Polymeric micelles loaded with carfilzomib increase tolerability in a humanized bone marrow-like scaffold mouse model

Abstract

Carfilzomib-loaded polymeric micelles (CFZ-PM) based on poly(ethylene glycol)-b-poly(N-2-benzoyloxypropyl methacrylamide) (mPEG-b-p(HPMA-Bz)) were prepared with the aim to improve the maximum tolerated dose of carfilzomib in a "humanized" bone marrow-like scaffold model. For this, CFZ-PM were prepared and characterized for their size, carfilzomib loading and cytotoxicity towards multiple myeloma cells. Further, circulation and tumor & tissue distribution of fluorescently labeled micelles were determined. Tolerability of CFZ-PM versus the clinical approved formulation - Kyprolis® - was assessed. CFZ-PM presented small diameter below 55 nm and low PDI < 0.1. Cy7-labeled micelles circulated for extended periods of time with over 80% of injected dose in circulation at 24 h after intravenous injection and 1.3% of the injected dose of Cy7-labeled micelles accumulated in myeloma tumor-bearing scaffolds. Importantly, CFZ-PM were well tolerated whereas Kyprolis® showed adverse effects. Kyprolis® dosed at the maximum tolerated dose, as well as CFZ-PM, did not show therapeutic benefit, while multiple myeloma cells showed sensitivity in vitro, underlining the importance of the bone marrow crosstalk in testing novel formulations. Overall, this work indicates that PM are potential drug carriers of carfilzomib.

Keywords: Bone Marrow Microenvironment; Carfilzomib; Drug delivery; Multiple Myeloma; Polymeric micelles; Proteasome inhibitor.

Graphical abstract

Fig. 1

Chemical structure of (A) carfilzomib and (B) mPEG-b-p(HPMA-Bz) copolymer (Total Mn = 22 kDa, mPEG of 5 kDa) prepared with HPMA-Bz as monomer and mPEG2-ABCPA as initiator.

Fig. 2

Schematic representation of “humanized” BM-like scaffold (huBMsc) xenograft model and treatment schedule. Eight weeks after implantation of bone-like scaffolds (4 per mouse) seeded with hMSCs, MM.1S cells were inoculated into the scaffolds. Treatments started twelve days after MM.1S cells inoculation. Formulations were i.v. administered twice a week, with a total of five injections per mouse. Bioluminescence imaging was performed every week up to four weeks from start of the treatment.

Fig. 3

Viability of MM cell lines after incubation with free carfilzomib, carfilzomib formulations and empty formulations for 48 h at 37°C and 5% CO₂. (A) MM.1S, (B) L363 and (C) UM-9 cells were treated in triplicates with increasing concentrations of carfilzomib solubilized in DMSO (CFZ free), in sulfobutylether β-cyclodextrin (CFZ-CD) or in polymeric micelles (CFZ-PM); (D) MM.1S, L363 and UM-9 cells incubated with empty polymeric micelles, at polymer concentrations between 0 and 100 µg/mL. After incubation, beetle luciferin was added to the wells at a final concentration of 3 mM. Ten minutes later, luminescence was detected. Percentage of viable cells was calculated compared to control (untreated) wells. Data is expressed as mean ± SD, n≥3.

Fig. 4

Blood circulation kinetics and distribution in tumor-bearing scaffolds & tissues of Cy7-labeled polymeric micelles on a "humanized" bone marrow-like scaffold model. (A) Circulation of Cy7-PM in blood at different timepoints after injection (1, 2, 4 and 24 h), expressed as % of Injected dose (ID) present in circulation. Blood collected immediately after injection was considered as 100% ID. Fluorescence intensity in plasma samples was measured in Odyssey® imager; (B) Images of scaffolds and tissues 4 and 24 h after i.v. injection of Cy7-PM. Tissues were excised and imaged using BiospaceLab Photon Imager; (C) Scaffolds and tissue distribution of Cy7-PM at 4 and 24 h after i.v. injection. Cy7-PM accumulation was determined after homogenization of the scaffolds and tissues. Fluorescence present in the homogenates was measured with Odyssey® imager. Data is expressed as mean ± SD, n=3 per time point, n(scaffolds) > 5 per time point.

Fig. 5

Effect of carfilzomib loaded polymeric micelles versus carfilzomib in sulfobutylether β-cyclodextrin on tumor growth of a "humanized" bone marrow-like scaffold model. Mice bearing four human bone containing scaffolds were inoculated with luciferase labeled MM1.S cells into the scaffolds. Twelve days after inoculation of tumor cells, animals were treated with PBS, CFZ-CD or CFZ-PM, both at 4 mg/kg. Arrows represent i.v. injections. Bioluminescence imaging (BLI) was performed weekly. (A) Relative body weight of the mice throughout the experiment. ɣ - body weight of 3 out of 4 mice and α – body weight of 2 out of 4 mice; (B) Percentage of tumor growth compared to the day when treatment started. Tumor growth % was determined through analysis of BLI of each scaffold as counts per minute (cpm), and 100% tumor growth considered on day 0 – start of the treatment; (C) BLI images of representative mice 7 days (top panels), 14 days (middle panels) and 21 days (bottom panels) after treatment initiation. Fifty percent of animals treated with CFZ-CD needed to be culled from the experiment due to toxicity. Data are presented as mean ± SEM, n=4 per group.