Hi, I'm Riccardo Serra, postdoc fellow in the Anterior Lab at Johns Hopkins, and today I'm going to present the local delivery of a novel biodegradable thermosensitive base loaded with olaparib to improve radiotherapy and prolonged survival in GBM. I have no disclosures. I first would like to give a brief introduction to the local delivery and its limitations in treating brain tumors. Three of the main limitations of the current approaches are the rigid structure of the wafer disc that sometimes allows for small gaps between the surface of the disc and the tumor section bed to form, and also the burst release of the loaded drug in the first days after the implantation, as well as the limited drug choice, which is currently available at BCNU, for example, in Gliedel. So for the future, we will try to develop new tools to deliver chemotherapeutics to the drug and also expand the number and types of drugs available. These are the goals of our study. To develop a new biodegradable thermosensitive base that can better adapt to the resection walls, to test in vivo and in vitro these new combinations, trying to overcome drug resistance and also increase DNA damage and the effects of radiation therapy. And then in the future, we're also planning to translate these findings into clinical practice through a phase one and two clinical trial that will start soon in the UK. And on the left, you can also see our first study in collaboration with the University of Nottingham in the UK, where we developed and successfully tested a combination of etoposide and TMZ in a rodent model of gliosarcoma. And these are our methods. We first tried to assess the cytotoxicity and anti-proliferative and anti-clonogenic effect of this combination of etoposide, TMZ, and LAPRIB. And then we turned to flow cytometry, trying to quantify the apoptotic effect on both human-derived and rodent cell lines. And we also assessed the AAF and PARP expression on 10 patient-derived lines that were derived from different areas of the tumor, from the necrotic core, from the margin, and from also the five ALA positive and negative regions. With immunofluorescence, we also assessed the AAF translocation to the nucleus after treatment and the expression of H2AX, which is a common DNA damage marker. And then we obviously developed this combination in vitro, and we also tested the drug release in vitro. We then turned in vivo, and we tested in a 9L rodent model of gliosarcoma the efficacy of this combination and of the combination of the paste with XRT. And we assessed also with immunohistochemistry and mass spectrometry the markers of apoptosis, PARP expression, tumor proliferation, and also the tissue concentration of chemotherapeutics. These are our results, and you can see in vitro the increase in apoptotic double-positive cells after combination therapy compared to single-treatment arms and to the control group. And then we turned to our patient-derived tumor tissues with either five ALA positive margins or negative margins, and also the rim, the core, and the invasive region. These lines were obtained with cell-sorting technologies, and we noticed an increase in PARP expression and also in AIF expression in the five ALA positive-invasive margins. With immunofluorescence, we then evaluated the nuclear translocation of AIF from the mitochondria, and you can see the top row, which is control, and then the middle and bottom rows are the triple-combination therapies, and you can see the translocation from the cytoplasm to the cell nucleus after treatment. Finally, we also investigated the expression of DNA damage markers, such as H2AX, and you can see all these positive foci in the cell nuclei, both in the olaparib-etoposide-XRT combination and even more in the olaparib-TMC-XRT combination compared to control. The next phase was the in-vitro safety study. I would like to first show this nice panel from Ian Souk, our artist at Johns Hopkins, and you can see also here how the drug is injected into the resection cavity, and then also the effect of each single drug on the DNA and the radiotherapy on the bottom left. This is our treatment schedule. You can see the implantation of 9L at day 0, the surgical resection and paste injection at day 4, as well as the XRT administration on day 5, and then the either oral TMC or IP olaparib administration over the following two weeks. This is our Kaplan-Meier with all the different groups, with 10% olaparib and 20% olaparib, and then our reference group, which was the surgery TMZ and XRT for obvious reasons, and our untreated control on the extreme left, which is that black line that you can see. These are our combinations, so local olaparib plus XRT, local olaparib plus TMZ and XRT, and local olaparib and etoposide plus XRT. You can see that all these combinations were able to achieve higher median overall and long-term survival than surgery TMZ and XRT, and obviously than control. So in summary, we were able to show in vitro the induction of apoptosis and cell death after the use of these combinations, as well as higher PARP expression and AIF translocation in 5 ALA positive tumor margins. Significant DNA damage was also shown with both Western blotting and immunofluorescent analysis. And then in vivo, we achieved significantly superior survival after combination therapy compared to controls and also to TMZ surgery and XRT groups. There's definitely some limitations to our study. One is the use of a rodent model of clitoris sarcoma to investigate survival, and also the short- and long-term toxicity that need to be investigated better, possibly in a non-human primary model. So in conclusion, we developed a biodegradable, thermosensitive-based inputting antibody olaparib TMZ or etoposide. We showed the combinatorial effect of PARP and AIF both in commercial and patient-derived lines and also on these 5 ALA tumor regions through different techniques. And then we were also able to achieve longer median and overall survival with combination therapy compared to control and surgery TMZ and XRT. And also, we didn't see any sign of toxicity in these groups. So for the future, we're planning to investigate even better in vivo these combinations with other novel chemotherapeutic agents and novel compounds, and also to run more safety and efficacy studies, possibly in non-human primates and then ultimately in human GBM patients. With this, I would like to thank our group at the Anterian Neurosurgical Lab at Johns Hopkins, Dr. Grelick, and also my mentors, Dr. Bremer and Tyler. And our collaborators, obviously, at University of Nottingham, and in particular Dr. Smith and Dr. Raman. And I would also like to thank you for your attention.

local delivery

biodegradable base

olaparib

radiotherapy

glioblastoma