Field of the Invention

The invention relates to the use of CDP-choline (cytidine diphosphate choline, citicoline) in the prevention and treatment of Bronchopulmonary Dysplasia (BDP).

The invention particularly relates to the use of CDP-choline, an endogenous molecule, and of a pharmaceutically acceptable salt of the same for applying to pregnant women with threatened premature labor in the health sector, in the pharmaceutical industry, in the clinic, and the use of the same in the preparation of a drug for the prevention and/or treatment of Bronchopulmonary Dysplasia, an important cause of morbidity in premature babies with no definitive treatment and a subject of several studies.

Present State of the Art

Although Bronchopulmonary dysplasia (BPD) is one of the most common long-term complications of preterm birth, it is one of the major causes of morbidity and mortality in premature babies with its short and long term effects (Bhandari and McGrath, 2013). BPD development is a complex process which is influenced by many factors. Risk factors effective in the formation of BPD are prematurity, mechanical ventilation, hyperoxia, inflammation, antenatal factors, fluid overload, genetic effects and nutrition. Today, most of the approaches used in the treatment of BPD are directed to reducing the symptoms and they do not eliminate the disease. Appropriate mechanical ventilation methods, oxygen therapy, vasodilators, bronchodilators, diuretics, postnatal steroids, nitric oxide and antioxidant treatments as well as nutritional treatments are used. Since treatment methods have some side effects, treatment strategies of babies with BPD are determined by considering the severity of the disease and individual differences. However, despite all these options, none of the treatment strategies is effective by itself, and essentially the studies are carried out in order to prevent BPD development.

Studies with regard to the prevention of BPD most importantly focuses on pregnancy period, in other means, the prenatal period. Although today, antenatal steroid (betamethasone) reduces the frequency of Respiratory Distress Syndrome (RDS) at birth in premature babies by being administered to pregnant women who are likely to have premature labor between the 23-34 gestational week (GH), it is known that it does not have sufficient effectiveness in the prevention of BPD. Fetal lung development is accelerated pharmacologically with antenatal steroid administration.

Dexamethasone and betamethasone are used as active ingredients for fetal lung maturation. Although it is shown in the observational studies that the risk of cystic periventricular leukomalacia increases in babies of pregnant women using dexamethasone, it has been reported according to the recently published meta-analysis that intraventricular bleeding is less with the use of dexamethasone, and in this case, no recommendation can be made regarding the choice of steroid preparation containing the active substance (Baud et al., 1999; Brownfoot et al., 2008; Jobe and Soil, 2004).

Uncertainty about repeating antenatal steroid course still continues. It has been reported that 7 days after the first cycle, repeated doses to pregnant women who are at risk of preterm delivery reduce the risk of RDS in the first week after delivery, but these babies are born with a lower birth weight and it has no benefit in early childhood (Crowther et al., 2011).

In conclusion, the following is recommended in terms of antenatal steroid applications in the 2011 guideline of the American Association of Obstetricians and Gynecologists (ACOG Committee, 2011):

The steroid preparations to be used,

Betamethasone (12 mg), intramuscular, in two doses 24 hours-interval or

Dexamethasone (6 mg), intramuscular, in four doses 12 hours-interval or

- Administering a single course of antenatal steroids to pregnant women who are at risk of preterm birth within seven days at 24 ^th - 34 ^th weeks of gestation,

- Administering a single course of antenatal steroid to pregnant women with premature rupture of membranes before 32 ^nd week of gestation so as to reduce the risk of RDS, perinatal mortality and other morbidities,

Below 32 ^6/7 weeks of gestation, if more than two weeks have passed since the previous steroid cycle and if it is thought that the birth will take place one week later, a rescue dose of antenatal steroid can be administered. However, it was reported that antenatal steroid therapy applied with all these indications and approaches has no effect on BPD, although it reduces important morbidities such as RDS, necrotizing enterocolitis, intraventricular hemorrhage, sepsis in the first 48 hours, need for mechanical ventilation and death, according to the most recent Cochrane metaanalysis (Roberts D, Brown J, Medley N, Dalziel SR Cochrane Database Syst 2017; 3(3): CD004454).

Although postnatal use of glucocorticoids initially reduced the incidence of BPD, it has been removed from routine practice due to unwanted side effects in the long term (Shah et al., 2003). It is recommended to be administered systemically only after 3 weeks and as a short-term treatment in the treatment of BPD. The neurodevelopmental delay it causes is thought to be due to early and long-term use (Tin and Wiswell, 2008).

Nutritional deficiency in preterm babies negatively affects lung development and repair mechanisms. Nutritional supplements, protein and fatty acids should be supported so as to ensure sufficient growth in preterm infants (Carlo and Ambalavanan, 2011). Vitamin A levels are low in preterm infants (Dani and Poggi, 2012). It has been reported that the development of BPD decreased by 17% in preterms given vitamin A (Kennedy et al., 1997). When vitamin A is given orally to preterm infants, it was observed that no benefit is provided in reducing the incidence of BPD (Wardle etal., 2001).

In cases that preterm newborns with BPD are exposed to normal or excess hydration, they have difficulty tolerating it. In such cases, pulmonary edema caused by fluid overload can be reduced by administering short-term diuretic therapy to infants. Furosemide, which is used as a diuretic in the clinic, is administered as 1 mg/kg/dose intravenously (i.v.) twice a day or 2 mg/kg/dose orally for once. There shall be fluid and salt limitation in the treatment of preterm newborns, but sufficient fluid intake should be provided for the energy and metabolic needs required for growth (Bancalari, 2011). Long-term use of diuretics is also associated with various problems. After excessive diuretic use, side effects such as hyponatremia, hypokalemia, hypocalcemia, alkalosis, azotemia, and hypercalciuria can be observed (Carlo and Ambalavanan, 2011).

Airway hyper-reactivity and hypertrophy of airway smooth muscle are observed in preterm newborns with BPD. Airway resistance can be reduced by relaxing bronchial smooth muscle cells by bronchodilator application. Despite the side effects of beta 2 agonists like albuterol such as hypertension and tachycardia, it is frequently used in the clinic and shows its effect within 4-6 hours. The most commonly used anticholinergic drug is ipratropium bromide. Using these two drugs in combination is more effective than their effects alone and the side-effect profile is reduced (Carlo and Ambalavanan, 2011). Theophylline and caffeine, which are among systemically used methylxanthines, stimulate respiration with bronchodilation and exhibit mild diuretic and anti-inflammatory effects (Millar and Schmidt, 2004).

One of the important applications in the prevention and treatment of BPD is ventilation strategies. It is known that BPD begins with the first positive pressure applied in the delivery room in premature infants who do not have sufficient respiratory effort and need resuscitation. Measurable positive end-expiratory pressure and positive airway pressure (CPAP) application is considered as the most appropriate approach with high evidence value in order to minimize barotrauma, volutrauma, atelectotrauma and surfactant inactivation as a result of mechanical ventilation in the delivery room (Morley et al., 2008). Since CPAP application is more gentle and less invasive than other mechanical ventilation techniques, it is thought that it can reduce BPD (Narendran et al., 2003). Some researchers suggested that exogenous surfactant treatment should be initially administered and then treatment continued with CPAP in premature infants with respiratory distress. The blood gases should be monitored and targeted blood gas values should be kept in the range of pH 7.25-7.45, PaOs 60-80 mmHg and PaCOs 45-65 mmHg in order to prevent oxygen toxicity and reduce baro/volutrauma in newborns requiring PBV (Atici and Ozkan, 2011).

BPD develops as a result of alveolar hypoxemia due to the deterioration of ventilation and perfusion balance. The vascular development of the lung is impaired, and the oxygen amount of the perivascular areas of the lung decreases. Pulmonary artery pressure increases as a result of alveolar hypoxemia in infants with BPD, it was reported that while pulmonary hypertension, cor pulmonale, and right ventricular failure is observed, somatic development and brain development slows down (Nievas and Chernick, 2002). The present method is to administer oxygen with a nasal cannula while the baby is awake, such that arterial oxygen saturation (SaOs) is between 90-95% (Poets and Southall, 1994). If the infant has clinical and echocardiographic signs of pulmonary hypertension, then the SaOs should be kept around 95-96% (Albertine et al., 1999). If the weight gain of the patient decreases or stops by more than 20% in the following weeks despite sufficient caloric intake after oxygen therapy is stopped, it should be considered as strong evidence of significant intermittent hypoxemia and oxygen therapy should be restarted (Poets and Southall, 1994). Nitric oxide (NO) contributes to oxygenation by reducing pulmonary vascular resistance without reducing systemic blood pressure due to its vasodilatory effect (Christou and Brodsky, 2005). It has no acute effects on lung function and oxygenation when inhaled at low doses. However, it decreases FiOs and ventilation requirement by increasing oxygenation of premature babies with severe BPD (Carlo and Ambalavanan, 2011).

It is thought that new treatment options are needed for the prevention and treatment of BPD in the light of all these findings and experimental studies focus on new preventive treatment strategies.

CDP-choline (cytidine diphosphate choline, citicoline) is a nucleotide that structurally contains ribose, cytosine, pyrophosphate and choline groups. CDP-choline, which is produced endogenously in the body, occurs as an intermediate metabolite during the synthesis of phospholipids that constitute the structure of the cell membrane.

In the patent numbered EP1267892B1 , the use of CDP-choline in the treatment of alcohol withdrawal syndrome is disclosed. The patent numbered EP1267892B1 describes the use of CDP-choline in the prophylactic treatment of cerebral ischemia. There is no study in the literature regarding the use of CDP-choline in the prevention and treatment of BPD.

As a result, due to the abovementioned disadvantages and the insufficiency of the current solutions regarding the subject matter, a development is required to be made in the relevant technical field.

Brief Description of the Invention

The present invention relates to use of CDP-choline which fulfills the abovementioned requirements, eliminate all disadvantages and bring some additional advantages.

The main object of the invention is to prevent and/or treat Bronchopulmonary Dysplasia, which is an important cause of morbidity in newborn premature babies, by administering CDP-choline to pregnant mammals (human or animal) who are at risk of premature birth.

An object of the invention is to produce a drug containing CDP-choline for the prevention and/or treatment of Bronchopulmonary Dysplasia. Therefore, lung damage caused by hyperoxia in newborns will be prevented or reduced. The present invention is related with the use of CDP-choline or a pharmaceutically acceptable salt thereof in the preparation of a medication for the prevention and/or treatment of Bronchopulmonary Dysplasia in order to fulfill abovementioned objectives. In one embodiment of the invention; prenatal administration to pregnant women is recommended at a daily dose range of 100 mg/kg to 500 mg/kg, preferably equivalent to 300 mg/kg CDP-choline for the prevention of Bronchopulmonary Dysplasia in premature infants. In alternative embodiments, CDP-choline may also be combined with a steroid.

The structural and characteristic features of the present invention will be understood clearly by the following drawings and the detailed description made with reference to these drawings and therefore the evaluation shall be made by taking these figures and the detailed description into consideration.

Figures Clarifying the Invention

Figure 1 is a representative image of the protein bands obtained as a result of Westernblot.

Figure 2 is a graph of the percentage change of Bax/p-Actin protein ratio relative to the NSF group.

***p<0.001 compared to Normoxia+Saline group;

###p<0.001 compared to Hyperoxia+Saline group;

++p<0.01 compared to Hyperoxia+CDP-choline group; n=6 (mean value±standard error) in each group.

Figure 3 is a graph of the total amount of phospholipids (nmol/mg protein) measured in Lung homogenates.

**p<0,01,

***p<0.001 compared to Normoxia+Saline group;

###p<0.001 compared to Hyperoxia+Saline group;

+++p<0,001 compared to Hyperoxia+CDP-choline group. n=6 (mean value±standard error) in each group.

Figure 4 is a graph of Phosphatidylcholine (PC) amount (nmol/mg protein) measured in Lung homogenates.

*p<0,05,

***p<0.001 compared to Normoxia+Saline group;

###p<0.001 compared to Hyperoxia+Saline group;

+++p<0,001 compared to Hyperoxia+CDP-choline group. n=6 (mean value±standard error) in each group.

Figure 5 illustrates representative images of lung tissue morphology. Figure 6 is a graph of average radial alveolar count.

***p<0.001 compared to Normoxia+Saline group;

###p<0,001 compared to Hyperoxia+Saline group. n=6 (mean value±standard error) in each group.

Detailed Description of the Invention

In this detailed description, the inventive method is described only for clarifying the subject matter in a manner such that no limiting effect is created.

The present invention is related with the use of CDP-choline or a pharmaceutically acceptable salt thereof in the preparation of a medication for the prevention and/or treatment of Bronchopulmonary Dysplasia. The recommended daily dose within the scope of the invention is in the dose range of 100 mg/kg to 500 mg/kg, preferably equivalent to 300 mg/kg CDP-choline.

In the studies carried out by the inventors, the efficacy of CDP-choline treatment administered to the mother during pregnancy in reducing lung injury in neonatal rats exposed to hyperoxia for 10 days postpartum was analyzed. The results show that CDP- choline treatment administered during pregnancy increases total phospholipid and phosphatidylcholine levels and decreases the levels of Bax protein, an apoptotic marker, in newborn rats with hyperoxic lung damage. Furthermore, it was shown in the study that CDP-choline treatment improved lung histology in newborn rats and increased alveolarization, which was impaired due to hyperoxia. Each effect was statistically significant. These findings showed that, in addition to the enhancing effect of antenatal steroid on the lung maturation, antenatally administered CDP-choline reduces lung damage and therefore BPD with its positive effects on multiple etiological factors on the development of BPD by increasing the surfactant components, while preventing lung injury. The data obtained with CDP-choline administration were observed for the first time in the literature in addition to the steroids used in the prenatal period in the treatment of BPD and may provide benefit for premature babies in the clinical setting.

CDP-choline or citicoline is a nucleotide structurally containing ribose, cytosine, pyrophosphate and choline groups. CDP-choline, which is produced endogenously in the body, occurs as an intermediate metabolite during the synthesis of phospholipids that constitute the structure of the cell membrane. Acetylcholine synthesis stimulated by exogenously administered CDP-choline itself and its metabolites has important effects on cholinergic system activation and membrane phospholipids. CDP-choline administration stimulates the synthesis of such membrane phospholipids as phosphatidylcholine (PC), phosphatidylserine (PS) and phosphatidylethanolamine (PE) in the brain (Lopez-Coviella et al., 1995). Furthermore, it has been shown that CDP-choline inhibits phospholipase A2 enzyme activation and reduces PC destruction, in order to protect membrane integrity (Arrigoni et al., 1987).

Until today, there are two studies evaluating CDP-choline therapy in preterm infants with respiratory distress syndrome (Colombo et al., 1976; Valls I Soler et al., 1988). While parenteral administration of CDP-choline at a dose of 100 mg/kg for 7 days did not show a beneficial effect ( Valls I Soler et al., 1988), longer administration of 300 mg/kg CDP- choline has been reported to reduce the severity of respiratory distress and oxygen demand (Colombo et al., 1976). Furthermore, in the study recently carried out by Cetinkaya et aL, it was shown that CDP-choline treatment injected at a dose of 300 mg/kg for 10 days in neonatal rats exposed to hyperoxic lung injury improved lung histology, reduced proinflammatory cytokine levels, alleviated fibrosis, improved alveolarization and contributed to lung morphology, inhibited apoptosis and increased lung phospholipid amounts (Cetinkaya et al., 2013). The beneficial effects of the CDP-choline dose (300 mg/kg) decided on the basis of the above-mentioned studies for hyperoxic lung injury support our data and suggest that CDP-choline can be used in the clinical setting in order to prevent BPD.

Hyperoxic damage induces programmed cell death in the lung. Apoptotic processes are also among the most important aspects that contribute to BPD damage. In this sense, it is important to examine apoptotic markers in terms of evaluating the efficacy of treatment. In the studies carried out by the inventors, the levels of Bax protein as an apoptotic marker were examined. While Bax levels decreased significantly with CDP-choline administration, betamethasone treatment had only a limited effect on Bax levels, which was not statistically significant. Therefore, although betamethasone increases phospholipid levels, it was shown that it has no effect on the apoptotic process. In addition to these findings, treatment with a combination of CDP-choline and betamethasone exhibited a stronger effect than the effect of CDP-choline alone; the combination reduced the Bax protein to nearly basal levels. Oxygen toxicity, which is known to contribute to the formation of BPD in premature infants (Gien and Kinsella, 2011), leads to peroxidation of membrane lipids and formation of hydroxyl radicals. Proinflammatory cytokines leads to the breakdown of phosphatidylcholine by activating phospholipase A2 and phospholipase C (Adibhatla and Hatcher, 2005). Hydrolysis of phosphatidylcholine by phospholipase A2 results in the release of lysophosphatidylcholine, which inhibits the CTP-phosphocholine cytidyyltransferase enzyme. CDP-choline is a molecule known to reduce membrane damage by counteracting oxidative stress mechanisms (Adibhatla and Hatcher, 2005). Furthermore, CDP-choline administration increases phospholipid synthesis (Lopez- Coviella et al., 1995). In the studies carried out by the inventors, the enhancing effect of CDP-choline on lung phospholipid levels is possibly related to these two activities. It has been shown in previous studies that steroids have similar efficacy. For example, when steroids are given in antenatal term, they inhibit Phospholipase A2 enzyme activity in newborn lung tissue (Remesal et al., 2016). In addition, steroids stimulate phospholipid synthesis by increasing the activity of CTP-phosphocholine cytidylyl transferase enzyme (Hogan et al., 1996). In the light of this information, in studies carried out by the inventors, prenatally administered CDP-choline and betamethasone increased both total phospholipid and phosphatidylcholine levels.

Animal studies have shown that exposure to hyperoxia in the newborn period leads to structural changes in the lung, similar to the histology observed in infants with BPD. Lung histology after hyperoxia is characterized by decreased alveolar number, vascular enlargement and simplified structure of the distal lung (Roberts et al., 1983; Warner et al., 1988; Wilson et al., 1985). In the studies carried out by the inventors, alveolar septation decreased, distal air spaces were enlarged and simplified alveolar structures emerged as a result of hyperoxia. CDP-choline treatment improved distal lung histology, contributing to the formation of smaller and more numerous alveoli. The number of intact alveoli, which decreased as a result of hyperoxia, did not change with betamethasone treatment according to the radial alveolar count results. In animal experiments, it has been reported that glucocorticoids cause larger and simplified alveolar structures by reducing alveolarization (Grier and Halliday, 2004). Combination treatment of CDP-choline and betamethasone improved alveolarization however, this effect was not significantly different from the effect of CDP-choline alone. These findings support the previous reports that betamethasone application does not improve lung alveolarization. These data show that, on one hand antenatal steroids increase lung maturation, on the other hand antenatal CDP-choline increases lung alveolarization and decreases apoptosis in addition to phospholipid synthesis; their combination reduces hyperoxic lung injury in lungs of newborn rats. With the present invention, antenatal (prenatal) CDP-choline treatment combined with antenatal steroid, has been shown as a new treatment strategy to reduce other morbidities with similar pathogenesis in addition to BPD in these infants.

Experimental Studies:

In the experimental studies carried out by inventors, an attempt has been made to experimentally mimic BPD in premature infants by exposing newborn rats to hyperoxic lung injury.

Sprague-Dawley female rats with planned pregnancy were kept in temperature-controlled (22-240) rooms with a 12-hour light - 12-hour dark cycle, they were allowed for free food and water intake and separated into 4 groups:

Injected Pregnant Rat Groups:

- Mother Group 1 (Control group): The group that received intraperitoneal 0.9% saline (SF) (1 ml/kg) once a day on the 17th, 18th and 19th days of pregnancy.

- Mother Group 2 (CDP-choline group): The group that received intraperitoneal CDP- choline (300 mg/kg/ml dissolved in SF) once a day on the 17th, 18th and 19th days of pregnancy.

- Mother Group 3 (Steroid group): The group that received subcutaneous Betamethasone (Celestone Chronodose) (0.4 mg/kg/ml dissolved in saline) twice at 8 hour intervals on day 20 of pregnancy.

- Mother Group 4 (CDP-choline + steroid group): The group that received intraperitoneal CDP-choline (300 mg/kg/ml dissolved in SF) once a day on days 17, 18 and 19 of pregnancy, and subcutaneous Betamethasone (Celestone Chronodose) (0.4 mg/kg/ml dissolved in SF) twice a day at 8 hour intervals on day 20 of pregnancy.

Following spontaneous birth, newborn rats were kept with their mothers. When pregnant rats gave birth (postnatal day 0), randomly selected newborn rats on the day of birth were exposed to normoxia or hyperoxia (85-90% oxygen) in the experiment that continue from postnatal day 1 to day 11 . To create a hyperoxic lung injury model:

• Newborn rats were exposed to hyperoxia (85-90%) during the experiment by keeping them in a plexiglass chamber between postnatal days 1 -10.

• Oxygen concentration was checked three times a day (MiniOX 3000, Ohio Medical Corporation).

• Humidity was kept above 80% and CO2 was removed with soda-lime.

• Rats kept in room air (normoxia) and rats exposed to hyperoxia were kept in the same room throughout the experiment.

• Mothers that received the same injection of active substance kept in hyperoxia and normoxia were changed every 24 hours in order to prevent oxygen toxicity.

• Newborn rats were sacrificed on postnatal 11 ^th day and their lungs were used for analysis.

Newborn Experimental Groups:

- Control Normoxia Group (Normoxia+Saline): Newborn rats born to control group mothers and monitored in room air for 10 days

- Control Hyperoxia Group (Hyperoxia+Saline): Newborn rats born to control mothers and exposed to hyperoxia for 10 days

- CDP-choline Normoxia Group (Normoxia+CDP-choline): Newborn rats born to mothers that received CDP-choline and monitored at room air for 10 days

- CDP-choline Hyperoxia Group (Hyperoxia+CDP-choline): Newborn rats born to mothers that received CDP-choline and exposed to hyperoxia for 10 days

- Betamethasone Normoxia Group (Normoxia+Betamethasone): Newborn rats born to mothers that received Betamethasone and monitored at room air for 10 days

- Betamethasone Hyperoxia Group (Hyperoxia+Betamethasone): Newborn rats born to mothers that received betamethasone and exposed to hyperoxia for 10 days

- CDP-choline + Betamethasone Normoxia Group (Normoxia+Combination Therapy): Newborn rats born to mothers that received CDP-choline and Betamethasone and monitored at room air for 10 days

- CDP-choline + Betamethasone Hyperoxia Group (Hyperoxia+Combination Therapy): Newborn rats born to mothers that received CDP-choline and betamethasone and exposed to hyperoxia for 10 days

Both lungs of newborn rats were excised and stored at -800 after thoracotomy on postnatal 11 ^th day. The tissues were removed and homogenized in 4 ml of cold PBS on the day of analysis. The homogenates were centrifuged at 10.000 rpm for 5 min, and the analyses were carried out on supernatants. After the total protein amount of all samples was analyzed according to the Lowry method (Lowry et al., 1951 ), they were mixed with 1 :1 Laemmli buffer (Laemmli, 1970) and boiled at OC for 5 minutes. Loading volumes were calculated to ensure equal amounts of protein in each sample and samples were run by means of Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis method (SDS- PAGE; 4-20%; Mini Protean II, Bio-Rad, Hercules, CA, USA). After the execution step, the proteins in the gel were transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). Membranes were blocked for 30 min with a solution of 5% skim milk powder (Carnation, Glendale, CA, USA) dissolved in tris buffer and tween 20 (TBST). After the membranes were blocked, they were washed with TBST. The membranes were cut at the appropriate place and incubated with primary antibodies overnight because the molecular weights of the proteins to be analyzed were not close to each other. Anti-rabbit-BAX and positive control anti-mouse-p-Actin (1 :1000, Cell Signaling Technology, Danvers, MA, USA) were used as primary antibodies. After overnight incubation, the membranes were washed with TBST and incubated for 1 hour with HRP (Horse radish peroxidase)-linked rabbit anti IgG and mouse anti IgG secondary antibodies (1 :5000, Cell Signaling Technology, Danvers, MA, USA). The membranes that were washed with TBST again were incubated with enhanced chemiluminescence solution (Millipore, Billerica, MA, USA), protein bands were digitally visualized (Figure 1 ) with a Licor CDigit scanner (LI-COR Biotechnology, Lincoln, NE, USA) and images were analyzed. The density of the bands was compared using the software of the Licor CDigit system.

Phospholipid extraction from lung homogenates was performed according to the Folch method (Folch et al., 1957), and measurements were performed according to the measurement method of Lopez-Coviella et al. (Lopez-Coviella et al., 1995). In brief, after the lung tissues were homogenized in 4 ml of cold PBS, 2 ml of the homogenate was taken and 2 ml of chloroform-methanol (2:1 v/v) mixture and 1 ml of distilled water were added, and then vortexed well. The mixture was incubated at +4 ^c C overnight. The samples were centrifuged (10 min, 1000xg, +4G) and separated into two phases as organic phase (lower) and aqueous phase (upper). The upper phase was aspirated and respectively 300 pl and 1200 pl were taken from the organic phase for PC measurement from total phospholipid and phospholipid fractions.

The chloroform-methanol solvent of 300 pl of sample taken for total phospholipid analysis was evaporated and 100 pl of 70% HCIO4 was added onto the dried samples. Samples that have a light brown color were boiled at 1500, covered with balls, and boiled for 1.5 hours until their color became clear. KH2PO4 standards were prepared at 1 mM concentration and 10, 20, 50, 100, 200 pl were pipetted into glass tubes and the total volume was completed to 200 pl with distilled water. 200 pl of distilled water was added to the samples and 100 pl of HCIO4 was added to the standards so as to equalize the volumes of the samples and standards. 1 ml of distilled water, 200 pl of 5% ammonium molybdate and 300 pl of 15% ascorbic acid solution were added to all tubes and after incubation for 30 minutes, measurements were made by means of a spectrophotometer at 790 nm.

Thin layer chromatography method was used for PC analysis from phospholipid fractions. After 1200 pl of chloroform-methanol solvent of the sample was evaporated, 40 pl of methanol was added onto the dried sample and vortexed until it dissolved. The samples and PC standard were applied to the silica-coated TLC plates with a 2 cm space from the bottom. Silica-coated TLC plates were kept in tanks containing a mixture of chloroform/ethanol/triethylamine/deionized water (30:34:30:8) for 2 hours to allow the samples to run. After the running process was completed, petrolium ether containing 0.1% diphenylhexatriene was sprayed onto the plates, allowing the fractions to be seen under UV. The bands corresponding to the PC standard were taken into glass tubes by scraping the silica on the plate. 1 ml of methanol was added thereon and it was kept for 30 minutes for the lipids to pass into the methanol. After a short centrifugation, 750 pl was taken and dried. The amount of PC in each sample was measured in the same way as the total phospholipid measurement.

In another set of experiments, both the lungs and heart were visualized by thoracotomy on postnatal 11 ^th day in order to perform paraformaldehyde perfusion through cardiac route in anesthetized pups. Lungs were perfused first with SF and then with 0.1 mol/l PBS (Phosphate buffer saline) containing 4% paraformaldehyde (PFA) at a constant pressure of 5 cmH2O. After the lungs were kept in PFA solution for 1 day for fixation, the cryoprotectant was taken into 30% sucrose solution and kept the tissues were settled. Using the left lung taken from sucrose, 10 micron thick sections were taken with a cryostat. The preparations were stained with hematoxylin-eosin and examined under a light microscope.

Alveolar counting was performed on digital images (Figure 5) taken from the sections in order to evaluate alveolar development. A line was drawn from the center of the respiratory bronchiole to the nearest connective tissue septum and perpendicular to the epithelium. The alveoli through which this line passed were counted.

Bax Protein Ratio

The Bax/P-Actin protein ratio was calculated as a percentage of the average densities of the Bax and P-Actin protein bands, and presented as the percent change relative to animals in the Normoxia+Saline group (Figure 2):

It was found that the Bax/P-Actin protein levels of the drug-administered Normoxia groups were not significant compared to the Normoxia+Saline group (p>0.05).

Hyperoxia significantly increased Bax/P-Actin protein levels (p<0.001 ).

When compared with Hyperoxia+Saline group, there was no significant difference in the Hyperoxia+Betamethasone group in terms of Bax/P-Actin protein levels, while Bax/P- Actin protein levels were significantly decreased in Hyperoxia+CDP-choline and Hyperoxia+Combination Therapy groups (p<0,001).

Compared to the Hyperoxia+CDP-choline group, Bax/P-Actin protein levels were significantly decreased in the Hyperoxia+Combination Therapy group (p<0.01 ).

Total Phospholipid Amount

Comparing the total amount of phospholipid measured in the lung tissue homogenate between the groups (Figure 3):

Compared to the Normoxia+Saline group, the total phospholipid amount was significantly increased only in the Normoxia+Combination Therapy group among the other normoxia groups (p<0,01).

Total phospholipid amount was found to be significantly decreased in Hyperoxia+Saline group compared to Normoxia+Saline group (p<0,001 ). Total phospholipid amount was significantly increased in Hyperoxia+Betamethasone, Hyperoxia+CDP-choline and Hyperoxia+Combination Therapy groups compared to Hyperoxia+Saline group (p<0,001 ).

Compared to the Hyperoxia+CDP-choline group, the total amount of phospholipids was found to be significantly higher in the Normoxia+Betamethasone and Normoxia+Combination Therapy groups (p<0,001).

Phosphatidylcholine (PC) Amount

Comparing the amount of PC, a fraction of phospholipids from lung homogenates, between groups (Figure 4):

Compared to the Normoxia+Saline group, the amount of PC was significantly increased only in the Normoxia+Combination Therapy group among the other normoxia groups (p<0,05).

The amount of PC was found to be significantly decreased in the Hyperoxia+Saline group compared to the Normoxia+Saline group (p<0,001 ).

The amount of PC was significantly increased in the Hyperoxia+Betamethasone, Hyperoxia+CDP-choline and Hyperoxia+Combination Therapy groups compared to the Hyperoxia+Saline group (p<0,001 ).

When compared to the Hyperoxia+CDP-choline group, the amount of PC was found to be significantly increased in the Normoxia+Betamethasone and Normoxia+Combination Therapy groups (p<0,001).

Radial Alveolar Count Results

The mean radial alveolar count results reflecting the number of intact alveoli were compared between the groups exposed to hyperoxia and treated/untreated groups compared to the Normoxia+Saline group (Figure 6):

The number of intact alveoli was found to be significantly lower in the Hyperoxia+Saline group compared to the Normoxia+Saline group (p<0.001). The number of intact alveoli was significantly increased in the Hyperoxia+CDP-choline and Hyperoxia+Combination Therapy groups compared to the Hyperoxia+Saline group (p<0.001 ).

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