Pharmacokinetic modeling of an induction regimen for in vivo combined testing of novel drugs against pediatric acute lymphoblastic leukemia xenografts

Abstract

Current regimens for induction therapy of pediatric acute lymphoblastic leukemia (ALL), or for re-induction post relapse, use a combination of vincristine (VCR), a glucocorticoid, and L-asparaginase (ASP) with or without an anthracycline. With cure rates now approximately 80%, robust pre-clinical models are necessary to prioritize active new drugs for clinical trials in relapsed/refractory patients, and the ability of these models to predict synergy/antagonism with established therapy is an essential attribute. In this study, we report optimization of an induction-type regimen by combining VCR, dexamethasone (DEX) and ASP (VXL) against ALL xenograft models established from patient biopsies in immune-deficient mice. We demonstrate that the VXL combination was synergistic in vitro against leukemia cell lines as well as in vivo against ALL xenografts. In vivo, VXL treatment caused delays in progression of individual xenografts ranging from 22 to >146 days. The median progression delay of xenografts derived from long-term surviving patients was 2-fold greater than that of xenografts derived from patients who died of their disease. Pharmacokinetic analysis revealed that systemic DEX exposure in mice increased 2-fold when administered in combination with VCR and ASP, consistent with clinical findings, which may contribute to the observed synergy between the 3 drugs. Finally, as proof-of-principle we tested the in vivo efficacy of combining VXL with either the Bcl-2/Bcl-xL/Bcl-w inhibitor, ABT-737, or arsenic trioxide to provide evidence of a robust in vivo platform to prioritize new drugs for clinical trials in children with relapsed/refractory ALL.

Figure 1. Synergy between VCR, DEX and ASP against ALL cell lines in vitro.

Cell lines were exposed to VCR (open circles), DEX (open triangles), ASP (open squares), or the triple combination VXL (closed circles), at fixed ratios, and dose-responses were assessed using the DIMSCAN assay as described in Materials and methods. Fractional survival of treated vs. untreated control cells is shown. Each condition included 12 replicates and error bars represent standard deviation. The data shown are representative of two independent experiments.

Figure 2. In vivo sensitivity of ALL-19 to low dose VCR, DEX and ASP.

Female mice were engrafted with ALL-19 cells and treated with vehicle (A); DEX (5 mg/kg) (B); VCR (0.15 mg/kg) (C); and ASP (1000 U/kg) (D); as single agents or the combination of the three drugs at the same doses (VXL) (E). The %huCD45⁺ cells in PB of individual mice (A–E); control vehicle-treated mice (dashed lines); drug-treated mice (solid lines). Kaplan-Meier analysis of EFS (F) control (grey solid line), VCR (grey dashed line), DEX (black dashed line), ASP (black dotted line), VXL (solid black line). All events were leukaemia-related. Shaded boxes represent the treatment period.

Figure 3. In vivo sensitivity of ALL xenografts to VXL combination treatment.

Female mice were engrafted with: ALL-4 (A); ALL-7 (B); ALL-11 (C); or ALL-16 (D); and treated with a combination of VCR (0.15 mg/kg), DEX (5 mg/kg) and ASP (1000 U/kg). The %huCD45⁺ cells in PB of individual mice (left panel) and Kaplan-Meier analysis of EFS (right panel). Control vehicle-treated mice (dashed lines); drug-treated mice (solid lines). Shaded boxes represent the treatment period. No leukaemia related events were recorded for the drug treated group of ALL-16 engrafted mice.

Figure 4. LGD in response to VXL treatment in xenografts stratifies according to patient outcome.

Median LGD obtained by VXL treatment for a panel of ALL xenografts derived from long term survivors (Alive) and from patients who died of their disease (DOD). There is evidence that the two groups are different (p = 0.0159) by Mann-Whitney two tailed test.

Figure 5. Pharmacokinetic analysis of VCR, DEX and ASP in leukemias bearing NOD/SCID mice.

Engrafted female mice (ALL-19) were treated with VCR (0.15 mg/kg), DEX (5 mg/kg), ASP (1000 U/kg) or their combination (VXL) at the same doses. Three mice each were sacrificed at specified time points and drug concentrations in plasma for VCR (A); DEX (B); and ASP (C) in the single agent or combination treatment were assessed as described in Materials and Methods.

Figure 6. In vivo sensitivity of ALL xenografts to VXL and VXL/ABT-737 combination treatments.

Female mice were engrafted with: ALL-2 (A); ALL-8 (B); ALL-10 (C); or ALL-17 (D) and treated with a diluent vehicle (controls, dashed black lines), ABT-737 (25 mg/kg, solid grey lines), VXL combination (solid black lines), or VXL+ABT-737 quadruple combination (dashed grey lines). Engraftment kinetics indicated by %huCD45⁺ cells in PB of individual mice (left panel) and Kaplan-Meier survival curves (EFS) (right panel) are shown. Shaded boxes represent the treatment period. All events were leukemia-related except for 1 and 4 in the VXL/ABT-737-treated group of the ALL-8, and ALL-10, respectively. In the ALL-17 quadruple drug combination cohort all mice were culled due to leukemia or toxicity unrelated morbidity.

Figure 7. In vivo sensitivity of ALL xenografts to VXL and VXL/ATO combination treatments.

Female mice were engrafted with: ALL-4 (A); ALL-7 (B); ALL-8 (C); or ALL-19 (D) and treated with a diluent vehicle (controls, dashed black lines), ATO (2.5 mg/kg, solid grey lines), VXL combination (solid black lines), or VXL+ATO quadruple combination (dashed grey lines). Engraftment kinetics indicated by %huCD45⁺ cells in PB of individual mice (left panel) and Kaplan-Meier survival curves (EFS) (right panel) are shown. Shaded boxes represent the treatment period.