The ability of a panel of camptothecin derivatives to access the tumor compartment was evaluated to determine the mechanisms by which the architecture of solid tumors may act to limit their activity. Microregional localization and activity of members of the camptothecin class of topoisomerase I targeting agents, including topotecan, irinotecan, and irinophore C, a lipid-based nanoparticulate formulation of irinotecan, were evaluated over time in HCT116 and HT29 colorectal tumor xenografts. Using native drug fluorescence, their distributions in tissue cryosections were related to the underlying tumor vasculature, tumor cell proliferation, and apoptosis. Topotecan exhibited a relatively uniform tumor distribution; in tissue 100 mm away from vessels, it reached 94% AE 5% of levels seen around blood vessels, whereas irinotecan and irinophore C were found to reach only 41% AE 10% and 5% AE 2%, respectively. Surprisingly, all three agents were able to initially inhibit proliferation uniformly throughout the tumors, and it was their rate of washout (topotecan > irinotecan > irinophore C) that correlated with activity. To explain this discrepancy, we looked at SN38, the active metabolite of irinotecan, and found it to penetrate tissue similarly to topotecan. Hence, the poor access to the tumor compartment of irinotecan and irinophore C could be offset by their systemic conversion to SN38. It was concluded that all three agents were effective at reaching tumor cells, and that despite the poor access to the extravascular compartment of irinophore C, its extended plasma exposure and systemic conversion to the diffusible metabolite SN38 enabled it to effectively target solid tumors.

The presence of hypoxia in solid tumours is correlated with poor treatment outcome. We have developed a 3-D tissue engineered construct to quantitatively monitor oxygen penetration through tumour tissue using the exogenous 2-nitroimidazole bioreductive probe pimonidazole and phosphorescence quenching technologies. Using this in vitro model we were able to examine the influence of the biguanides metformin and phenformin, antimycin A and KCN, on the distribution and kinetics of oxygen delivery as prototypes of modulators of oxygen metabolism.

The presence of hypoxia in solid tumors is correlated with poor treatment outcome. Even small population of hypoxic cells within tumors can significantly decrease the efficacy of radiation therapy. One approach to treating hypoxia in tumours is to reduce overall tumor cell oxygen consumption by modulating mitochondrial respiration. This could reoxygenate hypoxic cells within the tumor and restore radiation sensitivity. To guide in vivo application we tested the effects of three respiratory chain inhibitors; the biguanides metformin and phenformin and rotenone on oxygen consumption in 3D tissue engineered discs. Experiments were carried out to evaluate the effect of respiratory inhibition on the hypoxic fraction of HCT116 and HT29 tissue discs using immunostaining with the hypoxic cell marker pimonidazole. An oxygen sensing phosphorescence lifetime probe assessed pO2 on the basal side of the disc as a function of time after inhibitors were exposed to the apical surface. Clonogenic assays were then performed to confirm the radiosensitization of hypoxic cells within the tissue discs. Pimonidazole staining and direct pO2 sensing confirmed that all three electron transfer inhibitors decrease the hypoxic fraction by reducing cellular oxygen consumption. Rotenone was found to completely eliminate the hypoxic fraction at nanomolar concentrations compared to metformin and phenformin which require milli to micromolar concentrations respectively. Furthermore, rotenone was found to have an effect on oxygen utilization within minutes of drug exposure compared to both metformin and phenformin, which took hours to be effective. In addition, clonogenics assays determined that all three electron transfer inhibitors radiosensitize tumor cells with rotenone being the most efficient compound on a molar basis. The mitochondrial inhibitors of complex I metformin, phenformin and rotenone decrease oxygen utilization by tumor cells in a concentration dependent manner resulting in reduction of the hypoxic fraction and increased radiosensitivity. Citation Format: Maria Jose Gandolfo, Alastair H. Kyle, Andrew I. Minchinton. Metabolic manipulation of hypoxia and radiotherapy response by electron transport inhibitors. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4314. doi:10.1158/1538-7445.AM2014-4314

We have evaluated distribution and micro-regional activity of members of the camptothecin class of topoisomerase I targeting agents including topotecan (TPT), irinotecan (IRN), Irinophore C (IrC), a liposomal formulation of IRN and SN38, the active metabolite of IRN. Despite having a similar mechanism of action these agents are used against different cancers and possess significantly different pharmacological and physicochemical properties. The native fluorescence of each agent was used to visualize its distribution in HCT116 & HT29 tissue cryosections that were subsequently processed via multiplexed staining to relate drug exposure to its effect on tumor cell status (proliferation, apoptosis) and distance from vasculature. TPT exhibited the most uniform tumor distribution but it also experienced a significantly faster tumor wash-out while IRN was retained longer in tumors but exhibited a larger concentration gradient in relation to tumor blood vessels. IrC, the high molecular weight liposomal formulation, exhibited the poorest tissue distribution but it also displayed longer tumor retention due to its extended plasma half-life. At a depth of 100 µm away from vessels drug levels reached 94 ± 5 % (TPT), 41 ± 10 % (IRN) and 4.8 ± 2 % (IrC) of maximal levels seen near vessels. In terms of vessel permeability, IRN and IrC did not extravasate equally from all vessels, ∼50% of vessels showed little permeation by the two drugs and, because of this, some microregions of the tumors received much higher exposure than others. However, despite the significant differences in their tissue distribution, all 3 agents were able to exert a complete suppression of cycling tumor cells at 4 hours following treatment. At 24 hours following TPT or IRN tumor proliferation status returned to normal levels indicating that neither drug was retained long enough to exert activity on out of cycle cells. In contrast IrC benefited from an extended plasma exposure and was able to inhibit proliferation over a period of several days. The time to 50% recovery in proliferation was ∼3 days for TPT and IRN versus 14 days for IrC. Tumor growth delay studies showed that both TPT and IRN benefited somewhat from a dose splitting schedule designed to specifically target proliferation at peak levels two days in row. However, this strategy was still outperformed by IrC. Tumor growth delay following 50 mg/kg drug qwx2 was 9.6 ± 3 days (IRN) versus 27.8 ± 3.8 days (IrC). To determine the root cause of the discrepancy between the poor distribution but good microregional activity of IRN and IrC an in vitro 3D tissue-disc model was employed. It was found that IRN tissue penetration was limited by its high degree of cellular accumulation. SN38, the active metabolite of IRN, did not accumulate to the same extent and did penetrate tissue effectively. Hence it was concluded that the poor tissue distribution observed for both IRN and IrC was offset by systemic conversion to SN38, which was able to exert a more uniform activity within tumors. Citation Format: Alastair H. Kyle, Maria Jose Gandolfo, Jennifer H.E. Baker, Andrew I. Minchinton. Camptothecins: Tissue penetration and implications for therapy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 827. doi:10.1158/1538-7445.AM2014-827