A platinum(IV) prodrug strategy to overcome glutathione-based oxaliplatin resistance

Abstract

Clinical efficacy of oxaliplatin is frequently limited by severe adverse effects and therapy resistance. Acquired insensitivity to oxaliplatin is, at least in part, associated with elevated levels of glutathione (GSH). In this study we report on an oxaliplatin-based platinum(IV) prodrug, which releases L-buthionine-S,R-sulfoximine (BSO), an inhibitor of glutamate-cysteine ligase, the rate-limiting enzyme in GSH biosynthesis. Two complexes bearing either acetate (BSO-OxOAc) or an albumin-binding maleimide (BSO-OxMal) as second axial ligand were synthesized and characterized. The in vitro anticancer activity of BSO-OxOAc was massively reduced in comparison to oxaliplatin, proving its prodrug nature. Nevertheless, the markedly lower intracellular oxaliplatin uptake in resistant HCT116/OxR cells was widely overcome by BSO-OxOAc resulting in distinctly reduced resistance levels. Platinum accumulation in organs of a colorectal cancer mouse model revealed higher tumor selectivity of BSO-OxMal as compared to oxaliplatin. This corresponded with increased antitumor activity, resulting in significantly enhanced overall survival. BSO-OxMal-treated tumors exhibited reduced GSH levels, proliferative activity and enhanced DNA damage (pH2AX) compared to oxaliplatin. Conversely, pH2AX staining especially in kidney cells was distinctly increased by oxaliplatin but not by BSO-OxMal. Taken together, our data provide compelling evidence for enhanced tumor specificity of the oxaliplatin(IV)/BSO prodrug.

Fig. 1. Synthetic route to platinum(IV) prodrugs BSO-OxOAc and BSO-OxMal.

The respective experimental conditions can be found in the Methods Section.

Fig. 2. UHPLC stability kinetic measurements of BSO-OxOAc.

The stability of the complex (1 mM) was measured in 100 mM phosphate buffer (pH 7.4; 20 °C) and the reduction in the presence of 10 mM AA in 500 mM phosphate buffer (pH 7.4; 20 °C).

Fig. 3. Platinum-SEC-ICP-MS traces of BSO-OxMal and BSO-OxOAc.

(a) shows BSO-OxMal and (b) BSO-OxOAc. The compounds were incubated in fetal calf serum at 37 °C and measured after 0, 1, and 24 h. The peaks at t_r > 6 min represent low molecular weight platinum, the peak at t_r ~4 min albumin-bound platinum. The quantitative analysis of the albumin binding is summarized in Supplementary Table 1.

Fig. 4. Impact of BSO-OxOAc as compared to oxaliplatin and cisplatin on the viability of the indicated cancer cell lines and their sublines with the respective acquired platinum drug resistance.

a Impact of a 72 h continuous drug exposure on the viability of human colon cancer cells HCT116 and a oxaliplatin-resistant subline HCT116/OxR, as well as the human ovarian cancer cell model A2780 and the cisplatin-resistant subline A2780/Cis. One representative experiment out of at least three performed in triplicate is shown. Data points are depicted as mean ± SD. b IC₅₀ values were derived from the dose-response curves as shown under (a) using the four-parameter logistic nonlinear regression model. Resistance factors were calculated by dividing the IC₅₀ values for the resistant subline by those of the respective sensitive parental cell model.

Fig. 5. Cellular uptake of BSO-OxOAc as compared to free oxaliplatin and impact of acquired oxaliplatin resistance.

a Parental HCT116 and oxaliplatin-resistant HCT116/OxR cells were exposed to the indicated concentrations of BSO-OxOAc and oxaliplatin for 3 h and cellular uptake quantified by ICP-MS. Data are depicted as mean ± SD. b Log-fold difference in the platinum accumulation between parental HCT116 and HCT116/OxR calculated from the data shown in (a). Statistical significance was tested using one-way ANOVA. *p < 0.05; ***p < 0.001.

Fig. 6. Impact of BSO-OxOAc as single agent and in combination with the reducing agents NAC (100 µM) and AA (50 µM) on the clonogenic potential of HCT116 cells and the subline with acquired oxaliplatin resistance (HCT116/OxR).

Sparsely seeded cells (1 × 10³/24-well plate well) were exposed for 10 days to the indicated compounds and derived cell clones stained with crystal violet, photographed, and results evaluated by ImageJ software as described in the Methods section. One respective experiment out of three performed in duplicate is shown under (a) and the respective evaluation under (b), depicted as mean ± SD. Statistical significance was tested using two-way ANOVA. In all cases: *p < 0.05; **p < 0.01; ***p < 0.001.

Fig. 7. Tissue/organ distribution of BSO-OxMal compared to free oxaliplatin.

Mice bearing CT26 allografts (n = 4 per group) were treated twice a week with equimolar concentrations of BSO-OxMal (23.5 mg/kg) or oxaliplatin (9 mg/kg) and sacrificed 24 h after the last drug dosing. Platinum contents of tumor tissue (a) and serum as compared to blood cells (b) was determined by ICP-MS. Statistical differences were tested by Student´s t-test. (c) Platinum levels in the indicated organs are given relative to the ones in the tumor tissue. Data are depicted as mean ± SD. Statistical significance was tested for all tissues/organs as compared to platinum levels in tumor tissue using one-way ANOVA. In all cases: *p < 0.05; ***p < 0.001.

Fig. 8. In vivo anticancer activity of BSO-OxMal and BSO-OxOAc compared to oxaliplatin.

Mice bearing CT26 allografts (n = 4 per group) were treated twice a week for two weeks (black arrows) with equimolar concentrations of BSO-OxMal (23.5 mg/kg), BSO-OxOAc (19.1 mg/kg) or oxaliplatin (9 mg/kg). Mean ± SEM (standard error of mean) (a) and individual (b) tumor volumes were assessed at the indicated time points by caliper measurements (maximal tumor growth inhibition of 61.2%, 51.3% and 51.3% at day 11, for BSO-OxMal, BSO-OxOAc and oxaliplatin respectively; tumor doubling time extended by 1.8-fold, 1.2-fold, and 1.3-fold, respectively). (c) Overall survival of mice based on FELASA guidelines was analyzed by Kaplan Meier curves. Statistical significance was tested using one-way ANOVA (a) or Mantel-Cox test (c). In all cases: *p < 0.05; **p < 0.01; ***p < 0.001.

Fig. 9. Effects of BSO-OxMal and oxaliplatin treatment.

Cancer proliferative activity (a) and GSH content (b) as well as DNA damage in tumor (c) and kidney (d) tissues. Mice bearing CT26 allografts (n = 4 per group) were treated twice a week with equimolar concentrations of BSO-OxMal (23.5 mg/kg) or oxaliplatin (9 mg/kg) and sacrificed 24 h after the last drug dosing. Immunohistochemical staining of Ki-67 (a) and the DNA damage parameter pH2AX (c) were quantified in 8–10 regions of interest (ROI) per tumor within random non-necrotic, viable cancer regions by ImageJ software. In (d) cells stained positively for pH2AX in the kidney were counted within 5 ROI per kidney section. Representative images are shown for tumor sections in Supplementary Fig. 14 and for organ sections in Supplementary Fig. 15. In (b) tumor GSH content of the respective cancer samples was quantified by a calorimetric method as described in the methods section. Data in (b, d) are depicted as mean ± SEM (standard error of mean). Statistical significance was tested using one-way ANOVA (a, c) or Student´s t test (b, d). In all cases: *p < 0.05; **p < 0.01; ***p < 0.001.

Fig. 10. NMR Numbering scheme of the platinum ligands.

The letters stand for B - BSO; M - maleimide and D - 1,2-diaminocyclohexane, respectively.