A Novel Nanomicellar Combination of Fenretinide and Lenalidomide Shows Marked Antitumor Activity in a Neuroblastoma Xenograft Model

Abstract

Purpose: Currently >50% of high-risk neuroblastoma (NB) patients, despite intensive therapy and initial partial or complete response, develop recurrent NB due to the persistence of minimal residual disease (MRD) that is resistant to conventional antitumor drugs. Indeed, their low therapeutic index prevents drug-dose escalation and protracted administration schedules, as would be required for MRD treatment. Thus, more effective and less toxic therapies are urgently needed for the management of MRD. To address this aim, we evaluated a new combination of fenretinide and lenalidomide, both endowed with antitumor activity and low-toxicity profiles. New nanomicelles were prepared as carriers for this combination to maximize bioavailability and accumulation at the tumor site because of the enhanced permeability and retention (EPR) effect.

Experimental design: New nanomicelles containing the fenretinide-lenalidomide combination (FLnMs) were prepared by a one-step method, providing high drug encapsulation and micelle dimensions suitable for tumor accumulation. Their administration to mice bearing human NB xenografts allowed us to evaluate their efficacy in comparison with the nanomicelles containing fenretinide alone (FnMs).

Results: Treatment by FLnMs significantly decreased the tumor growth of NB xenografts. FLnMs were more active than FnMs despite comparable fenretinide concentrations in tumors, and lenalidomide alone did not show cytotoxic activity in vitro against NB cells. The tumor mass at the end of treatment with FLnMs was predominantly necrotic, with a decreased Ki-67 proliferation index.

Conclusion: FLnMs provided superior antitumor efficacy in NB xenografts compared to FnMs. The enhanced efficacy of the combination was likely due to the antiangiogenic effect of lenalidomide added to the cytotoxic effect of fenretinide. This new nanomicellar combination is characterized by a low-toxicity profile and offers a novel therapeutic option for the treatment of high-risk tumors where the persistence of MRD requires repeated administrations of therapeutic agents over long periods of time to avoid recurrent disease.

Keywords: drugs combination; fenretinide; lenalidomide; nanomicelles; neuroblastoma.

Figure 1

Schematic representation of FLnMs composed of phospholipids and hydroxypropyl-beta-cyclodextrin in the outer layer and fenretinide + lenalidomide in the inner core.

Figure 2

Confocal Laser Scanning image of FLnMs (right) and FnMs (left) showing the presence of autofluorescent fenretinide within micelles (A). 2D representation of 125 depth-resolved slices separated by 10 nm (B). (a) trans-axial slice (XY plane) through FLnMs: (b) XZ cross-section of planes indicated by horizontal line. (c) YZ cross-sections of planes indicated by vertical line (Bar = 200 nm). (d) trans-axial slice (XY plane) through FnMs: (e) YZ cross-section of planes indicated by vertical line. (f) XZ cross-sections of planes indicated by horizontal line (Bar = 200 nm).

Figure 3

Differential scanning calorimetry of the drugs-semisolid phase mixtures: semisolid phase without drugs (SP), mixture with Fenretinide (SP + Fen), mixture with Fenretinide and Lenalidomide (SP + Fen + Len), pure Fenretinide (Fen), pure Lenalidomide (Len).

Figure 4

Leakage of fenretinide and lenalidomide from FLnMs in PBS (pH 7.4) containing 25% human plasma at 37° C. All data are the average of at least three different experiments ± SD.

Figure 5

Cytotoxic activity of FnMs and FLnMs at increasing fenretinide concentrations in BR6 cells (A). Cytotoxic activity of pure fenretinide and pure lenalidomide at increasing concentrations in BR6 cells (B). The cells were exposed at the indicated drug doses and cell viability was evaluated by Incucyte after 24, 48 and 72 hrs and indicated as percentage versus control cells (mean ± SD, n = 8).

Figure 6

Efficacy of FLnMs and FnMs on BR6 flank xenografts. Cells were implanted subcutaneously in the flank of each mouse. Mice (n=4 per arm) were treated intravenously via tail vein injections with FLnMs (30 mg/kg fenretinide and 17 mg/kg Lenalidomide) or FnMs (30 mg/kg fenretinide) 3x/week for 3 weeks when tumors reached a volume of 0.1 cm³. (A) Tumor growth inhibition. (B) Event-free survival curves of mice. (C) Body weight of mice during treatment. (mean ± SD, n = 4).

Figure 7

Plasma and tumor concentrations of fenretinide in mice treated with FLnMs and FnMs. Mice (n=3 per arm, per time point) were given a single dose of FLnMs (30 mg/kg fenretinide and 17 mg/kg Lenalidomide) or FnMs (30 mg/kg fenretinide) intravenously via tail vein. After 4, 24 and 48 h fenretinide levels were determined in plasma (A) and tumors (B). The mice treated with FLnMs (30 mg/kg fenretinide and 17 mg/kg Lenalidomide) or FnMs (30 mg/kg fenretinide) 3x/week for 3 weeks were sacrificed after 24 h from the last treatment and the concentrations of fenretinide were determined in plasma and tumors (C). (mean ± SD, n = 3).

Figure 8

Histology of tumor samples. H&E staining of BR6 tumor xenografts: untreated tumors (Control) show packed undifferentiated cells, tumors treated with fenretinide (FnMs) are less compact and rich in neuropil, treatments with the fenretinide–lenalidomide combination (FLnMs) provide loose cells mostly differentiated with scarce active cells as demonstrated by the scant nuclear staining. Ki-67 staining show proliferation activity in untreated tumors (Control) with a proliferation index >30%, a decrease in proliferation in FnMs-treated tumors (proliferation index <25%) and a minimum proliferation activity in FLnMs-treated tumors (proliferation index <3%). (mean ± SD, n = 4).