HK40010458B - A therapeutic combination comprising a pulmonary surfactant and a steroid for the treatment of evolving bpd - Google Patents

Description

Therapeutic combination comprising a pulmonary surfactant and a steroid for the treatment of progressive BPD

Technical Field

The present invention relates to compositions and methods for treating preterm labor. In particular, the present invention relates to the use of lung surfactants in combination with steroids (steroids) for the treatment of advanced bronchopulmonary dysplasia (evolving broncho-pulmony dyslasia) in preterm neonates (preterm nerves).

Background

The human lung is composed of a large number of small air sacs, called alveoli, in which gas is exchanged between the blood and the air space of the lung. 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 mainly composed of lipids and contains a small amount of various proteins. Lack of sufficient levels of this complex can lead to lung dysfunction. This syndrome is known as Respiratory Distress Syndrome (RDS) and commonly affects premature newborns.

The main method for treating RDS is to use exogenous lung surfactant extracted from animal lungReplacement therapy for substance preparations (preperations), known as modified natural surfactants. For example, the modified natural surfactant used in clinical practice is porcine lung phospholipid (poractant alfa) derived from porcine lung and sold under the following trademark:beractant (beractant) (B)Or) Bovactant)(both from bovine lung) and calfatant (calfatant)(from calf lung).

Currently, exogenous pulmonary surfactant is administered by intratracheal instillation as a suspension in saline solution to preterm infants kept intubated under mechanical ventilation with oxygen.

Although the treatment results in a greatly improved postpartum survival, children surviving from RDS are at high risk for bronchopulmonary dysplasia (BPD), a common and serious complication of preterm birth, associated with significant mortality, morbidity and medical resource utilization. Despite advances in prenatal and neonatal care, the incidence of disease is increasing. Management of BPD and its associated problems remains a major challenge for newborn and pediatricians. Various interventions (interventions) have been proposed to prevent and treat BPD, but many remain without evidence. Current treatment methods appear to reduce the severity of BPD, but have little effect on its incidence. BPD is a progressive lung injury process whose pathophysiology varies at different stages of the disease. Thus, its management is unlikely to be in the form of a single intervention, but rather a combinatorial approach employing different strategies for different factors and/or stages of the disease.

Thus, it is useful to classify BPD interventions in the three subsequent phases when designing an overall management plan. These are: i) prevention of BDP; ii) treatment of progressive BPD; iii) treatment of diagnosed BPD (see Bowen P et al, Pediatrics and Child Health 2013,24:1, 27-31).

BPD prevention in RDS affected newborns is controlled by systemic administration of corticosteroids prenatally or postnatally for a few hours. However, the effectiveness of postpartum corticosteroid administration is offset by possible adverse systemic effects such as hypertension, hyperglycemia, gastrointestinal complications, and neurodevelopmental disability.

As an alternative to systemic administration, delivery of corticosteroids by inhalation or intratracheal instillation has been proposed for the prevention of BDP.

For example, US2010-0317636 discloses a method of preventing BPD in infants suffering from respiratory distress syndrome by administering to the infant a combination of a corticosteroid and a lung surfactant having high local to systemic anti-inflammatory activity.

Yeh et al (Pediatrics 2008,121, (5), e1310-e1318) propose the use of pulmonary surfactantsAs a carrier, budesonide was instilled intratracheally. Dani et al (Peditr Pulmonol 2009,44,1159-Combined beclomethasone dipropionate.

However, by these methods, a large population of premature neonates will be exposed to corticosteroids, many of which have no benefit, if not they will develop BPD (see Bancalari E Am J Respir Crit Care Med 2016,193: 1, 12).

On the other hand, the efficacy of corticosteroids in BDP has been identified as a serious concern, since the disease is characterized by intense and persistent airway inflammation, fibrosis and smooth muscle hypertrophy.

Postpartum corticosteroids may find their place in treatment for advanced BPD during treatment as they will then be administered to patients in need thereof.

However, due to observed side effects or lack of clear indications of efficacy, it is currently not recommended to routinely perform systemic postpartum administration of dexamethasone and hydrocortisone.

In view of the above considerations, there remains a need to develop more compliant corticosteroid-based drugs for the treatment of progressive BPD in preterm newborns.

Furthermore, it would be advantageous to provide a medicament that can be administered locally by inhalation or intratracheal instillation.

Finally, it would be particularly advantageous to provide a medicament capable of promoting lung development.

Disclosure of Invention

The present invention relates to a lung surfactant in combination with budesonide, at a budesonide dose of 0.1 to 1.5mg/kg, for use in the treatment of advanced bronchopulmonary dysplasia (BPD) in preterm neonates.

Preferably, the combination of the invention increases mRNA expression of some protein indicators of lung maturation, more preferably mRNA expression of surfactant proteins SP-A, SP-B and SP-C.

Advantageously, the combination is administered from day 2 to day 28 of life, preferably from day 5 to day 15 of life, more preferably from day 7 to day 10 of life.

The invention also relates to the use of a lung surfactant in combination with budesonide in a dose of 0.1-1.5mg/kg for the manufacture of a medicament for the treatment of bronchopulmonary dysplasia (BPD) in preterm neonates. Advantageously, the combination is administered from day 2 to day 28 of life, preferably from day 5 to day 15 of life, more preferably from day 7 to day 10 of life.

The dose of budesonide is preferably 0.2 to 1.0 mg/kg.

The agents of the invention may be administered simultaneously, sequentially or separately, preferably simultaneously as a fixed combination.

In a specific embodiment, the medicament is in the form of a pharmaceutical composition for inhalation or intratracheal administration comprising the fixed combination.

In another embodiment, the invention relates to a method of treating progressive bronchopulmonary dysplasia comprising administering a lung surfactant to a preterm infant in need of such treatment in combination with budesonide at a dose of 0.1 to 1.5mg/kg, wherein the combination is administered from day 2 to day 28 of life, preferably from day 5 to day 15 of life, more preferably from day 7 to day 10 of life.

Definition of

The term "bronchopulmonary dysplasia (BPD)" refers to a chronic lung disorder, also known as Chronic Lung Disease (CLD), which is the result of unresolved or abnormally repaired lung injury.

BPD commonly occurs in ultra-low birth weight (VLBW) infants with persistent lung injury due to oxygen toxicity and barotrauma induced by mechanical ventilation early in life. The definition and classification of BPD has changed since the beginning of the description of BPD by Northway et al in 1961. The national institute for child health and human development (NICHD) defined BPD in the 2001 consensus statement. This definition uses supplemental oxygen requirements for 28 days, and then determines grade 3 severity, depending on the respiratory support required at post-menopausal age at 36 weeks (PMA) or at discharge from those born at <32 weeks gestation or at day 56 of life or discharge from those born at >32 weeks gestation.

According to recent definitions, BPD may be considered primarily as a failure of lung development (see Jobe A et al, Ped Res 1999,46, 641).

In 2001, Jobe A et al (Am J Respir Crit Care Med; 163 (7)) 1723-.

Mild BDP is defined as a disease requiring: oxygen supplementation for > 28 days, and room air supplementation at 36 weeks PMA or discharge (for <32 weeks of birth) or at 56 days or discharge (for >32 weeks of birth).

Moderate BDP is defined as a disease requiring: oxygen supplementation was > 28 days and required < 30% oxygen supplementation at 36 weeks PMA/discharge (<32 weeks) or at 56 days/discharge (for infants >32 weeks).

Severe BPD is defined as a disease requiring: supplemental oxygen was greater than or equal to 28 days and required greater than or equal to 30% oxygen at 36 weeks PMA/discharge (<32 weeks) or 56 days/discharge (<32 weeks) or required nasal CPAP or mechanical ventilation.

The term "progressive BPD", sometimes referred to as early BPD, refers to the initial phase of the chronic process leading to confirmed BDP and indicates a disease characterized by oxygen and/or ventilator dependence from day 7 to day 14 of life (Walsh MC et al Pediatrics 2006,117, S52-S56).

The term "modified natural surfactant" refers to a lipid extract of minced mammalian lungs. The hydrophilic proteins SP-A and SP-D are lost due to the lipid extraction process used in the manufacturing process. These preparations have variable amounts of the two hydrophobic surfactant-related proteins SP-B and SP-C and may contain non-surfactant lipids, proteins or other components depending on the extraction method.

The term "porcine lung phospholipids" refers to modified natural surface-active substances extracted from porcine lungs, which essentially consist of polar lipids, mainly phospholipids and the proteins SP-B and SP-C. Pork lung phospholipid trademarksProvided is a method.

The term "artificial" lung surfactant (artificial pure pulmonary surfactant) refers to a synthetic compound, mainly a simple mixture of phospholipids and other lipids, formulated to mimic the lipid composition and behavior of natural lung surfactants. They are free of lung surfactant proteins.

The term "reconstituted" lung surfactant "refers to an artificial lung surfactant to which has been added a lung surfactant protein/peptide isolated from an animal or a protein/peptide or lung surfactant protein analogue prepared by recombinant techniques such as those described in WO 95/32992, for example as described in WO 89/06657, WO 92/22315 and WO 00/47623.

The term non-invasive ventilation (NIV) procedure defines a mode of ventilation that supports breathing without the need for intubation, such as nasal continuous positive airway pressure (nasal CPAP). Other non-invasive ventilation procedures are Nasal Intermittent Positive Pressure Ventilation (NIPPV), High Flow Nasal Cannula (HFNC) and bi-level positive airway pressure (BiPAP).

The term "respiratory support" includes any intervention to treat a respiratory disease, including, for example, supplemental oxygen, mechanical ventilation, and administration of nasal CPAP.

The term "treating" refers to curing a disease or disorder in a patient, alleviating the symptoms of a disease or disorder in a patient, or alleviating the symptoms of a disease or disorder in a patient, such as BPD.

The term "preventing" refers to slowing the progression and/or delaying the onset of a disease or disorder, such as BPD, in a patient.

The term "preterm neonate" or preterm neonate includes Extremely Low Birth Weight (ELBW), ultra-low birth weight (VLBW), and Low Birth Weight (LBW) neonates at 24-35 weeks gestational age.

The term "fixed combination" refers to a combination in which the active substances are in a fixed quantitative ratio.

"pharmaceutically acceptable" is a term used herein to refer to a medium that does not produce an allergic or similar untoward reaction when administered to an infant.

The "surfactant activity" of a surfactant preparation is defined as the ability to reduce surface tension.

The in vitro efficacy of exogenous surface-active substance preparations is usually tested by measuring their ability to reduce surface tension using suitable devices such as Wilhelmy Balance, Pulsating Bubble surface activity meter, Bubble capture surface activity meter and Capillary surface activity meter.

The in vivo efficacy of the exogenous surfactant preparation was tested by measuring lung mechanics in an animal model of preterm birth according to known methods.

In the context of the present specification, the term "synergistic" means that the activity of the lung surfactant plus budesonide is higher than the activity of either the surfactant or budesonide alone.

The term "biosimilar product of porcine lung phospholipid" is used, which refers to a modified natural lung surfactant that is therapeutically comparable to porcine lung phospholipid but has at least 80% similarity in composition to porcine lung phospholipid and a viscosity of less than 15mPas (cP) at room temperature when suspended in an aqueous solution at a concentration of 80/mg/ml. The viscosity can be determined according to known methods.

Drawings

Figure 1-plan of such a complex series of interventions.

FIG. 2-oxygenation

FIG. 3-40 cmH measured from a pressure-volume curve₂Static lung gas volume of O.

FIG. 4-lung weight

FIG. 5-Lung gas volume versus Lung weight

FIG. 6-Lung Dry weight to Wet weight ratio

FIG. 7-mRNA indicators for SP-A, SP-B and SP-C for lung maturation

Detailed Description

The present invention is based, in part, on the unexpected discovery that budesonide at a dose of 0.1mg/kg to 1.5mg/kg can be combined with a lung surfactant, such as porcine lung phospholipid, to treat progressive bronchopulmonary dysplasia (BPD) without altering the activity of the surfactant.

The advantages of combining a lung surfactant with a claimed dose of budesonide will be apparent from the findings below.

In fact, it was surprisingly found that in a study conducted on preterm lambs with RDS undergoing nasal CPAP ventilation, lung surfactant such as porcine lung phospholipid in combination with budesonide significantly increased mRNA expression of certain protein indicators of lung maturation, whereas it was unexpected that lung surfactant alone caused a decrease in such mRNA expression.

The addition of budesonide also significantly increased lung gas volume and decreased lung weight relative to lung surfactant alone.

The reduction in lung weight itself (in turned) is associated with loss of water, indicating mesenchymal cell loss and maturation response.

Furthermore, the addition of budesonide to the surfactant reduces airway wall thickness and collagen deposition, both of which are indicators of insufficient lung maturation.

Since budesonide is a highly lipophilic corticosteroid, this may facilitate its mucosal absorption and uptake across the phospholipid cell membrane with negligible systemic absorption, making the combination safe for therapeutic use in preterm newborns.

On the other hand, lung surfactants may facilitate the diffusion of corticosteroids by the Marangoni effect (Marangoni effect), facilitating their distribution and thus the access to all lung areas of interest.

Any pulmonary surfactant currently in use, or hereafter developed for use in respiratory distress systems and other pulmonary diseases, is suitable for use in the present invention. These include modified natural, artificial and reconstituted lung surfactants.

Current modified natural lung surfactants include, but are not limited to, bovine lipid lung surfactant (BLES)^TMBLES Biochemicals, inc. london, Ont), calfatan (Infasurf)^TMForest Pharmaceuticals, St.Louis, MO), bofakrant (Alveofact)^TMThomae, Germany), bovine lung surfactant (Pulmony surfactant TA)^TMTokyo Tanabe, dayBen), pulmonis Sus domestica phospholipid (Guersu)^TMChiesi Farmaceutii SpA, Parma, Italy) and Beractant (Survanta)^TM，Abbott Laboratories，Inc.，Abbott Park，111)。

Examples of reconstituted surface-active substances include, but are not limited to, the compositions disclosed in EP 2152288, WO 2008/011559, WO 2013/120058, the product lucina ctant (Surfaxin)^TMWindtree-Discovery Laboratories inc., Warrington, Pa.) and a product having the composition disclosed in table 2 of example 2 of WO2010/139442, i.e.

1.5% SP-C33(leu) acetate;

0.2% Mini-B (leu) acetate; and

DPPC to POPG in a weight ratio of 50: 50.

The lung surfactant selected for use in the medicament of the invention may be the same as or different from the lung surfactant used for RDS. In a preferred embodiment, the same lung surfactant is used.

In a preferred embodiment, the lung surfactant is a modified natural lung surfactant.

More preferably the lung surfactant is a porcine lung phospholipid as defined above (Gulsu @)^TM) Or a biologically similar product thereof, because it has a very low viscosity, and can therefore be administered at high concentrations using small amounts of aqueous carrier.

In another embodiment, the lung surfactant is a reconstituted surfactant having the composition disclosed in table 2 of example 2 of WO 2010/139442.

The dose of lung surfactant to be administered will vary with the body weight and gestational age of the preterm neonate and the severity of the condition of the neonate. One skilled in the relevant art will be readily able to determine these factors and adjust the dosage accordingly.

Advantageously, the dose of lung surfactant may be 100-200 mg/kg.

In a preferred embodiment of the invention, a dose of 100-200mg/kg of pig lung phospholipids may be used.

In a preferred embodiment, the dose may be 100mg/kg, and in another preferred embodiment, the dose may be 200 mg/kg.

Advantageously, the dose of budesonide is from 0.1 to 1.5mg/kg, more advantageously from 0.2 to 1.0mg/kg, even more advantageously from 0.25 to 1.0 mg/kg.

In certain embodiments, when primarily aimed at affecting lung maturation, the dose of budesonide may be from 0.1 to 0.5mg/kg, while in other embodiments the dose of budesonide may be from 0.5 to 1.0 mg/kg.

Preferably, the combination of the invention is administered to a preterm neonate maintained under a non-invasive ventilation procedure, more preferably under nasal CPAP, even more preferably with a nasal device, under a pressure of 1 to 12cm of water.

The claimed doses of the combination of pulmonary surfactant and budesonide may be administered sequentially, separately or together. Advantageously, when the two active substances are administered together, they are administered as a fixed combination.

Thus, the present invention also relates to the use of a combination of the invention as a fixed combination for the preparation of a medicament for the treatment of progressive BPD.

The medicament may be in the form of a pharmaceutical composition.

The formulations may be administered in the form of a solution, dispersion, suspension or dry powder. Preferably, the composition comprises the claimed combination suspended in a suitable physiologically tolerable solvent.

More preferably, the formulation comprises an aqueous solution, preferably sterile, which may also contain a pH buffer and other pharmaceutically acceptable excipients, such as polysorbate 20, polysorbate 80 or sorbitan monolaurate as a wetting agent and sodium chloride as an isotonicity agent.

The formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, or may be stored in a frozen or freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier immediately prior to use.

Preferably, the formulation is provided as a sterile suspension in a buffered saline (0.9% w/v sodium chloride) aqueous solution in a disposable vial.

Administration of the claimed formulations can be carried out according to known methods, for example, by intratracheal instillation, spray administration or jet ultrasonic atomization or mesh-vibrating atomizers (mesh-vibrating atomizers) commonly available on the market.

When the formulation is administered by intratracheal instillation, different approaches may be used depending on the severity of the respiratory distress syndrome. For example, the claimed formulations may be administered via an endotracheal tube to a preterm neonate maintained under mechanical ventilation.

Alternatively, the formulation may be administered by using a microcatheter placed in the trachea and the neonate is supported by a specially designed nasal device (e.g. a mask, prongs or tube) according to a method known as nasal continuous positive airway pressure (nCPAP) according to the method described in WO 2008/148469.

The latter method can only use exogenous surface-active substances, such as porcine lung phospholipids, which have a low viscosity, since a high viscosity makes the passage of the surface-active substance through a thin catheter more difficult.

The volume of the aqueous solution in which the two combined active substances are suspended depends on the desired concentration.

Advantageously, the volume of the formulation should be no more than 5.0ml, preferably 4.5-2.0ml, more preferably 3.5-2.5 ml.

In other embodiments, when the lung surfactant and budesonide are administered separately, each active may be formulated separately. In this case, the two separate active substances do not have to be taken simultaneously, without any requirement.

In the case of such separate administration, the formulations of the two separate active substances can be packaged simultaneously in suitable container means. Such separate packaging of the components in suitable containers is also referred to as a kit.

Accordingly, the present invention also relates to a kit for the treatment of progressive bronchopulmonary dysplasia, comprising: a) a dose of 100-200mg/kg of a pulmonary surfactant and a pharmaceutically acceptable carrier or diluent in the first unit dosage form; b) budesonide in the second unit dosage form at a dose of 0.1 to 1.5mg/kg and a pharmaceutically acceptable carrier or diluent; c) container means containing said first and second dosage forms.

The combination of the invention, which can be administered postpartum to preterm newborns according to conditions which will be determined by the person skilled in the art, is suitable for the treatment of any form of progressive bronchopulmonary dysplasia.

The frequency of administration will vary with the size and gestational age of the preterm neonate, as well as the severity of the condition of the neonate and the route of administration. It will be readily ascertainable by one skilled in the relevant art.

For example, the medicament of the present invention may be administered once or twice daily.

Advantageously, the combination of the invention is administered from day 2 to day 28 of life, preferably from day 5 to day 15 of life, more preferably from day 7 to day 10 of life.

During the interval, the treatment may continue for a period of time deemed appropriate by the physician or other practitioner to achieve the therapeutic effect.

A preterm neonate in need of the present medicament may or may not exhibit Respiratory Distress Syndrome (RDS). In one embodiment, administration of the medicament of the invention is started in a newborn exhibiting RDS after treatment of this syndrome with a pulmonary surfactant or by other means (e.g. ventilation) or a combination thereof.

In certain embodiments, a neonate treated with a drug of the present invention requires respiratory support but does not necessarily exhibit respiratory distress syndrome. These infants were either not diagnosed with RDS or were not treated with RDS with lung surfactant.

All preterm neonates are suitable for administration of the medicament of the invention, including very low birth weight (ELBW), ultra low birth weight (VLBW) and Low Birth Weight (LBW) neonates at 24-35 weeks gestational age. Preferably, the drug is administered to VLBW newborns with severe RDS, which have a high incidence of BPD.

In general, since management of progressive BPD is unlikely to be a form of single intervention, but a combined approach, physicians should assess whether preterm newborns also require concomitant respiratory support and/or other suitable drugs, such as vitamin a and antibiotics.

The following examples illustrate the invention in more detail.

Examples

Example 1 in vitro evaluation of the surface Activity of porcine Lung Phospholipids by capillary surface Activity meters in the Presence of budesonide

The surface activity of porcine lung phospholipid (2ml, 1.0mg in the presence of budesonide) was evaluated by a capillary surface activity meter commercially available from caliia Medical, inc., USA, in comparison to porcine lung phospholipid alone.

Two samples were prepared: one from a vial of pulmonic phospholipid (1.5ml, 80mg/ml) diluted with saline to a concentration of 1mg/ml phospholipid and the other from a vial of pulmonic phospholipid (1.5ml, 80mg/ml) mixed with a vial of budesonide (2ml, 1.0mg) and diluted with saline to the same concentration (1mg/ml phospholipid). Samples of 0.5ml of both solutions were then evaluated in a capillary surfactant meter.

The principle of capillary surfactant meters is to simulate the terminal human airway. The sample was introduced into the narrow part of a glass capillary tube with an internal diameter of 0.25mm, similar to the terminal human airway. At one end, the capillary tube is connected to a bellows and a pressure sensor. When the bellows slowly compressed, the pressure rose and was recorded. The pressure increase causes the sample to be squeezed out of the narrow portion of the capillary. When air passes, the pressure suddenly drops. If the sample contains a well-functioning lung surfactant, the sample fluid will not return to the stenosed part. The steady flow obtained by the continuous compression of the bellows will not encounter resistance and the recorded pressure will be zero. On the other hand, if the sample does not contain a lung surfactant with good function, the sample liquid is repeatedly returned.

The behavior of porcine lung phospholipid in the presence of budesonide proved to be statistically indistinguishable from porcine lung phospholipid alone, indicating that the dose of budesonide did not affect the surface activity of the surfactant.

Example 2 in vivo evaluation of porcine pneumococcal phospholipid Activity in the Presence of budesonide in a lamb model of BPD

Experiments were conducted to study neonatal resuscitation and lung injury, evaluating surfactant and budesonide treatment for reduction of lung injury. This experiment was aimed at examining whether fetal lung stretch injury would modulate secondary ventilation-mediated injury 24 hours after intrauterine recovery.

This is a study to test the potential for fetal lung pretreatment or tolerance response. Surfactant treatment with or without budesonide after the initial stressful injury was performed to test whether steroids had anti-inflammatory effects that protected the fetal lungs. The animal groups included CPAP exposure and there was no initial exposure to compare with stretch injuries. These groups and their characteristics and treatments are given in table 1. The description in table 1 gives details of the intervention. Fig. 1 is a schematic diagram of a scenario for such a complex series of interventions.

TABLE 1

Description of the tables

CPAP: the animals were placed in 5cm H₂O CPAP was continued for 15 minutes as a control for anesthesia and surgery associated with injury intervention.

Fetal lung injury intervention: is that the head and chest of the animal are exposed; the 4.5mm endotracheal tube was fixed in the trachea. The fetus was then aerated with 100% humidified nitrogen, R30, IT 1 sec, PEEP 0, maximum pressure to 55cm H₂And O. The goal was to reach an estimated V of 7ml/kg at 4 minutes_tAn estimated V of 12ml/kg was reached at 8 minutes_tEstimated V of 15ml/kg at 12 minutes_t. The total aeration time was 15 minutes.

Pulmonary surfactant (Surf): in the CPAP or fetal lung injury prognosis, animals were treated with 100mg/Kg of Gulssu assuming a body weight of 3 Kg. The solid solution of the. The surfactant is administered through an endotracheal tube and mixed with the fetal lung fluid by a syringe. After surfactant treatment, the trachea is ligated to prevent loss of surfactant.

Budesonide (Bud): primicketto (Pulmicort Respules) (Astra Zeneca, Sweden) containing 0.5mg micronized budesonide in 1ml was mixed with solid soda saline to deliver 0.25 or 1.0mg/kg budesonide and surfactant in 10ml suspension.

24 hour ventilation test: the animal's head was again exposed and a 4.5mm endotracheal tube was placed. Fetal lung fluid was aspirated with a syringe, delivered to lambs and ventilated using: rate of 40, inspiration time of 0.45 seconds, PEEP of 5cmH₂O and a maximum peak inspiratory pressure of 40cm H₂O, 100% humidified oxygen.

The experiment was designed for 46 foetal sheep, with a final total of 44, since one ewe had no foetus and one twin was a single young animal (singleton). All other lambs were successfully used to complete the experimental procedure. Adjusting the number of animals per group to increase V_t15 animals in the lesion and surfactant groups, thereby increasing the statistical efficacy of those groups. Specific comments on important elements of the experimental design are as follows.

Although using 55cm H₂Maximum pressure of O, but ventilation impairment at 15 minutes for an estimated 15ml/kg tidal volume only reached volumes of 11-13ml/kg, indicating that the fetal lung is immature and deficient in surfactant.

·V_tThe injury and CPAP groups (assuming 3kg birth weight) were given 100mg/kg of either Gulsu or Gulsu budesonide, diluted to 10ml with saline. The trachea was ligated after treatment to ensure that the treatment stayed intrauterine in the lungs for 24 hours before assessing lung function (lumen).

Budesonide was used as 0.5mg/ml pramipexole in order to have a standardized sterile product for exposing the fetal lungs.

At 24 hours delivery after initial intervention and treatment, the endotracheal tube was placed and any free flowing fetal lung fluid was removed with a syringe. Large volumes of fluid are recovered from CPAP exposed lungs. Without fetal lung fluid, only a small amount of thick secretionsFrom V_tThe injured lung was aspirated.

A post-delivery ventilation period of 30 minutes was successful in all ventilated lambs. Some lungs have air collection (air collection) in the lung tissue and pleural sacs, but most pressure-volume curves are successful.

Results

There were no significant differences in gestational age or birth weight between groups (table 1). These experimental analyses were complex because there were 7 groups. No + no animals were used for tissue collection only for baseline measurements.

Oxygenation

The results are reported in figure 2.

When animals were ventilated with 100% oxygen, arterial PO was administered₂Oxygenation measured was very low for intact and surfactant untreated ventilated twins (ventilated twins) which were exposed to anesthesia only 24 hours prior to ventilation-mean 54 mmHg. This result confirmed that the lambs had immature lungs prior to any intervention. The damage abrogates the oxygenation response despite the surfactant with or without budesonide. 15 min CPAP + surfactant treatment resulted in an average PO₂Increased significantly to 380mmHg and addition of budesonide resulted in an average PO₂At 417mmHg, indicating that the addition slightly increased oxygenation.

In the non-aerated group, even though it was quite low, a slight trend of improvement with increasing budesonide concentration was seen by comparison of the median values of the groups.

The static lung gas volume measured from the pressure-volume curve was 40cmH₂O。

The results are reported in figure 3.

In ventilated twins, the maximum lung gas volume expressed relative to body weight measured after oxygen adsorption for an inflation measurement for lungs without air lungs is low. The CPAP + surfactant group lung gas volume/kg body weight increased significantly, budesonide increased significantly in volume relative to CPAP + surfactant. As for oxygenation, for V_tThe lung gas volume in the 15 injured animals had no major effect.

Lung weight

The results are reported in fig. 4.

Surprisingly, large changes in lung weight were observed in each group at necropsy. Ventilated twins, V_t15 lung weights of lesion + surfactant and CPAP + surfactant were similar. However, in each of the budesonide groups, the lung weight was lower, with the effect being greatest in the 1.0mg/kg budesonide group. The large changes in lung weight of the fetus exposed to maternal steroids have never been measured in other experiments, and therefore the very large changes in lung weight observed are quite unexpected.

Lung gas volume relative to lung weight

The results are reported in fig. 5.

Notably, although lung weight decreased for the CPAP + surfactant + budesonide group relative to the CPAP + surfactant group, lung gas volume increased. Thus, the lung volume to lung weight ratio is shown. This ratio emphasizes the combined effect of lighter lungs holding more gas. The CPAP + budesonide lungs hold more gas than the CPAP lungs. The ratio also shows that V_tThe 15+ surfactant lung retains less gas than the lung exposed to lung budesonide.

Dry to wet weight ratio of lung

The results are reported in fig. 6.

These large changes in lung weight associated with budesonide must be a loss of lung water primarily within 24 hours. Loss of water may indicate loss of mesenchymal cells and a maturation response.

mRNA indicators of lung maturation

mRNA was analyzed for Surfactant Proteins (SP) A, B, C and D.

It is well known that expression of surfactant proteins is a marker of lung maturation.

The results for SP-A, SP-B and SP-C are reported in FIG. 7.

The mRNA of the CPAP + surfactant group was consistently reduced relative to the untreated control and ventilated twins. The inhibition of the surfactant protein mRNA is quite unexpected. Interestingly, the combination of CPAP + surfactant with budesonide significantly increased the surfactant protein.

Example 3-formulation in the form of an aqueous suspension according to the invention.

Example 4 airway thickness determination

Both CPAP and mechanical ventilation are known to change the appearance and thickness of large and small airways.

In particular, in the absence of adequate lung maturation, an increase in wall thickness and an increase in collagen deposition is observed.

Therefore, the thickness of the large airways/bronchioles and collagen deposition were measured in lambs exposed to high oxygen according to the method reported in Wang H et al Am J Physiol. lung Cell Mol Physiol 2014,307, L295-L301.

Thickness was measured on blind sections, 3 times per slide image, 5 slide images per animal, 4 to 6 animals per group.

The results are reported in table 2.

Animals receiving mechanical ventilation have thickened small and large airways. Furthermore, quantification of collagen in the airways indicates increased collagen staining in animals receiving mechanical ventilation.

In contrast, a decrease in the CPAP + surfactant + budesonide group was observed, particularly significant for the CPAP + surfactant + budesonide 0.25mg/kg group.

TABLE 2

Claims (14)

1. Use of a lung surfactant in combination with budesonide in the manufacture of a medicament for the treatment of progressive bronchopulmonary dysplasia in a preterm neonate, wherein the lung surfactant is in a dose of from 100 to 200mg/kg, the budesonide is in a dose of from 0.1 to 1.5mg/kg and the medicament is to be administered from day 2 to day 28 of the life of the preterm neonate.

2. The use according to claim 1, wherein the medicament is administered from day 5 to day 15 of life.

3. Use according to claim 2, wherein the medicament is administered from day 7 to day 10 of life.

4. Use according to any one of claims 1 to 3, wherein the preterm neonate is maintained under a non-invasive ventilation procedure.

5. The use of claim 4, wherein the non-invasive ventilation procedure is nasal CPAP.

6. The use according to any one of claims 1-3 and 5, wherein the dose of budesonide is from 0.2 to 1.0 mg/kg.

7. Use according to any one of claims 1-3 and 5, wherein the dose of budesonide is from 0.1 to 0.5 mg/Kg.

8. Use according to any one of claims 1-3 and 5, wherein the dose of budesonide is from 0.5 to 1.0 mg/kg.

9. Use according to any one of claims 1-3 and 5, wherein the lung surfactant and budesonide are administered simultaneously, sequentially or separately.

10. Use according to any of claims 1-3 and 5, wherein the lung surfactant is porcine lung phospholipid.

11. Use according to any one of claims 1-3 and 5, wherein the medicament is administered as a pharmaceutical formulation by inhalation or intratracheal route.

12. The use according to claim 11, wherein the pharmaceutical formulation is in the form of an aqueous suspension comprising a pharmaceutically acceptable carrier.

13. Use according to any one of claims 1-3, 5 and 12, wherein the pulmonary surfactant and budesonide are administered simultaneously as a fixed combination and the pulmonary surfactant and budesonide are a suitable pharmaceutical formulation suspended in a suitable physiologically tolerable solvent.

14. The use according to claim 13, the pharmaceutical formulation comprising an aqueous solution comprising a pH buffer and other pharmaceutically acceptable excipients selected from polysorbate 20, polysorbate 80 or sorbitan monolaurate as wetting agent and sodium chloride as isotonicity agent.