Pilot dose-ranging of rhIGF-1/rhIGFBP-3 in a preterm lamb model of evolving bronchopulmonary dysplasia

Abstract

Background: Low levels of insulin-like growth factor-1 (IGF-1) protein in preterm human infants are associated with bronchopulmonary dysplasia (BPD). We used our preterm lamb model of BPD to determine (1) dosage of recombinant human (rh) IGF-1 bound to binding protein-3 (IGFBP-3) to reach infant physiologic plasma levels; and (2) whether repletion of plasma IGF-1 improves pulmonary and cardiovascular outcomes.

Methods: Group 1: normal, unventilated lambs from 128 days gestation through postnatal age 5 months defined normal plasma levels of IGF-1. Group 2: continuous infusion of rhIGF-1/rhIGFBP-3 (0.5, 1.5, or 4.5 mg/kg/day; n = 2) for 3 days in mechanically ventilated (MV) preterm lambs determined that 1.5 mg/kg/day dosage attained physiologic plasma IGF-1 concentration of ~125 ng/mL, which was infused in four more MV preterm lambs.

Results: Group 1: plasma IGF-1 protein increased from ~75 ng/mL at 128 days gestation to ~220 ng/L at 5 months. Group 2: pilot study of the optimal dosage (1.5 mg/kg/day rhIGF-1/rhIGFBP-3) in six MV preterm lambs significantly improved some pulmonary and cardiovascular outcomes (p < 0.1) compared to six MV preterm controls. RhIGF-1/rhIGFBP-3 was not toxic to the liver, kidneys, or lungs.

Conclusions: Three days of continuous iv infusion of rhIGF-1/rhIGFBP-3 at 1.5 mg/kg/day improved some pulmonary and cardiovascular outcomes without toxicity.

Impact: Preterm birth is associated with rapid decreases in serum or plasma IGF-1 protein level. This decline adversely impacts the growth and development of the lung and cardiovascular system. For this pilot study, continuous infusion of optimal dosage of rhIGF-1/rhIGFBP-3 (1.5 mg/kg/day) to maintain physiologic plasma IGF-1 level of ~125 ng/mL during mechanical ventilation for 3 days statistically improved some structural and biochemical outcomes related to the alveolar formation that would favor improved gas exchange compared to vehicle-control. We conclude that 3 days of continuous iv infusion of rhIGF-1/rhIGFBP-3 improved some physiological, morphological, and biochemical outcomes, without toxicity, in mechanically ventilated preterm lambs.

Figure 1.

Group 1 was plasma level of IGF-1 protein in normal, unventilated fetal and postnatal lambs. Panel A: 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). Panel B: 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 through 72h 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 Panel B were by two-way ANOVA and Holm- Šídák's multiple comparisons test, with α=0.05 (95%).

Figure 2.

RhIGF-1 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 treatments led to p-IGF-1-R 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 3.

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 respiratory gas exchange (Panels A-F), as well as systemic hemodynamic and heart rate outcomes (Panels G-J) relative to vehicle-control preterm lambs (open circles); however, no statistically significant differences (p>0.1) were detected.

Figure 4.

Group 2 alveolar formation in preterm lambs managed by invasive mechanical ventilation for 3d. Continuous iv infusion of rhIGF-1/rhIGFBP-3 (1.5 mg/Kg/day) led to somewhat better appearing terminal respiratory units (TRU; Panel B) and secondary septa (arrow in Panel D) relative to vehicle-control (Panels A and C). Panels A and B are the same magnification; see scale bar. Panels C and D are the same magnification; see scale bar. Distal airspace walls (arrowhead in Panels C and D) appear to have similar thickness between the two groups. Quantitative histology revealed no statistical differences (p>0.1) for radial alveolar count, secondary septal volume density, or distal airspace wall thickness between the rhIGF-1/rhIGFBP-3-treated preterm lambs and vehicle-control preterm lambs (Panel E-G).

Figure 5.

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-1/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 6.

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-1/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).