Poor Brain Penetration Research Articles

DDDR-33. TRANSCRIPTOMICS-BASED NOVEL DRUG DISCOVERY IN DIFFUSE MIDLINE GLIOMA

Abstract Introduction. Pediatric high-grade gliomas are the most common cause of cancer-related death in children, of which the most malignant and devastating tumors include diffuse midline glioma (DMG). Recent availability of tumor samples and advances in next-generation sequencing enable the profiling of thousands of molecular features in DMG. We employ a system-based approach that uses gene expression as a representation of the molecular state, providing an avenue for therapeutic discovery. Previously, we used this approach to identify repurposed candidates for DMG; however, the final candidate had poor brain penetration, prompting us to discover novel compounds with better brain penetration. To apply it to novel compound discovery, we need the expression profiles of all library compounds, which is impractical for millions of compounds. To overcome this, we have developed a platform, Gene expression profiles Predictor on chemical Structures (GPS), that incorporates machine learning algorithms which leverage existing drug-induced gene expression profiles to predict gene expression based on compound structure. methods. DMG RNA-Seq samples were acquired from St. Jude Children’s Research Hospital and Children’s Brain Tumor Network (CBTN). We developed deep learning autoencoder to search reference normal tissues from Genotype-Tissue Expression (GTEx). A DMG meta-signature was created and fed into GPS to predict drug candidates from the Enamine CNS library, seven of which were experimentally tested in vitro and the most promising one subsequently validated in vivo. Results. Cell proliferation analyzed by MTS assay showed three compounds with IC50< 50 µM in K27M-mutant DMG cell lines (SF8628, DIPG007, SU-DIPG36). Toxicity study showed no adverse effects in the mice treated with 100-200 mg/kg of the most promising compound (IC50 = 1.9 µM in DMG cells). Conclusion. Our novel computational framework leverages deep learning to fill the gap in drug discovery in DMG. Work is underway to determine antitumor activity, brain penetration (via HPLC) and survival benefit in the mice bearing DMG PDX treated with the lead compound.

Preclinical evaluation of targeted therapies for central nervous system metastases.

The central nervous system (CNS) represents a site of sanctuary for many metastatic tumors when systemic therapies that control the primary tumor cannot effectively penetrate intracranial lesions. Non-small cell lung cancers (NSCLCs) are the most likely of all neoplasms to metastasize to the brain, with up to 60% of patients developing CNS metastases during the disease process. Targeted therapies such as tyrosine kinase inhibitors (TKIs) have helped reduce lung cancer mortality but vary considerably in their capacity to control CNS metastases. The ability of these therapies to effectively target lesions in the CNS depends on several of their pharmacokinetic properties, including blood-brain barrier permeability, affinity for efflux transporters, and binding affinity for both plasma and brain tissue. Despite the existence of numerous preclinical models with which to characterize these properties, many targeted therapies have not been rigorously tested for CNS penetration during the discovery process, whereas some made it through preclinical testing despite poor brain penetration kinetics. Several TKIs have now been engineered with the characteristics of CNS-penetrant drugs, with clinical trials proving these efforts fruitful. This Review outlines the extent and variability of preclinical evidence for the efficacy of NSCLC-targeted therapies, which have been approved by the US Food and Drug Administration (FDA) or are in development, for treating CNS metastases, and how these data correlate with clinical outcomes.

Discovery of BI-9508, a Brain-Penetrant GPR88-Receptor-Agonist Tool Compound for In Vivo Mouse Studies.

Decreased activity and expression of the G-protein coupled receptor GPR88 is linked to many behavior-linked neurological disorders. Published preclinical GPR88 allosteric agonists all have in vivo pharmacokinetic properties that preclude their progression to the clinic, including high lipophilicity and poor brain penetration. Here, we describe our attempts to improve GPR88 agonists' drug-like properties and our analysis of the trade-offs required to successfully target GPR88's allosteric pocket. We discovered two new GPR88 agonists: One that reduced morphine-induced locomotor activity in a murine proof-of-concept study, and the atropoisomeric BI-9508, which is a brain penetrant and has improved pharmacokinetic properties and dosing that recommend it for future in vivo studies in rodents. BI-9508 still suffers from high lipophilicity, and research on this series was halted. Because of its utility as a tool compound, we now offer researchers access to BI-9508 and a negative control free of charge via Boehringer Ingelheim's open innovation portal opnMe.com.

A phase 1, open-label, dose escalation and dose expansion study to evaluate the safety, tolerability, pharmacokinetics, and antitumor activity of PF-07799544 (ARRY-134) as a single agent and in combination with PF-07799933 BRAF dimer inhibitor, in participants 16 years and older with advanced solid tumors.

TPS3180 Background: The MAPK pathway plays a role in several key signaling and phosphorylation events that contribute to tumorigenesis. Inhibition of MEK, along with RAF kinases, has proven to be a successful transformative strategy for melanoma and other MAPK pathway-altered tumors. However, the duration of clinical benefit is limited by a narrow therapeutic index, de novo and acquired resistance and poor brain penetration. PF-07799544 is a next-generation, fully brain penetrant MEK inhibitor. PF-07799933 is an oral selective ATP-competitive small-molecule RAF kinase inhibitor that suppresses BRAF signaling in BRAF V600-mutant and non-V600-BRAF mutant tumors. It displays significantly less paradoxical activation than approved BRAFi and is not “pan RAF” as it spares non-BRAF mutated-containing RAF dimers. We describe here the design of the Phase 1 study of PF-07799544 as Monotherapy or in Combination with a next generation BRAF dimer inhibitor PF-07799933 in participants with BRAF mutated (BRAFm) advanced solid tumors. Methods: The purpose of this first-in-human study is to investigate the safety, tolerability, pharmacokinetics (PK), pharmacodynamics, and potential clinical benefits of PF-07799544 administered as a single agent in participants with advanced solid tumors (Phase 1a) and in combination with PF-07799933 in participants with BRAF Class 1, 2 and 3 mutated solid tumors with or without brain involvement (Phase 1b). Phase 1a will enroll participants with advanced solid tumors who have progressed on standard of care. The primary objective is to determine monotherapy MTD/RDE of PF-07799544. Once PK measurements indicate the potential for significant WT MEK mutation target coverage, participants with untreated and symptomatic brain metastases (bm) will be allowed to enroll. Phase 1b is comprised of multiple sub-studies, all of which evaluate PF-07799544 in combination with PF-07799933 in participants with BRAFm solid tumors who have progressed on standard of care therapies, including Class 1 BRAFi where indicated. Sub-study B: BRAFm melanoma (V600 and non-V600) will determine the MTDc/RDEc of the combination (Part 1 primary objective), followed by dose expansion cohorts (Part 2) (~80 participants): Cohort 1: BRAF V600 melanoma (asymptomatic and untreated bm permitted), Cohort 2: BRAF V600 melanoma (symptomatic bm), Cohort 3 BRAFm Class 2/3 melanoma (asymptomatic and untreated bm permitted); Sub-study C: BRAFm Class 2 or 3 advanced solid tumor Cohort 1 (asymptomatic and untreated bm permitted and Cohort 2 (symptomatic bm) (~20 each cohort). The main objectives of the dose expansion cohorts in all substudies, will be to evaluate anti-tumor activity, safety, PK and PD. Clinical trial information: NCT05538130 .

A Next-Generation BRAF Inhibitor Overcomes Resistance to BRAF Inhibition in Patients with BRAF-Mutant Cancers Using Pharmacokinetics-Informed Dose Escalation.

RAF inhibitors have transformed treatment for patients with BRAFV600-mutant cancers, but clinical benefit is limited by adaptive induction of ERK signaling, genetic alterations that induce BRAFV600 dimerization, and poor brain penetration. Next-generation pan-RAF dimer inhibitors are limited by a narrow therapeutic index. PF-07799933 (ARRY-440) is a brain-penetrant, selective, pan-mutant BRAF inhibitor. PF-07799933 inhibited signaling in vitro, disrupted endogenous mutant-BRAF:wild-type-CRAF dimers, and spared wild-type ERK signaling. PF-07799933 ± binimetinib inhibited growth of mouse xenograft tumors driven by mutant BRAF that functions as dimers and by BRAFV600E with acquired resistance to current RAF inhibitors. We treated patients with treatment-refractory BRAF-mutant solid tumors in a first-in-human clinical trial (NCT05355701) that utilized a novel, flexible, pharmacokinetics-informed dose escalation design that allowed rapid achievement of PF-07799933 efficacious concentrations. PF-07799933 ± binimetinib was well-tolerated and resulted in multiple confirmed responses, systemically and in the brain, in patients with BRAF-mutant cancer who were refractory to approved RAF inhibitors. Significance: PF-07799933 treatment was associated with antitumor activity against BRAFV600- and non-V600-mutant cancers preclinically and in treatment-refractory patients, and PF-07799933 could be safely combined with a MEK inhibitor. The novel, rapid pharmacokinetics (PK)-informed dose escalation design provides a new paradigm for accelerating the testing of next-generation targeted therapies early in clinical development.

Prophylactic treatment with the c-Abl inhibitor, neurotinib, diminishes neuronal damage and the convulsive state in pilocarpine-induced mice

The molecular mechanisms underlying seizure generation remain elusive, yet they are crucial for developing effective treatments for epilepsy. The current study shows that inhibiting c-Abl tyrosine kinase prevents apoptosis, reduces dendritic spine loss, and maintains N-methyl-d-aspartate (NMDA) receptor subunit 2B (NR2B) phosphorylated in invitro models of excitotoxicity. Pilocarpine-induced status epilepticus (SE) in mice promotes c-Abl phosphorylation, and disrupting c-Abl activity leads to fewer seizures, increases latency toward SE, and improved animal survival. Currently, clinically used c-Abl inhibitors are non-selective and have poor brain penetration. The allosteric c-Abl inhibitor, neurotinib, used here has favorable potency, selectivity, pharmacokinetics, and vastly improved brain penetration. Neurotinib-administered mice have fewer seizures and improved survival following pilocarpine-SE induction. Our findings reveal c-Abl kinase activation as a key factor in ictogenesis and highlight the impact of its inhibition in preventing the insurgence of epileptic-like seizures in rodents and humans.

Abstract CT178: Phase 1 trial of PF-07799933 (ARRY-440), a next-generation BRAF inhibitor, for BRAF-mutant cancers

Abstract BACKGROUND Approved BRAF inhibitors (BRAFi) have transformed the treatment of BRAF V600-mutant cancers. However, clinical benefit is limited by BRAF dimer-promoting resistance mutations, toxicity from paradoxical activation of signaling in BRAF wild-type cells, and poor brain penetration. Further, current RAF inhibitors are ineffective against non-V600 BRAF mutants. Next-generation agents that cause pan-RAF inhibition may more broadly target BRAF mutants but are limited by a narrow therapeutic index. PF-07799933 (ARRY-440) is a brain-penetrant BRAF-selective monomer/dimer inhibitor that spares ARAF and CRAF. METHODS We characterized PF-07799933 in patient (pt)-derived BRAF-mutant cancer cells in vitro and in vivo. We investigated PF-07799933 in pts with refractory BRAF-mutant solid tumors using a novel first-in-patient phase 1 design that enabled flexible, rapid dose escalation based on safety and pharmacokinetics (PK) assessments. RESULTS PF-07799933 inhibited signaling in vitro, disrupted BRAF-containing homo- and heterodimers, and caused less paradoxical signaling than approved agents. PF-07799933 alone and in combination with binimetinib inhibited tumor growth systemically and in the brain in mouse xenografts harboring de novo and acquired BRAF dimer-forming mutations. As of August 24, 2023 data cutoff, 30 pts with BRAF-mutant cancers started PF-07799933 treatment at 50 mg QD-450 mg BID as monotherapy (60%) or combination with binimetinib (27%) or cetuximab (all colorectal cancer [CRC]) (13%). Tumor types were melanoma (mel) (43%), CRC (17%), primary brain (PBT) (13%), thyroid (10%), and other (n=1/3% each). BRAF mutations were class I (V600E) (73%), Class II (13%), and class III (13%). All BRAF V600E+ cancer pts were previously treated with ≥1 approved BRAFi and all BRAF V600E+ mel pts also were previously treated with ≥1 immune checkpoint inhibitor. PF-07799933 was well-tolerated as monotherapy or combination. There were no DLTs and the MTD was not reached. Treatment-emergent adverse events (TEAEs) in ≥3 pts for monotherapy were fatigue (any grade 44%/grade ≥3 0%), headache (28%/0%), vision blurred (22%/6%), and lipase increased (16%/0%). The most common TEAEs for combination were peripheral edema (33%/0%), acneiform rash, diarrhea, and fatigue (each 28%/0%). The AE profile was consistent with less paradoxical signaling activation. PK-guided dose escalation enabled PF-07799933 efficacious concentrations to be reached by dose level 3 (225 mg BID), together with multiple confirmed responses in BRAF V600E+ cancer pts (n=4/7 at 225 mg BID ± binimetinib, includes 1 complete response [CR] in a BRAF V600E+ PBT pt) systemically, in the brain, and all after failure of approved BRAF inhibitors. A 5th confirmed response (2nd BRAF V600E+ PBT pt) occurred after data cutoff. This includes, to our knowledge, the first evidence of efficacy in a BRAF V600E+ pt with a dimerizing BRAF splice variant. A BRAF G466E+ (class III) adenoid cystic carcinoma pt had a molecular CR in ctDNA. CONCLUSION PF-07799933 treatment was associated with anti-tumor activity against BRAF V600- and non-V600-mutant cancers preclinically and in treatment-refractory pts. The novel, rapid PK-informed dose escalation design provides a new paradigm for accelerating testing of next-generation targeted therapies early in clinical development. TRIAL REGISTRATION NUMBER NCT05355701 Citation Format: Rona Yaeger, Meredith McKean, Rizwan Haq, Joseph T. Beck, Matthew H. Taylor, Jonathan Cohen, Daniel Bowles, Shirish M. Gadgeel, Catalin Mihalcioiu, Kyriakos P. Papadopoulos, Eli L. Diamond, Keren B. Sturtz, Gang Feng, Stefanie K. Drescher, Micaela B. Reddy, Bhaswati Sengupta, Arnab K. Maity, Suzy A. Brown, Anurag Singh, Eric N. Brown, Brian R. Baer, Jim Wong, Tung-Chung Mou, Wen I. Wu, Dean Kahn, Sunyana Gadal, Neal Rosen, John J. Gaudino, Patrice A. Lee, Dylan P. Hartley, Stephen Michael Rothenberg. Phase 1 trial of PF-07799933 (ARRY-440), a next-generation BRAF inhibitor, for BRAF-mutant cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT178.

Abstract 1658: CFT1946, a potent, selective BRAF V600X mutant-specific degrader demonstrates superior activity as a single agent to clinically approved BRAF inhibitors and standard of care combinations in preclinical models of BRAF V600X melanoma, CRC, NSCLC, and brain metastasis

Abstract Activating mutations in BRAF at residue V600 (typically V600E) lead to dysregulation of the MAPK pathway and occur in approximately 8% of all human cancers including 60% of melanoma, up to 12% of colorectal cancer (CRC), and 4% of NSCLC. Currently approved BRAF inhibitors (BRAFi) are selective for BRAF V600X mutant proteins and are typically used in combination with MEK inhibitors (MEKi) in melanoma and NSCLC, or anti-EGFR antibodies such as cetuximab in CRC. However, their activity is limited by primary or acquired resistance often mediated by RAF dimer-inducing mechanisms. Furthermore, progression of BRAF V600X melanoma after BRAFi/MEKi treatment frequently involves brain metastasis, and currently approved BRAFi have relatively poor brain penetration. CFT1946 is a potent, orally bioavailable, cereblon-based BiDACTM degrader that selectively degrades BRAF V600X mutant protein and is currently under investigation in a Phase I clinical trial. CFT1946 has the potential to overcome limitations of clinically approved BRAFi, as degradation of BRAF V600X should abrogate RAF dimer-driven resistance and paradoxical activation. Indeed, we have previously demonstrated that CFT1946 is efficacious in an A375 BRAF V600E/NRAS Q61K xenograft model of BRAFi resistant melanoma. Here we substantially expand our preclinical characterization of CFT1946 across multiple models of BRAF V600X-driven cancers including BRAF V600X-driven CRC and NSCLC, additional BRAFi-resistant melanoma models, and a brain metastatic melanoma model. Single agent CFT1946 outperformed the SOC encorafenib + cetuximab combination in a panel of BRAF-V600X CRC xenograft models. Pathway analysis in BRAF V600X CRC models revealed that CFT1946 suppresses EGFR-mediated MAPK pathway reactivation, a mechanism known to diminish the impact of approved BRAF inhibitors in this indication. CFT1946 also demonstrated regression in a BRAF-V600X NSCLC PDX model where the SOC dabrafenib + trametinib showed only modest tumor growth inhibition. Consistent with our previous results in the A375 melanoma model, single agent CFT1946 showed superior activity versus the SOC dabrafenib + trametinib combination in all additional melanoma models tested, with complete regression achieved using a CFT1946 + trametinib combination in a PDX model bearing a BRAF-V600E kinase duplication that showed minimal response to dabrafenib + trametinib. Finally, using an A375 intracranial model, treatment with CFT1946 gave robust, dose dependent efficacy and survival advantage over encorafenib. The promising activity of CFT1946 in a broad range of BRAF V600X preclinical models supports its ongoing clinical investigation in BRAF V600 mutant solid tumors (NCT05668585). Citation Format: Bridget Kreger, Jacob R. Stephenson, Mathew E. Sowa, Samantha A. Perino, Laura L. Poling, Eunju Hurh, Michael J. Thomenius, Yanke Liang, Stewart L. Fisher, Roy M. Pollock. CFT1946, a potent, selective BRAF V600X mutant-specific degrader demonstrates superior activity as a single agent to clinically approved BRAF inhibitors and standard of care combinations in preclinical models of BRAF V600X melanoma, CRC, NSCLC, and brain metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1658.

Radiosynthesis and In Vitro Evaluation of [11C]tozadenant as Adenosine A2A Receptor Radioligand.

Tozadenant (4-hydroxy-N-(4-methoxy-7-morpholinobenzo[d]thiazol-2-yl)-4-methylpiperidine-1-carboxamide) is a highly selective adenosine A2A receptor (A2AR) antagonist and a promising lead structure for the development of A2AR-selective positron emission tomography (PET) probes. Although several 18F-labelled tozadenant derivatives showed favorable in vitro properties, recent in vivo PET studies observed poor brain penetration and lower specific binding than anticipated from the in vitro data. While these findings might be attributable to the structural modification associated with 18F-labelling, they could also reflect inherent properties of the parent compound. However, PET studies with radioisotopologues of tozadenant to evaluate its cerebral pharmacokinetics and brain distribution are still lacking. In the present work, we applied N-Boc-O-desmethyltozadenant as a suitable precursor for the preparation of [O-methyl-11C]tozadenant ([11C]tozadenant) by O-methylation with [11C]methyl iodide followed by acidic deprotection. This approach afforded [11C]tozadenant in radiochemical yields of 18 ± 2%, with molar activities of 50-60 GBq/µmol (1300-1600 mCi/µmol) and radiochemical purities of 95 ± 3%. In addition, in vitro autoradiography in pig and rat brain slices demonstrated the expected striatal accumulation pattern and confirmed the A2AR specificity of the radioligand, making it a promising tool for in vivo PET studies on the cerebral pharmacokinetics and brain distribution of tozadenant.

Multifunctional Nanotheranostics for Overcoming the Blood–Brain Barrier

AbstractThe blood–brain barrier (BBB) is a tailored system of capillary endothelial cells intermixed with tight junctions and adherent junctions that regulates the transport of various materials and substances between the blood vasculature and the central nervous system (CNS). However, in cases of brain diseases, BBB's protective and regulatory effects hamper therapeutics from reaching affected sites in sufficient quantities. This has so far been a leading challenge in treating CNS diseases and disorders. For this problem to be overcome, recent research has sought to develop novel modalities to achieve efficient therapy and alleviate associated symptoms. Therefore, numerous strategies have operated in recent years to address the limitations of traditional and invasive methods, including poor brain penetration and serious side effects. As a desperately in‐demand technology, nanotheranostics has particularly shown promising results. Herein, this review reports recent advancements in CNS nanotheranostics and novel techniques and nanotechnology‐based strategies developed for treating neurodegenerative disorders. The study provides comprehensive data on the subject to be used for future studies for the therapy and management of CNS disorders and diseases.

Targeted Hyperbranched Nanoparticles for Delivery of Doxorubicin in Breast Cancer Brain Metastasis.

Breast cancer brain metastases (BM) are associated with a dismal prognosis and very limited treatment options. Standard chemotherapy is challenging in BM patients because the high dosage required for an effective outcome causes unacceptable systemic toxicities, a consequence of poor brain penetration, and a short physiological half-life. Nanomedicines have the potential to circumvent off-target toxicities and factors limiting the efficacy of conventional chemotherapy. The HER3 receptor is commonly expressed in breast cancer BM. Here, we investigate the use of hyperbranched polymers (HBP) functionalized with a HER3 bispecific-antibody fragment for cancer cell-specific targeting and pH-responsive release of doxorubicin (DOX) to selectively deliver and treat BM. We demonstrated that DOX-release from the HBP carrier was controlled, gradual, and greater in endosomal acidic conditions (pH 5.5) relative to physiologic pH (pH 7.4). We showed that the HER3-targeted HBP with DOX payload was HER3-specific and induced cytotoxicity in BT474 breast cancer cells (IC50: 17.6 μg/mL). Therapeutic testing in a BM mouse model showed that HER3-targeted HBP with DOX payload impacted tumor proliferation, reduced tumor size, and prolonged overall survival. HER3-targeted HBP level detected in ex vivo brain samples was 14-fold more than untargeted-HBP. The HBP treatments were well tolerated, with less cardiac and oocyte toxicity compared to free DOX. Taken together, our HER3-targeted HBP nanomedicine has the potential to deliver chemotherapy to BM while reducing chemotherapy-associated toxicities.

Development of a Dihydroquinoline-Pyrazoline GluN2C/2D-Selective Negative Allosteric Modulator of the N-Methyl-d-aspartate Receptor.

Subunit-selective inhibition of N-methyl-d-aspartate receptors (NMDARs) is a promising therapeutic strategy for several neurological disorders, including epilepsy, Alzheimer's and Parkinson's disease, depression, and acute brain injury. We previously described the dihydroquinoline-pyrazoline (DQP) analogue 2a (DQP-26) as a potent NMDAR negative allosteric modulator with selectivity for GluN2C/D over GluN2A/B. However, moderate (<100-fold) subunit selectivity, inadequate cell-membrane permeability, and poor brain penetration complicated the use of 2a as an in vivo probe. In an effort to improve selectivity and the pharmacokinetic profile of the series, we performed additional structure-activity relationship studies of the succinate side chain and investigated the use of prodrugs to mask the pendant carboxylic acid. These efforts led to discovery of the analogue (S)-(-)-2i, also referred to as (S)-(-)-DQP-997-74, which exhibits >100- and >300-fold selectivity for GluN2C- and GluN2D-containing NMDARs (IC50 0.069 and 0.035 μM, respectively) compared to GluN2A- and GluN2B-containing receptors (IC50 5.2 and 16 μM, respectively) and has no effects on AMPA, kainate, or GluN1/GluN3 receptors. Compound (S)-(-)-2i is 5-fold more potent than (S)-2a. In addition, compound 2i shows a time-dependent enhancement of inhibitory actions at GluN2C- and GluN2D-containing NMDARs in the presence of the agonist glutamate, which could attenuate hypersynchronous activity driven by high-frequency excitatory synaptic transmission. Consistent with this finding, compound 2i significantly reduced the number of epileptic events in a murine model of tuberous sclerosis complex (TSC)-induced epilepsy that is associated with upregulation of the GluN2C subunit. Thus, 2i represents a robust tool for the GluN2C/D target validation. Esterification of the succinate carboxylate improved brain penetration, suggesting a strategy for therapeutic development of this series for NMDAR-associated neurological conditions.

Abstract 3415: Preclinical characterization of a brain penetrant RAF inhibitor, BDTX-4933, targeting oncogenic BRAF Class I/II/III and RAS mutations

Abstract Mutations in BRAF and RAS are often oncogenic and lead to a constitutively active MAPK pathway that promotes aberrant cell proliferation and tumor growth. Currently approved BRAF inhibitors are selective against monomeric BRAF V600 mutants. These drugs are largely inactive against non-V600 dimeric BRAF mutants and have poor brain penetration. Although there is an FDA-approved KRAS G12C mutant-selective inhibitor, there are no approved inhibitors for cancer patients who harbor other (non-G12C) KRAS and NRAS mutations which promote tumor growth likely through constitutively active RAF dimers. There remains a high unmet clinical need for a highly CNS penetrant oral RAF inhibitor that targets a broad spectrum of BRAF mutations and constitutively active RAF dimers without paradoxical activation of the MAPK signaling pathway. BDTX-4933 is a potent, reversible, CNS penetrant RAF MasterKey inhibitor designed to target a large family of oncogenic BRAF mutations including BRAF monomers and RAF dimers. The compound inhibits not only all classes (I, II, and III) of BRAF mutations but also targets constitutively active RAF dimers promoted by upstream oncogenic MAPK pathway alterations, such as RAS mutations. In a panel of cancer cell lines that endogenously express BRAF or RAS mutations, BDTX-4933 demonstrates inhibition of the MAPK pathway signaling without paradoxical activation, resulting in potent inhibition of cellular proliferation. BDTX-4933 shows target engagement, inhibiting ERK phosphorylation, in tumor models in vivo, achieving strong anti-tumor efficacy and tumor regression across tumor models driven by either BRAF or RAS mutations. Furthermore, BDTX-4933 exhibits high CNS exposure leading to dose-dependent tumor growth inhibition, and survival benefit in mice implanted intracranially with xenograft BRAF mutant tumors. BDTX-4933 has a best-in-class profile to treat cancer patients harboring BRAF mutations or RAF dimer-promoting upstream genetic alterations. IND-enabling studies for BDTX-4933 are on-going. Citation Format: Yoon-Chi Han, Pui-Yee Ng, Luisa Shin Ogawa, Shao Ning Yang, Miao Chen, Noboru Ishiyama, Tai-An Lin, Elizabeth Buck. Preclinical characterization of a brain penetrant RAF inhibitor, BDTX-4933, targeting oncogenic BRAF Class I/II/III and RAS mutations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3415.

Belvarafenib penetrates the BBB and shows potent antitumor activity in a murine melanoma brain metastasis model.

Brain metastasis is a common complication in melanoma patients with BRAF and NRAS mutations and has a poor prognosis. Although BRAF inhibitors are clinically approved, their poor brain penetration limits their efficacy in brain metastasis. Thus, melanoma brain metastasis still requires better treatment. Belvarafenib, a pan-RAF inhibitor, has reported antitumor activity in melanoma with RAF and RAS mutations in animal models and patients. However, brain permeability and antitumor efficacy on brain metastasis have not been determined. This study confirmed the brain penetration of belvarafenib, the antitumor activity on BRAF and NRAS mutant melanoma, and the efficacy on melanoma within the brain. Belvarafenib strongly suppressed melanoma in BRAF V600E mutant A375SM tumor-bearing mice. It also significantly inhibited tumor growth in NRAS mutant SK-MEL-30 and K1735 tumor-bearing mice and synergized to enhance the antitumor activity combined with cobimetinib or atezolizumab. Belvarafenib was penetrated at considerable levels into the brains of mice and rats following oral administration. The exposure of belvarafenib in the brain was similar to or higher than that in plasma, and this high brain penetration differed significantly from that of other BRAF inhibitors with low brain penetration. Most importantly, belvarafenib strongly reduced tumor burden and markedly improved survival benefits in mice intracranially implanted with A375SM melanoma. These results demonstrated that belvarafenib, which has favorable BBB permeability, and potent antitumor activity on the tumors with BRAF/NRAS mutations, may be a promising therapeutic option for patients with BRAF/NRAS mutant melanoma brain metastasis.

EXTH-09. HSP90 INHIBITION AS A NOVEL THERAPY FOR DIFFUSE INTRINSIC PONTINE GLIOMA (DIPG)

Abstract Diffuse intrinsic pontine glioma (DIPG) is an aggressive pediatric brain tumor. The mean age of onset is 7-9 years with a median survival of 9 months following diagnosis. Histone 3 (H3) mutations (H3K27M) have been identified in approximately 80% of patients, representing an intriguing target, but how to do this is unclear. To address this, we performed a synthetic lethality drug screen of over 2400 compounds on isogenic cells expressing H3K27M and empty vector. 37 drugs were synthetically lethal with H3K27M, including HSP90 inhibitors. In cancer, HSP90 has a higher isoelectric point which can be targeted specifically by PU-H71. Using PU-H71, HSP90 complexed with oncogenic clients can be inhibited to reduce tumor cell viability. We tested PU-H71 on primary patient derived DIPG lines demonstrating caspase-3/7 mediated cell death within 24 hours (n=3) and an IC50 of 100-200 nM (n=5) at 72 hours. HSP90 is a molecular chaperone that supports protein activation and localization; thus, its inhibition may allow simultaneous targeting of multiple oncogenic pathways. To determine which pathways are targeted in DIPG we used PU-H71-conjugated beads to pull-down HSP90 complexed with client proteins. LC-MS/MS characterization of the HSP90 interactome from three DIPG cell lines (SU-DIPG XVII, SU-DIPGXXV, SU-DIPG 50) identified 339 overlapping proteins including MAPK3, SMS, SRM, CTSP1, OAT, GMPS, and PYG. Pathway enrichment analysis highlighted roles in cell cycle regulation and metabolic processes. Treating DIPG with PU-H71 successfully reduced tumor cell viability but PU-H71 has poor brain penetration. To overcome this, a molecular isoform of PU-H71 has been developed, PU-HZ151, which will be used to test the in vivo efficacy of HSP90 inhibition for DIPG.

Soluble guanylate cyclase stimulator riociguat improves spatial memory in mice via peripheral mechanisms

Soluble guanylate cyclase (sGC) - cyclic guanosine monophosphate (cGMP) signalling is important for healthy memory function and a healthy vascular system. Targeting sGC-cGMP signalling can therefore be a potential strategy to enhance memory processes. sGC can be targeted by using agonists, such as sGC stimulator riociguat. Therefore, this study aimed to target sGC using riociguat to investigate its acute effects on memory function and neuronal plasticity in mice. The effects of riociguat on long-term memory and a biperiden-induced memory deficit model for assessing short-term memory were tested in the object location task, and working memory was tested in the Y-maze continuous alternation task. Pharmacokinetic measurements were performed within brain tissue of mice, and hippocampal plasticity measures were assessed using western blotting. Acute oral administration with a low dose of 0.03 mg/kg riociguat was able to enhance working-, short-, and long-term spatial memory. Under cerebral vasoconstriction higher doses of riociguat were still effective on memory. Pharmacokinetic measurements revealed poor brain penetration of riociguat and its metabolite M−1. Increased activation of VASP was found, while no effects were found on other memory-related hippocampal plasticity measures. Memory enhancing effects of riociguat are most likely regulated by vascular peripheral effects on cGMP signalling. Yet, further research is needed to investigate the possible contribution of hemodynamic or metabolic effects of sGC stimulators on memory performance.

ApoE-mediated systemic nanodelivery of granzyme B and CpG for enhanced glioma immunotherapy.

The response of malignant glioma to immunotherapy remains gloomy due to its discrete immunological environment and poor brain penetration of immunotherapeutic agents. Here, we disclose that ApoE peptide-mediated systemic nanodelivery of granzyme B (GrB) and CpG ODN co-stimulates enhanced immunotherapy of murine malignant LCPN glioma model. ApoE peptide-functionalized polymersomes encapsulating GrB (ApoE-PS-GrB) could effectively penetrate the blood-brain barrier-mimicking endothelial cell monolayer in vitro and further be taken up by LCPN cells, inducing strong immunogenic cell death (ICD). The co-administration of ApoE-PS-GrB and ApoE-PS-CpG in orthotopic LCPN glioma-bearing mice co-stimulated cytokine production, maturation of dendritic cells (DCs), infiltration of cytotoxic T lymphocytes (CTLs) while reduction of regulatory T lymphocytes (Treg) and M2 phenotype macrophages in the tumor microenvironment, leading to greatly delayed tumor progression and significantly prolonged survival time compared with all controls. The ApoE-mediated systemic nanodelivery of GrB and CpG ODN opens a new pathway for potent immunotherapy of malignant glioma.

Olivetolic acid, a cannabinoid precursor in Cannabis sativa, but not CBGA methyl ester exhibits a modest anticonvulsant effect in a mouse model of Dravet syndrome

ObjectiveCannabigerolic acid (CBGA), a precursor cannabinoid in Cannabis sativa, has recently been found to have anticonvulsant properties in the Scn1a+/- mouse model of Dravet syndrome. Poor brain penetration and chemical instability of CBGA limits its potential as an anticonvulsant therapy. Here, we examined whether CBGA methyl ester, a more stable analogue of CBGA, might have superior pharmacokinetic and anticonvulsant properties. In addition, we examined whether olivetolic acid, the biosynthetic precursor to CBGA with a truncated (des-geranyl) form, might possess minimum structural requirements for anticonvulsant activity. We also examined whether olivetolic acid and CBGA methyl ester retain activity at the epilepsy-relevant drug targets of CBGA: G-protein-coupled receptor 55 (GPR55) and T-type calcium channels.MethodsThe brain and plasma pharmacokinetic profiles of CBGA methyl ester and olivetolic acid were examined following 10 mg/kg intraperitoneal (i.p.) administration in mice (n = 4). The anticonvulsant potential of each was examined in male and female Scn1a+/- mice (n = 17–19) against hyperthermia-induced seizures (10–100 mg/kg, i.p.). CBGA methyl ester and olivetolic acid were also screened in vitro against T-type calcium channels and GPR55 using intracellular calcium and ERK phosphorylation assays, respectively.ResultsCBGA methyl ester exhibited relatively limited brain penetration (13%), although somewhat superior to that of 2% for CBGA. No anticonvulsant effects were observed against thermally induced seizures in Scn1a+/- mice. Olivetolic acid also showed poor brain penetration (1%) but had a modest anticonvulsant effect in Scn1a+/- mice increasing the thermally induced seizure temperature threshold by approximately 0.4°C at a dose of 100 mg/kg. Neither CBGA methyl ester nor olivetolic acid displayed pharmacological activity at GPR55 or T-type calcium channels.ConclusionsOlivetolic acid displayed modest anticonvulsant activity against hyperthermia-induced seizures in the Scn1a+/- mouse model of Dravet syndrome despite poor brain penetration. The effect was, however, comparable to the known anticonvulsant cannabinoid cannabidiol in this model. Future studies could explore the anticonvulsant mechanism(s) of action of olivetolic acid and examine whether its anticonvulsant effect extends to other seizure types.

Dendrimer-2PMPA selectively blocks upregulated microglial GCPII activity and improves cognition in a mouse model of multiple sclerosis.

Cognitive impairment is a common aspect of multiple sclerosis (MS) for which there are no treatments. Reduced brain N-acetylaspartylglutamate (NAAG) levels are linked to impaired cognition in various neurological diseases, including MS. NAAG levels are regulated by glutamate carboxypeptidase II (GCPII), which hydrolyzes the neuropeptide to N-acetyl-aspartate and glutamate. GCPII activity is upregulated multifold in microglia following neuroinflammation. Although several GCPII inhibitors, such as 2-PMPA, elevate brain NAAG levels and restore cognitive function in preclinical studies when given at high systemic doses or via direct brain injection, none are clinically available due to poor bioavailability and limited brain penetration. Hydroxyl-dendrimers have been successfully used to selectively deliver drugs to activated glia.Methods: We attached 2-PMPA to hydroxyl polyamidoamine (PAMAM) dendrimers (D-2PMPA) using a click chemistry approach. Cy5-labelled-D-2PMPA was used to visualize selective glial uptake in vitro and in vivo. D-2PMPA was evaluated for anti-inflammatory effects in LPS-treated glial cultures. In experimental autoimmune encephalomyelitis (EAE)-immunized mice, D-2PMPA was dosed biweekly starting at disease onset and cognition was assessed using the Barnes maze, and GCPII activity was measured in CD11b+ hippocampal cells.Results: D-2PMPA showed preferential uptake into microglia and robust anti-inflammatory activity, including elevations in NAAG, TGFβ, and mGluR3 in glial cultures. D-2PMPA significantly improved cognition in EAE mice, even though physical severity was unaffected. GCPII activity increased >20-fold in CD11b+ cells from EAE mice, which was significantly mitigated by D-2PMPA treatment.Conclusions: Hydroxyl dendrimers facilitate targeted drug delivery to activated microglia. These data support further development of D-2PMPA to attenuate elevated microglial GCPII activity and treat cognitive impairment in MS.

Macrocyclic Immunoproteasome Inhibitors as a Potential Therapy for Alzheimer's Disease.

Previously, we reported that immunoproteasome (iP)-targeting linear peptide epoxyketones improve cognitive function in mouse models of Alzheimer's disease (AD) in a manner independent of amyloid β. However, these compounds' clinical prospect for AD is limited due to potential issues, such as poor brain penetration and metabolic instability. Here, we report the development of iP-selective macrocyclic peptide epoxyketones prepared by a ring-closing metathesis reaction between two terminal alkenes attached at the P2 and P3/P4 positions of linear counterparts. We show that a lead macrocyclic compound DB-60 (20) effectively inhibits the catalytic activity of iP in ABCB1-overexpressing cells (IC50: 105 nM) and has metabolic stability superior to its linear counterpart. DB-60 (20) also lowered the serum levels of IL-1α and ameliorated cognitive deficits in Tg2576 mice. The results collectively suggest that macrocyclic peptide epoxyketones have improved CNS drug properties than their linear counterparts and offer promising potential as an AD drug candidate.