Pharmacological Evaluation of a First-in-Class Hemoglobin Elevating Agent (HbEA) AND017 in a Mouse MDS Model of an Inducible Mutant c-Myc Knock-in

Background

Myelodysplastic syndromes (MDS) are a group of diseases of hematopoietic stem cells (HSCs) that occur primarily in the aging population either de novo or secondarily following therapy for unrelated cancers. Among the pan-cytopenia, anemia-related symptoms caused by ineffective erythropoiesis and reduced RBC and hemoglobin levels are a serious condition by most MDS patients that has yet to be resolved by transfusion and/or erythropoiesis-stimulating agents (ESAs).

Conditional knockout of c-Myc gene in blood leads to MDS-like phenotypes, including pan-cytopenia, micromegakaryocytes and severe anemia. Differentiation of erythrocytes is blocked at the erythroid progenitor stage. To mimic low c-Myc-driven MDS-like phenotypes in mice, we generated a knock-in (KI) mouse model by introducing a doxycycline-inducible double mutations c-Myc (T58A/S62E) expression cassette at Collagen 1A1 (Col1a1) locus. This KI allele was further combined with Vav1-Cre and ROSA-26-LSL-rtTA alleles in C57 mice. In this mouse model, Vav1-Cre expression induces cleavage in LSL casettte, resulting expression of rtTA. After doxycycline administration, rtTA binds to Tet promoter of c-Myc (T58A/S62E) cassette and promotesits expression, which results in similar MDS-like phenotypes in mice, specifically the macrocytic anemia (low RBC, Hb, Hct, and high MCV). Importantly, this anemia model does not respond to rEPO, thus providing as an ideal model to test EPO-independenthemoglobin elevating agent. AND017 is a first-in-class hemoglobin elevating agent (HbEA), which has been shown excellent efficacy in a wild type rat model, rat 5/6 nephrectomy chronic kidney disease (CKD) model, the Hbb^d3th β-thalassemia mouse model, and Townes sickle cell disease (SCD) mouse model. In this study, we evaluated hemoglobin elevating agent (HbEA), AND017, in our MDS mouse model.

Methods

Forty Myc mutant KI mice (female/F: 20; male/M: 20; 8-12 weeks) were randomly divided into 4 groups: G1, G2, G3 and G4 (5F + 5M in each group). Ten Vav1-Cre/LSL-tTA mice (female/F: 5; male/M: 5; 8-12 weeks) were in G5. Mice in all 5 groups were fed with chaws containing 200 mg/kg Doxycycline. After 4 weeks, mice in G1, G2, G3, G4 and G5 were orally dosed three times per week with vehicle, 10 mg/kg AND017, 30 mg/kg AND017, 1666 U/kg erythropoietin (EPO) and 30 mg/kg AND017 respectively for 28 days. RBC, HGB, and HCT were measured and calculated by a hematology system on days 0, 14, and 28, respectively. The level of statistical significance was set at 5% or P < 0.05; the data were compared to G1 at the same time point.

Results

AND017 was well tolerated in all groups. HGB levels (g/dL) for G1 to G5 were 9.67, 9.52, 9.54, 8.77 and 15.08 respectively on day 0; were 8.72, 10.97^****, 13.56^****, 9.76 and 18.48^**** on day 14; and were 8.19, 11.77^****, 14.91^****, 9.57 and 20.62^**** on day 28. The RBC numbers (×10⁶cells/μL) for G1 to G5 were 6.01, 6.03, 5.90, 5.65 and 9.61 on day 0, were 5.57, 6.33^***, 7.11^****, 5.91 and 11.39^**** on day 14; and were 5.27, 7.17^****, 8.46^****, 5.95 and 12.57^**** on day 28. The hematocrits (%) for G1 to G5 were 27.47, 28.12, 28.01, 26.15 and 43.78 on day 0; were 27.69, 34.36^****, 43.13^****, 30.14 and 53.57^**** on day 14; and were 27.13, 37.52^****, 47.41^****, 30.69 and 59.25^**** on day 28. (^*, ^**, ^***, and ^****: for p < 0.05, 0.01, 0.001, and 0.0001 respectively)

Conclusion

As a hemoglobin elevating agent (HbEA), AND017 elevated important several parameters related to anemia significantly and dose-dependently, including Hb levels, RBC numbers, and HCTs in a c-Myc mutant KI MDS mouse model. These results indicate that AND017 has the potential to be a safe, efficacious and convenient treatment for MDS patients in the future. Future clinical trials for AND017 in MDS are warranted.