HK1140420B - Reconstituted surfactants having improved properties - Google Patents

Description

Reconstituted surfactants with improved properties

The present invention relates to a reconstituted surfactant comprising a lipid carrier and a combination of a specific polypeptide analogue of the native surfactant protein SP-C and a polypeptide comprising a sequence of repeating units consisting of 3 to 8 various hydrophobic amino acid residues and one basic amino acid residue.

The invention also relates to corresponding pharmaceutical compositions and their use for the prevention and/or treatment of RDS and other respiratory diseases.

Background

The human lung is composed of a large number of small air pockets called alveoli, in which gas is exchanged between the blood and the air chambers of the lung. In healthy individuals, this exchange is mediated by the presence of a protein-containing surfactant complex that prevents lung collapse at the end of expiration.

The lung surfactant complex is composed mainly of lipids and contains a small amount of various proteins. The absence of sufficient levels of this complex leads to lung malfunction. This condition is known as respiratory distress syndrome RDS, which often affects premature infants.

The syndrome can be effectively treated with a modified natural surfactant preparation extracted from animal lungs.

Commercially available modified surfactant preparations are, for example, curosurf from porcine lung, Infasurf extracted from calf lung lavage and Survanta, a chemically modified natural bovine lung extract.

The major components of these surfactant preparations are phospholipids, such as 1, 2-dipalmitoyl-sn-glycero-3-phosphatidylcholine, commonly referred to as Dipalmitoylphosphatidylcholine (DPPC), Phosphatidylglycerol (PG), and the surfactant hydrophobins B and C (SP-B and SP-C).

Due to the disadvantages of surfactant preparations from animal tissue, such as complicated production and sterilization processes and the possibility of inducing immune responses, synthetic surfactants have been developed which mimic the composition of modified natural surfactants.

The synthetic surfactant is referred to as a reconstituted surfactant.

However, it has proven to be very complex to develop clinically effective reconstituted surfactants, since the natural hydrophobic proteins are too large to be synthesized, structurally complex and unstable in purified form.

In order to replace said natural hydrophobic proteins, some synthetic polypeptides have been proposed in the prior art which partly correspond to their sequence and analogues thereof and are disclosed, for example, in WO89/06657, WO92/22315, WO95/32992, US 6,660,833, EP 413,957, WO91/18015 and WO 00/47623.

However, according to the available literature, treatment with reconstituted surfactants in animal studies results in poor lung gas volume and alveolar patency at the end of expiration, and ventilation requires Positive End Expiratory Pressure (PEEP) in order to obtain comparable in vivo activity obtained with modified natural surfactants (Johansson J et al, J Appl Physiol 2003, 95, 2055-.

The available reconstituted surfactant formulations are virtually incapable of forming a stable phospholipid membrane in the alveoli at the end of expiration.

Thus, there is an unmet need for reconstituted surfactants with improved properties in terms of lung compliance.

In particular, there is a need for a reconstituted surfactant formulation that is capable of ensuring alveolar stability, and thus is capable of maintaining alveolar stability at the end of expiration without requiring ventilation with PEEP.

It has been found, and it is an object of the present invention, that in view of lung compliance, in particular in view of the ability to effectively maintain alveolar patency at the end of expiration without requiring ventilation by PEEP, in order to provide recombinant surfactant preparations with improved properties, specific polypeptide analogs of native SP-C protein may be advantageously combined with specific polypeptides comprising a sequence of alternating 3 to 8 various hydrophobic amino acid residues and one basic amino acid residue.

In a model of RDS wherein an immature neonate is treated with an exogenous surfactant preparation without PEEP, the combination of polypeptides acts on lung gas volume, which is an indicator of alveolar patency at the end of expiration.

Summary of The Invention

The present invention relates to a reconstituted surfactant comprising a lipid carrier and a combination of a specific polypeptide analogue of the native surfactant protein SP-C and a polypeptide comprising a sequence of repeating units consisting of 3 to 8, preferably 4 to 5, hydrophobic amino acid residues and one basic amino acid residue.

In particular, the present invention relates to a reconstituted surfactant comprising:

a) a lipid carrier;

b) a polypeptide of at least 20 amino acid residues and not more than 40 amino acid residues, having a sequence represented by the general formula (I)

F_eG_eI_fP_fS_gSPVHLKRX_aBX_bGALLΩ_pG_pL_p (I)

Wherein:

x is an amino acid residue independently selected from I, L and nL;

b is an amino acid residue independently selected from K, R, H, W, F, Y and Orn;

s is optionally substituted with an acyl group having 12 to 22 carbon atoms, preferably 16 carbon atoms, and attached to the side chain via an ester bond;

Ω is an amino acid residue selected from M, I, L and nL with M or sulfur atoms oxidized;

a is an integer having a value of 1 to 8;

b is an integer having a value of 1 to 19;

e. f, g and p are integers having a value of 0 or 1;

with the proviso that X_aBX_bIs a sequence having a maximum of 22 amino acids;

c) a polypeptide of at least 12 amino acid residues and not more than 60 amino acid residues comprising a sequence represented by the general formula (II)

B(U_aB)_x (II)

Wherein

U is an amino acid residue independently selected from L, I, nL, V, A, M and F;

b is an amino acid residue independently selected from K, R, H and Orn;

a is an integer having a value of 3 to 8, preferably 4 to 5; and is

x is an integer having a value of 2 to 6, preferably 3 to 4.

The invention also includes pharmaceutically acceptable salts of the polypeptides and their N-and/or C-terminally blocked derivatives, e.g. by acetylation and amidation.

The invention also provides pharmaceutical compositions comprising the reconstituted surfactants of the invention.

The invention further relates to the use of the aforementioned reconstituted surfactant as a medicament.

According to another aspect, the present invention relates to the use of the aforementioned reconstituted surfactants for the prevention and/or treatment of Respiratory Distress Syndrome (RDS), diseases involving surfactant deficiency or dysfunction and other respiratory diseases in preterm infants.

Yet another aspect of the present invention relates to a method for the prevention and/or treatment of Respiratory Distress Syndrome (RDS), other diseases involving surfactant deficiency or dysfunction and other respiratory diseases in premature infants, said method comprising administering an effective amount of a reconstituted surfactant as described hereinbefore.

Drawings

FIG. 1 shows the amino acid sequence of the human protein SP-C. Cys residues in native SP-C are palmitoylated.

Figure 2 shows a helical wheel representation of the polypeptide KL 4. The basic amino acid residue is rounded off.

Fig. 3 shows the results in terms of lung gas volume (ml/kg).

Figure 4 shows the results in terms of tidal volume (ml/kg) as a function of time/pressure.

Definition of

Respiratory function following in vivo treatment with exogenous surfactant preparations was performed by measuring two parameters:

i) tidal volume, an indicator of lung compliance, and

ii) lung gas volume, is an indicator of the degree of alveolar air expansion or patency at the end of expiration, and thus of the ability of the end of expiration to form a stable lecithin film in the alveoli.

As used herein, an effective amount for treating a particular disease is an amount sufficient to alleviate, or in some way alleviate, the symptoms associated with the disease. The amount will depend on the kind and severity of the disease and the condition of the patient (weight, sex, age).

As used herein, the term "reconstituted surfactant" means a lipid carrier to which a polypeptide analog of a surfactant protein prepared by recombinant techniques or synthetic methods has been added.

As used herein, the term "lipid carrier" means a mixture of phospholipids and optionally other lipid components, e.g. neutral lipids such as triacylglycerols, free fatty acids and/or cholesterol.

As used herein, the term "polypeptide analog of the native surfactant protein SP-C" includes polypeptides comprising an amino acid sequence in which one or more amino acids have been lost or replaced with other amino acids as compared to the native protein, so long as the polypeptide, in admixture with a lipid carrier, exhibits pulmonary surfactant activity.

The amino acid sequence is shown according to the three-letter code, with the amino acid with the free amino group on the left (amino-terminal end) and the amino acid with the free carboxyl group on the right (carboxyl-terminal end).

All amino acid residues identified herein are in the native L-configuration, and the sequences identified herein are recorded according to the standard abbreviations for the amino acid residues shown in the following control tables.

Comparison table

Amino acids Symbol

One letter Three letters

Glycine G Gly

L-proline P Pro

L-isoleucine I Ile

L-leucine L Leu

L-tyrosine Y Tyr

L-cysteine C Cys

L-Tryptophan W Trp

L-alanine A Ala

L-lysine K Lys

L-arginine, R Arg

L-Glutamine Q Glu

L-methionine M Met

L-serine S Ser

L-valine V Val

L-asparagine N Asn

L-aspartic acid D Asp

L-glutamic acid E Glu

L-histidine H His

L-threonine T Thr

L-phenylalanine F Phe

L-norleucine-nLeu

L-Ornithine-Orn

Detailed Description

The present invention relates to a reconstituted surfactant comprising a lipid carrier and a combination of a polypeptide of general formula (I) and a polypeptide comprising a sequence of repeating units consisting of 3 to 8, preferably 4 or 5, hydrophobic amino acid residues and one basic amino acid residue.

We have now found with certainty that in an RDS model in which immature neonates are treated with exogenous surfactant preparations without PEEP, the polypeptide of formula (I), in combination with the polypeptide of formula (II), acts favorably on lung gas volume, which is an indicator of alveolar patency at the end of expiration.

The claimed reconstituted surfactant formulation is capable of improving respiratory function, as expressed by tidal volume, to a degree comparable to that achieved following administration of the modified natural surfactant.

Advantageously, the analogue of the native protein SP-C is a polypeptide having at least 20 amino acid residues and not more than 40 amino acid residues and having a sequence represented by the general formula (I)

F_eG_eI_fP_fS_gSPVHLKRX_aBX_bGALLΩ_pG_pL_p (I)

Wherein:

x is an amino acid residue independently selected from I, L and nL;

b is an amino acid residue independently selected from K, R, H, W, F, Y and Orn;

s is optionally substituted with an acyl group having 12 to 22 carbon atoms, preferably 16 carbon atoms, and attached to the side chain via an ester bond;

Ω is an amino acid residue selected from M, I, L and nL with M or sulfur atoms oxidized;

a is an integer having a value of 1 to 8;

b is an integer having a value of 1 to 19;

e. f, g and p are integers having a value of 0 or 1;

with the proviso that X_aBX_bIs a sequence having a maximum of 22 amino acids.

Preferably, the polypeptide of formula (I) consists of at least 30 and not more than 35 amino acids, more preferably not more than 33 amino acids.

In particular embodiments, the polypeptide of formula (I) consists of 30 or 33 or 35 amino acids.

Preferably, the polypeptide analogue of the SP-C protein is represented by the general formula (Ia) wherein e and n are 0 and g is 1

I_fP_fSSPVHLKRX_aBX_bGALLΩ_pG_pL_p (Ia)

Wherein

X, B and Ω are as described above;

a is 1;

b is 14;

f and p are 0 or 1.

More preferably, the polypeptide analogue of the SP-C protein is represented by the general formula (Ib), wherein f is 1

IPSSPVHLKRX_aBX_bGALLΩ_pG_pL_p (Ib)

Wherein:

x, B, Ω, a, and b are as described above;

p is 0 or 1.

Even more preferably, the polypeptide analogue of the SP-C protein is represented by the general formula (Ic)

IPSSPVHLKRLKLLLLLLLLILLLILGALLΩ_pG_pL_p (Ic)

Wherein:

Ω is as described above

p is 0 or 1.

Examples of polypeptides of general formula (Ic) are described below:

IPSSPVHLKRLKLLLLLLLLILLLILGALLMGL (Id) (SEQ ID NO：1)

IPSSPVHLKRLKLLLLLLLLILLLILGALLIGL (Ie) (SEQ ID NO：2)

IPSSPVHLKRLKLLLLLLLLILLLILGALLLGL (If) (SEQ ID NO：3)

IPSSPVHLKRLKLLLLLLLLILLLILGALLnLGL (Ig) (SEQ ID NO：4)

IPSSPVHLKRLKLLLLLLLLILLLILGALL (Ih) (SEQ ID NO：5)

the polypeptide (Id) is also known in the art as SP-C33.

Most preferably, the SP-C analogue is a polypeptide selected from the group of polypeptides having the general formulae (Ie), (Ig) and (Ih).

Most preferred polypeptides have the general formula (If).

Advantageously, the polypeptide comprising a sequence of alternating between 3 and 8 hydrophobic amino acid residues and one basic amino acid residue of the sequence consists of at least 12 amino acid residues and not more than 60 amino acid residues, preferably of at least 20 and not more than 35, and is represented by the general formula (II)

B(U_aB)_x (II)

Wherein

U is an amino acid residue independently selected from the group consisting of L, I, nL, V, A, M and F;

b is an amino acid residue independently selected from K, R, H and Orn;

a is an integer having a value of 4 to 5, preferably 4; and

x is an integer having a value of 3 to 4, preferably 4.

A preferred group of polypeptides comprises or consists of the sequence wherein U is L or I, B is K or R, a is 4 or 5, preferably 4, and x is 3 or 4, preferably 4.

A particularly preferred polypeptide is that known in the art as KL₄And a polypeptide having a sequence described below

KLLLLKLLLLKLLLLKLLLLK (KL₄) (SEQ ID NO：6)

Another example of a particularly preferred polypeptide is a polypeptide having the sequence set forth below, herein designated KL₅The polypeptide of (1):

KLLLLLKLLLLLKLLLLLKLLLLLK (KL₅) (SEQ ID NO：7)

indeed KL has been found₅Shows that the charged residues are distributed around the entire helical circumference of the peptide, and KL₄Similarly.

Further examples of preferred polypeptides are those having the sequence (III):

FGIPSSPVHLKBX4BX4BX4BLGALLMGL(III)

an example of a polypeptide of formula (IIIIa) is the polypeptide known in the art as SP-C (LKS)

FGIPSSPVHLKRLLILKLLLLKILLLKLGALLMGL[SP-C(LKS)](SEQ IDNO：8)

The polypeptides of formulae (I) and (II) may be prepared by any known technique, such as those described in J.M.Steward and J.D.Young, "Solid phase peptide Synthesis", W.H.Freeman Co., san Francisco, 1969, and J.Meienhofer, Hormonal Proteins and Peptides, Vol.2, p.46, Academic Press (New York), 1983, for Solid phase peptide Synthesis, and E.Schroder and K.Kubke, "The Peptides", Vol.1, Academic Press (New York), 1965, for traditional solution Synthesis. An overview of polypeptide Synthesis techniques can be found in J.Stuart and J.D.Young, Solid Phase Peptide Synthesis, Pierce chemical company, Rockford, IL, 3 rd edition, Neurath, H. et al, p.104-237, Academic Press, New York, NY (1976).

Suitable protecting groups for use in these syntheses are to be found in the above references as well as in J.F.W.McOmie, protective groups in Organic Chemistry, Plenum Press, New York, NY (1973).

Generally, these methods involve the sequential addition of one or more amino acid residues or appropriately protected amino acid residues to a growing peptide chain. Typically, the amino or carboxyl group of the first amino acid residue is protected by a suitable, optionally removable, protecting group.

Using solid phase synthesis as an example, protected or derivatized amino acids are attached to an inert solid support through their unprotected carboxy or amino groups. The amino-or carboxy-protecting group is then selectively removed and the next amino acid in the sequence with the complementary group (amino or carboxy) appropriately protected is mixed and reacted under conditions suitable to form an amide bond with the residue already attached to the solid support. The amino-or carboxy-protecting group is then removed from this newly added amino acid residue, followed by the addition of the next (appropriately protected) amino acid, and so on.

After all desired amino acids are linked in the correct order, any remaining terminal and side-group protecting groups (and solid support) are removed sequentially or simultaneously to obtain the final polypeptide.

In particular, the polypeptides of the general formulae (I) and (III) can be prepared according to the process disclosed in WO00/47623, whereas the polypeptide consisting of the sequence of the general formula (II) can be prepared according to the process described in WO 92/22315.

The reconstituted surfactants of the invention can be prepared by mixing a solution or suspension of the polypeptides of general formulae (I) and (II) with a lipid, followed by drying the mixture, and in addition, they can be prepared by lyophilization or spray drying.

Preferably, the polypeptide of formula (I) and the polypeptide of formula (II) are present in the reconstituted surfactant of the invention as a fixed combination in fixed amounts and quantitative ratios.

The ratio of polypeptides of formulae (I) and (II) relative to the reconstituted surfactant may vary. Advantageously, each polypeptide may be present in an amount of 0.5 to 10% (w/w), preferably 1 to 5%, most preferably 1 to 3%, based on the weight of the surfactant.

Advantageously, the lipid carrier comprises phospholipids contained in natural lung surfactant preparations, such as Phosphatidylcholine (PC), such as Dipalmitoylphosphatidylcholine (DPPC) and palmitoyl oleoyl-phosphatidylcholine (POPC), and Phosphatidylglycerol (PG), such as Palmitoyl Oleoyl Phosphatidylglycerol (POPG) and dipalmitoyl phosphatidylglycerol (DPPG).

Other phospholipids that may be advantageously used are Phosphatidylinositol (PI), Phosphatidylethanolamine (PE), phosphatidylserine and Sphingomyelin (SM).

In particular embodiments, the lipid carrier may include further components, for example neutral lipids such as triacylglycerols, free fatty acids and/or cholesterol.

Advantageously, the reconstituted surfactant according to the invention contains 90 to 99% by weight of the lipid carrier, preferably 92 to 98%, more preferably 94 to 96%, and 1 to 10% by weight of the sum of the two peptides, preferably 2 to 8%, more preferably 4 to 6%.

In a particular embodiment of the invention, the reconstituted surfactant comprises 96% by weight of a lipid carrier, 2% by weight of a polypeptide of formula (I) and 2% by weight of a polypeptide of formula (II).

The phospholipid comprised in the lipid carrier preferably consists of DPPC and a palmitoyl oleoyl phospholipid selected from POPG or a mixture thereof with POPC in a weight ratio of 95: 5 to 50: 50, preferably 80: 20 to 60: 40. The weight ratio of DPPC to POPG is preferably in the range of 75: 25 to 65: 35, more preferably 68: 31. In the case of DPPC: POPG: POPC mixtures, the phospholipids are preferably used in a weight ratio of 60: 20 or 68: 15: 16.

In a preferred embodiment, the reconstituted surfactant comprises 1 to 5% by weight of the polypeptide of formula (Ia), 1 to 5% by weight of the polypeptide of formula (II) and a mixture of DPPC and POPG in a weight ratio of 68: 31.

In a more preferred embodiment, the reconstituted surfactant comprises 1.5 to 3% by weight of a polypeptide selected from (Ie), (If), (Ig) and (Ih), more preferably of polypeptide (If), 1.5 to 3% by weight of a peptide of formula (II) and a mixture of DPPC and POPG in a weight ratio of 68: 31.

Administration of the reconstituted surfactant of the invention is carried out routinely, preferably by intratracheal instillation (infusion or bolus) or by nebulization.

The effective amount of the reconstituted surfactant will vary depending on a number of different factors, including the mode of administration, the type and severity of the disease, and whether the treatment is prophylactic or therapeutic. In general, the dosage is from 0.01mg to 10g/kg body weight, preferably from 10 to 500mg/kg, more preferably from 40 to 200 mg/kg. Optimal dosages and dosing frequency can be readily determined by those skilled in the art.

The invention also relates to pharmaceutical compositions containing the reconstituted surfactants of the invention. The compositions are advantageously administered in the form of solutions, dispersions, suspensions or dry powders. Preferably, the composition comprises a reconstituted surfactant dissolved or suspended in a suitable solvent or resuspension medium.

Preferably, the pharmaceutical composition is supplied for use as a suspension in a buffered saline solution in a single use glass vial. Advantageously, the concentration of reconstituted surfactant (expressed as phospholipid content) is in the range of about 2 to about 160mg of active agent/ml, preferably 10 to 100mg/ml, more preferably 20 to 80 mg/ml.

To achieve a lower viscosity, the composition may further contain an electrolyte, such as calcium, magnesium and/or sodium salts (e.g., calcium chloride or sodium chloride).

The pharmaceutical composition according to the present invention is suitable for the prevention and/or treatment of Respiratory Distress Syndrome (RDS) or other diseases associated with surfactant deficiency or dysfunction in premature infants, including Acute Lung Injury (ALI), adult RDS (ards), fetal fecal aspiration syndrome (MAS), and bronchopulmonary dysplasia (BPD).

They can also be used for the prophylaxis and/or treatment of other respiratory diseases, such as pneumonia, bronchitis, COPD (chronic obstructive pulmonary disease), asthma and cystic fibrosis, and for the treatment of serous otitis media (glue ear).

The following examples illustrate the invention in more detail.

Examples

Use of the polypeptide SP-C33 and KL₄In vivo experiments with reconstituted surfactants.

The surfactant formulations were tested in preterm neonatal rabbits obtained by hysterectomy during the 27 day pregnancy. Experiments were performed without the use of Positive End Expiratory Pressure (PEEP).

As an analogue of SP-C, a polypeptide designated SP-C33 prepared as described in WO00/47623 was used.

As an analogue of the protein SP-B, the protein KL was used₄According to the teachings of WO 92/22315.

The animals are treated at birth with 200mg/kg (80mg/kg) of a reconstituted surfactant preparation containing 2% SP-C33 or 2% SP-C33+ 2% KL₄Combined with a phospholipid mixture consisting of DPPC: POPG in a 68: 31w/w ratio.

By using(80mg/ml) treated animals served as positive controls, while the untreated litters served as negative controls.

Immature neonatal rabbits were ventilated in parallel with a standardized sequence of peak insufflation pressures. To open the lungs, the pressure was first set to 35cm H₂O, for 1 minute. After this recovery operation, the pressure was reduced to 25cm H₂O, for 15 min, and further down to 20 and 15cm H₂And O. Finally, the pressure was again raised to 25cm H₂O, for 5 minutesThereafter, the lungs were re-inflated with nitrogen for 5 minutes, and then excised for gas volume measurement.

The lung gas volume and tidal volume, both expressed in ml/kg, were measured and the results given as median values are set forth in fig. 4 and 5, respectively.

FIG. 3 shows the use of a reagent containing 2% w/w SP-C33 and 2% KL₄The animals treated with the combined reconstituted surfactant formulation had higher lung gas capacity than the animals receiving 2% w/w SP-C33, indicating that 2% w/w KL was administered₄Addition to the SP-C33 surfactant produced an increase in lung gas volume.

The results demonstrate that the reconstituted surfactant of the invention provides better stabilization of the phospholipid membrane in the alveoli at the end of expiration than reconstituted surfactant formulations containing only the protein SP-C analogue.

Furthermore, figure 4 demonstrates that the claimed reconstituted surfactant formulation can significantly improve respiratory function as expressed in tidal volume.

Claims (13)

1. A reconstituted surfactant comprising a lipid carrier and a combination of an analogue of native surfactant protein SP-C and a polypeptide represented by sequence KLLLLKLLLLKLLLLKLLLLK (SEQ ID NO: 6), wherein the polypeptide analogue of native surfactant protein SP-C is represented by the following general formula

IPSSPVHLKRLKLLLLLLLLILLLILGALLΩ_pG_pL_p

Wherein:

Ω is an amino acid residue selected from M, I, L and nL with M or sulfur atoms oxidized; and is

p is 1.

2. A reconstituted surfactant according to claim 1 wherein the analogue of native surfactant protein SP-C is selected from the following polypeptides:

IPSSPVHLKRLKLLLLLLLLILLLILGALLMGL(Id)(SEQ ID NO：1)

IPSSPVHLKRLKLLLLLLLLILLLILGALLIGL(Ie)(SEQ ID NO：2)

IPSSPVHLKRLKLLLLLLLLILLLILGALLLGL(If)(SEQ ID NO：3)

IPSSPVHLKRLKLLLLLLLLILLLILGALLnLGL(Ig)(SEQ ID NO：4)。

3. a reconstituted surfactant according to claim 1 or 2 wherein the lipid carrier comprises a mixture of phospholipids.

4. A reconstituted surfactant according to claim 3 wherein the phospholipid mixture consists of Dipalmitoylphosphatidylcholine (DPPC) and a palmitoyl oleoyl phospholipid selected from Palmitoyl Oleoyl Phosphatidylglycerol (POPG) or consists of a mixture of dipalmitoyl phosphatidylcholine (DPPC) and Palmitoyl Oleoyl Phosphatidylglycerol (POPG) and Palmitoyl Oleoyl Phosphatidylcholine (POPC) in a weight ratio of 95: 5 to 50: 50.

5. A reconstituted surfactant according to claim 4 wherein the phospholipid mixture consists of DPPC and POPG in a weight ratio of 68: 31.

6. A pharmaceutical composition comprising a reconstituted surfactant according to any one of claims 1 to 5.

7. The pharmaceutical composition according to claim 6, in the form of a dispersion or a dry powder.

8. The pharmaceutical composition according to claim 7, wherein the dispersion is a solution or a suspension.

9. The pharmaceutical composition according to claim 8, wherein the suspension is an aqueous suspension.

10. A pharmaceutical composition according to claim 7 or 8, containing a reconstituted surfactant at a concentration of 2 to 160 mg/ml.

11. The pharmaceutical composition according to claim 10, wherein the concentration of the reconstituted surfactant is from 20 to 80 mg/ml.

12. Use of a reconstituted surfactant according to any one of claims 1 to 5 in the manufacture of a medicament for the prevention and/or treatment of Respiratory Distress Syndrome (RDS) in premature infants or the treatment or prevention of other disorders associated with surfactant deficiency or dysfunction.

13. The use according to claim 12, wherein the disorders associated with surfactant deficiency or dysfunction include Acute Lung Injury (ALI), adult rds (ards), fetal fecal aspiration syndrome (MAS), and bronchopulmonary dysplasia (BPD).