Abstract
Activation of prodrugs in tumors (e.g., by bioreduction in hypoxic zones) has the potential to generate active metabolites that can diffuse within the tumor microenvironment. Such “bystander effects” may offset spatial heterogeneity in prodrug activation but the relative importance of this effect is not understood. Here, we quantify the contribution of bystander effects to antitumor activity for the first time, by developing a spatially resolved pharmacokinetic/pharmacodynamic (SR-PK/PD) model for PR-104, a phosphate ester pre-prodrug that is converted systemically to the hypoxia-activated prodrug PR-104A. Using Green’s function methods we calculated concentrations of oxygen, PR-104A and its active metabolites, and resultant cell killing, at each point of a mapped three-dimensional tumor microregion. Model parameters were determined in vitro, using single cell suspensions to determine relationships between PR-104A metabolism and clonogenic cell killing, and multicellular layer (MCL) cultures to measure tissue diffusion coefficients. LC-MS/MS detection of active metabolites in the extracellular medium following exposure of anoxic single cell suspensions and MCLs to PR-104A confirmed that metabolites can diffuse out of cells and through a tissue-like environment. The SR-PK/PD model estimated that bystander effects contribute 30 and 50% of PR-104 activity in SiHa and HCT116 tumors, respectively. Testing the model by modulating PR-104A-activating reductases and hypoxia in tumor xenografts showed overall clonogenic killing broadly consistent with model predictions. Overall, our data suggest that bystander effects are important in PR-104 antitumor activity, although their reach may be limited by macroregional heterogeneity in hypoxia and reductase expression in tumors. The reported computational and experimental techniques are broadly applicable to all targeted anticancer prodrugs and could be used to identify strategies for rational prodrug optimization.
Highlights
Intra-tumor heterogeneity is a fundamental barrier to all targeted therapies [1]
DEVELOPMENT OF SR-PK/PD MODELS FOR THREE CELL LINES Using the approach illustrated in Figure 1, SR-PK/PD models were developed for SiHa and HCT116 cells, which have high and low expression of the aerobic PR-104A reductase aldoketo reductase 1C3 (AKR1C3) respectively [22], and for HCT116/sPOR#6 cells engineered to provide a high rate of PR-104A activation under hypoxia
We distinguished the intracellular and extracellular compartments in the SR-PK/PD model by using a continuum approximation in which transfer between the two compartments is represented by rate constants kei N and kie N, and it is assumed that metabolism is restricted to the intracellular compartment while diffusion is confined to the extracellular compartment (Figure 2)
Summary
One of the attractive features of prodrugs that are activated within tumors is their potential for decoupling targeting and pharmacodynamic effect through diffusion of active metabolites from prodrug-activating cells to surrounding untargeted cells. These bystander effects are thought to be important for monotherapy activity of targeted anticancer prodrugs [2,3,4], including hypoxia-activated prodrugs (HAP) activated by bioreduction in hypoxic regions [5,6,7]. Half-maximal inhibition of PR-104A cytotoxicity was found to require only ∼0.13 μM O2 in SiHa cell suspensions [20], which is well below that for half-maximal radiosensitization www.frontiersin.org
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