Therapeutic combinations containing pulmonary surfactant and steroids for the treatment of advanced BPD

Abstract

The present invention relates to compositions and methods comprising a pulmonary surfactant in combination with a corticosteroid for use in treating progressive bronchopulmonary dysplasia (BPD) in preterm newborns.

Description

[Technical Field]

[0001] Technical Field The present invention relates to compositions and methods for treating diseases of prematurity. In particular, the present invention relates to the use of pulmonary surfactant in combination with steroids to treat progressive bronchopulmonary dysplasia in premature newborns. [Background technology]

[0002] Background of the Invention The human lung is composed of numerous small air sacs called alveoli that exchange gas between the blood and the lung's air spaces. In healthy individuals, this exchange is mediated by the presence of protein-containing surfactant complexes that prevent the lungs from collapsing at the end of expiration.

[0003] The pulmonary surfactant complex is composed primarily of lipids and contains small amounts of various proteins. Absence of sufficient levels of this complex leads to pulmonary dysfunction, a condition called respiratory distress syndrome (RDS), which commonly affects preterm newborns.

[0004] The mainstay of treatment for RDS is replacement therapy with exogenous pulmonary surfactant preparations extracted from animal lungs, known as processed natural surfactant. For example, processed natural surfactants used in clinical practice include poractant alfa, derived from porcine lung and sold under the trademark Curosurf®, beractant (Surfacten® or Survanta®), bobactant (Alveofact®), and calfactant (Infasurf®), both derived from bovine lung.

[0005] Exogenous pulmonary surfactant is currently administered by intratracheal instillation as a suspension in saline solution to intubated preterm infants maintained under mechanical ventilation with oxygen.

[0006] Although the above-mentioned therapies have significantly increased postnatal survival rates, children who survive RDS are at high risk of developing bronchopulmonary dysplasia (BPD), a common and serious complication of prematurity associated with significant mortality, morbidity, and healthcare resource utilization. Despite advances in both prenatal and neonatal care, the incidence of this condition continues to rise. The management of BPD and its associated problems remains a major challenge for neonatologists and pediatricians. Numerous interventions have been proposed to prevent and treat BPD, but many are not yet evidence-based. Current treatments appear to reduce the severity of BPD but have had little impact on its incidence. BPD is a progressive process of lung injury, and its pathophysiology changes during different stages of the disease. Therefore, its management is unlikely to take the form of a single intervention, but rather a multidisciplinary approach with different strategies used to target different factors and / or stages of the disease.

[0007] For this reason, when designing an overall management plan, it is useful to categorize interventions for BPD in three successive phases: i) prevention of BPD, ii) treatment of progressive BPD, and iii) treatment of established BPD (see Non-Patent Document 1).

[0008] Prevention of BPD in newborns with RDS has been managed with systemic corticosteroids administered before or within the first few hours of birth. However, the effectiveness of postnatal corticosteroid administration is offset by potential adverse systemic effects (e.g., hypertension, hyperglycemia, gastrointestinal complications, and neurodevelopmental disorders).

[0009] As an alternative to systemic administration, delivery of corticosteroids by inhalation or intratracheal instillation has been proposed for the prevention of BDP.

[0010] For example, Patent Document 1 discloses a method for preventing BPD in infants suffering from respiratory distress syndrome by administering to the infant a combination of a corticosteroid with high local to systemic anti-inflammatory activity and a pulmonary surfactant.

[0011] Yeh et al. (Non-Patent Document 2) proposed intratracheal instillation of budesonide using the pulmonary surfactant Survanta® as a carrier, while Dani et al. (Non-Patent Document 3) proposed intratracheal instillation of beclomethasone dipropionate in combination with Cursourf®.

[0012] However, even through these approaches, a large proportion of the preterm neonate population will be exposed to corticosteroids, and many will not benefit, even though they would not otherwise develop BPD (see Non-Patent Document 4).

[0013] However, serious concerns have been raised about the efficacy of corticosteroids in established BDP, as the disease is characterized by intense and persistent airway inflammation, fibrosis, and smooth muscle hypertrophy.

[0014] In this way, corticosteroids are administered to patients in need thereof, and postnatal corticosteroids have been used as a therapy in the treatment of advanced BPD.

[0015] However, due to observed side effects or lack of clear signs of efficacy, systemic postnatal administration of dexamethasone and hydrocortisone is not currently routinely recommended. [Prior art documents] [Patent documents]

[0016] [Patent Document 1] US Patent Application Publication No. 2010 / 0317636 [Non-patent literature]

[0017] [Non-Patent Document 1] Bowen P et al., Pediatrics and Child Health 2013, 24:1, 27-31 [Non-patent document 2] Pediatrics 2008, 121(5), e1310-e1318 [Non-patent document 3] Pediatr Pulmonol 2009, 44, 1159-1167 [Non-patent document 4] Bancalari E, Am J Respir Crit Care Med 2016, 193:1, 12 Summary of the Invention [Problem to be solved by the invention]

[0018] In view of the above considerations, there remains a need to develop more compatible corticosteroid-based medications for the treatment of advanced BPD in preterm newborns.

[0019] Furthermore, it would be advantageous to provide a medicament that can be administered locally, either by inhalation or intratracheal instillation.

[0020] Finally, it would be particularly advantageous to provide a pharmaceutical agent that can promote lung development. [Means for solving the problem]

[0021] Summary of the Invention The present invention relates to a pulmonary surfactant in combination with budesonide in a dose comprised between 0.1 and 1.5 mg / kg for use in the treatment of progressive bronchopulmonary dysplasia (BPD) in preterm neonates.

[0022] Preferably, the combination of the present invention increases the mRNA expression of several protein indicators of lung maturation, more preferably the mRNA expression of surfactant proteins SP-A, SP-B and SP-C.

[0023] Advantageously, the combination is administered from day 2 to day 28 after birth, preferably from day 5 to day 15 after birth, more preferably from day 7 to day 10 after birth.

[0024] The present invention also relates to the use of a pulmonary surfactant in combination with budesonide in a dose comprised between 0.1 and 1.5 mg / kg in the manufacture of a medicament for the treatment of bronchopulmonary dysplasia (BPD) in premature newborns, the combination being advantageously 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.

[0025] Preferably, the dose of budesonide may be comprised between 0.2 and 1.0 mg / kg.

[0026] The medicaments of the present invention may be administered simultaneously, sequentially or separately, preferably simultaneously as a fixed combination.

[0027] In a particular embodiment, the medicament is in the form of a pharmaceutical composition for inhaled or intratracheal administration comprising the fixed combination.

[0028] In a further embodiment, the present invention relates to a method for treating progressive bronchopulmonary dysplasia, comprising administering to a premature newborn in need of such treatment a pulmonary surfactant in combination with budesonide at a dose comprised between 0.1 and 1.5 mg / kg, wherein the combination is administered from day 2 to day 28 of life, preferably from day 5 to day 15 of life, and more preferably from day 7 to day 10 of life.

[0029] definition The term "bronchopulmonary dysplasia (BPD)" refers to a chronic lung disorder, also known as chronic lung disease (CLD), that is the result of unresolved or abnormal repair of lung injury.

[0030] BPD typically occurs in very low birth weight (VLBW) infants who sustain lung injury as a result of oxygen toxicity and barotrauma caused by mechanical ventilation early after birth. The definition and classification of BPD have changed since its original description by Northway et al. in 1967. The National Institute of Child Health and Human Development (NICHD) defined BPD in a 2001 consensus statement. This definition uses supplemental oxygen for 28 days, followed by three levels of severity depending on the respiratory support required at 36 weeks postmenstrual age (PMA) or at hospital discharge for those born before 32 weeks' gestation, or at 56 days of age or at hospital discharge for those born after 32 weeks' gestation.

[0031] According to more recent definitions, BPD can be thought of primarily as a blockage of lung development (see Jobe A et al Ped Res 1999, 46, 641).

[0032] In 2001, Jobe A et al. (Am J Respir Crit Care Med; 163(7) 1723-1729) proposed a new definition that included specific criteria for "mild," "moderate," and "severe" BPD.

[0033] Mild BDP is defined as disease requiring supplemental oxygen for 28 days or more and room air at 36 weeks PMA or at discharge (for infants younger than 32 weeks at birth) or at 56 days or at discharge (for infants 32 weeks or older at birth).

[0034] Moderate BDP is defined as disease requiring supplemental oxygen for 28 days or more and less than 30% supplemental oxygen requirement at 36 weeks PMA / discharge (for infants under 32 weeks) or 56 days / discharge (for infants 32 weeks or older).

[0035] Severe BPD is defined as disease requiring supplemental oxygen for 28 days or more and 30% or more oxygen or nasal CPAP or mechanical ventilation at PMA 36 weeks / discharge (for those under 32 weeks) or at 56 days / discharge (for those over 32 weeks).

[0036] The term "progressive BPD," sometimes known as early BPD, refers to the early stages of the chronic process that leads to established BPD and refers to the disease characterized by oxygen and / or ventilator dependence between the 7th and 14th days of life (Walsh MC et al Pediatrics 2006, 117, S52-S56).

[0037] The term "processed natural surfactant" refers to a lipid extract of minced mammalian lungs. The lipid extraction process used in the manufacturing process results in the loss of the hydrophilic proteins SP-A and SP-D. 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.

[0038] The term "poractant alpha" refers to a processed natural surfactant extracted from pig lungs that consists essentially of polar lipids (primarily phospholipids) and the proteins SP-B and SP-C. Poractant alpha is available under the trademark Curosurf®.

[0039] The term "artificial" pulmonary surfactant refers to a simple mixture of synthetic compounds (mainly phospholipids and other lipids) formulated to mimic the lipid composition and behavior of natural pulmonary surfactants. They do not contain pulmonary surfactant proteins.

[0040] The term "reconstituted" pulmonary surfactant refers to an artificial pulmonary surfactant to which has been added pulmonary surfactant proteins / peptides isolated from animals, or proteins / peptides produced via recombinant technology such as those described in WO 95 / 32992, or synthetic pulmonary surfactant protein analogs such as those described in WO 89 / 06657, WO 92 / 22315 and WO 00 / 47623.

[0041] The term "noninvasive ventilation (NIV)" defines a ventilation modality that supports breathing without the need for intubation, such as nasal continuous positive airway pressure (nasal CPAP). Other noninvasive ventilation techniques are nasal intermittent positive pressure ventilation (NIPPV), high-flow nasal cannula (HFNC), and bilevel positive airway pressure (BiPAP).

[0042] The term "respiratory support" includes any intervention to treat respiratory disorders, including, for example, administration of supplemental oxygen, mechanical ventilation, and nasal CPAP.

[0043] The term "treatment" refers to use to cure, alleviate, or reduce the symptoms of a disease or condition (e.g., BPD) in a patient.

[0044] The term "prophylaxis" refers to use to slow the progression and / or delay the onset of a disease or condition (e.g., BPD) in a patient.

[0045] The term "preterm newborn", or preterm newborn, includes extremely low birth weight (ELBW), very low birth weight (VLBW) and low birth weight (LBW) newborns between 24 and 35 weeks of gestation.

[0046] The term "fixed combination" means a combination in which the active substances are in a fixed ratio of amounts.

[0047] "Pharmaceutically acceptable" is a term used herein to mean a medium that does not produce allergic or similar untoward reactions when administered to an infant.

[0048] "Surfactant activity" for surfactant preparations is defined as the ability to reduce surface tension.

[0049] The in vitro efficacy of exogenous surfactant preparations is commonly tested by measuring their ability to reduce surface tension using suitable devices such as a Wilhelmy balance, a Pulsating Bubble Surfactometer, a Captive Bubble Surfactometer, and a Capillary Surfactometer.

[0050] The in vivo efficacy of exogenous surfactant formulations is tested by measuring pulmonary mechanics in a preterm animal model according to known methods.

[0051] In the context of this specification, the term "synergistic" means that the activity of the pulmonary surfactant plus the activity of budesonide is greater than would be expected by the activity of either the surfactant or budesonide alone.

[0052] By the term "biosimilar of poractant alfa" is meant a processed natural pulmonary surfactant that is therapeutically equivalent to poractant alfa but has at least 80% compositional similarity with respect to poractant alfa and a viscosity of less than 15 mPas (cP) at room temperature when poractant alfa is suspended in aqueous solution at a concentration of 80 mg / ml. Viscosity can be determined according to known methods. [Brief explanation of the drawings]

[0053] [Figure 1] A protocol scheme for this complex series of interventions [Figure 2] Oxygen load [Figure 3] Static lung volume at 40 cm H2O measured from pressure-volume loops [Figure 4] lung weight [Figure 5] Lung volume relative to lung weight [Figure 6] Lung wet / dry weight ratio [Figure 7] mRNA indicators of lung maturation: SP-A, SP-B, and SP-C DETAILED DESCRIPTION OF THE INVENTION

[0054] Detailed Description of the Invention The present invention is based in part on the unexpected discovery that budesonide in doses comprised between 0.1 mg / kg and 1.5 mg / kg, in combination with a pulmonary surfactant, such as poractant alfa, can treat progressive bronchopulmonary dysplasia (BPD) without altering the surface activity of the surfactant.

[0055] The benefits of combining a pulmonary surfactant with budesonide at the claimed doses become apparent from the following findings.

[0056] Quite surprisingly, in a study of preterm lambs with RDS receiving nasal CPAP ventilation, it was found that pulmonary surfactants such as poractant alfa in combination with budesonide significantly increased the mRNA expression of several protein indicators of lung maturation, whereas, unexpectedly, pulmonary surfactant alone caused a decrease in such mRNA expression.

[0057] The addition of budesonide also significantly increases lung volume as well as decreases lung weight compared to pulmonary surfactant alone.

[0058] The loss of lung weight is in turn associated with loss of fluid, which indicates a loss of mesenchymal cells and a maturation response.

[0059] Furthermore, the addition of budesonide to surfactant reduces airway wall thickness and collagen deposition, both indicators of a lack of lung maturity.

[0060] Because budesonide is a highly lipophilic corticosteroid, this may favor its mucosal absorption and uptake across phospholipid cell membranes while minimizing systemic absorption, making the combination safe for treatment use in preterm neonates.

[0061] On the other hand, pulmonary surfactant may favor the diffusion of corticosteroids due to the Marangoni effect, which favors their distribution and therefore the reach of all involved lung regions.

[0062] Any pulmonary surfactant currently in use or hereafter developed for use in respiratory distress systems and other pulmonary conditions may be suitable for use in the present invention, including processed natural, artificial, and reconstituted pulmonary surfactants.

[0063] Current engineered natural pulmonary surfactants include, but are not limited to, bovine lipid pulmonary surfactant (BLES™, BLES Biochemicals, Inc., London, Ont.), calfactant (Infasurf™, Forest Pharmaceuticals, St. Louis, Mo.), bovacant (Alveofact™, Thomae, Germany), bovine pulmonary surfactant (Pulmonary Surfactant TA™, Tokyo Tanabe, Japan), poractant alfa (Curosurf™, Chiesi Farmaceutici SpA, Parma, Italy), and beractant (Survanta™, Abbott Laboratories, Inc., Abbott Park, 111).

[0064] Examples of reconstituted surfactants include the compositions disclosed in EP 2152288, WO 2008 / 011559, WO 2013 / 120058, the product lucinactant (Surfaxin™, Windtree-Discovery Laboratories Inc., Warrington, Pa.), and the product having the composition disclosed in Table 2 of Example 2 of WO 2010 / 139442, i.e. 1.5% SP-C33(leu) acetate; 0.2% Mini-B(leu) acetate; and DPPC:POPG in a 50:50 weight ratio These include, but are not limited to:

[0065] The pulmonary surfactant selected for use in the medicament of the present invention may be the same as or different from the pulmonary surfactant utilized in RDS, in a preferred embodiment the same pulmonary surfactant is used.

[0066] In a preferred embodiment, the pulmonary surfactant is an engineered natural pulmonary surfactant.

[0067] More preferably, the pulmonary surfactant is poractant alfa (Curosurf™) or a biosimilar thereof as defined above, since it is endowed with a very low viscosity and can therefore be administered in high concentrations using a small amount of aqueous carrier.

[0068] In another embodiment, the pulmonary surfactant is a reconstituted surfactant having a composition disclosed in Table 2 of Example 2 of WO 2010 / 139442.

[0069] The dose of pulmonary surfactant to be administered will vary depending on the weight and gestational age of the preterm newborn, as well as the severity of the newborn's condition. Those skilled in the art can readily determine these factors and adjust the dose accordingly.

[0070] Advantageously, the dose of pulmonary surfactant could be between 100 and 200 mg / kg.

[0071] In a preferred embodiment of the present invention, a dose comprised between 100 and 200 mg / kg of poractant alfa may be used.

[0072] In a preferred embodiment, the dose could be 100 mg / kg, while in another preferred embodiment, the dose could be 200 mg / kg.

[0073] Advantageously, the dose of budesonide is comprised between 0.1 and 1.5 mg / kg, more advantageously between 0.2 and 1.0 mg / kg, and even more advantageously between 0.25 and 1.0 mg / kg.

[0074] In certain embodiments, where the effect on lung maturation is primarily sought, the dose of budesonide may be comprised between 0.1 and 0.5 mg / kg, while in other embodiments the dose of budesonide may be comprised between 0.5 and 1.0 mg / kg.

[0075] Preferably, the combination of the present invention is administered to preterm newborns maintained under non-invasive ventilation at a pressure of 1 to 12 cm of water, more preferably maintained under nasal CPAP, even more preferably with a nasal device.

[0076] The claimed dosage combination of pulmonary surfactant and budesonide can be administered sequentially, separately, or together. Advantageously, when the two active agents are administered together, they are administered as a fixed combination.

[0077] Thus, the present invention also relates to the use of a combination of the invention as a fixed combination in the manufacture of a medicament for treating progressive BPD.

[0078] The medicament may be in the form of a pharmaceutical composition.

[0079] The formulations may be administered in the form of a solution, dispersion, suspension, or dry powder. Preferably, the compositions comprise the claimed combination suspended in a suitable physiologically acceptable solvent.

[0080] More preferably, formulations comprise aqueous solutions, preferably sterile, which may also contain pH buffering agents and other pharmaceutically acceptable excipients (e.g., polysorbate 20, polysorbate 80, or sorbitan monolaurate as a wetting agent, and sodium chloride as an isotonicity agent).

[0081] The formulations may be dispensed in unit-dose or multi-dose containers, for example, sealed ampoules and vials, and may be stored frozen or freeze-dried (lyophilized) requiring only the addition of the sterile liquid carrier immediately prior to use.

[0082] Preferably, the formulation is supplied as a sterile suspension in a buffered saline (0.9% w / v sodium chloride) aqueous solution in a single-use vial.

[0083] Administration of the claimed formulations can be carried out according to known methods, such as by intratracheal instillation, by spray administration, or by inhalation with jet ultrasound or commercially available mesh vibrating nebulizers.

[0084] When the formulation is administered by intratracheal instillation, different methods may be appropriate depending on the severity of the respiratory distress syndrome. For example, the claimed formulation may be administered to preterm newborns maintained under mechanical ventilation through an endotracheal tube.

[0085] Alternatively, the formulation may be administered using a thin catheter placed in the trachea and neonatal breathing supported through a specialized nasal device such as a mask, prongs, or tube according to the methodology known as nasal continuous positive airway pressure (nCPAP), in accordance with the procedures described in WO 2008 / 148469.

[0086] The latter approach would only be possible with exogenous surfactants such as poractant alfa, which have low viscosity, since high viscosity would make passage of the surfactant through a thin catheter more difficult.

[0087] The volume of aqueous solution in which the two combined active substances are suspended depends on the desired concentration.

[0088] Advantageously, the volume of the formulation should be less than or equal to 5.0 ml, preferably comprised between 4.5 and 2.0 ml, more preferably between 3.5 and 2.5 ml.

[0089] In other embodiments, when the pulmonary surfactant and budesonide are administered separately, the individual active agents may be formulated separately, in which case the two individual active agents do not necessarily have to be taken at the same time.

[0090] In the case of such separate administration, the formulations of the two individual active agents may be packaged together in one suitable container means. Such separate packaging of the components in suitable container means is also described as a kit.

[0091] Accordingly, the present invention also relates to a kit for treating progressive bronchopulmonary dysplasia, comprising: a) a pulmonary surfactant in a dose comprised between 100 and 200 mg / kg in a first unit dosage form and a pharmaceutically acceptable carrier or diluent; b) budesonide in a dose comprised between 0.1 and 1.5 mg / kg in a second unit dosage form and a pharmaceutically acceptable carrier or diluent; and c) container means for containing the first and second dosage forms.

[0092] The combinations of the present invention, which may be administered to premature newborns after birth according to conditions to be established by one skilled in the art, are suitable for treating any form of progressive bronchopulmonary dysplasia.

[0093] The frequency of administration will vary depending on the size and gestational age of the premature newborn, as well as the severity of the newborn's condition and the route of administration, and can be readily determined by one skilled in the art.

[0094] For example, the medicament of the present invention could be administered once or twice daily.

[0095] Advantageously, the combination of the invention is administered from day 2 to day 28 after birth, preferably from day 5 to day 15 after birth, more preferably from day 7 to day 10 after birth.

[0096] Within the above time intervals, the treatment may be continued for as long as a physician or other medical practitioner determines is appropriate to achieve a therapeutic effect.

[0097] A premature newborn in need of the medicament of the present invention may or may not exhibit respiratory distress syndrome (RDS). In one embodiment, administration of the medicament of the present invention is initiated in a newborn exhibiting RDS after treatment of such syndrome with pulmonary surfactant or by other means (e.g., ventilation), or a combination thereof.

[0098] In certain embodiments, the neonates to be treated with the medicaments of the present invention require respiratory assistance but do not necessarily exhibit respiratory distress syndrome. These infants have not been diagnosed with RDS or treated with pulmonary surfactant for RDS.

[0099] All preterm newborns, including extremely low birth weight (ELBW), very low birth weight (VLBW), and low birth weight (LBW) newborns with a gestational age of 24 to 35 weeks, may be eligible for administration of the medicament of the present invention. Preferably, the medicament is administered to VLBW newborns who suffer from severe RDS, which will have a higher incidence of BPD.

[0100] Broadly speaking, because management of progressive BPD is unlikely to be a single form of intervention, but rather a multidisciplinary approach, the internist will also assess whether the preterm neonate requires concomitant respiratory support and / or other appropriate medications (e.g., vitamin A and antibiotics).

[0101] The following examples further illustrate the invention. [Example]

[0102] Example 1 - In vitro evaluation of the surface activity of poractant alfa in the presence of budesonide using a capillary surface tensiometer

[0103] The surface activity of poractant alfa (2 ml, in the presence of 1.0 mg budesonide) compared to poractant alfa alone is assessed using a capillary surface tensiometer commercially available from Calmia Medical, Inc., USA.

[0104] Two samples are prepared: one from a vial of poractant alfa (1.5 ml, 80 mg / ml) diluted with saline to a concentration of 1 mg / ml in phospholipids, and the other from a vial of poractant alfa (1.5 ml, 80 mg / ml) mixed with a vial of budesonide (2 ml, 1.0 mg) and diluted to the same concentration (1 mg / ml phospholipid) with saline. 0.5 ml samples of both solutions are then evaluated in a capillary tensiometer.

[0105] The principle of the capillary surface tensiometer is to simulate the human small airways. A sample is introduced into the narrow end of a glass capillary with an inner diameter of 0.25 mm, similar to that of a small human airway. At one end, the capillary is connected to a bellows and a pressure transducer. By slowly compressing the bellows, the pressure increases and is recorded. The increasing pressure pushes the sample out of the narrow end of the capillary. As air passes through, the pressure suddenly decreases. If the sample contains a well-functioning pulmonary surfactant, the sample liquid does not return to the narrow end. The steady airflow obtained by continuously compressing the bellows is not resisted, and a pressure of zero is recorded. On the other hand, if the sample does not contain a well-functioning pulmonary surfactant, the sample liquid repeatedly returns.

[0106] The behavior of poractant alfa in the presence of budesonide was found to be statistically indistinguishable from that of poractant alfa alone, indicating that budesonide at these doses does not affect the surface activity of the surfactant.

[0107] Example 2 - In vivo evaluation of the activity of poractant alfa in the presence of budesonide in a lamb model of BPD

[0108] A study on neonatal resuscitation and lung injury using surfactant plus budesonide treatment to reduce lung injury was conducted to determine whether stretch injury to the fetal lung modulates second ventilation-mediated injury after 24 hours of intrauterine recovery.

[0109] This was a study to test for the possibility of fetal lung preconditioning or tolerance responses. Treatment with surfactant with or without budesonide after the initial stretch injury was intended to test whether this steroid has an anti-inflammatory effect that protects the fetal lung. Animal groups included CPAP exposure and no initial exposure for comparison with the stretch injury. These groups and their characteristics and treatments are shown in Table 1. The legend to Table 1 provides details about the interventions. Figure 1 is a schematic diagram of the protocol for this complex series of interventions.

[0110] [Table 1]

[0111] Table Description · CPAP: Animals were placed on 5 cm H2O CPAP for 15 minutes as a control for anesthesia and surgery related to the injury intervention.

[0112] Fetal Lung Injury Intervention: Yes = Animal's head and thorax are exposed; a 4.5 mm endotracheal tube is secured to the trachea. The fetus is then ventilated with 100% humidified nitrogen at R=30, IT=1 sec, PEEP=0, to a maximum pressure of 55 cm H2O. The goal is an estimated V of 7 ml / kg at 4 min, 12 ml / kg at 8 min, and 15 ml / kg at 12 min. t The total ventilation period was 15 minutes.

[0113] Pulmonary surfactant (Surf): After CPAP or fetal lung injury intervention, animals were treated with 100 mg / kg of Curosurf, assuming a body weight of 3 kg. Curosurf + budesonide was diluted to 10 ml with saline. Surfactant was administered via an endotracheal tube and mixed with fetal lung fluid via a syringe. After surfactant treatment, the trachea was ligated to prevent surfactant loss.

[0114] Budesonide (Bud): Pulmicort Respules (Astra Zeneca, Sweden), containing 0.5 mg of micronized budesonide per ml, was mixed with Curosurf plus saline to deliver 0.25 or 1.0 mg / kg budesonide and surfactant in a 10 ml suspension.

[0115] 24-hour ventilation study: The animal's head was re-exposed and a 4.5 mm endotracheal tube was placed. Fetal lung fluid was aspirated with a syringe, and the lamb was delivered and ventilated at a rate of 40, with an inspiratory time of 0.45 seconds, a PEEP of 5 cm H2O, and a maximum peak inspiratory pressure of 40 cm H2O with 100% humidified oxygen.

[0116] The experiment was designed for 46 fetal lambs; one ewe had no fetuses and one set of twins was a singleton, resulting in a final total of 44. The experimental procedure was successful for all other lambs. The number of animals per group was V to increase the statistical power of the groups. t Adjustments were made to increase the number of animals in the 15 injury and surfactant groups. Specific comments regarding important elements of the experimental design follow.

[0117] Ventilatory injury, targeting an estimated tidal volume of 15 ml / kg at 15 min, only achieved a volume of 11–13 ml / kg despite the use of a maximum pressure of 55 cm H2O, indicating that the fetal lungs were immature and surfactant deficient.

[0118] ·V t The injured and CPAP groups (estimated birth weight 3 kg) received 100 mg / kg Curosurf or Curosurf + budesonide diluted to 10 ml with saline. The trachea was ligated after treatment to ensure the treatment remained in the lungs for 24 hours in utero before assessment of pulmonary function.

[0119] Budesonide was used as 0.5 mg / ml Pulmicort Respules to obtain a standardized, sterile product for exposure of the fetal lungs.

[0120] At delivery, 24 hours after the initial intervention and procedure, an endotracheal tube was placed and all free-flowing fetal lung fluid was withdrawn with a syringe. A large amount of fluid was recovered from the lungs exposed to CPAP. There was no fetal lung fluid, only a small amount of thick secretions. t Aspirated from injured lung.

[0121] The 30-minute postpartum ventilation period was successful in all ventilated lambs. Although some lungs had air collections within the lung tissue and pleural blebs, the majority of pressure-volume curves were successful.

[0122] result There were no significant differences in gestational age or birth weight between groups (Table 1). Analysis of these experiments is complicated by the presence of seven groups. None + None animals are used only for tissue collection for baseline measurements.

[0123] Oxygen load The results are reported in Figure 2.

[0124] Oxygen tolerance, measured as arterial PO2 while the animals were ventilated with 100% oxygen, was very low, averaging 54 mmHg, in intact, non-surfactant-treated ventilated twins exposed to anesthesia only 24 hours before ventilation. This result demonstrates that the lambs had immature lungs before the intervention. Injury, whether with or without budesonide, abolished the oxygen tolerance response. A 15-minute CPAP plus surfactant treatment resulted in a significant increase in mean PO2 to 380 mmHg, and the addition of budesonide resulted in a mean PO2 of 417 mmHg, indicating that the addition slightly increased oxygen tolerance.

[0125] Although significantly lower in the non-ventilated group, a slight trend towards improvement in the presence of increased concentrations of budesonide can be seen from a comparison of the medians of the groups.

[0126] 40 cm H measured from the pressure-volume curve 2 Static lung volumes in O The results are reported in FIG.

[0127] Maximum lung volume, measured relative to body weight after oxygen adsorption for inflation measurements in air-free lungs, was lower in the ventilated twins. The CPAP + surfactant group showed a greater increase in lung volume / kg body weight, and budesonide significantly increased lung volume compared with CPAP + surfactant. Regarding oxygen loading, V t 15 In injured animals, lung volumes were not significantly affected.

[0128] lung weight The results are reported in FIG.

[0129] A surprising large variation in lung weight between groups was observed at necropsy. Lung weights were similar for ventilated twins, Vt15 injury + surfactant, and CPAP + surfactant. However, lung weights were lower in each budesonide group, with the 1.0 mg / kg budesonide group showing the greatest effect. The observed very large lung weight changes are rather unexpected, as large changes in lung weight due to fetal exposure to maternal steroids have never been measured in other experiments.

[0130] Lung volume relative to lung weight The results are reported in FIG.

[0131] Notably, lung weight decreased in the CPAP + surfactant + budesonide group compared to the CPAP + surfactant group, but lung volume increased. Thus, the ratio of lung volume to lung weight is shown. This ratio highlights the combined effect of lighter lungs holding more gas. CPAP + budesonide lungs held more gas than CPAP lungs. This ratio also correlates with V t This indicates that lungs exposed to 15+ surfactant retained less gas than those exposed to lung budesonide.

[0132] Lung wet / dry weight ratio The results are reported in FIG.

[0133] These large changes in lung weight associated with budesonide must be primarily due to loss of lung fluid over the 24 hour period, which could represent a loss of mesenchymal cells and maturation responses.

[0134] mRNA indicators of lung maturation mRNA was analyzed for surfactant proteins (SP) A, B, C, and D.

[0135] It is well known that the expression of surfactant proteins is a sign of lung maturation.

[0136] The results of SP-A, SP-B, and SP-C are reported in Figure 7 .

[0137] There was a consistent decrease in mRNA for the CPAP + surfactant group compared to unmanipulated controls and ventilated twins. This suppression of surfactant protein mRNA is quite unexpected. Interestingly, the combination of CPAP + surfactant with budesonide significantly increased surfactant protein.

[0138] Example 3 - Formulation in the form of an aqueous suspension according to the invention.

[0139] [Table 2]

[0140] Example 4 - Measurement of airway thickness The appearance and thickness of large and small airways are known to be altered by both CPAP and mechanical ventilation.

[0141] In particular, in the absence of proper lung maturation, increased wall thickening and increased collagen deposition are observed.

[0142] Therefore, the thickness of large airways / bronchioles and collagen deposition were measured in lambs exposed to hyperoxia according to the method reported in Wang H et al. Am J Physiol. Lung Cell Mol Physiol 2014,307,L295-L301.

[0143] Thickness measurements were made on blinded sections using three measurements per slide, five slides per animal, and four to six animals per group.

[0144] The results are reported in Table 2.

[0145] Mechanically ventilated animals had thickened small and large airways. Furthermore, quantification of collagen within the airways showed increased collagen staining in mechanically ventilated animals.

[0146] In contrast, a decrease was observed in the CPAP + surfactant + budesonide group, and was particularly pronounced in the CPAP + surfactant + budesonide 0.25 mg / kg group.

[0147] [Table 3]

Claims

1. 1. A pharmaceutical composition for the treatment of progressive bronchopulmonary dysplasia (progressive BPD) in preterm newborns, comprising: Pulmonary surfactant at a dose of 100 to 200 mg / kg (preterm newborn weight) Budesonide at a dose of 0.1 to 0.5 mg / kg (preterm newborn weight) Including, The pharmaceutical composition is administered once or twice daily from day 2 to day 28 after birth to the premature newborn.

2. The pharmaceutical composition of claim 1, which increases the mRNA expression of proteins SP-A, SP-B and SP-C.

3. 3. The pharmaceutical composition of claim 1 or 2, which is administered once or twice daily from day 5 to day 15 after birth of the preterm neonate.

4. 3. The pharmaceutical composition of claim 1 or 2, which is administered once or twice daily from day 7 to day 10 after birth of the preterm neonate.

5. The pharmaceutical composition according to any one of claims 1 to 4, wherein the premature newborn is maintained under non-invasive ventilation.

6. The pharmaceutical composition of claim 5, wherein the non-invasive ventilation is nasal CPAP.

7. The pharmaceutical composition according to any one of claims 1 to 6, wherein the pulmonary surfactant and budesonide are administered simultaneously, sequentially, or separately.

8. The pharmaceutical composition according to any one of claims 1 to 7, wherein the pulmonary surfactant is poractant alfa.

9. A pharmaceutical composition according to any one of claims 1 to 8, in the form of an aqueous suspension comprising a pharmaceutically acceptable carrier.

10. The pharmaceutical composition according to any one of claims 1 to 9, which is administered by inhalation or intratracheal route.

11. A pH buffer; other pharmaceutically acceptable excipients selected from polysorbate 20, polysorbate 80 or sorbitan monolaurate as wetting agents; Sodium chloride as an isotonic agent 11. The pharmaceutical composition of claim 10, comprising an aqueous solution comprising:

12. In the manufacture of a medicament for the treatment of progressive bronchopulmonary dysplasia (progressive BPD) in preterm newborns, Use of a pulmonary surfactant and budesonide, wherein the medicament is Pulmonary surfactant at a dose of 100 to 200 mg / kg (preterm newborn weight) Budesonide at a dose of 0.1 to 0.5 mg / kg (preterm newborn weight) Including, The use wherein the medicament is administered once or twice daily from day 2 to day 28 of life of the premature newborn.

13. In the manufacture of a medicament for the treatment of progressive bronchopulmonary dysplasia (progressive BPD) in preterm newborns, Use of a pulmonary surfactant, where The medicament comprises a pulmonary surfactant at a dose of 100 to 200 mg / kg (weight of preterm newborn), The medicament is administered separately, simultaneously, or sequentially with budesonide at a dose of 0.1 to 0.5 mg / kg (weight of the preterm newborn) once or twice daily from day 2 to day 28 of the preterm newborn's life. use.

14. In the manufacture of a medicament for the treatment of progressive bronchopulmonary dysplasia (progressive BPD) in preterm newborns, Use of budesonide, where The medicament comprises budesonide at a dose of 0.1 to 0.5 mg / kg (weight of preterm newborn), The medicament is administered separately, simultaneously, or sequentially with a pulmonary surfactant at a dose of 100 to 200 mg / kg (weight of the preterm newborn) once or twice daily from day 2 to day 28 after birth of the preterm newborn; use.

15. The use according to any one of claims 12 to 14, wherein the medicament is administered once or twice daily from day 5 to day 15 of life of the premature newborn.

16. The use according to any one of claims 12 to 14, wherein the medicament is administered once or twice daily from day 7 to day 10 after birth of the premature newborn.

17. The use according to any one of claims 12 to 16, wherein the preterm newborn is maintained under non-invasive ventilation.

18. 18. The use according to claim 17, wherein the non-invasive ventilation is nasal CPAP.

19. The use according to any one of claims 12 to 18, wherein the pulmonary surfactant is poractant alfa.

20. The use according to any one of claims 12 to 19, wherein the medicament is in the form of an aqueous suspension comprising a pharmaceutically acceptable carrier.

21. The use according to any one of claims 12 to 20, wherein the medicament is administered by inhalation or intratracheal route.

22. The medicine A pH buffer; other pharmaceutically acceptable excipients selected from polysorbate 20, polysorbate 80 or sorbitan monolaurate as wetting agents; Sodium chloride as an isotonic agent 22. The use of claim 21, comprising an aqueous solution comprising:

23. a) a pulmonary surfactant in a dose comprised between 100 and 200 mg / kg of patient body weight in a first unit dosage form and a pharmaceutically acceptable carrier or diluent; b) budesonide in a dose of 0.1 to 0.5 mg / kg patient weight and a pharmaceutically acceptable carrier or diluent in a second unit dosage form; c) container means for containing said first and second dosage forms; and for treating progressive bronchopulmonary dysplasia (progressive BPD) in a patient, comprising: kit.

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