RS20170169A1 - Improved reconstituted surfactant composition containing analogs of surfactant protein b (sp-b) and surfactant protein c (sp-c) - Google Patents

Abstract

The present invention relates to a reconstituted pulmonary surfactant containing a lipid carrier, a combination of an analog of the native surfactant protein SP-C polypeptide with a particular native surfactant polypeptide analog of protein SP-B. The invention also relates to pharmaceutical formulations thereof and to their use for the treatment or prophylaxis of RDS and other respiratory disorders.

Description

PROTEIN B SURFACTANTS (SP-B) AND PROTEIN C SURFACTANTS (SP-C)

TECHNICAL FIELD

The present invention relates to a reconstituted lung surfactant that is suitable for prophylaxis and/or treatment of respiratory distress syndrome (RDS) and other respiratory disorders.

BACKGROUND OF THE INVENTION

Human lungs consist of a large number of small air sacs, called alveoli, where gases are exchanged between the blood and the air spaces of the lungs. In healthy individuals, this exchange is mediated by the presence of a protein-containing surfactant complex that prevents the lungs from collapsing at the end of expiration.

The lung surfactant complex is composed primarily of lipids and contains smaller amounts of various proteins. The lack of adequate levels of this complex results in the malfunctioning of the lungs. This syndrome is called Respiratory Distress Syndrome (RDS) and it usually affects newborns.

The above syndrome is effectively treated with commercially available preparations of modified natural surfactant extracted from the lungs of animals, such as the gold standard preparation known as Curosurf.

The main ingredients of these surfactant preparations are phospholipids and surfactant hydrophobic proteins B and C (SP-B and SP-C).

Due to the disadvantages of surfactant preparations from animal tissue, such as the complication of the production process, and possible viral contamination and/or induction of immune reactions, synthetic surfactants are available in the art.

Said synthetic surfactants may simply be mixtures of synthetic compounds, primarily phospholipids and other lipids, and are known as "artificial" surfactants; although they have been used in clinical practice for many years, their effectiveness is not comparable to that of modified natural surfactant.

Artificial surfactants also containing surfactant proteins/peptides are currently under development. These are called either "reconstituted" surfactants or "bio-mimetic surfactants".

However, according to the available literature, none of the reconstituted surfactants developed have so far demonstrated efficacy in terms of lung compliance comparable to that of surfactants extracted from animals. Furthermore, they lead to depletion of lung volume and end-expiratory alveolar patency, and positive end-expiratory pressure (PEEP) ventilation is required to achieve in vivo activity comparable to that achieved with modified natural surfactants (Johansson J et al J Appl Physiol 2003, 95, 2055-2063; Davis AJ et al Am J Respir Crit Care Med 1998; 157, 553-559).

profile of modified natural surfactants because they contain only one protein (peptide) component.

On the other hand, in addition to good efficiency, surfactant compositions should also show a low viscosity to allow the preparation of a concentrated suspension in an aqueous medium. The ability to prepare a concentrated suspension in a small volume is indeed a feature of particular importance for their use in very low birth weight infants.

The peptide:phospholipid system is a rather complex mixture whose properties largely depend on the composition of the phospholipid mixture, as well as on the specific combination of phospholipids/peptides.

The presence of more peptides will affect the rheological properties of the composition, making the system even more complicated.

In the prior art, reconstituted surfactants contain SP-B and SP-C analogs that resemble human surfactant proteins disclosed, for example, in WO0076535, WO2008011559 and WO2008044109.

However, none of the compositions disclosed herein have been shown to possess optimal properties in terms of lung compliance as well as rheological properties, particularly viscosity.

Therefore, there is still a need for reconstituted preparations that have comparable efficacy to that of surfactants extracted from animal lungs, as well as optimal rheological properties enabling their easy delivery and distribution-distribution in the bronchoalveolar part of the lung after administration.

The present invention addresses that issue by providing a reconstituted surfactant composition with improved lung compliance and viscosity properties.

SUMMARY OF THE INVENTION

The present invention relates to a reconstituted surfactant containing:

a) from 1.2 to 1.8% by weight of the polypeptide analog of the native surfactant protein SP-C, which consists of the sequence represented by the formula IPSSPVHLKRLKLLLLLLLLILLLILGALLLLGL (SEQ ID NO:1);

b) from 0.1 to 0.5% by weight of the polypeptide analog of the native surfactant protein SP-B consisting of the sequence represented by the formula CWLCRALIKRIQALIPKGGRLLPQLVCRLVLRCS (SEQ ID NO:2); monounsaturated and saturated phospholipid in a weight ratio ranging from 45:55 to 55:45;

all amounts are calculated relative to the total weight of reconstituted surfactant.

The present invention also provides pharmaceutical compositions comprising the claimed reconstituted surfactant alone or in combination with one or more pharmaceutically acceptable

carrier.

The present invention also provides the use of the reconstituted surfactant as a medicament for which it is

protection requested.

In a further aspect, the invention provides the use of a reconstituted surfactant as claimed

protection for the prophylaxis and/or treatment of respiratory distress syndrome (RDS) and other respiratory disorders.

In addition, the invention provides the use of the claimed reconstituted surfactant for the manufacture of a medicament for the prophylaxis and/or treatment of respiratory distress syndrome (RDS) and other respiratory disorders.

The invention also provides a method for the prophylaxis and/or treatment of respiratory distress syndrome (RDS) and other respiratory disorders, said method comprising administering to a patient in need

such treatment with a therapeutically effective amount of the reconstituted surfactant mentioned above.

In a further embodiment, the present invention provides a method comprising administering a diluted reconstituted surfactant of the present invention to the lung via a lavage technique to remove

harmful material and/or inflammatory exudate, to expand the lungs and improve lung function.

reconstituted surfactant in the form of powder, in the form of the first unit dose; b) a pharmaceutically acceptable carrier, in the form of a second unit dose; and c) a container of means for holding the specified forms of the first and second doses.

DEFINITIONS

"Surfactant activity" for a surfactant preparation is defined as the ability to reduce surface tension.

The in vitro effectiveness of exogenous surfactant preparations is usually tested by measuring its ability to lower surface tension using a suitable apparatus such as the so-called "Wilhelmy Balance" and "Captive Bubble Surfactometer".

The in vivo efficacy of exogenous surfactant preparations is usually tested by measuring two parameters:

i) respiratory volume, which is an index of lung compliance and

ii) the volume of air in the lungs, which is an index of air expansion in the alveoli or patency at the end of exhalation, and therefore the ability to form a stable film of phospholipids in the alveoli at the end of exhalation.

A "therapeutically effective" amount as used herein refers to an amount of reconstituted surfactant capable of preventing, avoiding, reducing, or eliminating a respiratory disease or disorder associated with endogenous surfactant deficiency or dysfunction.

The term "pharmaceutically acceptable" or "physiologically tolerable" refers to compositions, media, solvents, salts capable of being administered to humans without producing undesirable physiological effects.

The term "native surfactant protein SP-C polypeptide analog" means a polypeptide having an amino acid sequence in which, compared to the native SP-C protein, amino acids are missing and/or replaced with other amino acids, so long as the polypeptide in mixture with phospholipids exhibits pulmonary surfactant activity (as demonstrated by in vitro and in vivo efficacy tests).

The term "polypeptide analog of the native surfactant protein SP-B", means a polypeptide having an amino acid sequence in which, compared to the native SP-B protein, amino acids are missing and/or replaced with other amino acids, so long as the polypeptide in admixture with phospholipids exhibits lung surfactant activity (as demonstrated by in vitro and in vivo efficacy tests).

"Recombinant", when used with reference, eg, to a cell, or nucleic acid, protein or vector, indicates that the cell, nucleic acid, protein or vector has been modified by introducing a heterologous nucleic acid or protein, or by altering the native nucleic acid or protein.

As used herein, the term "about" applied to the indicated value, indicates a variability of ± 1%.

Phospholipids are lipids in which one fatty acid has been replaced by a phosphate group and a simple organic molecule. The most common class of phospholipids that can be found in surfactant preparations are: phosphatidylcholines (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylinositol (PI), and phosphatidylserine (PS).

Glycerol residues from phospholipids are generally esterified with long-chain fatty acids, which in turn may be saturated (eg, myristic, palmitic, and stearic acids), monounsaturated (eg, oleic acid), or polyunsaturated (eg, linoleic and arachidonic acids).

Specifically, the types considered in the application are:

, , ,

saturated derivative, and 1-palmitoyl-2-oleyl-sn-glycero-3-phosphoglycerol, also known as palmitoyl-oleylphosphatidylglycerol (POPG), which is a monounsaturated derivative.

"Polypeptide", "peptide" and "protein" are used interchangeably herein and refer to a polymer of amino acid residues. _

Amino acid sequences are shown according to a single letter code with an amino acid bearing a free amino group at the left end (amino terminus) and an amino acid bearing a free carboxyl group at the right end (carboxy terminus).

All amino acid residues are identified here in the natural

The L-configuration and sequences identified herein are shown according to standard abbreviations for amino acid residues. For the avoidance of doubt, the amino acid derivative L-nor-leucine is designated herein as nLeu i

L-ornithine is abbreviated as Om.

PICTURES

Figure 1 shows the results in terms of tidal volumes (ml/kg) as a function of time/pressure of the reconstituted surfactant of the invention (N3) versus Curosurf® and untreated animals (n = number of animals).

Figure 2 shows the results in terms of lung air volume (ml/kg) of the reconstituted surfactant of the present invention (N3) versus Curosurf® and untreated animals (n = number of animals).

DETAILED DESCRIPTION OF THE INVENTION

An experimental multi-factorial design was constructed to investigate how viscosity is influenced by the relative amounts of phospholipids and SP-B and SP-C analogues in the surfactant compositions generically disclosed in WO 2008044109, as well as how these components interact with each other in reconstituted surfactants.

It was found that the amount of SP-B analogue has a significant effect on the viscosity of the surfactant composition, and thus that the SP-B analogue content should be kept as low as possible compatible with therapeutic efficiency, in order to reduce the viscosity of the reconstituted surfactant to acceptably low levels.

WO 2008044109 relates to the use of a DPPC:POPG ratio preferably equal to or greater than 7:3, in the presence of an SP-B analog.

WO 2008044109 does not discuss the viscosity of such surfactant compositions. It has now been found that when the amount of SP-B analog in the surfactant is minimized, a high ratio between desaturated phospholipids such as DPPC and unsaturated phospholipids such as POPG leads to an unacceptably high viscosity of the respective composition. However, this problem has been overcome by the preparation of) low SP-B surfactants in which the ratio of unsaturated phospholipids to saturated phospholipids is reduced below the ratio described in the prior art, preferably around 1:1, and these surfactant compositions have been shown to have surprisingly low viscosity, making them useful for all clinical applications.

In one embodiment, the present invention relates to a reconstituted surfactant containing:

a) from 1.2 to 1.8% by weight of the polypeptide analog of the native surfactant protein SP-C, which consists of the sequence represented by the formula IPSSPVHLKRLKLLLLLLLLILLLILGALLLLGL (SEQ ID NO:1);

b) from 0.1 to 0.5% by weight of the polypeptide analog of the native surfactant protein SP-B, which consists of the sequence represented by the formula CWLCRALIKRIQALIPKGGRLLPQLVCRLVLRCS (SEQ ID NO:2); monounsaturated and saturated phospholipid in a weight ratio ranging from 45:55 to 55:45;

all amounts are calculated relative to the total weight of reconstituted surfactant.

between 1.4 and 1.6%, and more preferably about 1.5% by weight of the total weight of the reconstituted surfactant. In the reconstituted surfactant of the present invention, the SP-B protein analog component preferably comprises between 0.2 and 0.4%, and more preferably about 0.2% by weight of the total weight of the reconstituted surfactant. The reconstituted surfactant of the present invention may advantageously include saturated phospholipids, such as dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylglycerol (DPPG), and monounsaturated phospholipids such as palmitoyloleoylphosphatidylcholine (POPC) and palmitoyloleoylphosphatidylglycerol (POPG).

A preferred saturated phospholipid for use in accordance with the present invention is DPPC, while a preferred monounsaturated phospholipid is POPG.

Advantageously, the combined weight of phospholipids constitutes at least 90%, preferably at least 95%, preferably at least 97.7%, more preferably 98.3% of the total weight of the reconstituted surfactant.

In one embodiment, the present invention relates to a reconstituted surfactant containing:

a) about 1.5% by weight of the polypeptide analog of the native surfactant protein SP-C consisting of the sequence represented by the formula IPSSPVHLKRLKLLLLLLLLILLLILGALLLLGL (SEQ ID NO:1);

b) about 0.2% by weight of the polypeptide analog of the native surfactant protein SP-B consisting of the sequence represented by the formula CWLCRALIKRIQALIPKGGRLLPQLVCRLVLRCS (SEQ ID NO:2);

c) 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1-palmitoyl-2-oleyl-sn-glycero-3-phosphoglycerol (POPG) in a weight ratio of about 50:50;

all amounts are calculated relative to the total weight of reconstituted surfactant.

In one specific embodiment, the present invention relates to a reconstituted surfactant composition that includes:

a) about 1.5% by weight of the polypeptide analog of the native surfactant protein SP-C consisting of the sequence represented by the formula IPSSPVHLKRLKLLLLLLLLILLLILGALLLLGL (SEQ ID NO:1);

b) about 0.2% by weight of the polypeptide analog of the native surfactant protein SP-B consisting of the sequence represented by the formula CWLCRALIKRIQALIPKGGRLLPQLVCRLVLRCS (SEQ ID NO:2);

c) about 49.15% by weight of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC); and

d) about 49.15% by weight of 1-palmitoyl-2-oleyl-sn-glycero-3-phosphoglycerol (POPG);

all amounts are calculated relative to the total weight of reconstituted surfactant.

In a preferred embodiment, the polypeptide of SEQ. ID BR:2 can be in the form of a disulfide linked molecule, whereby the intramolecular disulfide bonds are between two cysteine residues at positions 1- and 33- and/or between two cysteine residues at positions 4- and 27-.

Polypeptide of SEQ. ID NO:1 is listed in WO 2008044109 (incorporated herein in its entirety by reference) as SP-C33(Leu), while polypeptide SEQ. ID NO:2 is listed in WO 2008044109 as Mini-B(Leu), and its disulfide linked form as ox Mini-B(Leu).

In an even more preferred embodiment, the reconstituted surfactant of the present invention essentially consists of components a), b), c), and optionally d) in amounts for which protection is sought.

include, for example, salts of hydrochloric acid, acetic acid, and trifluoroacetic acid.

Preferably, both polypeptides are present in the composition in the form of acetate.

Also POPG may advantageously be present in the form of pharmaceutically acceptable salts, for example as the sodium salt (POPG Na).

Polypeptides from SEQ. ID NO:1 and SEC. ID BR:2 can be prepared according to synthetic procedures or recombinant techniques well known to a person skilled in the art.

An excellent summary of the many techniques available can be found 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 classical solution synthesis.

Polypeptides may preferably be prepared using the solid phase synthetic technique originally described by Merrifield, in J. Am. Chem. Soc. 85: 2149-2154 (1963). Other polypeptide synthesis techniques can be found, for example, in M. Bodanszky et al., Peptide Synthesis, John Wiley & Sons, 2d Ed., (1976) as well as other references known to those skilled in the art.

Suitable protecting groups for use in such synthesis will be found in the texts cited above, as well as in J.F.W. McOmie, Protective Groups in Organic Chemistry, Plenum Press, New York, NY (1973).

For example, both polypeptides can be prepared as described in WO2008044109.

Effective doses of the reconstituted surfactant of the present invention for the treatment of a disease, such as RDS, as described herein, vary with many different factors, including the type of disease, route of administration, weight and physiological condition of the patient, and whether the treatment is prophylactic or therapeutic.

In general, the dose consists of 0.01 mg to 10 g per kg of body weight, preferably from 0.1 to 1 g per kg of body weight, and the frequency of administration may vary depending on whether the treatment is prophylactic or therapeutic. Usually a dose of about 50 mg/kg, 100 mg/kg or 200 mg/kg is administered in a single dose. For use in infants, one or two applications are generally sufficient.

Although needs may vary, depending on the severity of the respiratory disease and/or other variables, determining the optimal ranges for effective doses is within the skill of one skilled in the art. The present invention also relates to pharmaceutical formulations containing the reconstituted surfactant of the present invention.

The mentioned formulations are given primarily in the form of solution, dispersion, suspension or dry powder. Preferably said compositions contain reconstituted surfactant dissolved or suspended in a suitable physiologically tolerant solvent or carrier resuspension, such as water or physiological saline aqueous solution (0.9% w/v NaCl).

The formulations of the present invention may contain aqueous solutions, preferably sterile, which may also contain pH buffering agents, diluents and other suitable additives.

Formulations may be presented in unit-dose or multiple-dose containers, for example in hermetically sealed ampoules and vials, and may be stored in frozen or freeze-dried so-called eva pods immediately prior to use.

Preferably, the reconstituted surfactant of the present invention is supplied as a sterile suspension in a buffered physiological saline solution in single-use glass vials.

Pharmaceutical formulations may be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of mixing polypeptides and phospholipids in the presence of an organic solvent. The solvent is then removed by dialysis or evaporation under nitrogen and/or exposure to vacuum or other suitable techniques well known to those skilled in the art, such as lyophilization.

Advantageously, the amount of residual solvent may be less than 0.1%, preferably less than 0.05%, more preferably less than 0.003%, even more preferably less than 0.001% by weight.

The resulting powder is then uniformly and intimately brought into contact with liquid carriers or finely divided solid carriers or both.

The mixture of polypeptides and phospholipids can be sterilized before removing the solvent, for example by sterile filtration. In other certain embodiments, the reconstituted surfactant composition is terminally sterilized according to procedures well known in the art.

Administration of the reconstituted surfactant of the invention is carried out in a manner known to a person skilled in the art, for example, by intratracheal instillation (by infusion or through a bite or through a catheter), by application by spray, or by nebulization.

As disclosed herein, the present invention contemplates the use of both concentrated and dilute surfactant formulations, depending on the particular use, as described hereinafter. Concentrated compositions are typically used for a "bite" type of application, while dilute surfactant compositions are typically used for an "irrigation" type of application.

Advantageously, the viscosity of said formulation is less than 20 cP, preferably less than 15 cP, as determined with a common viscometer available on the market, according to procedures well known in the art.

Advantageously, for the "bite" type of administration, the concentration of reconstituted surfactant by weight per ml of solution or suspension (after addition of liquid carrier) is in the range of about 0.1 to about 100 mg/ml, preferably between 5 and 80 mg/ml.

In a preferred embodiment of the present invention, when the reconstituted surfactant is administered by intratracheal injection as a suspension in physiological saline (0.9% w/v sodium chloride in water) the concentration is about 80 mg/ml.

Therefore, a preferred embodiment of the present invention relates to a pharmaceutical formulation in the form of an aqueous suspension in saline containing about 80 mg/ml of reconstituted surfactant composed of

a) about 1.5% by weight of the polypeptide analog of the native surfactant protein SP-C consisting of the sequence represented by the formula IPSSPVHLKRLKLLLLLLLLILILILGALLLLGL (SEQ ID NO:1);

b) about 0.2% by weight of the polypeptide analog of the native surfactant protein SP-B consisting of the sequence represented by the formula CWLCRALIKRIQALIPKGGRLLPQLVCRLVLRCS (SEQ ID NO:2);

c) about 49.15% by weight of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC); and

calculated in relation to the total weight of the reconstituted surfactant.

Typically the viscosity of said formulation is about 9 ± 3 cP at 25°C and 8 ± 3 cP at 37°C, as determined using a commercially available rotary viscometer.

When used for flush application, a typical surfactant concentration is from about 0.1 to 20 mg/ml, and more preferably about 0.5 to 10 mg/ml (in terms of mg of surfactant per ml of solution or suspension).

Since it depends on the concentration, the viscosity of diluted formulations should be even lower.

When used as a pharmaceutical treatment, formulations containing the reconstituted surfactant of the present invention may be administered either alone or optionally in combination with other compounds or compositions used in the treatment of respiratory diseases or disorders. For example, if a subject is being treated for a respiratory disorder caused by a bacterial infection, then the reconstituted surfactant of the present invention may be administered in conjugation with another compound used to treat the bacterial infection, such as an antibiotic.

Otherwise, in certain cases, for example, for the prevention of complications, such as bronchopulmonary dysplasia, the reconstituted surfactant of the present invention can be used in conjugation with corticosteroids, such as budesonide and beclomethasone dipropionate.

In certain embodiments, the reconstituted surfactant of the present invention and the resuspension carrier may be separately packaged simultaneously in a suitable container for the means. Such separate packaging of components in a suitable container for means is also described as a kit.

Therefore, this invention also relates to a kit, which includes: a) reconstituted surfactant of the subject invention in powder form, in the form of a first unit dose; b) a pharmaceutically acceptable carrier, in the form of a second unit dose; and c) a container for the means for holding said form of the first and second doses. Preferably, a pharmaceutically acceptable carrier is a physiological saline aqueous solution, preferably sterile.

As described herein, various methods for administering the reconstituted surfactant and its formulations of the present invention are available and well known to those skilled in the art.

Depending on the type of disease, e.g. infants or adults with respiratory distress syndrome, different treatment methods may be appropriate.

Typically, surfactant administered by endotracheal instillation in patients (eg, premature infants) is maintained on continuous or intermittent positive pressure ventilation (IPPV). Alternatively, surfactant can be administered using a thin catheter placed in the trachea and the patient's breathing is supported through specially designed nasal devices such as masks, arms or tubes using a methodology known as nasal Continuous Positive Airway Pressure (nCPAP).

The second approach would only be possible with a surfactant that has a low viscosity, because a high viscosity would make the passage of the surfactant through the thin catheter much more difficult.

In cases where the patient is suffering from a state of respiratory distress associated with pneumonia, pulmonary infection, and pulmonary contusion, certain treatment modalities may be recommended. In one such therapeutic procedure, lavage of the patient's lungs with the surfactant composition of the present invention is performed as one or more treatments.

, , , inhibits the development of symptoms or conditions associated with respiratory disease.

It is particularly useful for the prophylaxis and/or treatment of respiratory distress syndrome (RDS) in premature infants or other diseases associated with surfactant deficiency or dysfunction, including acute lung injury (ALI), adult RDS (ARDS), meconium aspiration syndrome (MAS), and bronchopulmonary dysplasia (BPD).

It may also be useful for the prophylaxis and/or treatment of other respiratory disorders such as chronic obstructive pulmonary disease (COPD), asthma, respiratory infection (eg, pneumonia, pneumocystitis carinii, cystic fibrosis, and respiratory syncytial virus), as well as for the treatment of otitis media (sticky ear). The following example illustrates the subject invention in more detail.

EXAMPLES

Example 1 - Effect of components on viscosity

A multi-factor experimental design was constructed to evaluate the effect of the components on the formulation in terms of viscosity. Different percentages of SP-C33 (leu) and ox-Mini-B (leu) polypeptides, as well as different ratios of DPPC and POPG, were tested.

All obtained mixtures were resuspended in physiological saline solution (0.9% w/v) at a concentration of 80 mg/ml.

Viscosity was determined at 25°C and 37°C using the so-called "VISCO STAR Plus (Fungilab)" viscometer applying a rotation speed of 100 r.p.m.

For the sake of comparison, the so-called "Curosurf<®">shows a viscosity that is between 6 and 10 mPas (1 mPas = 1 centipois).

The results are shown in Table 1.

The results show that at low concentrations of ox-Mini-B(leu) and in the presence of a low ratio between DPPC and POPG (column N3 in Table 1) the viscosity of the mixture is very low. These results show that by reducing the amount of SP-B analog in the surfactant and simultaneously reducing the concentration of saturated phospholipid relative to unsaturated phospholipid, a composition can be obtained with a viscosity low enough to be used for all surfactant applications, including use in concentrated form.

Example 2 - Characterization of reconstituted surfactant N3

A mixture of DPPC:POPG Na in the ratio 1:1, SPC-33 (Ieu) and ox Mini-B (Ieu) in percentage amounts recorded for composition N3 in Table 1 from Example 1 was dissolved in chloroform/ethanol 98:2 (v/v).

The solvent was evaporated, and the resulting powder was subsequently hydrated in 0.9% w/v Nad aqueous solution, with stirring, to obtain a surfactant concentration of 80 mg/ml.

The viscosity of the said formulation confirms that it is very low, for example, about 9 cP at 25°C and about 7 cP at 37°C. Moreover, it does not change after 6 months of storage at 5°C.

The formulation also appears chemically stable after six months of storage and the total amount of lysoform phospholipids detected by HPLC is less than 1% by weight.

Primer 3 - In vivo activity of reconstructed surfactants N3

The reconstituted surfactant from Example 2 was tested in premature rabbits, obtained by hysterectomy at a gestational age of 27 days. The experiments were performed without the application of positive and expiratory pressure (PEEP).

Animals receiving Curosurf<®> serve as positive controls and untreated pups in the same litter serve as negative controls.

All surfactant preparations are applied in a concentration of 80 mg/ml and in a standard dose of 2.5 ml/kg.

Immature newborn rabbits were ventilated in parallel with a standardized sequence of peak insufflation pressures. To open the lungs, the pressure was first set to 35 cmH2O for 1 min. After this maneuver recruitment, the pressure was lowered to 25 cmH2O for 15 minutes and further to 20 and 15 cmH2O.

Finally, the pressure was raised again to 25 cmH2O for 5 min, after which the lungs were ventilated for an additional 5 min with nitrogen and then excised for gas volume measurement.

Both tidal volume and lung volume of air, expressed as ml/kg, were measured and the obtained results recorded as median values are in Figures 1 and 2, respectively.

In Figure 1, it can be seen that animals treated with the reconstituted surfactant of the present invention show an improvement in tidal volume somewhat better than that achieved after administration of Curosurf<®>. This result suggests that the reconstituted artificial surfactant of the present invention may provide better clinical efficacy compared to the current gold standard in the field.

Regarding lung air volume, Figure 2 shows that the reconstituted surfactant of the present invention is able to increase a value comparable to that of the reconstituted surfactant tested in Example 3, of WO 2008044109 containing a higher ratio between DPPC and POPG (68:31).

Moreover, the magnitude of lung volume of air from the reconstituted surfactant of the present invention appears robust and reproducible.

Claims (9)

PATENT REQUESTS 1. A composition of reconstituted surfactant that includes: a) from 1.2 to 1.8% by weight of the polypeptide analog of the native surfactant protein SP-C consisting of the sequence represented by the formula IPSSPVHLKRLKLLLLLLLLILLLILGALLLLGL (SEC. ID NO:1); b) from 0.1 to 0.5% by weight of the polypeptide analog of the native surfactant protein SP-B consisting of the sequence represented by the formula CWLCRALIKRIQALIPKGGRLLPQLVCRLVLRCS (SEQ ID NO:2); c) monounsaturated and saturated phospholipid in a weight ratio ranging from 45:55 to 55:45, wherein said monounsaturated phospholipid is selected from the group consisting of palmitoyloleoylphosphatidylcholine (POPC) and palmitoyloleoylphosphatidylglycerol (POPG) and wherein said saturated phospholipid is selected from the group consisting of dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylglycerol (DPPG); all amounts are calculated relative to the total weight of reconstituted surfactant. 2. Reconstituted surfactant according to patent claim 1, which includes: a) about 1.5% by weight of the polypeptide analog of the native surfactant protein SP-C consisting of the sequence represented by the formula IPSSPVH LKRLKLLLLLLLLILLLILGALLLLGL (SEC. ID NO:1); b) about 0.2% by weight of the polypeptide analog of the native surfactant protein SP-B consisting of the sequence represented by the formula CWLCRALIKRIQALIPKGGRLLPQLVCRLVLRCS (SEQ ID NO:2); c) 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1-palmitoyl-2-oleyl-sn-glycero-3-phosphoglycerol (POPG) in a weight ratio of about 50:50; all amounts are calculated relative to the total weight of reconstituted surfactant. 3. Reconstituted surfactant according to patent claim 2, which includes: a) about 1.5% by weight of the polypeptide analog of the native surfactant protein SP-C consisting of the sequence represented by the formula IPSSPVH LKRLKLLLLLLLLILLLILGALLLLGL (SEC. ID NO:1); b) about 0.2% by weight of the polypeptide analogue of the native surfactant SP-B consisting of the sequence represented by the formula CWLCRALIKRIQALIPKGGRLLPQLVCRLVLRCS (SEC. ID NO:2); c) about 49.15% by weight of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC); and d) about 49.15% by weight of l-palmitoyl-2-oleyl-sn-glycero-3-phosphoglycerol (POPG); all amounts are calculated relative to the total weight of reconstituted surfactant. 4. The reconstituted surfactant according to any one of claims 1 to 3, wherein the polypeptide of SEQ. ID BR:2 in the form of a disulfide-bonded molecule with an intramolecular disulfide bond between two cysteine residues at positions 1- and 33- and/or between two cysteine residues at positions 4- and 27-. 5. A reconstituted surfactant according to any one of the preceding claims, wherein the POPG is in the form of a pharmaceutically acceptable salt. 6. The reconstituted surfactant according to claim 5, wherein the salt is a sodium salt. 1. A reconstituted surfactant according to any of the preceding claims, wherein each of said polypeptides is present in the form of a pharmaceutically acceptable salt. 2. The reconstituted surfactant according to claim 7, wherein the salt is an acetate salt. 3. A pharmaceutical formulation containing a reconstituted surfactant according to any one of claims 1 to 8, said formulation being in the form of a solution, dispersion, suspension or dry powder, optionally in combination with one or more pharmaceutically acceptable carriers. 4. Pharmaceutical formulation according to claim 9, wherein said formulation is in the form of an aqueous suspension. 5. Pharmaceutical formulation according to patent claim 10, wherein the reconstituted surfactant is contained in a concentration between 0.1 and 160 mg/ml in an aqueous suspension. 6. Kit, which contains; a) reconstituted surfactant according to any one of claims 1 to 8, in powder form in the form of a first unit dose; b) a pharmaceutically acceptable carrier in the form of a second unit dose; and c) means of storage means for keeping the mentioned first and second dosage form. 7. The reconstituted surfactant according to any one of claims 1 to 8, for use as a medicine. 8. The reconstituted surfactant according to any one of claims 1 to 8, for use in the treatment or prophylaxis of respiratory distress syndrome (RDS) in premature infants, or for the treatment or prophylaxis of other diseases associated with surfactant deficiency or dysfunction. 9. The reconstituted surfactant for use according to claim 14, wherein the disease includes adult RDS (ARDS), meconium aspiration syndrome (MAS) and bronchopulmonary dysplasia (BPD).