The acridone derivative MBLI-87 sensitizes breast cancer resistance protein-expressing xenografts to irinotecan

Abstract

The breast cancer resistance protein ABCG2 confers cellular resistance to irinotecan (CPT-11) and its active metabolite SN-38. We utilised ABCG2-expressing xenografts as a model to evaluate the ability of a non-toxic ABCG2 inhibitor to increase intracellular drug accumulation. We assessed the activity of irinotecan in vivo in SCID mice: irinotecan completely inhibited the development of control pcDNA3.1 xenografts, whilst only delaying the growth of ABCG2-expressing xenografts. Addition of MBLI-87, an acridone derivative inhibitor, significantly increased the irinotecan effect against the growth of ABCG2-expressing xenografts. In vitro, MBLI-87 was as potent as GF120918 against ABCG2-mediated irinotecan efflux, and additionally was specific for ABCG2. A significant sensitisation to irinotecan was achieved despite the fact that doses remained well below the maximum tolerated dose (due to the rather limited solubility of MBLI-87). This suggested that MBLI-87 is an excellent candidate to prevent drug efflux by ABCG2, without altering plasma concentrations of irinotecan and SN-38 after IP (intra-peritoneal) injections. This could constitute a useful strategy to improve drug pharmacology, to facilitate drug penetration into normal tissue compartments protected by ABCG2, and potentially to reverse drug resistance in cancer cells.

Fig. 1 –

In vitro irinotecan accumulation in control and ABCG2-expressing cells. Cells were loaded with 2 μM irinotecan in DMEM medium (without FBS) for 60 min at 37 °C in the absence or presence of either 5 μFM GF120418, 5 μM MBLI-87 or 10 μM fumitremorgin C. Intracellular irinotecan accumulation was quantified by HPLC MS/MS. Results were expressed as ng irinotecan/mg protein.

Fig. 2 –

(A) ABCG2 expression in xenografts. As soon as the tumour volume reached approximately 1800 mm³, mice were sacrificed, and crude membrane fractions were prepared from (T31–T110) individual xenografts. ABCG2 expression was quantified by immunoblotting with the monoclonal BXP21 antibody, as described in Section 2. (B) Effects of treatment with irinotecan and/or MBLI-87 on ABCG2 expression levels. ABCG2 expression was quantified as in A, and normalised to the alpha-tubulin content (ABCG2 expression levels/tubulin expression levels). Protein level ratios were expressed as arbitrary units (AU). The values are means ± S.E.

Fig. 3 –

Activity of MBLI-87, NANO and irinotecan, either alone or in combination, against control (A) and ABCG2-expressing (B and C) xenografts. Doses and schedules are detailed in Section 2. (A and B) The data at each time period were averaged. The tumour volume values were plotted against time. Differences with p values <0.05 were considered as statistically significant between two sets of xenograft volume (N = 6/group). (C) Mice were treated with either NANO or MBLI-87 in combination with irinotecan. The data were sets of tumour measurements (in sixes) at four time periods: first therapy period, first rest period, second therapy period and second rest period, for two parallel therapy conditions: MBLI-87-irinotecan (N = 5) and NANO-irinotecan (N = 6). The data at each time period were averaged and standard errors (SE) computed. A plot was made of tumour volume ± SE against time.

Fig. 4 –

Representative plots of irinotecan (A) and SN-38 (B) plasma concentrations as a function of time. Mice were treated by IP injection of irinotecan in combination with either MBLI-87 or NANO (5 mice per condition). HPLC MS/MS determined irinotecan and SN-38 plasma concentrations (ng/mL plasma) are represented as plots as a function of time. Irinotecan (A) or SN-38 (B) plasma concentration of each mouse is shown in the absence (red triangles/NANO-irinotecan) or presence of MBLI-87 (blue crosses/MBLI-87-irinotecan). Median values are either represented by a red line (NANO-irinotecan) or a blue line (MBLI-87-irinotecan).