(543a) Imipramine Blue-Doxorubicin Co-Loaded Nanoparticles Increase Survival In Glioblastoma Over Doxorubicin Alone In a Single Treatment

Introduction:  Though only accounting for 2% of new cancer cases, brain tumors are a large clinical problem with poor survival times.  Glioblastoma (GBM), the most common brain tumor, is a highly invasive tumor which infiltrates the brain. Invasion causes failure of common treatments as cells move away from the site or are missed by the treatment due to surgical or diffusional limitations. In this study we aim to incorporate a novel anti-invasive compound, Imipramine Blue (IB), with a common chemotherapeutic, Doxorubicin (DXR), in a single nanoparticle to enhance response to treatment in a rat model of glioblastoma. In this way, we test the hypothesis that inhibition of invasion will assist in the treatment paradigm of GBM.

Materials and Methods: Liposomal encapsulation of IB and Doxorubicin Liposomal nanoparticles were made from 10mol% cholesterol (Sigma), 85mol% DSPC (Avanti), 5mol% mPEG-DSPE (Avanti), dissolved in ethanol with 0.9 mg/ml IB, and hydrated with ammonium sulfate to create a gradient. DXR was remotely loaded and liposomes were concentrated to yield a final IB concentration of 1.6 mg/ml. Control liposomes were made without one or both components (IB, DXR, or control). In vitro treatment RT2 glioma cells were treated with liposomes (Control, IB, DXR, IB-DXR) in the same concentrations of IB and DXR, then measured after 72 hours by CCK8 assay (Dojindo). Viability was normalized to control. In vivo survival studies Male Fisher 344 rats were inoculated with RT2 glioma (250,000 cells) into the cortex (5-7 animals per group based on MRI confirmation of tumor at time of treatment). The tumor established for 7 days at which point MRI was performed using a Bruker 7T microMRI with T₂ weighted measurements. Animals were then injected with liposomes via tail vein (6.8 mg/kg DXR and 3 mg/kg IB). MRI was used to monitor progression of tumor and animals were euthanized upon display of signs of morbidity and histology performed for tumor morphology. Statistical Analysis One-way ANOVA was used for in vitro study analysis. Survival data was analyzed using Gehan-Breslow-Wilcoxon analysis.

Results and Discussion: Liposomes were approximately equivalent in size and lipid:drug ratio (average size of liposomes 160 nm). In vitro studies showed no synergistic effect between IB and DXR on the RT2 glioma. This is a desirable effect since our central hypothesis is that the anti-invasive effect of IB will assist the efficacy of DXR and not some other effect of the combination. This was also expected from previous microarray pathway analysis data of the effects of the drug. Survival studies revealed a significant increase in survival in the IB-DXR group over the DXR alone group to survive after treatment (Χ² (1) = 4.033, p<0.05) with a median survival time increase from 44.5 days to >210 days. Interestingly, at the end of the study (210 days), MRI revealed in the DXR animals evidence of tumor including blood brain barrier breach and inflammation whereas the co-treated animals had no evidence of tumor existence. This was confirmed by histology showing that the original tumor site in the co-treated animals was mostly scar tissue and remnant blood products. The use of this combination nanoparticle enhances survival significantly in just a single treatment which is unprecedented in the literature. Further, use of a single nanoparticle yields easier delivery to patient and reduced toxicity from possible accumulation of multiple treatments. Histological analysis confirming absence of tumor at the end of study further enhances the possibility of this compound combination for future trials and studies.

Conclusions: Integration of a novel anti-invasive Imipramine Blue significantly enhances the efficacy of Doxorubicin in malignant glioma after a single treatment.

Acknowledgments: Johannes Leisen, Alexander Ortiz, Safkat Alkindi, Daniel Brat.