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.

Pharmacokinetics of panobinostat: Inter-species difference in metabolic stability.

Panobinostat is a potent pan-HDAC inhibitor that has been tested in multiple studies for the treatment of brain tumors. There have been contrasting views surrounding its efficacy for the treatment of tumors in the CNS following systemic administration when examined in different models or species. We conducted experiments using three different mouse strains or genotypes to have a more comprehensive understanding of the systemic as well as the CNS distributional kinetics of panobinostat. Our study found that panobinostat experienced rapid degradation in vitro in FVB mouse matrices and a faster degradation rate was observed at 37{degree sign}C compared with room temperature and 4{degree sign}C, suggesting that the in vitro instability of panobinostat was due to enzymatic metabolism. Panobinostat also showed inter-strain and inter-species differences in the in vitro plasma stability; and was stable in human plasma. The objective of this study was to examine the in vitro metabolic stability of panobinostat in different matrices and assess the influence of that metabolic stability on the in vivo pharmacokinetics and CNS delivery of panobinostat. Importantly, the plasma stability in various mouse strains was not reflected in the in vivo systemic pharmacokinetic behavior of panobinostat. Several hypotheses arise from this finding, including: the binding of panobinostat to red blood cells, the existence of competing endogenous compounds to enzyme(s), the distribution into tissues with a lower level of enzymatic activity or the metabolism occurring in the plasma is a small fraction of the total metabolism in vivo Significance Statement Panobinostat showed different in vitro degradation in plasma from different mouse strains and genotypes. However, despite the differences surrounding in vitro plasma stability, panobinostat showed similar in vivo pharmacokinetic behavior in different mouse models. This suggests that the inter-strain difference in enzymatic activity did not affect the in vivo pharmacokinetic behavior of panobinostat and its CNS distribution in mice. This lack of translation between in vitro metabolism assays and in vivo disposition can confound drug development.

Delivery versus Potency in Treating Brain Tumors: BI-907828, a MDM2-p53 Antagonist with Limited BBB Penetration but Significant In Vivo Efficacy in Glioblastoma.

MDM2-p53 inhibition may be effective in glioblastoma (GBM). This study evaluates the pharmacokinetics/pharmacodynamics of BI-907828, a potent antagonist of MDM2, in GBM, and demonstrates a translational paradigm with a focus on a unified "Delivery - Potency - Efficacy" relationship in drug development for central nervous system(CNS) tumors. BI-907828 was tested for cytotoxicity and MDM2-p53 pathway inhibition. Systemic pharmacokinetics and transport mechanisms controlling CNS distribution were evaluated in mice. BI-907828 free fractions in cell media, mouse and human specimens were measured to determine "active" unbound concentrations. Efficacy measures, including overall survival and target expression were assessed in mouse orthotopic GBM xenografts. BI-907828 exhibited potent inhibition of MDM2-p53 pathway and promoted cell death in GBM TP53 wild-type cells. MDM2-amplified cells are highly sensitive to BI-907828, with an effective unbound concentration of 0.1 nmol/L. The CNS distribution of BI-907828 is limited by blood-brain barrier (BBB) efflux mediated by P-gp, resulting in a Kp,uu_brain of 0.002. Despite this seemingly "poor" BBB penetration, weekly administration of 10 mg/kg BI-907828 extended median survival of orthotopic GBM108 xenografts from 28 to 218 days (P < 0.0001). This excellent efficacy can be attributed to high potency, resulting in a limited, yet effective, exposure in the CNS. These studies show that efficacy of BI-907828 in orthotopic models is related to high potency even though its CNS distribution is limited by BBB efflux. Therefore, a comprehensive understanding of all aspects of the "Delivery - Potency - Efficacy" relationship is warranted in drug discovery and development, especially for treatment of CNS tumors.

Methodological and analytical considerations for intra-operative microdialysis.

BACKGROUND: Microdialysis is a technique that can be utilized to sample the interstitial fluid of the central nervous system (CNS), including in primary malignant brain tumors known as gliomas. Gliomas are mainly accessible at the time of surgery, but have rarely been analyzed via interstitial fluid collected via microdialysis. To that end, we obtained an investigational device exemption for high molecular weight catheters (HMW, 100 kDa) and a variable flow rate pump to perform microdialysis at flow rates amenable to an intra-operative setting. We herein report on the lessons and insights obtained during our intra-operative HMW microdialysis trial, both in regard to methodological and analytical considerations. METHODS: Intra-operative HMW microdialysis was performed during 15 clinically indicated glioma resections in fourteen patients, across three radiographically diverse regions in each patient. Microdialysates were analyzed via targeted and untargeted metabolomics via ultra-performance liquid chromatography tandem mass spectrometry. RESULTS: Use of albumin and lactate-containing perfusates impacted subsets of metabolites evaluated via global metabolomics. Additionally, focal delivery of lactate via a lactate-containing perfusate, induced local metabolic changes, suggesting the potential for intra-operative pharmacodynamic studies via reverse microdialysis of candidate drugs. Multiple peri-operatively administered drugs, including levetiracetam, cefazolin, caffeine, mannitol and acetaminophen, could be detected from one microdialysate aliquot representing 10 min worth of intra-operative sampling. Moreover, clinical, radiographic, and methodological considerations for performing intra-operative microdialysis are discussed. CONCLUSIONS: Intra-operative HMW microdialysis can feasibly be utilized to sample the live human CNS microenvironment, including both metabolites and drugs, within one surgery. Certain variables, such as perfusate type, must be considered during and after analysis. Trial registration NCT04047264.

Central Nervous System Distribution of Panobinostat in Preclinical Models to Guide Dosing for Pediatric Brain Tumors.

Achieving adequate exposure of the free therapeutic agent at the target is a critical determinant of efficacious chemotherapy. With this in mind, a major challenge in developing therapies for central nervous system (CNS) tumors is to overcome barriers to delivery, including the blood-brain barrier (BBB). Panobinostat is a nonselective pan-histone deacetylase inhibitor that is being tested in preclinical and clinical studies, including for the treatment of pediatric medulloblastoma, which has a propensity for leptomeningeal spread and diffuse midline glioma, which can infiltrate into supratentorial brain regions. In this study, we examined the rate, extent, and spatial heterogeneity of panobinostat CNS distribution in mice. Transporter-deficient mouse studies show that panobinostat is a dual substrate of P-glycoprotein (P-gp) and breast cancer resistant protein (Bcrp), which are major efflux transporters expressed at the BBB. The CNS delivery of panobinostat was moderately limited by P-gp and Bcrp, and the unbound tissue-to-plasma partition coefficient of panobinostat was 0.32 and 0.21 in the brain and spinal cord in wild-type mice. In addition, following intravenous administration, panobinostat demonstrated heterogeneous distribution among brain regions, indicating that its efficacy would be influenced by tumor location or the presence and extent of leptomeningeal spread. Simulation using a compartmental BBB model suggests inadequate exposure of free panobinostat in the brain following a recommended oral dosing regimen in patients. Therefore, alternative approaches to CNS delivery may be necessary to have adequate exposure of free panobinostat for the treatment of a broad range of pediatric brain tumors. SIGNIFICANCE STATEMENT: This study shows that the central nervous system (CNS) penetration of panobinostat is limited by P-gp and Bcrp, and its efficacy may be limited by inadequate distribution to the tumor. Panobinostat has heterogeneous distribution into various brain regions, indicating that its efficacy might depend on the anatomical location of the tumors. These distributional parameters in the mouse CNS can inform both preclinical and clinical trial study design and may guide treatment for these devastating brain tumors in children.

How Much is Enough? Impact of Efflux Transporters on Drug delivery Leading to Efficacy in the Treatment of Brain Tumors.

The lack of effective chemotherapeutic agents for the treatment of brain tumors is a serious unmet medical need. This can be attributed, in part, to inadequate delivery through the blood-brain barrier (BBB) and the tumor-cell barrier, both of which have active efflux transporters that can restrict the transport of many potentially effective agents for both primary and metastatic brain tumors. This review briefly summarizes the components and function of the normal BBB with respect to drug penetration into the brain and the alterations in the BBB due to brain tumor that could influence drug delivery. Depending on what is rate-limiting a compound's distribution, the limited permeability across the BBB and the subsequent delivery into the tumor cell can be greatly influenced by efflux transporters and these are discussed in some detail. Given these complexities, it is necessary to quantify the extent of brain distribution of the active (unbound) drug to compare across compounds and to inform potential for use against brain tumors. In this regard, the metric, Kp,uu, a brain-to-plasma unbound partition coefficient, is examined and its current use is discussed. However, the extent of active drug delivery is not the only determinant of effective therapy. In addition to Kp,uu, drug potency is an important parameter that should be considered alongside drug delivery in drug discovery and development processes. In other words, to answer the question - How much is enough? - one must consider how much can be delivered with how much needs to be delivered.

Low serum complement level is associated with higher mortality in tuberculous meningitis: a retrospective cohort study.

Purpose: We evaluated the associations between serum complement levels and tuberculous meningitis (TBM), bacterial meningitis (BM), and viral meningitis (VM), as well as the association between serum complement levels and mortality in TBM. Methods: Background information and blood/cerebrospinal fluid analysis results were collected from 2009 to 2019. Patients who had serum complement level data collected at admission and who were diagnosed with TBM (n = 97), BM (n = 31), or VM (n = 557) were enrolled. Results: Initial serum complement levels were significantly lower in the TBM group than the VM group in both the total population and the propensity score-matched population. In the TBM and VM groups, compared to patients with initial highest-quartile C4 level, patients in the lowest quartile (C4 < 24.3 mg/dL) had significantly greater odds of TBM diagnosis (odds ratio, 2.2; 95% confidence interval, 1.0-4.5; p = 0.038). In the TBM group, patients with the lowest-quartile C3 level (<96.9 mg/dL) experienced a significantly higher 90-day mortality rate compared to other TBM patients (hazard ratio, 19.0; 95% confidence interval, 2.1-167.4.5; p = 0.008). Conclusion: Both serum C3 and C4 levels were significantly lower in the TBM group than in the VM group. TBM patients with lower serum C3 level had a significantly higher mortality rate than those with higher C3 level.

WSD-0922, a novel brain-penetrant inhibitor of epidermal growth factor receptor, promotes survival in glioblastoma mouse models.

Background: Although the epidermal growth factor receptor (EGFR) is a frequent oncogenic driver in glioblastoma (GBM), efforts to therapeutically target this protein have been largely unsuccessful. The present preclinical study evaluated the novel EGFR inhibitor WSD-0922. Methods: We employed flank and orthotopic patient-derived xenograft models to characterize WSD-0922 and compare its efficacy to erlotinib, a potent EGFR inhibitor that failed to provide benefit for GBM patients. We performed long-term survival studies and collected short-term tumor, plasma, and whole-brain samples from mice treated with each drug. We utilized mass spectrometry to measure drug concentrations and spatial distribution and to assess the impact of each drug on receptor activity and cellular signaling networks. Results: WSD-0922 inhibited EGFR signaling as effectively as erlotinib in in vitro and in vivo models. While WSD-0922 was more CNS penetrant than erlotinib in terms of total concentration, comparable concentrations of both drugs were measured at the tumor site in orthotopic models, and the concentration of free WSD-0922 in the brain was significantly less than the concentration of free erlotinib. WSD-0922 treatment provided a clear survival advantage compared to erlotinib in the GBM39 model, with marked suppression of tumor growth and most mice surviving until the end of the study. WSD-0922 treatment preferentially inhibited phosphorylation of several proteins, including those associated with EGFR inhibitor resistance and cell metabolism. Conclusions: WSD-0922 is a highly potent inhibitor of EGFR in GBM, and warrants further evaluation in clinical studies.

Blood-brain barrier disruption defines the extracellular metabolome of live human high-grade gliomas.

The extracellular microenvironment modulates glioma behaviour. It remains unknown if blood-brain barrier disruption merely reflects or functionally supports glioma aggressiveness. We utilised intra-operative microdialysis to sample the extracellular metabolome of radiographically diverse regions of gliomas and evaluated the global extracellular metabolome via ultra-performance liquid chromatography tandem mass spectrometry. Among 162 named metabolites, guanidinoacetate (GAA) was 126.32x higher in enhancing tumour than in adjacent brain. 48 additional metabolites were 2.05-10.18x more abundant in enhancing tumour than brain. With exception of GAA, and 2-hydroxyglutarate in IDH-mutant gliomas, differences between non-enhancing tumour and brain microdialysate were modest and less consistent. The enhancing, but not the non-enhancing glioma metabolome, was significantly enriched for plasma-associated metabolites largely comprising amino acids and carnitines. Our findings suggest that metabolite diffusion through a disrupted blood-brain barrier may largely define the enhancing extracellular glioma metabolome. Future studies will determine how the altered extracellular metabolome impacts glioma behaviour.

Overcoming translational barriers in H3K27-altered diffuse midline glioma: Increasing the drug-tumor residence time.

Background: H3K27-altered diffuse midline glioma (DMG) is the deadliest pediatric brain tumor; despite intensive research efforts, every clinical trial to date has failed. Is this because we are choosing the wrong drugs? Or are drug delivery and other pharmacokinetic variables at play? We hypothesize that the answer is likely a combination, where optimization may result in a much needed novel therapeutic approach. Methods: We used in vitro drug screening, patient samples, and shRNA knockdown models to identify an upregulated target in DMG. A single small molecule protein kinase inhibitor with translational potential was selected for systemic and direct, loco-regional delivery to patient-derived xenografts (PDX) and genetically engineered mouse models (GEMM). Pharmacokinetic studies were conducted in non-tumor bearing rats. Results: Aurora kinase (AK) inhibitors demonstrated strong antitumor effects in DMG drug screens. Additional in vitro studies corroborated the importance of AK to DMG survival. Systemic delivery of alisertib showed promise in subcutaneous PDX but not intracranial GEMM and PDX models. Repeated loco-regional drug administration into the tumor through convection-enhanced delivery (CED) was equally inefficacious, and pharmacokinetic studies revealed rapid clearance of alisertib from the brain. In an effort to increase the drug to tumor residence time, continuous CED over 7 days improved drug retention in the rodent brainstem and significantly extended survival in both orthotopic PDXs and GEMMs. Conclusions: These studies provide evidence for increasing drug-tumor residence time of promising targeted therapies via extended CED as a valuable treatment strategy for DMG.

Murine Central Nervous System and Bone Marrow Distribution of the Aurora A Kinase Inhibitor Alisertib: Pharmacokinetics and Exposure at the Sites of Efficacy and Toxicity.

Important challenges in developing drugs that target central nervous system (CNS) tumors include overcoming barriers for CNS delivery and reducing systemic side effects. Alisertib, an aurora A kinase inhibitor, has been examined for treatment of several CNS tumors in preclinical and clinical studies. In this study, we investigated the distribution of alisertib into the CNS, the site of efficacy for brain tumors, and into the bone marrow, the site of dose-limiting toxicity leading to myelosuppression. Mechanisms influencing site-specific distribution, such as active transport mediated by the efflux proteins, p-glycoprotein (P-gp) and breast cancer resistance protein (Bcrp), were examined. Alisertib exposure to the brain in wild-type mice was less than 1% of that in the plasma, and was evenly distributed throughout various brain regions and the spinal cord. Studies using transporter knockout mice and pharmacological inhibition show that alisertib CNS distribution is influenced by P-gp, but not Bcrp. Conversely, upon systemic administration, alisertib distribution to the bone marrow occurred rapidly, was not significantly limited by efflux transporters, and reached higher concentrations than in the CNS. This study demonstrates that, given an equivalent distributional driving force exposure in plasma, the exposure of alisertib in the brain is significantly less than that in the bone marrow, suggesting that targeted delivery may be necessary to guarantee therapeutic efficacy with minimal risk for adverse events.Therefore, these data suggest that, to improve the therapeutic index when using alisertib for brain tumors, a localized regional delivery, such as convection-enhanced delivery, may be warranted. SIGNIFICANCE STATEMENT: The CNS penetration of alisertib is limited with uniform distribution in various regions of the brain, and P-gp efflux is an important mechanism limiting that CNS distribution. Alisertib rapidly distributes into the bone marrow, a site of toxicity, with a greater exposure than in the CNS, a possible site of efficacy. These results suggest a need to design localized delivery strategies to improve the CNS exposure of alisertib and limit systemic toxicities in the treatment of brain tumors.

Changes in the Brain in Temporal Lobe Epilepsy with Unilateral Hippocampal Sclerosis: An Initial Case Series.

Temporal lobe epilepsy (TLE) is a network disorder of the brain. Network disorders predominately involve dysregulation of hippocampal function caused by neuronal hyperexcitability. However, the relationship between the macro- and microscopic changes in specific brain regions is uncertain. In this study, the pattern of brain atrophy in patients with TLE and hippocampal sclerosis (HS) was investigated using volumetry, and microscopic changes in specific lesions were observed to examine the anatomical correspondence with specific target lesions using diffusion tensor imaging (DTI) with statistical parametric mapping (SPM). This retrospective cross-sectional study enrolled 17 patients with TLE and HS. We manually measured the volumes of the hippocampus (HC), amygdala (AMG), entorhinal cortex, fornix, and thalamus (TH) bilaterally. The mean diffusivity and fractional anisotropy of each patient were then quantified and analyzed by a voxel-based statistical correlation method using SPM8. In right TLE with HS, there was no evidence of any abnormal diffusion properties associated with the volume reduction in specific brain regions. In left TLE with HS, there were significant changes in the volumes of the AMG, HC, and TH. Despite the small sample size, these differences in conditions were considered meaningful. Chronic left TLE with HS might cause structural changes in the AMG, HC, and TH, unlike right TLE with HS.

Central Nervous System Delivery of the Catalytic Subunit of DNA-Dependent Protein Kinase Inhibitor Peposertib as Radiosensitizer for Brain Metastases.

Cytotoxic effects of chemotherapy and radiation therapy (RT) used for the treatment of brain metastases results from DNA damage within cancer cells. Cells rely on highly evolved DNA damage response (DDR) pathways to repair the damage caused by these treatments. Inhibiting these repair pathways can further sensitize cancer cells to chemotherapy and RT. The catalytic subunit of DNA-dependent protein kinase, in a complex with Ku80 and Ku70, is a pivotal regulator of the DDR, and peposertib is a potent inhibitor of this catalytic subunit. The characterization of central nervous system (CNS) distributional kinetics of peposertib is critical in establishing a therapeutic index in the setting of brain metastases. Our studies demonstrate that the delivery of peposertib is severely restricted into the CNS as opposed to peripheral organs, by active efflux at the blood-brain barrier (BBB). Peposertib has a low free fraction in the brain and spinal cord, further reducing the active concentration, and distributes to the same degree within different anatomic regions of the brain. However, peposertib is heterogeneously distributed within the metastatic tumor, where its concentration is highest within the tumor core (with disrupted BBB) and substantially lower within the invasive tumor rim (with a relatively intact BBB) and surrounding normal brain. These findings are critical in guiding the potential clinical deployment of peposertib as a radiosensitizing agent for the safe and effective treatment of brain metastases. SIGNIFICANCE STATEMENT: Effective radiosensitization of brain metastases while avoiding toxicity to the surrounding brain is critical in the development of novel radiosensitizers. The central nervous system distribution of peposertib, a potent catalytic subunit of DNA-dependent protein kinase inhibitor, is restricted by active efflux in the normal blood-brain barrier (BBB) but can reach significant concentrations in the tumor core. This finding suggests that peposertib may be an effective radiosensitizer for intracranial tumors with an open BBB, while limited distribution into normal brain will decrease the risk of enhanced radiation injury.

The influence of the blood-brain barrier in the treatment of brain tumours.

Brain tumours have a poor prognosis and lack effective treatments. The blood-brain barrier (BBB) represents a major hurdle to drug delivery to brain tumours. In some locations in the tumour, the BBB may be disrupted to form the blood-brain tumour barrier (BBTB). This leaky BBTB enables diagnosis of brain tumours by contrast enhanced magnetic resonance imaging; however, this disruption is heterogeneous throughout the tumour. Thus, relying on the disrupted BBTB for achieving effective drug concentrations in brain tumours has met with little clinical success. Because of this, it would be beneficial to design drugs and drug delivery strategies to overcome the 'normal' BBB to effectively treat the brain tumours. In this review, we discuss the role of BBB/BBTB in brain tumour diagnosis and treatment highlighting the heterogeneity of the BBTB. We also discuss various strategies to improve drug delivery across the BBB/BBTB to treat both primary and metastatic brain tumours. Recognizing that the BBB represents a critical determinant of drug efficacy in central nervous system tumours will allow a more rapid translation from basic science to clinical application. A more complete understanding of the factors, such as BBB-limited drug delivery, that have hindered progress in treating both primary and metastatic brain tumours, is necessary to develop more effective therapies.

Negligible Effect of Quercetin in the Pharmacokinetics of Sulfasalazine in Rats and Beagles: Metabolic Inactivation of the Interaction Potential of Quercetin with BCRP.

Breast cancer resistance protein (BCRP) mediates pharmacokinetic drug interactions. This study evaluated the potential of quercetin to inhibit and induce BCRP in vitro and in vivo. The inhibition of BCRP was investigated for quercetin and its metabolites using BCRP/mBcrp1-overexpressing MDCKII cells by flow cytometry. The induction of BCRP was investigated in LS174T cells using quantitative PCR. The expression of rat BCRP in rat small intestine, liver, and kidney was also measured after multiple administrations of quercetin in rats (50, 100, and 250 mg/kg, seven days). The in vivo pharmacokinetic changes of sulfasalazine following single or multiple administration of quercetin in rats and beagles were investigated. Although the induction effect of quercetin on BCRP was observed in vitro, the in vivo expression of rat BCRP was not changed by multiple quercetin administrations. Oral administration of quercetin did not affect the plasma concentration or pharmacokinetic parameters of sulfasalazine, regardless of dose and dosing period in either rats or beagles. In addition, the inhibitory effect of quercetin metabolites on BCRP/mBcrp1 was not observed. These results suggest that the in vivo drug interaction caused by quercetin via BCRP was negligible, and it may be related to the metabolic inactivation of quercetin for the inhibition of BCRP.

Methods for intratumoral microdialysis probe targeting and validation in murine brain tumor models.

BACKGROUND: Microdialysis is a well validated sampling technique that can be used for pharmacokinetic studies of oncological drugs targeting the central nervous system. This technique has also been applied to evaluate tumor metabolism and identify pharmacodynamic biomarkers of drug activity. Despite the potential utility of microdialysis for therapeutic discovery, variability in tumor size and location hamper routine use of microdialysis as a preclinical tool. Quantitative validation of microdialysis membrane location relative to radiographically evident tumor regions could facilitate rigorous preclinical studies. However, a widely accessible standardized workflow for preclinical catheter placement and validation is needed. NEW METHOD: We provide methods for a workflow to yield tailored placement of microdialysis probes within a murine intracranial tumor and illustrate in an IDH1-mutant patient-derived xenograft (PDX) model. This detailed workflow uses a freely available on-line tool built within 3D-slicer freeware to target microdialysis probe placement within the tumor core and validate probe placement fully within the tumor. RESULTS: We illustrate use of this workflow to validate microdialysis probe location relative to implanted IDH1-mutant PDXs, using the microdialysis probes to quantify levels of extracellular onco-metabolite D-2 hydroxyglutarate. COMPARISON WITH EXISTING METHODS: Previous methods have used 3D slicer to reliably measure tumor volumes. Prior microdialysis studies have targeted expected tumor locations without validation. CONCLUSIONS: The new method offers a streamlined and freely available workflow in 3D slicer to optimize and validate microdialysis probe placement within a murine brain tumor.

The association between serum sodium level and tuberculous meningitis compared with viral and bacterial meningitis.

We evaluated the association between hyponatremia and tuberculous meningitis (TBM) with the aim of providing additional information for differential diagnosis from other types of infectious meningitis, especially viral meningitis (VM). Cross-sectional and longitudinal data involving 5026 participants older than 18 years were analyzed in the total population and a propensity-matched population. The initial and lowest sodium levels and longitudinal changes in TBM, bacterial meningitis (BM), and VM patients were compared. Participants in the TBM group were enrolled when they were diagnosed as possible, probable, or definite TBM according to the Marais' criteria. The initial serum sodium level was significantly lower in TBM patients than in BM and VM patients (136.9 ± 5.9 vs. 138.3 ± 4.7 mmol/L, p < 0.001 for TBM vs. BM, and 139.0 ± 3.1, p < 0.001 for TBM vs. VM), and it decreased significantly more steeply to lower levels in both the TBM and BM patients compared with VM patients. The lowest serum sodium level was in the order of TBM < BM < VM patients, and the change was statistically significant in all subgroups (131.8 ± 6.4, 133.1 ± 5.1, 137.4 ± 3.7, respectively, p < 0.001). Participants with lower serum sodium level were more likely to have a diagnosis of TBM rather than VM, and this association was more pronounced for the lowest sodium level than the initial sodium level [OR 4.6 (95% CI 2.4-8.8, p < 0.001)]. These findings indicate that baseline and longitudinal evaluation of serum sodium level can provide information for differential diagnosis of TBM from BM or VM.

Heterogeneous delivery across the blood-brain barrier limits the efficacy of an EGFR-targeting antibody drug conjugate in glioblastoma.

BACKGROUND: Antibody drug conjugates (ADCs) targeting the epidermal growth factor receptor (EGFR), such as depatuxizumab mafodotin (Depatux-M), is a promising therapeutic strategy for glioblastoma (GBM) but recent clinical trials did not demonstrate a survival benefit. Understanding the mechanisms of failure for this promising strategy is critically important. METHODS: PDX models were employed to study efficacy of systemic vs intracranial delivery of Depatux-M. Immunofluorescence and MALDI-MSI were performed to detect drug levels in the brain. EGFR levels and compensatory pathways were studied using quantitative flow cytometry, Western blots, RNAseq, FISH, and phosphoproteomics. RESULTS: Systemic delivery of Depatux-M was highly effective in nine of 10 EGFR-amplified heterotopic PDXs with survival extending beyond one year in eight PDXs. Acquired resistance in two PDXs (GBM12 and GBM46) was driven by suppression of EGFR expression or emergence of a novel short-variant of EGFR lacking the epitope for the Depatux-M antibody. In contrast to the profound benefit observed in heterotopic tumors, only two of seven intrinsically sensitive PDXs were responsive to Depatux-M as intracranial tumors. Poor efficacy in orthotopic PDXs was associated with limited and heterogeneous distribution of Depatux-M into tumor tissues, and artificial disruption of the BBB or bypass of the BBB by direct intracranial injection of Depatux-M into orthotopic tumors markedly enhanced the efficacy of drug treatment. CONCLUSIONS: Despite profound intrinsic sensitivity to Depatux-M, limited drug delivery into brain tumor may have been a key contributor to lack of efficacy in recently failed clinical trials.

Taxotere Induces Dephosphorylation of MET in Patient-derived Tumor Models.

BACKGROUND/AIM: Although molecular targeting therapy is an attractive treatment for cancer, resistance eventually develops in most cases. Here, we evaluated chemotherapeutic efficacy on non-small cell lung cancer (NSCLC) with acquired resistance to epidermal growth factor receptor inhibitors mechanistically. MATERIALS AND METHODS: Antitumor effects of taxotere were evaluated using multiple models, including xenograft, and patient-derived models developed from adenocarcinoma cancer patients. Protein expressions were analyzed after drug treatment. RESULTS: Taxotere inhibited tumor growth of NSCLC cells harboring drug resistance, and reduced the expression of phosphorylated MET proto-oncogene, receptor tyrosine kinase (MET). A tumor-inhibitory effect of taxotere was also demonstrated in vivo in xenografts in mice, patient-derived primary lung tumor cells and patient-derived xenograft with concomitant repression of phosphorylated MET expression. Chemotherapeutic and MET-targeting drug exhibited a synergistic cell growth-inhibitory effect. CONCLUSION: These results suggest that the anticancer drug taxane may be an adjuvant for lung tumors exhibiting enhanced signaling of MET networks.

Optimized liquid chromatography-tandem mass spectrometry for Otaplimastat quantification in rat plasma and brain tissue.

An optimized liquid chromatography-tandem mass spectrometry method for simple and sensitive quantification of Otaplimastat in rat plasma and brain tissue was developed and validated. Protein precipitation with acetonitrile was selected for sample preparation method based on recovery and matrix effect. The chromatographic separation of the sample was performed on a reverse-phase AQ column with an isocratic mobile phase consisting of 10 mM ammonium acetate (pH 4.0) and acetonitrile (50:50, v/v). The analyte was quantified by multiple reaction monitoring with a Waters Quattro micro™ API mass spectrometer. The lower limits of quantification were 20 ng/mL in plasma and 2 ng/g in brain, with the relative standard deviation % of 7.6 and 8.0% for plasma and brain samples, respectively. Acceptable intra-day and inter-day precisions and accuracies were obtained. Otaplimastat was sufficiently stable under all relevant analytical conditions, including a temperature of 4°C for 24 hr, room temperature 20°C for 24 hr, -80°C for 10 days and three freeze-thaw cycles (each at -80°C for 24 hr), for rat plasma and brain tissue. The validated method was successfully used to measure Otaplimastat concentrations in rat plasma and brain samples.