Page 179 - Haematologica March 2020

KS99 alone or in combination in AML
Figure 5. Preclinical efficacy of KS99 in acute myeloid leukemia (AML) mouse models. (A) Pharmacokinetics of KS99 (2.5 mg/kg body weight) concentration in plasma of NSG mice (n=3 per time point) measured after 0.5, 3, 6 and 24 hours (h) of treatment. Results are mean±standard error of the mean (SEM). (B) Mice engrafted with luciferase-expressing MV4-11 (MV4-11-Luc) cells subcutaneously were treated either with vehicle control (DMSO) or KS99 (2.5 mg/kg). Bioluminescence imaging (BLI) and quantification of radiance as a surrogate for tumor of mice (n=4) over the time course of the study were assessed. (C) Tumors were isolated and weighed at the termination of the study. (D) U937-bearing NSG mice (n=3-4) were treated either with vehicle control (DMSO) or KS99 (2.5 mg/kg). Bar graph indicates the percentage of human CD45+ cells in the bone marrow of mice. The insert is the body weight of mice throughout the study. Points represent individual mice (mean±SEM). (E-G) Luciferase-expressing U937 (U937-Luc)-bearing NRG mice (n=3) were treated either with vehicle control (DMSO), KS99 (2.5 mg/kg), Cytarabine (Ara-C, 50 mg/kg) or combination of KS99 and Ara-C. (E and F) Bioluminescence imaging signals of mice over the time course of the study. (G) Bar graph depicts a number of human CD45+ cells in the bone marrow of mice as detected using flow cytometry (top) and representative flow cytometric analysis of bone marrow cells (bottom). *P<0.05; **P<0.01; ***P<0.001 were assessed by two-way ANOVA analysis. (H and I) Luciferase-expressing C1498 (C1498-Luc)-bear- ing albino C57BL/6 mice (n=3) were treated either with vehicle control (DMSO), KS99 (2.5 mg/kg), Cytarabine (Ara-C, 50 mg/kg) or a combination. (H) Quantification of BLI signals of mice over the time course of the study. (I) Kaplan-Meier survival analysis of animals. Results are mean±SEM. NS: not significant; *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001, unpaired t-test.
ity in primary human AML cells was tested by ALDE- FLOUR assay as described above. Briefly, cells were treat- ed with DMSO, ibrutinib (10 mM) or KS99 (3 mM) for 48 h, harvested and subjected to flow cytometer. Both KS99 and ibrutinib treatments caused a decrease in the ALDH+ subpopulation in all primary human AML cases studied (Figure 4D and E).
KS99 reduces the leukemic burden in preclinical acute myeloid leukemia animal models and improves the survival of animals
The study by Pandey et al. has shown the efficacy of KS99 at 1 mg/kg body weight in multiple myeloma.20 In the current work, we determined the maximum-tolerated dose (MTD) of KS99 in NSG mice to be 2.5 mg/kg via an intraperitoneal (i.p.) route. Next, a single dose pharmaco- kinetic (PK) study was performed in NSG mice. Mice were injected with KS99 (2.5 mg/kg), sacrificed at various time
points post-dosing (n=3), and plasma levels were analyzed by LC-MS/MS. PK data showed a maximum of 49 ng/mL (105.1 nM) plasma drug concentration and rapid elimina- tion from the systemic circulation after 8 h (Figure 5A).
To evaluate the preclinical efficacy of KS99 in vivo, sub- cutaneous (s.c.) and disseminated xenograft mouse mod- els were used. For the subcutaneous xenograft model, MV4-11-Luc cells were injected subcutaneously into NSG mice. Once visible tumors had been established, mice were treated with either vehicle control or KS99 (2.5 mg/kg) three times a week (Online Supplementary Figure S5A). Data showed a 65% decrease in bioluminescent sig- nals in KS99-treated mice compared to the vehicle. Tumor sizes and weight (approx. 70%) were lower than vehicle- treated animals (Figure 5C).
To extend our findings in a subcutaneously implanted AML xenograft model, the efficacy of KS99 was further evaluated in a disseminated mouse model using human
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