(T1330-05-30) Permeability Enhancer Incorporated Oral Nano-Suspension of BRD4 Degrader for Glioblastoma Treatment

Purpose: The scarcity of existing and novel therapies for brain cancer has significantly affected the survival rate of glioblastoma (GBM) patients. This accounts for a poor prognosis of GBM indicating a median survival rate below 15 months and less than 5% of the five-year survival rate. With the over-escalating need of new therapeutics due to existing treatment drawbacks, another active field in development is protein degraders i.e., proteolysis targeting chimeras (PROTACs). PROTACs have emerged as a breakthrough technology that targets overexpressed protein moieties by recruiting E3 ubiquitin ligase machinery. BRD4 degrader, one of the cereblon-based PROTAC is responsible for BRD4 protein degradation. Herein, we have used BRD4 degrader-ARV 825 comprising OTX015 combined with an E3 ubiquitin ligase with an ethoxy spacer. It has molecular weight of 923.43 making it more difficult for solubilization. It exhibits topological polar surface area 233Ų restricting its permeability. Nanosuspension (NS) is a unique system consists of drugs and stabilizers having nano-range particle size, generally ranging from 10 to 1000 nm. For this purpose, nanoformulation could be a safe and most effective approach for the enhancement of oral bioavailability of ARV-825 (Fig. 1a). Herein, we employed sodium lauryl sulphate (SLS) and Polyvinylpyrrolidone (PVP K-30) to enhance the solubility by electrostatic interaction and stearic stabilization of nanosuspension. In addition, Salcoprazoate sodium (SNAC), an FDA approved permeation enhancer was incorporated to improve the permeability. The current study aimed to develop, optimize, and characterize oral nanosuspension of BRD4 degrader- ARV 825 using combination of stabilizers.

Methods: BRD4 degrader loaded nanosuspension (A-NS) was formulated using the wet media milling top-down dual centrifugation technique. The A-NS was prepared by using suitable concentrations of SLS, PVP K-30 and SNAC as stabilizers. The nano-milling was carried out using 0.3 mm Zirconium beads in NETZSCH DeltaVita® as shown in Fig. 1 (b). The nano milling process was carried out at 1500 RPM and temperature of -15℃ for 1 hr. After milling, the A-NS was analyzed by Malvern zeta sizer for dynamic light scattering (DLS) particle size, Poly dispersibility index (PDI) and zeta potential. The RP-HPLC method was used to determine the drug retention of BRD4 degrader into A-NS. Different cryoprotectants were screened for freeze-drying including trehalose, Kleptose HPB, mannitol, and lysine acetate. Suitable combination ratios were employed to get stable A-NS upon reconstitution. The freeze dried (FZ) A-NS was further characterized for the drug content using RP-HPLC method. The drug release of BRD4 degrader and A-NS was carried out in pH 1.2 maintained at 37℃ at 5, 10, and 30 mins. Cytotoxicity assessment of optimized surfactants was also evaluated using crystal violet staining in Caco-2 cells. In-vitro cytotoxicity study of BRD4 degrader was evaluated in U 87 MG cells using crystal violet assay. Ongoing studies are permeability study, in vitro migration assay, 3D spheroid assay, and western blot assay.

Results: An effective nano system for the oral delivery of BRD4 degrader was developed using SNAC, SLS and PVP K-30 (Fig. 2 a). The analytical RP-HPLC method revealed 91.23 ± 2.48 % encapsulation efficiency of A-NS at retention time of 4.9 ± 0.12 min. (Fig. 2 b). A-NS depicted a hydrodynamic diameter and polydispersity index of 204 ± 0.210 nm and 0.16 with zeta potential of -25.7 mV as shown in Fig. 2 (c) and (d). The particle size and PDI were also analyzed for the FZ A-NS upon reconstitution. The resulting hydrodynamic diameters and PDI were found to be 302.1±0.28 nm and 0.354±0.18 for Trehalose and Kleptose HPB (1:1) and 253.2±0.17 nm and 0.244±0.12 for mannitol with lysine acetate (4:1) Fig. 2 (e) and (f). Different cryoprotectants played significant roles in drug retention of the BRD4 degrader upon reconstitution. The A-NS with Trehalose and Kleptose HPB resulted in 83.15±0.32 % and mannitol with lysine acetate depicted 75.68±0.23 % as shown in Fig 2 (g). The in-vitro drug release of A-NS was found to be 2.5 folds higher as compared to pure drug at 15 min Fig. 3 (a). The toxicity study on Caco-2 cells revealed that the amount of SNAC incorporated into A-NS was found to be significantly safer than SLS. BRD4 degrader showed IC50 of 0.27 ± 0.12 µM in U 87 MG cells (Fig. 3 c).

Conclusion: This study entails a great promise of proteolysis targeting chimera-loaded nanosuspension as a novel therapeutic intervention for GBM patients. We have explored the potency of a novel class of molecule loaded in a nanosized drug-delivering system for the enhancement of solubility and permeability.


Fig. 1. (a) Purpose of Nanoformulation (b) Preparation of BRD4 degrader loaded Nanosuspension (A-NS) using dual centrifuge media milling method



Fig. 2. (a) Representation of BRD4 loaded NS before milling and after milling (b) HPLC chromatogram of A-NS, Physical characterization of A-NS (c) Dynamic light scattering graph demonstrating unimodal particle size distribution of A-NS (d) Zeta Potential, Characterization of A-NS after freeze drying with different cryoprotectants (e) Particle size (nm) (f) Poly-dispersibility Index (g) Drug Retained (%)



Fig. 3. (a) In-vitro drug release of BRD4 degrader and A-NS at pH 1.2
(b) Crystal violet-stained images for cytotoxicity of excipients incorporated into A-NS in Caco-2 cells
(c) In vitro cytotoxicity of BRD4 degrader in U-87 MG cells