Source: Dissertation Abstracts International, Volume: 78-11(E), Section: B.

Adviser: W. Mark Saltzman.

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High-grade gliomas (HGG) are devastating intracranial tumors that occur in both adults and children. An estimated 14,000 adults and 1,000 children will be diagnosed in 2016 with HGG. In the most malignant forms of this disease, less than five percent of adults and one percent of children will survive for five years after diagnosis. The greatest barrier to treatment efficacy in nearly all HGG is local recurrence after standard treatment with radiation therapy (RT), chemotherapy, and/or surgical resection. These high rates of recurrence have prompted a number of clinical studies focused on combining novel targeted agents with RT and chemotherapy as a means to sensitize tumor cells to RT and chemotherapy-induced DNA damage. Unfortunately, to date, these studies have largely been unsuccessful.

This project focuses on addressing four major issues associated with these trials. First, candidate radiosensitizers are often tested without rigorous in vitro assessment of either the mechanism or the synergy of their radiosensitizing properties, and thus translation of these therapies to clinic is frequently unsuccessful; second, drug penetration into intracranial tumors is severely limited by the blood brain barrier (BBB); third, even when candidate drugs are able to penetrate the BBB, they often require extraordinarily high and toxic doses of systemic therapies to achieve therapeutic intracranial levels; and finally, even if therapeutically-relevant intracranial levels are achieved, drugs are rapidly cleared from the brain, limiting the duration of their effects and requiring serial re-administration. To overcome these issues, this project proposed a new approach to the treatment of HGG which combines fractionated RT with local convection-enhanced delivery (CED) of radiosensitizer-loaded nanoparticles (NPs) to inhibit DNA repair following RT-induced DNA damage, thus decreasing tumor recurrence and ultimately increasing overall survival.

To test this approach, a collection of small molecule inhibitors of several key double-strand break (DSB) repair and DNA damage response proteins were chosen. Poly(lactic acid)-polyethylene glycol (PLA-PEG) NPs were engineered to meet a set of parameters that were hypothesized to be associated with the greatest efficacy as RT response modifiers: (1) high efficiencies of drug loading into NPs, (2) minimal loss of function as DSB repair inhibitors compared to the un-encapsulated molecules, (3) sustained release for periods of weeks or longer, and (4) extensive brain penetration when administered by CED. In vitro testing of these candidate formulations led to the selection of VE822-loaded NPs. VE822 is a small molecule inhibitor of ATR (Ataxia Telangiectasia and Rad3-related protein), a protein which plays a key role in the homologous recombination (HR) pathway of DSB repair. VE822 was efficiently encapsulated (>40%) into PLAPEG NPs and demonstrated a slow and sustained release for more than 14 days in artificial cerebrospinal fluid. The VE822-NPs demonstrated potent, dose-dependent inhibition of the homologous recombination (HR) DNA repair pathway, leading to significant radiosensitization of tumor cells in vitro. CED of VE822-NPs was associated with effective intracranial distribution and sustained intracranial release of the drug over at least 10 days. One dose of VE822-NPs and a 5 day course of fractionated RT led to prolonged in vivo survival compared to free VE822 in a rat intracranial allograft model.

To further quantify the mechanism by which the NPs confer this sustained intracranial release, a descriptive model was developed to assess the contribution of various NP characteristics to the intracranial drug retention profile. The results of this model suggested that the most significant factor in determining intracranial drug retention was the drug's release rate from the NP. To this extent, a new tri-block co-polymer, poly(w-pentadecalactone-co-p-dioxanone)PEG (poly(PDL-co-DO)-PEG) was synthesized and formulated into NPs capable of effective brain distribution via CED administration. Additionally, the release profiles of radiosensitizers from these NPs could be tuned by varying the ratios of the PDL and DO units in the polymer. The most promising of these formulations used a polymer with 71% PDL in the hydrophobic block and conferred the highest radiosensitizer encapsulation (>40% for VE822, equivalent to PLA-PEG NPs) as well as the smallest burst release (13% for VE822 compared to 36% for PLA-PEG NPs) and slowest linear release rate (1.4 nmol/d/mg NP for VE822 -- equivalent to PLA-PEG NPs). This formulation was found to confer intracranial drug retention levels that were ~2x those of the original PLA-PEG NP formulations over the course of 7 days following CED administration, suggesting a successful rational design improvement on NPs engineering for sustained intracranial delivery of radiosensitizers.

These findings serve as both proof-of-concept for a new multimodal approach to the treatment of HGG, and pre-clinical evidence supporting further development of newly engineered radiosensitizing-NPs for the treatment of intracranial tumors.