by SEQ ID NO: 2 It is to provide a composition and a method for preparing the same.

Another object of the present invention is to provide a pharmaceutical composition for preventing or treating respiratory distress syndrome comprising the pulmonary surfactant composition as an active ingredient.

Another object of the present invention is to provide a kit comprising a container means containing the pulmonary surfactant composition and a dosage form thereof.

As one aspect for achieving the above object, the present invention is an analogue of a surfactant protein SP-C consisting of some polypeptides of the surfactant protein SP-B consisting of the sequence represented by SEQ ID NO: 1 and the sequence represented by SEQ ID NO: 2 It provides a pulmonary surfactant composition comprising a polypeptide.

The present invention relates to a synthetic pulmonary surfactant, not using the entire sequence of each of SP-B and SP-C, but by including a partial sequence of SP-B and/or a similar sequence of SP-C to the existing pulmonary surfactant. It was first identified by the present inventors to prepare a synthetic pulmonary surfactant having a pulmonary surfactant superior to that of animal-derived pulmonary surfactant.

In the present invention, "surface activity" refers to the ability to lower the surface tension. The in vitro efficacy of exogenous surfactants can generally be measured using appropriate devices such as Wilhelmy Balance and Captive Bubble Surfactometer to lower the surface tension.

In the present invention, the "pulmonary surfactant or lung surfactant" is a mixture of protein and lipid that coats the inner surface of the alveoli to enable normal breathing. Because of its surfactant properties, pulmonary surfactants significantly reduce the surface tension at the air-liquid interface of the alveoli, reducing the pressure required for alveolar expansion and reducing the effort of respiration.

In the present invention, "surfactant protein SP-B partial polypeptide" is a kind of surfactant protein, and refers to a partial amino acid sequence of SP-B, which is an essential protein related to lipids in a pulmonary surfactant. The SP-B polypeptide is hydrophobic like membrane proteins, but does not exist in the cell membrane. The SP-B polypeptide may be a natural polypeptide derived from an animal or human, or may be an artificially synthesized polypeptide. Preferably, the amino acid sequence of a part of the C (carbon)-terminus of the natural SP-B polypeptide derived from human is artificially It may be a polypeptide synthesized by. Specifically, it may be represented by the sequence of RMLPQLVCRLVLRCSMD (SEQ ID NO: 1).

In the present invention, the "surfactant protein SP-C analog polypeptide" is also a kind of surfactant protein, in which a part of the amino acid sequence of SP-C, a protein related to lipids in the lung surfactant, is deleted and/or substituted, or another amino acid sequence It includes all the polypeptides exhibiting the added pulmonary surfactant. The SP-C polypeptide may be a natural polypeptide derived from an animal or human, or may be an artificially synthesized polypeptide, but preferably may be a polypeptide artificially synthesized of the amino acid sequence of a natural SP-C analog polypeptide derived from human. have. Specifically, it may be represented by the sequence of CPVHLKRLLLLLLLLLLLLLLLL (SEQ ID NO: 2).

In the present invention, "polypeptide", "peptide" and "protein" may be used interchangeably to refer to a polymer of amino acid residues, and the amino acid sequence is a free amino acid group (amino terminal) on the left, and a carboxyl group (carboxy terminal) on the right. It can be represented by short letters.

In addition, the composition may further contain a phospholipid in addition to the polypeptide.

In the present invention, "phospholipid" is a lipid in which one fatty acid is substituted by a phosphate and a simple organic molecule. Examples of the most common phospholipids that can be found in surfactant agents include phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylinositol (PI), and phosphatidylserine (PS). The glycerol moiety of the phospholipid is primarily esterified with long chain fatty acids, which can be resaturated (e.g. myristic acid, palmitic acid and stearic acid), monounsaturated (e.g., oleic acid), or It can be polyunsaturated (linoleic acid and arachidonic acid). Specific examples are dipalmitoylphosphatidylcholine (DPPC), dilauroylphosphatidylglycerol (DLPG), dimyristoylphosphatidylglycerol (DMPG), dipalmitoylphosphatidylglycerol (DPPG), distearoylphosphatidylglycerol (DSPG), palma Toyl-oleoyl-phosphatidyl glycerol (POPG), etc. may be mentioned, but the present invention is not limited thereto. Phospholipids that may be included in the lung surfactant composition of the present invention may be dipalmitoylphosphatidylcholine (DPPC), phosphatidylglycerol (PG), and/or palmitic acid (PA), but are not limited thereto.

In addition, some polypeptides of the surfactant protein SP-B or an analog polypeptide of the surfactant protein SP-C may be contained in an amount of 1 to 5% by weight of the total composition of the pulmonary surfactant.

It is apparent in the art that the pulmonary surfactant composition of the present invention corresponds to a mixture of phospholipids and/or proteins, and that physical properties of the pulmonary surfactant prepared according to their content ratio can be remarkably changed. In particular, International Publication No. 2008-044109 discloses how the viscosity is affected by the relative amounts of phospholipids and SP-B and SP-C analogs in the generally described surfactant composition, and how the components mutually influence among synthetic surfactants. It is intended to study whether or not the amount of the SP-B analog significantly affects the viscosity of the surfactant composition, and the content of the SP-B analog should be kept as low as possible within a range that does not affect the therapeutic efficacy. .

Therefore, properties may be different depending on the content of some of the polypeptides of the surfactant protein SP-B and/or the analog polypeptide of the surfactant protein SP-C, which is an important factor to be considered when preparing a lung surfactant composition.

In the present invention, some of the polypeptides of the surfactant protein SP-B or the analog polypeptide of the surfactant protein SP-C may be included in 1 to 5% by weight, preferably about 2% by weight, of the total composition of the pulmonary surfactant, respectively. However, it is not limited thereto.

Human natural pulmonary surfactants contain 10 to 11 mg of SP-B, or 22 to 34 mg of SP-C per 1 mmol of phospholipid, and commercially available animal-derived pulmonary surfactants contain 0 to 5.4 mg of SP-B, depending on the product. Alternatively, considering that it contains 1 to 11.6 mg of SP-C, some polypeptides of the surfactant protein SP-B or an analog polypeptide of the surfactant protein SP-C are each more than 5% by weight of the total composition of the lung surfactant. When included in an amount or in an amount less than 1% by weight, there is a problem that the function of the surface-active protein is deteriorated or lost.

In addition, some of the polypeptides of the surfactant protein SP-B or the analog polypeptide of the surfactant protein SP-C may be included in an amount of 1 to 5 parts by weight based on 100 parts by weight of phospholipid, respectively. In the example of the present invention, a pulmonary surfactant was prepared by setting the weight ratio of DPPC/PG/PA/SP-B/SP-C to 75:25:10:2.24:2.24 (w/w).

In addition, some of the polypeptides of the surfactant protein SP-B: the analog polypeptide of the surfactant protein SP-C may be included in a weight ratio of 1:1 to 1:10, and preferably in a weight ratio of 1:1. Since there is a problem that the viscosity of the lung surfactant increases when the content of some of the polypeptides of the surfactant protein SP-B is high compared to the analog polypeptide of the surfactant protein SP-C, it is preferable to maintain the above range.

As another aspect, the present invention provides a pharmaceutical composition for preventing or treating respiratory distress syndrome comprising the pulmonary surfactant composition as an active ingredient.

Pulmonary surfactants comprising some polypeptides of the surfactant protein SP-B consisting of the sequence represented by SEQ ID NO: 1 of the present invention and an analog polypeptide of the surfactant protein SP-C consisting of the sequence represented by SEQ ID NO: 2 are conventionally used in natural lungs. It was confirmed that it can be effectively used in the prevention or treatment of respiratory distress syndrome by confirming that it has superior pulmonary surfactant activity (surface tension, diffusion rate, adsorption rate, etc.) than surfactants (eg, Surfacten ^{® ).}

In the present invention, "prevention" includes all actions of inhibiting or delaying the onset of respiratory distress syndrome by administration of the composition of the present invention, and "treatment" is improved by the composition of the present invention or Includes all behaviors that change to the advantage.

In the present invention, "dyspnea syndrome" is a disease that causes respiratory failure due to insufficient production of pulmonary surfactant, and is the most common disease that occurs in newborns, especially premature infants.

The pharmaceutical composition comprising the pulmonary surfactant composition of the present invention may further include an appropriate carrier, excipient, or diluent commonly used in the preparation of pharmaceutical compositions. At this time, the content of the waste surfactant included in the composition is not particularly limited thereto, but may include 0.01% by weight to 100% by weight, preferably 1% by weight to 50% by weight, based on the total weight of the composition.

The pharmaceutical composition is any one selected from the group consisting of tablets, pills, powders, granules, capsules, suspensions, solutions, emulsions, syrups, sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried agents, and suppositories. It can have a formulation of. In the case of formulation, it is prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants that are usually used. Preparations for parenteral administration, such as injections, may include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. As the non-aqueous solvent and the suspension solvent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate may be used. As a base for suppositories, witepsol, macrogol, tween 61, cacao butter, laurin paper, glycerogelatin, and the like may be used.

As another aspect, the present invention provides a method for preventing or treating respiratory distress syndrome, comprising administering the pharmaceutical composition to a suspected subject of respiratory distress syndrome.

In the present invention, the suspected individual of respiratory distress syndrome includes all animals including humans who have or may develop respiratory distress syndrome, and by administering the pharmaceutical composition of the present invention to an individual suspected of respiratory distress syndrome, the individual is efficiently It can be cured. The respiratory distress syndrome is as described above.

In the present invention, "administration" means introducing the pharmaceutical composition of the present invention to an individual suspected of having respiratory distress syndrome by any suitable method, and the route of administration may be administered through various routes as long as it can reach the target tissue. Specifically, it may be administered by parenteral administration such as injection, but is not limited thereto.

The pharmaceutical composition comprising the pulmonary surfactant of the present invention can be administered to a patient (e.g., a premature infant) by continuous infusion by a conventional method or by intrabronchial injection to the patient under continuous or intermittent positive pressure breathing (IPPV). In addition, it can be administered by using a thin catheter, mask, prongs, etc. placed inside the trachea, or by continuous positive pressure (nCPAP).

The prevention or treatment method of the present invention may include administering the pharmaceutical composition in a pharmaceutically effective amount.

In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is the type and severity of the individual, age, sex, type of disease , Drug activity, sensitivity to the drug, time of administration, route of administration and rate of excretion, duration of treatment, factors including drugs used concurrently, and other factors well known in the medical field. The composition of the present invention may be administered as an individual therapeutic agent or administered in combination with other therapeutic agents, and may be administered sequentially or simultaneously with a conventional therapeutic agent. For example, if an individual is undergoing treatment for a respiratory disease caused by a bacterial infection, the surfactant of the present invention may be administered together with other compounds used to treat a bacterial infection such as antibiotics, and bronchopulmonary formation In order to prevent complications such as the above, the surfactant of the present invention may be administered together with a corticosteroid such as budesonide and beclomethasone dipropionate. And can be administered single or multiple. It is important to administer an amount capable of obtaining the maximum effect in a minimum amount without side effects in consideration of all of the above factors, and can be easily determined by a person skilled in the art. The preferred dosage of the composition of the present invention varies depending on the condition and weight of the patient, the degree of disease, the form of the drug, the route and duration of administration, and the total amount of daily dosage suitable for use may be determined by the treating physician within the range of correct medical judgment.

In another aspect, the present invention provides a kit comprising the waste surfactant composition and a container means for storing the waste surfactant composition.

The kind of the kit of the present invention is not particularly limited, and a kit in a form commonly used in the art may be used. Specifically, the waste surfactant composition may be included in the container means, but is not limited thereto. In addition, the kit may further include a pharmaceutically acceptable carrier, and the pharmaceutically acceptable carrier may be specifically sterilized physiological saline, but is not limited thereto. The synthetic waste surfactant composition of the present invention and the pharmaceutically acceptable carrier may be packaged in a form contained in a separate container, or contained in a container divided into one or more compartments.

In another aspect, the present invention provides (a) a partial polypeptide of the surfactant protein SP-B consisting of the sequence represented by SEQ ID NO: 1, an analog polypeptide of the surfactant protein SP-C consisting of the sequence represented by SEQ ID NO: 2, Mixing phospholipids and fatty acids; And (b) it provides a method for preparing the waste surfactant composition comprising the step of evaporating after dissolving the mixture. The phospholipid may be dipalmitoylphosphatidylcholine (DPPC) and/or phosphatidylglycerol (PG), and the fatty acid may be palmitic acid.

The pulmonary surfactant composition of the present invention not only has superior pulmonary surfactant performance than conventional animal-derived pulmonary surfactants, but also can be economically mass-produced by building a large-scale synthesis system.

1 is a diagram showing the surface diffusion rate in a modified Wilhelmy balance.
2 is a diagram showing the surface adsorption rate in a modified Wilhelmy balance.
3 is a diagram showing a surface tension area chart in a modified Wilhelmy balance.
4 is a diagram showing the difference in lung volume according to the pressure between the term control group and the immature control group.
5 is a diagram showing the difference in lung volume according to the pressure between the control group and each experimental group.
6 is a photograph of the pathological findings of the lung tissue of the control group and each experimental group observed through H&E staining.
7 is a diagram comparing and calculating the airized area (%) in the alveoli of the control group and each experimental group.

Hereinafter, the present invention will be described in more detail through examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Example: Preparation of artificial synthetic lung surfactant formulation

Part of the sequence of the carbon-side end portion of human SP-B (SP-B: RMLPQLVCRLVLRCSMD: SEQ ID NO: 1) and a polypeptide analog of human SP-C (SP-C: CPVHLKRLLLLLLLLLLLLLLLLLL: SEQ ID NO: 2) were added to Anygen Co., Ltd. It was synthesized on request. Based on this, dipalmitoyl-phosphatidylcholine (DPPC), phosphatidylglycerol (PG), palmitic acid (PA) phospholipids, (i) SP-B polypeptide of SEQ ID NO: 1, (ii) SP-C polypeptide of SEQ ID NO: 2 The analogue and (iii) the SP-B polypeptide of SEQ ID NO: 1 and the SP-C polypeptide analog of SEQ ID NO: 2 were mixed, respectively. Subsequently, the three synthetic surfactant formulations were designated as Synsurf-1, Synsurf-2, and Synsurf-3, respectively (Table 1).

Product name SP formulation (% by weight of the total composition) Mixing ratio (composition ratio) Synsurf-1 SP-B (2% by weight) DPPC:PG:PA: SP-B = 75:25:10:2.24 (w/w) Synsurf-2 SP-C (2% by weight) DPPC:PG:PA: SP-C = 75:25:10:2.24 (w/w) Synsurf-3 SP-B/SP-C (2% by weight each) DPPC:PG:PA: SP-B : SP-C = 75:25:10:2.24:2.24 (w/w)

Synthetic waste surfactant was prepared more specifically as follows. Surfactant proteins were prepared in the composition ratios described above, dissolved in a small amount of trifluoroacetic acid, and then mixed with DPPC/PG/PA in a ratio of 75:25:10 as a mass ratio, and CHCl ₃ After dissolving in a /CH ₃ OH (2:1, v/v) solution, it was dissolved in a 10% ethanol solution for better mixing, and then allowed to stand at a temperature of 40-45° C. for 15 minutes to evaporate. Finally, freeze-dried to obtain Synsurf-1, 2 and 3, which are white powders.

Experimental example 1: Inspection of surface physical properties of artificial synthetic lung surfactant

Whether Synsurf-1, 2, and 3 synthesized in the above examples have the same surface physical properties with Surfacten ^® (Mitsubishi Tanabe, Japan), which is a formulation having the best surface physical properties among the previously commercialized and used waste surfactants. Was compared.

Specifically, it was compared through the Modified Wilhelmy balance test, and the criteria of the surface area characteristics of the ideal artificial pulmonary surfactant formulation suggested by Fujiwara et al. are shown in Table 2 below (Modified from Fujiwara T, Robertson B. Pharmacology of exogenous surfactant.In: Robertson B, Van Golde LMG, Batenburg JJ, editors.Pulmonary surfactant.2 ed., Amsterdam: Elservier, 1992:561-92.).

1.Pulsating bubble surfactometer 1) The minimum surface tension is less than 10 mN/m 2) The maximum surface tension is about 30 mN/m 2. Modifided Wilhelmy balance 1) Fast diffusion: less than 10 seconds to reach the equilibrium surface tension of 24-27 mN/n 2) Rapid adsorption: The time to reach the surface tension of 27-30 mN/m (surface pressure 42-45 mN/m) is less than 1 minute 3) Minimum surface tension of less than 10mN/m at 20-30% of surface compression 4) Reproducible surface tension-maximum surface tension of 27-30 mN/m on the domain diagram 5) Very low surface compressibility (less than 0.03 m/Mm) at a surface tension of 10 mN/m

Experimental example 1-1: Measurement of maximum and minimum surface tension

When making microbubbles and vibrating them in a PBS (Pulsating bubble surfactometer) machine (GENERAL TRANSCO, INC, USA), the minimum and maximum surface tension (ST) of microbubbles (min-ST, max-ST) ) Was measured. The standard of surface tension as an ideal artificial lung surfactant formulation is Min-ST of 10 mN/m or less and Max-ST of 30 mN/m.

As a result, as shown in Table 3 below, except for Synsurf-1, Surfacten ^® , Synsurf-2 and Synsurf-3 have a minimum surface tension of 10 nM/m or less, and a maximum surface tension of about 30 nM/m. It was confirmed that it had an appropriate surface tension by showing the results of the formulation having the effect of.

Minimum surface tension (mN/m) Surface tension (mN/m) 1 minute 5 minutes 1 minute 5 minutes Surfacten ^® 6.2±0.9 5.5±0.4 33.8±1.5 32.8±1.6 Synsurf-1 16.2±0.9 16.7±0.6 27.6±1.8 28.7±1.5 Synsurf-2 8.1±1.1 7.9±1.0 33.0±1.7 33.1±1.6 Synsurf-3 8.0 ±0.9 7.1±0.8 34.6±0.8 34.5±1.0

Experimental example 1-2: Modified Wilhelmy balance test

A modified Wilhelmy balance (Acoma, Japan) machine was used to perform a ST-area diagram (hysteresis curve), a surface spreading test, and a surface adsorption test.

As a result, (i) in the surface diffusion test, it was confirmed that Synsurf-2, 3 and Surfacten ^® showed similar fast diffusion rates (Fig. 1), and (ii) in the surface adsorption test, Synsurf-3 was the fastest adsorption rate. After that, Surfacten ^® , Synsurf-2, Synsurf-1 showed a high speed in the order of (Fig. 2), (iii) all four hysteresis curves in the surface tension area chart (Fig. 3). ). However, the minimum surface tension (min-ST) was the ^{lowest in Surfacten ®} , and then Synsurf-2 and Synsurf-3 showed similar levels.

In conclusion, it was confirmed that Synsurf-2 and Synsurf-3 of the present invention have similar or superior surface activity effects to ^{Surfacten ®, a commercially available animal-derived lung surfactant.}

Surfacten ^® Synsurf-1 Synsurf-2 Synsurf-3 Surface diffusivity (equilibrium surface tension reached in less than 10 seconds) (mN/m) 27 ^* 43 27 ^* 27 ^* Surface adsorption (surface tension reached in less than 1 minute) (mN/m) 28 ^* 35 29 ^* 27 ^* Minimum surface tension at 20-30% surface compression (mN/m) 8 ^* 12 9 ^* 9 ^* Reproducible surface tension-maximum surface tension on area diagram (mN/m) 28 ^* 47 36 36 Surface compressibility at 10 mN/m surface tension (mN/m) 0.03 ^* 0.05 0.03 ^* 0.03 ^{* *} Means that it has suitable properties for an ideal artificial lung surfactant.

Experimental example 2: animals ( in vivo ) Dosing experiment

For animal testing, a cesarean section was performed on pregnant rabbits to extract term rabbit fetuses (31 days gestation) and preterm rabbit fetuses (27 days gestation period). Tracheostomy was performed on the fetus rabbit. ) Was performed and forced breathing of 0.3-0.5cc of air was performed 30 times.

The fetus rabbit was divided into a control group and a test group as follows. Specifically, 1) control of preterm infants who did not administer anything through tracheostomy tube in immature fetal rabbits (preterm control, Preterm), 2) control of term infants who did not administer anything through tracheostomy tube in term fetal rabbits (term control, Term) , 3) Group administered with ^{Surfacten ®} through tracheostomy tube in immature fetal rabbits ^{(Surfacten ®} administered group), 4) Group administered with Synsurf-1 in immature fetal rabbits (Synsurf-1 administered group), 5) In immature fetal rabbits Synsurf-2 was administered group (Synsurf-2 administered group), 6) Synsurf-3 was administered to immature fetal rabbits (Synsurf-3 administered group).

In the Surfacten ^® administration group, Synsurf-1 administration group, Synsurf-2 administration group, and Synsurf-3 administration group, each of the formulations Surfacten ^® , Synsurf-1, Synsurf-2, Synsurf-3 was administered at a total dose of 100 mg/kg every 30 seconds. It was divided into times and injected into the lungs through the airway. After injection, artificial respiration was performed 30 times for even distribution of the lungs.

Example 2-1: Measurement of the volume during exhalation in the pressure-volume curve of the lungs

When pressure was applied to the lungs of each of the above groups at a constant rate, the change in the volume increase of the lungs according to the increase in pressure was observed, and this was expressed as a pressure-volume curve. Among these, the exhaled volume (ml/kg) at each exhalation pressure was measured and compared.

As a result, as shown in Figure 4, it was confirmed that the difference in lung volume was shown between the term control group and the immature control group. In addition, as shown in FIG. 5 ^{, when comparing the Surfacten ®} administration group, Synsurf-1 administration group, Synsurf-2 administration group, and Synsurf-3 administration group based on the immature control group, there was no significant difference between the immature control group and the Synsurf-1 administration group. However (p=0.4811), there was a significant difference from the Synsurf-2 administration group, Synsurf-3 administration group, and Surfacten ^® administration group (p=0.0068, 0.0031, <0.0001, respectively). When comparing the volume size of each administration group, the Synsurf-3 administration group was the best, followed by the Synsurf-2 administration group, and Surfacten ^® administration group, in that order, and it was confirmed that the pulmonary surface activity of Synsurf-3 was the most excellent.

Example 2-2: Analysis of aerated area in the pathologic alveoli

After measuring the pressure-volume curve, _{a constant pressure of 10 cmH 2} O was applied and the end of the tracheostomy was blocked, and then fixed in formalin fixative. H&E (hematoxylin and eosin) staining was performed on the left and right lung tissue samples to complete the pathological sample. After taking ×100-fold pictures of the pathological specimen, using the ImageJ software program (V 1.48 for Windows, NIH, USA) on a computer, the percentage of aerated area (%) of the total alveolar area of the ×100-fold field of view ) Was measured. Each of the left and right lungs was performed in two tissues, one for each fetal rabbit individual, and the measured values between each group were compared.

As a result, as shown in FIGS. 6 and 7, compared to the immature control ^{group, Surfacten ®} administration group, Synsurf-1 administration group, Synsurf-2 administration group, and Synsurf-3 administration group all showed significantly greater results (p<0.0001), Compared to the Surfacten ^® administration group, there was no significant difference between the Synsurf-1 administration group and the Synsurf-2 administration group (p=0.7289, 0.7224, respectively), but the value of the Synsurf-3 administration group was significantly higher (p=0.0202). This suggests that Synsurf-3 has the best effect as a lung surfactant.

From the above description, those skilled in the art to which the present invention pertains will understand that the present invention can be implemented in other specific forms without changing the technical spirit or essential features thereof. In this regard, the embodiments described above are illustrative in all respects and should be understood as non-limiting. The scope of the present invention should be construed that all changes or modifications derived from the meaning and scope of the claims to be described later rather than the above detailed description and equivalent concepts are included in the scope of the present invention.

<110> University-Industry Cooperation Group of Kyung Hee University <120> Synthetic Pulmonary Surfactant Composition Comprising Analogues of Surfactant Protein B and Surfactant Protein C <130> KPA150012-KR-D1 <160> 2 <170> KoPatentIn 3.0 <210> 1 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> SP-B polypeptide <400> 1 Arg Met Leu Pro Gln Leu Val Cys Arg Leu Val Leu Arg Cys Ser Met 1 5 10 15 Asp <210> 2 <211> 23 <212> PRT <213> Artificial Sequence <220> <223> SP-C polypeptide analogue <400> 2 Cys Pro Val His Leu Lys Arg Leu Leu Leu Leu Leu Leu Leu Leu Leu 1 5 10 15 Leu Leu Leu Leu Leu Leu Leu 20

Claims (12)

A partial polypeptide of a surfactant protein SP-B comprising the sequence represented by SEQ ID NO: 1, an analog polypeptide of a surfactant protein SP-C comprising the sequence represented by SEQ ID NO: 2, a phospholipid and a fatty acid, Activator composition.
The composition of claim 1, wherein the phospholipid is dipalmitoyl phosphatidylcholine (DPPC) or phosphatidyl glycerol (PG).
The composition of claim 1, wherein the fatty acid is palmitic acid.
2. The composition of claim 1, wherein the composition comprises from 1 to 5% by weight of the total lung surfactant composition of a polypeptide of the surfactant protein SP-B or an analogous polypeptide of the surfactant protein SP-C.
The composition of claim 1, wherein the composition comprises a portion of the polypeptide of the surfactant protein SP-B and the analogous polypeptide of the surfactant protein SP-C in a weight ratio of 1: 1 to 1:10.
A pharmaceutical composition for preventing or treating respiratory distress syndrome comprising the pulmonary surfactant composition according to any one of claims 1 to 5 as an active ingredient.
7. The pharmaceutical composition according to claim 6, wherein the respiratory distress syndrome occurs in premature infants or newborn infants.
A kit comprising the pulmonary surfactant composition of any one of claims 1 to 5 and container means for storing the same.
(a) mixing a partial polypeptide of a surfactant protein SP-B comprising the sequence of SEQ ID NO: 1, an analog polypeptide of a surfactant protein SP-C comprising the sequence of SEQ ID NO: 2, a phospholipid and a fatty acid; And
(b) dissolving and evaporating the mixture. &lt; RTI ID = 0.0 &gt; 11. &lt; / RTI &gt;
10. The method of claim 9, wherein the phospholipid is dipalmitoyl phosphatidylcholine (DPPC) or phosphatidylglycerol (PG).
10. The method of claim 9, wherein the fatty acid is palmitic acid.
10. The method of claim 9, wherein a part of the polypeptide of the surfactant protein SP-B and the analogous polypeptide of the surfactant protein SP-C are contained in a weight ratio of 1: 1 to 1:10.

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