Statistical Assessment of Drug Synergy from In Vivo Combination Studies Using Mouse Tumor Models

Abstract

Drug combination therapy is a promising strategy for treating cancer; however, its efficacy and synergy require rigorous evaluation in preclinical studies before going to clinical trials. Existing methods have limited power to detect synergy in animal studies. Here, we introduce a novel approach to assess in vivo drug synergy with high sensitivity and low false discovery rate. It can accurately estimate combination index and synergy score under the Bliss independence model and the highest single agent (HSA) model without any assumption on tumor growth kinetics, study duration, data completeness and balance for tumor volume measurement. We show that our method can effectively validate in vitro drug synergy discovered from cell line assays in in vivo xenograft experiments, and can help to elucidate the mechanism of action for immune checkpoint inhibitors in syngeneic mouse models by combining an anti-PD-1 antibody and several tumor-infiltrating leukocytes depletion treatments. We provide a unified view of in vitro and in vivo synergy by presenting a parallelism between the fixed-dose in vitro and the 4-group in vivo combination studies, so they can be better designed, analyzed, and compared. We emphasize that combination index, when defined here via relative survival of tumor cells, is both dose and time dependent, and give guidelines on designing informative in vivo combination studies. We explain how to interpret and apply Bliss and HSA synergies. Finally, we provide an open-source software package named invivoSyn that enables automated analysis of in vivo synergy using our method and several other existing methods. Significance: This work presents a general solution to reliably determine in vivo drug synergy in single-dose 4-group animal combination studies.

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“…To rule out the impact of tumor size on anti-CTLA4 and nobiletin therapy, we selected tumors of similar size to those treated with IgG for anti-CTLA4 and/or nobiletin treatment, and the mice were given nobiletin (25 mg kg −1 ) daily and/or anti-CTLA4 (200 μg/mouse) once every 3 days. The tumor volume of each mouse was measured daily and combined with the invivoSyn method ( 46 ), the results showed that nobiletin and anti-CTLA4 combination therapy synergistically and most notably reduced tumor growth and tumor weight ( Fig. 7A and B ; Supplementary Fig.…”