Central Nervous System Exposure Research Articles

Cerebrospinal fluid distribution and pharmacokinetics of ponatinib in Ph1+ acute lymphoblastic leukemia

AbstractTyrosine kinase inhibitors efficacy in central nervous system (CNS) disease remains uncertain. Ponatinib was studied for CNS distribution in 16 patients with Philadelphia-positive acute lymphoblastic leukemia. Cerebrospinal fluid concentrations fell below the 40 nM threshold, suggesting suboptimal CNS exposure.

Pharmacokinetic Modeling of the Effect of Tariquidar on Ondansetron Disposition into the Central Nervous System

PurposeSerotonin (5-HT3) receptor antagonists are promising agents for treatment of neuropathic pain. However, insufficient drug exposure at the central nervous system (CNS) might result in lack of efficacy. The goal of this study was to evaluate the impact of administration of a Pgp inhibitor (tariquidar) on ondansetron exposure in the brain, spinal cord, and cerebrospinal fluid in a wild-type rat model.MethodsOndansetron (10 mg/kg) and tariquidar (7.5 mg/kg) were administered intravenously, plasma and tissue samples were collected and analyzed by HPLC. A mathematical model with brain, spinal cord, cerebrospinal fluid and two systemic disposition compartments was developed to describe the data.ResultsThe results demonstrate that tariquidar at 7.5 mg/kg resulted in a complete inhibition of Pgp efflux of ondansetron in the brain and spinal cord. The compartmental model successfully captured pharmacokinetics of ondansetron in wild type and Pgp knockout (KO) animals receiving the drug alone or in wild type animals receiving the ondansetron and tariquidar combination.ConclusionsThe study provided important quantitative information on enhancement of CNS exposure to ondansetron using co-administration of Pgp Inhibitor in a rat model, which will be further utilized in conducting a clinical study. Tariquidar co-administration resulted in ondansetron CNS exposure comparable to observed in Pgp KO rats. Results also highlighted the effect of tariquidar on plasma disposition of ondansetron, which may not be dependent on Pgp inhibition, and should be evaluated in future studies.

Ubiquity of Synthetic Phenolic Antioxidants in Children's Cerebrospinal Fluid from South China: First Evidence for Their Penetration across the Blood-Cerebrospinal Fluid Barrier.

Synthetic phenolic antioxidants (SPAs) and relevant transformation products (TPs) are potentially neurotoxic pollutants to which humans are widely exposed. However, their penetration behavior across the brain barrier and associated exposure to the central nervous system (CNS) remain unknown. This study is the first to investigate a wide range of 30 SPAs and TPs, including emerging SPAs, in matched serum and cerebrospinal fluid (CSF) samples from children in Guangzhou, China. Sixty-two children of either sex aged <14 years with nonbloody CSF and complete clinical information were included. The findings demonstrated the ubiquitous occurrence of many SPAs and TPs, particularly BHT, 2,4-di-tert-butylphenol (DBP), AO 1010, AO 1076, BHT-Q, and BHT-quinol, not only in serum but also in the CSF. Median total concentrations of SPAs and TPs were up to 22.0 and 2.63 ng/mL in serum and 14.5 and 2.11 ng/mL in CSF, respectively. On calculating the penetration efficiencies across the blood-CSF barrier (BCSFB) (RCSF/serum, CCSF/Cserum) for selected SPAs and TPs, their RCSF/serum values (median 0.52-1.41) were highly related to their physicochemical properties, indicating that passive diffusion may be the potential mechanism of BCSFB penetration. In addition, the RCSF/serum values were positively correlated with the barrier permeability index RAlb (AlbuminCSF/Albuminserum), indicating that barrier integrity is an important determinant of BCSFB penetration. Overall, these results will improve our perception of human internal exposure to SPAs and lay a solid foundation for assessing the risk of CNS exposure to various SPAs.

Central Nervous System Distributional Kinetics of Selected Histone Deacetylase Inhibitors.

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

Predictions of Bedaquiline Central Nervous System Exposure in Patients with Tuberculosis Meningitis Using Physiologically based Pharmacokinetic Modeling.

The use of bedaquiline as a treatment option for drug-resistant tuberculosis meningitis (TBM) is of interest to address the increased prevalence of resistance to first-line antibiotics. To this end, we describe a whole-body physiologically based pharmacokinetic (PBPK) model for bedaquiline to predict central nervous system (CNS) exposure. A whole-body PBPK model was developed for bedaquiline and its metabolite, M2. The model included compartments for brain and cerebrospinal fluid (CSF). Model predictions were evaluated by comparison to plasma PK time profiles following different dosing regimens and sparse CSF concentrations data from patients. Simulations were then conducted to compare CNS and lung exposures to plasma exposure at clinically relevant dosing schedules. The model appropriately described the observed plasma and CSF bedaquiline and M2 concentrations from patients with pulmonary tuberculosis (TB). The model predicted a high impact of tissue binding on target site drug concentrations in CNS. Predicted unbound exposures within brain interstitial exposures were comparable with unbound vascular plasma and unbound lung exposures. However, unbound brain intracellular exposures were predicted to be 7% of unbound vascular plasma and unbound lung intracellular exposures. The whole-body PBPK model for bedaquiline and M2 predicted unbound concentrations in brain to be significantly lower than the unbound concentrations in the lung at clinically relevant doses. Our findings suggest that bedaquiline may result in relatively inferior efficacy against drug-resistant TBM when compared with efficacy against drug-resistant pulmonary TB.

Pharmacological Characterization of SDX-7320/Evexomostat: A Novel Methionine Aminopeptidase Type 2 Inhibitor with Anti-tumor and Anti-metastatic Activity.

Methionine aminopeptidase type 2 (METAP2) is a ubiquitous, evolutionarily conserved metalloprotease fundamental to protein biosynthesis which catalyzes removal of the N-terminal methionine residue from nascent polypeptides. METAP2 is an attractive target for cancer therapeutics based upon its over-expression in multiple human cancers, the importance of METAP2-specific substrates whose biological activity may be altered following METAP2 inhibition, and additionally, that METAP2 was identified as the target for the anti-angiogenic natural product, fumagillin. Irreversible inhibition of METAP2 using fumagillin analogues has established the anti-angiogenic and anti-tumor characteristics of these derivatives; however, their full clinical potential has not been realized due to a combination of poor drug-like properties and dose-limiting central nervous system (CNS) toxicity. This report describes the physicochemical and pharmacological characterization of SDX-7320 (evexomostat), a polymer-drug conjugate of the novel METAP2 inhibitor (METAP2i) SDX-7539. In vitro binding, enzyme, and cell-based assays demonstrated that SDX-7539 is a potent and selective METAP2 inhibitor. In utilizing a high molecular weight, water-soluble polymer to conjugate the novel fumagillol-derived, cathepsin-released, METAP2i SDX-7539, limitations observed with prior generation, small molecule fumagillol derivatives were ameliorated including reduced CNS exposure of the METAP2i, and prolonged half-life enabling convenient administration. Multiple xenograft and syngeneic cancer models were utilized to demonstrate the anti-tumor and anti-metastatic profile of SDX-7320. Unlike polymer-drug conjugates in general, reductions in small molecule-equivalent efficacious doses following polymer conjugation were observed. SDX-7320 has completed a phase I clinical safety study in patients with late-stage cancer and is currently being evaluated in multiple phase Ib/II clinical studies in patients with advanced solid tumors.

Mechanistic Modeling of Intrathecal Chemotherapy Pharmacokinetics in the Human Central Nervous System.

The pharmacokinetics of intrathecally administered antibody or small-molecule drugs in the human central nervous system (CNS) remains poorly understood. This study aimed to provide mechanistic and quantitative perspectives on the CNS pharmacokinetics of intrathecal chemotherapy, by using a physiologically based pharmacokinetic (PBPK) modeling approach. A novel CNS PBPK model platform was developed and verified, which accounted for the human CNS general anatomy and physiologic processes governing drug distribution and disposition. The model was used to predict CNS pharmacokinetics of antibody (trastuzumab) and small-molecule drugs (methotrexate, abemaciclib, tucatinib) following intraventricular injection or intraventricular 24-hour infusion, and to assess the key determinants of drug penetration into the deep brain parenchyma. Intraventricularly administered antibody and small-molecule drugs exhibited distinct temporal and spatial distribution and disposition in human CNS. Both antibody and small-molecule drugs achieved supratherapeutic or therapeutic concentrations in the cerebrospinal fluid (CSF) compartments and adjacent brain tissue. While intrathecal small-molecule drugs penetrated the deep brain parenchyma to a negligible extent, intrathecal antibodies may achieve therapeutic concentrations in the deep brain parenchyma. Intraventricular 24-hour infusion enabled prolonged CNS exposure to therapeutically relevant concentrations while avoiding excessively high and potentially neurotoxic drug concentrations. CNS PBPK modeling, in line with available clinical efficacy data, confirms the therapeutic value of intrathecal chemotherapy with antibody or small-molecule drugs for treating neoplastic meningitis and warrants further clinical investigation of intrathecal antibody drugs to treat brain parenchyma tumors. Compared with intraventricular injection, intraventricular 24-hour infusion may mitigate neurotoxicity while retaining potential efficacy.

Evaluating the photocatalytic performance of MOF coated on glass for degradation of gaseous styrene under visible light

Styrene is a volatile organic compound with various applications, especially in the plastics and paint industries. Exposure to it leads to symptoms such as weakness, suppression of the central nervous system, and nausea, and prolonged exposure to it increases the risk of cancer. Its removal from the air is a topic that researchers have considered. Various methods such as absorption, membrane separation, thermal and catalytic oxidation, biofiltration have been used to remove these compounds. The disadvantages of these compounds include the need for high energy, production of secondary pollutants, large space, providing environmental conditions (temperature and humidity) and long time. The photocatalyst process is considered as an advanced process due to the production of low and safe secondary pollutants. MOFs are nanoparticles with unique photocatalytic properties that convert organic pollutants into water and carbon dioxide under light irradiation and in environmental conditions, which prevent the production of secondary pollutants. The present study aimed to investigate the efficiency of MIL100 (Fe) nanoparticles coated on glass in removing styrene vapor from the air. Surface morphology, crystal structure, pore size, functional groups, and chemical composition of the catalyst were analyzed by SEM, XRD, BET, FTIR, and EDX analysis. The effect of parameters such as initial pollutant concentration, temperature, time, relative humidity, and nanoparticle concentration was evaluated as effective parameters in the removal process. Based on the results, MIL100 (Fe) 0.6 g/l with an 89% removal rate had the best performance for styrene removal. Due to its optimal removal efficiency, it can be used to degrade other air pollutants.

Design, Synthesis, Pharmacology, and In Silico Studies of (1S,2S,3S)-2-((S)-Amino(carboxy)methyl)-3-(carboxymethyl)cyclopropane-1-carboxylic Acid (LBG30300): A Picomolar Potency Subtype-Selective mGlu2 Receptor Agonist.

Metabotropic glutamate (Glu) receptors (mGlu receptors) play a key role in modulating excitatory neurotransmission in the central nervous system (CNS). In this study, we report the structure-based design and pharmacological evaluation of densely functionalized, conformationally restricted glutamate analogue (1S,2S,3S)-2-((S)-amino(carboxy)methyl)-3-(carboxymethyl)cyclopropane-1-carboxylic acid (LBG30300). LBG30300 was synthesized in a stereocontrolled fashion in nine steps from a commercially available optically active epoxide. Functional characterization of all eight mGlu receptor subtypes showed that LBG30300 is a picomolar agonist at mGlu2 with excellent selectivity over mGlu3 and the other six mGlu receptor subtypes. Bioavailability studies on mice (IV administration) confirm CNS exposure, and an in silico study predicts a binding mode of LBG30300 which induces a flipping of Tyr144 to allow for a salt bridge interaction of the acetate group with Arg271. The Tyr144 residue now prevents Arg271 from interacting with Asp146, which is a residue of differentiation between mGlu2 and mGlu3 and thus could explain the observed subtype selectivity.

Abstract A089: NST-628 is a potent, fully brain-penetrant, RAS/MAPK pathway molecular glue inhibitor with efficacy in CNS tumor models

Abstract Alterations in the RAS/RAF/MEK/ERK signaling cascade are common across multiple solid tumor types and aberrant signaling of the pathway is a driver for RAS- and RAF-driven cancers. Apart from approved mutation selective inhibitors for BRAF Class I and KRAS G12C mutations, other mutations of RAS and RAF are not directly addressable by currently approved inhibitors and there is a need for inhibitors with superior efficacy, durability and tolerability for the MAPK pathway in RAS- and RAF-driven cancers. Currently approved inhibitors of the RAS-MAPK pathway also have limited central nervous system (CNS) exposure and an area of significant unmet patient need exists for patients with primary and metastatic tumors harboring RAS-MAPK pathway alterations. NST-628 has CNS permeability, with a Kpu,u &amp;gt;1, that is far higher than trametinib or VS-6766 (Kpu,u of 0.11 and 0.18 respectively). The predicted effective human half-life of NST-628 (~9h) is amenable to daily dosing in the clinic. In vivo, NST-628 leads to a dose-dependent inhibition of the MAPK pathway in murine brain tissue[KS1] as measured by phospho-ERK. In an intracranial luciferase-tagged SK-MEL-2 xenograft model (NRAS Q61R), NST-628 leads to tumor regressions with a daily dosing regimen (3 mg/kg QD). Tumor regressions as measured by bioluminescent imaging (BLI) were only seen with NST-628 (50%) in the SK-MEL-2 model, whereas trametinib and VS-6766 showed minimal efficacy due to low exposure in the CNS. In the NF-1 mutant MeWo (NF1 Q1336*) melanoma intracranial xenograft model (luciferase tagged), NST-628 led to tumor regressions at two dosing regimens (1, 3 mg/kg QD) and tumor stasis at lower doses (0.3 mg/kg) as measured by BLI. In contrast, the brain penetrant type II RAF inhibitor DAY101 (formerly TAK580) showed no efficacy in the MeWo model and resulted in paradoxical pathway hyper-activation, as determined by enhanced PD response and tumor growth. Only NST-628 effectively inhibited MAPK signaling as measured by DUSP6 transcript in a time- and dose-dependent manner. Overall, survival of mice harboring MeWo intracranial tumors was also significantly increased versus vehicle control and DAY101 treatment groups. These data collectively support best-in-class potential of NST-628 and initiation of clinical trials in patients with solid tumors harboring RAS- and RAF-mutations, including those presenting clinically with brain metastases and primary intracranial tumors. Citation Format: Meagan B Ryan, Chun Li, Yongxin Han, Klaus P Hoeflich, Michael R Hale, Margit Hagel. NST-628 is a potent, fully brain-penetrant, RAS/MAPK pathway molecular glue inhibitor with efficacy in CNS tumor models [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A089.

Hypertension: smoking or quit. Clinical difficulties

The review describes the combination of hypertension (HTN) and smoking in the same person. The prevalence of this combination in the Russian population is high due to the widespread prevalence of both HTN and smoking. There are following unidirectional negative impact of both factors on vascular system: endothelium, microcirculation, large vessel stiffness, contributes to higher blood pressure among such patients and leads to early target organ damage, increased cardiovascular and death risk. Smoking cessation is the main effective measure of primary and secondary prevention among smoking patients with HTN. The review provides links to well-known articles and the results of studies showing the benefits of smoking cessation. However, it is not easy for smoking patients with hypertension to quit smoking: on the one hand, nicotine addiction, similar to drugs, through central nervous system exposure, makes the habit stable, while on the other hand, the low motivation of such patients due to young age and the absence of current smoking consequences, which complicates working with such patients. Knowledge of algorithms for working with smoking patients and methods of non-drug support can help practitioners supporting such patients at the stage of smoking cessation.

Comparison of pharmaceutical properties and biological activities of prednisolone, deflazacort, and vamorolone in DMD disease models.

Duchenne muscular dystrophy (DMD) is a progressive disabling X-linked recessive disorder that causes gradual and irreversible loss of muscle, resulting in early death. The corticosteroids prednisone/prednisolone and deflazacort are used to treat DMD as the standard of care; however, only deflazacort is FDA approved for DMD. The novel atypical corticosteroid vamorolone is being investigated for treatment of DMD. We compared the pharmaceutical properties as well as the efficacy and safety of the three corticosteroids across multiple doses in the B10-mdx DMD mouse model. Pharmacokinetic studies in the mouse and evaluation of p-glycoprotein (P-gP) efflux in a cellular system demonstrated that vamorolone is not a strong P-gp substrate resulting in measurable central nervous system (CNS) exposure in the mouse. In contrast, deflazacort and prednisolone are strong P-gp substrates. All three corticosteroids showed efficacy, but also side effects at efficacious doses. After dosing mdx mice for two weeks, all three corticosteroids induced changes in gene expression in the liver and the muscle, but prednisolone and vamorolone induced more changes in the brain than did deflazacort. Both prednisolone and vamorolone induced depression-like behavior. All three corticosteroids reduced endogenous corticosterone levels, increased glucose levels, and reduced osteocalcin levels. Using micro-computed tomography, femur bone density was decreased, reaching significance with prednisolone. The results of these studies indicate that efficacious doses of vamorolone, are associated with similar side effects as seen with other corticosteroids. Further, because vamorolone is not a strong P-gp substrate, vamorolone distributes into the CNS increasing the potential CNS side-effects.

Dosimetry of the PIM1 Pion Beam at the Paul Scherrer Institute for Radiobiological Studies of Mice.

Significant past work has identified unexpected risks of central nervous system (CNS) exposure to the space radiation environment, where long-lasting functional decrements have been associated with multiple ion species delivered at low doses and dose rates. As shielding is the only established intervention capable of limiting exposure to the dangerous radiation fields in space, the recent discovery that pions, emanating from regions of enhanced shielding, can contribute significantly to the total absorbed dose on a deep space mission poses additional concerns. As a prerequisite to biological studies evaluating pion dose equivalents for various CNS exposure scenarios of mice, a careful dosimetric validation study is required. Within our ultimate goal of evaluating the functional consequences of defined pion exposures to CNS functionality, we report in this article the detailed dosimetry of the PiMI pion beam line at the Paul Scherrer Institute, which was developed in support of radiobiological experiments. Beam profiles and contamination of the beam by protons, electrons, positrons and muons were characterized prior to the mice irradiations. The dose to the back and top of the mice was measured using thermoluminescent dosimeters (TLD) and optically simulated luminescence (OSL) to cross-validate the dosimetry results. Geant4 Monte Carlo simulations of radiation exposure of a mouse phantom in water by charged pions were also performed to quantify the difference between the absorbed dose from the OSL and TLD and the absorbed dose to water, using a simple model of the mouse brain. The absorbed dose measured by the OSL dosimeters and TLDs agreed within 5-10%. A 30% difference between the measured absorbed dose and the dose calculated by Geant4 in the dosimeters was obtained, probably due to the approximated Monte Carlo configuration compared to the experiment. A difference of 15-20% between the calculated absorbed dose to water at a 5 mm depth and in the passive dosimeters was obtained, suggesting the need for a correction factor of the measured dose to obtain the absorbed dose in the mouse brain. Finally, based on the comparison of the experimental data and the Monte Carlo calculations, we consider the dose measurement to be accurate to within 15-20%.

An LC-ESI-MS/MS method for determination of ondansetron in low-volume plasma and cerebrospinal fluid: Method development, validation, and clinical application

Ondansetron is used in clinical settings as an antiemetic drug. Although the animal studies showed its potential effectiveness also in treating neuropathic pain, the results from humans are inconclusive. The lack of efficacy of ondansetron in a subset of patients might be due to the overexpression of P-glycoprotein, which could result in low concentrations of ondansetron in the central nervous system (CNS). A surrogate of the CNS exposure might be drug concentration in the cerebrospinal fluid (CSF), especially in humans, as assessing the drug disposition directly in the patient’s brain would be challenging. The study aimed to develop a sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to determine concentrations of ondansetron in human K3EDTA plasma and CSF. Ondansetron was extracted from biological matrices by liquid-liquid extraction. The quantification was performed on a Sciex QTRAP 6500+ mass spectrometer with labeled ondansetron as an internal standard. The calibration range was 0.25–350 ng/mL in plasma and 0.025–100 ng/mL in CSF; for both matrices, 25 µL of samples was required for the assays. The method was validated according to the FDA and EMA guidelines and showed acceptable results. A pilot study confirmed its suitability for clinical samples: after 4–16 mg of intravenous ondansetron, the determined concentrations in plasma were 1.22–235.90 ng/mL, while in CSF - 0.018–11.93 ng/mL. In conclusion, the developed method fulfilled all validation requirements and can be applied to pharmacokinetic studies assessing the CNS ondansetron exposure in humans. The method’s advantages, such as a low volume of matrix and a wide calibration range, support its use in a study in which rich sampling and various drug doses are expected.

Discovery of a brain-sparing GIRK1/4 inhibitor for pharmacological cardioversion of atrial fibrillation

Atrial fibrillation (AF) is the most common cardiac arrhythmia, and a significant risk factor for ischemic stroke and heart failure. Marketed anti-arrhythmic drugs can restore sinus rhythm, but with limited efficacy and significant toxicities, including potential to induce ventricular arrhythmia. Atrial-selective ion channel drugs are expected to restore and maintain sinus rhythm without risk of ventricular arrhythmia. One such atrial-selective channel target is GIRK1/4 (G-protein regulated inwardly rectifying potassium channel 1/4). Here we describe 14b, a potent GIRK1/4 inhibitor developed to cardiovert AF to sinus rhythm while minimizing central nervous system exposure – an issue with preceding GIRK1/4 clinical candidates.

The Blood-Brain Barrier: Implications for Experimental Cancer Therapeutics.

The blood-brain barrier is critically important for the treatment of both primary and metastatic cancers of the central nervous system (CNS). Clinical outcomes for patients with primary CNS tumors are poor and have not significantly improved in decades. As treatments for patients with extracranial solid tumors improve, the incidence of CNS metastases is on the rise due to suboptimal CNS exposure of otherwise systemically active agents. Despite state-of-the art surgical care and increasingly precise radiation therapy, clinical progress is limited by the ability to deliver an effective dose of a therapeutic agent to all cancerous cells. Given the tremendous heterogeneity of CNS cancers, both across cancer subtypes and within a single tumor, and the range of diverse therapies under investigation, a nuanced examination of CNS drug exposure is needed. With a shared goal, common vocabulary, and interdisciplinary collaboration, the field is poised for renewed progress in the treatment of CNS cancers.

Efficacy evaluation of a novel vectorized anti‐tau antibody, targeting a C‐terminal phospho‐tau epitope, using systemic dosing of a blood brain barrier penetrant AAV capsid in mouse models of tauopathies.

AbstractBackgroundAnti‐tau immunotherapy has become a promising therapy for Alzheimer’s disease (AD) and tauopathies. With the hypothesis that tau pathology spreads via cell‐to‐cell transmission, including trans‐synaptic propagation, success of anti‐tau immunotherapy relies, in part, on the identification of efficacious antibodies and their delivery to affected or vulnerable brain regions with sufficient or enhanced exposure in the CNS. We have previously demonstrated broad distribution and expression of vectorized anti‐tau antibodies in the mouse brain using a blood brain barrier penetrant capsid, VOY101, administered intravenously (IV).MethodSeveral novel anti‐tau antibodies that met the target profile have been generated and are being evaluated in vivo (Liu et al, submitted as a separated abstract in AAIC 2022).ResultOne of the antibodies discovered, antibody 1, exhibits strong affinity for PHF‐tau, demonstrates specific binding to tau pathology on brain sections of AD and PSP patients, and potently prevents PHF seeding and propagation in vitro and in vivo. This antibody recognizes a phospho‐specific epitope in the C‐terminal region of tau and shows significant reduction of tau pathology in an AD‐PHF induced P301S hippocampal seeding and propagation model. Furthermore, we have vectorized antibody 1 into an AAV expression vector with by BBB penetrant capsid and are evaluating it in two independent mouse models of tauopathy.ConclusionThis gene therapy‐based approach has potential advantages over traditional passive immunization, including 1) continuous expression of antibody in the central nervous system (CNS) after a single gene therapy administration compared to repetitive administrations of high dose of antibody by passive immunotherapy; 2) increased CNS exposure of tau antibody relative to passive immunotherapy; and 3) the potential to target intracellular tau aggregates which are less effectively accessed by passively delivered antibody. These results add to accumulating evidence that systemic dosing of a vectorized anti‐tau antibody using a BBB‐penetrant AAV capsid results in reduced tau pathology and may represent a new single‐dose therapeutic strategy for treating various tauopathies.

DDDR-37. GB5121 IS A NOVEL, IRREVERSIBLE, COVALENT BTK INHIBITOR WITH HIGH SELECTIVITY AND CNS-PENETRANCE FOR TREATMENT OF CNS MALIGNANCIES

Abstract Inhibitors of Bruton’s tyrosine kinase (BTK) are approved treatments for several B cell lymphomas. However, they are characterized by modest selectivity and/or limited central nervous system (CNS) exposure which is important in instances of CNS disease. GB5121 is an orally available, selective, irreversible, covalent small molecule BTK inhibitor that was designed to address these limitations. Here, GB5121 was profiled for selectivity, potency, inactivation kinetics and CNS penetrance in comparison to ibrutinib. GB5121 pharmacodynamic properties were evaluated using both cell-free enzymatic and cell-based functional assays, showing nM potency for BTK inhibition and rapid BTK inactivation kinetics (Kinact/Ki) in both peripheral and CNS tissue, a critical parameter for irreversible inhibitors. In a kinome scan, GB5121 exhibited high kinase selectivity against 349 kinases with only TEC/TXK demonstrating &amp;gt; 50% inhibition at 1 µM; GB5121 did not inhibit phosphorylation of EGFR in a cell-based assay. When compared with other BTK inhibitors, GB5121 showed superior CNS target occupancy using a probe-based ELISA measuring free BTK in the brain of mice receiving 3 daily oral doses. GB5121 also demonstrated significantly higher brain to plasma ratio in mice with an intact blood brain barrier. In non-human primates, a 1:1 brain to plasma concentration ratio was demonstrated for GB5121 for up to 8 hours with both oral (30 mg/kg) and IV (2 mg/kg) doses. Together, these features differentiate GB5121 from both FDA-approved BTK inhibitors, as well as those currently under clinical investigation. Our data supports the use of GB5121 in clinical trials where BTK is a known driver of malignancies, including CNS lymphoma. This research has been previously presented at the AACR Annual Meeting 2022.

A Biodistribution Study of the Radiolabeled Kv1.3-Blocking Peptide DOTA-HsTX1[R14A] Demonstrates Brain Uptake in a Mouse Model of Neuroinflammation.

The voltage-gated potassium channel Kv1.3 regulates the pro-inflammatory function of microglia and is highly expressed in the post-mortem brains of individuals with Alzheimer's and Parkinson's diseases. HsTX1[R14A] is a selective and potent peptide inhibitor of the Kv1.3 channel (IC50 ∼ 45 pM) that has been shown to decrease cytokine levels in a lipopolysaccharide (LPS)-induced mouse model of inflammation. Central nervous system exposure to HsTX1[R14A] was previously detected in this mouse model using liquid chromatography with tandem mass spectrometry, but this technique does not report on the spatial distribution of the peptide in the different brain regions or peripheral organs. Herein, the in vivo distribution of a [64Cu]Cu-labeled DOTA conjugate of HsTX1[R14A] was observed for up to 48 h by positron emission tomography (PET) in mice. After subcutaneous administration to untreated C57BL/6J mice, considerable uptake of the radiolabeled peptide was observed in the kidney, but it was undetectable in the brain. Biodistribution of a [68Ga]Ga-DOTA conjugate of HsTX1[R14A] was then investigated in the LPS-induced mouse model of neuroinflammation to assess the effects of inflammation on uptake of the peptide in the brain. A control peptide with very weak Kv1.3 binding, [68Ga]Ga-DOTA-HsTX1[R14A,Y21A,K23A] (IC50 ∼ 6 μM), was also tested. Significantly increased uptake of [68Ga]Ga-DOTA-HsTX1[R14A] was observed in the brains of LPS-treated mice compared to mice treated with control peptide, implying that the enhanced uptake was due to increased Kv1.3 expression rather than simply increased blood-brain barrier disruption. PET imaging also showed accumulation of [68Ga]Ga-DOTA-HsTX1[R14A] in inflamed joints and decreased clearance from the kidneys in LPS-treated mice. These biodistribution data highlight the potential of HsTX1[R14A] as a therapeutic for the treatment of neuroinflammatory diseases mediated by overexpression of Kv1.3.

The 46.1 Antibody Mediates Neurotensin Uptake into the CNS and the Effects Depend on the Route of Intravenous Administration.

Central nervous system (CNS) exposure to blood-borne biotherapeutics is limited by the restrictive nature of the brain vasculature. In particular, tightly sealed endothelial cells of the blood–brain barrier (BBB) prevent the uptake of protein and gene medicines. An approach to increase the bioavailability of such therapeutics is harnessing the BBB endothelial cells’ own receptor-mediated transcytosis (RMT) mechanisms. Key to this process is a targeting ligand that can engage a BBB-resident RMT receptor. We recently identified an antibody, named 46.1, that accumulates in the mouse brain after intravenous injection. To further characterize the brain targeting and penetrating properties of clone 46.1, we conjugated neurotensin (NT) to an scFv-Fc form of the antibody (46.1-scFv-Fc-LongLinker-NT). While centrally administered NT decreases the core body temperature and locomotor activity, effects attributed to two spatially segregated brain areas, systemically administered NT has limited effects. Hence, NT can be used as a model therapeutic payload to evaluate the brain penetration of BBB-targeting antibodies and their capability to accumulate in discrete brain areas. We demonstrate that intravenously administered 46.1-scFv-Fc-LL-NT can elicit transient hypothermia and reduce drug-induced hyperlocomotion, confirming that 46.1 can deliver drug cargo to the CNS at pharmacologically relevant doses. Interestingly, when two intravenous administration routes in mice, retro-orbital and tail vein, were compared, only retro-orbital administration led to transient hypothermia. We further explored the retro-orbital route and demonstrated that the 46.1-scFv-Fc-LL-NT could enter the brain arterial blood supply directly from the retro-orbital/cavernous sinus. Taken together, the 46.1 antibody is capable of transporting drug cargo into the CNS, and at least of a portion of its CNS accumulation occurs via the cavernous sinus–arterial route.