Poor Blood-brain Barrier Penetration Research Articles

Homogeneity of antibody-drug conjugates critically impacts the therapeutic efficacy in brain tumors.

SUMMARYGlioblastoma multiforme (GBM) is the most aggressive and fatal disease of all brain tumor types. Most therapies rarely provide clinically meaningful outcomes in the treatment of GBM. Although antibody-drug conjugates (ADCs) are promising anticancer drugs, no ADCs have been clinically successful for GBM, primarily because of poor blood-brain barrier (BBB) penetration. Here, we report that ADC homogeneity and payload loading rate are critical parameters contributing to this discrepancy. Although both homogeneous and heterogeneous conjugates exhibit comparable in vitro potency and pharmacokinetic profiles, the former shows enhanced payload delivery to brain tumors. Our homogeneous ADCs provide improved antitumor effects and survival benefits in orthotopic brain tumor models. We also demonstrate that overly drug-loaded species in heterogeneous conjugates are particularly poor at crossing the BBB, leading to deteriorated overall brain tumor targeting. Our findings indicate the importance of homogeneous conjugation with optimal payload loading in generating effective ADCs for intractable brain tumors.

Penicillin- and third-generation cephalosporin-resistant strains of Streptococcus pneumoniae meningitis: Case report and literature review

BackgroundTreatment of patients with penicillin-resistant S. pneumoniae (PRSP) is complicated because of the relatively poor blood-brain barrier penetration of effective antimicrobials. Our case: A previously healthy 70-year-old woman, a traveler from China to Japan, was admitted to our hospital with fever and loss of consciousness. She has no history of pneumococcal vaccination. She was diagnosed with bacterial meningitis due to penicillin-and third-generation cephalosporin-resistant strains of S. pneumoniae. The patient was successfully treated with a combination therapy of vancomycin (VCM) and levofloxacin (LVFX) and recovered without any neurological sequelae. As the treatment of penicillin-and third-generation cephalosporin-resistant strains of S. pneumoniae meningitis remains unclear, we conducted a review of the reported cases of meningitis caused by penicillin- and cephalosporin-resistant S. pneumoniae. MethodWe performed a search using the keywords “penicillin-resistant Streptococcus pneumoniae,” “meningitis,” and “pneumococcal meningitis”. We searched the electronic databases PubMed, Embase, and Ichushi from their inception to March 2020. Subsequently, two authors independently reviewed the resulting database records, retrieved full texts for eligibility assessment, and extracted data from these cases. ResultWe identified 18 papers describing thirty-five cases of penicillin- and cephalosporin-resistant S. pneumoniae meningitis including our case. The patient's characteristics were; median age: 50 years, men:50%, 85% of cases received combination regimens of antibiotics: Ceftroriaxone (CTRX) plus VCM (20 cases), CTRX plus VCM plus rifampicin (RFP) (two cases), CTRX plus linezolid (one case), fluoroquinolones (two cases), carbapenems (six cases), Thirty-five percent received steroids. Twenty-four percent of patients died. Twenty-six percent of patients complicated neurological sequalae. ConclusionCombination therapy including VCM plus LVFX could be a treatment option.

Role of Oxytocin and Vasopressin in Neuropsychiatric Disorders: Therapeutic Potential of Agonists and Antagonists.

Oxytocin (OT) and vasopressin (AVP) are hypothalamic neuropeptides classically associated with their regulatory role in reproduction, water homeostasis, and social behaviors. Interestingly, this role has expanded in recent years and has positioned these neuropeptides as therapeutic targets for various neuropsychiatric diseases such as autism, addiction, schizophrenia, depression, and anxiety disorders. Due to the chemical-physical characteristics of these neuropeptides including short half-life, poor blood-brain barrier penetration, promiscuity for AVP and OT receptors (AVP-R, OT-R), novel ligands have been developed in recent decades. This review summarizes the role of OT and AVP in neuropsychiatric conditions, as well as the findings of different OT-R and AVP-R agonists and antagonists, used both at the preclinical and clinical level. Furthermore, we discuss their possible therapeutic potential for central nervous system (CNS) disorders.

Screening novel CNS drug candidates for P-glycoprotein interactions using the cell line iP-gp: In vitro efflux ratios from iP-gp and MDCK-MDR1 monolayers compared to brain distribution data from mice

Drug efflux by P-glycoprotein (P-gp, ABCB1) is considered as a major obstacle for brain drug delivery for small molecules. P-gp-expressing cell monolayers are used for screening of new drug candidates during early states of drug development. It is, however, uncertain how well the in vitro studies can predict the in vivo P-gp mediated efflux at the blood–brain barrier (BBB). We previously developed a novel cell line of porcine origin, the iP-gp cell line, with high transepithelial resistance and functional expression of human P-gp. The aim of the present study was to evaluate the applicability of the cell line for screening of P-gp interactions of novel drug candidates. For this purpose, bidirectional fluxes of 14 drug candidates were measured in iP-gp cells and in MDCK-MDR1 cells, and compared with pharmacokinetic data obtained in male C57BL/6 mice. The iP-gp cells formed extremely tight monolayers (>15 000 Ω∙cm2) as compared to the MDCK- MDR1 cells (>250 Ω∙cm2) and displayed lower Papp,a-b values. The efflux ratios obtained with iP-gp and MDCK-MDR1 monolayers correlated with Kp,uu,brain values from the in vivo studies, where compounds with the lowest Kp,uu,brain generally displayed the highest efflux ratios. 12 of the tested compounds displayed a poor BBB penetration in mice as judged by Kp,uu less than 1. Of these compounds, nine compounds were categorized as P-gp substrates in the iP-gp screening, whereas analysis of data estimated in MDCK-MDR1 cells indicated four compounds as potential substrates. The results suggest that the iP-gp cell model may be a sensitive and useful screening tool for drug screening purposes to identify possible substrates of human P-glycoprotein.

Role of Laser Interstitial Thermal Therapy in the Management of Primary and Metastatic Brain Tumors.

Laser interstitial thermal therapy (LITT) is a minimally invasive treatment option for brain tumors including glioblastoma, other primary central nervous system (CNS) neoplasms, metastases, and radiation necrosis. LITT employs a fiber optic coupled laser delivery probe stabilized via stereotaxis to deliver thermal energy that induces coagulative necrosis in tumors to achieve effective cytoreduction. LITT complements surgical resection, radiation treatment, tumor treating fields, and systemic therapy, especially in patients who are high risk for surgical resection due to tumor location in eloquent regions or poor functional status. These factors must be balanced with the increased rate of cerebral edema post LITT compared to surgical resection. LITT has also been shown to induce transient disruption of the blood-brain barrier (BBB), especially in the peritumoral region, which allows for enhanced CNS delivery of anti-neoplastic agents, thus greatly expanding the armamentarium against brain tumors to include highly effective anti-neoplastic agents that have poor BBB penetration. In addition, hyperthermia-induced immunogenic cell death is another secondary side effect of LITT that opens up immunotherapy as an attractive adjuvant treatment for brain tumors. Numerous large studies have demonstrated the safety and efficacy of LITT against various CNS tumors and as the literature continues to grow on this novel technique so will its indications.

Focused Ultrasound-Mediated Blood-Brain Barrier Opening Enhances Panobinostat Efficacy in a Murine Diffuse Intrinsic Pontine Glioma Model

Purpose/Objective(s)Diffuse intrinsic pontine glioma (DIPG) is the most lethal brain tumor in children with no effective treatment options to date. The prognosis remains poor with a median overall survival of less than 1 year. Recent in vitro drug screening studies found the histone deacetylase (HDAC) inhibitor, panobinostat, to be highly effective against DIPG. However, as panobinostat has poor blood-brain barrier (BBB) penetration, phase 1 clinical trials using systemic administration of panobinostat have been unsuccessful. Therefore, improved efficacy will require enhancement of delivery into the brain. Focused ultrasound (FUS), a non-ionizing acoustic form of radiation, has been shown to safely and non-invasively open the BBB, which can increase drug delivery. In this study, we hypothesize that FUS-mediated BBB opening (BBBO) can enhance the delivery of panobinostat for a therapeutic benefit in DIPG.Materials/MethodsA syngeneic DIPG model was established by injecting mouse DIPG cells (PDGFB+, H3.3K27M, p53−/−) intracranially. A single-element, spherical-segment FUS transducer (center frequency: 0.5 MHz) driven by a function generator through a power amplifier was used with concurrent microbubble injection to perform BBBO. We then utilized magnetic resonance imaging to evaluate BBBO and tumor progression.ResultsWe first demonstrated that FUS-mediated BBBO is safe and feasible in mice with DIPG tumors by MRI and passive cavitation detection. Our DIPG cell line was shown to be very sensitive to panobinostat in cell viability assay (IC50 = 0.012 μM). The combined treatment of FUS-mediated BBBO and panobinostat showed benefits in both local control and overall survival.ConclusionThe current results demonstrate that FUS could increase the treatment efficacy of panobinostat in a DIPG animal model due to the improved targeted dose of systemic panobinostat in the DIPG tumors. We believe these data establish a strong preclinical rationale to assess the combination of FUS-mediated BBB opening with panobinostat in pediatric patients with DIPG. Diffuse intrinsic pontine glioma (DIPG) is the most lethal brain tumor in children with no effective treatment options to date. The prognosis remains poor with a median overall survival of less than 1 year. Recent in vitro drug screening studies found the histone deacetylase (HDAC) inhibitor, panobinostat, to be highly effective against DIPG. However, as panobinostat has poor blood-brain barrier (BBB) penetration, phase 1 clinical trials using systemic administration of panobinostat have been unsuccessful. Therefore, improved efficacy will require enhancement of delivery into the brain. Focused ultrasound (FUS), a non-ionizing acoustic form of radiation, has been shown to safely and non-invasively open the BBB, which can increase drug delivery. In this study, we hypothesize that FUS-mediated BBB opening (BBBO) can enhance the delivery of panobinostat for a therapeutic benefit in DIPG. A syngeneic DIPG model was established by injecting mouse DIPG cells (PDGFB+, H3.3K27M, p53−/−) intracranially. A single-element, spherical-segment FUS transducer (center frequency: 0.5 MHz) driven by a function generator through a power amplifier was used with concurrent microbubble injection to perform BBBO. We then utilized magnetic resonance imaging to evaluate BBBO and tumor progression. We first demonstrated that FUS-mediated BBBO is safe and feasible in mice with DIPG tumors by MRI and passive cavitation detection. Our DIPG cell line was shown to be very sensitive to panobinostat in cell viability assay (IC50 = 0.012 μM). The combined treatment of FUS-mediated BBBO and panobinostat showed benefits in both local control and overall survival. The current results demonstrate that FUS could increase the treatment efficacy of panobinostat in a DIPG animal model due to the improved targeted dose of systemic panobinostat in the DIPG tumors. We believe these data establish a strong preclinical rationale to assess the combination of FUS-mediated BBB opening with panobinostat in pediatric patients with DIPG.

Benzothiazolium Derivative-Capped Silica Nanocomposites for β-Amyloid Imaging In Vivo

Alzheimer’s disease (AD) is a neurodegenerative disease, and β-amyloid (Aβ) is believed to be a causative factor in AD pathology. The abnormal deposition of Aβ is believed to be responsible for progression of AD. In order to facilitate the imaging of Aβ in vivo, suitable probe molecules with a near-infrared emission wavelength that can penetrate the blood–brain barrier (BBB) were utilized. The commercial fluorescent probe thioflavin-T (ThT) is used to image Aβ; however, because of its short emission wavelength and poor BBB penetration, ThT can only be used in vitro. With this research, based on ThT, we design three fluorescent probes (SZIs) having a longer emission wavelength in order to image Aβ aggregates. SZIs with different numbers of double bonds respond to Aβ aggregates. The SZIs have a structure similar to ThT, and as such, the SZIs are also unable to penetrate the BBB. To deal with the problem, we develop nanocomposites (MSN-Lf@SZIs) to deliver SZIs into the brain of AD mouse and image Aβ successfully. These new nanocomposites are able to deliver the dyes into the brain and facilitate Aβ imaging in vivo.

Abstract 49: Brain penetrant HDAC inhibitor RG2833 induces DIPG cell death and synergizes with lomustine

Abstract Diffuse intrinsic pontine glioma (DIPG) is a universally fatal pediatric brain tumor. The pan-histone deacetylase (HDAC) inhibitor panobinostat showed great activity against DIPG in pre-clinical models, but has poor blood brain barrier penetration and demonstrated significant toxicity in clinical trials. RG2833 (RGFP109) is a selective HDAC1/3 inhibitor with established brain penetration. In clinical trials, the Cmax(plasma) of RG2833 was 32uM. RG2833 demonstrated cytotoxicity against temozolomide-resistant glioblastoma and downregulated the NFĸB pathway. Because this pathway is overexpressed in DIPG and may play a role in DIPG cell growth and survival, we hypothesized that RG2833 would kill DIPG cells. Treatment of DIPG cell lines with RG2833 as a single agent inhibits cell growth in the 5 to 10μM range (MTS assay for HSJD007 p=0.0004 10μM vs DMSO, JHH-DIPG1 p=0.001 10μM vs DMSO, SF-7761 p=0.04 10μM vs DMSO, SU-DIPG13 p=0.01 10μM vs DMSO by t-test). RG2833 induces apoptosis by 48 hours as measured by Western blot for cPARP and cleaved caspase 3 immunofluorescence (HSJD007 p<0.003 8μM vs DMSO, JHH-DIPG1 p=0.0026 10μM vs DMSO by t-test). RG2833 also slows cell proliferation as measured by Western blot for phospho-Rb and immunofluorescence for BrdU (HSJD007 p=0.008 8μM vs DMSO, JHH-DIPG1 p=0.0002 10μM vs DMSO by t-test). Western blot confirmed a dose-dependent increase in histone 3 acetylation with RG2833 treatment. We detected increased acetylated p65 and decreased expression of the NFĸB regulated pro-survival genes BCL2, BCL-xL, and XIAP with RG2833 treatment in vitro. Treatment of DIPG flank tumor mouse models with RG2833 alone for 5 days suppressed tumor growth (p< 0.005 by t-test). Combination treatment with RG2833 and lomustine was synergistic against DIPG cell lines in vitro (ZIP synergy scores by SynergyFinder for JHH-DIPG1 17.8 and HSJD007 17.7). We will next assess combination treatment in vitro and in vivo for apoptotic effects. This data indicates that selective HDAC inhibitor RG2833 may be a promising therapeutic candidate for DIPG. Citation Format: Katherine K. Barnett, Orlandi Novak, Charles Eberhart, Eric Raabe. Brain penetrant HDAC inhibitor RG2833 induces DIPG cell death and synergizes with lomustine [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 49.

EPCT-23 PRE-CLINICAL STUDY OF FOCUSED ULTRASOUND-MEDIATED BLOOD-BRAIN BARRIER OPENING AND PANOBINOSTAT FOR DIFFUSE INTRINSIC PONTINE GLIOMA TREATMENT

Diffuse intrinsic pontine glioma (DIPG) is the lethal high-grade brain tumor in children with no effective treatment options to date. Despite excessive clinical trials, the prognosis remains poor, with a median overall survival (mOS) of less than 1 year. Genomic studies of DIPG tissue have identified highly recurrent mutations in genes encoding histone H3 resulting in the substitution of lysine to methionine at position 27 (K27M), which is found in approximately 80% of DIPG. Recent drug screening studies identified the histone deacetylase (HDAC) inhibitors panobinostat as a highly effective drug against DIPG in vitro. However, due to the poor Blood-Brain Barrier (BBB) penetration of systemic administration, to enhance the delivery of panobinostat to improve treatment efficacy is needed. Focused ultrasound (FUS) has been shown to be able to safely and non-invasively open BBB to enhance drug delivery. Hence, in this study, we hypothesize that FUS-mediated BBBO (BBBO) can enhance the delivery of panobinostat for a therapeutic benefit in DIPG. Herein we established the syngeneic DIPG model by intracranially injecting mouse DIPG cells (PDGFB+, H3.3K27M, p53−/−) and used FUS and microbubbles to open BBB and enhance the panobinostat delivery. Magnetic resonance (MR) imaging was utilized to evaluate BBBO and tumor progression. We first demonstrated that FUS-mediated BBB-opening is safe and feasible to mice with DIPG tumors by MR imaging and passive cavitation detection. Moreover, this DIPG cell line is very sensitive to panobinostat in in vitro cytotoxicity assay. The combined treatment of FUS-mediated BBBO and panobinostat showed benefits in both local control and overall survival. The current results demonstrated FUS could increase the treatment efficacy of panobinostat to DIPG animals may be due to the increase of targeted delivery of systemic panobinostat to DIPG tumors in brainstem.

Outcomes of first, second, and third-generation anaplastic lymphoma kinase (ALK) inhibitors in non-small cell lung cancer brain metastases (NSCLCBM).

2034 Background: Non-small cell lung cancer (NSCLC) is the most common cause of brain metastases. ALK, which codes for tyrosine kinase receptors, is rearranged in 4-7% of NSCLC. First-generation ALK inhibitors have restricted efficacy due to poor blood-brain barrier (BBB) penetration and ALK-resistant tumor mutations. Second-generation ALK inhibitors have shown better BBB penetration, while third-generation ALK inhibitors were efficacious even against ALK-resistant mutations. In this retrospective study, we investigated the overall survival (OS) and progression-free survival (PFS) in NSCLCBM patients treated with first, second, and third-generation ALK inhibitors. Methods: NSCLCBM patients between 2010 and 2019 were evaluated. We analyzed data regarding molecular marker status, systemic therapies, and date of progression. OS was defined as the start date of systemic therapy to the date of last follow-up or death. The Cox proportional model was used to estimate OS and PFS. Results: A total of 90 patients had ALK gene rearrangement. 16 ALK positive patients received first-generation ALK inhibitor (crizotinib), with a median age of 59.2 years, 50% of the cohort being female and 83.3% being white. Another 17 patients received second-generation (alectinib, ceritinib, brigatinib) and third-generation ALK inhibitors (lorlatinib), with a combined median age of 52.2 years and a cohort of 52.6% females and 72.2% white patients. The 5-year OS rate was 49% (95% confidence interval (CI) = 24%, 71%) for first-generation ALK inhibitors and 76% (95% CI = 40%, 92%) for second and third-generation ALK inhibitors (p-value (p) = 0.019). The median PFS (mPFS) for patients who received first-generation ALK inhibitors was 45.3 months and for those who received second or third-generation ALK inhibitors was 180.1 months. The respective 5-year PFS rate was 43% (95% CI = 19%, 65%) and 72% (95% CI = 42%, 89%). Conclusions: Newer generations of targeted therapies in NSCLCBM have improved BBB penetration and effectiveness against resistant mutations. We determined that there was a significant 5-year OS benefit in patients who received second and third-generation ALK inhibitors compared to first-generation ALK inhibitors, and a respective trend towards significant PFS benefit in newer-generation ALK inhibitors when compared to first-generation. These results are encouraging, but the effect on intracranial lesion size and response rates should be examined in the future.[Table: see text]

Improved Antiglioblastoma Activity and BBB Permeability by Conjugation of Paclitaxel to a Cell-Penetrative MMP-2-Cleavable Peptide.

In order to solve the problems of receptor promiscuity and poor blood‐brain barrier (BBB) penetration in the treatment of glioblastomas (GBM), a novel dual‐functional nanocomplex drug delivery system is developed based on the strategy of peptide‐drug conjugates. In this study, SynB3‐PVGLIG‐PTX is designed and screened out by matrix metalloproteinase‐2 (MMP‐2), to which it exhibits the best affinity. The MMP‐2‐sensitive peptide (PVGLIG) and a cell‐penetration peptide (SynB3) are combined to form a dual‐functional peptide. Moreover, as a drug‐peptide nanocomplex, SynB3‐PVGLIG‐PTX exhibited a high potential to form an aggregation with good solubility that can release paclitaxel (PTX) through the cleavage of MMP‐2. From a functional perspective, it is found that SynB3‐PVGLIG‐PTX can specifically inhibit the proliferation, migration, and invasion of GBM cells in vitro in the presence of MMP‐2, in contrast to that observed in MMP‐2 siRNA transfected cells. Further investigation in vivo shows that SynB3‐PVGLIG‐PTX easily enters the brain of U87MG xenograft nude mice and can generate a better suppressive effect on GBM through a controlled release of PTX from SynB3‐PVGLIG‐PTX compared with PTX and temozolomide. Thus, it is proposed that SynB3‐PVGLIG‐PTX can be used as a novel drug‐loading delivery system to treat GBM due to its specificity and BBB permeability.

RARE-10. ADAMANTINOMATOUS CRANIOPHARYNGIOMA RESIDES OUTSIDE THE BLOOD BRAIN BARRIER

BACKGROUNDAdamantinomatous craniopharyngioma (ACP) is a devastating skull-base tumor believed to derive from epithelial remnants of the primordial craniopharyngeal duct (Rathke’s pouch), which gives rise to the anterior pituitary gland. Genetically engineered mouse models of ACP demonstrate that perturbation of the fetal anterior pituitary can generate tumors analogous to ACP. Clinical and preclinical data indicate that IL-6 blockade may contribute to ACP tumor control, with the most common agent being the humanized monoclonal antibody, tocilizumab. This agent demonstrated poor blood-brain barrier (BBB) penetration in primates. We present findings from two children enrolled on a phase 0 clinical trial (NCT03970226) of a single dose of preoperative intravenous tocilizumab prior to resection of newly diagnosed ACP.METHODSBlood samples were obtained at multiple timepoints. Serum was isolated via ficoll separation. Tumor tissue and cyst fluid were obtained 4–6 hours following the single IV dose of tocilizumab. Tissue was snap-frozen. Tumor was homogenized in RIPA buffer. Free tocilizumab in serum, cyst fluid, and tumor tissue was measured using enzyme-linked immunosorbent assay (ELISA) against a standard curve.RESULTSBoth patients in this trial demonstrated clinically relevant levels of tocilizumab (≥ 4µg/mL) in serum, cyst fluid, and tumor tissue, compared to undetectable levels in control samples.CONCLUSIONACP resides outside BBB protection. In addition to demonstrating the feasibility of systemic delivery of tocilizumab, these findings indicate that other large molecules, including those known to have poor BBB penetration, may be systemically delivered as part of an antitumor regimen in the treatment of ACP.

DIPG-71. SELECTIVE HDAC INHIBITOR RG2833 INDUCES DIPG CELL DEATH VIA DOWNREGULATION OF THE NFĸB PATHWAY

Histone deacetylase (HDAC) inhibitor panobinostat demonstrated activity against diffuse intrinsic pontine glioma (DIPG) in vitro, but its efficacy in vivo was limited by toxicity and poor blood brain barrier penetration. RG2833 (RGFP109) is a selective HDAC1/3 inhibitor that has established brain penetration. In clinical trials, the Cmax (plasma) of RG2833 was 32uM. RG2833 demonstrated cytotoxicity against temozolomide-resistant glioblastoma and downregulated the NFĸB pathway. Because this pathway is overexpressed in DIPG and may play a role in DIPG cell growth and survival, we hypothesized that RG2833 would kill DIPG cells. Treatment of DIPG cell lines with RG2833 as a single agent suppresses cell proliferation in the 5–10μM range (MTS assay for HSJD007 p=0.0004 10μM vs DMSO, JHH-DIPG1 p=0.001 10μM vs DMSO, SF-7761 p=0.04 10μM vs DMSO, SU-DIPG13 p=0.01 10μM vs DMSO by t-test). RG2833 induces apoptosis by 48 hours as measured by Western blot for cPARP and cleaved caspase 3 immunofluorescence (HSJD007 p<0.003 8μM vs DMSO, JHH-DIPG1 p=0.0026 10μM vs DMSO by t-test). RG2833 also slows cell proliferation as measured by Western blot for pRb and immunofluorescence for BrdU (HSJD007 p=0.008 8μM vs DMSO, JHH-DIPG1 p=0.0002 10μM vs DMSO by t-test). Western blot confirmed a dose-dependent increase in histone 3 acetylation with RG2833 treatment at 5 hours. We detected increased acetylated p65 and decreased expression of the NFĸB regulated pro-survival genes BCL2, BCL-xL, and XIAP with RG2833 treatment. Together, this data shows that HDAC inhibitor RG2833 may be a promising therapeutic candidate for DIPG via downregulation of the NFĸB pathway.

Blood-Brain Barrier Modulation to Improve Glioma Drug Delivery.

The blood–brain barrier (BBB) is formed by brain microvascular endothelial cells that are sealed by tight junctions, making it a significant obstacle for most brain therapeutics. The poor BBB penetration of newly developed therapeutics has therefore played a major role in limiting their clinical success. A particularly challenging therapeutic target is glioma, which is the most frequently occurring malignant brain tumor. Thus, to enhance therapeutic uptake in tumors, researchers have been developing strategies to modulate BBB permeability. However, most conventional BBB opening strategies are difficult to apply in the clinical setting due to their broad, non-specific modulation of the BBB, which can result in damage to normal brain tissue. In this review, we have summarized strategies that could potentially be used to selectively and efficiently modulate the tumor BBB for more effective glioma treatment.

Lipid Nanoparticles Improve the Uptake of α-Asarone Into the Brain Parenchyma: Formulation, Characterization, In Vivo Pharmacokinetics, and Brain Delivery.

Treatment of brain-related diseases is one of the most strenuous challenges in drug delivery research due to numerous hurdles, including poor blood-brain barrier penetration, lack of specificity, and severe systemic toxicities. Our research primarily focuses on the delivery of natural therapeutic compound, α-asarone, for the treatment of brain-related diseases. However, α-asarone has poor aqueous solubility, bioavailability, and stability, all of which are critical issues that need to be addressed. This study aims at formulating a lipid nanoparticulate system of α-asarone (A-LNPs) that could be used as a brain drug delivery system. The physicochemical, solid-state properties, stability, and in vitro and in vivo studies of the A-LNPs were characterized. The release of α-asarone from the A-LNPs was prolonged and sustained. After intravenous administration of A-LNPs or free α-asarone, significantly higher levels of α-asarone from the A-LNPs were detected in murine plasma and brain parenchyma fractions, confirming the ability of A-LNPs to not only maintain a therapeutic concentration of α-asarone in the plasma, but also transport α-asarone across the blood-brain barrier. These findings confirm that lipid nanoparticulate systems enable penetration of natural therapeutic compound α-asarone through the blood-brain barrier and may be a candidate for the treatment of brain-related diseases.

Abstract 1721: Hypoxia penetrating ultra-small nanoparticle for anti-cancer effect in Glioblastoma

Abstract Glioblastoma multiforme (GBM) is a lethal brain tumor currently without successful treatment options. Chemotherapeutic agents are the frontline in the management of GBM. Nonetheless, accessibility of the brain tumor is greatly impeded by the poor blood-brain barrier (BBB) penetration of drugs, which needs immediate attention. Ultra-small multifunctional nanoparticle offers a great promise in overcoming the delivery barriers to GBM. The primary goal of our research is to develop BBB targeted nanoparticle that can selectively deliver drugs to GBM and trigger cell death. We developed Anti-GBM drug, such as temozolomide (TMZ) encapsulated biocompatible, ultra-small nanoparticle using Lactoferrin (LAC) protein. Further, we chemically conjugated LAC with tumor hypoxia biomarker homing molecule, Acetazolamide (ATZ) to obtain LAC-ATZ nanoparticle. To validate our hypothesis of hypoxia targeting, we evaluated the carbonic anhydrase-IX (CAIX) receptor expression in human brain tumor section. The IHC data demonstrated significant overexpression of CAIX in tumor tissue compared to healthy human brain tissue section. Literatures have reported overexpression of LRP-1 in endothelial blood vessel of GBM tumor. Thus, use of LAC-ATZ will enhance its penetration across the BBB and delivery of TMZ in hypoxic core of GBM. To achieve the effective BBB penetration, we engineered LAC-ATZ@TMZ nanoparticle that demonstrated homogenous ultra-small particle size of average diameter of 20-30 nm using TEM analysis. The SDS-PAGE analysis demonstrated the conjugation of LAC with ATZ. Our preliminary data in GBM cells, U87MG suggested the dose dependent cell uptake of DiD-fluorescence labeled LAC-ATZ nanoparticle. The nanoparticles are selective in targeting the U87MG cell when compared to healthy microglial BV2 cells. Thus, it is supporting the potential of LAC-ATZ nanoparticle for targeting the GBM tumor with limited effect in healthy brain tissue. We developed conditioned BBB cell culture model to demonstrate the penetration of LAC-ATZ across the BBB. Based on the exciting preliminary outcome in this study builds a strong rational of using LAC-ATZ nanoparticle for overcoming the bottle neck barrier of BBB with improved anti-GBM response. Keywords: Glioblastoma multiforme (GBM), blood-brain barrier (BBB), Lactoferrin (LAC), Acetazolamide (ATZ) Citation Format: Mohd A. Rauf, Katyayani Tatiparti, Samaresh Sau, Arun K. Iyer. Hypoxia penetrating ultra-small nanoparticle for anti-cancer effect in Glioblastoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1721.

Novel, non-nitrocatechol catechol-O-methyltransferase inhibitors modulate dopamine neurotransmission in the frontal cortex and improve cognitive flexibility.

Cognitive impairment is a primary feature of many neuropsychiatric disorders and there is a need for new therapeutic options. Catechol-O-methyltransferase (COMT) inhibitors modulate cortical dopaminergic function and have been proposed as potential cognitive enhancers. Unfortunately, currently available COMT inhibitors are not good candidates due to either poor blood-brain barrier penetration or severe toxicity. To address the need for safe, brain-penetrant COMT inhibitors, we tested multiple novel compounds in a set of preclinical in vivo efficacy assays in rats to determine their ability to inhibit COMT function and viability as potential clinical candidates. We measured the change in concentration of dopamine (DA) metabolites in cerebrospinal fluid (CSF) from the cisterna magna and extracellular fluid (ECF) from the frontal cortex produced by our novel compounds. Additionally, we tested the effects of our brain-penetrant COMT inhibitors in an attentional set-shifting assay (ASST). We benchmarked the performance of the novel COMT inhibitors to the effects produced by the known COMT inhibitor tolcapone. We found that multiple COMT inhibitors, exemplified by LIBD-1 and LIBD-3, significantly modulated dopaminergic function measured as decreases in homovanillic acid (HVA) and increases in 3,4-Dihydroxyphenylacetic acid (DOPAC), two DA metabolites, in CSF and the frontal cortex. Additionally, we found thatLIBD-1 significantly improved cognitive flexibility in the ASST, an effect previously reported following tolcapone administration. These results demonstrate that LIBD-1 is a novel COMT inhibitor with promising in vivo activity and the potential to serve as a new therapy for cognitive impairment.

Evaluation of sodium valproate loaded nanoparticles in acute and chronic pentylenetetrazole induced seizure models

Background and purposeEfficacy of sodium valproate in epilepsy is limited by its poor blood brain barrier penetration and side effects. Nanoparticles may offer a better drug delivery system to overcome these limitations. This study evaluated the efficacy of sodium valproate encapsulated in nanoparticles in pentylenetetrazole (PTZ) induced acute and kindling models of seizures in male Wistar rats. MethodsPoly lactic-co-glycolic acid (PLGA) based, polysorbate 80 stabilized sodium valproate loaded nanoparticles (nano sodium valproate) and rhodamine loaded nanoparticles (RLN) were formulated by double emulsion- solvent evaporation method and characterized for their size, shape, zeta potential and drug loading percentage. RLN was used to demonstrate blood brain barrier (BBB) permeability of nanoparticles. Serum drug levels were estimated using high performance liquid chromatography. The efficacy of standard sodium valproate (300 mg/kg) and nano sodium valproate (∼300, ∼150 and ∼75 mg/kg of sodium valproate) were evaluated in experimental animal models of seizures along with their effects on behavioral and oxidative stress parameters. Drugs were administered 60 min before PTZ in acute model. In the kindling model, drugs were administered every day while PTZ was administered on alternate days 60 min after drug administration. All the study drugs/compounds were administered intraperitoneally. ResultsRLN were observed to be clustered in cortex which implied that the nanoparticles crossed BBB. Both standard sodium valproate and nano sodium valproate reached therapeutic serum level at 15 min and 1 h, but were undetectable in serum at 24 h. In acute PTZ (60 mg/kg) model, nano sodium valproate (∼300 mg/kg of sodium valproate) and standard sodium valproate showed protection against seizures till 6 h and 4 h, respectively. There were significant behavioral impairment and oxidative stress with standard sodium valproate in acute model as compared to nano sodium valproate at 6 h. In kindling model, induced with PTZ (30 mg/kg, every alternate day for 42 days), complete protection from seizures was observed with nano sodium valproate (∼150 mg/kg and ∼75 mg/kg of sodium valproate) and standard sodium valproate (300 mg/kg). Similarly, significant protection from behavioral impairment and oxidative stress was observed with standard sodium valproate and nano sodium valproate as compared to PTZ. ConclusionWhen compared to conventional therapy, nano sodium valproate showed protection from seizures at reduced doses and for a longer duration in animal models of epilepsy. This study suggests the potential of nano sodium valproate in the treatment of epilepsy.

Abstract 3120: The novel MEK inhibitor binimetinib decreases cell growth and induces apoptosis in AT/RT

Abstract Atypical teratoid/rhabdoid tumors are a class of malignant CNS tumors disproportionately affecting children with limited therapeutic options. Our group has previously demonstrated that the MAP kinase pathway is altered in a large proportion of these tumors and the MEK inhibitor selumetinib decreases cell growth and promotes apoptosis of AT/RT cells. One of the major drawbacks of selumetinib is its poor blood-brain barrier penetration. Binimetinib is a novel MEK inhibitor currently in pediatric phase II clinical trials for low grade glioma that is thought to have improved brain penetration. Here we demonstrate nanomolar levels of binimetinib successfully decreases cell proliferation and induces apoptosis (2-3 fold induction of cleaved Parp) in AT/RT cell lines, suggesting binimetinib could offer a potential avenue for treating these deadly tumors. Citation Format: Shubin Shahab, Jeffrey Rubens, Charles Eberhart, Eric Raabe. The novel MEK inhibitor binimetinib decreases cell growth and induces apoptosis in AT/RT [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3120.

Design of a brain-penetrant CDK4/6 inhibitor for glioblastoma.

CDK4 and CDK6 are kinases with similar sequences that regulate cell cycle progression and are validated targets in the treatment of cancer. Glioblastoma is characterized by a high frequency of CDKN2A/CCND2/CDK4/CDK6 pathway dysregulation, making dual inhibition of CDK4 and CDK6 an attractive therapeutic approach for this disease. Abemaciclib, ribociclib, and palbociclib are approved CDK4/6 inhibitors for the treatment of HR+/HER2- breast cancer, but these drugs are not expected to show strong activity in brain tumors due to poor blood brain barrier penetration. Herein, we report the identification of a brain-penetrant CDK4/6 inhibitor derived from a literature molecule with low molecular weight and topological polar surface area (MW = 285 and TPSA = 66 Å2), but lacking the CDK2/1 selectivity profile due to the absence of a basic amine. Removal of a hydrogen bond donor via cyclization of the pyrazole allowed for the introduction of basic and semi-basic amines, while maintaining in many cases efflux ratios reasonable for a CNS program. Ultimately, a basic spiroazetidine (cpKa = 8.8) was identified that afforded acceptable selectivity over anti-target CDK1 while maintaining brain-penetration in vivo (mouse Kp,uu = 0.20-0.59). To probe the potency and selectivity, our lead compound was evaluated in a panel of glioblastoma cell lines. Potency comparable to abemaciclib was observed in Rb-wild type lines U87MG, DBTRG-05MG, A172, and T98G, while Rb-deficient cell lines SF539 and M059J exhibited a lack of sensitivity.