Extracellular Fluid Samples Research Articles

EXTH-99. SYSTEMIC AND BRAIN PHARMACOKINETICS OF A GABAA RECEPTOR AGONIST, AMLAL-101, AS AN INVESTIGATIONAL THERAPEUTIC FOR THE TREATMENT OF PRIMARY AND METASTATIC BRAIN CANCERS

Abstract PURPOSE AMLAL-101 is a novel agent which preferentially targets α3, α5 subtypes of ɣ-amino butyric acid receptors and shows anti-tumor activity against disparate cancer types. AMLAL-101 is being advanced as an ‘add-on’ to potentiate treatment of primary and metastatic brain cancers. However, AMLAL-101 must penetrate the blood-brain barrier (BBB) and show sufficient brain retention. The primary purpose of this study was to determine the plasma pharmacokinetics (PK) and quantitative estimate of the BBB permeability of AMLAL-101. METHODS We performed intracranial microdialysis, employing jugular vein cannulated Sprague-Dawley rats which facilitated simultaneous serial blood and brain extracellular fluid (ECF) sampling. AMLAL-101 was injected i.p. at 5 mg/kg and serial blood and brain ECF samples collected up to 10 h post-dosing. Plasma and ECF samples were analyzed by LC/MS-MS and plasma and ECF concentration vs time PK profiles determined. In vivo recovery analysis was performed using retrodialysis and rapid equilibrium dialysis employed to determine the extent of protein binding. RESULTS AMLAL-101 plasma protein binding was 85% and in vivo recovery from ECF was 25%. AMLAL-101 peak concentration (Cmax) in plasma and brain ECF were 15 µM and 13.8 µM, respectively. The plasma and brain ECF area under the concentration (AUC0-10) were 27.5 h.µg/mL and 24.10 h.µg/mL, respectively. The brain partitioning of unbound AMLAL-101 (Kp,uu; determined either as a ratio of brain ECF Cmax:unbound plasma Cmax or brain ECF AUC: unbound plasma AUC), were 6.13 and 4.13, respectively. The elimination half-life of AMLAL-101 was 3 h for both brain ECF and plasma. CONCLUSIONS These results suggest that AMLAL-101 has the requisite BBB permeability required for brain cancer therapeutics. AMLAL-101 shows significant brain retention when compared to a chemically similar agent that does not show anti-cancer activity, which may contribute to efficacy of AMLAL-101 as an anti-tumor agent for treatment of brain cancers.

Disruption of Glutamate Homeostasis in the Brain of Rat Offspring Induced by Prenatal and Early Postnatal Exposure to Maternal High-Sugar Diet.

A high-calorie diet has contributed greatly to the prevalence of overweight and obesity worldwide for decades. These conditions also affect pregnant women and have a negative impact on the health of both the woman and the fetus. Numerous studies indicate that an unbalanced maternal diet, rich in sugars and fats, can influence the in utero environment and, therefore, the future health of the child. It has also been shown that prenatal exposure to an unbalanced diet might permanently alter neurotransmission in offspring. In this study, using a rat model, we evaluated the effects of a maternal high-sugar diet on the level of extracellular glutamate and the expression of key transporters crucial for maintaining glutamate homeostasis in offspring. Glutamate concentration was assessed in extracellular fluid samples collected from the medial prefrontal cortex and hippocampus of male and female offspring. Analysis showed significantly increased glutamate levels in both brain structures analyzed, regardless of the sex of the offspring. These changes were accompanied by altered expression of the EAAT1, VGLUT1, and xc− proteins in these brain structures. This animal study further confirms our previous findings that a maternal high-sugar diet has a detrimental effect on the glutamatergic system.

BIMG-20. METABOLIC BIOMARKERS IN MICRODIALYSATE OF IDH-1 MUTANT TUMORS

Glioblastoma (GBM) is a common deadly malignant brain cancer of the central nervous system, with a median survival of 12–15 months. Scientific advancements are lacking in developing effective therapies for both primary GBM, as well as secondary GBMs, that typically originate as malignant transformation of lower-grade isocitrate dehydrogenase (IDH) 1-mutant tumors. The unique metabolomic profile of IDH1-mutant tumors presents opportunities to develop biomarker signatures of therapeutic efficacy. Microdialysis is an extracellular fluid sampling collection technique utilizing a perfused semipermeable catheter to permit diffusion of molecules between brain interstitium and the perfusate. We hypothesized that microdialysis may identify a metabolomics-based biomarker response to therapy in IDH1-mutant tumors. To test this hypothesis, orthotopic xenografts were generated from patient-derived xenografts (PDX) harboring mutant IDH-1 (R132H). Perfusates were collected from intra-cranial tumors in athymic nude mice sampled at baseline and 72h post treatment with temozolomide (TMZ), an oral alkylating agent used to treat IDH1-mutant gliomas, compared with vehicle treatment. Perfusates were analyzed via untargeted metabolomic profiling using liquid chromatography-mass spectrometry. Tumor specific metabolites such as (D)-2 hydroxyglutarate, were detected in microdialysate from IDH-1 mutant tumor bearing mice compared to non-tumor bearing mice. We also found high levels of metabolites such as 5-methylthioadenosine, and dimethylarginine and wide range of amino acids in microdialysate from IDH-1 mutant tumor bearing mice. TMZ treatment induced changes to metabolites in creatine and histidine metabolism. Our results indicate that microdialysis is a feasible technology to identify metabolomics-based biomarkers in IDH1-mutant gliomas and their response to therapy. We suggest that in vivo intratumoral microdialysis over several days could yield metabolic pharmacodynamic biomarkers of value to therapeutic translation for IDH-mutant gliomas.

Intratumoral drug distribution of adavosertib in patients with glioblastoma: Interim results of phase I study.

2568 Background: Wee1 is a key regulator of the G2/M checkpoint and is frequently overexpressed in glioblastoma (GB). Adavosertib is a first-in-class oral, small molecule inhibitor of Wee1 that acts primarily as a DNA damage sensitizer. A phase I clinical trial was conducted to evaluate its safety and establish the recommended phase II dosing. Studies were undertaken to evaluate whether potentially therapeutic concentrations of the drug are achieved in recurrent tumor and adjacent non-enhancing brain regions with presumed intact blood-brain barrier (BBB). Methods: Twelve patients received five daily doses of adavosertib pre-operatively at either the maximum tolerated dose (MTD) for concurrent radiation or adjuvant temozolomide. Tissue from contrast enhancing (CE) and non-enhancing (NE) brain regions was obtained for analysis during surgical resection. A second stage is being conducted using microdialysis (MD) to facilitate continuous sampling of extracellular fluid (ECF) and measuring free drug concentrations in: normal-appearing brain, contrast enhancing tumor, and a peritumoral T2 hyperintense area. The concentration of total adavosertib in plasma and tissue homogenates and free drug in ECF were determined by validated LC/MS/MS methods. Results: Geometric mean concentrations of adavosertib after a 200 mg dose were 644 ng/mL and 119 ng/mL in CE and NE tissue specimens, respectively (6 patients). At the 425 mg dose, the mean concentrations were 3,576 ng/mL in CE tissue and 885 ng/mL in NE tissue (6 patients). MD was performed in 2 patients. Samples from functional MD catheters were collected from NE brain in patient no. 1 and from two NE areas and a FLAIR hyperintense region in patient no. 2, with the following results in the table. Conclusions: The total drug concentration in tissue samples was notably lower in regions of the brain with a relatively intact BBB as compared to contrast enhancing tissue. Concentrations of adavosertib measured by MD vary markedly depending on catheter location. Free drug levels in ECF within brain with a functional BBB, although considerably lower than total drug levels in tissue, were 2-10 times below the previously reported IC50 for antiproliferative activity against sensitive GB cell lines (127 ng/mL). Whether or not the target of the drug is effectively inhibited at these concentrations remains to be demonstrated. Clinical trial information: NCT01849146 . [Table: see text]

TMIC-42. LOCAL TISSUE METABOLOMICS BASED BIOMARKERS OF RESPONSE TO THERAPY FOR GLIOBLASTOMA

Abstract Glioblastoma (GBM) is a common deadly malignant brain cancer of the central nervous system (CNS), with a median survival of 12–15 months. Scientific advancements are lacking in developing effective therapies for both primary GBM, as well as secondary GBMs, that typically originate as malignant transformation of lower-grade isocitrate dehydrogenase (IDH) 1-mutant tumors. The unique metabolomic profile of IDH1-mutant tumors may present opportunities to develop biomarker signatures of therapeutic efficacy. Microdialysis is an extracellular fluid sampling collection technique utilizing a perfused semipermeable catheter to permit diffusion of molecules between brain interstitium and the perfusate. We hypothesized that microdialysis may identify a metabolomics-based biomarker response to therapy in IDH1-mutant tumors. To test this hypothesis, orthotopic xenografts were generated from two patient-derived GBM lines harboring mutations in IDH1. Perfusates were collected from intra-cranial tumors in aythmic nude mice sampled at baseline and 72h post treatment with temozolomide, an oral alkylating agent used to treat IDH1-mutant gliomas, compared with vehicle treatment, and TMZ-treated non-tumor bearing animals. Perfusates were analyzed via unsupervised metabolomic profiling using both gas and liquid chromatography-mass spectrometry (GC/LC-MS). Tumor specific metabolites such as carnitine and pyruvic acid were detected in microdialysate from tumor bearing mice compared to non-tumor bearing mice. Microdialysis is a feasible technology to identify metabolomics-based biomarker in IDH1-mutant PDX. This work is complemented by parallel analysis of non-IDH1-mutant and TMZ resistant xenografts to yield predictive in vivo tissue biomarkers of drug responsiveness translatable to clinical practice.

Construction and characterization of a transmembrane eukaryotic expression vector based on the membrane domain structure of TNF-α.

The aim of the present study was to construct a fast-acting, eukaryotic expression vector in eukaryotic cells based on transmembrane-tumor necrosis factor-α (TM-TNF-α) structure. Two types of recombinant eukaryotic expression vectors were constructed, pcDNA3.1-TM-enterokinase-TNF-α and pcDNA3.1-TM-Factor Xa-TNF-α, according to the TNF-α transmembrane segments. Following the generation of these vectors, mouse embryonic 3T3 fibroblasts were transfected and reverse transcription-polymerase chain reaction and western blotting analyses were used to analyze mTNF-α mRNA and protein expression levels, respectively, in total cellular protein extracts and extracellular fluid. The biological activity of TNF-α in the extracellular fluid was then measured using an MTT assay. The vectors were successfully constructed, and mRNA and fusion proteins were detected in the 3T3 cells. Among the fusion proteins, the one observed in pcDNA3.1-TM-FactorXa-TNF-α-transfected 3T3 cells remained as a transmembrane protein. In addition, treatment of L929 cells with TNF-α derived extracellular fluid samples from pcDNA3.1-TM-FactorXa-TNF-α-transfected 3T3 cells was associated with a dose-dependent reduction in in cell-specific activity. The results indicate that proteins expressed using pcDNA3.1-TM-FactorXa-TNF-α vectors form transmembrane proteins. In addition, the results indicate that, only when coupled with FactorXa activity, the extracellular region of TM-TNF-α forms s-TNF-α, and the controlled expression of the fusion protein is initiated.

Quantification of Temozolomide in Nonhuman Primate Fluids by Isocratic Ultra-High Performance Liquid Chromatography-Tandem Mass Spectrometry to Study Brain Tissue Penetration Following Intranasal or Intravenous Delivery.

A sensitive and selective ultra-high performance liquid chromatography-tandem mass spectrometric method was developed for the quantification of temozolomide (TMZ) in nonhuman primate (NHP) plasma, cerebrospinal fluid (CSF), and brain extracellular fluid (ECF) following microdialysis. Ethyl acetate was used to extract the plasma and CSF samples, using theophylline as the internal standard (IS). ECF samples were diluted with acetonitrile prior to analysis. TMZ was separated on a Waters UPLC® BEH C18 column with an isocratic mobile phase of ammonium acetate (10 mM)-0.1% formic acid/acetonitrile (30:70, v/v) in a positive-ion multi pie reaction monitoring mode (m/z 195.5 →137.6 for TMZ; m/z 181.5→124.2 for IS). The retention time of TMZ and theophylline was 0.45 min with a total run time of 2.5 min. The method was validated over the range from 5-2000 ng/mL in NHP plasma, CSF, and ECF with respect to linearity, accuracy, precision, selectivity, and stability. This method was successfully applied toward the measurement of pharmacokinetic samples following various routes of drug administration.

A novel method to detect in vivo oxidant production reveals increased susceptibility to high‐fat diet induced penile oxidative stress in GSNOR knockout mice (LB688)

Excessive production of reactive oxygen species (ROS) has consistently been described as a cause of erectile dysfunction. However, direct measurement of ROS in the penis has remained elusive due to the short half life of ROS and limited accessibility to appropriate tissue beds in humans or animals. Binding of nitric oxide to a cysteine thiol is termed S‐nitrosylation, which can mediate function of many proteins. S‐nitrosylated glutathione reductase (GSNOR) is a major mediator of de‐nitrosylation, which can impact reactive oxygen/nitrogen homeostasis. The objective of this study was to optimize a new technique to measure in vivo ROS production in the mouse penis, and to investigate the effect of a high‐fat diet on penile ROS production in wild type (WT) and GSNOR knockout (KO) mice. WT and GSNOR KO mice were fed a standard chow or high‐fat diet for 3 or 6 weeks (n=3/group). A linear microdialysis probe (3mm membrane, 20kDa cutoff) was inserted into the shaft of the penis of mice under ketamine/xylazine anesthesia, allowing sampling of extracellular fluid. To measure extracellular ROS, probes were perfused with saline containing 100 µM Amplex Ultrared and 1 U/ml horseradish peroxidase. Dialysate fluorescence was measured at the outlet with a Turner Designs (TD‐700) fluorometer at 510Ex/590Em. Mean hydrogen peroxide (H2O2) concentrations were: WT (chow = 0.664, 3wk HFD = 0.666, 6wk HFD = 1.202 µM), GSNOR KO (chow = 0.735, 3wk HFD = 1.349, 6wk = 1.389 µM). Following 3 weeks of HFD, mean penile H2O2 was increased 103% in GSNOR KO vs. WT mice. It can be concluded that lack of GSNOR accelerates the development of oxidative stress in the penis in response to a high‐fat diet.Grant Funding Source: Supported by a trainee grant from the Sexual Medicine Society of North America

The pharmacokinetics of letrozole in brain and brain tumor in rats with orthotopically implanted C6 glioma, assessed using intracerebral microdialysis

Emerging evidence suggests that primary and metastatic brain tumors may be sensitive to hormonal manipulations. However, the pharmacokinetics of compounds against such targets in the brain and, more importantly, in the brain tumor are not well characterized. Here, we investigated the pharmacokinetics of letrozole, a third-generation aromatase inhibitor, in the normal brain and in orthotopically implanted C6 glioma in Sprague-Dawley rats. Intracerebral microdialysis was employed to determine the concentrations of unbound letrozole in the brain extracellular fluid (ECF) while simultaneously collecting blood samples (via jugular vein) to assess plasma levels of letrozole. Letrozole was administered intravenously at doses of 4, 6, 8 and 12mg/kg, and ECF and blood samples were collected over 8h. For assessing normal versus tumoral brain pharmacokinetics, letrozole (4 or 8mg/Kg; i.v.) was administered 10days after implantation of C6 glioma in the brain. Dual-probe intracerebral microdialysis was employed for assessing ECF samples from tumor-free and tumor-bearing regions of the brain. Normal brain ECF and plasma C max and AUC0-8h increased linearly with letrozole doses up to 8mg/kg dose, but at 12mg/kg, the pharmacokinetics were nonlinear. The relative brain distribution coefficients, AUCECF/AUCplasma (ub), were 0.3-0.98. The tumoral uptake of letrozole was 1.5- to 2-fold higher relative to tumor-free region. Thus, letrozole permeability across the blood brain barrier is high, and the exposure to the brain is dose dependent. Furthermore, the brain tumoral letrozole levels are markedly higher than those in the tumor-free regions, which underscore potential selectivity of its activity against tumor cells.

Abstract 3360: The pharmacokinetics of letrozole in brain and brain tumor in rats (orthotopically implanted C6 glioma) assessed using microdialysis.

Abstract The presence of blood-brain or blood-tumor barrier is well known to restrict access of drugs to CNS malignancies. For most drugs, there is limited quantitative information pertaining to the time course of drug absorption, distribution and elimination and the resulting drug exposure. In this study, we employed intracerebral microdialysis to access the extracellular fluid (ECF) to assess the pharmacokinetics of letrozole in brain and primary glioma in rats. Intracerebral microdialysis (perfusion rate; 2 μl/min) was performed in female rats to determine the unbound letrozole ECF concentrations. Serial ECF samples were collected over a 30 min period up to 8 hrs following drug administration. Blood samples were simultaneously collected through the jugular vein cannulation. Letrozole concentrations in extracted plasma and dialysate samples were analyzed employing HPLC. In vivo relative recovery was estimated to be 9.5%. Normal brain versus plasma pharmacokinetic study employed four doses (4, 6, 8 & 12 mg/kg; i.v.). For tumoral pharmacokinetic studies, C6 glioma cells were orthotopically implanted in the right striatum while the left hemisphere served as control. Ten days after tumor implantation, dual probe microdialysis was performed under the aforementioned experimental conditions following letrozole administration at 4 and 8 mg/kg doses. Pharmacokinetic analyses were carried out using WinNonlin 6.2 (Pharsight Inc., CA). Normal brain ECF and plasma peak concentrations (Cmax) and the Area Under the Curve (AUC0-8hr) increased linearly with increase in doses of letrozole administration up to 8mg/kg dose. However, at higher dose of 12mg/kg brain and plasma AUC0-8hr increased non-linearly. The relative brain distribution coefficient, calculated as (AUCbrain ecf/AUCplasma), was 0.122 at the lowest dose of 4mg/kg letrozole, whereas, 0.3 for the other three doses. The distribution of letrozole into the brain was rapid with the time to attain the peak levels (Tmax) ranging between 1.3-1.5 hr for the four doses. Furthermore, the tumoral uptake of letrozole was 1.5 - 2 folds higher relative to tumor-free region of the brain for the two doses of letrozole, 4 and 8 mg/kg. Histological evaluation confirmed the presence and restriction of glioma in the right hemisphere, and the passage of the microdialysis probe in the tumor mass. Our extensive in vivo pharmacokinetic study represents the first quantitative assessment of letrozole exposure to the brain and brain tumor tissues. These studies clearly demonstrate a rapid and high partitioning of letrozole to the brain with higher accumulation in the tumoral region of the brain. Further pharmacokinetic and pharmacodynamics modeling and simulations of the observed unbound ECF concentrations are in progress to assess clinical implications of our findings. Citation Format: Nimita Dave, Xiaoyang Qi, Gary G. Gudelsky, Pankaj B. Desai. The pharmacokinetics of letrozole in brain and brain tumor in rats (orthotopically implanted C6 glioma) assessed using microdialysis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3360. doi:10.1158/1538-7445.AM2013-3360

A novel method to detect in vivo oxidant production reveals microvascular endothelial dysfunction linked to NADPH oxidase in obese human skeletal muscle

Excessive production of reactive oxygen species (ROS) contributes to the development of endothelial dysfunction and progression of vascular diseases that are observed in obesity. Three microdialysis probes were inserted into the vastus lateralis of young, sedentary lean (LN; n=7), overweight (OW; n=8) and obese (OB; n=7) individuals, allowing sampling of extracellular fluid. To measure extracellular ROS, probes were perfused with saline containing 5 mM ethanol, 100 μM Amplex Ultrared, 1 U/ml horseradish peroxidase, and 10 U/ml superoxide dismutase, with or without 300 μM apocynin (Apo; an NADPH oxidase inhibitor). Dialysate fluorescence was measured at the outlet. Endothelial function was assessed with addition of acetylcholine (ACh) to the perfusate, with local blood flow calculated from the ethanol outflow:inflow ratio. Extracellular ROS was elevated in OB 3.2‐fold over OW, and 5.6‐fold over LN (p < 0.05), which was prevented by Apo (p < 0.01). ACh‐stimulated blood flow was depressed 60% in OB vs. OW, and 68% vs. LN (p = 0.01). Apo augmented ACh‐stimulated blood flow only in OB (p = 0.02). Microvascular endothelial dysfunction was evident in OB, which was largely reversed by NADPH oxidase inhibition. It can be concluded that the substantial elevation in ROS in OB was predominantly driven by NADPH oxidase.Supported by a doctoral student grant from the American College of Sports Medicine.

Elevated glucose metabolism in the amygdala during an inhibitory avoidance task

There is a long-standing debate as to whether the memory process of consolidation is neurochemically similar to or the same as the set of processes involved in retrieval and reconsolidation of that memory. In addition, although we have previously shown that initial memory processing in the hippocampus causes a drainage of hippocampal glucose because of increased local metabolic demand, it is unknown what metabolic changes occur elsewhere in the brain or during subsequent processing of a previously consolidated memory. Male Sprague Dawley rats (3 months old) were implanted with unilateral microdialysis cannulae and in vivo microdialysis of amygdala extracellular fluid (ECF) was performed during both (i) initial learning and (ii) retrieval 24h later of an aversively motivated avoidance memory task. ECF samples were analyzed for glucose, lactate, pyruvate and glutamate. Results showed close similarity between increases in local glycolysis seen during both consolidation and retrieval, but also suggested that there may perhaps be a difference in amygdalar oxidative phosphorylation stimulated by the two processes. Hence, our data suggest that memory formation places similar metabolic demands across neural systems, and that consolidation may be metabolically different from retrieval.

The impact of bevacizumab on temozolomide concentrations in intracranial U87 gliomas

An important question in the sequencing of anti-cancer therapies in patients with glioblastoma (GBM) is whether concurrent anti-angiogenesis therapies improve or impair brain concentrations of concomitantly administered cytotoxic therapies. The purpose of this study is to assess the intratumoral disposition of temozolomide (TMZ) via microdialysis before and after bevacizumab in an intracranial GBM xenograft model. Microdialysis probes were placed within tumor and contralateral brain in athymic rats bearing U87 intracerebral gliomas. TMZ (50 mg/kg oral) was administered 10 days thereafter. Extracellular fluid (ECF) was collected for 6 h. BEV was administered (10 mg/kg IV), and TMZ was re-dosed (50 mg/kg oral) 36 h thereafter with additional ECF collection. All ECF samples were assessed for TMZ concentration with liquid chromatography-tandem mass spectrometry. Tumor TMZ mean area under the concentration-time curve (AUC(0-∞)) was 3.35 μg h/mL pre-BEV. Post-BEV, tumor mean TMZ AUC(0-∞) was 3.98 μg h/mL. In non-tumor brain, mean TMZ AUC(0-∞) pre-BEV was 3.22 μg h/mL and post-BEV was 3.34 μg h/mL. There were no statistically significant changes in TMZ pharmacokinetics before or after BEV in the athymic rat U87 intracranial glioma model. BEV and TMZ are being investigated as a combination therapy in several ongoing studies for patients with glioma. These data reassuringly suggest that BEV does not significantly change the ECF tumor concentrations of TMZ in either tumor-bearing or normal brain when dosed 36 h prior to TMZ.

The Prognostic Value of Brain Extracellular Fluid Nitric Oxide Metabolites After Traumatic Brain Injury

Nitric oxide (NO) is a compound with both protective and damaging effects on neurons. Quantification of NO metabolites in humans is limited by sample contamination with blood. In vivo cerebral microdialysis may offer an alternative approach as sampling of extracellular fluid (ECF) adjacent to neurons becomes possible. We investigate the prognostic value of brain ECF NO metabolites in patients with traumatic brain injury (TBI). A prospective case cohort of 195 ECF samples collected from 11 cases over 4 days following TBI was collected. Nitrate and nitrite concentrations ([NO x ]) were quantified using a vanadium-based colorimetric assay. Early ECF [NO x ] (<48 h post TBI) were significantly higher in non-survivors (median 59.2 μmol/l, n = 7) compared to survivors (23.3 μmol/l, n = 4) (P = 0.04). Late (48-96 h) ECF [NO x ] remained higher in non-survivors (47.9 μmol/l) compared to survivors (23.0 μmol/l) but this was not significant (P = 0.29). Receiver operator characteristic analysis shows an optimized cutoff level for ECF [NO x ] of 26.5 μmol/l measured <48 h post TBI for predicting non-survival (sensitivity 100%, specificity 75%). Early ECF NO x concentrations are of prognostic value after TBI. ECF NO x may be a useful biomarker for treatment trials targeted at nitric oxide metabolism.

In vivo monitoring of neuronal loss in traumatic brain injury: a microdialysis study

Traumatic brain injury causes diffuse axonal injury and loss of cortical neurons. These features are well recognized histologically, but their in vivo monitoring remains challenging. In vivo cortical microdialysis samples the extracellular fluid adjacent to neurons and axons. Here, we describe a novel neuronal proteolytic pathway and demonstrate the exclusive neuro-axonal expression of Pavlov’s enterokinase. Enterokinase is membrane bound and cleaves the neurofilament heavy chain at positions 476 and 986. Using a 100 kDa microdialysis cut-off membrane the two proteolytic breakdown products, extracellular fluid neurofilament heavy chains NfH476−986 and NfH476−1026, can be quantified with a relative recovery of 20%. In a prospective clinical in vivo study, we included 10 patients with traumatic brain injury with a median Glasgow Coma Score of 9, providing 640 cortical extracellular fluid samples for longitudinal data analysis. Following high-velocity impact traumatic brain injury, microdialysate extracellular fluid neurofilament heavy chain levels were significantly higher (6.18 ± 2.94 ng/ml) and detectable for longer (>4 days) compared with traumatic brain injury secondary to falls (0.84 ± 1.77 ng/ml, <2 days). During the initial 16 h following traumatic brain injury, strong correlations were found between extracellular fluid neurofilament heavy chain levels and physiological parameters (systemic blood pressure, anaerobic cerebral metabolism, excessive brain tissue oxygenation, elevated brain temperature). Finally, extracellular fluid neurofilament heavy chain levels were of prognostic value, predicting mortality with an odds ratio of 7.68 (confidence interval 2.15–27.46, P = 0.001). In conclusion, this study describes the discovery of Pavlov’s enterokinase in the human brain, a novel neuronal proteolytic pathway that gives rise to specific protein biomarkers (NfH476−986 and NfH476−1026) applicable to in vivo monitoring of diffuse axonal injury and neuronal loss in traumatic brain injury.

Intracerebral microdialysis during deep brain stimulation surgery

BackgroundThis report describes the use of microdialysis in conjunction with deep brain stimulation (DBS) surgery to assess extracellular levels of neurotransmitters within the human basal ganglia (BG). Electrical stimulation of the subthalamic nucleus (STN) is an efficacious treatment for advanced Parkinson's disease, yet the mechanisms of STN DBS remain poorly understood. Measurement of neurotransmitter levels within the BG may provide insight into mechanisms of DBS, but such an approach presents technical challenges. MethodsAfter microelectrode recordings confirmed location of STN, a custom microdialysis guide cannula was inserted. A CMA (Stockholm, Sweden) microdialysis probe was then positioned to the same depth as the microrecording electrode in STN or 2mm inferiorly to record in the substantia nigra. The catheter was perfused at a rate of 2.0μL/min with a sterile mock CSF solution and samples of extracellular fluid were collected at regular intervals. Dialysate samples were analyzed using high-pressure liquid chromatography (HPLC) detection procedures for quantitation of glutamate, gamma-aminobutyric acid (GABA), and dopamine. ResultsLevels of neurotransmitters were reliably identified in dialysate samples using HPLC. By monitoring concentrations of glutamate, GABA and dopamine, we were able to demonstrate what seemed to be a steady state baseline within approximately 30min. ConclusionMicrodialysis during DBS surgery is a feasible method for assessing levels of glutamate, GABA and dopamine within the human BG. Obtaining a steady state baseline of neurotransmitter levels appears feasible, thus making future studies of intraoperative microdialysis during DBS meaningful.

Evaluation of the distribution of enrofloxacin by circulating leukocytes to sites of inflammation in dogs

To determine the effect of WBC accumulation on the concentration of enrofloxacin in inflamed tissues in dogs. 6 adult Bloodhounds. Dogs were instrumented bilaterally with tissue chambers. Peripheral WBCs collected from each dog were exposed in vitro to radiolabeled enrofloxacin ((14)C-ENR). Inflammation was induced with carrageenan in 1 chamber. Ten hours later, treated cells were administered IV to each dog such that (14)C-ENR was delivered at a mean +/- SD dosage of 212 +/- 43 microg. Samples of extracellular fluid from inflammation and control chambers and circulating blood were then collected before (baseline) and for 24 hours after WBCs were administered. Samples were centrifuged to separate WBCs from plasma (blood) or chamber fluid. Radiolabeled enrofloxacin was scintigraphically detected and pharmacokinetically analyzed. Comparisons were made between extra- and intracellular chamber fluids by use of a Student paired t test. (14)C-ENR was not detectable in plasma, peripheral WBCs, control chambers, or baseline samples from inflammation chambers. However, (14)C-ENR was detected in extra- cellular fluid from inflammation chambers (mean +/- SD maximum concentration, 2.3 +/- 0.5 ng/mL) and WBCs (maximum concentration, 7.7 +/- 1.9 ng/mL). Mean disappearance half-life of (14)C-ENR from extracellular fluid and WBCs from inflammation chambers was 26 +/- 10 hours and 17 +/- 6 hours, respectively. WBCs were responsible for the transport and release of (14)C-ENR at sites of inflammation. Accumulation of drug by WBCs might increase the concentration of drug at the site of infection, thus facilitating therapeutic success.

POTENTIATION OF 3,4-METHYLENEDIOXYMETHAMPHETAMINE-INDUCED 5-HT RELEASE IN THE RAT SUBSTANTIA NIGRA BY CLORGYLINE, A MONOAMINE OXIDASE A INHIBITOR

1. It is well established that the commonly used recreational drugs 3,4-methylenedioxymethamphetamine (MDMA, 'ecstasy') and para-methoxyamphetamine (PMA) facilitate the release and prevent the reuptake of 5-hydroxytryptamine (5-HT, serotonin). Although these drugs have similar potencies for their abilities to increase the release and inhibit the re-uptake of 5-HT, PMA has greater potency as an inhibitor of monoamine oxidase (MAO)-A. 2. The present study compared the abilities of PMA and MDMA to increase extracellular 5-HT concentrations in animals with functional MAO-A and when MAO-A activity was inhibited by clorgyline. 3. Samples of extracellular fluid from rat substantia nigra were collected using microdialysis and then analysed for 5-HT and 5-hydroxyindol acetic acid (5-HIAA) by high-performance liquid chromatography coupled with electrochemical detection. The 5-HT-mediated effects on body temperature and behaviour were also recorded. Rats were pretreated with saline or 10 mg/kg, i.p., clorgyline and, 24 h later, injected with 10 mg/kg MDMA, PMA or saline. 4. Both MDMA and PMA produced significant increases in extracellular 5-HT concentrations (482 +/- 83 and 726 +/- 287%, respectively; P < 0.05). Rats treated with PMA and MDMA displayed significantly increased 5-HT-related behavours (P < 0.05). Furthermore, only MDMA was capable of producing additional significant increases in 5-HT concentrations (1033 +/- 131%; P < 0.01) when coadministered with clorgyline. 5. The results of the present study suggest that PMA and MDMA are similar in their abilities to increase extracellular 5-HT levels in animals with functional MAO-A activity. However, coadministration of these substituted amphetamines with an MAO-A inhibitor causes significant potentiation in the ability to increase extracellular levels of 5-HT for MDMA, but not PMA.

Intracerebral microdialysis for determination of the neuropharmacokinetics and neuropharmacodynamics of temozolomide in patients with primary or metastatic brain tumors

2074 Background: Microdialysis (MD) is a technique for continuously analyzing concentrations of endogenous chemicals and drugs in the extracellular fluid (ECF) of a body tissue. The purpose of this study is to apply MD to the determination of the neuropharmacokinetics (nPK) and neuropharmacodynamics (nPD) of temozolomide (TMZ) following systemic administration. Methods: After surgical debulking, a MD catheter is placed in peritumoral brain tissue. Artificial cerebrospinal fluid (CSF) is continuously perfused at a rate of 1 μl/min. Fused CT/MRI scans confirm correct placement of the catheter in non-enhancing brain. Starting 24 hrs after surgery, patients are given a dose of TMZ (150 mg/m2), and serial ECF and plasma samples are collected over 24 hrs. TMZ concentrations are determined by LC/MS/MS. In vitro recovery of TMZ via the MD catheter has been determined to be 87±5.5% at a flow rate of 1 μl/min. Prior to and 24 hrs post TMZ, additional ECF samples are collected for analysis of glucose, glutamate, lactate, and pyruvate, which are measured using a CMA 600 Analyzer®. Results: Four patients have been enrolled. There have been no grade 3 or 4 catheter-related adverse events. Mean peak TMZ concentrations in brain ECF and plasma are 0.4±0.2 and 5.5±1.4 μg/ml, respectively. Mean TMZ AUC in brain ECF and plasma are 3.2±1.6 and 18.2±4.6 μg/mLxhr, with an average ECF/plasma AUC ratio of 18±4%. Mean basal levels of glucose, glutamate, lactate, and pyruvate in ECF are 0.4±0.3, 34±66, 3±4, 69±62 mM, respectively. No consistent changes were seen in these nPD markers 24 hrs after TMZ. Conclusions: Concentrations of TMZ in brain ECF obtained by MD are similar to published data of TMZ concentrations in the CSF (Clin Cancer Res 2004;10:3728–36). Baseline values of markers of brain tissue metabolism are consistent with previous data in patients with brain tumors (J Neuro-Oncol 2003; 61:151–60). Intracerebral MD is an important tool that can be applied to nPK/nPD studies of new and targeted agents for the treatment of brain tumors. (Supported by CA01727) No significant financial relationships to disclose.

Pharmacokinetics of gemcitabine in tumor and non-tumor extracellular fluid of brain: an in vivo assessment in rats employing intracerebral microdialysis

Gemcitabine is a pyrimidine nucleoside analogue anticancer agent that has shown promising anti-tumor activity in several experimental models of brain tumor. However, the pharmacokinetic behavior of gemcitabine in the central nervous system, especially in brain tumors is currently not well understood. In this study we evaluated the gemcitabine brain extracellular fluid (ECF) in normal rats and in ECF obtained from tumor- and tumor-free regions of glioma-bearing rats, to better understand the availability of the drug to brain and brain tumors. The brain ECF pharmacokinetics of gemcitabine were investigated employing intracerebral microdialysis following intravenous administration of 10, 25 and 100 mg/kg doses in male Sprague-Dawley rats. In the second phase of the study, gemcitabine (25 mg/kg) was intravenously administered in rats implanted with C6 gliomas and ECF samples were simultaneously obtained from the tumor and tumor-free regions of the brain. Serial blood samples were obtained for evaluating the plasma pharmacokinetics of gemcitabine. Non-compartmental approach was employed for the analyses of the brain ECF and plasma pharmacokinetics of gemcitabine. Following intravenous administration, gemcitabine rapidly distributed into rat brain. At doses equivalent to 10, 25 and 100 mg/kg, the brain ECF gemcitabine AUC (area under the plasma concentration--time curve measured over the last sampling time point) values were 2.46 +/- 0.7, 3.20 +/- 1.1, and 9.06 +/- 3.0 microg h/ml, respectively. The brain ECF concentrations of gemcitabine declined in parallel with plasma concentrations. At the three doses evaluated, the relative brain distribution coefficient (AUC brainECF/AUC plasma) of gemcitabine ranged from 0.07 to 0.09 suggesting limited gemcitabine availability to brain tissues. Studies on C6 glioma-bearing rats revealed that following an intravenous dose of 25 mg/kg, the AUC values in the tumor-free and tumor-brain regions were 4.52 +/- 2.4, and 9.82 +/- 3.3 microg h/ml, respectively. Thus, the AUC of gemcitabine in the tumor ECF was on average 2.2-fold greater than the corresponding value in the tumor-free ECF of the brain. Plasma pharmacokinetics of gemcitabine remained unaltered in tumor-bearing animals, when compared to plasma pharmacokinetics in healthy animals. Our findings suggest that the overall brain exposure to gemcitabine is likely to be low as evident from the relative brain distribution coefficient of <0.1. However, the exposure is likely to be considerably higher in the brain tumor relative to tumor-free regions of the brain. The higher drug levels in brain tumor compared to the non-tumor region may facilitate selectively higher cytotoxicity against brain tumor cells.