WO2023242439A1 - Vascular stabilisation (preterm infants) - Google Patents

Abstract

A method of treatment or prophylaxis comprising stabilising blood pressure and/or flow in a preterm infant (also referred to a neonate herein) 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 parenterally.

Description

VASCULAR STABILISATION (PRETERM INFANTS) BACKGROUND The preterm infants as a patient population are some of the most delicate, vulnerable and difficult to treat. Some of these infants have only 4 tablespoons of blood in their whole body. This leads to large variations in parameters, such as blood pressure measured in this population. It is also very difficult to do clinical trials in this patient population, for example sampling and administration is not easy, even a saline drip has the potential to cause a brain haemorrhage. Many drugs are not licensed for use in these infants and the dose has not been established adequately. In addition, drugs often have a different half-life in these patients. What is more reaching statical significance is not straightforward because of the variability between individual patients. The review Dempsey et al 2015 discusses the challenges of treating low blood pressure in preterm infants. There is no established clinical protocol that is the standard of care. Thus, the choice of intervention remains unresolved. The majority of clinicians administer an inotrope/vasopressor agent, such as dopamine. However, this is not without problems. Interestingly one finding was that preterm babies with normotensive blood pressure prior to treatment had a worse outcome than infants who were hypotensive and not treated. The present inventors have worked with the premature infants continuously for many, many years and have developed expertise based on clinical research, which not only includes measuring parameters but also empirical observation. The present inventors have established that in preterm infants rather than worrying about a number per se, where the blood flow and pressure is functioning adequately infants have the ability to urinated consistently. Urine output can easily be measure by weighing diapers. Thus the ability to urinate can be used as indicator for the status blood pressure/flow. In preterm infants with low systemic mean blood pressure the ability to urinate is profoundly affected. If the blood flow/pressure is low the capillaries in the kidney restrict to try and create adequate pressure to function. This response of the kidneys causes further problems and puts pressure on the whole system. Thus, there is an intrinsic relationship between blood flow/pressure and the ability to urinate. A standard response to this inability to urinate is to administer a vasopressor, such as dopamine. If the preterm infant needs further assistance to urinate a diuretic is administered. The inventors have data to suggest that IGF-1/IGFBP treatment may help to regulate fluctuation in blood flow and/or pressure. It is hypothesised that IGF-1/IGFBP treatment may stimulate releases of endogenous vasopressors, such as aldosterone. What is more there are a number of improvements in preterm infants treated with a complex of IGF-1/IGFBP because they are discernibly more robust than untreated preterm infants. So much so that “blinded” carers can establish within 3 days, which infants are receiving treatment in comparison to untreated infants. This manifests itself in general systemic effects that are very evident to the trained observer, for example ability to urinate unassisted, the stabilised breathing, improved food digestion etc. It translates in practice to an easier to care for baby and reduces the mortality in the treated population. A plethora of data has been generated and analysed, and a strong underlying trend seems to be stabilisation of blood flow/pressure may fundamentally underpin many of these observations. 50% glycerol administered systemically generates hyperosmolarity leading to disturbance of metabolic systems, changes in glucose levels and significant changes in blood flow including mean arterial pressure (in particular more variable and/or higher pressure), for example in preterm rabbit pups increased cerebral blood flow is observed. These changes cause a number of disfunctions in preterm infants, such as intraventricular haemorrhage. This hyperosmolarity and increased blood flow to the brain renders rabbit pups/patients (such as preterm infants) susceptible to metabolic dysfunction, cardiovascular irregularities, increased blood flow to the brain and perhaps systemic vascular collapse. Untreated rabbit pups have significantly higher mortality than pups treated with IGF-1/IGFBP-3. This suggests that treatment stabilises or assists patients (such as preterm infants) in regulating blood flow pressure, which in turn confers a significant and surprising mortality benefit. Whilst not wishing to be bound by theory, it now considered that if the blood flow/pressure can be stabilised, the oxygen can reach the tissues, waste can be removed, and thereby prevent damage of specific tissue. This in turn may reduce inflammatory response, aid digestion, and generally create wellbeing in the preterm infant. The smallest birth weight babies, not necessarily the youngest babies, benefit the most. These are the babies that would normally die without treatment. The preterm infants may also have increased spleen weight. Interestingly the spleen is involved in filtering the blood, fighting infection, recycling useful components, such as haemoglobin. Whilst these concepts may appear to a person outside the field to be amorphous, they are fundamentally important to health and survival. They translate to a real positive impact on mortality rates in the treated infants. SUMMARY OF THE DISCLOSURE The disclosure is summarised in the following paragraphs. 1. A method of treatment or prophylaxis comprising stabilising blood pressure and/or flow (vascular bed stabilisation) in a preterm infant (also referred to a neonate herein) 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 parenterally. 1A. A composition of IGF-1 and an IGF binding protein (such as IGFBP-3) for example as a complex, such as parenterally, for use in the treatment or prophylaxis of stabilising blood pressure and/or flow (vascular bed stabilisation) in a preterm infant. 1B. A composition of IGF-1 and an IGF binding protein (such as IGFBP-3) for example as a complex, such as parenterally, for use in the manufacture of a medicament for the treatment or prophylaxis of stabilising blood pressure and/or flow (vascular bed stabilisation) in a preterm infant. 2. A method or composition for use according to any preceding paragraph wherein the blood pressure and/or flow is stabilised without increasing vascular resistance. 3. A method or composition for use according to any preceding paragraph, wherein the preterm infant is not hypovolaemic, before treatment. A method or composition for use according to any preceding paragraph, wherein the blood flow is stabilised. A method or composition for use according to any preceding paragraph, wherein central blood flow is stabilised. A method or composition for use according to any preceding paragraph, wherein peripheral blood flow is stabilised. A method or composition for use according to any preceding paragraph, wherein the blood pressure (such as mean system blood pressure, in particular arterial pressure) is stabilised. A method or composition for use according to any preceding paragraph, wherein systolic pressure is stabilised, for example in the range 60 to 90mmHg (eg 66, 61, 62, 63, 64, 65, 70, 75, 80, 85, 86, 87, 88, 89 or 90), such as where the systolic pressure is higher than an untreated preterm infant (in particular in the period up to 72 hours post birth). A method or composition for use according to any preceding paragraph, wherein diastolic 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, such as 31, 32, 33, 34, 35, 40, 45, 50, 55, 56, 57, 58, 59 to 60. A method or composition for use according to any preceding paragraph, wherein tissue perfusion is stabilised, for example treated preterm infant is pink. A method or composition for use according to any preceding paragraph, wherein the treated preterm (is more robust in that it) has a lower heart rate in comparison to untreated preterm infants (or average thereof), for example a bpm in the range about 120 to 180, for example on average 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175 or 180. A method or composition for use according to any preceding paragraph, wherein the treated preterm does not require intervention for low blood pressure, for example does not require a vasopressor, such as dopamine. A method or composition for use according to any preceding paragraph, wherein the blood pressure of a treated preterm infant does not drop below 30mmHg. A method or composition for use according to any preceding paragraph, wherein the preterm infant has a blood pressure in the range 30 to 50mmHg, during treatment, for example 30, 31, 32, 33, 34, 35, 40, 45, 46, 47, 48, 49 or 50 mmHg, such as 30, 35, 40, 45 or 50 mmHg. A method or composition for use according to any preceding paragraph, wherein the preterm infant has hypertension, before treatment is initiated. A method or composition for use according to any preceding paragraphs, wherein the preterm infant receiving said treatment/prophylaxis is more robust than a preterm infant without the treatment/prophylaxis. A method or composition for use according to paragraph 16, wherein more robust is adequate temperature regulation. A method or composition for use according to paragraph 16 or 17, wherein more robust is a good level of activity (motor activity), for example than a corresponding untreated patient. A method or composition for use according to any one of paragraphs 16 to 18, wherein more robust is adequate urine output, for example in the 2 to 5mg/Kg/hour (2, 3, 4, 5mg/Kg/hour) such as in the first 72 hours of life. 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). A method or composition for use according to any one of paragraphs 16 to 20, wherein more robust is minimal assistance with breathing. A method or composition for use according to paragraph 21, wherein the infant is on mechanical ventilation. A method or composition for use according to paragraph 21 or 22, wherein the treated preterm infant stays within the predefined parameters for gases, for example the mechanical ventilator does not require adjustment by a carer. A method or composition for use according to any preceding paragraph (such as paragraph16 to 22), 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. A method or composition for use according to any preceding paragraph (such as paragraph 16 to 23), wherein the treated preterm infant has a peak inspiratory pressure (cmH20) that is lower than untreated preterm infants (or an average thereof), for example in the range 45 and 60 (such as 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 or 60) in particular in the first 72 hours of life. A method or composition for use according to any preceding paragraph, 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. A method or composition for use according to any preceding paragraph, wherein the treated preterm infant has no residual food in their digestion at the next feeding time. A method according to any preceding paragraph, wherein the treated preterm infant can absorb higher amounts of IV dextrose than an untreated preterm infant, for example at least 2.9mL/Kg/h for each 12 hour period. A method or composition for use according to any preceding paragraph, wherein the treated preterm infant requires lower amounts of IV saline than an untreated preterm infant, for example 1.5mL/Kg/h for each 12 hour period (especially in the first 36 hours of life). A method or composition for use according to any preceding paragraph, wherein the treated preterm infant has higher levels of total bilirubin than an untreated preterm infant, and for example does not have raised levels of direct bilirubin. 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. A method or composition for use according to any preceding paragraph, wherein preterm infants are in the range 23 to 34 weeks post gestation (such as 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, or 34), when treatment is initiated. A method or composition for use according to any preceding paragraph, 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. 34. A method or composition for use according to any preceding paragraph, wherein the infant, for example a preterm infant, is treated by subcutaneous administration, for example bolus subcutaneous administration, in particular for at least 1 week, for example 2 to 6 weeks, such as 2, 3, 4, 5 or 6 weeks. 35. A method or composition for use according to any preceding paragraph, wherein the infusion is initiated within 24 hours of birth, for example within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 1516, 17, 18, 19, 20, 21, 22, 23 and 24 hours of birth, in particular within 1, 2 or 3 hours of birth, more specifically within 1 hour of birth. 36. A method or composition for use according to any preceding paragraph, wherein a treated infant has reduced incidence of hypoxic ischemic encephalopathy, in comparison to an infant without treatment. 37. A method or composition for use according to any preceding paragraphs, wherein treated preterm infants have a lower mortality rate than untreated preterm infants. 38. A method or composition for use according to any preceding paragraphs, where the composition comprises equimolar amounts of IGF-1 and IGFBP-3. 39. A method or composition for use according to any preceding paragraphs, wherein the 200 to 500µg/Kg/24hours of complex are administered, such as 200, 250, 300, 325, 350, 375, 400, 425, 450, 475 or 500µg/Kg/24hours, in particular 400µg/Kg/24hours. 40. A method or composition for use according to any preceding paragraphs, wherein the 55 to 110µg/Kg/24hours of IGF-1 (or the equivalent thereof) are administered, such as 55, 56, 57, 58, 59, 60 , 65, 70, 75, 80, 85, 90, 95, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109 or 110µg/Kg/24hours of IGF-1. 41. 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 on average 28, 29, 30, 31, 32, 33, 34, 35, 40, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 101, 102, 103, 104, 105, 106, 107, 108 or 109 ng/mL. 42. A method or composition for use according to any preceding paragraphs, wherein the preterm infant has a reduced incidence or susceptibility to sepsis, for example in comparison to a preterm infant without treatment. 43. 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,. 44. A method or composition for use according to any preceding paragraph, wherein the infant has birth weight of 2.2 pounds or less (1Kg or less). 45. A method or composition for use according to any preceding paragraph, wherein the infant is in the prone position. In one embodiment preterm infants treated according to the present disclosure have reduced tissue damage. In one embodiment respiratory severity score is reduced in treated infants, for example is 3 or less, such as 2 (in particular from day 2 or 3 of treatment or life respectively). This may be considered evidence of improved robustness. It is hypothesised that respiratory severity score per Kg has the potential to be a marker associated with the development of pulmonary hypotension. 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). This may be considered evidence of improved robustness. In one embodiment the A-a gradient is lower in the treated patients (neonates) than untreated patients, for example 30 or less in treated patients. 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 be considered evidence of improved robustness. In one embodiment the oxygen index is lower in treated patients (neonates) than untreated patients, for example about 3.5 or less. 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 treated patients (neonates) have a P/F ratio of 300 or above, for example in the first three days of 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 350, 355, 360, 365, 370, 375, 380, 385, 390, 395 or 400, in particular about400) for example from day 4 of treatment or life. In one embodiment treated patients (neonates) are not hypoxemic. In one embodiment treated patients (neonates) require less saline intravenously than untreated patients. In one embodiment treated patients (neonates) have stabilised sodium levels. In one embodiment treated patients (neonates) have stabilised potassium levels. In one embodiment treated patients (neonates) have stabilised calcium levels. In one embodiment treated patients (neonates) are able to metabolise more parenteral glucose, than untreated patients. In one embodiment treated patients (neonates) are able to urinate more than untreated patients, for example in the first the 12 to 24 hours after birth. In one embodiment treated patients (neonates) have reduced mortality. In one embodiment treated patients (neonates) have increased total bilirubin, for example 24 to 72 hours post birth. In one embodiment treated patient (neonates) do not have increased plasma bilirubin (direct). Increased total bilirubin without increased plasma bilirubin may allow better digestion in neonates because bilirubin is employed bile which is used to digest food. However, bilirubin increases can also be a sign of increased metabolism of red blood cells and/or reduced liver function. In one embodiment treated patients (neonates) do not have hypotension. In one embodiment treated patients (neonates) maintain physiologic systemic perfusion pressure. In one embodiment treated patients (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. In one embodiment treated patients (neonates) have one or more improved physiological parameters. In one embodiment treated patients (neonates) have one or more improved morphological elements. In one embodiment treated patients (neonates) have one or more improved biochemical parameters. In one embodiment treated patients (neonates) have a larger surface density for capillary endothelial cells in comparison to untreated patients. In one embodiment treated patients (neonates) have larger airspace epithelial cells in comparison to untreated patients. 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 one embodiment treated patients (neonates) have improved structural thinning of saccular walls in comparison to untreated patients, for example elevated levels of caspase-3 involved in apoptosis may be indirect evidence of the same. In one embodiment treated 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 embodiment treated patients (neonates) have higher aortic pressure. In one embodiment treated patients (neonates) have lower peak inspiratory pressure. In one embodiment the treatment according to the present disclosure is used in combination with a further therapy, for example a therapy employed in a preterm infant, such as surfactant therapy. In one embodiment there are no toxic effects. In one embodiment respiratory mechanics, such as measured by R, Cdyn, and/or 20/Cdyn remain unchanged. In one embodiment the composition is administered subcutaneously, e.g.3 times a day. In one independent aspect the invention extends to treatment of an infant with congenital heart defects such as patent ductus arteriosus, including a preterm infant and/or an infant born closer to full term gestation. Surprisingly the present inventors have established that infants treated (for at least 3 days, for example at 4 or 5 days) according to the present disclosure are much more robust and require less intervention than infants who do not receive the treatment. More robust as employed herein refers to one or more the following: more stable in one or more the following urinating, breathing, digestion; require less medical intervention (e.g. less medication); less breathing support; better digestion; less overall care; more activity; tissue is perfused; better temperature control; more motor activity, and combinations of two or more of the same, in comparison to a corresponding infant that did not receive treatment according to the present disclosure. Amazingly, these observations translate to a lower mortality rate in the treated infants and in particular the lowest birth weight infants have improved survival rates. DETAILED DISCLOSURE 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. 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. Some neonates do not grow well in the womb. Even though they may be “older” they are small for their gestational age. The treatment according to the present disclosure is particularly useful for these infants, such as those in the lower quartile of birth weight. Stabilising blood pressure as employed herein is a fit for purpose test: is the infant 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 maintained in the range 30 to 60mgHg. 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 be 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 on one or more key biological functions. For example, is the infant urinating, perfused, and/or breathing adequately etc. In one embodiment the disclosure is not for the treatment of bronchopulmonary dysplasia. A corresponding infant as employed herein is an infant with corresponding parameters, for example gestational age, weight and the like, generally without treatment. 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 respirator 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 FIO2 is set to attain target hemoglobin oxygen saturation of 90-94% (PaO260-90 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 PaCO2 between 45 and 60 mmHg (such as 45, 50, 55, 60), for example 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. Calculated oxygenation index (OI) = [(Paw x FiO2)/PaO2], P/F (PaO2/FiO2) ratio. Alveolar-arterial (A-a) gradient = [((F O /100) x (640-47))-(PaCO /0.8)-PaO ]. 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 mL) and the volume 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, for example 60, 65, 70, 75, 80, 85 or 90 mg/dL. Vascular Bed stabilization as employed herein refers to the stabilization/maturation of the intricate network of minute blood vessels pervade the tissue to carry the blood thereto, with minimal restriction, for example where constriction is avoided and vessels remain open and able to function. Vascular resistance (sometimes referred to as peripheral vascular resistance or systemic vascular resistance, SVR) is the resistance in the circulatory system that is used to create blood pressure, the flow of blood and is also a component of cardiac function. When blood vessels constrict (vasoconstriction) this leads to an increase in SVR. When blood vessels dilate (vasodilation), this leads to a decrease in SVR. If referring to resistance within the pulmonary vasculature, this is called pulmonary vascular resistance (PVR). Vascular resistance is used to maintain organ perfusion. In certain disease states, such as congestive heart failure, there is a hyper-adrenergic response, causing an increase in peripheral vascular resistance. Prolonged increases in blood pressure affect several organs throughout the body. In conditions such as shock, there is a decrease in vascular resistance thus causing decreased organ perfusion which leads to organ malfunction. Peripheral vascular resistance is mediated locally by metabolites, and over a distance on a neuro-hormonal level, therefore, many different components may become altered leading to changes in peripheral vascular resistance. Hypovolemia is a state of low extracellular fluid volume, generally secondary to combined sodium and water loss. Peripheral blood flow as employed herein refers to the transport of blood, blood flow distribution, exchange between blood and tissue, and storage of blood (venous system). Central blood flow is everything other than peripheral blood flow. Blood pressure (BP) is the pressure of circulating blood against the walls of blood vessels. Most of this pressure results from the heart pumping blood through the circulatory system. When used without qualification, the term "blood pressure" refers to the pressure in a brachial artery, where it is most commonly measured. Blood pressure is usually expressed in terms of the systolic pressure (maximum pressure during one heartbeat) over diastolic pressure (minimum pressure between two heartbeats) in the cardiac cycle. It is measured in millimeters of mercury (mmHg) above the surrounding atmospheric pressure, or in kilopascals (kPa). In pregnancy, it is the fetal heart and not the mother's heart that builds up the fetal blood pressure to drive blood through the fetal circulation. The blood pressure in the fetal aorta is approximately 30 mmHg at 20 weeks of gestation, and increases to approximately 45 mmHg at 40 weeks of gestation. The average blood pressure for full-term infants: ^ Systolic 65–95 mmHg ^ Diastolic 30–60 mmHg Mean blood pressure (MBP also referred to as mean systemic pressure) is generally calculated as follows: MBP= diastolic blood pressure (DBP) + 1/3 [systolic blood pressure (SBP) – DBP]. Mean arterial pressure (MAP) is an average calculated blood pressure in an individual during a single cardiac cycle. Methods of estimating MAP vary. A common calculation is to take one-third of the pulse pressure (the difference between the systolic and diastolic pressures), and add that amount to the diastolic pressure. MAP is altered by cardiac output and systemic vascular resistance. It is used clinically to estimate the risk of cardiovascular diseases, where a MAP of 90 mmHg or less is low risk, and a MAP of greater than 96 mmHg in adults represents "stage one hypertension" with increased risk. Variants of venous pressure include: ^ Central venous pressure, which is a good approximation of right atrial pressure, which is a major determinant of right ventricular end diastolic volume. (However, there can be exceptions in some cases). ^ The jugular venous pressure (JVP) is the indirectly observed pressure over the venous system. It can be useful in the differentiation of different forms of heart and lung disease. ^ The portal venous pressure is the blood pressure in the portal vein. It is normally 5– 10 mmHg in adults. Pulmonary pressure is the pressure in the pulmonary arteries, and normally is about 15 mmHg in adults at rest. Increased blood pressure in the capillaries of the lung causes pulmonary hypertension, leading to interstitial edema if the pressure increases to above 20 mmHg (in adults) and to pulmonary edema at pressures above 25 mmHg (in adults). Systolic Blood Pressure is the maximum blood pressure during contraction of the ventricles. Diastolic pressure is the minimum pressure recorded just prior to the next contraction. 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 FiO2 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 FIO2. Natural air includes 21% oxygen, which is equivalent to FIO2 of 0.21. Oxygen-enriched air has a higher FIO2 than 0.21; up to 1.00 which means 100% oxygen. FIO2 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: P_AO₂, P_EO₂, and P_IO₂ are the expired, and inspired gas, respectively, and VD/Vt is the

tidal volume. In medicine, the F_IO₂ 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 (FiO2) 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 FiO2. This is reflected in the formula as a decrease in the numerator or an increase in the denominator - thus lowering the OI. Typically, an OI threshold is set for when a neonate should be placed on ECMO, for example >40. The equation is: FiO2: Fraction of inspired Mean airway pressure, in

PaO2 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 = PAO2 – PaO2 PAO2 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 (PAO2) is not easily measured directly; instead, it is estimated using the alveolar gas equation: PAO2 = (Patm – PH2O) FiO2 – PaCO2/RQ The 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. P/F ratio equals the arterial pO2 (“P”) from the ABG divided by the FIO2 (“F”) – the fraction (percent) of inspired oxygen that the patient is receiving expressed as a decimal (40% oxygen = FIO2 of 0.40). A P/F Ratio less than 300 indicates acute respiratory failure in adults. P/F ratio has been validated and used in the context of ARDS (acute respiratory distress syndrome) for many years, where acute respiratory failure is called “acute lung injury.” A P/F ratio < 300 indicates mild ARDS, < 200 is consistent with moderate ARDS and < 100 is severe ARDS. The P/F ratio indicates what the pO2 would be on room air. SpO2 translated to PO2. The arterial pO2 measured by arterial blood gas (ABG) is the definitive method for calculating the P/F ratio. However, when the pO2 is unknown because an ABG is not available, the SpO2 measured by pulse oximetry can be used to approximate the pO2, as shown in the Table below. It is important to note that estimating the pO2 from the SpO2 becomes unreliable when the SpO2 is 98% - 100%. Conversion of SpO2 to pO2 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 FIO2 (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 FIO2 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 also. “IGF-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 et al., 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 et al., 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 Binkert et al., 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 µg/Kg/24hours of the IGF-1/IGFBP-3 complex. In one embodiment, the method or composition comprises 200 to 500µg/Kg/24hours of the IGF-1/IGFBP-3 complex, for example 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475 or 500 µg/Kg/24hours of the IGF-1/IGFBP-3 complex. In one embodiment, the method or composition comprises 400µg/Kg/24hours of the IGF-1/IGFBP-3 complex. In one embodiment, the method or composition comprises 55 to 110 µg/Kg/24hours of IGF-1, such as 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105 or 110 µg/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 US63/353,139 filed 17 June 2022, and US63/376,172 filed 19 September 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 120d gestation) to adolescent lambs (about 150d of age), IGF-1 protein level progressively increased (Pearson r=0.540; p=0.004). Figure 1B shows (Group 2) plasma level of IGF-1 protein in preterm lambs managed by invasive mechanical ventilation for 3d. Continuous iv infusion of rhIGF-1/rhIGFBP-3 (1.5 mg/Kg/day; black squares) attained the target plasma 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-1/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 vehicle- control preterm lambs. Statistical analyses for were by two-way ANOVA and Holm- Šídák's multiple comparisons test, with ^= 0.05 (95%). Figure 1C-E shows plasma IGF-1 protein level for various IV doses. Figure 2 rhIGF-1/rhIGFBP-3 led to phosphorylation of IGF-1 receptor (IGF-1-R) in sheep endothelial cells in vitro. Panel A: The response was concentration-dependent. Panel B: Only 50 ng/mL and 100 ng/mL rhIGF1/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 3A-D shows (Group 2) physiological parameters for preterm lambs managed by invasive mechanical ventilation for 3d. Continuous iv infusion of rhIGF-1/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-F shows respiratory gas exchange physiological parameters for preterm lambs (Group 2) managed by invasive mechanical ventilation for 3 days. Continuous iv infusion of rhIGF-1/rhIGFBP-3 (1.5 mg/Kg/day; blacks squares). Figure 5 shows respiratory severity score for 7 days lamb study. Figure 6 shows A-a gradient for 7 day lamb study. Figure 7 shows oxygen index for 7 day lamb study. Figure 8 shows P/F ratio for 7 day lamb study. Figure 9 shows S/F ratio for 7 day lamb study. EXAMPLES Example 1 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 dosage of rhIGF-1/rhIGFBP-3 required to attain physiological level of IGF-1 in plasma (~125 ng/mL). This 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-1/rhIGFBP-3 during MV for 3d significantly improved some pulmonary and cardiovascular outcomes (p<0.1). Also, rhIGF-1/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-1/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-1/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 rhIGF-1/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 1d, 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. Am 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-1/rhIGFBP-3 or Vehicle (Control) in Preterm Lambs During Invasive Mechanical Ventilation for 3 Days We verified in vitro that rhIGF-1/rhIGFBP-3 led to downstream signaling by sheep vascular endothelial cells (ATCC- Manassas, VA). Cells were seeded at 60,000/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 ^L lysis buffer (10 mM HEPES (4-(2-hydroxyethyl)-1-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 µL of cell lysate and 5 µL acceptor mix were added to a PerkinElmer white half area plate and incubated for 1h in the dark at room temperature. Five µL of donor mix was added and incubated in the dark for 1h at room temperature. The plate was read on an Envision instrument. A pilot dosage-finding pharmacokinetic study of continuously infused rhIGF-1/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 t_1/2 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-1/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 mL 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 Dräger 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. FIO2 was adjusted to attain target hemoglobin oxygen saturation of 90-94% (PaO260-90 mmHg) by pulse oximetry (Model SurgiVet V9200IBP/Temp, Smith Medical ASD, Inc., St. Paul, MN). Peak inspiratory pressure was adjusted to attain a target PaCO2 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 (OI) [(Paw x FiO2)/PaO2], P/F (PaO2/FiO2) ratio, and Alveolar-arterial (A-a) gradient [((F O /100) x (640-47))-(PaCO /0.8)-PaO ]. 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 O 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 injury (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 end- inspiration (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 cmH2O. 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 capillary 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-1/rhIGFBP-3, we used two-way ANOVA (treatment and time), followed by post-hoc Holm-Šídák's multiple comparisons test for physiological results, with ^=0.1 (90%), except as noted for Figure 1. We used one-tailed parametric t-test ( ^=0.1) for morphological outcomes and one-tailed non-parametric tests ( ^=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 unventilated term lambs (Figure 1A). Effect of Continuous Infusion of rhIGF-1/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-1/rhIGFBP-3-treated preterm lambs’ gestation age was 1d younger and delivery weight was about 0.5 kg lower than for the vehicle-control preterm lambs. Female:male distribution was not equal between the rhIGF- 1/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 (Figure1B; “predose” level was ~100 ng/mL; not statistically different). Subsequently, plasma IGF-1 protein level diverged between the rhIGF-1/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-1/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-1/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 2A). Only 50 ng/mL and 100 ng/mL rhIGF1/rhIGFBP-3 treatments led to levels above background at all timepoints tested (Figure 3B). Control (bovine serum albumin) treatment did not lead to signal elevation across all time points and dose ranges. Physiological parameters for respiratory gas exchange are presented in Figure 4. Results are shown for 12h epochs of postnatal age during the 3d of MV. Targets were SaO2 range 90-94% (PaO2 range 60-90 mmHg) for oxygenation and PaCO2 range 45-60 mmHg for ventilation. Although numerical results favored rhIGF-1/rhIGFBP-3 treatment, no statistical differences were detected between the rhIGF-1/rhIGFBP-3-treated versus vehicle-control preterm lambs for the applied FiO2 or PIP to sustain the oxygenation and ventilation targets, respectively (Figures 4A-D). OI and A-a gradient were significantly improved in rhIGF-1/rhIFGBP-3 treated lambs (Table 2).

Plasma pH and bicarbonate also were not different between the rhIGF-1/rhIGFBP-3-treated and vehicle-control preterm lambs over the 3d study period (Figures 4E 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-1/rhIGFBP-3-treated preterm lambs compared to the vehicle- control preterm lambs (data not shown). The systemic hemodynamic parameters shown in Figure 3 are numerically better for the rhIGF- 1/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-1/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.

Fluid balance parameters are summarized in Table 4.

Average results a e 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-1/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-1/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).

Histological examples of alveolar architecture were similar between the rhIGF-1/rhIGFBP-3-treated and vehicle-control preterm lambs at the end of 3d of MV. 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-1/rhIGFBP-3-treated preterm lambs. Quantitative histological indices of alveolar formation revealed no statistical differences between the rhIGF-1/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 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). Protein abundance in lung parenchyma was assessed semi-quantitatively. Statistical difference was detected for cleaved caspase 3, for which the relative protein abundance was significantly greater for the rhIGF-1/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. 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. 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-1/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 of rhIGF-1/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 rhIGF-1/rhIGFBP-3 in mechanically ventilated preterm lambs (n=6). Continuous infusion of rhIGF-1/rhIGFBP-3 during MV for 3d statistically improved some pulmonary and cardiovascular outcomes compared to vehicle-control preterm lambs. Systemic hypotension did not occur in rhIGF-1/rhIGFBP-3-treated preterm lambs (0/6), whereas 2/6 vehicle- control preterm lambs required dopamine to maintain physiologic systemic perfusion pressure. Also, rhIGF-1/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-1/IGFBP-3-treated preterm lambs compared to vehicle-control preterm lambs at the end of the 3d study. Another result is that rhIGF- 1/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-1/rhIGFBP-3 improved some physiological, morphological, and biochemical outcomes, without toxicity, in mechanically ventilated preterm lambs. 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-1/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-1/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- 1/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. An important observation in our study is that neither liver function nor renal function indices were adversely affected during 3d of continuous intravenous infusion of rhIGF-1/rhIGFBP- 3. Similarly, infusion did not adversely affect respiratory gas exchange or cardiovascular physiology. 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 rhIGF- 1/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-1/rhIGFBP-3. Three-day duration of continuous infusion of rhIGF-1/rhIGFBP-3 may have been insufficient for IGF- 1’s morphogenic effects. Example 2 Pre-term Lamb Model 7 days 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 of rhIGF-1/rhIGFBP-3 (1.5 mg/Kg/d, iv; n=9). Respiratory severity score, oxygenation index (Oi), SpO₂/FiO₂ (S/F) ratio, PaO₂/FiO₂ (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-1/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 rhIGF1/rhIGFBP-3 during 7d of mechanical ventilation improved respiratory gas exchange indices in preterm lambs.

Claims

CLAIMS: 1. A method of treatment or prophylaxis comprising stabilising blood pressure and/or flow in a preterm infant by administering a therapeutic amount of a composition comprising IGF-1 and an IGF binding protein. 1A A composition of IGF-1 and an IGF binding protein (such as IGFBP-3) for use in the treatment or prophylaxis of stabilising blood pressure and/or flow in a preterm infant. 1B A composition of IGF-1 and an IGF binding protein for use in the manufacture of a medicament for the treatment or prophylaxis of stabilising blood pressure and/or flow in a preterm infant.

2. A method or composition for use according to any one of claims 1, 1A or 1B, wherein the blood pressure and/or flow is stabilised without increasing vascular resistance.

3. A method or composition for use according to any one of claims 1, 1A, 1B and 2, wherein the preterm infant is not hypovolaemic, before treatment.

4. A method or composition for use according to any one of claims 1, 1A, 1B, and 2 to 3, wherein the blood flow is stabilised.

5. A method or composition for use according to claim 4, wherein central blood flow is stabilised.

6. A method or composition for use according to claims 4 or 5, wherein peripheral blood flow is stabilised, for example in comparison to corresponding untreated infant.

7. A method or composition for use according to any one of claims 1, 1A, 1B and 2 to 6, wherein the blood pressure (such as mean system blood pressure) is stabilised.

8. A method or composition for use according to any one of claims 1, 1A, 1B and 2 to 7, wherein systolic pressure is stabilised, for example in the range 60 to 90mmHg, such as where the systolic pressure is higher than an untreated preterm infant (in particular in the period up to 72 hours post birth).

9. A method or composition for use according to any one of claim 1, 1A, 1B and 2 to 8, wherein diastolic 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.

10. A method or composition for use according to any one of claims 1, 1A, 1B and 2 to 9, wherein tissue perfusion is stabilised, for example treated preterm infant is pink.

11. A method or composition for use according to any one of claims 1, 1A, 1B and 2 to 10, wherein a treated preterm has a lower heart rate in comparison to untreated preterm infants (or average thereof), for example a bpm in the range about 120 to 180.

12. A method or composition for use according to any one of claims 1, 1A, 1B and 2 to 11, wherein the treated preterm does not require intervention for low blood pressure, for example does not require a vasopressor, such as dopamine.

13. A method or composition for use according to any one of claims 1, 1A, 1B and 2 to 12, wherein the blood pressure of a treated preterm infant does not drop below 30mmHg.

14. A method or composition for use according to any one of claims 1, 1A, 1B and 2 to 13, wherein the preterm infant has a blood pressure in the range 30 to 50mmHg, during treatment, for example 30, 35, 40, 45 or 50 mmHg.

15. A method or composition for use according to any one of claims 1, 1A, 1B and 2 to 14, wherein the preterm infant has hypertension, before treatment is initiated.

16. A method or composition for use according to any one of claims 1, 1A, 1B and 2 to 15, wherein the preterm infant receiving said treatment/prophylaxis is more robust than a preterm infant without the treatment/prophylaxis.

17. A method or composition for use according to claim 16, wherein more robust is adequate temperature regulation.

18. A method or composition for use according to claims 16 or 17, wherein more robust is a good level of activity (motor activity).

19. A method or composition for use according to any one of claims 16 to 18, 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). .

20. A method or composition for use according to any one of claims 16 to 19, wherein more robust is minimal assistance with breathing.

21. A method or composition for use according to claim 20, wherein the infant is on mechanical ventilation.

22. A method or composition for use according to claims 20 or 21, wherein the treated preterm infant stays with the predefined parameters for gases, for example the instruct does not require adjustment by a career.

23. A method or composition for use according to any one of claims 1, 1A, 1B and 2 to 22, 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.

24. A method or composition for use according to any one of claims 1, 1A, 1B and 2 to 23, wherein the treated preterm infant has a peak inspiratory pressure (cmH20) that is lower than untreated preterm infants (or an average thereof), for example in the range 45 and 60, in particular in the first 72 hours of life.

25. A method or composition for use according to any one of claims 1, 1A, 1B and 2 to 24, wherein the preterm infant does not lose weight in the first few days of life, for example days 1 to 3.