HK1042500B - Artificial peptides having surface activity and the use thereof in the preparation of artificial surfactant - Google Patents

Description

Artificial peptide with surface activity and application thereof in preparing artificial surface active substance

The present invention provides novel artificial peptides having surface activity. In particular, the present invention provides SP-C analogs that are particularly effective in reducing the surface tension of the gas-liquid interface once combined with a suitable lipid.

Thus, the peptides of the invention, in combination with a lipid, and optionally in combination with SP-B or an active analogue thereof or a substitute for SP-B, are useful in the preparation of artificial surfactants useful in the treatment of Respiratory Distress Syndrome (RDS), other surfactant deficiencies or dysfunctions, associated lung diseases such as pneumonia, bronchitis, asthma, meconium aspiration syndrome and other diseases such as serous otitis media (otitis media).

Background

Pulmonary surfactants can reduce the surface tension of the gas-liquid interface in the lining of the alveoli, preventing the lungs from collapsing during terminal exhalation. Surfactant deficiency is a common condition in premature infants and causes Respiratory Distress Syndrome (RDS), and this disease can be effectively treated with natural surfactants extracted from the lungs of animals (Fujiwara, T. and Robertson B. (1992) in Robertson, B., van Golde, L.M.G., and Batenburg, B. (eds.). Lung surfactants: from molecular biology to clinical practice Amsterdam, Elsevier, pp. 561-. The main constituents of these surfactant preparations are phospholipids such as 1, 2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), Phosphatidylglycerol (PG) and hydrophobic surface-active proteins B and C (SP-B and SP-C). Is C-type (Ca)²⁺-dependent) collectins and are believed to play ｃA major role in the host defense system, the hydrophilic surfactant proteins SP- ｃA and SP-D, which are normally not present in these surfactant preparations due to the use of an organic solvent extraction step.

SP-B and SP-C account for only about 1-2% of the weight of the surface-active substance, but they are still able to improve the surface activity significantly compared to pure lipid preparations (Curstedt, T. et al (1987) J. Eur. biochem. 168, 255-42; Takahashi, A., Nemoto, T. and Fujiwara, T. (1994) Acta Paediatr. Jap.36, 613-618). The primary and secondary structures of SP-B and SP-C and the tertiary structure of SP-C in solution have been determined (see FIG. 4). SP-B consists of two identical polypeptide chains of 79 amino acids which are linked to interchain disulfide bridges (Curstedt, T. et al (1990) Proc. Natl. Acad. Sci. USA 87, 2985. 2989; Johansson, J., Curstedt, T. and J _ rnvall, H. (1991) biochemistry 30, 6917. sup. 6921). Each monomer chain has three intrachain disulfide bridges and at least four amphipathic helices with one polar and one non-polar surface through which SP-B can interact with and bring two lipid bilayers into close proximity (Andersson, M. et al (1995) FEBS Lett.362, 328-332). SP-C is a lipoprotein composed of 35 amino acid residues with an alpha-helical domain between residues 9-34 (Johansson, J. et al (1994) biochemistry 33, 6015-. The helix is composed primarily of valyl residues and is embedded in the lipid bilayer, oriented parallel to the lipid acyl chain (Vandenbussche et al (1992) J. European J. biochem. 203, 201-209). Two palmitoyl groups are covalently linked to cysteine residues at positions 5 and 6 of the N-terminal part of the peptide (Curstedt, T. et al (1990) Proc. Natl. Acad. Sci. USA 87, 2985-2989). The two conserved positively charged residues at positions 11 and 12, arginine and lysine, may interact with the negatively charged head group of the lipid membrane, thus increasing its stiffness. Since the C-terminus contains only small or hydrophobic residues, making this moiety potentially more susceptible in the phospholipid bilayer, the stiffness of the lipid-peptide interaction may decrease towards the C-terminus. SP-C is thought to affect the thickness and fluidity of the surrounding lipids via a highly stable poly-valyl helix (Johansson, J. and Curstedt, T. (1997) J. European J. Biol. Chem. 244, 675-693).

State of the art

Due to some drawbacks of surfactant preparations obtained from animal tissues, such as their limited availability and the possibility that they contain infectious agents and may induce immune responses, attempts have been made to prepare artificial surfactants (Johansson, J. and Curstedt, T. (1997) J. Eur. biochem. 244, 675-693; Johansson, J. et al (1996) acta Paediata tr.85, 642-646), usually from synthetic lipids and hydrophobic proteins.

Previous work has demonstrated that synthetic SP-C cannot fold into the alpha-helix conformation necessary for optimal surface activity as native peptides (Johansson, J. et al (1995) J. Biochem. 307, 535-541) and therefore cannot properly interact with surfactant lipids.

Thus, synthetic SP-C analogs do not fold as do natural peptides and do not interact properly with surfactant lipids. To circumvent this problem, several schemes have been proposed to modify the sequence, for example by replacing all helical Val residues in native SP-C with Leu, which strongly supports the alpha-helixA conformation. The corresponding transmembrane analog, SP-C (Leu), shows good spreading at the gas-liquid interface when combined with DPPC: PG: PA (68: 22: 9) (w/w). However, the maximum surface tension value (γ) during compression of the scalloped surface_max) Gamma significantly higher than natural surface active substances_max. Furthermore, it is not possible to prepare lipid-peptide mixtures at concentrations above about 20mg/ml, probably due to the formation of peptide oligomers (Nilsson, G. et al (1998) J. European biochem.255, 116-124). In addition, biologically active polyleucine SP-C analogs of varying lengths have been synthesized (Takei, T. et al (1996) biopharmaceutical bulletin 19, 1550-. In later studies, neither self-oligomerization nor problems in producing high lipid concentration samples were reported.

Different publications address the problem of providing peptide analogues of natural surfactant peptides and offer a number of different solutions. In these publications, WO9321225, EP733645, WO9617872 in the name of Tokyo Tanabe disclose peptide analogues of native SP-C, which are usually different from the native peptide with respect to the sequence of the N-terminal part.

Patent applications WO8906657 and WO9222315 by the Scripps institute disclose SP-B analogs having alternating hydrophobic and hydrophilic amino acid residues. Peptides with alternating leucine and lysine residues (KL4) are particularly claimed.

Clercx A. et al, J. European biochem. 229, 465-72, 1995 disclose peptides of varying lengths corresponding to the N-terminus of porcine SP-C and hybrid peptides derived from porcine SP-C and bacteriorhodopsin.

Johansson j. et al journal of biochemistry 307, 535: 41, 1995 discloses synthetic peptides that differ from the natural porcine SP-C by substitutions of some amino acids.

WO89/04326 in the name of California Biotechnology-Byk Gulden and WO91/18015 in the name of California Biotechnology-Scios Nova disclose SP-C analogs containing an initial N-terminal sequence in which the two Cys of the native SP-C are replaced by two Ser.

Description of the invention

It has now been found that SP-C analogue peptides combine the following characteristics: i) substitution of Val residues with other neutral and hydrophobic residues, ii) substitution of Cys residues with Ser residues, iii) substitution of some neutral amino acid residues with bulky or polar residues, show particularly useful properties for reducing surface tension. In particular, it has been found that the latter feature allows the avoidance of self-oligomerization due to the positive charge imparted by polar residues or steric hindrance imparted by bulky substituents.

According to a first aspect, the present invention provides SP-C analogues of the general formula (I) having the following one-letter amino acid code:

F_eG_fIPZZPVHLKR(X_aB)_n(X_bB)_n(X_cB)_mX_dGALLMGL (I)

wherein:

x is an amino acid selected from the group consisting of: v, I, L, Nle (norleucine);

b is an amino acid selected from the group consisting of: ornithine, K, I, W, F, Y, Q, N;

z is an amino acid selected from the group consisting of: s, C, F, wherein the Ser or Cys residue is optionally linked to an acyl group containing 12 to 22 carbon atoms via an ester or thioester bond;

a is an integer of 1 to 19

b is an integer of 1 to 19

c is an integer of 1 to 21

d is an integer of 0 to 20

e is 0 or 1

f is 0 or 1

n is 0 or 1

m is 0 or 1

With the following conditions:

n+m＞0，

f≥e；

(X_aB)_n(X_bB)_n(X_cB)_mX_dis a sequence having a maximum of 22 amino acids, preferably 10 to 22.

Preferred peptides of formula (I) have the following sequence:

(Ia)FGIPSSPVHLKRX₄BX₄BX₄BXGALLMGL

(Ib)FGIPSSPVHLKRX₅BX₅BX₄GALLMGL

(Ic)FGIPSSPVHLKRX₄BX₁₁GALLMGL

(Id)FGIPSSPVHLKRX₈BX₇GALLMGL

(Ie)FGIPSSPVHLKRX₁₁BX₄GALLMGL

in the sequences (Ia) - (Ic), preference is given to those in which B ═ Lys or Phe and X ═ Leu, Ile or Nle.

According to a preferred embodiment, the peptides of formulae (Ia) to (If) have the following sequences, respectively:

FGIPSSPVHLKRLLIL KLLLLKILLLKLGALLMGL[SP-C(LKS)]

FGIPSSPVHLKRLLILLKLLLLIKLLILGALLMGL[SP-C(LKS)1]

FGIPSSPVHLKRLLILKLLLLLILLLILGALLMGL[SP-C(LKS)2]

FGIPSSPVHLKRLLILLLLLKLILLLILGALLMGL[SP-C(LKS)3]

FGIPSSPVHLKRLLILLLLLLLIKLLILGALLMGL[SP-C(LKS)4]

FGIPSSPVHLKRLLILFLLLLFILLLFLGALLMGL[SP-C(LFS)]

in a more preferred embodiment of the invention, the Ser residue is covalently linked to an acyl group containing 12 to 22 carbon atoms.

The peptides of formula (I) may be prepared by synthetic or recombinant techniques.

Conventional syntheses are described, for example, in: schroeder et al, peptide, Vol.1, academic Press, 1965; bodanszky et al, peptide Synthesis, Interscience publishers, 1996; peptide from Baramy and Merrifield: analysis, Synthesis, biology Vol.2, Chapter 1, academic Press, 1980. The techniques include peptide synthesis in solid phase, in solution, organic chemical synthesis, or any combination thereof.

S-or O-acylated peptides are preferably synthesized by treating non-acylated peptides with an acid chloride in pure trifluoroacetic acid, as described in Yousefi-Salakdeh et al, J. Biochem.1999, 343, 557-562. After synthesis and purification, the synthetic peptides were characterized by biochemical and biophysical methods as reported in the "examples" section below.

The peptides of the invention were evaluated for their activity in reducing surface tension in combination with lipids and phospholipids, SP-B, SP-B analogs, or SP-B surrogates. In particular, these peptides have been combined with DPPC (1, 2-dipalmitoyl-sn-glycero-3-phosphocholine)/PG (phosphatidylglycerol)/PA (palmitic acid) with or without SP-B, its active analogs and polymyxins.

The results of the pulse-bubble surfactant test clearly show that the synthetic peptide of the present invention strongly compresses the cylindrical curved surface at the minimum and maximum surface tensions (gamma.) during the compression_minAnd gamma_max) To a value comparable to that obtained using surfactants of natural origin.

Particularly useful results are obtained by adding SP-B or an active analogue thereof to the mixed peptide/lipid-phospholipid. Furthermore, it has been surprisingly found that polymyxin, and in particular polymyxin B, acts as a substitute for SP-B and that the results obtained with its addition are comparable to those obtained with SP-B.

According to a second aspect, the present invention provides a synthetic surface active substance comprising one or more peptides of formula (I) in admixture with a lipid and/or phospholipid and optionally SP-B, an active derivative thereof or a polymyxin. Suitable lipids/phospholipids may be selected from phosphatidylcholine (preferably DPPC), PG, PA, triacylglycerols, sphingomyelin.

In a more preferred embodiment of the invention, a surfactant mixture comprising a peptide wherein the palmitoyl chain is covalently linked to the Ser residue via an O-bond should be used. It has been found that the surface active substance mixture containing dipalmitoylated form of the reference peptide (SP-c (leu)) exhibits higher surface membrane stability and an increased size of the surface associated lipid reservoir as measured by the trapped bubble system compared to the mixture containing the corresponding non-palmitoylated peptide. In samples containing 5% dipalmitoylated peptide,. gamma._minBelow 1.5mN/m and these films are very stable, since the surface tension does not increase by 0.5mN/m within 10 minutes at a constant bubble volume. In contrast, gamma for non-palmitoylated peptides_minAbout 5mN/m, the film was observed to be less stable at low surface tension with frequent air bubble rattling. Moreover, after subphase depletion of the non-palmitoylated peptide containing sample, after several adsorption steps, it lost its ability to reach near zero stable surface tension, while with dipalmitoylated peptide, there was no deterioration in membrane quality even after 10 more expansion steps and incorporation of reservoir materials equivalent to two or more monolayers. The increased surface activity of dipalmitoylated peptides was also confirmed by a pulsed bubble surfactant meter. Furthermore, the presence of acyl groups was found to also reduce the tendency to form oligomers. This finding is very important because peptide oligomerization has been found to hinder the preparation of mixtures at concentrations above 20mg/ml during the preparation of artificial surface-active substances (Nilsson et al, J. Eur. biochem. 1998, 255, 116-124).

The synthetic surface-active substance may be prepared by mixing a solution or suspension of the peptide and the lipid and then drying the mixture.

In this case, the dry mixture may be suspended, dispersed or administered as such to a patient in need of treatment for surfactant deficiency.

The synthetic surface-active substance is preferably administered intratracheally or by aerosol. The latter form of administration requires mixing the small particle surface active substance with a suitable inert propellant. Other forms of administration, such as stable solutions/suspensions of atomized or sprayed surface active substances, are also included within the scope of the present invention.

According to a further aspect, the invention provides the use of the peptides for the preparation of a surfactant which is useful in all conditions of adult or neonatal surfactant deficiency or dysfunction, associated lung diseases such as pneumonia, bronchitis, asthma, meconium aspiration syndrome and other diseases such as serous otitis media (glue ear).

Typically, in the treatment of respiratory distress syndrome, which often affects premature infants, the surfactant is preferably administered by intratracheal administration.

The following examples describe the invention in more detail.

Example 1

Peptide Synthesis and purification

SP-C analogs, SP-C (LKS), were synthesized in an applied biosystems 430A instrument using stepwise solid phase techniques and t-butoxycarbonyl chemistry (Kent, S.B.H. (1988) Ann. Rev. biochem. 57, 957-. Cleavage of the resin-peptide bond and deprotection of the side chain was performed in anhydrous hydrogen fluoride/methoxybenzene/dimethylsulfide 10: 1(v/v/v) at 0 ℃ for 1.5 hours. The protecting group and scavenger were removed by repeated extractions with diethyl ether, followed by 3: 1(v/v) extraction of the peptide from the resin with dichloromethane/trifluoroacetic acid (TFA), followed by rotary evaporation. The crude peptide extract was redissolved at a concentration of 100mg/ml in chloroform/methanol 1: 1(v/v) with 5% water. A10 mg aliquot was applied to a Sephadex LH-60 column (40X 1cm) in the same solvent (Curstedt, T. et al (1987) J. European biochem. 168, 255-262). Fractions of 2.5ml were collected and the absorbance at 214 and 280nm was measured. Identification and quantification was performed by amino acid analysis.

For acylation, the purified peptide (typically about 5mg) was dried, dissolved in distilled TFA (100. mu.l) and acid chloride (10-20 equivalents compared to the peptide) was added. After 10 minutes the reaction was quenched with 80% aqueous ethanol (1.9 ml). The purification of acyl peptides was: the column was first chromatographed over Lipidex 5000 in ethylene dichloride/methanol 1: 4(v/v) and then passed through a C18 column by reverse phase HPLC using a linear gradient of 2-propanol/0.1% TFA to 60% (aqueous) methanol/0.1% TFA or 75% (aqueous) ethanol/0.1% TFA.

Example 2

Biochemical characterization

The purity of the peptides was checked by Sodium Dodecyl Sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) (Phast-system, Pharmacia, Sweden) and by reverse phase High Performance Liquid Chromatography (HPLC) (Gustafsson, M. et al (1997) J. Biochem. 326, 799-one 806) using a C18 column and a linear gradient of 60% aqueous methanol/0.1% TFA and isopropanol/0.1% TFA.

The molecular weight was determined by matrix assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (Lasermat 2000, Finnigan MAT) calibrated with angiotensin (Mr 3326.8).

The secondary structure of the peptide was studied using Circular Dichroism (CD) spectroscopy (Jasco-720 Jasco, Japan). After dissolution with Trifluoroethanol (TFE), spectra at 260-184nm were recorded at a scanning speed of 20nm/min and a resolution of 2 data points/nm. Calculating the residual molar ellipticity in kdeg xcm²And/dmol. The content of helical structures was estimated using the molar ellipticity at 208 and 222nm (Barrow, C.J. et al (1992) journal of molecular biology 225, 1075-.

Studies of the secondary structure of SP-C (LKS) using CD spectroscopy showed spectra typical of alpha-helical peptides and estimated alpha-helical content of about 75% from the 208nm and 222nm minima. Stepwise dilution with water until 12% TFE, the secondary structure remained stable, provided the peptide was soluble in pure TFE.

SDS-PAGE of SP-C (LKS) showed a single band similar to native SP-C, although SP-C lacking Lys in its helical portion (Leu) formed oligomers. In contrast to our experience with SP-C (Leu)/lipid mixtures, which are difficult to dissolve at concentrations above 20mg/ml (Nilsson, G. et al (1998) J. Eur. biochem.255, 116. 124.), SP-C (LKS)/lipid mixtures with a lipid concentration of 80mg/ml and a polypeptide/lipid ratio of 0.03 can be prepared.

Example 3

Preparation of peptide/lipid mixtures

DPPC, PG and PA were all purchased from Sigma Chemical Co, (St Louis, MO). These lipids were mixed in chloroform/methanol 98: 2(v/v) in the ratio DPPC: PG: PA 68: 22: 9(w/w/w) or DPPC/PG 7: 3 (w/w). The surfactant preparation is prepared by adding SP-C (LKS) alone or SP-C (LKS) and SP-B to each lipid mixture in a total polypeptide/lipid weight ratio of 0-0.05. The mixtures are evaporated under nitrogen and resuspended at a lipid concentration of 10-80mg/ml in 150mmol/l NaCl or 10mmol/l containing 140mmol/l NaCl and 2.0mmol/l CaCl₂10mmol/l Hepes buffer (pH 6.9). Freezing and sonication (50W, 48kHz) were repeated until a homogeneous suspension was obtained. Sometimes, these final suspensions were incubated at 45 ℃ for 1 hour.

The pH of the surfactant preparation suspended in 150mmol/l NaCl is 3.5-5.5. This lower pH-value of 3.5-4.5 was observed in formulations containing SP-B. Since native SP-B is purified using acidified organic solvents (Curstedt, T. et al (1987) J. Eur. biochem. 168, 255-262), small amounts of acid may remain in the formulation. By suspending the surface-active substance preparation in a suspension containing 140mmol/l NaCl and 2mmol/l CaCl₂In Hepes buffer (pH6.9), a pH close to physiological was obtained (Table 1). When DPPC/PG 7: 3(w/w) is used as a lipid mixture, compared to the corresponding formulation in unbuffered salineγ_maxOr gamma_minThere was no change. However, when PA is included in the lipid mixture, γ is at higher pH_maxAnd gamma_minBoth increased (tables 1 and 2).

Table 1: surface Properties of Artificial surfactant formulations in physiological saline solutions

The assay is performed directly after sample preparation or after incubation at 45 ℃ for 1 hour. The phospholipid concentration in NaCl150mmol/l was 10 mg/ml. Recordings were obtained over different periods of time using a pulsed bubble surfactant meter at 37 ℃, 50% surface compression and a rate of 40 weeks/min. These values are the mean (standard deviation) of 3-5 determinations. Abbreviations are defined herein.

Surface-active substance preparation			Surface tension (mN/m)
										SP-C(LKS)(％w/w)	SP-B(％w/w)	Phospholipids	Temperature of culture	7.5 seconds		1 minute		5 minutes
γmin	γmax	γmin	γmax	γmin	γmax
						3333333333	--22--2210.5	DPPC/PG/PADPPC/PG/PADPPC/PG/PADPPC/PG/PADPPC/PGDPPC/PGDPPC/PGDPPC/PGDPPC/PGDPPC/PG	-45℃-45℃-45℃-45℃-45℃					＜1＜1＜1＜1128(3)2(1)3(3)＜14(2)	41(1)41(1)33(2)34(1)39(5)35(5)31(1)29(3)24(4)29(1)	＜1＜1＜1＜114(4)9(3)2(1)1(1)1(2)4(3)	41(1)41(1)33(2)34(2)42(4)39(5)31(3)33(2)26(4)29(2)	＜1＜1＜1＜19(5)6(4)2(1)1(0)＜13(1)	41(0)41(1)33(2)25(2)42(2)42(3)33(1)36(1)31(1)34(2)

Table 2: surface Properties of Artificial surfactant formulations in buffered saline solutions

For NaCl 140mmol/l and CaCl₂The Hepes buffer (pH6.9) contained 2.0mmol/lA sample of phospholipid at 10mg/ml was assayed. Recordings were obtained over different periods of time using a pulsed bubble surfactant meter at 37 ℃, 50% surface compression and a rate of 40 weeks/min. These values are the mean (standard deviation) of 3-5 determinations. Abbreviations are defined herein.

Surface-active substance preparation			Surface tension (mN/m)
						SP-C(LKS)(％w/w)	SP-B(％w/w)	Phospholipids	7.5 seconds	1 minute	5 minutes
γmin γmax	γmin γmax	γmin γmax
			3333	-2-2	DPPC/PG/PADPPC/PG/PADPPC/PGDPPC/PG				4(1) 44(2)3(3) 38(3)15(3) 39(4)2(2) 26(3)	5(2) 47(2)4(4) 40(2)16(2) 42(3)1(1) 29(3)	7(2) 50(1)3(3) 44(2)13(3) 44(3)＜1 35(1)

Example 4

Preparation of phospholipid mixtures of SP-C (LKS) and polymyxin B

DPPC and PG were purchased from Sigma Chemical Co. (St Louis, Mo.). These phospholipids were mixed in chloroform/methanol 98: 2(v/v) at the ratio DPPC/PG 7: 3 (w/w). By adding SP-C (LKS) to the phospholipid mixture at a total polypeptide/phospholipid weight ratio of 0.03. The mixtures were evaporated under nitrogen and resuspended at room temperature in a medium containing 140mmol/l NaCl and 2.0mmol/l CaCl₂10mmol/l Hepes buffer (pH6.9) or the same buffer containing 0.01% polymyxin B (PxB) (Sigma Chemical Co, St Louis, Mo.). Freezing and sonication (50W, 48kHz) were repeated until a homogeneous suspension was obtained. The final phospholipid concentration was 10mg/ml for both formulations. Addition of PxB makes gamma_maxAnd gamma_minBoth decreased and the best surface activity was obtained (table 3).

Table 3: surface Properties of the Artificial surface-active substances with or without polymyxin B

Recordings were obtained at 37 ℃, 50% surface compression and a rate of 40 weeks/min for different periods of time using a pulsed bubble surfactant meter. These values are the mean (SD) of 5-11 determinations. Abbreviations are defined herein.

Surface-active substance preparation			Surface tension (mN/m)
						SP-C(LKS)(％w/w)	PxB(％w/w)	Phospholipids	7.5 seconds	1 minute	5 minutes
γmin γmax	γmin γmax	γmin γmax
			33	-l	DPPC/PGDPPC/PG				15(3) 39(4)3(2) 29(3)	16(2) 42(3)2(2) 31(4)	13(3) 44(3)1(1) 34(2)

Example 5

Biophysical characterization

Surface extension kinetics were measured at about 34-37 ℃ using a William surface balance (Biegler, Vienna, Austria). The surface tension was monitored for 10 minutes using a 20ml150mmol/1 NaCl low phase 1mm deep platinum plate attached to a strain gauge and inserted into a Tacron bath. A total of 1mg of lipid suspension was added as droplets onto the low phase at 4cm from the platinum plate.

Kinetic measurements of 3 wt% SP-C (LKS) in DPPC/PG, 7: 3(w/w) using a William balance showed a rapid spread after 3 seconds with a surface tension of 28mN/m (FIG. 2). It spreads slightly more slowly with 1 wt% SP-C (LKS) in the same lipid mixture. There was no significant change in the extension rate or equilibrium surface tension after the addition of 2 wt% SP-B (FIG. 2). No increase was observed after incubation of the mixture at 45 ℃ for 1 hour (data not shown). Similar results were obtained using DPPC: PG: PA, 68: 22: 9(w/w/w) as the lipid mixture (data not shown).

The kinetic surface tension was recorded using a pulsed bubble surfactant meter (surfactometer International, Toronto, canada) during 50% repeated compressions of the bubble surface at 37 ℃ and at a frequency of 40 weeks/min. All assays were continued for 5 minutes at a lipid concentration of 10 mg/ml. The pressure gradient across the bubble wall is measured at specific time intervals and used to calculate the minimum (. gamma.) value_min) And max (γ)_max) Surface tension at bubble size.

3 wt% SP-C (LKS) in DPPC: PG: PA, 68: 22: 9(w/w/w) at minimum bubble radius (. gamma.) on a pulsed bubble surfactant basis_min) Generates a surface tension of less than 1mN/m, whereas γ is observed with 3 wt% SP-C (LKS) in DPPC: PG, 7: 3(w/w)_min9-14mN/m (Table 1). In both cases at the maximum bubble radius (γ)_max) The surface tension was about 40 mN/m. Gamma conferred to both lipid formulations by addition of 2 wt% SP-B_maxA value of 31 to 33mN/m and gamma_minIs 0-2 mN/m. These values are very similar to those obtained for surfactant preparations isolated from natural sources (Robertson, B. et al (1990) prog. Respir, Res.25, 237-. These formulations had no significant effect on surface activity when incubated at 45 ℃ for 1 hour (table 1). Reduction of the amount of SP-B to 0.5 wt% in 3 wt% SP-C (LKS) in DPPC: PG 7: 3(w/w) tends to increase γ_minAlthough the results did not reach statistical significance (table 1). In contrast to SP-B, 2 wt% KL4(Cochrane, C.G., and Revak, S.D. (1991) Science 254, 566-_maxIt still remained relatively high, 41-42 mN/m.

Example 6

Comparison between mixtures containing dipalmitoylated and non-palmitoylated reference peptides

To each lipid mixture prepared from DPPC/PG/PA 68: 22: 9 w/w/w was added 3% w/wSP-C (Leu) or dipalmitoylated SP-C (Leu) to prepare the surfactant preparation. The mixtures were evaporated under nitrogen and resuspended in 150mmol/l NaCl at a lipid concentration of 10 mg/ml. In the samples where the SP-B substitute was also used, 1% w/w polymyxin B was added.

The mixture containing dipalmitoylated SP-C (Leu), with or without polymyxin B, showed a significant increase, in particular a decrease of 5 minutes in gamma_maxAnd gamma of early time interval_minAnd (5) carrying out the following steps.

Table 4: surface property(s)

The surface tension of the mixture was obtained using a pulsed bubble surfactant meter. After 2 minutes of equilibration, recordings were obtained at 37 ℃, 50% surface compression and a rate of 40 weeks/minute for different periods.

Surface-active substance preparation			Surface tension (mN/m)
						SP-C(Leu)(wt％)	Dipalmitoylated SP-C (Leu) (% by weight)	PxB(wt％)	7.5 seconds	1 minute	5 minutes
γmin γmax	γmin γmax	γmin γmax
			11	11	1-1				11 393 371 340 29	6.2 393 381 350 34	2 420 401 360 35

Example 7

In vivo assays

The therapeutic effect of surfactant on mechanical properties of an immature lung was evaluated in 9 preterm newborn rabbits of 27 days gestational age. At birth, these animals were tracheotomized, 5 of which received 2 times 2.5ml/kg of an artificial surfactant containing DPPC, PG and SP-C (LKS), with or without polymyxin B (as above) through a tracheal cannula. The total phospholipid concentration of the exogenous surface active substance is 40 mg/ml. The 2 animals used as negative controls received no tracheal material and the other 2 animals used as positive controls were treated with the same dose of modified natural surfactant (Curosurf, Chiesi farmaceci Spa, Parma, Italy) diluted to 40 mg/ml. 1 animal was treated with a mixture of DPPC and PG (same concentration as above) in saline at a dose of 2.5 ml/kg. All animals were kept in a body plethysmograph box at 37 ℃ and 100% oxygen was bubbled in parallel for 60 minutes with Servo Ventilator 900B (Siemens-Elema, Solna, Sweden) set at 40 minutes and 50% inspiratory time. Tidal volume is measured using a respiration rate meter connected to each plethysmograph cartridge. These animals were ventilated with a standardized tidal volume of 8-10ml/kg and without terminal positive expiratory pressure (PEEP). Lung-chest compliance is defined as the ratio of tidal volume to peak inspiratory pressure, expressed in ml/cm water kg.

The compliance in animals treated with the artificial surfactant, in particular animals receiving a surfactant containing polymyxin B, was significantly improved compared to untreated control animals. Clearly, this improvement appears to be superior to that seen after treatment with similar doses of modified natural surfactant (figure 3).

Brief Description of Drawings

FIG. 1, amino acid sequence and helical circular display of SP-C and analogs thereof

The sequence for human SP-C was taken from Johansson, J. et al (1988) FEBS Lett.232, 61-64 and the sequence for SP-C (Leu) was taken from Nilsson, G. et al (1998) Eur.J.biochem.255, 116-. SP-C (LKS) is based on the primary structure of SP-C, but all Val residues at positions 16-28 except position 17 have been replaced with Leu residues, Lys residues have been introduced at positions 17, 22 and 27, and palmitoylated Cys at positions 5 and 6 have been replaced with Ser.

FIG. 2 surface extension of synthetic surfactant preparation

(iii) extended kinetics of 3 wt% SP-C (LKS) (filled squares, solid line) and 3 wt% SP-C (LKS) +2 wt% SP-B (open triangles, dashed line). All formulations were measured at a concentration of DPPC/PG, 7: 3(w/w) in 150mmol/l NaCl at 10 mg/ml. The recordings were obtained on a wilhelmy balance, with each data point being the average of 3 different recordings.

FIG. 3 in vivo results

Lung-chest compliance of 5 preterm newborn rabbits (gestational age 27 days) ventilated at a normalized tidal volume of 8-10ml/kg without terminal expiratory positive pressure (PEEP). Compliance was significantly improved in treated animals. It appears that the addition of polymyxin b (pxb) increases the effect of the artificial surfactant. The phospholipid concentration was the same in all the surfactant preparations, i.e.40 mg/ml.

Sequence listing

<110>Chiesi Farmaceutici spa

<120> artificial peptide having surface activity and use thereof in the preparation of artificial surface active substances

<130>chiesi

<140>

<141>

<150>MI99A000275

<151>1999-02-12

<160>6

<170>PatentIn Ver.2.1

<210>1

<211>35

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400>1

Phe Gly Ile Pro Ser Ser Pro Val His Leu Lys Arg Leu Leu Ile Leu

1 5 10 15

Lys Leu Leu Leu Leu Lys Ile Leu Leu Leu Lys Leu Gly Ala Leu Leu

20 25 30

Met Gly Leu

35

<210>2

<211>35

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400>2

Phe Gly Ile Pro Ser Ser Pro Val His Leu Lys Arg Leu Leu Ile Leu

1 5 10 15

Leu Lys Leu Leu Leu Leu Ile Lys Leu Leu Ile Leu Gly Ala Leu Leu

20 25 30

Met Gly Leu

35

<210>3

<211>35

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400>3

Phe Gly Ile Pro Ser Ser Pro Val His Leu Lys Arg Leu Leu Ile Leu

1 5 10 15

Lys Leu Leu Leu Leu Leu Ile Leu Leu Leu Ile Leu Gly Ala Leu Leu

20 25 30

Met Gly Leu

35

<210>4

<211>35

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400>4

Phe Gly Ile Pro Ser Ser Pro Val His Leu Lys Arg Leu Leu Ile Leu

1 5 10 15

Leu Leu Leu Leu Lys Leu Ile Leu Leu Leu Ile Leu Gly Ala Leu Leu

20 25 30

Met Gly Leu

35

<210>5

<211>35

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400>5

Phe Gly Ile Pro Ser Ser Pro Val His Leu Lys Arg Leu Leu Ile Leu

1 5 10 15

Leu Leu Leu Leu Leu Leu Ile Lys Leu Leu Ile Leu Gly Ala Leu Leu

20 25 30

Met Gly Leu

35

<210>6

<211>35

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400>6

Phe Gly Ile Pro Ser Ser Pro Val His Leu Lys Arg Leu Leu Ile Leu

1 5 10 15

Phe Leu Leu Leu Leu Phe Ile Leu Leu Leu Phe Leu Gly Ala Leu Leu

20 25 30

Met Gly Leu

35

Claims (18)

1. An SP-C analogue according to the one-letter amino acid code of the general formula (I):

F_eG_fIPZZPVHLKR(X_aB)_n(X_bB)_n(X_cB)_mX_dGALLMGL (I)

wherein:

x is an amino acid selected from the group consisting of: I. l, norleucine;

b is an amino acid selected from the group consisting of: K. w, F, Y, respectively;

z is S and is optionally linked to an acyl group containing 12 to 22 carbon atoms via an ester linkage;

a is an integer of 1 to 19

b is an integer of 1 to 19

c is an integer of 1 to 21

d is an integer of 0 to 20

e is 0 or 1

f is 0 or 1

n is 0 or 1

m is 0 or 1

With the following conditions:

-n+m＞0；

-f≥e；

-(X_aB)_n(X_bB)_n(X_cB)_mX_dis a sequence having a maximum of 22 amino acids.

2. SP-C analog according to claim 1, wherein (X)_aB)_n(X_bB)_n(X_cB)_mX_dIs a sequence of 10-22 amino acids.

3. SP-C analogue according to claim 1, having formula (Ia):

(Ia)FGIPSSPVHLKRX₄BX₄BX₄BXGALLMGL。

4. SP-C analogues according to claim 1, having formula (Ib):

(Ib)FGIPSSPVHLKRX₅BX₅BX₄GALLMGL。

5. SP-C analogues according to claim 1, having formula (Ic):

(Ic)FGIPSSPVHLKRX₄BX₁₁GALLMGL。

6. SP-C analogue according to claim 1, having the formula (Id):

(Id)FGIPSSPVHLKRX₈BX₇GALLMGL。

7. SP-C analogue according to claim 1, having formula (Ie):

(Ie)FGIPSSPVHLKRX₁₁BX₄GALLMGL。

8. SP-C analogue according to any one of claims 1 to 7, wherein the Ser residue is acylated with a palmitoyl group.

9. SP-C analog according to any of claims 1 to 7, wherein B is Lys or Phe and X is Leu, Ile or Nle.

10. SP-C analogues according to claim 9, selected from:

SP-C(LKS)FGIPSSPVHLKRLLIL KLLLLKILLLKLGALLMGL

SP-C(LKS)₁FGIPSSPVHLKRLLILLKLLLLIKLLILGALLMGL

SP-C(LKS)₂FGIPSSPVHLKRLLILKLLLLLILLLILGALLMGL

SP-C(LKS)₃FGIPSSPVHLKRLLILLLLLKLILLLILGALLMGL

SP-C(LKS)₄FGIPSSPVHLKRLLILLLLLLLIKLLILGALLMGL

SP-C(LFS)FGIPSSPVHLKRLLILFLLLLFILLLFLGALLMGL。

11. a synthetic surfactant comprises at least one SP-C analogue of formula (I) in admixture with a lipid and a phospholipid.

12. The synthetic surfactant of claim 11, wherein the mixture lipid/phospholipid comprises DPPC, PG, PA.

13. A synthetic surface active substance according to any one of claims 11-12, further comprising SP-B or an analogue thereof or a polymyxin.

14. The synthetic surface-active substance according to any of claims 11 to 12, in the form of a solution, dispersion, suspension, dry powder.

15. The synthetic surface-active substance according to any of claims 11 to 12, further comprising a polymyxin.

16. The synthetic surfactant of claim 15, wherein said polymyxin is polymyxin B.

17. Use of an SP-C analogue according to any one of claims 1 to 8 for the preparation of a synthetic surfactant for the treatment of all conditions of lung surfactant deficiency or dysfunction or serous otitis media.

18. Use according to claim 17, wherein the pulmonary surfactant deficiency is respiratory distress syndrome.