Developmental Differences Between Newborn and Adult Mice In Response To Romiplostim

Thrombocytopenia affects 20-35% of infants admitted to Neonatal Intensive Care Units. The incidence of thrombocytopenia is inversely proportional to gestational age, and approaches 70% among the most preterm neonates (birth weight <1,000 grams). Preterm infants also have the highest incidence of bleeding of any age group, with 25-31% developing intracranial hemorrhage. Currently, platelet (plt) transfusions are the only therapeutic option for thrombocytopenic neonates. In the last 5 years, two thrombopoietin (TPO) mimetics, romiplostim (ROM) and eltrombopag, received FDA approval for the treatment of adults with ITP. Based on the severity and duration of thrombocytopenia, 10% of thrombocytopenic neonates could benefit from TPO-mimetic therapy. Our prior in vitro studies demonstrated that human neonatal megakaryocyte (MK) progenitors are significantly more sensitive to TPO than adult progenitors (Pastos et al., Blood, 2006; Liu et al., Blood, 2011). This study was designed to compare the in vivo responses of newborn vs. adult mice to ROM. Based on prior observations, we hypothesized that newborn pups would be more sensitive to TPO-mimetics than adult mice.

As a first step, healthy adult C57BL/6 mice were given a single subcutaneous (SC) injection of 0.1% BSA (control) or ROM at a dose of 10, 30, 100, or 300 ng/g body weight. Newborn mice on post-natal day 1 (P1) received a single SC injection of either 0.1% BSA or ROM at a dose of 30 or 300 ng/g. Plt count and immature plt fraction (IPF) were measured on the day of injection and every other day for 14 days. The baseline plt count in adult mice was 1,184±204 x10³/µL. Adult mice treated with ROM (n=3-4 per group) exhibited a dose-dependent increase in plt count and IPF, which peaked on day 5 in those receiving lower ROM doses (10 and 30 ng/g), and on day 7 in those receiving higher ROM doses (100 and 300 ng/g). On day 7, adult mice treated with ROM 300 ng/g had a 4.2-fold increase in plt count compared to BSA controls (6,733±511 vs. 1,600±216 x10³/µL, respectively; p<0.0001). Newborn mice (P1) had significantly lower baseline plt counts (624±130 x10³/µL; p<0.0001) compared to adults, and similarly responded to ROM injection with a dose-dependent increase in plt count that peaked on day 5. However, plt counts on post-natal day 5 (P5) were 1,020±198 x10³/µL for newborn mice treated with ROM 30 ng/g and 1,355±137 x10³/µL for newborn mice treated with ROM 300 ng/g (n=17 per group), representing less than a 2-fold increase over BSA treated pups (701±119 x10³/µL).

To evaluate the effect of ROM on megakaryopoiesis, a subset of adult and newborn mice treated with 0.1% BSA or ROM 300 ng/g (n=3-4 per group) were euthanized on day 5 after injection. Liver, spleen, and bone marrow (BM) MKs were immunohistochemically stained for von Willebrand factor and quantified as described (Hu Z et al., Neonatology, 2010). Overall, ROM-treated adult mice had significantly increased numbers of MKs compared to controls in BM (2.3-fold increase; p=0.0002) and spleen (3.9-fold increase; p=0.006). ROM-treated newborn mice exhibited non-significant increases in MK numbers in BM (2.2-fold increase; p=0.19), spleen (1.6-fold increase; p=0.35), and liver (1.4-fold increase; p=0.31).

Because newborn C57BL/6 mice transition from fetal liver to adult BM hematopoiesis during the first 10 to 14 days of life and the BM is not well formed until P10, we injected newborn mice at P5 (instead of P1) and evaluated the response to ROM. Similar to the younger group, P5 mice treated with ROM 300 ng/g reached peak platelet counts at P11, but the plt count was only 1.4-fold higher than BSA control animals (1,340±440 vs. 927±151 x10³/µL, respectively; p=0.19).

In conclusion, this study indicated that newborn mice are less responsive to ROM than adult mice. This was a surprising finding, given that human neonatal MK progenitors have been consistently shown to be more sensitive to TPO than adult MK progenitors. The reasons underlying the modest in vivo response of neonates are unclear, but might be related to the transition in hematopoietic sites that occurs during this period in murine development (corresponding to the second trimester of human gestation), high baseline thrombopoietic demands associated with rapid growth, potential pharmacokinetic factors, or developmental differences in the splenic or BM microenvironments of newborn and adult mice.