Poor Blood-brain Barrier Penetration Research Articles

Blood-brain barrier permeable carbon nano-assemblies for amyloid-β clearance and neurotoxic attenuation

Abnormal amyloid β-protein (Aβ42) fibrillation is a key event in Alzheimer's disease (AD), and photodynamic therapy (PDT) possesses great potential in modulating Aβ42 self-assembly. However, the poor blood-brain barrier (BBB) penetration, low biocompatibility, and limited tissue penetration depth of existing photosensitizers limit the progress of photo-oxidation strategies. In this paper, novel indocyanine green-modified graphene quantum dot nano-assemblies (NBGQDs-ICGs) were synthesized based on a molecular assembly strategy of electrostatic interactions for PDT inhibition of Aβ42 self-assembly process and decomposition of preformed fibrils under near-infrared light. Combining the small-size structure of graphene quantum dots and the near-infrared light-responsive properties of ICGs, the NBGQDs-ICGs could achieve BBB penetration under 808nm irradiation. More importantly, the neuroprotective mechanism of NBGQDs-ICG was studied for the first time by AFM, which effectively weakened the adhesion of Aβ42 aggregates to the cell surface by blocking the interaction between Aβ42 and the cell membrane, and restored the mechanical stability and adhesion of the neuron membrane. Meanwhile, NBGQDs-ICG promoted phagocytosis of Aβ42 by microglia. In addition, the good biocompatibility and stability ensured the biosafety of NBGQDs-ICG in future clinical applications. We anticipate that such multifunctional nanocomponents may provide promising avenues for the development of novel AD inhibitors.

Exploring How Antibody Format Drives Clearance from the Brain.

Monoclonal antibodies (mAbs) have high binding specificity and affinity, making them attractive for treating brain diseases. However, their effectiveness is limited by poor blood-brain barrier (BBB) penetration and rapid central nervous system (CNS) clearance. Our group identified blood-brain barrier modulator (BBBM) peptides that improved mAb penetration across the BBB into the brain. In this study, we investigated the pharmacokinetics of a mAb delivered to the brain using BBBMs after intravenous (IV) administration and explored the impact of antibody format (size, neonatal Fc receptor (FcRn) binding, hyaluronic acid binding) on brain clearance following direct injection into the central nervous system (CNS) via intracerebroventricular (ICV) injection. IRDye800CW-labeled antibodies were administered into C57BL/6 mice via ICV or IV injection, and organ concentrations were measured after various time points. When a mAb was coadministered with a BBBM peptide, the permeation of mAb across the BBB was increased compared to mAb alone at early time points; however, the mAb was cleared within 2 h from the brain. ICV experiments revealed that an antibody Fab fragment had a higher brain exposure than a mAb, and that a Fab fused to a hyaluronic acid binding domain (Fab-VG1) showed remarkable improvement in brain exposure. These findings suggest that BBBMs and antibody format optimization may be promising strategies for enhancing brain retention of therapeutic antibodies.

Prolonged complete response to adjuvant tepotinib in a patient with newly diagnosed disseminated glioblastoma harboring mesenchymal-epithelial transition fusion.

The prognosis of patients with glioblastoma (GBM) remains poor despite current treatments. Targeted therapy in GBM has been the subject of intense investigation but has not been successful in clinical trials. The reasons for the failure of targeted therapy in GBM are multifold and include a lack of patient selection in trials, the failure to identify driver mutations, and poor blood-brain barrier penetration of investigational drugs. Here, we describe a case of a durable complete response in a newly diagnosed patient with GBM with leptomeningeal dissemination and PTPRZ1-MET fusion who was treated with tepotinib, a brain-penetrant MET inhibitor. This case of successful targeted therapy in a patient with GBM demonstrates that early molecular testing, identification of driver molecular alterations, and treatment with brain-penetrant small molecule inhibitors have the potential to change the outcome in select patients with GBM.

4-Amidophenol Quinone Methide Precursors: Effective and Broad-Scope Nonoxime Reactivators of Organophosphorus-Inhibited Cholinesterases and Resurrectors of Organophosphorus-Aged Acetylcholinesterase.

Acetylcholinesterase (AChE) inhibition by organophosphorus (OP) compounds poses a serious health risk to humans. While many therapeutics have been tested for treatment after OP exposure, there is still a need for efficient reactivation against all kinds of OP compounds, and current oxime therapeutics have poor blood-brain barrier penetration into the central nervous system, while offering no recovery in activity from the OP-aged forms of AChE. Herein, we report a novel library of 4-amidophenol quinone methide precursors (QMP) that provide effective reactivation against multiple OP-inhibited forms of AChE in addition to resurrecting the aged form of AChE after exposure to a pesticide or some phosphoramidates. Furthermore, these QMP compounds also reactivate OP-inhibited butyrylcholinesterase (BChE) which is an in vivo, endogenous scavenger of OP compounds. The in vitro efficacies of these QMP compounds were tested for reactivation and resurrection of soluble forms of human AChE and BChE and for reactivation of cholinesterases within human blood as well as blood and brain samples from a humanized mouse model. We identify compound 10c as a lead candidate due to its broad-scope efficacy against multiple OP compounds as well as both cholinesterases. With methylphosphonates, compound 10c (250 μM, 1 h) shows >60% recovered activity from OEt-inhibited AChE in human blood as well as mouse blood and brain, thus highlighting its potential for future in vivo analysis. For 10c, the effective concentration (EC50) is less than 25 μM for reactivation of three different methylphosphonate-inhibited forms of AChE, with a maximum reactivation yield above 80%. Similarly, for OP-inhibited BChE, 10c has EC50 values that are less than 150 μM for two different methylphosphonate compounds. Furthermore, an in vitro kinetic analysis show that 10c has a 2.2- and 92.1-fold superior reactivation efficiency against OEt-inhibited and OiBu-inhibited AChE, respectively, when compared to an oxime control. In addition to 10c being a potent reactivator of AChE and BChE, we also show that 10c is capable of resurrecting (ethyl paraoxon)-aged AChE, which is another current limitation of oximes.

296 Towards Nose-to-brain Inhibition of NFB: Activity of a Novel p65 Inhibitor in Glioblastoma and Feasibility of Intranasal Delivery With a Near-infrared Fluorescent HMC-Chitosan Nanoparticle

INTRODUCTION: Constitutive NFκB inflammatory signaling is a driving force of tumor progression, immune evasion, and therapeutic resistance in glioblastoma (GBM). CRL1101 is a novel small molecule inhibitor of RelA (p65), a transcription factor of NFκB-mediated gene expression. Previously, we have shown that heptamethine carbocyanine (HMC) conjugated nanoparticles target GBM tumor sites via organic anion transporter proteins. The activity of CRL1101 in GBM has not been explored. Poor blood-brain barrier (BBB) penetration and theoretical toxicities related to p65 inhibition pose challenges to its implementation in vivo. METHODS: GBM transcriptomic analysis was performed using Gene Profiling Interactive Analysis. In vitro cytotoxicity and migration assays were performed in U87 and GL261. Western blot was used to confirm the mechanism of CRL1101. Quantitative PCR was used to assess the relative expression of NFκB-regulated genes. HMC-chitosan-CRL1101 nanoparticles were synthesized by cross-linking chitosan with HMC and their uptake was visualized using immunocytochemistry. For survival analysis, we implanted GL261 cells into immunocompetent mice and treated intranasally with HMC-chitosan-CRL1101. RESULTS: RelA is overexpressed in GBM relative to normal brain tissue. CRL1101 alone effectively kills U87 cells (IC50 = 16.0 uM) and prevents migration in vitro (p < 0.0001) by disabling the nuclear translocation of RelA. Treatment with CRL1101 was associated with a decrease in IL-8 (p = 0.01) and PD-L1 (p = 0.44) expression. HMC-chitosan-CRL1101 demonstrated an IC50 of 25.23 uM in U87. Near-infrared imaging confirmed intranasal delivery of HMC-chitosan-CRL1101 across the BBB in vivo. CONCLUSIONS: RelA-targeted NFκB inhibition with CRL1101 is a promising immunotherapeutic strategy in GBM. Intranasal nanoparticle delivery is a practical route to bypass the BBB and minimize systemic toxicities in mouse models of GBM, with expected survival benefit in vivo.

Abstract 5702: BTX-9341, a bifunctional degrader of CDK4 and CDK6 for glioblastoma multiforme

Abstract Glioblastoma multiforme (GBM) is an aggressive form of brain cancer with limited treatment options. GBM has a high frequency of dysregulation of the CDKN2A-cyclin D-CDK4/CDK6 signaling node and should subsequently be sensitive to CDK4/6 inhibition. Despite this, the CDK4/6 inhibitors that are approved for HR+/HER2- breast cancer have shown limited efficacy in GBM due to poor blood brain barrier (BBB) penetration. BTX-9341 is a Cereblon (CRBN) mediated CDK4/6 bifunctional degrader that we have developed for HR+/HER2- breast cancer. This degrader shows good exposure in brain tissues with a high brain to plasma ratio. Given the exposure in the brain, we explored the in vitro and in vivo efficacy of BTX-9341 in GBM cell line and xenograft models. GBM cell lines treated with BTX-9341 showed up to 86% degradation of CDK4 and CDK6 with DC50s <1nM. CDK4/6 phosphorylates the protein RB which releases the transcription factor E2F, inducing the expression of genes which promote cell cycle progression. We examined RB phosphorylation by immunoblot and cell cycle progression by propidium iodide staining followed by flow cytometry. BTX-9341 was potent in all downstream assays, with phospho-RB IC50s <10nM, and G0/G1 cell cycle arrest at concentrations as low as 10nM. We used a 2D colony formation assay (CFA) to assess inhibition of proliferation by cell cycle arrest. BTX-9341 potently inhibited cell proliferation with CFA IC50s of 13-100nM in GBM cell lines. BTX-9341 displays excellent pharmacokinetic properties with good oral bioavailability and high brain to plasma ratios which allowed for oral dosing in both subcutaneous and orthotopic xenograft studies. We examined tumor growth inhibition efficacy in two GBM xenograft models: an intracranial U87 model and a subcutaneous U118 model and saw robust tumor growth inhibition in both models with BTX-9341 treatment. The U118 cells have partially unmethylated MGMT, which lead to a loss of efficacy of temozolomide over time, while BTX-9341 maintained inhibition of tumor growth. This indicates that BTX-9341 may be effective in patient populations for which temozolomide is ineffective. The U87 intracranial xenograft model also showed dose-dependent tumor growth inhibition upon treatment with BTX-9341, indicating that BTX-9341 can cross the BBB and reach concentrations high enough to execute cell proliferation inhibition effects. BTX-9341 treated mice also had much better survival than abemaciclib treated mice, the only CDK4/6i which has BBB penetration. These results show that BTX-9341 displays excellent single agent activity in vitro and in vivo in GBM models. The xenograft data indicates that BTX-9341 can inhibit growth of MGMT methylated and unmethylated GBM cell lines, and that BTX-9341 can inhibit tumor growth in the brain more effectively than BBB penetrant CDK4/6i. Together this data shows that BTX-9341 may be a promising candidate for treating GBM. Citation Format: Hannah Majeski, Kirti Chahal, Akinori Okano, Angela Pasis, Casey Carlson, Arvind Shakya, Qiao Liu, Shenlin Huang, Aparajita Hoskote Chourasia, Leah Fung. BTX-9341, a bifunctional degrader of CDK4 and CDK6 for glioblastoma multiforme [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 5702.

Efficacy of a Combination Therapy with Laronidase and Genistein in Treating Mucopolysaccharidosis Type I in a Mouse Model.

Mucopolysaccharidosis type I (MPS I) is a lysosomal storage disorder caused by α-L-iduronidase deficiency. The standard treatment, enzyme replacement therapy with laronidase, has limited effectiveness in treating neurological symptoms due to poor blood-brain barrier penetration. An alternative is substrate reduction therapy using molecules, such as genistein, which crosses this barrier. This study evaluated the effectiveness of a combination of laronidase and genistein in a mouse model of MPS I. Over 12 weeks, MPS I and wild-type mice received laronidase, genistein, or both. Glycosaminoglycan (GAG) storage in visceral organs and the brain, its excretion in urine, and the serum level of the heparin cofactor II-thrombin (HCII-T) complex, along with behavior, were assessed. The combination therapy resulted in reduced GAG storage in the heart and liver, whereas genistein alone reduced the brain GAG storage. Laronidase and combination therapy decreased liver and spleen weights and significantly reduced GAG excretion in the urine. However, this therapy negated some laronidase benefits in the HCII-T levels. Importantly, the combination therapy improved the behavior of female mice with MPS I. These findings offer valuable insights for future research to optimize MPS I treatments.

Development and validation of UV–Vis spectrophotometry-colorimetric method for the specific quantification of rivastigmine tartrate from separable effervescent microneedles: Ex vivo and in vivo applications in complex biological matrices

Rivastigmine Tartrate is an acetylcholinesterase inhibitor commonly used in Alzheimer treatment. Due to its hydrophilicity, short plasma elimination half-time, and poor blood-brain barrier penetration, developing a new drug delivery system for RT administration is essential. Accordingly, microneedles offer several advantages and could be a promising drug delivery system for RT administration. One of those types of MNs with numerous advantages is separable-effervescent microneedles. To support the formulation development, an analytical method using UV–Vis spectrophotometry was validated in phosphate buffered saline media and various biological matrices, such as rat skin, brain, and plasma to measure the amount of RT in the SEMNs preparations. The developed analytical method was validated under ICH guidelines. The results showed that the instrument could specifically detect the RT in the preparations without being interfered with by other compounds. The calibration curves in various biological matrices were linear ≥ 0.9985. The LOD and LLOQ values of the PBS media biological matrices were found to be 1.5 and 4.8 µg/mL, 1.2 and 3.7 µg/mL, 0.42 and 1.27 µg/mL, and 0.38 and 1.16 µg/mL, respectively. All the accuracy and precision values of this developed method were within acceptable ranges. Additionally, the extraction recovery was prominent after undergoing the protein precipitation process. As a result, the analytical method was proven to be valid and was successfully applied to quantify the amount of RT in the SEMNs formulation, in vitro and ex vivo permeation, along with in vivo pharmacokinetic studies.

PRMT5 inhibition shows in vitro efficacy against H3K27M-altered diffuse midline glioma, but does not extend survival in vivo

H3K27-altered Diffuse Midline Glioma (DMG) is a universally fatal paediatric brainstem tumour. The prevalent driver mutation H3K27M creates a unique epigenetic landscape that may also establish therapeutic vulnerabilities to epigenetic inhibitors. However, while HDAC, EZH2 and BET inhibitors have proven somewhat effective in pre-clinical models, none have translated into clinical benefit due to either poor blood–brain barrier penetration, lack of efficacy or toxicity. Thus, there remains an urgent need for new DMG treatments. Here, we performed wider screening of an epigenetic inhibitor library and identified inhibitors of protein arginine methyltransferases (PRMTs) among the top hits reducing DMG cell viability. Two of the most effective inhibitors, LLY-283 and GSK591, were targeted against PRMT5 using distinct binding mechanisms and reduced the viability of a subset of DMG cells expressing wild-type TP53 and mutant ACVR1. RNA-sequencing and phenotypic analyses revealed that LLY-283 could reduce the viability, clonogenicity and invasion of DMG cells in vitro, representing three clinically important phenotypes, but failed to prolong survival in an orthotopic xenograft model. Together, these data show the challenges of DMG treatment and highlight PRMT5 inhibitors for consideration in future studies of combination treatments.

The Effect of Levofloxacin Addition on the Outcome of Tuberculous Meningitis Patients: A Systematic Review and Meta-Analysis

Background: Tuberculous meningitis (TBM) is one of tuberculosis's deadliest extra-pulmonary manifestations. Although early initiation of anti-tuberculosis drugs can reduce mortality and morbidity, poor blood-brain barrier penetration hampered their effectiveness. Levofloxacin is an anti-tuberculosis drug with good BBB penetration. Objective: We aim to explore whether levofloxacin addition to the TBM patients’ regimen has a potential benefit to improve their outcomes. Methods: The literature search was done on PubMed, Google Scholar, and ProQuest databases without publication date limits to identify studies investigating the effect of augmenting levofloxacin in the outcome of TBM patients. The primary outcome of this study was to analyze the impact of these regimens in decreasing the risk of death and neurological deficit. The articles were collected using the PRISMA diagram, critically appraised using PICO analysis, then the data were analyzed using Review Manager 5.4.1 software with a Fixed Effect Model. The results were expressed as odds ratio (OR). Results: Four randomized controlled trials with a total of 930 patients were identified. Two trials compared the effectiveness of levofloxacin addition only, whereas the other two used the regimen containing the increased dose of rifampicin alongside levofloxacin addition. Based on the analysis, neither levofloxacin addition only nor increasing rifampicin dose with levofloxacin addition had a significant impact on the mortality of TBM patients (OR=0.55; 95% CI 0.19-1.59; p=0.27, OR=1.01; 95% CI 0.74-1.36; p=0.97, respectively). Conclusion: Additional Levofloxacin on TBM treatment shows no significant improvement in patient mortality. Routine levofloxacin use in TBM is discouraged due to limited evidence.

Site-oriented conjugation of poly(2-methacryloyloxyethyl phosphorylcholine) for enhanced brain delivery of antibody.

Antibody therapeutics are limited in treating brain diseases due to poor blood-brain barrier (BBB) penetration. We have discovered that poly 2-methacryloyloxyethyl phosphorylcholine (PMPC), a biocompatible polymer, effectively facilitates BBB penetration via receptor-mediated transcytosis and have developed a PMPC-shell-based platform for brain delivery of therapeutic antibodies, termed nanocapsule. Yet, the platform results in functional loss of antibodies due to epitope masking by the PMPC polymer network, which necessitates the incorporation of a targeting moiety and degradable crosslinker to enable on-site antibody release. In this study, we developed a novel platform based on site-oriented conjugation of PMPC to the antibody, allowing it to maintain key functionalities of the original antibody. With an optimized PMPC chain length, the PMPC-antibody conjugate exhibited enhanced brain delivery while retaining epitope recognition, cellular internalization, and antibody-dependent cellular phagocytic activity. This simple formula incorporates only the antibody and PMPC without requiring additional components, thereby addressing the issues of the nanocapsule platform and paving the way for PMPC-based brain delivery strategies for antibodies.

Structure-based design of nanobodies that inhibit seeding of Alzheimer’s patient–extracted tau fibrils

Despite much effort, antibody therapies for Alzheimer's disease (AD) have shown limited efficacy. Challenges to the rational design of effective antibodies include the difficulty of achieving specific affinity to critical targets, poor expression, and antibody aggregation caused by buried charges and unstructured loops. To overcome these challenges, we grafted previously determined sequences of fibril-capping amyloid inhibitors onto a camel heavy chain antibody scaffold. These sequences were designed to cap fibrils of tau, known to form the neurofibrillary tangles of AD, thereby preventing fibril elongation. The nanobodies grafted with capping inhibitors blocked tau aggregation in biosensor cells seeded with postmortem brain extracts from AD and progressive supranuclear palsy (PSP) patients. The tau capping nanobody inhibitors also blocked seeding by recombinant tau oligomers. Another challenge to the design of effective antibodies is their poor blood-brain barrier (BBB) penetration. In this study, we also designed a bispecific nanobody composed of a nanobody that targets a receptor on the BBB and a tau capping nanobody inhibitor, conjoined by a flexible linker. We provide evidence that the bispecific nanobody improved BBB penetration over the tau capping inhibitor alone after intravenous administration in mice. Our results suggest that the design of synthetic antibodies that target sequences that drive protein aggregation may be a promising approach to inhibit the prion-like seeding of tau and other proteins involved in AD and related proteinopathies.

Therapeutic avenues for targeting treatment challenges of diffuse midline gliomas

Diffuse midline glioma (DMG) is the leading cause of brain tumor-related deaths in children. DMG typically presents with variable neurologic symptoms between ages 3 and 10. Currently, radiation remains the standard therapy for DMG to halt progression and reduce tumor bulk to minimize symptoms. However, tumors recur in almost 100% of patients and thus, DMG is still considered an incurable cancer with a median survival of 9-12 months. Surgery is generally contraindicated due to the delicate organization of the brainstem, where DMG is located. Despite extensive research efforts, no chemotherapeutic agents, immune therapies, or molecularly targeted therapies have been approved to provide survival benefit. Furthermore, the efficacy of therapies is limited by poor blood-brain barrier penetration and inherent resistance mechanisms of the tumor. However, novel drug delivery approaches, along with recent advances in molecularly targeted therapies and immunotherapies, have advanced to clinical trials and may provide viable future treatment options for DMG patients. This review seeks to evaluate current therapeutics at the preclinical stage and those that have advanced to clinical trials and to discuss the challenges of drug delivery and inherent resistance to these therapies.

Abstract 6427: Selective-HDAC inhibitor relieves the suppressive immune microenvironment in the brain to impede breast cancer brain metastasis

Abstract Brain metastasis (BrM) incidence is rising due to longer survival resulted from better systemic control of cancer, counting for approximately 20% of patients with cancers. Major cancer types, including lung, breast, and skin cancers, have a high rate to develop BrM with a 1-year survival rate of less than 20% for symptomatic patients. The overall response rate for immune checkpoint inhibitors (ICI) is merely 10-30% in patients with neurological symptoms. Developing novel combinatorial therapeutic strategies based on mechanistic understanding is urgently needed to further improve the efficacy of current immunotherapies in BrM patients. We recently found that BrMs in patients frequently have severe MHC-I loss. BrM patients' scRNA-seq data from available datasets show that MHC-I expression level is negatively correlated with HDAC level in breast cancer brain metastasis. On the other hand, HDAC inhibitors (HDACi) are epigenetic modulators which have been indicated to recover MHC-I expression loss that contributes to immune invasion. HDACi has been indicated to enhance ICI efficacy in primary tumors by reprogramming the tumor microenvironment (TME). However, the immunomodulatory effect of HDACi is unclear in the unique brain immune microenvironment (BrIME), which is characterized as immunosuppressive. Here, we report that although the selective-HDACi Entinostat has a relatively poor blood-brain barrier (BBB) penetration compared to pan-HDACi Panobinostat, Entinostat induced a comparable level of histone acetylation in the brain to that of Panobinostat. Furthermore, combinatorial treatment consisting of HDACi Entinostat/Panobinostat and anti-PD1 reduced BrM outgrowth of EO771 mammary tumor cell-induced BrM in synergetic mouse model. We explored the impact of HDACi on cell surface MHC-I expression by flow cytometry. Entinostat restored MHC-I expression levels on both brain-seeking human and murine breast and lung cancer cell lines, indicating a general epigenetic regulation on MHC-I expression by inhibition of HDACs. To dissect the impact of HDACi on the BrIME, immune profiling of BrM was performed by mass cytometry (CyTOF). Notably, Entinostat induced a dramatic increment of a specific antigen-presenting cell (APC) cluster in BrM-bearing BrIME with increased ratio of M1 proinflammatory macrophages: M2 tumor-associated macrophages (TAMs) and decreased Tregs. Our results suggest that Entinostat can upregulate MHC-I for more effective antigen presentation and enhance anti-tumor immunity in BrIME. Citation Format: Shao-Ping Yang, Yimin Duan, Xiangliang Yuan, Lin Zhang, Patrick Zhang, Dihua Yu. Selective-HDAC inhibitor relieves the suppressive immune microenvironment in the brain to impede breast cancer brain metastasis. [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 6427.

Abstract P1-11-03: VRN101099, Brain Permeable HER2 Kinase Inhibitor, Shows the Anti-tumor Activity in Preclinical Models of HER2-positive Cancers

Abstract The brain is a common target organ for breast cancer metastasis, and the risk of brain metastasis is usually high for patients with HER2-positive breast cancer. While T-DXd showed positive results in HER2-positive breast cancer brain metastasis (BCBM), its activity and blood-brain barrier (BBB) permeability in active BCBM need to be further validated (TUXEDO-1 included 15 patients with active brain metastasis). Tucatinib plus Trastuzumab and Capecitabine triplet showed efficacy in patients with brain metastasis, but Tucatinib alone has poor BBB penetration. There is still an unmet medical need for active HER2-positive BCBM. VRN101099 is a highly selective kinase inhibitor of HER2 (Eurofins scanMAX Kinase Profiling, S-score (35) of 0.01). VRN101099 has single- to double-digit nanomolar (IC50) cellular potency in HER2-dependent cancer cells, and Ba/F3 cells expressing HER2 wild type or mutations with selectivity over wildtype EGFR. VRN101099 inhibited proliferation of BT474 with 3.6 nM IC50 but HaCaT with 829.2 nM IC50. VRN101099 binds HER2 kinase by forming a covalent bond to the Cys805 and its irreversible inhibition resulted in a longer target resident time than Tucatinib, confirmed by in vitro washout experiments. Robust in vivo activity of VRN101099 was observed in HER2-positive BT474 xenograft models. Moreover, once daily oral administration of VRN101099 significantly regressed intracranial BT474 tumor, demonstrating greater efficacy than twice daily oral administration of Tucatinib. These results were well explained by the superior brain to plasma exposure of VRN101099 to Tucatinib. Also, VRN101099 showed high exposure in the target organ, the fat pad, which potentiated better clinical translation. These anti-tumor efficacies are correlated with pharmacodynamic responses, as confirmed by decreased HER2, AKT, and ERK phosphorylation. In summary, VRN101099 is a brain penetrant, orally bioavailable, irreversible, and highly selective inhibitor of HER2 with therapeutic potential in HER2-positive BCBM. These data support the clinical development of VRN101099 in HER2-driven cancers. Citation Format: Yikyung Ko, Jihye yoo, Hong-ryul Jung, Hyerim Lim, YeongDeok Lee, Se Hyuk Kim, Dong Guk Shin, Serin Cho, Myung hoe Heo, Haelee Kim, Ha Yeon Cho, Ah Reum Han, Eunhwa Ko, Hwan Geun Choi, Deakwon Kim, Sunghwan Kim. VRN101099, Brain Permeable HER2 Kinase Inhibitor, Shows the Anti-tumor Activity in Preclinical Models of HER2-positive Cancers [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P1-11-03.

Molecular insights into the mechanism of sugar-modified enkephalin binding to opioid receptors

Opioid receptors (delta, kappa, and mu) belong to the G protein-coupled receptor (GPCR) superfamily. They are responsible for pain perception - being activated by opioid peptides such as enkephalins, endorphins and dynorphins and by opiates, such as morphine. Enkephalins are naturally occurring endogenous pentapeptides with the amino acid sequence Tyr-Gly-Gly-Phe-Leu/Met. Both enkephalins are potent agonists of the delta receptor, and to a lesser extent the mu receptor, with little to no effect on the kappa receptor. Like most small peptides, enkephalins are easily catabolised via enzymatic degradation and show poor blood-brain barrier penetration. The attachment of sugars to peptides increases their penetration of the blood-brain barrier but also may affect interactions with receptors. In this study, the [Leu5]enkephalin and [Leu5]enkephalin containing the β-D-glucuronic acid were investigated to explain how the presence of sugar moiety in the peptide molecule influences its interaction with the opioid receptors. In conclusion, the conjugation of an enkephalin molecule with the glucuronic acid has a direct and strong impact on the receptor-ligand interactions. The enhancement of ligand binding is much stronger in the delta receptor than in the mu receptor; thus, enkephalin conjugated with glucuronic acid shows greater selectivity toward the delta opioid receptor than the original peptide.

Synergistic effects of the galanin analog 810-2 with the antiseizure medication levetiracetam in rodent seizure models.

The use of many antiseizure medications (ASMs) is limited due to pharmacoresistance and dose-limiting side effects, suggesting an unmet need for novel therapeutic approaches. The neuropeptide galanin reduces seizures in several preclinical seizure and epilepsy models, but its clinical utility is limited due to rapid metabolism and poor blood-brain barrier penetration. The lead galanin analog 810-2 is systemically bioavailable and reduces seizures when administered alone. Further development of this analog, with the potential for use as an add-on therapy in patients with epilepsy, requires a better understanding of the use of this analog in combination with approved ASMs. We sought to evaluate 810-2 in combination with commonly used ASMs in rodent models of seizures. The mouse 6-Hz seizure assay was used to test efficacy of 810-2 in combination with levetiracetam (LEV), valproic acid (VPA), or lacosamide (LCM) using a 1:1 dose ratio in isobolographic studies. Further characterization was performed for the combination of 810-2 and LEV in the mouse corneal kindling and rat 6-Hz assays. Whereas the combination of 810-2 with VPA and LCM yielded additive interactions, the combination of 810-2 with LEV demonstrated a synergistic interaction in the mouse 6-Hz assay. Supra-additive effects were also observed in the mouse corneal kindling and rat 6-Hz assays for this combination. The combination of 810-2 with LEV suggests the potential for this galanin analog to be further developed as an add-on therapy for patients with epilepsy, particularly when coadministered with LEV.

Aspartic Acid-Modified Phospholipids Regulate Cell Response and Rescue Memory Deficits in APP/PS1 Transgenic Mice.

Misfolding and accumulation of amyloid-β (Aβ) to form senile plaques are the main neuropathological signatures of Alzheimer's disease (AD). Decreasing Aβ production, inhibiting Aβ aggregation, and clearing Aβ plaques are thus considered an important strategy for AD treatment. However, numerous drugs cannot enter the AD clinical trials due to unsatisfactory biocompatibility, poor blood-brain barrier penetration, little biomarker impact, and/or low therapeutic indicators. Here, a pair of chiral aspartic acid-modified 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (l- and d-Asp-DPPE) are prepared to build stabilized chiral liposomes. We find that both l- and d-liposomes are able to rescue Aβ aggregation-induced apoptosis, oxidative stress, and calcium homeostasis, in which the effect of d-liposomes is more obvious than that of l-ones. Furthermore, in AD model mice (APPswe/PS1d9 double-transgenic mice), chiral liposomes not only show biosafety but also strongly improve cognitive deficits and reduce Aβ deposition in the brain. Our results suggest that chiral liposomes, particularly, d-liposomes, could be a potential therapeutic approach for AD treatment. This study opens new horizons by showing that liposomes will be used for drug development in addition to delivery and targeting functions.

Spray Dried Mucoadhesive Microparticles of Donepezil with Chitosan and Carbopol in Alzheimer’s Disease

Background: The traditional oral formulation for Alzheimer’s disease treatment has the drawbacks of first-pass metabolism, plasma protein binding, and poor blood-brain barrier penetration. This study was conducted to establish the nasal route of administration for donepezil formulations in Alzheimer’s disease. Materials and Methods: Donepezil mucoadhesive microparticles synthesized by spray-drying and evaluated for Infrared spectroscopy, differential scanning calorimetry, and scanning electron microscopy. The ex vivo study was done with Franz’s diffusion cell using goat nasal mucosa. The in vivo study was performed on the Albino rat’s nasal route for determining drug concentration by HPLC analysis in brain tissue at single-point evaluation. Results: The microparticles were with optimum size with no drug-polymer interaction in Infrared spectroscopy and Differential scanning calorimetry. Scanning electron microscopy exhibited morphology of spherical or ellipsoid microparticles with efficient drug entrapment. The percentage drug release for chitosan microparticles was 66.57 to 85.74 and for carbopol microparticles was 69.54 to 91.53 in the ex vivo permeability study. In vivo studies showed that drug concentrations of 110.48% to 114.92% for chitosan batches and 111.87% to 142.08% for carbopol batches were superior to controls. Conclusion: Ex vivo permeability study revealed drug release patterns with as high as 85.74% ±0.02 for DCH2 formulation and 91.53±0.3% for DC3 formulation. In in vivo study formulation DCH2 displayed drug concentration 110.87±6.87% and DC3 shown 129.51±9.82% over the control batch which is conclusive for improved drug delivery of donepezil through mucoadhesive microparticles for the nose to brain targeting in Alzheimer’s disease.

Abstract 4113: Response to amivantamab, a bispecific EGF and MET receptor directed antibody, in a patient with an atypical EGFR mutated (G719X) non-small cell lung cancer (NSCLC) with leptomeningeal disease who progressed on osimertinib

Abstract Amivantamab is a bispecific antibody against EGF and MET receptors, approved for patients with locally advanced or metastatic NSCLC harboring EGFR exon 20 insertion after disease has progressed on or after platinum-based chemotherapy. Existing data is insufficient in establishing its efficacy on other rare EGFR mutated subtypes, central nervous system (CNS), or its resistance to 3rd generation tyrosine kinase inhibitor (TKI), osimertinib. Here, we report a case showing a good response on single agent amivantamab in an atypical EGFR mutation (G719A, exon18 substitution) NSCLC that progressed to CNS involvement on osimertinib. A 67-year-old male presented with stage IVB squamous cell carcinoma of lung with osseous metastases. CT chest, abdomen, and pelvis revealed a speculated 7.7 cm right upper lung lesion with hilar and subcarinal lymphadenopathies. MRI brain and spine showed multiple bone metastases without CNS involvement. Tissue biopsy confirmed poorly differentiated squamous cell carcinoma with PD-L1 IHC 75%. Tissue next generation sequencing (NGS) showed EGFR G719A. He was started on osimertinib. Two months later, he was hospitalized for drug-induced pneumonitis which prompted off osimertnib. CT angiogram chest showed new diffuse pericardial thickening and nodularity, suggestive of progressive disease. Two cycles of chemotherapy were delivered followed by immunotherapy with ipilimumab and nivolumab. In 10 weeks, progression of disease was revealed in MRI brain demonstrating new parenchymal and leptomeningeal metastases. Amivantamab monotherapy was initiated with discontinuation of immunotherapy. The patient tolerated amivantamab without major complications. His performance status remained the same before and after amivantamab. He denied fatigue, anorexia, nausea or vomiting, however, endorsed rash, which was managed with hydrocortisone cream. Repeat scans in 6 weeks showed decreased leptomeningeal enhancement, and reduction in the size of parenchymal lesions, lung mass, and lymphadenopathies. The highest variant allele fraction from circulating tumor DNA NGS assay from Guardant360 was significantly improved on amivantamab from 25.6% (EGFR G719A), at time of diagnosis, to non-detectable. Amivantamab monotherapy has shown an encouraging outcome in a patient with an atypical EGFR mutated (G719X) NSCLC with leptomeningeal disease who progressed on osimertinib. Our case has shown significant response on CNS involvements, which is contrary to known poor blood-brain barrier penetration of amivantamab. This supports current trial evaluating the efficacy of amivantamab for NSCLC with rare EGFR mutations such as G719X, and ones progressed on 3rd Gen TKI treatment. Additional studies evaluating the efficacy of amivantamab on CNS metastasis are warranted. Citation Format: Jinah Kim, Horyun Choi, Yeun Ho Lee, Leeseul Kim, Young Kwang Chae. Response to amivantamab, a bispecific EGF and MET receptor directed antibody, in a patient with an atypical EGFR mutated (G719X) non-small cell lung cancer (NSCLC) with leptomeningeal disease who progressed on osimertinib [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4113.