Schedule-dependent potentiation of chemotherapy drugs by the hypoxia-activated prodrug SN30000

Abstract

Hypoxia-activated prodrugs (HAPs) are hypothesized to improve the therapeutic index of chemotherapy drugs that are ineffective against tumor cells in hypoxic microenvironments. SN30000 (CEN-209) is a benzotriazine di-N-oxide HAP that potentiates radiotherapy in preclinical models, but its combination with chemotherapy has not been explored. Here we apply multiple models (monolayers, multicellular spheroids and tumor xenografts) to identify promising SN30000/chemotherapy combinations (with chemotherapy drugs before, during or after SN30000 exposure). SN30000, unlike doxorubicin, cisplatin, gemcitabine or paclitaxel, was more active against cells in spheroids than monolayers by clonogenic assay. Combinations of SN30000 and chemotherapy drugs in HCT116/GFP and SiHa spheroids demonstrated hypoxia-and schedule-dependent potentiation of gemcitabine or doxorubicin in growth inhibition and clonogenic assays. Co-administration with SN30000 suppressed clearance of gemcitabine in NIH-III mice, likely due to SN30000-induced hypothermia which also modulated extravascular transport of gemcitabine in tumor tissue as assessed from its diffusion through HCT116 multicellular layer cultures. Despite these systemic effects, the same schedules that gave therapeutic synergy in spheroids (SN30000 3 h before or during gemcitabine, but not gemcitabine 3 h before SN30000) enhanced growth delay of HCT116 xenografts without increasing host toxicity. Identification of hypoxic and S-phase cells by immunohistochemistry and flow cytometry established that hypoxic cells initially spared by gemcitabine subsequently reoxygenate and re-enter the cell cycle, and that this repopulation is prevented by SN30000 only when administered with or before gemcitabine. This illustrates the value of spheroids in modeling tumor microenvironment-dependent drug interactions, and the potential of HAPs for overcoming hypoxia-mediated drug resistance.

Keywords: Hypoxia-activated prodrugs; SN30000 (CEN-209); gemcitabine; multicellular spheroids; tumor hypoxia.

Figure 1.

Clonogenic survival of HCT116 monolayers and spheroids exposed to SN30000 or clinical chemotherapeutic drugs. HCT116 monolayers (5 × 10⁴ cells/well) and spheroids (one spheroid/well) grown under 20% O₂ in 96-well plates were exposed to a range concentrations of SN30000 (A) for 2 h under 5% O₂ or cisplatin (B), doxorubicin (C), or gemcitabine (D) for 2 h under 20% O₂. Immediately after drug exposure, 2 wells of monolayers or 8 spheroids were pooled for each group and dissociated to single cell suspensions for clonogenic survival assay. Values are mean ± SEM for 2–4 replicate determinations of plating efficiency from each pool for spheroids, and from 4 pools for monolayers. The curves are fitted to the clonogenic cell kill data using monoexponential without minimum (A, B), bi-exponential (C, representing sensitive and resistant subpopulations, without minimum) and monoexponential (D, with minimum representing a completely resistant subpopulation).

Figure 2.

Clonogenic survival of cells in spheroids in response to SN30000 and/or chemotherapeutic drugs under 5% and 20% O₂. HCT116/GFP (A) or SiHa (B) spheroids were exposed to SN30000 (25 µM under 5% O₂, or 100 µM under 20% O₂), chemotherapy drug (25 µM cisplatin, 5 µM doxorubicin, 10 µM gemcitabine), combinations of SN30000 before (SN-Drug) or after (Drug-SN) chemotherapy drugs with 1 h interval between, or simultaneously (SN+Drug). Each drug exposure was for 2 h. Immediately after the final drug treatment, 8 spheroids were pooled for each group and dissociated to single cell suspensions for clonogenic survival assay. The bold horizontal lines represent the predicted combined effect from adding the log cell kill for the individual drugs under 5%, (solid lines) or 20% O₂ (dashed lines). Values are mean ± SEM for 2–4 replicate determinations of plating efficiency from each pool.

Figure 3.

HCT116/GFP spheroid growth delay induced by SN30000 and chemotherapeutic drugs. HCT116/GFP spheroids were exposed to SN30000 (25 µM under 5% O₂, or 100 µM under 20% O₂, for 2 h) and chemotherapy drugs (25 µM cisplatin for 2 h, 5 µM doxorubicin for 2 h and 10 µM gemcitabine for 2 h) under 5% O₂ (A) and 20% O₂ (B), using the same schedules as in Figure 2. Immediately after the final drug exposure, spheroids were cultured in fresh medium in a standard incubator (20% O₂) and growth was monitored every second day by measuring fluorescence intensity of HCT116/GFP spheroids. Values are mean ± SEM, n = 16 spheroids. (C) Representative bright-field images of spheroids after treatment on day 4 with SN30000 and/or gemcitabine under 5% O₂ are illustrated. P values for statistically significant differences between gemcitabine only and the combination groups were determined with one-way ANOVA. * P < 0.05; ** P < 0.01; *** P < 0.001.

Figure 4.

Implications of SN30000-induced hypothermia in mice. (A) Mouse body temperature response to the drug treatments. Mice were injected i.p. with saline, SN30000 (130 mg/kg), gemcitabine (100 mg/kg), SN30000 3 h before (SN-3hr-Gem) or after (Gem-3hr-SN) gemcitabine, or gemcitabine 5 min before SN30000 (Gem-5min-SN). Values are mean ± SEM for three mice. Plasma pharmacokinetics of gemcitabine (B), SN30000 and its 1-oxide metabolite (C) after dosing as in (A). Values are mean ± SEM, n ≥ 3 mice. *** P < .001 between co-administration and gemcitabine alone groups. (D) Diffusion of gemcitabine, (300 µM), urea and mannitol through HCT116 multicellular layers under 32℃ or 37℃. Values are mean ± SEM, n = 4 replicates. Mannitol curves are offset on the time axis for clarity. Flux of gemcitabine was significantly greater at 32℃ (P = .005) than at 37℃, while flux of urea was not significantly different at the 2 temperatures (P = .072).

Figure 5.

HCT116 tumor growth delay induced by SN30000 and gemcitabine. Mice bearing HCT116 xenografts were randomized when tumors reached 9 mm diameter and treated as described in the legend of Figure 4 except that dosing was repeated 7 days later (arrows in A). (A) Tumor volume and (B) percentages of mice with tumor volume less than 3-times the pre-treatment volume. P values for statistically significant differences between gemcitabine only and the combination groups were determined with the LogRank test. (C) Days for tumor to reach 3-times pre-treatment volume versus body weight loss at nadir. Values represent mean ± SEM for 6–8 mice. Weight loss in drug treated groups was not statistically significant (one-way ANOVA).

Figure 6.

S-phase and hypoxic fractions of tumors in response to SN30000 and gemcitabine. Mice bearing HCT116 tumors (mean diameter 9 mm) were dosed i.p. with PIMO (60 mg/kg, to label initially hypoxic cells) 2 h before the drug treatments as described in the legend of Figure 4. 45 h after PIMO injection, mice were dosed i.p. with a second hypoxia marker, EF5 (60 mg/kg, to label newly hypoxic cells) and EdU (50 mg/kg, to label S-phase cells) 3 h before tumor excision. Half of each tumor sample was used for immunohistochemistry (A). The remainder was dissociated to single cell suspensions for ex-vivo clonogenic survival assay (B) and flow cytometry (C-G). (A) Merged false-colour images for PIMO (green), EF5 (gold), EdU (red) with H33342 nuclear counterstain (blue). (B) Clonogens per gram of tumor. (C) % newly hypoxic cells (EF5+) at 45–48 h; (D) % originally hypoxic cells (PIMO+) still present at 48 h; (E) % S-phase in well-oxygenated cells (EdU+, EF5-); (F) originally hypoxic cells that have survived and re-oxygenated by 48 h (PIMO+, EF5-) as a percentage of total cells; (G) originally hypoxic cells that are in S-phase at 48 h (PIMO+, EdU+) as a percentage of total PIMO+ cells; Values are mean ± SEM for 6–8 mice. * P < 0.05, ** P < 0.01.