Drug administration into mice

PXS-LOX_1 (referred to as PXS-5446 in our catalogue) was mixed with pure olive oil at a concentration of 5 mg/mL. GATA-1low mice received intra-peritoneal injections of either PXS-LOX_1 (n = 8, 5 males and 3 females) or vehicle (olive oil, n = 9, 5 males and 4 females) at a dose of 15 mg/kg four times a week for nine weeks and were then sacrificed for analysis. Spleen and femurs were harvested for analysis at sacrifice. At the beginning of experiments (drug administration), mice of 15 to 16 weeks old were used. At this mouse age we expect initiation of fibrosis[23], also based on our earlier study[24]. This dosage of PXS-LOX_1 was chosen as it provided inhibition of LOX activity.

PXS-LOX_2 (referred to as PXS-5505A in our catalogue) has improved properties and was given orally. PXS_LOX-2 was dissolved in phosphate-buffered saline (PBS) at a concentration of 1 mg/mL, aliquoted in single-use volumes and frozen at −20°C. At the day of the treatment, aliquots were thawed at room temperature and administered by oral gavage. Animals were treated at the dose of 5mg/kg, once a day 4 times a week for a total of 8 weeks. Treatment groups consisted of JAK2V617F/PXS-LOX_2 (n = 7, 4 males and 3 females), JAK2V617F/vehicle (PBS, n = 6, 4 males and 2 females), C57BL6J/PXS-LOX_2 (η = 11, 5 males and 6 females), C57BL6J /vehicle (PBS, n = 10, 4 males and 6 females). Animals were at 15 to 17 weeks of age at the start of the treatment. Peripheral blood counts were analyzed monthly, including at pre-treatment, and weight was measured bi-weekly, including at pre-treatment. At the end of the treatment, plasma was obtained from EDTA-anticoagulated peripheral blood collected by cardiac puncture, aliquoted, and frozen at −80 °C. Abdominal artery and ear flaps were collected and frozen immediately on liquid nitrogen and stored at −80 °C. Spleen was harvested, weighted and separated into frozen samples for hydroxyproline analysis and fixed samples for histology. Femurs were fixed for histological analysis.