Local controlled delivery of anti-neoplastic RNAse to the brain

Abstract

Purpose: Antineoplastic RNAse proteins, also known as Amphibinases, have been shown effective against various solid tumors but were found selectively neurotoxic to Purkinje cells in the cerebellum. This work describes the use of a waxy biodegradable poly(ricinoleic-co-sebacic acid) for the local controlled delivery of cytotoxic amphibinases in the parietal lobe of the brain in an attempt to overcome cerebellar neuronal toxicity while affecting glioma cells.

Methods: Amphibinase analogues were encapsulated in poly(ricinoleic-co-sebacic acid) formulations using mix-melt technology and loaded onto surgical foam. In-vitro release was monitored by BCA colorimetry and by RNAse specific bioactivity. The implants were inserted into rat brains bearing 9L glioma to assess toxicity and efficacy.

Results: The various formulations showed extended linear release for several weeks with minimal burst effect. Best in-vivo efficacy was obtained with ACC7201 containing implants, resulting in the extension of the median survival from 13 to 18 days with 13% long-term survivors.

Conclusion: Antineoplastic proteins were released from a p(SA-RA) polyanhydride implants in a controlled manner, providing efficacy against 9L glioma, while evading neurotoxicity in the cerebellum. The controlled release of Amphibinases forms the potential for a new therapy against brain tumors.

Fig. 1

Structure of poly(sebacic acid-co-ricinoleic acid anhydride) (poly(SA-RA) 3:7)

Fig. 2

Protein release from poly(RA:SA)7:3. Polymer. (187.5 mg) was melted and cooled to ambient temperature. Protein was dispersed in Tetraglycol and mixed with the molten polymer. Surgical gelfoam was saturated with the formulation and put on dry ice to harden. Pieces of 30 mg were cut, weighed and suspended in PBS at 37°C for controlled release measurements. Release was analysed with Total Protein Assay (Pierce). 2% Ranpirnase w/w polymer; Amphinase in ratio 4%, 10%, and 20% w/w polymer.

Fig. 3

Protein bioactivity from controlled release. Controlled release measured by specific RNAse activity with the RNAse alert kit. Ranpirnase in ratio of 2% w/w polymer (A), Amphinase in ratio of 4% w/w polymer (B) ACC7201 in ratio of 0.5% w/w polymer (C).

Fig. 4

In-vitro cell toxicity. 9L cells were seeded in 96-well plate at 3000 cells per well and incubated for 24 hours. The cells were then treated with compounds for 72 hours and assayed by MTT for growth inhibition. A. Ranpirnase and ACC7201, comparison of cytotoxic effects on 9L glioma cells in-vitro. B. Amphinase cytotoxic effect in-vitro on 9L glioma cells. C. Dose-response correlations.

Fig. 5

In-vivo safety study. Postmortem H&E staining of cerebellum cross-sections, 3 days after polymer implantation (pieces of 30 mg) with A) Ranpirnase 5% w/w and B) ACC7201 conjugate 5% w/w poly(SA-RA)3:7. White arrows indicate Purkinje cell deletion along the granular layer of the cerebellum. C) H&E staining of Purkinje cells in cerebellum upon implantation of Amphinase 10% w/w poly(SA-RA)3:7. D) ACC7201 1% w/w poly(SA-RA) 3:7. Pieces of formulation containing gelfoam were intracranially implanted. Black arrows point to intact Purkinje cells.

Fig. 6

Efficacy of locally delivered Ranpirnase, Amphinase and ACC7201 in the intracranial 9L glioma rodent model. Rodent 9L gliosarcoma was excised from the flanks of carrier rats and cut into 1-mm³ pieces, which were then implanted into the brain. The piece tumor tissue was implanted first and then the polymeric formulation was inserted adjacent to it. The polymeric formulation was implanted either simultaneously or 5 days after implantation of the tumor piece. Implants (30 mg) contained A) Ranpirnase 0.05% w/w w/w poly(SA-RA), B) Amphinase 5% w/w poly(SA-RA) 3:7. and C) ACC7201 4% w/w (n=16 per group).

Fig. 7

EGFR expression: cerebellum vs 9L-glioma. Immunohistochemical staining for Epithelial Growth Factor Receptor (EGFR) in A) cross section of cerebellum; B) cross section of 9L glioma, implanted intracranially in the parietal lobe.