Bystander effects, pharmacokinetics, and linker-payload stability of EGFR-targeting antibody-drug conjugates Losatuxizumab vedotin and Depatux-M in glioblastoma models.

PURPOSE: Antibody-drug conjugates (ADCs) are targeted therapies with robust efficacy in solid cancers, and there is intense interest in using EGFR-specific ADCs to target EGFR-amplified glioblastoma (GBM). Given the molecular heterogeneity of GBM, bystander activity of ADCs may be important for determining treatment efficacy. In this study, the activity and toxicity of two EGFR-targeted ADCs, Losatuxizumab vedotin (ABBV-221) and Depatuxizumab mafodotin (Depatux-M), with similar auristatin toxins, were compared in GBM patient-derived xenografts (PDXs) and normal murine brain following direct infusion by convection enhanced delivery (CED). METHODS: EGFRviii-amplified and non-amplified GBM PDXs were used to determine in vitro cytotoxicity, in vivo efficacy, and bystander activities of ABBV-221 and Depatux-M. Non-tumor bearing mice were used to evaluate pharmacokinetics and toxicity of ADCs using LC-MS/MS and immunohistochemistry. RESULTS: CED improved intracranial efficacy of Depatux-M and ABBV-221 in three EGFRviii-amplified GBM PDX models (Median survival: 125 to >300 days vs 20-49 days with isotype-control AB095). Both ADCs had comparable in vitro and in vivo efficacy. However, neuronal toxicity and CD68+ microglia/macrophage infiltration were significantly higher in brains infused with ABBV-221, with the cell-permeable MMAE, as compared to Depatux-M, with the cell-impermeant MMAF. CED infusion of ABBV-221 into brain or incubation of ABBV-221 with normal brain homogenate resulted in significant release of MMAE, which is consistent with linker instability in the brain microenvironment. CONCLUSION: EGFR-targeting ADCs are promising therapeutic options for GBM when delivered intra-tumorally by CED. However, the linker and payload for the ADC must be carefully considered to maximize the therapeutic window.

Central Nervous System Distributional Kinetics of Selected Histone Deacetylase Inhibitors.

Histone deacetylase expression and activity are often dysregulated in central nervous system (CNS) tumors, providing a rationale for investigating histone deacetylase inhibitors (HDACIs) in selected brain tumor patients. Although many HDACIs have shown potential in in vitro studies, they have had modest efficacy in vivo This lack of activity could be due to insufficient CNS exposure to the unbound drug. In this study, we investigated the systemic pharmacokinetics and subsequent CNS distribution of two potent HDACIs, vorinostat and quisinostat, in the murine model. Both compounds undergo in vitro degradation in mouse plasma, requiring precautions during sample processing. They also have short half-lives in vivo, in both plasma and CNS, which may lead to diminished efficacy. Transgenic transporter-deficient mouse models show that the CNS delivery of vorinostat was not limited by the two major blood-brain barrier efflux transporters, p-glycoprotein and breast-cancer-resistance protein. Vorinostat had an unbound CNS tissue-to-plasma partition coefficient of 0.06 {plus minus} 0.02. Conversely, the exposure of unbound quisinostat in the brain was only 0.02 {plus minus} 0.001 of that in the plasma, and the CNS distribution of quisinostat was limited by the activity of p-glycoprotein. To gain further context for these findings, the CNS distributional kinetics for vorinostat and quisinostat were compared to another hydroxamic acid HDACI, panobinostat. A comprehensive understanding of the CNS target exposure to unbound HDACI, along with known potencies from in vitro testing, can inform the prediction of a therapeutic window for HDACIs that have limited CNS exposure to unbound drug and guide targeted dosing strategies. Significance Statement This study indicates that quisinostat and vorinostat are susceptible to enzymatic degradation in the plasma, and to a lesser degree, in the target CNS tissues. Employing techniques that minimize the post-sampling degradation in plasma, brain and spinal cord, accurate CNS distributional kinetic parameters for these potentially useful compounds were determined. A knowledge of CNS exposure (Kp,uu), time to peak, and duration can inform dosing strategies in preclinical and clinical trials in selected CNS tumors.

Pharmacokinetics of panobinostat: Inter-species difference in metabolic stability.

Panobinostat is a potent pan-HDAC inhibitor that has been tested in multiple studies for the treatment of brain tumors. There have been contrasting views surrounding its efficacy for the treatment of tumors in the CNS following systemic administration when examined in different models or species. We conducted experiments using three different mouse strains or genotypes to have a more comprehensive understanding of the systemic as well as the CNS distributional kinetics of panobinostat. Our study found that panobinostat experienced rapid degradation in vitro in FVB mouse matrices and a faster degradation rate was observed at 37{degree sign}C compared with room temperature and 4{degree sign}C, suggesting that the in vitro instability of panobinostat was due to enzymatic metabolism. Panobinostat also showed inter-strain and inter-species differences in the in vitro plasma stability; and was stable in human plasma. The objective of this study was to examine the in vitro metabolic stability of panobinostat in different matrices and assess the influence of that metabolic stability on the in vivo pharmacokinetics and CNS delivery of panobinostat. Importantly, the plasma stability in various mouse strains was not reflected in the in vivo systemic pharmacokinetic behavior of panobinostat. Several hypotheses arise from this finding, including: the binding of panobinostat to red blood cells, the existence of competing endogenous compounds to enzyme(s), the distribution into tissues with a lower level of enzymatic activity or the metabolism occurring in the plasma is a small fraction of the total metabolism in vivo Significance Statement Panobinostat showed different in vitro degradation in plasma from different mouse strains and genotypes. However, despite the differences surrounding in vitro plasma stability, panobinostat showed similar in vivo pharmacokinetic behavior in different mouse models. This suggests that the inter-strain difference in enzymatic activity did not affect the in vivo pharmacokinetic behavior of panobinostat and its CNS distribution in mice. This lack of translation between in vitro metabolism assays and in vivo disposition can confound drug development.

Aberrant ATM signaling and homology-directed DNA repair as a vulnerability of p53-mutant GBM to AZD1390-mediated radiosensitization.

ATM is a key mediator of radiation response, and pharmacological inhibition of ATM is a rational strategy to radiosensitize tumors. AZD1390 is a brain-penetrant ATM inhibitor and a potent radiosensitizer. This study evaluated the spectrum of radiosensitizing effects and the impact of TP53 mutation status in a panel of IDH1 wild-type (WT) glioblastoma (GBM) patient-derived xenografts (PDXs). AZD1390 suppressed radiation-induced ATM signaling, abrogated G0-G1 arrest, and promoted a proapoptotic response specifically in p53-mutant GBM in vitro. In a preclinical trial using 10 orthotopic GBM models, AZD1390/RT afforded benefit in a cohort of TP53-mutant tumors but not in TP53-WT PDXs. In mechanistic studies, increased endogenous DNA damage and constitutive ATM signaling were observed in TP53-mutant, but not in TP53-WT, PDXs. In plasmid-based reporter assays, GBM43 (TP53-mutant) showed elevated DNA repair capacity compared with that in GBM14 (p53-WT), whereas treatment with AZD1390 specifically suppressed homologous recombination (HR) efficiency, in part, by stalling RAD51 unloading. Furthermore, overexpression of a dominant-negative TP53 (p53DD) construct resulted in enhanced basal ATM signaling, HR activity, and AZD1390-mediated radiosensitization in GBM14. Analyzing RNA-seq data from TCGA showed up-regulation of HR pathway genes in TP53-mutant human GBM. Together, our results imply that increased basal ATM signaling and enhanced dependence on HR represent a unique susceptibility of TP53-mutant cells to ATM inhibitor-mediated radiosensitization.

Differential Distribution of the DNA-PKcs Inhibitor Peposertib Selectively Radiosensitizes Patient-derived Melanoma Brain Metastasis Xenografts.

Radioresistance of melanoma brain metastases limits the clinical utility of conventionally fractionated brain radiation in this disease, and strategies to improve radiation response could have significant clinical impact. The catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) is critical for repair of radiation-induced DNA damage, and inhibitors of this kinase can have potent effects on radiation sensitivity. In this study, the radiosensitizing effects of the DNA-PKcs inhibitor peposertib were evaluated in patient-derived xenografts of melanoma brain metastases (M12, M15, M27). In clonogenic survival assays, peposertib augmented radiation-induced killing of M12 cells at concentrations ≥100 nmol/L, and a minimum of 16 hours exposure allowed maximal sensitization. This information was integrated with pharmacokinetic modeling to define an optimal dosing regimen for peposertib of 125 mpk dosed just prior to and 7 hours after irradiation. Using this drug dosing regimen in combination with 2.5 Gy × 5 fractions of radiation, significant prolongation in median survival was observed in M12-eGFP (104%; P = 0.0015) and M15 (50%; P = 0.03), while more limited effects were seen in M27 (16%, P = 0.04). These data support the concept of developing peposertib as a radiosensitizer for brain metastases and provide a paradigm for integrating in vitro and pharmacokinetic data to define an optimal radiosensitizing regimen for potent DNA repair inhibitors.

Altering distribution profile of palbociclib by its prodrugs.

Palbociclib, a cyclin-dependent kinase (CDK) 4/6 inhibitor, is currently used clinically for treating hormone receptor-positive and human epidermal growth factor receptor 2 negative breast cancer. Additionally, it has the potential to be utilized in the treatment of various tumors, including malignant glioblastoma. Previous research has indicated that palbociclib is a substrate for two efflux transporters, P-glycoprotein (P-gp; MDR1) and breast cancer-resistant protein (BCRP), which restrict the brain exposure of palbociclib. In the present study, our objective was to alter the brain distribution pattern of palbociclib by creating and assessing two novel prodrugs through in vitro, in situ, and in vivo evaluations. To this end, we synthesized two prodrugs of palbociclib by attaching it to the tyrosine promoiety at the para- (PD1) and meta-(PD2) position via a carbamate bond. We hypothesized that the prodrugs could bypass efflux transporter-mediated drug resistance by leveraging the l-type amino acid transporter (LAT1) to facilitate their transport across the blood-brain barrier (BBB) and into cancer cells, such as glioma cells that express LAT1. The compounds PD1 and PD2 did not show selective binding and had limited inhibitory effects on LAT1 in three cell lines (MCF-7, U87-MG, HEK-hLAT1). However, PD1 and PD2 demonstrated the ability to evade efflux mechanisms, and their in vitro uptake profiles were comparable to that of palbociclib, indicating their potential for effective cellular transport. In in situ and in vivo studies, brain uptake was not significantly improved compared to palbociclib, but the pharmacokinetic profiles showed encouraging enhancements. PD1 exhibited a higher AUCbrain/plasma ratio, suggesting safer dosing, while PD2 showed favorable long-acting pharmacokinetics. Although our prodrug design did not significantly improve palbociclib brain delivery due to the potential size limitation of the prodrugs, the study provides valuable insights for future prodrug development and drug delivery strategies targeting specific transporters.

Delivery versus Potency in Treating Brain Tumors: BI-907828, a MDM2-p53 Antagonist with Limited BBB Penetration but Significant In Vivo Efficacy in Glioblastoma.

MDM2-p53 inhibition may be effective in glioblastoma (GBM). This study evaluates the pharmacokinetics/pharmacodynamics of BI-907828, a potent antagonist of MDM2, in GBM, and demonstrates a translational paradigm with a focus on a unified "Delivery - Potency - Efficacy" relationship in drug development for central nervous system(CNS) tumors. BI-907828 was tested for cytotoxicity and MDM2-p53 pathway inhibition. Systemic pharmacokinetics and transport mechanisms controlling CNS distribution were evaluated in mice. BI-907828 free fractions in cell media, mouse and human specimens were measured to determine "active" unbound concentrations. Efficacy measures, including overall survival and target expression were assessed in mouse orthotopic GBM xenografts. BI-907828 exhibited potent inhibition of MDM2-p53 pathway and promoted cell death in GBM TP53 wild-type cells. MDM2-amplified cells are highly sensitive to BI-907828, with an effective unbound concentration of 0.1 nmol/L. The CNS distribution of BI-907828 is limited by blood-brain barrier (BBB) efflux mediated by P-gp, resulting in a Kp,uu_brain of 0.002. Despite this seemingly "poor" BBB penetration, weekly administration of 10 mg/kg BI-907828 extended median survival of orthotopic GBM108 xenografts from 28 to 218 days (P < 0.0001). This excellent efficacy can be attributed to high potency, resulting in a limited, yet effective, exposure in the CNS. These studies show that efficacy of BI-907828 in orthotopic models is related to high potency even though its CNS distribution is limited by BBB efflux. Therefore, a comprehensive understanding of all aspects of the "Delivery - Potency - Efficacy" relationship is warranted in drug discovery and development, especially for treatment of CNS tumors.

Methodological and analytical considerations for intra-operative microdialysis.

BACKGROUND: Microdialysis is a technique that can be utilized to sample the interstitial fluid of the central nervous system (CNS), including in primary malignant brain tumors known as gliomas. Gliomas are mainly accessible at the time of surgery, but have rarely been analyzed via interstitial fluid collected via microdialysis. To that end, we obtained an investigational device exemption for high molecular weight catheters (HMW, 100 kDa) and a variable flow rate pump to perform microdialysis at flow rates amenable to an intra-operative setting. We herein report on the lessons and insights obtained during our intra-operative HMW microdialysis trial, both in regard to methodological and analytical considerations. METHODS: Intra-operative HMW microdialysis was performed during 15 clinically indicated glioma resections in fourteen patients, across three radiographically diverse regions in each patient. Microdialysates were analyzed via targeted and untargeted metabolomics via ultra-performance liquid chromatography tandem mass spectrometry. RESULTS: Use of albumin and lactate-containing perfusates impacted subsets of metabolites evaluated via global metabolomics. Additionally, focal delivery of lactate via a lactate-containing perfusate, induced local metabolic changes, suggesting the potential for intra-operative pharmacodynamic studies via reverse microdialysis of candidate drugs. Multiple peri-operatively administered drugs, including levetiracetam, cefazolin, caffeine, mannitol and acetaminophen, could be detected from one microdialysate aliquot representing 10 min worth of intra-operative sampling. Moreover, clinical, radiographic, and methodological considerations for performing intra-operative microdialysis are discussed. CONCLUSIONS: Intra-operative HMW microdialysis can feasibly be utilized to sample the live human CNS microenvironment, including both metabolites and drugs, within one surgery. Certain variables, such as perfusate type, must be considered during and after analysis. Trial registration NCT04047264.

Central Nervous System Distribution of Panobinostat in Preclinical Models to Guide Dosing for Pediatric Brain Tumors.

Achieving adequate exposure of the free therapeutic agent at the target is a critical determinant of efficacious chemotherapy. With this in mind, a major challenge in developing therapies for central nervous system (CNS) tumors is to overcome barriers to delivery, including the blood-brain barrier (BBB). Panobinostat is a nonselective pan-histone deacetylase inhibitor that is being tested in preclinical and clinical studies, including for the treatment of pediatric medulloblastoma, which has a propensity for leptomeningeal spread and diffuse midline glioma, which can infiltrate into supratentorial brain regions. In this study, we examined the rate, extent, and spatial heterogeneity of panobinostat CNS distribution in mice. Transporter-deficient mouse studies show that panobinostat is a dual substrate of P-glycoprotein (P-gp) and breast cancer resistant protein (Bcrp), which are major efflux transporters expressed at the BBB. The CNS delivery of panobinostat was moderately limited by P-gp and Bcrp, and the unbound tissue-to-plasma partition coefficient of panobinostat was 0.32 and 0.21 in the brain and spinal cord in wild-type mice. In addition, following intravenous administration, panobinostat demonstrated heterogeneous distribution among brain regions, indicating that its efficacy would be influenced by tumor location or the presence and extent of leptomeningeal spread. Simulation using a compartmental BBB model suggests inadequate exposure of free panobinostat in the brain following a recommended oral dosing regimen in patients. Therefore, alternative approaches to CNS delivery may be necessary to have adequate exposure of free panobinostat for the treatment of a broad range of pediatric brain tumors. SIGNIFICANCE STATEMENT: This study shows that the central nervous system (CNS) penetration of panobinostat is limited by P-gp and Bcrp, and its efficacy may be limited by inadequate distribution to the tumor. Panobinostat has heterogeneous distribution into various brain regions, indicating that its efficacy might depend on the anatomical location of the tumors. These distributional parameters in the mouse CNS can inform both preclinical and clinical trial study design and may guide treatment for these devastating brain tumors in children.

How Much is Enough? Impact of Efflux Transporters on Drug delivery Leading to Efficacy in the Treatment of Brain Tumors.

The lack of effective chemotherapeutic agents for the treatment of brain tumors is a serious unmet medical need. This can be attributed, in part, to inadequate delivery through the blood-brain barrier (BBB) and the tumor-cell barrier, both of which have active efflux transporters that can restrict the transport of many potentially effective agents for both primary and metastatic brain tumors. This review briefly summarizes the components and function of the normal BBB with respect to drug penetration into the brain and the alterations in the BBB due to brain tumor that could influence drug delivery. Depending on what is rate-limiting a compound's distribution, the limited permeability across the BBB and the subsequent delivery into the tumor cell can be greatly influenced by efflux transporters and these are discussed in some detail. Given these complexities, it is necessary to quantify the extent of brain distribution of the active (unbound) drug to compare across compounds and to inform potential for use against brain tumors. In this regard, the metric, Kp,uu, a brain-to-plasma unbound partition coefficient, is examined and its current use is discussed. However, the extent of active drug delivery is not the only determinant of effective therapy. In addition to Kp,uu, drug potency is an important parameter that should be considered alongside drug delivery in drug discovery and development processes. In other words, to answer the question - How much is enough? - one must consider how much can be delivered with how much needs to be delivered.

WSD-0922, a novel brain-penetrant inhibitor of epidermal growth factor receptor, promotes survival in glioblastoma mouse models.

Background: Although the epidermal growth factor receptor (EGFR) is a frequent oncogenic driver in glioblastoma (GBM), efforts to therapeutically target this protein have been largely unsuccessful. The present preclinical study evaluated the novel EGFR inhibitor WSD-0922. Methods: We employed flank and orthotopic patient-derived xenograft models to characterize WSD-0922 and compare its efficacy to erlotinib, a potent EGFR inhibitor that failed to provide benefit for GBM patients. We performed long-term survival studies and collected short-term tumor, plasma, and whole-brain samples from mice treated with each drug. We utilized mass spectrometry to measure drug concentrations and spatial distribution and to assess the impact of each drug on receptor activity and cellular signaling networks. Results: WSD-0922 inhibited EGFR signaling as effectively as erlotinib in in vitro and in vivo models. While WSD-0922 was more CNS penetrant than erlotinib in terms of total concentration, comparable concentrations of both drugs were measured at the tumor site in orthotopic models, and the concentration of free WSD-0922 in the brain was significantly less than the concentration of free erlotinib. WSD-0922 treatment provided a clear survival advantage compared to erlotinib in the GBM39 model, with marked suppression of tumor growth and most mice surviving until the end of the study. WSD-0922 treatment preferentially inhibited phosphorylation of several proteins, including those associated with EGFR inhibitor resistance and cell metabolism. Conclusions: WSD-0922 is a highly potent inhibitor of EGFR in GBM, and warrants further evaluation in clinical studies.

Blood-brain barrier disruption defines the extracellular metabolome of live human high-grade gliomas.

The extracellular microenvironment modulates glioma behaviour. It remains unknown if blood-brain barrier disruption merely reflects or functionally supports glioma aggressiveness. We utilised intra-operative microdialysis to sample the extracellular metabolome of radiographically diverse regions of gliomas and evaluated the global extracellular metabolome via ultra-performance liquid chromatography tandem mass spectrometry. Among 162 named metabolites, guanidinoacetate (GAA) was 126.32x higher in enhancing tumour than in adjacent brain. 48 additional metabolites were 2.05-10.18x more abundant in enhancing tumour than brain. With exception of GAA, and 2-hydroxyglutarate in IDH-mutant gliomas, differences between non-enhancing tumour and brain microdialysate were modest and less consistent. The enhancing, but not the non-enhancing glioma metabolome, was significantly enriched for plasma-associated metabolites largely comprising amino acids and carnitines. Our findings suggest that metabolite diffusion through a disrupted blood-brain barrier may largely define the enhancing extracellular glioma metabolome. Future studies will determine how the altered extracellular metabolome impacts glioma behaviour.

Overcoming translational barriers in H3K27-altered diffuse midline glioma: Increasing the drug-tumor residence time.

Background: H3K27-altered diffuse midline glioma (DMG) is the deadliest pediatric brain tumor; despite intensive research efforts, every clinical trial to date has failed. Is this because we are choosing the wrong drugs? Or are drug delivery and other pharmacokinetic variables at play? We hypothesize that the answer is likely a combination, where optimization may result in a much needed novel therapeutic approach. Methods: We used in vitro drug screening, patient samples, and shRNA knockdown models to identify an upregulated target in DMG. A single small molecule protein kinase inhibitor with translational potential was selected for systemic and direct, loco-regional delivery to patient-derived xenografts (PDX) and genetically engineered mouse models (GEMM). Pharmacokinetic studies were conducted in non-tumor bearing rats. Results: Aurora kinase (AK) inhibitors demonstrated strong antitumor effects in DMG drug screens. Additional in vitro studies corroborated the importance of AK to DMG survival. Systemic delivery of alisertib showed promise in subcutaneous PDX but not intracranial GEMM and PDX models. Repeated loco-regional drug administration into the tumor through convection-enhanced delivery (CED) was equally inefficacious, and pharmacokinetic studies revealed rapid clearance of alisertib from the brain. In an effort to increase the drug to tumor residence time, continuous CED over 7 days improved drug retention in the rodent brainstem and significantly extended survival in both orthotopic PDXs and GEMMs. Conclusions: These studies provide evidence for increasing drug-tumor residence time of promising targeted therapies via extended CED as a valuable treatment strategy for DMG.

Murine Central Nervous System and Bone Marrow Distribution of the Aurora A Kinase Inhibitor Alisertib: Pharmacokinetics and Exposure at the Sites of Efficacy and Toxicity.

Important challenges in developing drugs that target central nervous system (CNS) tumors include overcoming barriers for CNS delivery and reducing systemic side effects. Alisertib, an aurora A kinase inhibitor, has been examined for treatment of several CNS tumors in preclinical and clinical studies. In this study, we investigated the distribution of alisertib into the CNS, the site of efficacy for brain tumors, and into the bone marrow, the site of dose-limiting toxicity leading to myelosuppression. Mechanisms influencing site-specific distribution, such as active transport mediated by the efflux proteins, p-glycoprotein (P-gp) and breast cancer resistance protein (Bcrp), were examined. Alisertib exposure to the brain in wild-type mice was less than 1% of that in the plasma, and was evenly distributed throughout various brain regions and the spinal cord. Studies using transporter knockout mice and pharmacological inhibition show that alisertib CNS distribution is influenced by P-gp, but not Bcrp. Conversely, upon systemic administration, alisertib distribution to the bone marrow occurred rapidly, was not significantly limited by efflux transporters, and reached higher concentrations than in the CNS. This study demonstrates that, given an equivalent distributional driving force exposure in plasma, the exposure of alisertib in the brain is significantly less than that in the bone marrow, suggesting that targeted delivery may be necessary to guarantee therapeutic efficacy with minimal risk for adverse events.Therefore, these data suggest that, to improve the therapeutic index when using alisertib for brain tumors, a localized regional delivery, such as convection-enhanced delivery, may be warranted. SIGNIFICANCE STATEMENT: The CNS penetration of alisertib is limited with uniform distribution in various regions of the brain, and P-gp efflux is an important mechanism limiting that CNS distribution. Alisertib rapidly distributes into the bone marrow, a site of toxicity, with a greater exposure than in the CNS, a possible site of efficacy. These results suggest a need to design localized delivery strategies to improve the CNS exposure of alisertib and limit systemic toxicities in the treatment of brain tumors.

Convection enhanced delivery of EGFR targeting antibody-drug conjugates Serclutamab talirine and Depatux-M in glioblastoma patient-derived xenografts.

Background: EGFR targeting antibody-drug conjugates (ADCs) are highly effective against EGFR-amplified tumors, but poor distribution across the blood-brain barrier (BBB) limits their efficacy in glioblastoma (GBM) when administered systemically. We studied whether convection-enhanced delivery (CED) can be used to safely infuse ADCs into orthotopic patient-derived xenograft (PDX) models of EGFRvIII mutant GBM. Methods: The efficacy of the EGFR-targeted ADCs depatuxizumab mafodotin (Depatux-M) and Serclutamab talirine (Ser-T) was evaluated in vitro and in vivo. CED was performed in nontumor and tumor-bearing mice. Immunostaining was used to evaluate ADC distribution, pharmacodynamic effects, and normal cell toxicity. Results: Dose-finding studies in orthotopic GBM6 identified single infusion of 2 µg Ser-T and 60 µg Depatux-M as safe and effective associated with extended survival prolongation (>300 days and 95 days, respectively). However, with serial infusions every 21 days, four Ser-T doses controlled tumor growth but was associated with lethal toxicity approximately 7 days after the final infusion. Limiting dosing to two infusions in GBM108 provided profound median survival extension of over 200 days. In contrast, four Depatux-M CED doses were well tolerated and significantly extended survival in both GBM6 (158 days) and GBM108 (310 days). In a toxicity analysis, Ser-T resulted in a profound loss in NeuN+ cells and markedly elevated GFAP staining, while Depatux-M was associated only with modest elevation in GFAP staining. Conclusion: CED of Depatux-M is well tolerated and results in extended survival in orthotopic GBM PDXs. In contrast, CED of Ser-T was associated with a much narrower therapeutic window.

Central Nervous System Distribution of the Ataxia-Telangiectasia Mutated Kinase Inhibitor AZD1390: Implications for the Treatment of Brain Tumors.

Effective drug delivery to the brain is critical for the treatment of glioblastoma (GBM), an aggressive and invasive primary brain tumor that has a dismal prognosis. Radiation therapy, the mainstay of brain tumor treatment, works by inducing DNA damage. Therefore, inhibiting DNA damage response (DDR) pathways can sensitize tumor cells to radiation and enhance cytotoxicity. AZD1390 is an inhibitor of ataxia-telangiectasia mutated kinase, a critical regulator of DDR. Our in vivo studies in the mouse indicate that delivery of AZD1390 to the central nervous system (CNS) is restricted due to active efflux by P-glycoprotein (P-gp). The free fraction of AZD1390 in brain and spinal cord were found to be low, thereby reducing the partitioning of free drug to these organs. Coadministration of an efflux inhibitor significantly increased CNS exposure of AZD1390. No differences were observed in distribution of AZD1390 within different anatomic regions of CNS, and the functional activity of P-gp and breast cancer resistance protein also remained the same across brain regions. In an intracranial GBM patient-derived xenograft model, AZD1390 accumulation was higher in the tumor core and rim compared with surrounding brain. Despite this heterogenous delivery within tumor-bearing brain, AZD1390 concentrations in normal brain, tumor rim, and tumor core were above in vitro effective radiosensitizing concentrations. These results indicate that despite being a substrate of efflux in the mouse brain, sufficient AZD1390 exposure is anticipated even in regions of normal brain. SIGNIFICANCE STATEMENT: Given the invasive nature of glioblastoma (GBM), tumor cells are often protected by an intact blood-brain barrier, requiring the development of brain-penetrant molecules for effective treatment. We show that efflux mediated by P-glycoprotein (P-gp) limits central nervous system (CNS) distribution of AZD1390 and that there are no distributional differences within anatomical regions of CNS. Despite efflux by P-gp, concentrations effective for potent radiosensitization are achieved in GBM tumor-bearing mouse brains, indicating that AZD1390 is an attractive molecule for clinical development of brain tumors.

Central Nervous System Delivery of the Catalytic Subunit of DNA-Dependent Protein Kinase Inhibitor Peposertib as Radiosensitizer for Brain Metastases.

Cytotoxic effects of chemotherapy and radiation therapy (RT) used for the treatment of brain metastases results from DNA damage within cancer cells. Cells rely on highly evolved DNA damage response (DDR) pathways to repair the damage caused by these treatments. Inhibiting these repair pathways can further sensitize cancer cells to chemotherapy and RT. The catalytic subunit of DNA-dependent protein kinase, in a complex with Ku80 and Ku70, is a pivotal regulator of the DDR, and peposertib is a potent inhibitor of this catalytic subunit. The characterization of central nervous system (CNS) distributional kinetics of peposertib is critical in establishing a therapeutic index in the setting of brain metastases. Our studies demonstrate that the delivery of peposertib is severely restricted into the CNS as opposed to peripheral organs, by active efflux at the blood-brain barrier (BBB). Peposertib has a low free fraction in the brain and spinal cord, further reducing the active concentration, and distributes to the same degree within different anatomic regions of the brain. However, peposertib is heterogeneously distributed within the metastatic tumor, where its concentration is highest within the tumor core (with disrupted BBB) and substantially lower within the invasive tumor rim (with a relatively intact BBB) and surrounding normal brain. These findings are critical in guiding the potential clinical deployment of peposertib as a radiosensitizing agent for the safe and effective treatment of brain metastases. SIGNIFICANCE STATEMENT: Effective radiosensitization of brain metastases while avoiding toxicity to the surrounding brain is critical in the development of novel radiosensitizers. The central nervous system distribution of peposertib, a potent catalytic subunit of DNA-dependent protein kinase inhibitor, is restricted by active efflux in the normal blood-brain barrier (BBB) but can reach significant concentrations in the tumor core. This finding suggests that peposertib may be an effective radiosensitizer for intracranial tumors with an open BBB, while limited distribution into normal brain will decrease the risk of enhanced radiation injury.

Brain barriers virtual: an interim solution or future opportunity?

BACKGROUND: Scientific conferences are vital communication events for scientists in academia, industry, and government agencies. In the brain barriers research field, several international conferences exist that allow researchers to present data, share knowledge, and discuss novel ideas and concepts. These meetings are critical platforms for researchers to connect and exchange breakthrough findings on a regular basis. Due to the worldwide COVID-19 pandemic, all in-person meetings were canceled in 2020. In response, we launched the Brain Barriers Virtual 2020 (BBV2020) seminar series, the first stand-in virtual event for the brain barriers field, to offer scientists a virtual platform to present their work. Here we report the aggregate attendance information on two in-person meetings compared with BBV2020 and comment on the utility of the virtual platform. METHODS: The BBV2020 seminar series was hosted on a Zoom webinar platform and was free of cost for participants. Using registration- and Zoom-based data from the BBV2020 virtual seminar series and survey data collected from BBV2020 participants, we analyzed attendance trends, global reach, participation based on career stage, and engagement of BBV2020. We compared these data with those from two previous in-person conferences, a BBB meeting held in 2018 and CVB 2019. RESULTS: We found that BBV2020 seminar participation steadily decreased over the course of the series. In contrast, live participation was consistently above 100 attendees and recording views were above 200 views per seminar. We also found that participants valued BBV2020 as a supplement during the COVID-19 pandemic in 2020. Based on one post-BBV2020 survey, the majority of participants indicated that they would prefer in-person meetings but would welcome a virtual component to future in-person meetings. Compared to in-person meetings, BBV2020 enabled participation from a broad range of career stages and was attended by scientists in academic, industry, and government agencies from a wide range of countries worldwide. CONCLUSIONS: Our findings suggest that a virtual event such as the BBV2020 seminar series provides easy access to science for researchers across all career stages around the globe. However, we recognize that limitations exist. Regardless, such a virtual event could be a valuable tool for the brain barriers community to reach and engage scientists worldwide to further grow the brain barriers research field in the future.

The influence of the blood-brain barrier in the treatment of brain tumours.

Brain tumours have a poor prognosis and lack effective treatments. The blood-brain barrier (BBB) represents a major hurdle to drug delivery to brain tumours. In some locations in the tumour, the BBB may be disrupted to form the blood-brain tumour barrier (BBTB). This leaky BBTB enables diagnosis of brain tumours by contrast enhanced magnetic resonance imaging; however, this disruption is heterogeneous throughout the tumour. Thus, relying on the disrupted BBTB for achieving effective drug concentrations in brain tumours has met with little clinical success. Because of this, it would be beneficial to design drugs and drug delivery strategies to overcome the 'normal' BBB to effectively treat the brain tumours. In this review, we discuss the role of BBB/BBTB in brain tumour diagnosis and treatment highlighting the heterogeneity of the BBTB. We also discuss various strategies to improve drug delivery across the BBB/BBTB to treat both primary and metastatic brain tumours. Recognizing that the BBB represents a critical determinant of drug efficacy in central nervous system tumours will allow a more rapid translation from basic science to clinical application. A more complete understanding of the factors, such as BBB-limited drug delivery, that have hindered progress in treating both primary and metastatic brain tumours, is necessary to develop more effective therapies.