Our next speaker is Dr. Zvankin, title of his talk is ABC Transporter Inhibition Plus Vessel Permeability Reduction Enhances the Efficacy of CED in DIPG. All right, two more. Thank you very much for having me. My name is A. Zvankin, and today I'll be discussing enhancing the efficacy of convection-enhanced delivery using desatinib using ABC transporter inhibition with teriquidar in H3K27M mutant DIPG. I have no disclosures. So first a brief overview of DIPG and a rationale for doing the study. So diffuse intrinsic pontine glioma represents 10 to 15 percent of all CNS tumors in children. It's the main cause of death in children with brain tumors, and it's not a surgical disease process. It's highly infiltrative, and it occurs in a highly eloquent area of the brain, and thus it's inoperable. I included a Kaplan-Meier curve in the right-hand corner of a group that attempted to risk stratify patients according to prognostically relevant groups, and the take-home message of this graph is really the very steep slope of that Kaplan-Meier curve. The median survival after diagnosis is nine months. Ninety percent of these children will die within two years of diagnosis, so the prognosis is quite dismal. And despite a lot of translational success, very little of that is actually translated into improved outcomes in clinical trials. And a large barrier to that is the blood-brain barrier. That's for two reasons. One is the principle of physiologic exclusion. So substances, drugs that we put into systemic circulation to attempt to treat these patients, are barred by the conspiracy of the astrocytes and the capillary endothelium that keep the drug from meaningfully getting into the tumor in many cases. And finally, there's the presence of ABC transporters, or ATP binding cassette transporters, which are present in the in the cells themselves, and use ATP to pump any drug that we put into the tumor back out into systemic circulation. And what that means is it's not necessarily a problem of a paucity of appropriate targets or even a paucity of agents for those targets. It could be that our lack of clinical success in this particular disease entity is attributed at least in part to our failure in getting the drugs that we're delivering to meaningfully interact with the tumor tissue. So with that in mind, our group has made some some prior observations. So in these tumors that overexpress PDGF, one appealing drug that that we use is dasatinib because it targets the PDGF receptor. And what we've noted in the past is that mice that have ABC transporters knocked out, we see upregulated concentrations of dasatinib in normal brain tissue when that dasatinib is administered systemically. And secondly, when you administer a single dose of systemic dasatinib, again peripherally in these mice that that have DIPGs, you actually see an increased amount of apoptosis within the tumor tissue compared to wild-type mouse. So what that indicates to us is that most likely ABC transporters are relevant in producing clinical outcomes and increased times of drug-tumor interaction. So with that in mind, that leads us to three questions. Is there a benefit to mechanically bypassing the blood-brain barrier using convection enhanced delivery of dasatinib, particularly compared with systemic administration? And will pretreating with a peripherally delivered ABC transporter inhibitor that is pharmacologically inhibiting ABC transporters with teriquidar or decreasing blood-brain barrier disruption with dexamethasone improve the efficacy of CED? And finally, can we gain insight into the kinetics or the mechanism of these events? So a brief note on our tumor model. So we use an R-Cas-TVA tumor model which requires a transgenic mouse. It co-expresses the nestin-TVA receptor. It's p53 floxed and the luciferase promoter is stop-floxed. The generation of the tumor involves generating plasmids which have replication-competent avian leukosis virus that's conjugated to the PDGF genes, the CRE system, as well as the H3K27M mutation. These are injected into chicken fibroblasts which are in turn stereotactically injected into the pons of our transgenic model. What that yields is, after the viroids are produced by the cells and then infect nestin-producing cells, is a tumor with constitutive PDGF secretion, deficient p53. It harbors the H3K27M mutation and it expresses luciferase. It looks like this and histologically it is equivalent to human DIPG. And gene expression analyses have also shown that it is similar to human DIPG. So using this tumor model, we wanted to answer the question of whether or not we can improve survival using either systemic or convection-enhanced delivery of dasatinib. And our CED platform is quite standard. We use the LZP pumps that are capable of infusing over a period of two weeks two micromolar dasatinib directly into the pontine tumors of these animals. And we compared that to, in addition to control groups, mice that were given dasatinib intraperitoneally. And indeed we did see that treating with dasatinib increases survival. We got roughly 10 days of survival benefit using either convection-enhanced delivery or intraperitoneal injection of dasatinib over our control groups, including our vehicle. So the next question we wanted to answer is, well, can we take a step back, look at our in vitro model, and then use teriquidar or use ABC transport inhibition with teriquidar in order to amplify the effects of the dasatinib that we're delivering. And so looking at our MTS assays of our DIPG cell lines in vitro with the H3K27M mutation, we saw that, yeah, there was a time-dependent effect of decreased cell viability and proliferation in cells that were cultured in the presence of dasatinib. And even more pertinently, when you added teriquidar to the mix, we saw an even greater increase in the deficit in proliferation and viability of our assays. So this did show that teriquidar can be effective, at least in vitro. So next we wanted to translate that to our animal model. Again, we used convection enhanced delivery. This time, instead of implanting pumps, we used a single dose infusion convection enhanced delivery. In a cohort of those animals, we pre-treated half an hour prior to that infusion of convection enhanced delivery with teriquidar and dexamethasone. And immediately after that infusion, the animals were subjected to MRI. The dasatinib was co-administered with gadolinium, and we used the enhancement pattern as a surrogate for the distribution pattern, as well as the dispersion on the MRI images. And what you see is images of non-pre-treated animals. You see a nice circular region of interest that's immediately adjacent to our catheter tip placement, and around that you see this hyperintensity blooming around our catheter tip, which over time receded. In animals that were pre-treated, and this is another illustrative MRI, we saw a large area of hypo-intensity corresponding to a high concentration of infusate at that area, which then over time yielded to the same hyperintensity that receded over time. And looking at that region of interest and how long it took for the intensity to peak, that is the time to peak of that intensity, what we saw is that in the non-pre-treated animals, it was about 1,500 seconds to peak. In the pre-treated animals, that time to peak was quite delayed by close to 75% or about 18 minutes, showing that the dispersion was delayed by pre-treatment with an ABC transporter inhibitor. And when we took a look at the histologic sections from the same animals with DIPG and assayed them for cleaved caspase 3 expression, what we found was that, in fact, yes, the animals treated with dexamethasone and triquidar had increased amounts of cleaved caspase 3 expression histologically, correlating to a greater amount of apoptosis in those tumors. And that was borne out by a survival study. So we saw that pre-treatment with dexamethasone and triquidar extended survival in the DIPG-bearing mice that we treated with dasatinib. So we did get a treatment effect with the dasatinib alone, 39 days post-injection, but we extended that by roughly 10 days by pre-treating with dexamethasone and triquidar, and that's about 18 days greater than our controls. And so to go and circle back to the questions that we posed originally, is there a benefit to mechanically bypassing the blood-brain barrier by convection-enhanced delivery? Well, yeah, we get a survival effect with convection-enhanced delivery, but we get a similar survival effect with systemic administration of the same drug. However, by pre-treating with triquidar and inhibiting ABC transporters, as well as tightening the blood-brain barrier with dexamethasone, we were able to improve survival even further in our convection-enhanced delivery treatment model. And can we gain insight into the kinetics? The MRI data that we showed, as well as the histologic data, suggest that that can be attributed to a greater amount of apoptosis within a tumor due to delaying the dispersion of the drug and keeping it in contact with the tumor cells for longer. And with that, thank you very much to all the folks mentioned here, in particular my PA, Dr. Thompson.

convection-enhanced delivery

diffuse intrinsic pontine glioma

blood-brain barrier

ABC transporters

dasatinib