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PURPOSE AMLAL-101 is a novel agent which preferentially targets α3, α5 subtypes of ɣ-amino butyric acid receptors and shows anti-tumor activity against disparate cancer types. AMLAL-101 is being advanced as an ‘add-on’ to potentiate treatment of primary and metastatic brain cancers. However, AMLAL-101 must penetrate the blood-brain barrier (BBB) and show sufficient brain retention. The primary purpose of this study was to determine the plasma pharmacokinetics (PK) and quantitative estimate of the BBB permeability of AMLAL-101. METHODS We performed intracranial microdialysis, employing jugular vein cannulated Sprague-Dawley rats which facilitated simultaneous serial blood and brain extracellular fluid (ECF) sampling. AMLAL-101 was injected i.p. at 5 mg/kg and serial blood and brain ECF samples collected up to 10 h post-dosing. Plasma and ECF samples were analyzed by LC/MS-MS and plasma and ECF concentration vs time PK profiles determined. In vivo recovery analysis was performed using retrodialysis and rapid equilibrium dialysis employed to determine the extent of protein binding. RESULTS AMLAL-101 plasma protein binding was 85% and in vivo recovery from ECF was 25%. AMLAL-101 peak concentration (Cmax) in plasma and brain ECF were 15 µM and 13.8 µM, respectively. The plasma and brain ECF area under the concentration (AUC0-10) were 27.5 h.µg/mL and 24.10 h.µg/mL, respectively. The brain partitioning of unbound AMLAL-101 (Kp,uu; determined either as a ratio of brain ECF Cmax:unbound plasma Cmax or brain ECF AUC: unbound plasma AUC), were 6.13 and 4.13, respectively. The elimination half-life of AMLAL-101 was 3 h for both brain ECF and plasma. CONCLUSIONS These results suggest that AMLAL-101 has the requisite BBB permeability required for brain cancer therapeutics. AMLAL-101 shows significant brain retention when compared to a chemically similar agent that does not show anti-cancer activity, which may contribute to efficacy of AMLAL-101 as an anti-tumor agent for treatment of brain cancers.

Standard-of-care for melanoma brain metastases is moving to stereotactic radiosurgery (SRS) with immunotherapy. Although this combined approach shows improved patient survival, the responses are not durable. SRS can also cause adverse reactions such as radiation necrosis, which can result in significant morbidity and even mortality. Lowering SRS dose without negatively impacting its effectiveness would therefore be highly beneficial clinically. Our published data suggests that when treating syngeneic mouse model B16F10 melanoma tumors, the radiation dose can be reduced and its effectiveness can be improved, by activating intrinsic melanoma GABAA receptor (GABAAR) activity using a novel agent, AMLAL-101. This agent acts to enhance GABAAR mediated chloride transport in melanoma cells, which in turn triggers mitochondrial dysregulation associated with tumor regression in mice. While AMLAL-101 alone reduces tumor growth, combining it with radiation results in near complete regression of the irradiated tumor, as well as shrinking of tumor not in the radiation field due to an immune response, i.e., a potent abscopal effect. AMLAL-101 may also function to regulate immune cell infiltration into the tumor microenvironment. Analysis of AMLAL-101 treated tumors show increased infiltration into the tumor microenvironment of: (1) polyfunctional (TNFα/IFN-γ) CD8+ T cells; and (2) tumor-associated macrophages (TAMs) of the tumor inhibitory M1-phenotype, possibly due to IFN-γ stimulation. We posit that activating GABAAR with AMLAL-101 promotes both a ‘direct’ anti-tumor response and an ‘indirect’, immune-driven anti-tumor activity in metastatic melanoma tumors. AMLAL-101 may address an urgent unmet need for a non-toxic therapeutic that can potentiate radiation and mitigate its toxicity profile to positively impact clinical outcomes of patients with brain metastatic disease treated with radiotherapy.

Most advanced-stage non-small cell lung cancer (NSCLC) patients have brain metastases that render a dismal prognosis. Treatment of metastatic brain lesions from NSCLC and other tumor types include radiation as part of a multimodal treatment regimen. Challenges in the application of radiotherapy include overcoming radiation resistance and reducing associated co-morbidities. Non-toxic therapeutics capable of sensitizing tumors to radiation are needed to improve survival and mitigate radiation side-effects. Many CNS and solid systemic tumors express ligand-gated ion channels, which may contribute to tumor growth. Leveraging ion channels is therefore a potential way of diminishing the spread of cancer. We find that NSCLC and its brain metastases express subunits of the type-A GABA-gated chloride channel or GABAA receptor. Importantly, patient-derived NSCLC cells have functional GABAA receptors. We identified a brain penetrant, small molecule activator of GABAA receptors (AMLAL-101), which alone impairs the viability of both primary NSCLC cells and brain metastatic cells. In addition, AMLAL-101 combined with radiation is a highly potent inducer of NSCLC cell death and clonogenic arrest. Using a human ex vivo model of NSCLC-on-chip, we assessed the efficacy and toxicity of AMLAL-101 relative to Docetaxel, an antimicrotubular agent used in treating advanced NSCLC. AMLAL-101 is as potent as Docetaxel but does not exhibit its toxic side effects. AMLAL-101 also potentiates radiation in vivo, significantly reducing lung adenocarcinoma xenograft tumor growth in mice, equivalent to docetaxel plus radiation. Mechanistically, AMLAL-101 activates GABAA receptors in NSCLC and synergizes with radiation by inducing an autophagic response that includes: (i) stabilization of Beclin-1, BNIP3L/NIX, and GABARAP; (ii) ATG7 upregulation; and (iii) utilization of ubiquitin-binding protein p62. Activating GABAA receptors in NSCLC and other tumor types may improve radiation efficacy and mitigate its toxic side effects in treating brain metastases.

PURPOSE Despite significant advances in multimodal approaches for the treatment of glioblastoma (GBM), the outcome for the patients remains dismal. Previous research from our lab has shown that aromatase (CYP19A1), an enzyme that catalyzes in situ production of estrogens, is markedly elevated in GBM tissues. Additionally, letrozole (LTZ), an aromatase inhibitor with extensive record of efficacious use in breast cancer, exhibited significant activity against GBM in preclinical studies. The purpose of this study was to investigate the mechanism(s) of LTZ activity in GBM. METHODS Patient-derived GBM cells with varying aromatase expression and other genotypic/phenotypic characteristics were employed including aromatase expressing G43, G75, G76, BT-142-gfp-luc and aromatase-null G80 cells. The effects of LTZ on cell viability, neurosphere growth, DNA damage (assessed by measuring phosphorylated histone product ỴH2A.X using flow cytometry) and induction of apoptosis (caspase3/7 activity) were assessed. The effects of exogenous addition of estradiol (E2) was also examined. RESULTS LTZ treatment (72 hours) caused a marked reduction in viability and growth of all GBM cells (IC50 ranging from 0.08 to 0.7 µM) except in G80 cells where LTZ had no effects. LTZ treatment also resulted in inhibition of E2 synthesis in a time and dose-dependent fashion. Furthermore, in aromatase expressing cells LTZ treatment induced apoptosis as measured by increase in Caspase-3/7 activity and triggered significant DNA double strand break as determined by ỴH2A.X formation. The addition of exogenous E2 (250 pg/ml) abrogated the effects of LTZ on cell viability, DNA damage and induction of apoptosis. CONCLUSIONS Our study suggests that the cytotoxic effects of LTZ against GBM are mediated, at least partially, by the inhibition of aromatase activity which results in estrogen deprivation. These mechanistic insights are important to facilitate repurposing LTZ as a novel anti-GBM drug.

Glioblastoma multiforme (GBM) is a highly malignant primary brain tumor. The current standard of care for GBM is the Stupp protocol which includes surgical resection, followed by radiotherapy concomitant with the DNA alkylator temozolomide; however, survival under this treatment regimen is an abysmal 12–18 months. New and emerging treatments include the application of a physical device, non-invasive ‘tumor treating fields’ (TTFs), including its concomitant use with standard of care; and varied vaccines and immunotherapeutics being trialed. Some of these approaches have extended life by a few months over standard of care, but in some cases are only available for a minority of GBM patients. Extensive activity is also underway to repurpose and reposition therapeutics for GBM, either alone or in combination with the standard of care. In this review, we present select molecules that target different pathways and are at various stages of clinical translation as case studies to illustrate the rationale for their repurposing-repositioning and potential clinical use.

PURPOSE AMP-activated protein kinase (AMPK) is a molecular hub for cellular metabolic control. Recent evidence suggests that AMPK is a “druggable” novel target for the treatment of Glioblastoma Multiforme (GBM). However, AMPK-inhibitory compounds are largely limited to compound C, which has a poor selectivity profile. SBI-0206965 is a diaminopyrimidine derivative that directly inhibits AMPK with 40-fold greater potency and markedly lower kinase promiscuity than compound C. The current studies provide insights into systemic pharmacokinetics and plasma to brain partitioning of SBI-0206965. METHODS We conducted an intracerebral microdialysis study employing jugular vein-cannulated Sprague Dawley rats (males, 6- 8 weeks). Serial brain extracellular fluid (ECF) and venous blood samples were collected up to 10 hrs following intraperitoneal administration of SBI-0206965 (25 mg/kg). These samples were then quantitated for SBI-0206965 levels using a LC/MS method (Thermo Scientific LTQ-FT™, Ionization: Electrospray Ionization; positive ion). PK analysis was performed using the Non–Compartmental Analysis (Phoenix® WinNonlin 8.2 Certara USA, Inc.). RESULTS Plasma and ECF peak concentrations (Cmax) were 7.15 µM and 0.68 µM, whereas the time to peak (Tmax) were 0.5 and 1 hr, respectively. The plasma and brain ECF elimination half-lives were 1.5 and 3 hours, respectively. Plasma protein binding of SBI-0206965 was 82%. A comparison of the brain ECF Cmax and area under the curve (AUC) to corresponding plasma values suggested that the brain partitioning of the compound was 10-18%. When corrected for unbound fraction in plasma the AUC ratio was 0.86. Thus, these studies show that SBI-0206965 has adequate brain penetration. Further studies are now in progress to assess selectivity of SBI-0206965 for AMPK expressing cell lines, efficacy against patient-derived GBM and PK in tumor-bearing mice. CONCLUSION Results from this study will help to design optimal dosing regimen of SBI-0206965 in our efforts to explore AMPK as a GBM-specific drug target.

Tissue Factor, the initiator of the extrinsic pathway of coagulation, and its isoform alternatively spliced Tissue Factor (asTF) are often overexpressed in cancer cells. Previously, we have shown that RabMab1, a rabbit monoclonal antibody specific for human asTF, disrupts the binding of asTF to beta-integrins and thereby inhibits the growth of breast cancer and pancreatic ductal adenocarcinoma (PDAC) cell lines in vitro and in vivo. Here, we report on humanization of RabMab1, assessment of its binding characteristics, and determination of its in vivo properties. The variable regions of the heavy and light chains of hybridoma derived RabMab1 were cloned and used to generate a rabbit/human chimera (cRabMab1). Substitutions of species-specific residues were then carried out to produce humanized RabMab1 (hRabMab1; IgG1 isotype). Antigen binding was assessed via anti-human IgG Fc capture biosensor assays. cRabMab1 was found to have a KD of 4.24 nM whereas hRabMab1 was found to have a KD in the low picomolar range, which could not be precisely measured due to an extremely slow off-rate. The in vivo half-lives of each were determined to be 280 and 908 hours, respectively. Orthotopic co-implantation of cRabMab1 with high-grade, human PDAC cell line Pt45.P1 in athymic nude mice resulted in tumors that were 82% and 92% smaller than tumors in the vehicle and IgG control group groups, respectively, with no statistical difference between vehicle and IgG isotype control groups. Intravenous administration of hRabMab1 slowed the growth of pre-formed orthotopic Pt45.P1 tumors in athymic nude mice by 64% and 61% compared to vehicle and isotype control groups, respectively; again, there was no difference between vehicle and IgG isotype control groups. Immunohistochemical analysis of tumor tissue revealed a statistically significant 68% reduction in neovascularization (CD31), a 58% reduction in M2-polarized macrophages (CD206), and a 24% reduction in proliferating cells (Ki67) in the hRabMab1 cohort. RNAseq analysis of tumor tissue revealed that components of the focal adhesion system were the most affected by hRabMab1 treatment. In addition, a significant suppression of pathways that promote mitosis and the cell cycle was seen in hRabMab1-treated tumors. By qRT-PCR, we validated a greater than 82% reduction in the expression of HBEGF, a gene encoding a cell surface mitogen, and a greater than 68% reduction in the expression of STN1, a gene encoding a component of the CST-complex, a telomere replication and maintenance structure. This is the first proof-of-concept study whereby a novel biologic that inhibits asTF has been used as a systemically administered single agent, with encouraging results. Because hRabMab1 has a favorable pharmacokinetic profile and is able to suppress PDAC tumor cell growth in vivo, it is an attractive candidate for further clinical development. Citation Format: Clayton S. Lewis, Aniruddha Karve, Kateryna Matiash, Timothy Stone, Jingxing Li, Jordan Wang, Henri Versteeg, Bruce Aronow, Syed Ahmad, Pankaj Desai, Vladimir Bogdanov. Preclinical in vivo characterization of a first-in-class humanized antibody targeting alternatively spliced tissue factor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 908.

Tissue Factor, the initiator of the extrinsic pathway of coagulation, and its isoform alternatively spliced Tissue Factor (asTF) are often overexpressed in cancer cells. While asTF is only minimally procoagulant, it activates both the extracellular mechanical and intracellular signaling functions of beta-integrins. This leads to activation of multiple MAPK pathways thereby promoting increases in proliferation, migration, and angiogenesis. Previously, we have shown that RabMab1, a rabbit monoclonal antibody specific for human asTF, disrupts the binding of asTF to beta-integrins and thereby inhibits the growth of breast cancer and pancreatic ductal adenocarcinoma (PDAC) cell lines in vitro and in vivo. Here, we report on humanization of RabMab1, assessment of its binding characteristics, and determination of its in vivo properties. The variable regions of the heavy and light chains of hybridoma derived RabMab1 were cloned and used to generate a rabbit/human chimera (cRabMab1). Substitutions of species-specific residues were then carried out to produce humanized RabMab1 (hRabMab1; IgG1 isotype). Antigen binding was assessed via anti-human IgG Fc capture biosensor assays. cRabMab1 was found to have a KD of 4.24 nM whereas hRabMab1 was found to have a KD in the low picomolar range, which could not be precisely measured due to an extremely slow off-rate. The in vivo half-lives of each were determined to be 280 and 908 hours, respectively. Orthotopic co-implantation of cRabMab1 with high-grade, human PDAC cell line Pt45.P1 in athymic nude mice resulted in tumors that were 82% and 92% smaller than tumors in the vehicle and IgG control group groups, respectively, with no statistical difference between vehicle and IgG isotype control groups. Intravenous administration of hRabMab1 slowed the growth of pre-formed orthotopic Pt45.P1 tumors in athymic nude mice by 64% and 61% compared to vehicle and isotype control groups, respectively; again, there was no difference between vehicle and IgG isotype control groups. Immunohistochemical analysis of tumor tissue revealed a statistically significant 68% reduction in neovascularization (CD31), a 58% reduction in M2-polarized macrophages (CD206), and a 24% reduction in proliferating cells (Ki67) in the hRabMab1 cohort. RNAseq analysis of tumor tissue revealed a significant suppression of pathways that promote mitosis and the cell cycle. By qRT-PCR, we validated a greater than 82% reduction in the expression of HBEGF, a gene encoding a cell surface mitogen, and a greater than 68% reduction in the expression of STN1, a gene encoding a component of the CST-complex, a telomere replication and maintenance structure. This is the first proof-of-concept study whereby a novel biologic that targets asTF has been used as a systemically administered single agent, with encouraging results. Because hRabMab1 has a favorable pharmacokinetic profile and is able to suppress PDAC tumor growth in vivo, it comprises an attractive candidate for further clinical development. Citation Format: Clayton S. Lewis, Aniruddha Karve, Kateryna Matiash, Timothy Stone, Pankaj Desai, Vladimir Y. Bogdanov. Preclinical in vivo characterization of a first-in-class humanized antibody targeting alternatively spliced Tissue Factor [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2020 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2020;80(22 Suppl):Abstract nr PO-041.