The present disclosure relates to treatment premature infants to promote lung development, maturation and/or elaboration of tissue structure, in particular in the treatment of new bronchopulmonary dysplasia. In a separate aspect there is provided treatment of premature infants to reduce the incidences of respiratory distress syndrome.

BACKGROUND

The following is an extract from the NORD (National Organisation for Rare Disorders) website under bronchopulmonary dysplasia:

Bronchopulmonary dysplasia (BPD) is a chronic respiratory disease that most often occurs in low-weight or premature infants who have received supplemental oxygen or have spent long periods of time on a breathing machine (mechanical ventilation), such as infants who have acute respiratory distress syndrome. BPD can also occur in older infants who experience abnormal lung development or some infants that have had an infection before birth (antenatal infection) or placental abnormalities (such as preeclampsia). Antenatal steroid treatment prior to preterm birth and early treatment with surfactant have reduced the need for high levels of respiratory support after birth.

Affected infants may have rapid, laboured breathing and bluish discoloration of the skin due to low levels of oxygen in the blood (cyanosis). Infants are not born with BPD, the condition results from damage to the lungs

...This damage most often occurs in infants who have required extended treatment with supplemental oxygen or breathing assistance with a machine (mechanical ventilation) such as infants who are born prematurely and have acute respiratory distress syndrome.

When infants receive mechanical ventilation, a tube is inserted through the windpipe and the machine pushes air into the lungs, which are often underdeveloped in premature infants. In some cases, the levels of oxygen required for an affected infant to survive are higher than normally would be found in the air we breathe. Over time, the constant pressure from the ventilator and the excess oxygen levels can damage the delicate tissues of an infant's lungs causing inflammation and scarring.

However, infants born very prematurely have lungs that are at the saccular stage, i.e. under developed and not well equipped for breathing. These infants do not have inflammation and scarring that is in classic BPD but still have difficulty breathing.

The under-developed lung can lead to an inability to oxygenate tissue, which can be described clinical as acute respiratoiy distressed syndrome (ARDS), a life-threating conditions where the lungs cannot provide the bodies vital organs with enough oxygen. This is about respiratory gas exchange and is an acute syndrome as opposed to long term damage and chronic illness.

Surprisingly the present inventors have established maturation and differentiation of lung tissue can stimulated by generating therapeutic levels of IGF-1 rapidly after birth. This leads to significant improvements in lung structure elaboration.

Thus in one aspect the present invention provides method of stimulating maturation of the lungs, to provide more structure. Surprisingly the present inventors have also established that treating preterm infants with a complex of IGF-1 and an IGF binding protein (such as IGFBP-3) increases the levels of caspase-3 in the lungs. This in turn leads to increased apoptosis and gas exchange. Thus, this complex is administered in the first few days after birth and in combination with positive airway pressure and/or mechanical ventilation the infants breathing is significantly stabilised and incidences of respiratory distress syndrome are reduced.

What is more the inventors have very carefully measured key respiratoiy parameters in preterm infant animal models and have established that, within two to three days of treatment the stress on the lungs is reduced, for example one or more of the following benefit(s) is /are present: a lower respiratory severity score (a measure of the difficult "breathing"); a lower alveolar-arterial (A-a) gradientfwhich measures the difference between the oxygen concentration in the alveoli and arterial system); better diffusion (for example a lower barrier to diffusion) which may be measured by P/F ratio or a proxy thereof, such as S/F ratio; reduced lung stiffness; and/or reduced oxygenation index (which may be a marker for neonatal outcomes including mortality).

Thus, in one aspect the present disclosure relates to treatment of the sickest patient population, namely those receiving positive airway pressure and/or mechanical ventilation, in particular mechanical ventilation

SUMMARY OF THE INVENTION

1. A method for stimulation of maturation and/or differentiation of lung tissue in a preterm infant by administering a therapeutic amount of a composition comprising IGF-1 and an IGF binding protein (such as IGFBP-3) for example as a complex, such as parenteral administration.

IA. A composition comprising IGF-1 and an IGF binding protein (such as IGFBP-3) for example as a complex, for use in the stimulation of maturation and/or differentiation of lung tissue in a preterm infant, for example parenteral administration.

IB. A composition comprising IGF-1 and an IGF binding protein (such as IGFBP-3), for example as complex, for use in the manufacture of a medicamentfor the stimulation of maturation and/or differentiation of lung tissue in a preterm infant, for example parenteral administration.

IC. A method of treatment or prophylaxis of respiratory distress syndrome in a preterm infant (also referred to a neonate herein), a low birth weight baby, an infant with hyaline membrane disease or surfactant deficiency disease by administering a therapeutic amount of a composition comprising IGF-1 and an IGF binding protein (such as IGFBP-3) for example as complex, in combination with a positive airway pressure and/or mechanical ventilation, in particular parenteral administration.

ID. A composition comprising IGF-1 and an IGF binding protein (such as IGFBP-3), for example as complex, for use in the treatment or prophylaxis of respiratory distress syndrome in a preterm infant (also referred to a neonate herein), a low birth weight baby, an infant with hyaline membrane disease or surfactant deficiency disease, wherein the composition is to be administered in combination with a positive airway pressure and/or mechanical ventilation in particular parenteral administration. IE. A composition comprising IGF-1 and an IGF binding protein (such as IGFBP-3) for example as complex, for use in the manufacture of a medicament for treatment or prophylaxis of respiratory distress syndrome in a preterm infant (also referred to a neonate herein), a low birth weight baby, an infant with hyaline membrane disease or surfactant deficiency disease, wherein the composition is to be administered in combination with a positive airway pressure and/or mechanical ventilation in particular parenteral administration.

2. A method or composition for use according to any preceding paragraph, wherein the preterm infant is born at the saccular stage of lung development.

3. A method or composition for use according to any preceding paragraph, wherein there is lung maturation and/or differentiation, for example within 3 days of initiating treatment.

4. A method or composition for use according to any preceding paragraph, wherein there is increased apoptosis in epithelial lung tissue.

5. A method or composition for use according to paragraph 4, wherein increased apoptosis results from increased caspase-3 levels.

6. A method or composition for use according to any preceding paragraph, wherein treated patients (for example neonates) have one or more improved morphological elements.

7. A method or composition for use according to any preceding paragraph, wherein treated patients (for example neonates) have one or more improved physiological elements.

8. A method or composition for use according to any preceding paragraph, wherein treated patients (for example neonates) have one or more improved biochemical parameters.

9. A method or composition according to any preceding paragraph, wherein the treated patients (for example neonates) have a larger surface density for capillary endothelial cells in comparison to untreated patients.

10. A method or composition for use according to any preceding paragraph, wherein the treated patients (for example neonates) maintain physiologic systemic perfusion pressure.

11. A method or composition for use according to any preceding paragraph, wherein treated patients (for example neonates) have a larger airspace epithelial cells in comparison to untreated patients.

12. A method or composition for use according to any preceding paragraph, wherein the treated patients (for example neonates) have improved gas exchange, for example a tendency for improved gas exchange or at least one or more properties are improved that support gas exchange.

13. A method or composition for use according to any preceding paragraph, wherein there is thinning of saccular walls, in treated patients (for example neonates).

14. A method or composition for use according to any preceding paragraph, wherein the treated patients (such as neonates) have PCNA relative protein abundance with a numerically lower value than untreated patients.

15. A method or composition for use according to any preceding paragraph, wherein treated patients (for example neonates) have higher oxygen saturation levels than untreated patients, for example as measured by pulse oximetry. 16. A method or composition for use according to any preceding paragraph, wherein the treated patients (for example neonates) have lower peak inspiratoiy pressure than untreated patients.

17. A method or composition for use according to any preceding paragraph, wherein tissue perfusion is stabilised, for example treated preterm infant is pink.

18. A method or composition for use according to any preceding paragraph, wherein the treated preterm infant stays within the predefined parameters for gases, for example the breathing apparatus/machine/instrumentdoes not require adjustmentor requires minimal adjustment by a carer.

19. A method or composition for use according to any preceding paragraphs, wherein the treated preterm infant has a fractional inspired oxygen level that is lower than untreated preterm infants (or an average thereof), for example 0.21, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9 or 0.95, in particular in the first 36 hours of life.

20. A method or composition for use according to any preceding paragraphs, wherein the treated preterm infant has a peak inspiratory pressure (cmfhO) that is lower than untreated preterm infants (or an average thereof), for example in the range 45 and 60 mmHg, such as 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 or 60 cmH20, in particular in the first 72 hours of life.

21. A method or composition for use according to any preceding paragraphs, wherein the preterm infant is 23 to 34 weeks post gestation, when treatment is initiated, for example 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33 or 34 weeks.

22. A method or composition for use according to any preceding paragraphs, wherein the preterm infants are treated by infusion, for example continuous infusion, in particular for at least 1 week, for example 2 to 6 weeks, such as 2, 3, 4, 5 or 6 weeks.

23. A method or composition for use according to paragraph 7, wherein the infusion/treatmentis initiated within 24 hours of birth, for example within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 16, 17, 18, 19, 20, 21, 22, 23 and 24 hours of birth, such as within 1, 2 or 3 hours of birth, in particular within 1 hour of birth.

24. A method or composition for use according to any preceding paragraphs, wherein the infant requires minimal assistance with breathing.

25. A method or composition for use according to any preceding paragraph (save where mechanical ventilation is specified), wherein the preterm infant has a nasal canula.

26. A method or composition for use according to any preceding paragraph (save where mechanical ventilation is specified), wherein the preterm infant has continuous positive airway pressure.

27. A method or composition for use according to any preceding paragraphs, wherein the infant is on mechanical ventilation.

28. A method or composition for use according to any preceding paragraph, wherein blood flow is stabilised.

29. A method or composition for use according to any preceding paragraph, wherein central blood flow is stabilised. 30. A method or composition for use according to any preceding paragraph, wherein peripheral blood flow is stabilized, for example in comparison to a corresponding infant without treatment

31. A method or composition for use according to any preceding paragraph, wherein blood pressure (such as mean system blood pressure) is stabilised, for example the blood pressure does not drop below 3 OmmHg.

32. A method or composition for use according to any preceding paragraph, wherein the preterm infant has a blood pressure in the range 30 to SOmmHg, during treatment, for example 30, 35, 40, 45 or 50 mmHg.

33. A method or composition for use according to any preceding paragraph, wherein diastolic pressure is stabilised, for example in the range 60 to 90mmHg, for example 60, 65, 70, 75, 80, 85, 90mmHg or such as where the diastolic pressure is higher than an untreated preterm infant (in particular in the period up to 72 hours post birth).

34. A method or composition for use according to any preceding paragraph, wherein systolic pressure is stabilised, for example is higher than untreated preterm infants (in particular in the period up to 72 hours post birth), such as stabilised in the range 30 to 60mmHg, in particular 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60mmHg.

35. A method or composition for use according to any preceding paragraph, wherein the blood pressure and/or flow is stabilised without increasing vascular resistance.

36. A method or composition for use according to any preceding paragraph, wherein the preterm infant receiving said treatment/prophylaxis is more robust than a preterm infant without the treatment/prophylaxis.

37. A method or composition for use according to paragraph 36, wherein more robust is a lower heart rate in comparison to untreated preterm infants (or average thereof), for example a bpm in the range about 120 to 180, such as 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175 or 180 bpm.

38. A method or composition for use according to paragraph 36 or 37, wherein more robust is adequate temperature regulation.

39. A method or composition for use according to any one of paragraphs 36 to 38, wherein more robust is a good level of activity (motor activity).

40. A method or composition for use according to any one of paragraphs 36 to 39, wherein more robust is adequate urine output, for example in the 2 to 5mg/Kg/hour (such as in the first 72 hours of life) in particular 2, 2.5, 3, 3.5, 4, 4.5 or 5mg/Kg/hour.

41. A method or composition for use according to any preceding paragraph, wherein the urine output of a treated preterm infant is higher than an untreated infant (at least in the first 12 hours of life).

42. A method or composition for use according to any preceding paragraphs, wherein the preterm infant does not lose weight in the first few days of life, for example days 1 to 3, such as day 1, 2, or 3. 43. A method or composition for use according to any preceding paragraph, wherein the treated preterm infant does not go into anaerobic metabolism, for example as measured by lactic acid levels.

44. A method or composition for use according to any preceding paragraph, wherein treated preterm infants have a lower mortality rate than untreated preterm infants.

45. A method or composition for use according to any preceding paragraph, where the composition comprises equimolar amounts of IGF-1 and IGFBP-3.

46. A method or composition for use according to any preceding paragraphs, wherein 200 to 500|ig/Kg/24 hours of complex are administered, for example 200, 250, 300, 350, 400, 450 or 500|ig/Kg/24 hours, in particular 400pg/Kg/24 hours.

47. A method or composition for use according to any preceding paragraph, wherein the 55 to 110|ig/Kg/24hours of IGF-1 are administered, for example 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105 or 110|ig/Kg/24hours.

48. A method or composition for use according to any preceding paragraphs, wherein serum levels of IGF-1 are maintained with the range 28 to 109ng/mL, for example 28, 29, 30, 31, 32, 33, 34, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 101, 102, 103, 104, 105, 106, 107, 108 109ng/mL.

49. A method or composition for use according to any preceding paragraphs, wherein the composition is administered by infusion, for example by continuous infusion.

50. A method or composition for use according to any preceding paragraph, wherein the composition is administered subcutaneously, for example as a depot injection, such as once, twice or three times a day.

51. A method or composition for use according to any preceding paragraph, wherein the preterm infant has a reduced levels of systemic inflammation, for example in comparison to a preterm infant without treatment

52. A method or composition for use according to any preceding paragraph, wherein the infant has birth weight of 2.2 pounds or less (lKg or less],

53. A method or composition for use according to any preceding paragraph, wherein the infant is in the prone position.

54. A method or composition for use according to any preceding paragraph wherein the complex is used in a combination therapy, for example in combination with therapies normally employed in preterm infants and neonates, such as surfactant replacement therapy.

In one embodiment alveolar formation is improved in treated infants, for example more alveolar and/or more elaborate alveolar.

In one embodiment the capillary surface density in the treated infants is improved, for example by at least 10%. Thus, in one embodiment lung capillaries are increased.

In one embodiment lung epithelial surface is increased in treated infants, for example by 10, 15, 20, 25, 30, 35, 40, 45 or 50% or more.

In one embodiment radial alveolar count is increased in treated infants, for example at least 25, 30, 35, 40, 45, 50, 55, 60, 64, 70, 75, 80,85, 90, 95, 100% or more. In one embodiment the value of volume density of secondary septa is increased in treated infants, for example at least, 10, 11, 12, 13, 14,15 or 16% or more.

In one embodiment the distal airspace wall thickness in um is reduced in treated infants.

In one independent aspect or embodiment, there is provided a method of treatment or prophylaxis of acute respiratoiy failure and/or acute respiratory injury in a preterm (also referred to as a neonate herein] comprising administering a therapeutic amount of a composition comprising IGF-1 and an IGF binding protein (such as IGFBP-3], for example as complex, such as 1:1 complex.

In one independent aspect or embodiment there is provided a composition comprising IGF- 1 and an IGF binding protein (such as IGFBP-3], for example as complex for use in the treatment or prevention of hypoxemic respiratoiy failure, for example in infants with an under developed lung, particular at the saccular stage.

In one independent aspect or embodiment there is provided a composition comprising IGF- 1 and an IGF binding protein (such as IGFBP-3], for example as complex for use in preventing or minimising multiple organ dysfunction syndrome, for example induced acute respiratory distress, for example in infants with an under developed lung, particular at the saccular stage.

In one independent aspect or embodiment there is provided a composition comprising IGF- 1 and an IGF binding protein (such as IGFBP-3], for example as complex for use in treatment or prevention of acute lung injury, for example in infants with an under developed lung, particular at the saccular stage.

In one embodiment respiratory severity score is reduced in treated infants, for example is 3 or less, such as 2 (in particular from day or 3 of treatment or life respectively].

In one embodiment the A-a gradient is reduced in treated infants, for example is 50 or less, such as 25 or less (in particular from day 2 of treatment], such as 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or 0 (including 10 or less, such as by day 4 of treatment and/or about 0 such as by day 5 of treatment or life].

In one embodiment the oxygenation index is reduced in treated infants, for example 4 or less (in particular from day 3 of treatment or life], such as 2.5 or 2. This may also be considered evidence of improved robustness.

In one embodiment the P/F ratio is increased in treated infants, for example is at least 300 (including where averages are maintained at 300 or above], such as 350 (in particular from day 4 of treatment or life].

In one embodiment the S/F ratio is increased in treated infants, for example is at least 350 (including where averages are maintained at 350 or above], such as about400 (in particular from day 4 of treatment or life].

In one embodiment treated patients (neonates] have improved structural thinning of saccular walls in comparison to untreated patients.

In one embodimenttreated patients (neonates] have a PCNA relative protein abundance that is numerically lower in comparison to untreated patients.

In one embodiment treated patients (neonates] have higher oxygen saturation levels than untreated patients, for example as measured by pulse oximetry. In one embodimenttreated patients (neonates] have higher aortic pressure.

In one embodiment treated patients (neonates] have lower peak inspiratory pressure. In one embodiment respiratory mechanics, such as measured by R, Cdyn, and/or 20/Cdyn remain unchanged.

In embodiment the patient populations is low birth weight babies, for example those who have not grown adequately in the womb and/or infants born with a gestational age of 22 to 26 weeks, such as 22, 23, 24, 25 or 26, in particular 23 to 25 or 26. This patient population particularly benefits from treatment In fact treatment may make the difference between survival and nonsurvival for these patients. Thus, in one embodiment the lowest birth weight babies benefit the most In one embodiment the lowest birth weight are the lowest quartile and include infant with low weight or gestational age.

In one embodiment there are no toxic effects.

In one embodiment the IGF-1 composition is administered subcutaneously, for example as a depot injection, for example once, twice or three times each day.

In one embodiment, the IGF-1 levels are raised to therapeutic levels by continuous infusion.

In one embodiment, the IGF-1 levels are raised to therapeutic levels by continuous infusion and then the administration is changed to subcutaneous (once, twice or three times a day].

In one embodiment continuous infusion is used intermittently with subcutaneous administration in accordance with a treatment protocol.

In one embodiment the present treatment is employed in a combination therapy, for example with other treatments usually employed in preterm infants, such as surfactant therapy.

DETAILED DISCLOSURE

Bronchopulmonary dysplasia [BPD] as used herein refers to a form of chronic lung disease that typically affects preterm infants. Preterm infants generally have under-developed lungs, which necessitates the use of mechanical ventilation. Unfortunately, because their lungs are vulnerable, the high amounts of inhaled oxygen and pressure introduced by mechanical ventilation may overstretch the alveoli, resulting in inflammation and scarring to the tissues of the airway and alveoli. This in turn affects the proper development of the alveoli - fewer and larger alveoli develop compared to usual and the interstitium is thickened. This produces a vicious cycle where the infant is increasingly reliant on mechanical ventilation and is unable to wean from artificial ventilation.

New bronchopulmonaiy dysplasia (new BPD] as used herein refers to a form of BPD whereby there is less inflammation and scarring than in classic BPD. In new BPD there is abnormal lung development resulting in an under-developed or immature lung. For example, there is alveolar simplification that includes fewer secondary septa, reduced capillary growth, and smaller surface density for capillary endothelial cells and airspace epithelial cells.

Respiratoiy distress syndrome as employed herein refers to a breathing disorder that in particular afflicts newborn babies, especially preterm infants (although it can also affect full-term infants]. The syndrome occurs when the lungs are not fully developed, as a result of which the lungs fail to manufacture sufficient amounts of surfactant Surfactant is a fluid that helps to keep the alveoli open; a lack of surfactant causes the alveoli to collapse with each breath. This leads to cellular damage, resulting in the accumulation of damaged cells in the airway, which in turn makes breathing even more difficult

Acute respiratory distress syndrome (ARDS) includes acute onset of disease, chest radiograph demonstrating bilateral pulmonary infiltrates, lack of significant left ventricular dysfunction and Pao2/Fio2 (PF) ratio <300 for ALI (acute lung injury) or <200 for ARDS.

Hypoxemic respiratory failure (HFR) happens when there is not enough oxygen transport into the blood (hypoxemia). Heart and lung conditions are the most common causes. Hypoxemic respiratory failure is also called hypoxic respiratory failure. Hypoxemic respiratoiy failure is associated with increased risk of mortality, morbidity, and worse neurological outcomes. Oxygenation index (01) is routinely used as an indicator of severity of HRF in neonates, with an arbitrary cutoff of 15 or less for mild HRF, between 16 and 25 for moderate HRF, between 26 and 40 for severe HRF, and more than 40 for veiy severe HRF.

Caspase-3 is a member of the cysteine-aspartic acid protease (caspase) family. Caspase-3 plays a central role in cellular apoptosis. It is activated by caspases-8, 9 and 10 and its role in turn is to cleave and activate caspases-6 and 7. Caspase-3 is typically referred to as an executioner caspase because of its role in co-ordinating the degradation of cytoskeletal proteins and the destruction of other cellular structures.

Thinning of saccular walls, in part by apoptosis of mesenchymal (interstitial) cells, is necessary to establish a thin diffusion barrier for oxygen and carbon dioxide. Increased caspade-3 may result in thinner membranes and thereby better diffusion.

A corresponding infant as employed herein is an infant with corresponding parameters, for example gestational age, weight and the like, for example which is usually untreated.

Less or minimal intervention (including minimal assistance with breathing) as employed herein refers to the amount of support from a carer required by a treated infant being less. For example, when an infant’s breathing is stable then less adjustments have to be made to instruments to keep the respiratory gases with the predefined parameters. This is not about whether the infant requires a nasal canula (the least invasive support), continuous positive airway pressure (which helps keep the lungs inflated using a higher air pressure than a standard nasal canula); or mechanical ventilation. Instead, it is about the number of adjustments that have to be made to keep the breathing within a predefined "desirable" range.

In one embodiment synchronized intermittent mandatory ventilation with pressure- controlled, with warmed and humidified gas is employed.

In one embodiment F1O2 is set to attain target hemoglobin oxygen saturation of 90-94% (Path 60-90 mmHg, such as 60, 65, 70, 75, 80, 85, 90mmHg), for example by pulse oximetry (Model SurgiVet V9200IBP/Temp, Smith Medical ASD, Inc., St. Paul, MN).

In one embodiment peak inspiratory pressure is set to attain a target PaCC>2 between 45 and 60 mmHg (such as 45, 50, 55, 60), resulting in pH between 7.25-7.35 (such as 7.25. 7.26, 7.27, 7.28, 7.29, 7.30, 7.31, 7.32, 7.33, 7.34 or 7.35).

In one embodiment target expiratory tidal volume, measured by the ventilator, in the range 5 to 7 mL/Kg, such as 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9 or 7. Fractional inspired oxygen as employed herein is an estimation of the oxygen content a person inhales and is thus involved in gas exchange at the alveolar level. Understanding oxygen delivery and interpreting Fi02 values are imperative for the proper treatment of patients with hypoxemia. It is the molar or volumetric fraction of oxygen in the inhaled gas. Medical patients experiencing difficulty breathing are provided with oxygen-enriched air, which means a higher- than-atmospheric F/O2. Natural air includes 21% oxygen, which is equivalent to F/O2 of 0.21. Oxygen-enriched air has a higher F/O2 than 0.21; up to 1.00 which means 100% oxygen. F/O2 is typically maintained below 0.5 even with mechanical ventilation, to avoid oxygen toxicity, but there are applications when up to 100% is routinely used. The abbreviated alveolar air equation is:

PAO ₂ , P _E 0 ₂ , and P1O2 are the partial pressures of oxygen in alveolar, expired, and inspired gas, respectively, and VD/Vtis the ratio of physiologic dead space over tidal volume.

In medicine, the F/O2 is the assumed percentage of oxygen concentration participating in gas exchange in the alveoli.

Oxygenation index is a calculation used in intensive care medicine to measure the fraction of inspired oxygen (Fi02) and its usage within the body.

A lower oxygenation index is better - this can be inferred by the equation itself. As the oxygenation of a person improves, they will be able to achieve a higher PaO2 at a lower Fi02. This would be reflected on the formula as a decrease in the numerator or an increase in the denominator - thus lowering the 01. Typically an 01 threshold is set for when a neonate should be placed on ECMO, for example >40.

The equation is:

FzO2: Fraction of inspired oxygen, in percent;

Mean airway pressure, in mmHg; and

Pa02 Partial pressure of oxygen in arterial blood, in mmHg.

A-a gradient has important clinical utility as it can help narrow the differential diagnosis for hypoxemia. The A-a gradient calculation is as follows:

A-a Gradient = PA02 - PaO2

PA02 representing alveolar oxygen pressure and

PaO2 representing arterial oxygen pressure.

The arterial oxygen pressure (PaO2) can be directly assessed with an arterial blood gas test (ABG) or estimated with a venous blood gas test (VBG). The alveolar oxygen pressure (PA02) is not easily measured directly; instead, it is estimated using the alveolar gas equation:

PA02 = (Patm - PH2O) Fi02 - PaCO2/RQ

As explained above oxygenation index is calculated as 01 = MAP x Fio2 x 100 / Pao2, where MAP indicates mean airway pressure and Fio2 indicates fraction of inspired oxygen.

Oxygenation index higher than 40 is used as a criterion for consideration of extracorporeal membrane oxygenation. Oxygenation index has also been proposed as a predictive marker for neonatal outcomes, including mortality.

Limitations of 01 include the need for an indwelling arterial catheter for frequent sampling and that it is an intermittent measurement of oxygenation status by nature.

P/F ratio is a powerful objective tool to identify acute hypoxemic respiratory failure at any time while the patient is receiving supplemental oxygen, a frequent problem faced by documentation specialists where no room air ABG (arterial blood gas] is available or pulse ox readings seem equivocal.

The following is a definition of A-a gradient by Hantzidiamantis PJ et al Physiology, Alveolar to Arterial Oxygen Gradient

The A-a gradient, or the alveolar-arterial gradient, measures the difference between the oxygen concentration in the alveoli and arterial system. The A-a gradient has important clinical utility as it can help narrow the differential diagnosis for hypoxemia. The A-a gradient calculation is as follows:

A-a Gradient = PA02 - Pa02.

With PA02 representing alveolar oxygen pressure and Pa02 representing arterial oxygen pressure. The arterial oxygen pressure [Pa02] can be directly assessed with an arterial blood gas test [ABG] or estimated with a venous blood gas test [VBG], The alveolar oxygen pressure [PA02] is not easily measured directly; instead, it is estimated using the alveolar gas equation:

PA02 = (Patm - PH20] Fi02 - PaC02/RQ

In a perfect system, no A-a gradient would exist: oxygen would diffuse and equalize across the capillary membrane, and the pressures in the arterial system and alveoli would be equal (resulting in an A-a gradient of zero]. However, there is a physiologic V/Q mismatch in the lungs due to heterogeneity in apical vs. basilar perfusion and ventilation. This mismatch is, in part, responsible for the slight difference in oxygen tension between the alveoli and arterial blood. So there exists a physiologic A-a gradient that changes based on a patient's age. The expected A-a gradient can be estimated with the following equation:

A-a gradient = (Age + 10] / 4

The value calculated for a patient's A-a gradient can assess if their hypoxia is due to the dysfunction of the alveolar-capillary unit, for which it will elevate, or due to another reason, in which the A-a gradient will be at or lower than the calculated value using the above equation.

The P/F ratio equals the arterial p02 ("P"] from the ABG divided by the FI02 ("F"] - the fraction (percent] of inspired oxygen that the patient is receiving expressed as a decimal (40% oxygen = FI02 of 0.40],

A P/F Ratio less than 300 indicates acute respiratoiy failure.

P/F ratio < 300 is equivalent to a p02 < 60 mm Hg on room air

P/F ratio < 250 is equivalent to a p02 < 50 mm Hg on room air

P/F ratio < 200 is equivalent to a p02 < 40 mm Hg on room air

S/F ratio is a proxy for P/F ratio. The arterial p02measured by arterial blood gas (ABG] is the definitive method for calculating the P/F ratio. However, when the p02 is unknown because an ABG is not available, the SpO2 measured by pulse oximetry can be used to approximate the p02, as shown in the Table below. It is important to note that estimating the p02 from the SpO2 becomes unreliable when the SpO2 is 98% - 100%. Conversion of SpO2 to p02

SpO2 pO2 (percent) (mm Hg) SpO2 pO2 (percent) (mm Hg)

86 51 92 64

87 52 93 68

88 54 94 73

89 56 95 80

90' 58 96 90

91 60 97 110

A nasal cannula provides oxygen at adjustable flow rates in litres of oxygen per minute (L/min or "LPM"). The actual FI02 (percent oxygen) delivered by nasal cannula is somewhat variable and less reliable than with a mask, but can be estimated as shown in the Table below. The

FI02 derived from nasal cannula flow rates can then be used to calculate the P/F ratio.

Flow Rate FIO2 Flow Rate FIO2

1 L/min 24% 4 L/min 36%

2 L/min 28% 5 L/min 40%

3 L/min 32% 6 L/min 44%

Assumes room air is 20% and each L/min of oxygen = +4%.

Hypoxemic is where oxygen levels in the blood are lower than normal.

Some values given for adults may also apply to neonates.

Preterm infant (or neonate) as employed herein refers to an infant born before 40 weeks of gestation, for example with a gestational age of 37 weeks or less, such as 22 to 37 weeks (gestational age), in particular 23, 24, 25, 26, 27, 28, 29, 30, 31, 33, 34, 35 or 36 weeks. At the present time neonates born at 22 weeks gestation are at the cusp of what can be saved, in that the majority may not respond to resuscitation. Having said that neonates born at 22 weeks have survived.

In one embodiment the disclosure relates to very preterm infants. Very preterm infants as employed herein refers to 32 weeks or less gestation age, for example 31 weeks or less, 30 weeks or less. 29 weeks or less, 28 weeks or less, 27 weeks or less, 26 weeks or less, 25 weeks or less, 24 weeks or less, 23 weeks or less or 22 weeks.

Generally, the preterm infant is human.

The lowest weight (not necessarily the most premature) infants may benefit the most from the therapy of the present disclosure, for example infants lKg or less. A low birthweight baby may be a baby with poor growth in the womb (intrauterine growth restriction-IUGR). In one independent aspect the treatment according to the present disclosure is employed in a low birth weight infant regardless of whether said infant is born prematurely.

Stabilising blood pressure as employed herein is a fit for purpose test, as the infant is able to urinate, preferably without intervention using a vasopressor and/or a diuretic. Thus, in one embodiment the stabilised blood pressure stabilises one or more systemic functions in the infant.

In one embodiment stabilised blood pressure is stabilisation of mean systemic pressure.

In one embodiment the stabilisation minimises incidences of or prevents hypertension. In one embodiment the disclosure does not relate to hypertension.

In one embodiment stabilisation minimises or prevents hypotension, for example a mean systemic pressure below 30mgHg. Thus, in one embodiment mean systemic pressure is in maintained in the range 30 to 60mgHg, for example 30, 35, 40, 45, 50, 55 or 60 mgHg.

In one embodiment the disclosure reduces the need for intervention, for example treatment with a vasopressor and/or diuretic.

Blood flow as employed herein refers to movement of blood in the infant When the blood flow is good and stabilised it reaches all the organs and tissues, for example infants are perfused and pink. This blood flow then supports the function of the different organs, such as the kidneys, lungs, stomach etc. Meaningful blood pressure readings may not be feasible in some infants, for example the amount of blood may so small that the pressure readings using instruments are anomalous. Nevertheless, these infants benefit from the treatment of the present disclosure. Thus, blood flow as employed herein is a fit for purpose test based one or more key biological functions - Is the infant urinating, perfused, breathing adequately etc, and preferably all of the same.

In one embodiment the disclosure is not for the treatment of bronchopulmonaiy dysplasia, as defined herein (i.e. traditional BPD).

A corresponding infant as employed herein is an infant with corresponding parameters, for example gestational age, weight and the like.

Less or minimal intervention as employed herein refers to the amount support from a career required by a treated infant being less. For example, when an infant’s breathing is stable then less adjustments have to be made to instruments to keep the respirator gases with the predefined parameters. This is not about whether the infant is a nasal canula (the least invasive support), continuous positive airway pressure (which helps keep the lungs inflated using a higher air pressure than a standard nasal canula); or mechanical ventilation. Instead, it is about the number of adjustments that have to be made to keep the breathing within a predefined "desirable" range.

In one embodiment synchronized intermittent mandatory ventilation with pressure- controlled, with warmed and humidified gas is employed.

In one embodiment F1O2 is set to attain target hemoglobin oxygen saturation of 90-94% (Path 60-90 mmHg) by pulse oximetry (Model SurgiVet V9200IBP/Temp, Smith Medical ASD, Inc., St Paul, MN).

In one embodiment peak inspiratory pressure is set to attain a target PaCC>2 between 45 and 60 mmHg, resulting in pH between 7.25-7.35.

In one embodiment target expiratory tidal volume, measured by the ventilator, in the range 5 to 7 mL/Kg.

In one embodiment calculated oxygenation index (01) [(Paw x FjO2)/PaO2], P/F (PaO2/FjO2) ratio, and Alveolar-arterial (A-a) gradient [((F 0 /100) x (640-47))-(PaCO /0.8)-Pa0 ]. At about 5,000 ft elevation, barometric pressure is about 640 mmHg.

In one embodiment the alveolar-arterial (A-a) gradient in a treated infant is improved over a corresponding untreated infant.

Orogastric feeding may be started at ~3h of postnatal life (3 mb) and the volume was gradually increased as tolerated, with target over the first week of postnatal life of ~20 to 100 kcal/Kg/d, such as 40, 45, 50, 55, 60, 65 or 70 kcal/Kg/d.

Parenteral dextrose may be infused to maintain plasma glucose between 60 and 90 mg/dL. "I GF-I" refers to insulin-like growth factor I from any species, including bovine, ovine, porcine, equine, and human, preferably human, and, if referring to exogenous administration, from any source, whether natural, synthetic, or recombinant, provided that it will bind IGF binding protein at the appropriate site. IGF-I can be produced recombinantly, for example, as described in PCT publication WO 95/04076.

An "IGFBP" or an "IGF binding protein" refers to a protein or polypeptide from the insulin-like growth factor binding protein family and normally associated with or bound or complexed to IGF-I whether or not it is circulatory (i.e., in serum or tissue). Such binding proteins do not include receptors. This definition includes IGFBP-1, IGFBP-2, IGFBP-3, IGFBP-4, IGFBP-5, IGFBP-6, Mac 25 (IGFBP-7), and prostacyclin-stimulating factor (PSF) or endothelial cell-specific molecule (ESM-1), as well as other proteins with high homology to IGFBPs. Mac 25 is described, for example, in Swisshelm et al., Proc. Natl. Acad. Sci. USA, 92: 4472-4476 (1995) and Oh et al., J. Biol. Chem., 271: 30322-30325 (1996). PSF is described in Yamauchi etal., Biochemical Journal, 303: 591-598 (1994). ESM-1 is described in Lassalle et al., J. Biol. Chem., 271: 20458-20464 (1996). For other identified IGFBPs, see, e.g., EP 375,438 published Jun. 27, 1990; EP 369,943 published May 23, 1990; WO 89/09268 published Oct 5, 1989; Wood et al., Molecular Endocrinology, 2: 1176-1185 (1988); Brinkman et al., The EMBO J., 7: 2417-2423 (1988); Lee et al., Mol. Endocrinol., 2: 404-411 (1988); Brewer etal., BBRC, 152: 1289-1297 (1988); EP 294,021 published Dec. 7, 1988; Baxter et al., BBRC, 147: 408-415 (1987); Leung et al., Nature, 330: 537-543 (1987); Martin et al., J. Biol. Chem., 261: 8754-8760 (1986); Baxter et al., Comp. Biochem. Physiol., 91B: 229-235 (1988); WO 89/08667 published Sep. 21, 1989; WO 89/09792 published Oct 19, 1989; and Binkertetal., EMBO J., 8: 2497- 2502 (1989).

"IGFBP-3" refers to insulin-like growth factor binding protein 3. IGFBP-3 is a member of the insulin-like growth factor binding protein family. IGFBP-3 may be from any species, including bovine, ovine, porcine and human, in native-sequence or variant form, including but not limited to naturally-occurring allelic variants, in particular human. IGFBP-3 may be from any source, whether natural, synthetic or recombinant, provided that it will bind IGF-I at the appropriate sites. IGFBP-3 can be produced recombinantly, as described in PCT publication WO 95/04076.

Therapeutic composition, as used herein, is defined as comprising IGF-I or an analogue thereof, in combination with its binding protein, such as IGFBP-3 or an analogue thereof. In some embodiments, the IGF-1 is recombinantly produced. In some embodiments, the IGFBP-3 is recombinantly produced. In some embodiments, the IGF-1 and the IGFBP-3 are complexed prior to administration to the subject In some embodiments, the IGF-1 and IGFBP-3 are complexed in equimolar amounts.

The therapeutic composition may also contain other substances such as water, minerals, carriers such as proteins, and other excipients known to one skilled in the art.

In one embodiment, the method or composition comprises 100 to 600 pg/Kg/24hours of the IGF-l/IGFBP-3 complex. In one embodiment, the method or composition comprises 200 to 500|ig/Kg/24hours of the IGF-l/IGFBP-3 complex, for example 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475 or 500 pg/Kg/24hours of the IGF-l/IGFBP-3 complex. In one embodiment, the method or composition comprises 400pg/Kg/24hours of the IGF-l/IGFBP-3 complex. In one embodiment, the method or composition comprises 55 to 110 pg/Kg/24hours of IGF-1, such as 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105 or 110 pg/Kg/24hours.

In the context of this specification "comprising" is to be interpreted as "including". Embodiments of the invention comprising certain features/elements are also intended to extend to alternative embodiments "consisting" or "consisting essentially" of the relevant elements/features.

Where technically appropriate, embodiments of the invention may be combined.

Technical references such as patents and applications are incorporated herein by reference.

Any embodiments specifically and explicitly recited herein may form the basis of a disclaimer either alone or in combination with one or more further embodiments.

Subject headings herein are employed to divide the document into sections and are not intended to be used to construe the meaning of the disclosure provided herein.

The background section contains technical information relating to the invention and may be employed as basis for amendment.

The present specification claims priority from US63/353,186 & 63/353,251 filed 17 June 2022, US63/376,195 & 63/376,220 filed 19 September 2022, and US63/376,557 filed on 21 Sep 2022, incorporated herein by reference. These documents can be used as basis for correction.

Individual numerical values in the Examples may be isolates and used as basis for amendment of the claims without reference to the other parameters recited in said Example.

The present invention is further described by way of illustration only in the following examples.

FIGURES

Figure 1A shows (Group 1) shows plasma level of IGF-1 protein in normal, unventilated fetal and postnatal lambs. During normal development from fetal lambs (about 12 Od gestation) to adolescent lambs (about 150d of age), IGF-1 protein level progressively increased (Pearson r=0.540; p=0.004).

Figure IB shows (Group 2) plasma level of IGF-1 protein in preterm lambs managed by invasive mechanical ventilation for 3d. Continuous iv infusion ofrhIGF-l/rhIGFBP-3 (1.5 mg/Kg/day; black squares) attained the targetplasma level of about 125 ng/mL for the last 48h of the 72h study, whereas for vehicle-control preterm lambs (white circles), plasma IGF-1 protein level significantly decreased to about 30 ng/mL for the last 48h of the 72h study period. Symbols for statistical differences: single line = significantly lower level from 12 through72h for the vehicle-control preterm lambs compared to this group’s "predose" level; * = significantly higher level from 12 through 72h for the rhIGF-l/rhIGFBP-3-treated preterm lambs compared to this group’s "predose" level; double lines = significantly greater for the rhIGF-1/rhIGFBP- 3-treated preterm lambs compared to the matched hour’s level for the vehiclecontrol preterm lambs. Statistical analyses for were by two-way ANOVA and Holm- Sidak's multiple comparisons test, with oc=0.05 (95%).

Figure 1C-E shows plasma IGF-1 protein level for various IV doses. Figure 2A-F shows respiratory gas exchange physiological parameters for preterm lambs (Group

2) managed by invasive mechanical ventilation for 3d. Continuous iv infusion of rhIGF-l/rhIGFBP-3 (1.5 mg/Kg/day; blacks squares).

Figure 3A-D shows (Group 2) physiological parameters for preterm lambs managed by invasive mechanical ventilation for 3d. Continuous iv infusion of rhIGF-l/rhIGFBP-3 (1.5 mg/Kg/day; blacks squares) led to somewhat better systemic hemodynamic and heart rate outcomes [Panels A-D} relative to vehicle-control preterm lambs (open circles); however, no statistically significant differences were detected.

Figure 4A-B RhIGF-l/rhIGFBP-3 led to phosphoiylation of IGF-1 receptor (IGF-l-R) in sheep endothelial cells in vitro. Panel A: The response was concentration-dependent. Panel B: Only 50 ng/mL and 100 ng/mL rhIGFl/rhIGFBP-3 treatments led to IGF-1 level above background at all timepoints tested. Control (BSA, bovine serum albumin) treatment did not lead to signal elevation across all time points and dose ranges.

Figure 5 Shows the respiratory severity score for 7 day lamb study.

Figure 6 Shows the A-a gradient for 7 day lamb study.

Figure 7 Shows the oxygenation index for 7 day lamb study.

Figure 8 Shows the P/F ratio for 7 day lamb study.

Figure 9 Shows the S/F ratio for 7 day lamb study.

Figure 10A-D shows Group 2 alveolar capillary growth in preterm lambs managed by invasive mechanical ventilation for 3d. Immunohistochemistry was used to label capillary endothelial cells (brown color in [Panels A and B; the panels are the same magnification; see scale bar). Quantitative histology [Panels C and D) showed that continuous iv infusion of rhIGF-l/rhIGFBP-3 (1.5 mg/Kg/day; black squares) led to significantly greater capillary surface density (p<0.1) and epithelial surface density (p<0.1) compared to vehicle-control (white circles).

Figure 11A-D shows (Group 2) alveolar formation in preterm lambs managed by invasive mechanical ventilation for 3d. Continuous iv infusion of rhIGF-l/rhIGFBP-3 (1.5 mg/Kg/day) led to somewhat better appearing terminal respiratory units (TRU; Panel B) and more buds of secondary septa (arrow in Panel D) relative to vehiclecontrol [Panels A and C). Panels A and B are the same magnification; see scale bar. Panels C and D are same magnification; see scale bar. Distal airspace walls (arrowhead in Panels C and D) appear to have similar thickness between the two groups.

Figure 12A-C shows (Group 2) semi-quantitative normalized protein abundance by immunoblot in lung parenchyma in preterm lambs managed by invasive mechanical ventilation for 3d. Continuous iv infusion of rhIGF-l/rhIGFBP-3 (1.5 mg/Kg/day; black squares) led to significantly greater abundance of cleaved caspase-3 [Panel B; p<0.1) compared to the vehicle-control (white circles). No statistical differences were detected for normalized protein abundance of proliferating cell nuclear antigen (PCNA; Panel A) or fetal liver kinase-1 (Flk-1 (VEGF-R2; Panel C).

Figure 13 Shows quantitative morphological results show that indices of alveolar formation are significantly better (* p<0.05 by unpaired t-test) in rhIGF-l/rhIGFBP-3-treated preterm lambs compared to control preterm lambs, both groups of which were mechanically ventilated for 7 days.

EXAMPLES

This study was designed to first define the developmental levels of plasma IGF-1 in lambs.

Two groups of lambs were used. The first group (Group 1) was used to determine normal plasma IGF-1 protein level during fetal and postnatal development in unventilated lambs. The second group (Group 2) was used to determine plasma levels of IGF-1 protein during 3d of mechanically ventilated (MV) lambs.

Initially we determined the optimal dosage of rhIGF-l/rhIGFBP-3 required to attain physiological level of IGF-1 in plasma (~125 ng/mL).

The optimal dosage (1.5 mg/Kg/d; n=6) was subsequently used for a pilot randomized, placebo-controlled study versus vehicle-control (n=6). Both sets of preterm lambs were managed by MV (mechanical ventilation) for 3 days.

Continuous infusion of rhIGF-l/rhIGFBP-3 during MV for 3d significantly improved some pulmonary and cardiovascular outcomes (p<0.1).

Also, rhIGF-l/rhIGFBP-3-treated preterm lambs maintained their weight, whereas vehicle-control preterm lambs lost weight from day of life 1 through day of life 3 (p<0.1).

Furthermore, some structural and biochemical outcomes related to alveolar formation were statistically significantly better in the rhIGF-l/rhIGFBP-3-treated preterm lambs compared to vehicle-control preterm lambs at the end of the 3 day study (p<0.1).

Another result is that rhIGF-l/rhIGFBP-3 infusion did not adversely affect the liver and kidneys of the preterm lambs. The data shows that 3 days of continuous iv infusion of rhlGF- l/rhIGFBP-3 improved some pulmonary and cardiovascular outcomes, without toxicity, in mechanically ventilated preterm lambs.

Methods

Protocols adhered to APS/NIH guidelines for humane use of animals for research and were prospectively approved by the IACUC at the University of Utah Health Sciences Center.

Plasma IGF-1 Protein Level in Normal Unventilated Lambs during Development

Plasma levels of IGF-1 protein were measured by endpoint ELISA, using a human IGF-1 ELISA kit (Mediagnost; Reutlinger, Germany), the reagents for which cross-react with IGF-1 from many species, including sheep. Plasma samples were acid-dissociated from binding proteins prior to analysis of free IGF-1. IGF-1 levels were extrapolated from a standard curve derived from recombinant human IGF-1.

Surgical preparation. To determine normal developmental changes in IGF-1, plasma samples were analyzed for 3-5 lambs/age in female and male normal unventilated fetal lambs at ~128d gestation (saccular stage of lung development; ~28 wk human equivalent), ~131d gestation (~29 wk human equivalent), ~135d (~36 wk human equivalent), unventilated lambs born at term (~150d gestation), and spontaneously breathing term-born lambs at Id, 2 months (weaning from ewe’s milk; 1-2 yr human equivalent), and 5 months (~6 yr human equivalent) postnatal age. Methods for surgical delivery of fetal lambs and term lambs are reported by our laboratory Dahl MJ et al. [2018] Former-preterm lambs have persistent alveolar simplification at 2 and 5 months corrected postnatal age. 21m J Physiol Lung Cell Mol Physiol 315, L816-L833.

Collection of fetal blood samples, time-dated anesthetized (ketamine; 10 mg/Kg, im; isoflurane ~2.5%, inhaled] and intubated pregnant ewes that carried one fetus or twin fetuses were used. Fetal lambs were not exposed to antenatal steroids. While the placental circulation was maintained, the fetal lambs were intubated with a cuffed endotracheal tube (3.5 to 4.0 French], which was plugged to prevent drainage of lung liquid and to prevent breathing. Catheters were inserted into a common carotid artery and external jugular vein for plasma sampling. Term newborn lambs (about 24h old) were anesthetized (ketamine; 10 mg/Kg, im; isoflurane ~2.5%, inhaled) and intubated for insertion of catheters into a common carotid artery and external jugular vein for plasma sampling, which was done after the term lambs recovered from anesthesia (~48h of life).

Effect of Continuous Infusion of rhIGF-l/rhIGFBP-3 or Vehicle (Control) in Preterm Lambs During Invasive Mechanical Ventilation for 3 Days

We verified in vitro that rhIGF-l/rhIGFBP-3 led to downstream signaling by sheep vascular endothelial cells (ATCC- Manassas, VA). Cells were seeded at60,000/well in a 96- well tissue culture plate. The next day, cells were washed once and left overnight in serum-free Dulbecco's Modified Eagle Medium (Invitrogen-City, state). After serum starvation, the cells were treated with 0, 10, 50, or 100 ng/mL human IGF1 (R&D systems) or bovine serum albumin (Fisher Scientific-city state) for 5, 10, or 30 min. Cells were washed and then lysed on ice for 30 min in 100 pL lysis buffer (10 mM HEPES (4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid), 0.5% Triton-x 100), with halt protease and phosphatase inhibitor cocktail (Fisher Scientific). Cell lysates were analyzed for pIGF- 1R, according to manufacturer’s instructions, using AlphaLISA SureFire Ultra kit (Perkin Elmer catalog #ALSU-PLGFR-A500). Briefly, 10 pL of cell lysate and 5 pL acceptor mix were added to a PerkinElmer white half area plate and incubated for lh in the dark at room temperature. Five pL of donor mix was added and incubated in the dark for lh at room temperature. The plate was read on an Envision instrument.

A pilot dosage-finding pharmacokinetic study of continuously infused rhIGF-l/rhIGFBP-3 (Mecasermin rinfabate) was done to define the optimal dosage required to achieve a plasma concentration of ~125 ng/mL during MV for 3d. Continuous infusion was employed because it was determined that IGF-1 has a ti/2 in plasma of ~2h in lambs. The tested dosage range of 0.5, 1.5, or 4.5 mg/Kg/d bracketed the physiological range of plasma IGF-1 levels in normal fetal and term lambs determined from the first study. Two preterm lambs were treated with each dosage. Once the optimal dosage was identified (1.5 mg/Kg/d), four more preterm lambs were studied with this dose (n=6). Six more ventilated preterm lambs were treated with vehicle (continuous iv infusion of sterile saline). The latter ten preterm lamb studies were assigned to rhIGF-l/rhIGFBP-3 treatment or vehicle treatment by a blinded selection before surgical delivery to minimize bias.

Surgical preparation and neonatal intensive care. Methods for delivering preterm lambs are reported by our laboratory, with differences noted below. Briefly, time-dated pregnant ewes (singletons or twins) were studied at ~131d of gestation (saccular stage of lung development). The pregnant ewes were given an intramuscular injection of dexamethasone phosphate (6 mg; Vedco, Inc., St Joseph, MO) at ~48h, and ~24h before Cesarean-section delivery. At delivery, we intubated the fetal lambs with a cuffed endotracheal tube (3.5 to 4.0 French), through which 10 mb of lung liquid were aspirated and replaced with Infasurf® (3 mL/Kg; ONY Biotech, Amherst, NY).

Preterm lambs were resuscitated through the endotracheal tube, using a programmed resuscitation box. The lambs were weighed, placed prone on a veterinary sling atop a radiantly heated NICU bed, and connected to a Drager ventilator (model VN500, Lubeck, Germany). Sedation was the same for all ventilated preterm lambs (pentobarbital as needed and buprenorphine every 6h). Lambs were supported with synchronized intermittent mandatory ventilation that was pressure-controlled, with warmed and humidified gas. F1O2 was adjusted to attain target hemoglobin oxygen saturation of 90-94% (Path 60-90 mmHg) by pulse oximetry (Model SurgiVet V9200IBP/Temp, Smith Medical ASD, Inc., St. Paul, MN). Peak inspiratory pressure was adjusted to attain a target PaCCh between 45 and 60 mmHg, resulting in pH between 7.25-7.35. Target expiratory tidal volume, measured by the ventilator, was 5 to 7 mL/Kg. We calculated oxygenation index (01) [(Paw x FiO2)/PaO2], P/F (PaO2/FiO2) ratio, and Alveolar-arterial (A-a) gradient [((F 0 /100) x (640-47))-(PaCO /0.8)-Pa0 ]. Salt Lake City is at about 5,000 ft elevation so barometric pressure is about 640 mmHg.

Orogastric feeding of ewe’s colostrum (Kid & Lamb Colostrum Replacement, Land 0 Lakes, Arden Hills, MN) was started at~3h of postnatal life (3 mL) and the volume was gradually increased as tolerated, with target over the first week of postnatal life of ~60 kcal/Kg/d. Parenteral dextrose was infused to maintain plasma glucose between 60 and 90 mg/dL. Arterial blood and urine samples were collected every 24h to measure plasma levels of IGF-1 protein, as well as indicators of liver and kidney injuiy (analyzed at Associated Regional and University Pathologists (ARUP) Laboratories, Salt Lake City), respectively.

Terminal Tissue Collection of the Lung from Preterm Lambs

At the end of MV for 3d, blood samples were collected before the preterm lambs were given heparin (1000 U, intravenously) followed by 5 mg/Kg of pentobarbital. Lambs were subsequently given 60 mg/Kg pentobarbital sodium solution intravenously (Beuthanasia solution, Ovation Pharmaceuticals, Inc., Deerfield, IL). The chest was opened, the trachea was ligated at endinspiration (to minimize atelectasis), and the lungs and heart were removed. The whole left lung was insufflated with 10% buffered neutral formalin at a static pressure of 25 cmH20. Fixed-lung displacement volume was measured by suspension in formalin before the lung was stored in fixative (4°C, 24h). Paraffin-embedded tissue blocks were prepared for histology and quantitative histology, including quantitative immunohistochemistry to assess structural indices of alveolar formation and alveolar capillaiy growth. The right caudal lobe of the lung was used for molecular analyses (snap- frozen in liquid nitrogen and stored at -80°C). We used systematic, uniform, and random, protocols for unbiased sampling of lung tissue.

Data Analysis

Physiological variables and quantitative histology results are summarized as mean ± standard deviation (standard deviation, SD) or mean (interquartile range, IQR), as shown in the tables and figures. Statistical analyses were done using GraphPad (Prism, v9). Because this was a pilot study designed to identify potential advantageous outcomes at the end of 3d of treatment with the optimized dosage of rhIGF-l/rhIGFBP-3, we used two-way ANOVA (treatment and time), followed by post-hoc Holm-Sidak's multiple comparisons test for physiological results, with oc=0.1 (90%), except as noted for Figure 1. We used one-tailed parametric t-test (oc=0.1) for morphological outcomes and one-tailed non-parametric tests (oc=0.1) for immunoblot results.

Results

Plasma IGF-1 Protein Level in Normal Unventilated Lambs during Development

Plasma IGF-1 protein level increased from ~75 ng/mL in normal unventilated fetal lambs to ~220 ng/mL through 5 months postnatal age in normal un ventilated term lambs (Figure 1A).

Effect of Continuous Infusion of rhIGF-l/rhIGFBP-3 or Vehicle (Control) in Preterm Lambs During Invasive Mechanical Ventilation (MV) for 3d

Demographic characteristics for this randomized, placebo-controlled study are summarized in Table 1.

Gestational age, birth weight, and ending weight were not statistically different between the two sets of ventilated preterm lambs. Nonetheless, at operative delivery, the rhIGF-l/rhIGFBP-3- treated preterm lambs’ gestation age was Id younger and delivery weight was about 0.5 kg lower than for the vehicle-control preterm lambs. Femaleimale distribution was not equal between the rhIGF-l/rhIGFBP-3-treated and vehicle-control-treated preterm lambs. This was not possible because treated or vehicle-control group assignment occurred before operative delivery of fetuses.

Plasma IGF-1 protein level at the beginning of each study was the same for both sets of ventilated preterm lambs (FigurelB; "predose" level was ~100 ng/mL; not statistically different). Subsequently, plasma IGF-1 protein level diverged between the rhIGF-l/rhIGFBP-3-treated and vehicle-control-treated preterm lambs over the 3d study period. For preterm lambs treated with 1.5 mg/Kg/d rhIGF-l/rhIGFBP-3, plasma IGF-1 protein level doubled (220±60 ng/mL) at 12h of continuous infusion compared to this set’s pretreatment baseline level (103±63 ng/mL; p<0.05). The IGF-1 protein level plateaued at ~140 ng/mL for the last 24h of the 3d study period (p<0.05 compared to this set’s pretreatment baseline level). For the vehicle-treated preterm lambs, by comparison, plasma IGF-1 protein level significantly decreased from the set’s baseline level (91±40 ng/mL) to a nadir of ~30 ng/mL for the last 48h of the 3d study period (36±25 ng/mL at 60 and 72h; p<0.05).

Recombinant IGF-l/rhIGFBP-3 led to phosphorylation of IGF-1R in sheep endothelial cells in vitro, as shown in Figure 2. The response was concentration-dependent (Figure 4A). Only 50 ng/mL and 100 ng/mL rhIGFl/rhIGFBP-3 treatments led to levels above background at all timepoints tested (Figure 4B). Control (bovine serum albumin) treatment did not lead to signal elevation across all time points and dose ranges.

Histological examples of alveolar architecture are shown in Figure 10 and were similar between the rhIGF-l/rhIGFBP-3-treated and vehicle-control preterm lambs at the end of 3d of MV. As expected, after 3d of MV, the vehicle-control preterm lambs had terminal respiratory units (TRU) that were distended and simplified. That is, the parenchyma was thick and cellular, and few buds of alveolar secondary septa were evident. TRU architecture appeared somewhat more developed, with short buds of secondary septa being more evident in the lung of the rhIGF-l/rhIGFBP-3- treated preterm lambs. Quantitative histological indices of alveolar formation revealed no statistical differences between the rhIGF-l/rhIGFBP-3-treated and vehicle-control preterm lambs for radial alveolar count, secondary septal volume density, or distal airspace wall thickness.

Histological examples of alveolar capillary endothelial cell identification by immunohistochemistry are shown in Figure 11 and was performed using immunostained sections of lung tissue to quantify indices of alveolar capillary growth and counterstain to identify epithelial cells. Stereological assessment of surface density detected statistically significantly larger surface density for capillary endothelial cells and airspace epithelial cells for the rhIGF-1/rhIGFBP- 3- group compared to the vehicle-control preterm lambs (p<0.1) (see Figures 11C and D).

Protein abundance in lung parenchyma was assessed semi-quantitatively and shown in Figure 12. Statistical difference was detected for cleaved caspase 3, for which the relative protein abundance was significantly greater for the rhIGF-l/rhIGFBP-3-treated preterm lambs compared to the vehicle control preterm lambs (p<0.1). Otherwise, no statistical differences were detected for protein abundance of proliferating cell nuclear antigen or fetal liver kinase- 1 (Flk-1) between the two groups.

The systemic hemodynamic parameters shown in Figure 3 are numerically better for the rhIGF-l/rhIGFBP-3-treated preterm lambs; however, results were not statistically different from the vehicle-control preterm lambs (Figure 3A-D, respectively). Consistent with other cardiovascular parameters, none of the six rhIGF-l/rhIGFBP-3-treated preterm lambs required dopamine to maintain mean aortic pressure >35 mmHg (Table 3), whereas two of the six vehicle-control preterm lambs required dopamine infusion, beginning on the first day of life and continuing for the 3d study period (4-7 mcg/Kg/min) to support systemic mean blood pressure.

Physiological parameters for respiratory gas exchange are presented in Figure 2. Results are shown for 12h epochs of postnatal age during the 3d of MV. Targets were SaCh range 90-94% (Path range 60-90 mmHg) for oxygenation and PaCCh range 45-60 mmHg for ventilation. Although numerical results favored rhIGF-l/rhIGFBP-3 treatment, no statistical differences were detected between the rhIGF-l/rhIGFBP-3-treated versus vehicle-control preterm lambs for the applied FiC>2 or PIP to sustain the oxygenation and ventilation targets, respectively (Figures 2A-D). OI and A-a gradient were significantly improved in rhIGF-l/rhIFGBP-3 treated lambs (Table 2). Plasma pH and bicarbonate also were not different between the rhIGF-l/rhIGFBP-3-treated and vehicle-control preterm lambs over the 3d study period (Figures 2E and F, respectively). No differences were detected in the amount of pentobarbital (mg/Kg/12h, iv) or buprenorphine (mcg/Kg/12h, iv) between the rhIGF-l/rhIGFBP-3-treated preterm lambs compared to the vehiclecontrol preterm lambs (data not shown).

Fluid balance parameters are summarized in Table 4. Average results are reported for 12h epochs. No statistical differences were detected for intravenous infusion of saline or dextrose, total fluid intake, enteral milk intake, or urine output between the rhIGF-l/rhIGFBP-3- treated versus vehicle-control preterm lambs.

Liver function and renal function test values are summarized in Tables 5 and 6, respectively. Plasma samples were taken while fetal lambs had their umbilical cord intact (‘pre’ in Table 5), and at'24h’ and ‘72h’. Liver function was assessed by measurement of plasma alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (AP), total bilirubin, and direct bilirubin. Indicators of kidney function were urine output, creatinine, blood urea nitrogen, lactate, and urine microalbumin. No differences were detected between the rhIGF-l/rhIGFBP-3-treated versus the vehicle-control preterm lambs. The levels were within reference limits for fetal lambs, adult sheep, and adult humans (Tables 5 and 6).

Table 5. Group 2 Liver function test values of preterm Iambs managed by mechanical ventilation for 3d

Discussion

Effective preventative strategies to improve long-term lung function and structure, and cardiovascular physiology after preterm birth followed by prolonged respiratory management in the neonatal intensive care setting remain a major challenge.

Using a large-animal model that emulates preterm birth and prolonged respiratory management, without hyperoxia, in a neonatal intensive care setting and allows a variety of assessments, from feeding tolerance and growth, respiratory gas exchange, cardiovascular physiology, and structural and biochemical indices relevant to alveolar formation to indices of liver and kidney function.

The present study is the preterm model is a non-lethal model that uses fetal lambs delivered at about 85% of gestation (saccular stage of lung development; equivalent to about 28 weeks gestation in humans]. Also, the duration of mechanical ventilation and exposure to rhlGF- l/rhIGFBP-3 was short, lasting only 3d for this pilot study. Nonetheless, the sheep endothelial cell experiments in vitro showed that downstream signaling was triggered by rhIGF-l/rhIGFBP-3. Three-day duration of continuous infusion of rhIGF-l/rhIGFBP-3 may have been insufficient for IGF- l’s morphogenic effects.

Our study was designed to first define the developmental levels of plasma IGF-1 protein during normal fetal and postnatal life in unventilated normal lambs. During normal development, IGF-1 protein level in plasma increased from ~75 ng/mL in unventilated fetuses (~128d gestation] to ~220 ng/mL in unventilated lambs (5 months postnatal age; ~6 yr human equivalent]. Next, we designed a pilot study to test effectiveness of rhIGF-l/rhIGFBP-3 continuous intravenous infusion to improve pulmonary and cardiovascular outcomes, using preterm lambs that were managed by MV for 3d. For this test, we first found that IGF-1 protein level decreased significantly after birth during 3d of MV in vehicle-control preterm lambs, similar to preterm infants. We subsequently established an optimal dosage ofrhIGF-l/rhIGFBP-3 (1.5 mg/Kg/d) to maintain physiologic plasma IGF-1 level of ~125 ng/mL. We used that dosage to evaluate the pulmonary and cardiovascular physiological, and lung structural and biochemical effects of 3 days’ continuous infusion of rhlGF- l/rhIGFBP-3 in mechanically ventilated preterm lambs (n=6). Continuous infusion of rhlGF- l/rhIGFBP-3 during MVfor 3d statistically improved some pulmonary and cardiovascular outcomes compared to vehicle-control preterm lambs. Systemic hypotension did not occur in rhlGF- l/rhIGFBP-3-treated preterm lambs (0/6), whereas 2/6 vehicle-control preterm lambs required dopamine to maintain physiologic systemic perfusion pressure. Also, rhlGF- l/rhIGFBP-3-treated preterm lambs maintained their weight, whereas vehicle-control preterm lambs lost weight from day of life 1 through day of life 3 (p<0.1). Furthermore, some structural and biochemical outcomes related to alveolar formation that would favor improved gas exchange were statistically better in the IGF-l/IGFBP-3-treated preterm lambs compared to vehiclecontrol preterm lambs at the end of the 3d study.

Angiogenesis is an important process for appropriate alveolar formation in the lung. This process is diminished in preterm infants with BPD and animal models of BPD, causing alveolar simplification that includes reduced capillary growth in the lung. A potentially important underlying molecular mechanism may be low plasma IGF-1 protein levels in preterm infants with BPD. Also, a recent study in rat pups showed that daily intraperitoneal treatment with 0.02 to 20 mg/Kg ofrhlGF- l/rhIGFBP-3 for 14d preserved lung structure in a dose-dependent manner and prevented right ventricular hypertrophy in antenatal and postnatal models of BPD. That study also showed that exogenous IGF-1 enhanced pulmonary vascular endothelial cell growth and tube formation.

Our study showed that stereological indices of capillaiy endothelial cell and airspace epithelial cell surface density were statistically larger in the lungs of the rhIGF-l/rhIGFBP-3-treated group compared to the vehicle- control preterm lambs (p<0.1). Therefore, therapeutic strategies such as exogenous IGF-1 treatment that enhance growth of endothelial and epithelial cells may provide a novel approach toward prevention of BPD following early diagnosis of high-risk preterm infants.

This study’s immunoblot results provide some insight into the impact of IGF-1 on lung outcomes. The statistically greater abundance of cleaved caspase 3 protein detected in parenchymal tissue of the lung of the rhIGF-l/rhIGFBP-3-treated preterm lamb. Thinning of saccular walls, in part by apoptosis of mesenchymal (interstitial) cells, is necessary to establish a thin diffusion barrier for oxygen and carbon dioxide. In this regard, greater abundance of cleaved caspase 3 in the lung of the rhIGF-l/rhIGFBP-3-treated preterm lambs is consistent with improving structural thinning of saccular walls. The current study did not detect significant decrease in proliferating cell nuclear antigen (PCNA) protein abundance.

Nonetheless, PCNA relative protein abundance was numerically lower in the rhIGF-l/rhIGFBP-3- treated preterm lambs.

These results will require further investigation because IGF-1 signaling, while typically protective against apoptosis, also is proapoptotic. Perhaps in the context of the immature lung stressed by preterm birth and MV with oxygen-rich gas, etc., IGF-1 signaling may shift the balance of apoptosis versus proliferation among cells in the lung.

Another result is that rhIGF-l/rhIGFBP-3 infusion did not adversely affect the liver and kidneys of the preterm lambs. We conclude from this pilot study that 3d of continuous iv infusion of rhIGF-l/rhIGFBP-3 improved some physiological, morphological, and biochemical outcomes, without toxicity, in mechanically ventilated preterm lambs.

Example 2 Pre-term Lamb 7 Day Study

Preterm lambs (~128d gestation; saccular stage lung development) were divided into two groups, both of which were mechanically ventilated for 7d. Group 1 was given continuous infusion of saline (vehicle control, iv; n=8). Group 2 was given continuous infusion ofrhIGF-l/rhIGFBP-3 (1.5 mg/Kg/d, iv; n=9). Respiratory severity score, oxygenation index (Oi), SpCh/FiCh (S/F) ratio, PaCh/FiCh (P/F) ratio, alveolar-arterial (A-a) gradient, resistance (R), dynamic compliance (Cdyn) and last 20% end expiratory compliance (20/Cdyn) were measured.

Preterm lambs treated with rhIGF-l/rhIGFB-3 had statistically better respiratory severity score, A- a gradient, S/F ratio, P/F ratio, and oxygenation index (Figure 5 to 9). Continuous infusion of rhIGFl/rhIGFBP-3 during 7d of mechanical ventilation improved respiratory gas exchange indices in preterm lambs.

Example 3 Per-term Lamb 7 Day Study Morphological Results

Preterm lambs (128d; term ~150d; ~28w human gestation) were exposed to antenatal steroids, perinatal surfactant, and resuscitated and supported by mechanical ventilation for 7d (Drager VN500, PC-SIMV mode). Physiological targets were Path 60-90 mmHg, PaCCh 45-60 mmHg, O2 saturation 88-92%, pH 7.25-7.35. The control group received vehicle (IGF-1 diluent in saline; continuous iv infusion; n=8; 4F 4M). The treated group received rhIGF-l/rhIGFBP-3 (1.5 mg/Kg/d, iv; n=9; 4F 5M). We used morphometry and stereology to quantify structural indices of alveolar formation.

Radial alveolar count and secondary septal volume density were significantly greater (* p<0.05) in the IGF-l-treated group compared to the control group (Fig 13). Distal airspace wall thickness was not statistically different between the groups (Fig 13). No differences were detected between females and males.

We conclude that rhIGF-l/rhIGFBP-3 improved indices of alveolar formation in preterm lambs that were mechanically ventilated for 7 days.