Cerebrospinal Fluid Exposure Research Articles

Comparative CNS Pharmacology of the Bruton's Tyrosine Kinase (BTK) Inhibitor Tolebrutinib Versus Other BTK Inhibitor Candidates for Treating Multiple Sclerosis.

Tolebrutinib is a covalent BTK inhibitor designed and selected for potency and CNS exposure to optimize impact on BTK-dependent signaling in CNS-resident cells. We applied a translational approach to evaluate three BTK inhibitors in Phase 3 clinical development in MS with respect to their relative potency to block BTK-dependent signaling and exposure in the CNS METHODS: We used in vitro kinase and cellular activation assays, alongside pharmacokinetic sampling of cerebrospinal fluid (CSF) in the non-human primate cynomolgus to estimate the ability of these candidates (evobrutinib, fenebrutinib, and tolebrutinib) to block BTK-dependent signaling inside the CNS. In vitro kinase assays demonstrated that tolebrutinib reacted with BTK 65-times faster than evobrutinib, while fenebrutinib, a classical reversible antagonist with a Ki value of 4.7 nM and slow off-rate (1.54 x 10-5 s-1), also had an association rate 1760-fold slower (0.00245 μM-1 * s-1). Estimates of cellular potency were largely consistent with the in vitro kinase assays, with an estimated IC50 of 0.7 nM for tolebrutinib against 33.5 nM for evobrutinib and 2.9 nM for fenebrutinib. We then observed that evobrutinib, fenebrutinib, and tolebrutinib achieved similar levels of exposure in non-human primate CSF after oral doses of 10 mg/kg. However, tolebrutinib CSF exposure (4.8 ng/mL) (kp,uu CSF=0.40) exceeded the IC90 (the estimated concentration inhibiting 90% of kinase activity) value, while evobrutinib (3.2 ng/mL) (kp,uu CSF=0.13) and fenebrutinib (12.9 ng/mL) (kp,uu CSF=0.15) failed to reach the estimated IC90 values. Tolebrutinib was the only candidate of the three that attained relevant CSF exposure in non-human primates.

Therapeutic activity of a new KRASG12C inhibitor JMKX001899 in brain metastases: Preclinical models of KRASG12C-mutant non-small cell lung cancer.

8630 Background: Development of therapies targeting KRAS have transformed KRAS from undruggable to druggable. However, patients with KRASG12C-mutant non-small cell lung cancer (NSCLC) with brain metastasis (BM) have a poor prognosis. JMKX001899, a new potent oral small-molecule KRASG12C inhibitor, irreversibly and selectively binds KRASG12C, locking it in its inactive state. Its antitumor activity in the intracranial is unclear. Methods: In order to verify the activity of new KRASG12C inhibitor JMKX001899 in KRASG12C-mutant NSCLC with BM, murine models were employed for investigating the efficacy. 5-6 weeks old female Balb/c nude mice were implanted intracranially with H358-Luc and H23-Luc cells. Baseline bioluminescent imaging (BLI) was used to monitor the tumor burden and total flux was calculated by Living Image. Ten days after implantation, oral once-daily dosing of vehicle, sotorasib (AMG510) at 100 mg/kg, 30 mg/kg and JMKX001899 at 100 mg/kg, 30 mg/kg, 10 mg/kg was started and continued for 21 days. The tumor burden was monitored by BLI weekly. The mice were weighed per 3 days. Results: At the 30mg/kg and 100mg/kg dose level, the mean cerebrospinal fluid (CSF)-to-unbound plasma ratios (Kp,uu) was 0.18-0.50, which determined JMKX001899 CSF exposure was sufficient to mediate regression tumors in experimental BM models. In mice bearing intracranial xenografts of H358-Luc and H23-Luc, oral JMKX001899 100mg/kg once daily treatment for 21 days resulted in significant inhibition of brain tumor growth revealed by BLI, compared with vehicle and AMG510. H358-Luc xenografts responded particularly well to the JMKX001899 treatment, showed by the ratio of 14 days and 0 days total bioluminescence flux (vehicle vs. AMG510 100mg/kg vs. JMKX001899 100mg/kg, 0.85 vs. 0.89 vs. 0.01, p < 0.05). In addition, JMKX001899 at 100mg/kg once daily demonstrated an increase in over survival. JMKX001899 100 mg/kg once daily treatment was well tolerated with minimal sign of overt toxicity or animal weight loss. Conclusions: The preclinical models in vivo suggest the new KRASG12C inhibitor JMKX001899 penetrates the central nervous system and show great inhibition of KRASG12C-mutant NSCLC with BM, which demonstrates therapeutic activity of JMKX001899 in BM. These data support further development of JMKX001899 in patients with KRASG12C-mutant NSCLC with BM.

Central Nervous System Antimicrobial Exposure and Proposed Dosing for Anthrax Meningitis.

The high mortality of systemic anthrax is likely a consequence of the severe central nervous system inflammation that occurs in anthrax meningitis. Effective treatment of such infections requires, at a minimum, adequate cerebrospinal fluid (CSF) antimicrobial concentrations. We reviewed English medical literature and regulatory documents to extract information on serum and CSF exposures for antimicrobials with in vitro activity against Bacillus anthracis. Using CSF pharmacokinetic exposures and in vitro B. anthracis susceptibility data, we used population pharmacokinetic modeling and Monte Carlo simulations to determine whether a specific antimicrobial dosage would likely achieve effective CSF antimicrobial activity in patients with normal to inflamed meninges (ie, an intact to markedly disrupted blood-brain barrier). The probability of microbiologic success at achievable antimicrobial dosages was high (≥95%) for ciprofloxacin, levofloxacin (500 mg every 12 hours), meropenem, imipenem/cilastatin, penicillin G, ampicillin, ampicillin/sulbactam, doxycycline, and minocycline; acceptable (90%-95%) for piperacillin/tazobactam and levofloxacin (750 mg every 24 hours); and low (<90%) for vancomycin, amikacin, clindamycin, and linezolid. Prompt empiric antimicrobial therapy of patients with suspected or confirmed anthrax meningitis may reduce the high morbidity and mortality. Our data support using several β-lactam-, fluoroquinolone-, and tetracycline-class antimicrobials as first-line and alternative agents for treatment of patients with anthrax meningitis; all should achieve effective microbiologic exposures. Our data suggest antimicrobials that should not be relied on to treat suspected or documented anthrax meningitis. Furthermore, the protein synthesis inhibitors clindamycin and linezolid can decrease toxin production and may be useful components of combination therapy.

Developing Topics.

E2814 is a human IgG1 antibody that binds to microtubule binding region of tau (MTBR-tau), a critical domain for the seeding and spreading of tau pathology in Alzheimer's Disease (AD). It is being developed for the treatment of AD by stopping tau propagation and slowing the associated cognitive decline. E2814 has been evaluated in healthy and Dominantly Inherited Alzheimer's Disease (DIAD) participants for safety, pharmacokinetics (PK), and cerebrospinal fluid (CSF) target engagement. Seventy-two healthy participants were enrolled in single and multiple ascending dose cohorts of Study 001 (NCT04231513) and received randomized, double-blind, placebo-controlled treatment of E2814. Intravenous infusion of single and multiple doses (once every 4 weeks for three times) were administered. In Study 103 (NCT04971733), 7 DIAD participants with confirmed mutations and mild to moderate cognitive impairment were enrolled and received open-label treatment of E2814. Four sequential doses were evaluated as intravenous infusion every 4 weeks. Serum and CSF PK, and CSF target engagement (by analyzing both bound and unbound MTBR-tau299 and MTBR-tau354 [containing the epitopes of E2814]) were measured in both healthy and DIAD participants, as well as safety assessments. CSF MTBR-tau243 recapitulating AD tau pathology was also measured in DIAD participants using methods previously described (Horie et al., J Prev Alz Dis 2022). E2814 was safe and well-tolerated over a wide dose range. No infusion-related reactions or other clinically significant safety signals were observed with single or repeated dose administration for up to 15 months. E2814 showed a dose-related increase in serum and CSF exposure with CSF-to-serum ratio of ∼0.2%, comparable between healthy and DIAD participants. CSF target engagement was demonstrated by concentration-related and sustained MTBR-tau299 and MTBR-tau354 binding in healthy and DIAD participants. More importantly, E2814 elicited a rapid and robust reduction on MTBR-tau243, a neurofibrillary tangle-specific biomarker, in DIAD participants. E2814 demonstrated promising safety, PK and CSF target engagement profile in DIAD participants, and showed evidence of downstream effects on tangle-specific biomarker MTBR-tau243. Data supports further evaluation in the ongoing Ph2/3 DIAN-TU Tau NexGen Platform Study in DIAD (NCT05269394) and future exploration in sporadic AD.

TMIC-41. LATERAL VENTRICLE PROXIMITY OF GLIOBLASTOMA IS ASSOCIATED WITH LOCALIZED MALIGNANCY-PROMOTING MOLECULAR SIGNATURES IN IMAGE-GUIDED BIOPSIES

Abstract Glioblastoma (GBM) is the most devastating and common form of primary brain cancer in adults. Tumor location plays a significant role in patient prognosis; in particular, GBM tumors contacting the lateral ventricles (LVs) are more aggressive than LV-distal counterparts. This may be due to interaction of tumors with unique features of the LV microenvironment, including the cerebrospinal fluid (CSF) and neural progenitor cells (NPCs) of the subventricular zone. Despite LV contact having a role on tumor malignancy, studies using bulk tumor samples to identify molecular contributors to LV-contacting prognosis have been only moderately successful, in large part due to intratumoral heterogeneity. Here, we leverage intraoperative surgical navigation to collect matched biopsies of adult LV-contacting primary GBM at locations proximal (&amp;lt; 2 cm) and distal ( &amp;gt; 2 cm) from the LV. Matched samples were collected from ten patients with equal sex distribution and an age range of 46 to 81 years. RNA sequencing of biopsies revealed a localized transcriptional signature of gene expression in LV-proximal locations. These genes include those identified to be associated with GBM malignancy and upregulated in primary GBM cells by CSF exposure or co-culture with NPCs, such as SERPINA3, CD44, and CTSB, as well as many newly identified genes. Implicated biological pathways involved in this transcriptional signature involve inflammation, chemotaxis, Notch signaling, angiogenesis, integrin signaling, and more. The top 50 upregulated genes by LV-proximity of GBM were used to cluster patient transcriptomes from the TCGA GBM database and evaluate the contribution to patient outcome. This analysis revealed a cluster with significantly extended overall survival that displayed a general downregulation of LV-proximity genes. Ultimately, these studies provide evidence that regional tumoral analysis is essential in identifying molecular signatures associated with microenvironmental influence, and that LV-contacting GBM has regional gene expression changes in many genes associated with malignancy.

EXTH-56. CSF PENETRATION COMPARISON OF TWO ORAL FORMULATIONS OF APL-101 IN A PRE-CLINICAL NON-HUMAN PRIMATE MODEL

Abstract Vebreltinib (APL-101) is a potent c-Met inhibitor which has shown efficacy in murine pre-clinical models and is under clinical trial evaluation for several cancer types, including adult high-grade glioma. The capsule formulation (APL-101-Cap) was reformulated as a pill (APL-101-Pill) with the expectation of increasing bioavailability. We determined and compared the plasma pharmacokinetics (PK) and cerebrospinal fluid (CSF) penetration of APL-101-Cap and APL-101-Pill in a non-human primate (NHP) model. METHODS: Seven animals, previously developed as serial CSF access models, were utilized across all studies. One NHP was studied with both formulations. Both formulations were administered orally as a single drug/single dose in serial studies, followed by serial plasma and CSF sample collections from 0-96 hours. Dosages: APL-101-Cap 3.25 mg/kg (n = 3) or 6.5 mg/kg (n = 4) and APL-101-Pill 5 mg/kg (n = 4). The resulting plasma concentrations from each formulation were quantified by LC-MS/MS. PK parameters were calculated via noncompartmental methods, and results were analyzed to assess CNS penetration (AUCCSF:AUCPlasma) and compare formulations. RESULTS: Mean (range) for plasma and CSF PK exposure [AUCLAST (hr*ng/mL)] reported. APL-101-Cap plasma exposure, regardless of dose, was more variable than APL-101-Pill. APL-101-Pill had lower exposure in both plasma (AUC 1893.0 (841-2865)] and CSF [AUC 27.7 (5.0-65.0)] than APL-101-Cap regardless of dose [(3.25 mg/kg): plasma AUC 9621.0 (1331-23132) and CSF AUC 688.0 (24-1352); 6.5 mg/kg: plasma AUC 6208.0 (1424-19800) and CSF AUC 606.0 (34-1179)]. CNS penetrance was higher with APL-101-Cap (3.2% [0.5-5.8%] at 3.25mg/kg; 4.2% (2.4-6%) at 6.5 mg/kg), but also more variable when compared to APL-101-Pill (1.2% [0.6-2.3%]). For APL-101-Cap, one animal at 3.25 mg/kg and two animals at 6.25 mg/kg had undetectable CSF concentrations. CONCLUSION: In this NHP model, single-dose oral administration of APL-101-Pill resulted in lower, but less variable plasma and CSF exposure compared to APL-101-Cap. APL-101-Cap CSF exposure was unaffected by dose escalation.

Comparative CNS Pharmacology of Tolebrutinib Versus Other BTK Inhibitor Candidates for Treating MS (S46.007)

<h3>Objective:</h3> Compare three BTK inhibitors in Phase 3 development in multiple sclerosis (MS) according to relative potencies and CNS exposure. <h3>Background:</h3> Modulating CNS neuroinflammation represents a paradigm shift for treating MS. Tolebrutinib is a covalent BTK inhibitor designed and selected to optimize both potency and exposure to modulate BTK signaling within the CNS. <h3>Design/Methods:</h3> We used in vitro kinase assays, cellular activation assays, and pharmacokinetic (PK) sampling of cerebrospinal fluid (CSF) in the non-human primate (NHP) cynomolgus to compare tolebrutinib, evobrutinib, fenebrutinib under identical conditions. We then evaluated the CSF PK of tolebrutinib in healthy volunteers. <h3>Results:</h3> Kinase assays demonstrated that tolebrutinib reacted with BTK 65-times faster than evobrutinib (K_inact/K_i = 4.37 × 10⁻³ and 6.82 × 10⁻⁵ nM⁻¹*s⁻¹, respectively). Fenebrutinib, a reversible antagonist had a K_i value of 4.7 nM. Because of the slow off-rate (1.54 × 10⁻⁵ s⁻¹), the forward rate was also very slow (3.28 × 10³ M⁻¹ * s⁻¹). Estimated cellular potency (using B cell activation) was consistent with kinase data (IC50 = 0.7 nM, 34.5 nM, and 2.9 nM, respectively). In NHP, we observed similar levels of exposure in the CSF after oral doses of 10 mg/kg for each candidate. However, tolebrutinib CSF exposure (4.8 ng/mL) (kp, uu CSF=0.40) exceeded the IC90, while evobrutinib (3.2 ng/mL) (kp, uu CSF=0.13) and fenebrutinib (12.9 ng/mL) (kp, uu CSF=0.15) failed to reach the IC50. In healthy volunteers, a single oral dose of 60 or 120 mg resulted in bioactive CSF levels between 2 and 4 hrs. (0.51 and 1.03 ng/mL, respectively). <h3>Conclusions:</h3> Next-generation drug candidates in MS must achieve pharmacologically-relevant concentrations in the CNS to address the existing treatment gap. In these NHP experiments, tolebrutinib was the only BTK inhibitor to show bioactive CSF levels, adding to the evidence that tolebrutinib has potential to slow disability accumulation via modulating neuroinflammation. <b>Disclosure:</b> Timothy J. Turner has received personal compensation for serving as an employee of Sanofi. Timothy J. Turner has received stock or an ownership interest from Sanofi. Timothy J. Turner has received intellectual property interests from a discovery or technology relating to health care. Dr. NICOLAS has nothing to disclose. Mrs. Brun has nothing to disclose. Dimitry Ofengeim has received personal compensation for serving as an employee of Sanofi. Dimitry Ofengeim has received stock or an ownership interest from Sanofi. Dimitry Ofengeim has received personal compensation in the range of $500-$4,999 for serving as a Study Section Reveiwer with NIH.

Antiretroviral Levels in the Cerebrospinal Fluid: The Effect of Inflammation and Genetic Variants.

Neurocognitive impairments are common in people living with HIV. Some conditions, such as chronic inflammation, astrocyte infection and an impaired blood-brain barrier (BBBi), along with host genetic variants in transporter genes, may affect antiretroviral (ARV) exposure in the cerebrospinal fluid (CSF). The aim of this study was to evaluate ARV CSF penetration according to compartmental inflammation, BBB permeability and single-nucleotide polymorphisms (SNPs) in drug transporter encoding genes. CSF neopterin (ELISA), plasma and CSF ARV concentrations (HPLC) and host genetic variants in ABCC2, HNF4α, SLCO1A2 and SLC22A6 (real-time PCR) were measured. Bi- and multivariate analyses were performed for single ARV and classes. We included 259 participants providing 405 paired plasma and CSF samples. CSF/plasma ratios (CPR) showed an increase for NRTIs and nevirapine with low penetrations for the majority of ARVs. At bi-variate analysis, several associations, including the effect of BBBi (emtricitabine, raltegravir), age (zidovudine and darunavir), and high CSF neopterin (NRTIs and border-line for PIs) were suggested. An association was found between genetic variants and integrase strand transfer (ABCC2 and HNF4α), non-nucleoside reverse transcriptase inhibitors (SLCO1A2), and protease inhibitors (SLC22A6). At multivariate analysis age, gender, BMI, and altered BBB were independent predictors of nucleoside reverse transcriptase CSF concentrations; age (for protease inhibitors) and body mass index and altered BBB (integrase strand transfer inhibitors) were also associated with ARV CSF exposure. We describe factors associated with CSF concentrations, showing that demographic, BBB integrity and, partially, genetic factors may be predictors of drug passage in the central nervous system.

Effect of Isavuconazole on Plasma and CSF Levels of Ibrutinib and Plasma Levels of Liposomal Doxorubicin Among Patients Receiving Teddi-R for CNS Lymphoma

Background: Ibrutinib is an oral BTK inhibitor that is highly effective for B-cell lymphomas and chronic graft versus host disease. Escalating doses of ibrutinib were highly active and achieved good CNS penetration when added to temozolomide, etoposide, liposomal doxorubicin, dexamethasone, rituximab (TEDDI-R) in primary CNS lymphoma (PCNSL) but was associated with aspergillosis (Lionakis Cancer Cell 2017). To prevent aspergillosis, we added the antifungal agent isavuconazole (isa) to the TEDDI-R regimen, but it is a moderate inhibitor of CYP3A4 and inhibits ibrutinib clearance. We performed a new dose escalation study of ibrutinib with concomitant isavuconazole and TEDD-R in PCNSL with a dose expansion cohort. In a separate study, we tested fixed doses of ibrutinib with TEDD-R in secondary CNS lymphoma (SCNSL) with pharmacokinetic (PK) sampling from plasma and cerebrospinal fluid (CSF). Herein we present the PK of ibrutinib in plasma and CSF from both ongoing studies along with plasma PK of liposomal doxorubicin. Methods: Patients with untreated and relapsed/refractory PCNSL enrolled onto the phase 1 trial of TEDDI-R with isa in a standard 3+3 fashion testing three dose levels of ibrutinib: DL1=280 mg, DL2=420 mg, and DL3=560 mg. Patients with untreated and relapsed/refractory SCNSL were enrolled onto a phase 2 trial of TEDDI-R with isa and a fixed dose of ibrutinib 560 mg. Isa was started ≥3 days before ibrutinib and continued throughout combination therapy of 4 to 6 cycles. At the first dose of ibrutinib, patients with PCNSL underwent plasma and CSF sampling at the following timepoints: pre-dose, then 1 hr, 2 hr, 4 hr, 6 hr, 8 hr, 10-18 hr (one sample in this timeframe), and 24 hr post-dose. Patients with SCNSL underwent plasma sampling at pre-dose, 1 hr, and 10-18 hr (one sample in this timeframe) after the first ibrutinib dose. All patients also had steady-state (≥4 prior ibrutinib doses) plasma and CSF sampling at pre-dose and 2 hr post-dose. Plasma sampling for liposomal doxorubicin (dose range 30-50 mg/m2) occurred at pre-dose and at 5-8 hours post-dose. Concentrations were measured using LC-MS/MS (Frontage Laboratories and NCI). PK parameters were estimated using non-compartmental methods. Results: 55 consecutive patients (30 PCNSL, 25 SCNSL) were included for analysis. Among the 30 patients with dense first-dose sampling, the Tmax was 2.05 hr (SD 1.06, n=30), T1/2 8.3 hr (median 6.2 hr, SD 6.4, n=26), volume of distribution 13121 L (median 10756, SD 11422, n=24), and clearance 1560 L/hr (median 1093, SD 1397, n=24) (patients with r2<0.9 excluded). There was wide interpatient variability in ibrutinib PK. In both the plasma and CSF, ibrutinib 560 mg + isa had a higher AUC, Cmax, and steady-state peak concentration than 560 mg alone. Compared to 700 mg and 840 mg alone, 560 mg + isa had a similar AUC and Cmax in the plasma, but a higher steady-state peak plasma concentration. In the CSF, 560 mg + isa had a similar AUC and Cmax but higher steady-state peak concentration compared to 700 mg alone, and a higher AUC and Cmax but similar steady-state peak concentration compared to 840 mg alone (Tables 1 and 2). Peak (5-8 hours post-dose) plasma concentrations of liposomal doxorubicin were not significantly different with isa versus without isa (dose-normalized geometric mean ratio 0.94, 95% CI 0.80-1.08, n=82 data points). Conclusions: Isavuconazole increases the plasma and CSF exposure of ibrutinib approximately 2-fold, allowing for similar or improved CNS penetration at 560 mg compared to what was previously observed with ibrutinib 700 mg and 840 mg without isavuconazole. Further development of the TEDDI-R regimen utilizes ibrutinib 560 mg as the target dose along with isavuconazole. Plasma concentrations of liposomal doxorubicin were not significantly affected by isavuconazole. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

BIOM-15. GLIOBLASTOMA CELLS SHOW ELEVATED CHITINASE 3-LIKE PROTEIN 1 GENE EXPRESSION IN RESPONSE TO HUMAN CEREBROSPINAL FLUID WITH INCREASED MIGRATION AND EPITHELIAL-MESENCHYMAL TRANSITION RELATED ACTIVITY

Abstract Glioblastoma (GBM) is the most common and malignant primary brain tumor in adults. GBM tumors proximal to the lateral ventricles have a worse prognosis with multifocality, recurrence, and lower overall survival. Studies suggest this increased aggressiveness is attributed to cerebrospinal fluid (CSF), as we have previously observed transcriptomic changes in GBM cells upon CSF exposure. GlioVis database reveals chitinase 3-like protein 1 (CHI3L1), a glycoprotein secreted by immune and cancer cells, is associated with poor prognosis and co-expressed with pro-inflammatory and tissue remodeling genes. We suggest exposure to CSF elevates CHI3L1 expression, consequently impacting malignancy-associated gene expression in patient-derived GBM lines. We first evaluated if patient CSF influences the expression of CHI3L1. GBM cells were treated with artificial CSF, non-cancer CSF, or cancer CSF for 72 hours. qPCR revealed significant increases in CHI3L1 (p&amp;lt; 0.0001), SPP1, and CD44 expression in cancer CSF groups compared to artificial CSF. CHI3L1 expression at the protein level is also increased by cancer CSF when profiled using Western Blot. Additionally, ELISA results indicated significantly higher levels of CHI3L1 in CSF samples obtained from patients with high-grade tumors compared to non-cancer samples. Next, we assessed if exogenous CHI3L1 stimulates GBM malignancy at the cellular level. An effective concentration of recombinant human CHI3L1 was determined through Alamar Blue assay, showing 500 ng/ml CHI3L1 increases viability in primary patient cell lines compared to non-treated media. Recombinant CHI3L1 also increases cell migration in Transwell assay. qPCR revealed Vimentin (p&amp;lt; 0.001) and Twist (p&amp;lt; 0.0001) expression significantly increases while E-cadherin (p&amp;lt; 0.001) significantly decreases in CHI3L1-treated groups, consistent with EMT and migratory activity. Ultimately, this study shows CHI3L1 is a CSF-induced factor in GBM and could be a therapeutic target as it enhances tumor malignancy by increasing proliferation, migration, and malignancy-promoting gene expression.

Abstract 1841: MRTX849 inhibits P-gp and demonstrates CNS exposure in mouse models and cancer patients and demonstrates antitumor activity in intracranial mouse models of lung cancer brain metastasis

Abstract MRTX849 is a covalent, mutant selective, KRAS G12C inhibitor in development for cancer patients harboring this mutation. KRAS G12C is mutated in ~14% of lung adenocarcinoma and KRAS G12C inhibitors adagrasib and sotorasib have demonstrated clinical activity. While early data are encouraging, approximately one-third of KRAS-mutant non-small cell lung cancer (NSCLC) patients develop brain metastases and this remains a significant unmet medical need. P-glycoprotein 1 (P-gp)-mediated efflux is a major mechanism for the active transport of small molecules out of the CNS. MRTX849 is a P-gp inhibitor and inhibits its own efflux at plasma exposure levels achieved in humans at the 600 mg BID dose level resulting in achieving appreciable drug levels in cerebrospinal fluid (CSF). Total and free-fraction adjusted plasma concentrations of MRTX849 were 8.6 μM and 43 nM; respectively, 8 hours post administration of a clinically relevant oral dose of 200 mg/kg to mice. In addition, the CSF concentration, a measure of the free brain concentration, was 52 nM, which is above the cellular pERK1/2 IC50 value measured at 24 hours in cancer cell lines (~5 nM). Administration of 100 mg/kg MRTX849 to mice also resulted in CSF exposure above the cellular IC50 and a CSF/plasma (free-fraction adjusted) partition coefficient (Kp,uu) value of 0.4. To evaluate the tumor growth inhibition following oral administration of MRTX849 in an orthotopic model of lung cancer brain metastasis, immunocompromised mice were intracranially implanted with luciferase-labeled human NSCLC LU99 cells. Lower bioluminescence imaging (BLI)-based tumor flux was observed for the 100 mg/kg BID MRTX849-treated mice compared to vehicle and an 88% reduction in bioluminescence signal compared to baseline was observed suggesting strong tumor regression and consistent with significantly longer survival in treated mice. In addition, MRTX849 treated brain tumors demonstrated 85% reduced ERK phosphorylation. Similarly, treatment with MRTX849 resulted in potent tumor regression and significant survival extension in another intracranial KRAS G12C model of NSCLC using LU65 cells. CSF levels were determined at steady-state in two patients in the MRTX849-001 Phase1/2 clinical trial. The CSF/plasma (free fraction adjusted) Kp,uu value was 0.47 with patient CSF levels achieved consistent with CSF levels observed in responding mouse models (24-35 nM). These data demonstrate MRTX849 crosses the blood brain barrier in preclinical models and cancer patients. In addition, antitumor activity observed in mouse models of brain metastases provides rationale for exploring the utility of MRTX849 for the treatment of patients harboring KRAS G12C mutant lung cancer with brain metastases. Citation Format: Kazuhide Shimizu, Jill Hallin, Mohini Singh, Naema Nayyar, Matthew R. Strickland, Aaron C. Burns, Cornelius Cilliers, Lauren Hargis, Peter A. Olson, Matthew A. Marx, Priscilla K. Brastianos, Hiroaki Wakimoto, James G. Christensen. MRTX849 inhibits P-gp and demonstrates CNS exposure in mouse models and cancer patients and demonstrates antitumor activity in intracranial mouse models of lung cancer brain metastasis [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 1841.

The CSF Vancomycin Concentration in Patients With Post-operative Intracranial Infection Can Be Predicted by the WBCs to Total Cells Ratio and the Serum Trough Concentration.

BackgroundThe pharmacokinetics of vancomycin in cerebrospinal fluid (CSF) is an important basis for evaluating the bactericidal effect. The accuracy of using serum vancomycin concentrations only to estimate the CSF concentrations remains controversial, may lead to underdosing.ObjectivesThe aims of this study were to evaluate the vancomycin exposure in CSF, investigate the factors affecting the vancomycin blood–brain barrier (BBB) penetration, and to establish the prediction model of vancomycin concentration in CSF.MethodsEligible patients were included and given a standard dose of vancomycin. At the fifth dose, the blood and CSF samples were collected 0.5 h before the start of infusion of vancomycin, and 1, 2, 3, and 8 h from the start of infusion, and were measured by the enzyme-multiplied immunoassay technique using the Siemens Viva-E Drug Testing System.ResultsThe AUCCSF/serum of patients with intracranial infection was higher than that of patients without (p = 0.001). The CSF concentration was relatively stable between dosing periods (p = 0.095). The area under the concentration–time curve (AUC) ratio of CSF to serum (AUCCSF/serum) in patients with intracranial infection ranged from 15.1 to 80.1% (33.23 ± 19.31%; median, 26.25%). The CSF vancomycin AUC levels were affected by the serum trough concentration (B: 5.23 ± 2.36, t = 2.22, p = 0.039), and were mainly affected by the CSF white blood cells (WBCs)/total cells (B: 113.96 ± 35.10, t = 3.25, p = 0.004) (Y = −17.86 + 5.23 × serum trough concentration + 113.96 × CSF [WBCs/total cells]; R2 = 0.473, F = 8.542, p = 0.002).ConclusionsAfter intravenous administration of vancomycin, the CSF concentration curve was fluctuated gently. The CSF vancomycin concentration in patients with postoperative intracranial infection can be predicted by the WBCs to total cells ratio and the serum trough concentration, and help to adjust the administration of vancomycin.

Scientific rationale for a higher dose of nusinersen.

ObjectiveThe long‐term favorable safety profile of nusinersen provides an opportunity to consider a higher dose. We report on the relationships between nusinersen cerebrospinal fluid (CSF) exposure, biomarker levels, and clinical efficacy.MethodsThe analyses used data from the CS3A and ENDEAR studies of nusinersen in participants with infantile‐onset spinal muscular atrophy (SMA). Steady‐state CSF trough (C trough) levels, plasma phosphorylated neurofilament heavy chain (pNF‐H) levels, body weight, and Children's Hospital of Philadelphia Infant Test of Neuromuscular Disorders (CHOP INTEND) scores were selected as parameters of interest. A validated population pharmacokinetic (PK) model was applied to predict the nusinersen CSF C trough. PK/pharmacodynamic (PK/PD) models used nusinersen CSF C trough measurements, which were time‐matched with CHOP INTEND scores.ResultsHigher nusinersen CSF exposure was associated with a greater decrease in pNF‐H levels and greater efficacy, as measured by change in the CHOP INTEND score from baseline. These findings indicate a dose–response relationship between CSF nusinersen levels and treatment response. The higher dose is predicted to lead to approximately a 2.4‐fold increase in nusinersen CSF levels with fewer loading doses. PK/PD modeling indicates that a higher concentration of nusinersen may predict an additional 5‐point increase in CHOP INTEND score beyond that observed with 12 mg.InterpretationOur data indicate that a higher dose of nusinersen may lead to additional clinically meaningful improvement in efficacy when compared with the currently approved 12‐mg dose. The efficacy, safety, and PK of a higher nusinersen dose are currently under investigation in the ongoing phase 2/3 DEVOTE study (NCT04089566).

Restoration of Nusinersen Levels Following Treatment Interruption in People With Spinal Muscular Atrophy: Simulations Based on a Population Pharmacokinetic Model.

BackgroundNusinersen is approved for the treatment of spinal muscular atrophy. The most common approved dosing regimen is four intrathecal loading doses of nusinersen 12 mg; the first three are administered at 14-day intervals followed by a fourth dose 30 days later, and then 12-mg maintenance doses are administered every 4 months thereafter. Interruption of nusinersen treatment in the maintenance dosing phase might occur for a number of clinical reasons.ObjectiveThe objective of this report is to describe dosing regimens that allow for the most rapid restoration of steady-state concentrations of nusinersen in the cerebrospinal fluid (CSF) following a treatment interruption during maintenance dosing.MethodsPopulation pharmacokinetic models using integrated pharmacokinetic data from ten nusinersen clinical trials that included a broad range of participants with spinal muscular atrophy treated with intrathecal nusinersen were used to investigate different durations of treatment interruptions during maintenance treatment. Potential dosing regimens for re-initiation of nusinersen were evaluated, with the goal of achieving the quickest restoration of steady-state nusinersen CSF concentrations without exceeding maximal CSF exposures observed during the initial loading period.ResultsOur pharmacokinetic modeling indicates the following regimen will lead to optimal restoration of nusinersen CSF levels after treatment interruption: two doses of nusinersen should be administered at 14-day intervals following treatment interruptions of ≥ 8 to < 16 months since the last dose, and three doses of nusinersen at 14-day intervals for treatment interruptions of ≥ 16 to < 40 months since the last maintenance dose, with subsequent maintenance dosing every 4 months in both instances. After treatment interruptions of ≥ 40 months, the full loading regimen will rapidly restore nusinersen CSF levels.ConclusionsProlonged treatment interruptions lead to suboptimal CSF levels of nusinersen. The optimal regimen to restore nusinersen CSF levels depends on the interval since the last maintenance dose was administered.

Efficacy of Nilotinib in Patients With Moderately Advanced Parkinson Disease

There is a critical need for careful and independent validation of reported symptomatic efficacy and dopaminergic biomarker changes induced by nilotinib in Parkinson disease (PD). To assess safety and tolerability of nilotinib in participants with moderately advanced PD. Secondary and exploratory objectives were to assess its affect on PD disability, pharmacokinetics, cerebrospinal fluid (CSF) penetration, and biomarkers. This was a 6-month, multicenter, randomized parallel-group, double-blind, placebo-controlled trial. Recruitment was from November 20, 2017, to December 28, 2018, and follow-up ended on September 9, 2019. The study was conducted at 25 US sites. The study approached 173 patients, of whom 48 declined, 125 were screened, and 76 who received a stable regimen of PD medications were enrolled (39% screen failure). Participants were randomized 1:1:1 to placebo, 150-mg nilotinib, or 300-mg nilotinib once daily orally for 6 months, followed by 2-month off-drug evaluation. The primary outcomes were safety and tolerability. The tolerability end point was defined as the ability to complete the study while receiving the assigned dose. An active arm was considered tolerable if the percentage of participants meeting the tolerability end point for that group was not significantly lower than the percentage observed in the placebo group. Secondary outcomes included change in PD disability (Movement Disorder Society Unified Parkinson's Disease Rating Scale [MDS-UPDRS], Part II OFF/ON). Exploratory outcomes included serum and CSF pharmacokinetic profile, and CSF dopaminergic biomarkers. At baseline, mean (SD) participants' age was 64.6 (7.5) years, 52 were male (68%), mean (SD) disease duration was 9.9 years (4.7), MDS-UPDRS Part 1-3 OFF score was 66.4 (19.3), ON score was 48.4 (16.2), and Montreal Cognitive Assessment score was 27.1 (2.2). The number of participants who completed the study receiving the assigned dose were 21 (84%), 19 (76%), and 20 (77%) in the placebo, 150-mg, and 300-mg arms, respectively. Both active doses had acceptable safety profile. The most common reasons for drug suspension were asymptomatic, dose-dependent elevations of amylase, and/or lipase. Nilotinib, 150 mg and 300 mg, exhibited worse MDS-UPDRS-3 ON scores compared with placebo, achieving significance for nilotinib, 300 mg, at month 1 (P < .01). There was no difference in the change of MDS-UPDRS-3 OFF from baseline to 6 months between groups (P = .17). Cerebrospinal fluid/serum ratio of nilotinib concentration was 0.2% to 0.3%. There was no evidence of treatment-related alteration of dopamine metabolites in the CSF. While we demonstrated acceptable safety and tolerability of nilotinib in our cohort, the low CSF exposure and lack of biomarkers effect combined with the efficacy data trending in the negative direction indicate that nilotinib should not be further tested in PD. ClinicalTrials.gov Identifier: NCT03205488.

A single‐ and multiple‐ascending dose study to evaluate the safety and pharmacokinetics of oral PU‐AD, an epichaperome inhibitor to treat Alzheimer’s disease

AbstractBackgroundBreakdown of regulatory pathways designed to prevent aggregation and accumulation of disease‐associated proteins contributes to or causes Alzheimer’s Disease (AD). Under these conditions epichaperomes form and take control of pathological pathways. PU‐AD is a small‐molecule, orally bioavailable inhibitor specific to epichaperomes with little or no effect on normal cells, and has shown dramatic beneficial effects in animal models of AD. We describe the first‐in‐man trial of PU‐AD.ObjectivesThis Phase 1, double‐blind, placebo‐controlled, single and multiple‐ascending dose study evaluated the safety and pharmacokinetics (PK) of single and multiple doses of oral PU‐AD.MethodsCohorts of 8 subjects were randomly assigned to active treatment or placebo in a 6:2 ratio. Single doses of oral PU‐AD were 10 mg, 20 mg, and 30 mg in healthy subjects ages 18‐60 years, and daily doses of 20 mg and 30 mg daily for 7 days in healthy subjects &gt;59 years old. Safety was evaluated through observation of adverse event incidence and severity; and changes from baseline in clinical laboratory test results, vital signs, physical examinations, or electrocardiogram results. Plasma PK parameters of PU‐AD were determined, and cerebrospinal fluid (CSF) was sampled for PU‐AD concentration and for biomarkers of neurodegenerative disease in the multiple‐dose cohorts.ResultsPU‐AD was well‐tolerated, with an adverse event profile comparable to that of placebo. No adverse events were serious, severe, or led to withdrawal of dosing. Additionally, there were no other clinically significant safety findings to report. PK was dose‐proportional between 10 mg to 30 mg and showed minimal accumulation with repeated dosing. CSF analyses will be presented.ConclusionPU‐AD, a novel and selective epichaperome inhibitor shown to be efficacious in animal AD models, warrants further evaluation in an AD population. This first‐in‐man trial demonstrated that PU‐AD is well‐tolerated, has favorable plasma PK and CSF exposure, and is a candidate for advancement into Phase 2A trials in AD populations to evaluate target engagement and pharmacological effects.

Abstract 1574: The role of human cerebrospinal fluid in glioblastoma malignancy

Abstract Glioblastoma (GBM), also known as grade IV astrocytoma, is the most common and devastating primary cancer of the central nervous system in adults. Despite the most advanced therapeutic strategies combining surgery, chemotherapy, and radiation, survival expectancy of GBM patients averages 14 months, with a recurrence rate of almost 100%. Remarkably, tumor location has great implications in the prognosis of GBM. Approximately 50-60% of GBM tumors infiltrates or contacts the subventricular zone (SVZ). These patients have significantly worse outcomes, in terms of median overall survival, time to progression and reoccurrence. The reason for the worse prognosis of SVZ-infiltrating tumors is unknown. The SVZ is the largest neurogenic niche in the adult brain, here the differentiation and migration of SVZ cells is regulated by direct contact with the cerebrospinal fluid (CSF). We suggest that CSF contribute to enhance GBM malignant characteristics in SVZ-infiltrating tumors. This study evaluated the impact of CSF exposure on GBM cells proliferation, migration in vitro and in vivo, as well as changes in gene expression profile. We demonstrated that CSF affects the malignancy of GBM cells by increasing their ability to proliferate and migrate. GBM cells stimulated with cancer CSF, non-cancer CSF, or control conditions at different time points (24-72 hrs) were evaluated for viability (by Alamar Blue assay) and proliferation rate (Ki67+ immunostaining), as well as for migratory capabilities (by means of transwell assay and time-lapse microscopy). We recapitulated these CSF effects in vivo using an in vivo model in which both female and male nude mice were implanted with GBM cells in suspension with human cancer or non-cancer CSF in an hydrogel as vehicle (n=8 mice per group). Tumor growth was followed via IVIS imaging and then studied by measuring tumor area on H&amp;E slides and immunohistochemistry staining for Ki67, 21 days after tumor implantation. Animals receiving cancer CSF presented with greater tumor size and Ki67 proliferation index. To further dissect the mechanisms behind these observations, we performed a transcriptome analysis of CSF-treated GBM cells. At the molecular level we observed that cancer CSF induced changes in pathways regulating apoptosis, survival, integrins signaling, angiogenesis, glucose metabolism and response to inflammatory stimuli, all of which might be responsible for the increase in GBM malignancy. Taken together, this study provides direct evidence that CSF is an important player in determining tumor growth and invasion through the activation of complex gene expression patterns characteristic of a malignant phenotype. Understanding the interaction of brain tumors and CSF may lead to new therapeutic strategies that will target the CSF components in GBM patients. Citation Format: Anna Carrano, Jordan Phillipps, Montserrat Lara-Velazquez, Natanael Zarco, Paola Suarez-Meade, Kaisorn Chaichana, Alfredo Quinones-Hinojosa, Yan Asmann, Hugo Guerrero-Cázares. The role of human cerebrospinal fluid in glioblastoma malignancy [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 1574.

Brain Shuttle Neprilysin reduces central Amyloid-β levels.

Reducing Amyloid β (Aβ) in the brain is of fundamental importance for advancing the therapeutics for Alzheimer`s disease. The endogenous metallopeptidase neprilysin (NEP) has been identified as one of the key Aβ-degrading enzymes. Delivery of NEP to the brain by utilizing the Brain Shuttle (BS) transport system offers a promising approach for clearing central Aβ. We fused the extracellular catalytic domain of NEP to an active or inactive BS module. The two BS-NEP constructs were used to investigate the pharmacokinetic/pharmacodynamics relationships in the blood and the cerebrospinal fluid (CSF) in dose-response and multiple dosing. As previously shown, NEP was highly effective at degrading Aβ in blood but not in the CSF compartment after systemic administration. In contrast, the NEP with an active BS module led to a significant CSF exposure of BS-NEP, followed by substantial Aβ reduction in CSF and brain parenchyma. Our data show that a BS module against the transferrin receptor facilitates the transport of an Aβ degrading enzyme across the blood-brain barriers to efficiently reduce Aβ levels in both CSF and brain.

Plasma and cerebrospinal fluid pharmacokinetics of the DNA methyltransferase inhibitor, 5-azacytidine, alone and with inulin, in nonhuman primate models.

Epigenetic modifiers are being investigated for a number of CNS malignancies as tumor-associated mutations such as isocitrate dehydrogenase mutations (IDH1/IDH2) and H3K27M mutations, which result in aberrant signaling, are identified. We evaluated the CNS exposure of the DNA methyltransferase inhibitor, 5-azacytidine (5-AZA), in preclinical nonhuman primate (NHP) models to inform its clinical development for CNS tumors. 5-AZA and 5-AZA+Inulin pharmacokinetics (PK) were evaluated in NHPs (n = 10) following systemic (intravenous [IV]) and intrathecal (intraventricular [IT-V], intralumbar [IT-L], and cisternal [IT-C]) administration. Plasma, cerebrospinal fluid (CSF), cortical extracellular fluid (ECF), and tissues were collected. 5-AZA levels were quantified via ultra-high-performance liquid chromatography with tandem mass spectrometric detection assay and inulin via ELISA. PK parameters were calculated using noncompartmental methods. After IV administration, minimal plasma exposure (area under the curve [AUC] range: 2.4-3.2 h*µM) and negligible CSF exposure were noted. CSF exposure was notably higher after IT-V administration (AUCINF 1234.6-5368.4 h*µM) compared to IT-L administration (AUCINF 7.5-19.3 h*µM). CSF clearance after IT administration exceeded the mean inulin CSF flow rate of 0.018 ± 0.003 ml/min as determined by inulin IT-V administration. 5-AZA IT-V administration with inulin increased the 5-AZA CSF duration of exposure by 2.2-fold. IT-C administration yielded no quantifiable 5-AZA ECF concentrations but resulted in quantifiable tissue levels. IT administration of 5-AZA is necessary to achieve adequate CNS exposure. IT administration results in pronounced and prolonged 5-AZA CSF exposure above the reported IC50 range for IDH-mutated glioma cell lines. Inulin administered with 5-AZA increased the duration of exposure for 5-AZA.

Effects of a new magnesium-rich artificial cerebrospinal fluid on contractile 5-hydroxytryptamine and endothelin receptors in rat cerebral arteries

ABSTRACTObjectives Cerebral vasospasm after subarachnoid haemorrhage (SAH) is associated with cerebrovascular contractile receptor upregulation resulted from haemolysis in the subarachnoid space. This study developed a new magnesium-rich artificial cerebrospinal fluid (MACSF) formula and investigated its effects on receptor-mediated contraction in rat basilar arteries.Methods Clear and haemorrhagic cerebrospinal fluid (CSF) were collected from patients with hydrocephalus or SAH. MACSF was freshly prepared using clinical intravenous injections. Rat basilar arteries were segmented and incubated with clear CSF, haemorrhagic CSF or MACSF. The contractile responses were studied by myograph. The messenger ribonucleic acid (mRNA) and protein expression of 5-hydroxytryptamine 1B (5-HT1B), endothelin subtype B (ETB) and endothelin subtype A (ETA) receptors were evaluated by real-time polymerase chain reaction (PCR) and Western blot analyses.Results Haemorrhagic CSF exposure shifted the contractile curves induced by 5-hydroxytryptamine (5-HT), sarafotoxins 6c (S6c) and endothelin-1 (ET-1) leftward with increased maximal contraction values. Furthermore, mRNA and protein expression were markedly elevated for 5-HT1B, ETB and ETA receptors on arteries exposed to haemorrhagic CSF. However, the contractile responses to 5-HT, S6c or ET-1 and expression of 5-HT1B, ETB and ETA receptors in rat cerebral arteries exposed to MACSF remained unaffected compared to those exposed to clear CSF. Besides, unlike normal saline which can inactive in-vitro vessels, MACSF can maintain their physiological activity.Conclusion Haemorrhagic CSF induces upregulation of 5-HT1B, ETB and ETA receptors in rat cerebral arteries. However, MACSF can maintain in-vitro rat basilar arteries in good physiological activity and normal expression of contractile 5-HT and ET receptors.