Table of Contents 0007 - DOSE OPTIMIZATION OF PYRAZINAMIDE BASED ON DERMAL MICRODIALYSIS IN WISTAR RATS FOR CUTANEOUS LEISHMANIASIS TREATMENT .................................................. 4 0008 - IMPROVED BLOOD-BRAIN BARRIER DISTRIBUTION: EFFECT OF BORNEOL ON THE BRAIN PHARMACOKINETICS OF KAEMPFEROL IN RATS BY IN VIVO MICRODIALYSIS SAMPLING ............................................................................................................................................. 5 0009 - PENETRATION OF QUINIDINE AT THE BLOOD-BRAIN INTERFACES ................................ 6 0010 - LINKING PHARMACOKINETICS TO CNS DOPAMINE D2 RECEPTOR OCCUPANCY ......... 7 0011 - PERITONEAL MICRODIALYSIS IN PATIENTS SURGICALLY TREATED FOR PERITONITIS SHOWS DIFFERENCES BETWEEN UPPER AND LOWER PERFORATIONS OF THE GASTROINTESTINAL TRACT - A PILOT STUDY ............................................................................... 8 0012 - BIOFILM INFECTION LOWERS CIPROFLOXACIN LUNG PENETRATION ............................ 9 0013 - EXTRACELLULAR N-ACETYLASPARTATE IN TRAUMATIC BRAIN INJURY .................... 10 0014 - COMBINING MICRODIALYSIS AND PHARMACOKINETIC MODELLING ............................ 11 0015 - A STRATEGY OF IN-VIVO MICRODIALYSIS FOR PRECLINICAL PHARMACOKINETIC STUDY .................................................................................................................................................. 12 0016 - MICRODIALYSIS MONITORING OF CEREBROSPINAL FLUID, SUBCUTANEOUS TISSUE AND BRAIN TISSUE REVEALS TEMPORAL PATTERNS AND CORRELATIONS OF GLUCOSE AND LACTATE. .................................................................................................................................... 13 0017 - CONGENITAL ABDOMINAL WALL DEFECT ......................................................................... 14 0018 - MICRODIALYSIS FOR THERAPEUTIC DRUG MONITORING IN INFANTS ......................... 15 0019 - DOSE OPTIMIZATION FOR MULTIDRUG RESISTANT AND EXTENSIVELY DRUG RESISTANT TUBERCULOSIS BASED ON LUNG MICRODIALYSIS IN PATIENTS ........................ 16 0020 - P-GP IMPACT ON CIPROFLOXACIN PLASMA AND TISSUES EXPOSURE ....................... 17 0021 - SUCCINATE PERFUSED FOCALLY ENTERS THE TRICARBOXYLIC ACID CYCLE AND POTENTIATES CEREBRAL METABOLISM IN TRAUMATIC BRAIN INJURY PATIENTS: A 13C-LABELLED MICRODIALYSIS AND HIGH-RESOLUTION NMR STUDY. .......................................... 18 0022 - DERMAL MICRODIALYSIS OF THE TOPICAL ANTIBIOTIC RETAPAMULIN TO QUANTIFY SKIN CONCENTRATIONS AND EVALUATE ITS PHARMACOKINETICS IN WISTAR RATS ........ 19 0023 - DEVELOPMENT OF A DYNAMIC IN VITRO MICRODIALYSIS SYSTEM .............................. 20 0024 - MUSCLE METABOLISM AND CYCLIN A1 IN FACIOSCAPULOHUMERAL MUSCULAR DYSTROPHY-1 (FSHD-1) .................................................................................................................... 21 0025 - IS RELATIVE RECOVERY INFLUENCED BY DRUG COMBINATIONS? .............................. 22 0026 - CONTINUOUS MONITORING WITH SURFACE MICRODIALYSIS OF THE STOMACH AFTER GASTROESOPHAGEAL RESECTION .................................................................................. 23 0027 - NEUROCHEMICAL INVESTIGATION OF SEIZURE-INDUCED OXIDATIVE STRESS USING MICRODIALYSIS SAMPLING.............................................................................................................. 24 0028 - PROTEIN ADSORPTION TO MICRODIALYSIS MEMBRANES STUDIED BY QUANTITATIVE MASS SPECTROMETRY ......................................................................................... 25 0029 - DEVELOPMENT OF AN ONLINE MICRODIALYSIS-MICROCHIP ELECTROPHORESIS WITH LED-INDUCED FLUORESCENCE DETECTION SYSTEM FOR MONITORING EXCITATORY AMINO ACID NEUROTRANSMITTERS IN TRAUMATIC BRAIN INJURY ........................................ 26 0030 - IN VITRO MICRODIALYSIS OF A THERAPEUTIC MONOCLONAL ANTIBODY .................. 27 0031 - AGREEMENT OF DOUBLE MEASUREMENTS WHEN DETERMINING SOFT TISSUE CONCENTRATIONS OF LINEZOLID IN NORMAL WEIGHT AND MORBIDLY OBESE PATIENTS BY MICRODIALYSIS ............................................................................................................................ 28 0032 - LOCAL DRUG DELIVERY IN NEUROBLASTOMA ................................................................. 29

0033 - EARLY DETECTION OF PANCREATIC FISTULA AFTER PANCREATICODUODENECTOMY ..................................................................................................... 30 0034 - POSTOPERATIVE MICRODIALYSIS CATHETER MONITORING SUCCESSFULLY DETECTS COMMON COMPLICATIONS AFTER PANCREATIC TRANSPLANTATION ................. 31 0035 - CHANGES IN METABOLIC MARKERS AND MICROVASCULAR BLOOD FLOW IN THE SKIN DURING LOCAL INSULIN DELIVERY AND AFTER AN ORAL GLUCOSE LOAD ................. 32 0036 - MONITORING LIVER GRAFTS IN PEDIATRIC RECIPIENTS ................................................ 33 0037 - TISSUE METABOLISM IN VASOSPASM THERAPY WITH SPASMOLYTICS ...................... 34 0038 - A COMPARISON BETWEEN 1 MDA AND 100 KDA MICRODIALYSIS PROBES ON CEREBRAL CHEMOKINE AND CYTOKINE CAPTURE AFTER EXPERIMENTAL TRAUMATIC BRAIN INJURY ..................................................................................................................................... 35 0039 - EXPLORING FACTORS CAUSING LOW BRAIN PENETRATION OF THE OPIOID PEPTIDE DAMGO ................................................................................................................................................. 36 0040 - COMBINED MICROPET IMAGING AND MICRODIALYSIS SAMPLING OF OXYCODONE AS A STEP IN TRANSLATION OF PET DATA TO UNBOUND CONCENTRATIONS ............................ 37 0041 - SPME, A COMPLEMENTARY TOOL FOR MICRODIALYSIS FOR IN-VIVO INVESTIGATIONS: RAT BRAIN AND PLANT SAMPLING ................................................................ 38

0005 - ISCHEMIA, MITOCHONDRIAL DYSFUNCTION AND EVALUATION OF GLOBAL ENERGY STATE – FOCUS ON THE LP RATIO Carl-Henrik Nordström Dept. of neurosurgery, Odense University hospital, Odense, Denmark

Introduction: The lactate/pyruvate (LP) ratio obtained from intracerebral microdialysis reflects cytoplasmatic redox state. The latter is determined by the oxidative metabolism which in turn is primarily dependent on adequate cerebral perfusion/oxygen delivery and mitochondrial function. Under clinical conditions an increase in LP ratio is mainly observed during ischemia and post-ischemic mitochondrial dysfunction. As therapy differs in the two conditions it is of clinical importance to diagnose and separate them bedside. Intracerebral microdialysis gives biochemical information of a narrow zone of tissue surrounding the catheter. In addition it would be of importance to receive information regarding global cerebral energy state in many clinical conditions.

Methods: The experimental studies were performed in pigs (N=27) under general anesthesia. Clinical data were obtained from patients with severe brain trauma (N=213), subarachnoid hemorrhage (N=55), bacterial meningitis (N=15), cerebral infarcts (N=44) and during open heart surgery (N=10).

Results: The pattern of the biochemical variables (lactate, pyruvate and the LP ratio) obtained during routine intracerebral microdialysis may be used to discriminate between ischemia and mitochondrial dysfunction. During ischemia pyruvate is reduced below control level while it is increased or within normal limits in mitochondrial dysfunction. In addition it is possible to evaluate global cerebral energy state from monitoring the LP ratio of the cerebral venous outflow.

Conclusion: The biochemical information obtained during routine intracerebral microdialysis discriminates between ischemia and mitochondrial dysfunction. The information obtained bedside may be used to choose adequate therapy. The LP ratio obtained from microdialysis of cerebral venous outflow reflects global cerebral energy state. As the technique does not need insertion of an intracerebral catheter it may be used in general intensive care of patients with various serious cerebral disorders.

0007 - DOSE OPTIMIZATION OF PYRAZINAMIDE BASED ON DERMAL MICRODIALYSIS IN WISTAR RATS FOR CUTANEOUS LEISHMANIASIS TREATMENT Nivea Voelkner, Alexander Voelkner, Peter Kima, Hartmut Derendorf University of Florida, Gainesville, FL, USA

Skin lesions are clinical symptoms correlated with infections caused by cutaneous Leishmaniasis. Pyrazinamide is an antibiotic usually effective in treating infections caused by Mycobacterium tuberculosis used by oral administration that helps to shorten the duration of current chemotherapy regimens for tuberculosis. Studies suggests pyrazinamide effective against stages of parasites and the drug distribution that reaches the target site is unknown. Nowadays, models can characterize parasitic growth, parasitic killing by antibiotics and immune system. The aim of this study was to correlate pharmacokinetics (PK) and pharmacodynamics (PD) parameters to optimize the dosing regimen of pyrazinamide for cutaneous Leishmaniasis. PK/PD parameters were correlated to model and simulate outcomes of various dosing regimens. Currently, microdialysis is a minimally-invasive sampling technique that allows for the measurement of unbound drug concentration in selected tissues. Following intravenous (i.v.) bolus injection of pyrazinamide in healthy Wistar rats model, unbound concentrations were measured by dermal microdialysis sampling and compared to plasma concentrations. Time-kill curves were performed over 48 hours in triplicate, using macrophages infected with amastigotes-like parasites from Leishmania (Leishmania) amazonensis in the concentrations range of 10-100 µg/ml. The results indicate a linear PK for pyrazinamide with mean percentages of unbound concentrations dispersed in the dermis per concentration administered were 0.82 ± 0.31 and 0.84 ± 0.25 for 25 and 50 mg/kg doses, respectively. Biologically active pyrazinamide levels in the skin were not significantly different compared to unbound plasma concentrations. The employment of pyrazinamide in the doses 50 µg/ml and 100 µg/ml exhibited faster decrease in the infection rate percentage after 24 hours post-infection. The EC50 of pyrazinamide was estimated to be 13.7 µg/ml for amastigotes within macrophages. Our result revealed that a reasonable dosing strategy for pyrazinamide might be equal to 50 mg/kg given every 12 hours in immediate release.

0008 - IMPROVED BLOOD-BRAIN BARRIER DISTRIBUTION: EFFECT OF BORNEOL ON THE BRAIN PHARMACOKINETICS OF KAEMPFEROL IN RATS BY IN VIVO MICRODIALYSIS SAMPLING Qi Zhang, Dong Wu, Juan Wu, Qunlin Zhang Anhui Medical University, Hefei, China

Introduction: In vitro and in vivo studies have focused on the anti-Alzheimer effect of kaempferol (KA). However, little is known about its brain pharmacokinetic profile. The accumulated amount of KA in brain is very low because of the protection of blood-brain barrier (BBB). According to the basic theories of traditional Chinese medicine, borneol (BO) is called an "upper guiding drug", which can guide other components to the targeting tissues or organs in the upper part of the body, especially in the brain.

Methods: The probes for blood and brain sampling were implanted within the jugular vein/right atrium and right hippocampus of SD rat, respectively. Rats were intravenous administered of KA (25 mg/kg) alone or combined with BO (15, 30 mg/kg) via caudal vein. The blood and brain microdialysates were collected every 15 min for 180 min and every 30 min for 180-300 min. A selective and sensitive high performance liquid chromatography-chemiluminescence method was developed for the determination of unbound KA in microdialysates.

Results: KA quickly crossed the BBB to enter the extracellular fluid of hippocampus and reached the maximum concentration of 0.11 μg/mL within 30 min. The brain bioavailability and brain delivery of KA evidently increased with the co-administration of 15 and 30 mg/kg of BO. The AUC0-inf of KA in brain increased 1.84 and 2.19 times, and the Cmax of KA in brain increased 2.09 and 3.18 times than that without BO. The brain-to-blood distribution ratio of KA increased by 48.68% and 57.97% compared with that without BO.

Conclusions: BO can enhance the BBB permeability and improve the transportation of KA to brain. The dose-dependent effect of BO on the brain pharmacokinetic parameters of KA was observed. This co-administration strategy can be designed to enhance the brain accumulation of other neuropsychiatric medications.

0009 - PENETRATION OF QUINIDINE AT THE BLOOD-BRAIN INTERFACES Franciska Erdö1,2, István Sziráki2, Mirabella Sike2, Péter Trampus2, Petra Molnár3 1Pázmány Péter Catholic University, Faculty of Information Technology and Bionics, Budapest, Hungary, 2Solvo Biotechnology, Budaörs, Hungary, 3University of Szeged, Szeged, Hungary

Introduction: Quinidine (QND) is a widely used P-gp transporter substrate both in in vitro and in vivo. Its brain exposure is enhanced by the co-administration of the P-gp inhibitor PSC-833 in rats and mice. The function and localization of Bcrp1, the other principal cerebral efflux transporter, are different in the blood-brain barrier (BBB) and in the blood-cerebrospinal fluid barrier (BCSFB). The aim of our study was to compare the time-concentration profiles of QND in brain extracellular fluid (ECF) and in CSF in presence or absence of the inhibitor.

Method: Blood, brain ECF and CSF concentrations of QND were monitored by triple-probe microdialysis. QND (5 mg/kg) and PSC-833 (2x2 mg/kg) were administered intravenously in rats.

Results: After a peak at 60 min, the ECF and CSF concentrations of QND converged to the baseline at 3.5 hours in the control animals. Contrary, in the PSC-833 treated animals the QND concentrations decreased only moderately in the frontal cortex and did not reach the baseline in the lateral ventricle within the observation period. In control animals the real concentrations of QND in CSF and ECF were similar. However, after the combination treatment, the ECF levels markedly increased, while the CSF levels increased only moderately. Also the characteristics of the curves were different.

Conclusions: Our results indicate that effect of chemical blocking of P-pg at the BBB and BCSFB resulted in increased drug levels both in ECF and CSF. However, the elimination of QND from the brain tissue was much slower than from the CSF. The observations that despite the different orientation of P-gp in BBB and BCSFB there is some correlation between the changes in the ECF and CSF concentrations of QND suggest that at least for compounds of decent passive permeability there is a substantial transport between these compartments.

0010 - LINKING PHARMACOKINETICS TO CNS DOPAMINE D2 RECEPTOR OCCUPANCY Yin Cheong Wong, Rob van Wijk, Robin Hartman, Willem van den Brink, Oscar Natan, Wilhelmus de Witte, Dirk-Jan van den Berg, Elizabeth de Lange Leiden Academic Centre for Drug Research/Pharmacology, Leiden University, Leiden, The Netherlands

Introduction: Traditionally, receptor occupancy (RO) determination requires the use of radiolabelled tracer which has several limitations. We aim to develop an improved method by simultaneous measurements of CNS pharmacokinetics (PK) and RO profiles using a LC-MS based method with non-radiolabelled tracer.

Methods: The disposition and dopamine D2 receptor occupancy (D2RO) of remoxipride, a D2-selective antagonist, at the target site striatum (ST) were investigated. In Experiment 1, rats received either remoxipride or saline intravenous infusion (10-min), with brain tissues and blood collected at different time points. In Experiment 2, two microdialysis probes were implanted, one in ST and the other in cisterna magna (CM), to measure the unbound remoxipride concentrations in ST extracellular fluid (brainECF) and cerebrospinal fluid (CSF) respectively. For all rats, non-radiolabelled raclopride (D2 tracer) was injected 15 min before sacrifice. D2RO was determined as the difference in ST-to-cerebellum raclopride ratio between the remoxipride and saline groups. Remoxipride and raclopride in all samples were simultaneously quantified by LC-MS.

Results: The elimination phases of unbound plasma, brainECF, CSF and total ST tissue had comparable slopes, indicating equilibration of remoxipride between these compartments. While after infusion remoxipride was monotonically eliminated from brainECF, D2RO reached the maximum (70%) slowly at 100 min and then declined slowly to 50% at 240 min. Simple Emax model, which estimates RO from drug concentration and receptor affinity (Kd) and assumes equilibrium state, was not adequate. Drug-receptor association and dissociation rates (kon and koff) are factors that determines the time needed to reach equilibrium receptor binding, and models are being constructed to simulate the influence of these binding kinetics on RO.

Conclusions: Remoxipride PK and its D2RO time profiles were successfully elucidated. However, relationship between target site concentrations and D2RO was not direct. Mechanistic modeling is underway to testify whether incorporating binding kinetics would further improve the RO prediction precision.

0011 - PERITONEAL MICRODIALYSIS IN PATIENTS SURGICALLY TREATED FOR PERITONITIS SHOWS DIFFERENCES BETWEEN UPPER AND LOWER PERFORATIONS OF THE GASTROINTESTINAL TRACT - A PILOT STUDY Jonas Emil Sabroe1, Anne Reiss Axelsen1, Mark Bremholm Ellebæk1, Bjarne Dahler Eriksen2, Niels Qvist1 1Department of Surgery, Odense University Hospital, Odense, Denmark, 2Department of Anaesthesiology and Intensive Care, Odense University Hospital, Odense, Denmark

Background: Secondary peritonitis is a condition associated with high morbidity and mortality. Continuous postoperative monitoring of patients to ensure timely intervention to treat complications without delay is important for survival and outcome. We aimed to 1) investigate potential differences in postoperative intraperitoneal biomarker levels between patients with upper and lower gastrointestinal tract lesion, and 2) compare postoperative biomarker levels between complicated and uncomplicated patients.

Method: We included a total of 15 consecutive patients operated for upper (n=7) and lower (n=8) gastrointestinal tract perforation. A microdialysis catheter was placed intraperitoneally in each patient. Samples were collected every 4th hour for up to 7 postoperative days. Samples were analysed for concentrations of glucose, lactate, pyruvate and glycerol.

Results: Microdialysis results showed that patients with upper gastrointestinal tract lesions had significantly higher levels of postoperative intraperitoneal glucose and glycerol concentrations, as well as lower lactate/pyruvate ratios and lactate/glucose ratios. In the group with perforation of the lower gastrointestinal tract, those patients with a complicated course showed lower levels of postoperative intraperitoneal glucose concentration and glycerol concentration and higher lactate/pyruvate ratios and lactate/glucose ratios than those patients with an uncomplicated course.

Conclusion: Patients with upper and lower gastrointestinal tract lesions showed differences in postoperative biomarker levels. A difference was also seen between patients with complicated and uncomplicated postoperative courses.

0012 - BIOFILM INFECTION LOWERS CIPROFLOXACIN LUNG PENETRATION Bruna G. S. Torres1, Priscila M. Bernardi1, Victória E. Helfer2, Alexandre Macedo1, Teresa Dalla Costa1 1Pharmceutical Sciences Graduate Program, Federal University of Rio Grande do Sul, Porto Alegre, Brazil, 2College of Pharmacy, Federal University of Rio Grande do Sul, Porto Alegre, Brazil

Introduction: Usually free plasma levels of antimicrobials are used as surrogate for free concentrations in infected tissues. Pseudomonas aeruginosa, however, can establish a mucoid biofilm chronic lung infection with increased antibiotic resistance. This study aimed to evaluate the influence of P. aeuginosa biofilm pneumonia on free concentrations of the antipseudomonal fluoroquinolone ciprofloxacin.

Methods: Protocol approved by UFRGS Ethics in Animal Use Committee (24140). A rat model of P. aeuginosa biofilm pneumonia was used1. Anesthetized and ventilated healthy and infected Wistar rats (n = 6/group) had the carotid artery cannulated for blood sampling and a microdialysis probe (CMA 20, Ringer solution 1.5 µL/min) inserted into the lung for free drug concentrations sampling. Animals received a single i.v. bolus dose of ciprofloxacin (20 mg/kg) and were sampled up to 12 h. Ciprofloxacin was quantified by HPLC-fluorescence2. Retrodialysis in vivo probes' recovery was 16.1 ± 3.7%. Data were analyzed by non-compartmental approach and parameters compared by Student's t-test (α = 0.05).

Results: Statistical differences were observed in plasma clearance (1.59 ± 0.41 and 0.89 ± 0.44 L/h/kg) and elimination rate constant (0.23 ± 0.04 and 0.14 ± 0.08 h-1) but not in volume of distribution (5.08 ± 0.68 and 5.61 ± 1.08 L/kg) of healthy and infected rats, respectively. Pulmonary penetration was for times lower in infected (fT = 0.44) than in healthy (fT = 1.69) rats with no difference in the elimination rate constant (0.22 ± 0.06 h-1 and 0.23 ± 0.03 h-1).

Conclusions: P. aeruginosa biofilm infection reduces ciprofloxacin lung and increases drug plasma exposure indicating that plasma concentrations are not adequate to determine dosing regimens in biofilm pneumonias.

Acknowledgements: CNPq/Brazil and CAPES/Brazil.

References

• 1. Johansen, H. K., HØiby, N. 1999. Handbook of Animal Models of Infection, 517-526. • 2. Zimmermann, E.S., Torres, B.G.S., Dalla Costa, T. 2016. Biomedical Chromatography 30,

330-336.

0013 - EXTRACELLULAR N-ACETYLASPARTATE IN TRAUMATIC BRAIN INJURY Richard Shannon1, Susan van der Heide1, Eleanor Carter3, Ibrahim Jalloh1, David Menon2,3, Peter Hutchinson1,2, Keri Carpenter1,2 1Division of Neurosurgery, Department of Clinical Neurosciences, University of Cambridge, Cambridge, UK, 2Wolfson Brain Imaging Centre, Department of Clinical Neurosciences, University of Cambridge, Cambridge, UK, 3Division of Anaesthesia, Department of Medicine, University of Cambridge, Cambridge, UK

Introduction: N-acetylaspartate (NAA) is an amino acid derivative primarily located in the neurons of the adult brain. The function of NAA is incompletely understood. Decrease in brain tissue NAA is presently considered symptomatic and a potential biomarker of acute and chronic neuropathological conditions. In this study we have used microdialysis to investigate the behaviour of extracellular NAA (eNAA) levels in patients after a traumatic brain injury (TBI).1

Methods: Sampling was performed using cerebral microdialysis catheters (M Dialysis 71) perfused at 0.3 µl/min. eNAA was measured in microdialysates by HPLC, in 30 patients with severe TBI, and, for comparison, in radiographically ‘normal' areas of brain in 6 non-TBI neurosurgical patients. We established a temporal eNAA profile in 8 additional patients with severe TBI. Microdialysate concentrations of glucose, lactate, pyruvate, glutamate and glycerol were measured on an ISCUS clinical microdialysis analyser.

Results: We show that the temporal profile of microdialysate eNAA was characterised by a distinct rise 24 h after injury, followed by a steady decline; beyond 70 h post-injury average levels were 40 % lower than those measured in non-TBI patients. There was a significant inverse correlation between concentrations of eNAA and pyruvate; eNAA showed significant positive correlations with glycerol and the lactate/pyruvate (L/P) ratio measured in microdialysates.

Conclusions: The results of this on-going study suggest that changes in eNAA after TBI relate to the release of intracellular components possibly due to neuronal death or injury, as well as to adverse brain energy metabolism.

Reference: 1. Shannon, R.J., van der Heide, S., Carter, E.L., Jalloh, I., Menon, D.K., Hutchinson, P.J., Carpenter, K.L.H. 2015. J. Neurotrauma, Online ahead of print.

0014 - COMBINING MICRODIALYSIS AND PHARMACOKINETIC MODELLING Florian Slimano1, Zoubir Djerada2, Laurence Van Gulick1, Sylvie Brassart-Pasco1, Sylvain Dukic1 1MEDyC Research Unit, UMR CNRS/URCA n°7369, University of Reims, Reims, France, 2Department of pharmacology, EA3801, University Hospital of Reims, Reims, France

Introduction: YSNSG is a synthetic peptide targeting αvβ3 integrin which demonstrated a promising activity in-vitro and in-vivo against melanoma tumour. In order to determine pharmacokinetic parameters and predictive active doses in central nervous system (CNS) and subcutaneous tissue (SC), we conducted a microdialysis study coupled with pharmacokinetic modelling and Monte Carlo simulation.

Methods: After a recovering period of surgical procedures, a microdialysis probe was inserted in the caudate (CMA/11) and in subcutaneous tissue (CMA/20). Plasma samples and dialysates for the determination of YSNSG were drawn between 0 and 300 min after YSNSG intravenous administration of 10 mg/kg. YSNSG were measured by UPLC-MS/MS. A nonlinear mixed-effect modelling approach implemented in Monolix® v4.2.2 was performed to study the pharmacokinetic profile of YSNSG. Model selection and evaluation were based on usual diagnostic plot, precision and information criteria.

Results: Two models of 2-compartment with additional microdialysis compartment, parameterized as constant rate (elimination k and distribution k12/k21, k13/k31) and volumes (central V1 and peripheral microdialysis compartment V3), with zero-order input were selected to describe dialysate concentrations in CNS and SC. Inter-individual variability (IIV) was described by exponential terms and residual variability by a combined additive and proportional error model. Individual AUC (plasma and tissues) were derived for each animal by using the Empirical-Bayes-Estimates of the individual parameters. Tissue/plasma and brain/plasma area under the curve (AUC) ratio were respectively 72.0±26.0% and 1.3±1.8%. The regimens needed to achieve in-vitro predetermined target concentration in tissues were studied by Monte Carlo simulations using Monolix® v4.2.2.

Conclusions: In our study, the combination between microdialysis and pharmacokinetic modelling provides precious data in YSNSG preclinical development. YSNSG pharmacokinetic parameters show promising results in term of subcutaneous disposition. However, AUC ratio reflects a low rate of brain disposition with substantial IIV. Further investigations like encapsulation are currently conducted.

0015 - A STRATEGY OF IN-VIVO MICRODIALYSIS FOR PRECLINICAL PHARMACOKINETIC STUDY Tung-Hu Tsai1,2 1National Yang-Ming University, Taipei, Taiwan, 2Taipei City Hospital, Taipei, Taiwan

Microdialysis is a sampling tool which has been applied in the in-vivo preclinical pharmacokinetic and pharmacodynamic studies. The basic principle of microdialysis is passive diffusion based on the Fick's law, and the driving force is a concentration gradient which allows the small molecules pass through a semi-permeable membrane. According to the clinical pharmacology, the protein-unbound fraction is the pharmacological active form, which is available for absorption, distribution, metabolism and elimination. Besides, only protein-unbound form of the drug molecule can be delivered to the target sites for pharmacodynamic actions. Single or multiple microdialysis probes has been applied in the preclinical experimental animal for blood, brain, muscle, liver, kidney ...etc. multiple targets. The microdialysis systems consisted of a micro-perfusion pump, a microfraction collector and the appropriate microdialysis probes. The dialysis probes for blood (10 mm in length), brain (3 mm in length) and bile (7 cm in length) were made of silica glass capillary tubing arranged in a concentric design. For enterohepatic circulation study, the bile duct of the donor rat was cannulated proximal to the liver and the other end of tube was inserted through the bile duct into the duodenum of the recipient rat. The regional brain distribution, the portion of drug that passes through the blood-brain barrier, and hepatobiliary excretion can be defined by the area under the curve (AUC) ratio of brain-to-blood (AUCbrain/AUCblood) and bile-to-blood (AUCbile/AUCblood). Furthermore, the pros and cons of microdialysis will be discussed, including the detailed surgical techniques in animal experiments from rat blood, liver and bile duct for the analysis of protein-unbound drug. In conclusion, the strategy of multiple sampling in a single animal largely reduces the use of experimental animals and receives more reliable data for preclinical pharmacokinetic study.

0016 - MICRODIALYSIS MONITORING OF CEREBROSPINAL FLUID, SUBCUTANEOUS TISSUE AND BRAIN TISSUE REVEALS TEMPORAL PATTERNS AND CORRELATIONS OF GLUCOSE AND LACTATE. Eric Thelin, Johan Jakobsson, Cecilia Åkerlund, Mikael Svensson, Bo-Michael Bellander, David Nelson Karolinska Institutet, Stockholm, Sweden

Introduction: The microdialysis tool facilitates analysis of biochemistry in selected compartments with a high temporal resolution. Glucose and lactate play a key part in cerebral metabolism, although the temporal flow between compartments has not been fully elucidated, especially not during pathological conditions such as traumatic brain injury (TBI). Our aim was to analyze the temporal trend of the flow of the metabolites glucose, lactate, pyruvate and glycerol in severe TBI patients.

Material and Methods: We included patients that had been monitored with the CMA64 iView catheter with hourly cerebrospinal fluid (CSF, "global microdialysis") samples, CMA70 microdialysis catheter for hourly brain extracellular fluid (ECF)- and subcutaneous (SC) samples as well as arterial blood gases for blood levels of lactate and glucose. Intracranial pressure was monitored using extraventricular catheters. Linear estimation was used to adjust for sampling time differences. Cross-correlation tables were used to illustrate the data and time lag.

Results: We were able to include n=14 severe TBI patients. In total, approximately 1500 hours were monitored. In the brain ECF, lactate and pyruvate levels were driven heavily by glucose levels (r=0.3, respectively). Interestingly, lactate and pyruvate levels were strongly correlated (SC=0.5), while the lactate:pyruvate had a weak negative correlation, indicating a relatively higher levels of pyruvate. Glucose flows more freely between compartments (r=0.38-0.23), while lactate seems more regulated (r=0.2-0.0). The lag in flow of glucose between compartments was low, and probably due to methodological causes. Glycerol levels were only weakly correlated between the different compartments.

Conclusion: In severe TBI patients, glucose is heavily driving the concentrations of lactate and pyruvate, which strongly correlated in several compartments. Glucose flows freely between compartment, while lactate does not, making arterial lactate unreliable and stressing the need for focal monitoring. This set-up allows for kinetic research to further analyze metabolites following pathological conditions.

0017 - CONGENITAL ABDOMINAL WALL DEFECT Kirsten Risby2, Mark Bremholm Ellebæk1, Marianne Skytte Jakobsen3, Steffen Husby3, Niels Qvist1 1Surgical Department A, Odense University Hospital, Odense, Denmark, 2Hans Christian Andersen Children's Hospital, Odense University Hospital, Odense, Denmark, 3Department of Pediatrics, Kolding Hospital, Kolding, Denmark

Introduction: The aim of the study was to investigate the safety and clinical implication of intraperitoneal microdialysis (MD) in newborns operated on for congenital abdominal wall defect.

Methods: A total of 13 infants underwent intraperitoneal microdialysis (9 with gastroschisis and 4 with omphalocele). MD samples were collected every four hours and the concentrations of lactate, glycerol, glucose and pyruvate were measured. The results of MD were compared between the group of infants with gastroschisis and the group with omphalocele. The duration of parenteral nutrition and tube feeding were compared for high and low levels of intraperitoneal lactate, glycerol, and glucose and lactate/pyruvate ratio respectively. High and low levels were defined as above or below the median value on day one.

Results: Results from intraperitoneal MD showed a significantly higher mean lactate concentration in the group of infants with gastroschisis compared with the group of infants with omphalocele. The median values were 6.19 mmol/l and 2.19 mmol/l, respectively (P=0.006). The results from MD in the six infants in the gastroschisis group who underwent secondary closure after Silo treatment were similar to those who underwent primary closure. None of the infants with omphalocele received parenteral nutrition whereas all of the infants with gastroschisis did. There was no significant difference in duration of parenteral nutrition or tube feeding, respectively, when comparing the gastroschisis children with high versus low intraperitoneal lactate values. Placement of the MD catheter in the intraperitoneal cavity was feasible and without any major complications.

Conclusion: Intraperitoneal MD is a safe procedure and an applicable method in surveillance of inflammatory changes in the peritoneal cavity in infants after operation for congenital abdominal wall defect. The true clinical value of the method in infants with congenital wall defect remains unknown.

0018 - MICRODIALYSIS FOR THERAPEUTIC DRUG MONITORING IN INFANTS Sebastian Schroepf1, Daniela Burau2, Hans-Georg Muench1, Hartmut Derendorf3, Dieter Adam4, Charlotte Kloft2 1Dept. of Pediatrics, Dr. von Hauner Children’s Hospital, Munich, Germany, 2Dept. Clinical Pharmacy and Biochemistry, Institute of Pharmacy, Freie Universitaet Berlin, Berlin, Germany, 3Dept. of Pharmaceutics, College of Pharmacy, University of Florida, Gainesville, USA, 4PaediaCRO GmbH, Munich, Germany

Introduction: Plasma-based Therapeutic Drug Monitoring (TDM) of antibiotic agents in infants and neonates is used to decrease the risk of therapy failure (underdosing) or adverse effects (overdosing). However, blood drawing is a painful procedure and available blood volume for repetitive analysis is limited in this population. This translational feasibility study was set up to prove the suitability of subcutaneous microdialysis (µD) as alternative for TDM in infants.

Methods: In vitro recovery and delivery experiments were performed to set optimal conditions for relative recovery (RR) determination of vancomycin (VAN). CMA 63 microdialysis catheters (membrane length 10 mm; mdialysis) were used for in vitro and in vivo experiments. Feasibility of bed side µD was evaluated in 9 infants with VAN as antibiotic treatment on a neonatal intensive-care unit. Sampling interval was 30 min over a period of 24 hours. Retrodialysis was used for catheter calibration using at least a 10-fold concentration of expected plasma trough level of VAN. Analysis of microdialysate samples using LC-MS/MS was established according to EMA guidelines [1].

Results: Optimal settings for bed side µD identified in vitro were, e. g. Ringer's solution as adequate perfusate with a flow rate of 1 µL/min. With comparable RRs in delivery and recovery of VAN, retrodialysis appeared suitable for in vivo catheter calibration. Subcutaneous µD was well tolerated by all patients, no adverse events related to µD were noted in this vulnerable population. Due to long washout period of VAN, retrodialyisis was needed to be performed after µD measurements. VAN was successfully quantified in microdialysate samples from subcutaneous interstitial fluid.

Conclusions: In vitro characterisation of the analyte is essential prior to in vivo µD to ensure conditions for catheter calibration. As attractive alternative to plasma-based TDM, subcutaneous µD is applicable for individual concentration-time profiles in neonatal patients.

References [1] European Medicines Agency (EMA): Guideline on bioanalytical method validation (2012)

0019 - DOSE OPTIMIZATION FOR MULTIDRUG RESISTANT AND EXTENSIVELY DRUG RESISTANT TUBERCULOSIS BASED ON LUNG MICRODIALYSIS IN PATIENTS Tobias Heinrichs1, Aline Barth1, Russell Ryan Kempker2, Charles Arthur Peloquin1, Hartmut Derendorf1 1College of Pharmacy, University of Florida, Gainesville, FL, USA, 2Division of Infectious Diseases, Emory School of Medicine, Atlanta, GA, USA

Introduction: The global emergence of multidrug-resistant tuberculosis (MDR TB) is an enormous public health threat and major barrier to effective TB control. In 2013, the World Health Organization (WHO) estimated 480,000 new cases of MDR TB worldwide and 210,000 MDR TB-related deaths, and stated we are "off track" in controlling the epidemic. We hypothesize that the concentrations of pyrazinamide (PZA), moxifloxacin (MXF) and levofloxacin (LEVO) are lower inside pulmonary tuberculous lesions, the ultimate site of action, compared to serum. Lower drug concentrations in cavitary lesions may lead to development and amplification of resistance and ultimately to treatment failure. In addition, we assert that optimal drug concentrations will be associated with a faster time to culture conversion and less development of acquired drug resistance. Microdialysis was used to investigate lung penetration of the drugs.

Methods: 1. We developed a microdialysis methodology for the drug quantification in pulmonary tissue ex vivo. To calibrate the probes we employed the No-Net Flux technique ex vivo in a preclinical study in rabbits. In a clinical study, LEVO cavitary concentrations were measured among 12 MDR-TB patients undergoing adjunctive surgical therapy. 2. Furthermore, we investigated the possibility of simultaneous measurement of PZA and MXF using microdialysis in vitro.

Results: 1. Free tissue concentrations were slightly lower as compared to free serum concentrations for patients receiving daily doses of 750 mg LEVO (p=0.3922, ratio[tissue/serum]: 0.838). 2. In vitro, microdialysis recoveries were not altered when MXF and PZA were investigated simultaneously (PZA: recovery_single drug: 97.7% vs recovery_combination: 95.6%; MXF: recovery_single drug: 113.7% vs recovery_combination: 113.7%).

Conclusion: 1. Free extracellular LEVO concentrations inside the TB lesion are comparable with free serum drug concentrations. 2. Microdialysis testing in vitro supports the application of this tool for measuring PZA and MXF in combination from surgically excised cavitary TB lesions.

0020 - P-GP IMPACT ON CIPROFLOXACIN PLASMA AND TISSUES EXPOSURE Estevan Sonego Zimmermann1, Bruna Gaelzer Silva Torres1, Camila Néris dos Santos2, Stephan Schmidt3, Chakriadhar Lagishetty3, Teresa Dalla Costa1 1Pharmaceutical Sciences Graduate Program, Federal University of Rio Grande do Sul, Porto Alegre, Brazil, 2College of Pharmacy, Federal University of Rio Grande do Sul, Porto Alegre, Brazil, 3Center for Pharmacometrics & Systems Pharmacology, School of Pharmacy, University of Florida at Lake Nona, Orlando, USA

Introduction: Efflux transporters such as P-glycoprotein (P-gp) might play a role on ciprofloxacin tissue distribution and could represent the main reason for sub-therapeutic concentration at the infection site, contributing to therapeutic failures1. In this work, ciprofloxacin lung and prostate concentrations were determined by microdialysis in presence and absence of tariquidar, a P-gp inhibitor.

Methods: Protocol approved by UFRGS Ethics in Animal Use Committee (211609). Four Wistar rats groups were investigated (n = 6-8/group): two control groups (plasma and tissues) received ciprofloxacin (7 mg/kg i.v.) alone and two inhibitor-groups received ciprofloxacin 30 minutes after tariquidar (15 mg/kg i.v.) administration. Blood was sampled from cannulated carotid artery and CMA 20 probes sampled ciprofloxacin in prostate and lung up to 12 h. Non-compartimental analysis (NCA) and population PK analysis (popPK) were performed.

Results: A four compartment model was able to simultaneously characterize and predict concentrations in plasma and in the interstitial fluid of prostrate and lung showing the best curve fitting, precision of parameter estimates and model stability. Significant differences were observed in ciprofloxacin total plasma clearances from control and inhibitor groups (CL = 0.313 L/h and CLTAR = 0.223 L/h) indicating that tariquidar decreased the fluoroquinolone clearance by interfering with the drug active renal secretion. No differences were observed in lung and prostate exposure in the tariquidar groups that shared the same inter-compartmental distribution rate constants K12 (1.62 h-1), K21 (8.47 h-1), K13 (2.84 h-1), K31 (6.22 h-1), K14 (1.06 h-1), K41 (0.61 h-1) with the control groups.

Conclusion: Our results indicate that ciprofloxacin transport mediated by efflux transporters is negligible in lung and prostate but have impact on renal active secretion after intravenous administration. The popPK model developed can be useful for ciprofloxacin's regimen optimization.

Acknowledgements: CNPq/Brazil and CAPES/Brazil.

References

• 1. BRILLAULT, J., et. al. 2010. Antimicrobial Agents Chemotherapy, 543-545.

0021 - SUCCINATE PERFUSED FOCALLY ENTERS THE TRICARBOXYLIC ACID CYCLE AND POTENTIATES CEREBRAL METABOLISM IN TRAUMATIC BRAIN INJURY PATIENTS: A 13C-LABELLED MICRODIALYSIS AND HIGH-RESOLUTION NMR STUDY. Ibrahim Jalloh1, Adel Helmy1, Duncan Howe1, Richard Shannon1, Peter Grice1, Andrew Mason1, Clare Gallagher2, Matthew Stovell1, Susan van der Heide1, Michael Murphy3, John Pickard1, David Menon1, Adrian Carpenter1, Peter Hutchinson1, Keri Carpenter1 1University of Cambridge, Cambridge, UK, 2University of Calgary, Calgary, Canada, 3MRC Mitochondrial Biology Unit, Cambridge, UK

Background and Aim: After traumatic brain injury (TBI), complex cerebral processes include energy perturbations. Correlating with unfavourable outcome, high brain extracellular lactate/pyruvate (L/P) ratio suggests hypoxic metabolism or mitochondrial dysfunction: glucose converted to pyruvate generating a low ATP yield, then lactate dehydrogenase (LDH) converts pyruvate plus NADH to lactate plus NAD+, allowing more glycolysis. We investigated whether focal administration of succinate, a tricarboxylic acid (TCA) cycle intermediate that interacts directly with the mitochondrial electron transport chain, could improve cerebral metabolism.

Methods: We used microdialysis to perfuse disodium 2,3-13C2 succinate (12 mmol/L) for 24h into a total of 9 sedated TBI patients' brains and simultaneously collected the microdialysates for measurement of biomarkers of brain metabolism (ISCUS analyzer; 9 patients) and analysis of 13C-labelling in metabolites by 13C NMR spectroscopy (6 patients) to ascertain metabolic pathways.

Results: Products of 2,3-13C2 succinate were 2,3-13C2 malate and 2,3-13C2 glutamine indicating TCA cycle metabolism, and 2,3-13C2 lactate suggesting TCA cycle spinout of pyruvate (by malic enzyme or phosphoenolpyruvate carboxykinase and pyruvate kinase) then LDH-mediated conversion to lactate. Compared with baseline, succinate perfusion significantly decreased the L/P ratio due to increased pyruvate while lactate showed little change. Succinate perfusion significantly decreased concentrations of glutamate and glucose.

Conclusions: Succinate perfusion potentiated brain chemistry focally. Lower L/P ratio suggests better cellular redox whereby cytosolic NADH is recycled to NAD+ by mitochondrial shuttles (malate-aspartate and/or glycerol 3-phosphate), diminishing need for LDH conversion of pyruvate to lactate. Lower glutamate concentration may also be due to the malate-aspartate shuttle. The fall in glucose concentration suggests better utilisation. This study demonstrates for the first time in TBI human brain that direct supplementation of the TCA cycle potentiates brain chemistry, judged by established microdialysis biomarkers as well 13C-labelling patterns in metabolites. Whether these changes will translate into better ATP generation requires further investigation.

0022 - DERMAL MICRODIALYSIS OF THE TOPICAL ANTIBIOTIC RETAPAMULIN TO QUANTIFY SKIN CONCENTRATIONS AND EVALUATE ITS PHARMACOKINETICS IN WISTAR RATS Alexander Voelkner1, Nivea Voelkner1, Frederico Martins2, Hartmut Derendorf1 1University of Florida, Gainesville, FL, USA, 2University of Sao Paulo, Sao Paulo, Brazil

Introduction: Retapamulin is approved to treat superficial skin infections caused by methicillin-susceptible Staphylococcus aureus (MSSA). Systemic exposure is low after topical administration and skin concentration profiles and pharmacokinetic (PK) parameters are not reported in literature. The purpose was to determine free skin concentrations and PK parameters of retapamulin.

Methods: The study comprised three treatment groups with five animals each. Group 1 was administered an IV bolus of 5 mg/kg retapamulin. Study groups 2 and 3 received 0.1 mg/cm2

retapamulin ointment on tape-stripped and intact skin, respectively. Two linear microdialysis catheters (Mdialysis 66, 30 mm, 20kDa) were inserted into the skin prior to ointment application. MD recovery was determined by retrodialysis. Blood and microdialysis (MD) samples were collected at different time points and analyzed using LC-MS/MS. Non-compartmental and population PK analysis were performed in Phoenix WinNonlin 6.3 and NONMEM 7.3.

Results: In vivo MD recovery ranged from 77.0-94.2%. Rapid distribution (Q=2.31 L/h) and fast elimination of retapamulin (t1/2=2.2 h) was observed after IV bolus administration. Equilibrium of the free drug concentrations between plasma and skin was reached after approximately three hours (fAUCplasma/fAUCskin 0.677). Exposure in tape-stripped skin was higher if compared to intact skin (fAUC0-∞/D 1,793.3 vs. 29.3 ng*h/mL/mg). Maximum skin concentration in the tape-stripped group (303.5 ng/mL) exceeded reported minimum inhibitory concentrations (MIC) and was sub-MIC for intact skin (8.9 ng/mL). Systemic exposure after application on tape-stripped skin was approximately 4% and could not be determined in intact skin due to concentrations below the quantification limit.

Conclusions: Retapamulin exposure in tape-stripped skin was more than 60-fold higher than in intact skin. Concentrations exceeded MICs suggesting that the drug may be effective to treat deeper skin infections. The microdialysis technique also helped to establish PK parameters and may aid to design future studies and predict the drug’s safety and efficacy.

0023 - DEVELOPMENT OF A DYNAMIC IN VITRO MICRODIALYSIS SYSTEM Christine Weiser, Tania Fuhrmann-Selter, Charlotte Kloft Freie Universitaet Berlin, Berlin, Germany

Introduction: In vitro microdialysis (µD) is highly recommended to optimise µD settings for subsequent in vivo µD studies, e.g. proof-of-concept (applicability and feasibility) studies and particularly in case of analytes with special characteristics (e.g. lipophilic). Until now, in vitro µD investigations have been performed in static in vitro µD system (IVMS) with standardised and physiological-like experimental conditions, which did not allow to continuously adjust analyte concentrations. Our objective was to develop a dynamic IVMS, enabling to mimic continuous concentration-time profiles of analytes at the target site in in vivo µD studies.

Methods: The dynamic IVMS consists of a double layer glass flask forming two compartments. The outer compartment is filled with circulating warm water (from a heated water bath) heating the inner compartment. The inner compartment is filled with analyte-containing medium. Tubes connect the latter compartment with a pump, enabling specific flow rates. Magnetic stirring guarantees homogenous analyte concentration within the inner compartment. Beside of temperature in the inner compartment, investigated by a thermometer, the validation covered continuous stirring, continuous pumping and a steady in- and outflow of the medium fluid to/from the inner compartment. Validation was performed for 12 h.

Results: The temperature of the medium fluid in the inner compartment was continuously held at 37° C. The magnetic stir bar was stirred steadily at 700 rpm leading to a homogeneous medium in the inner compartment. The pump worked constantly and the in- and outflow corresponded to the pump rate of 0.15-0.25 mL/min during the investigated time period.

Conclusions: A dynamic IVMS was successfully developed and the performance validated by the above described parameters over 12 h. The validation procedure showed that the dynamic IVMS is a robust system with the potential to perform in vitro µD under physiological-like and pharmcokinetic conditions.

0024 - MUSCLE METABOLISM AND CYCLIN A1 IN FACIOSCAPULOHUMERAL MUSCULAR DYSTROPHY-1 (FSHD-1) Michael Boschmann, Anna Pakula, Joanna Schneider, Anja Mähler, Simone Spuler Charité Universitätsmedizin Berlin, ECRC, Berlin, Germany

Introduction: FSHD-1 is autosomal-dominant inherited and caused by a deletion in a repetitive element on the long arm of chromosome 4, known as D4Z4 region. Due to D4Z4 contractions, chromatin is relaxed and a number of normally silenced genes become activated. Affected muscles are often fat-infiltrated. There are indications for a disturbed regulation of cellular energy metabolism in affected muscles in vitro.

We wanted to look 1) for differences in gene expression patterns in skeletal muscle of FSHD patients vs. other muscular disorders (e.g. caveolinopathy, dysferlinopathy) and healthy controls, and 2) for disturbances in systemic and muscle metabolism at rest and after an oral glucose load (OGL). We hypothesized that altered muscle cell cycle functions and energy metabolism contribute to FSHD-1 pathophysiology.

Methods: Muscle (Vastus lateralis) biopsies were taken from 7 patients and 7 controls for cDNA microarray, RT-PCR, immunohistochemistry and Western blot analyses to assess RNA and protein expression in muscle cell lines and tissue. Muscle fibers diameter was calculated on cryosections. Systemic and local (Vastus lateralis muscle) metabolism was studied in 15 patients and 15 healthy controls by calorimetry and microdialysis, respectively, before and during an OGL.

Results: Only cyclin A1 RNA and protein expression was up-regulated and this exclusively in FSHD-1. Mean muscle fiber diameter was increased and resting and postprandial carbohydrate oxidation were lower in FSHD vs. controls (women>men). Muscle tissue perfusion was also lower in FSHD (women=men). In men, higher resting and postprandial muscle Lac/Pyr ratios indicated mitochondrial dysfunction.

Conclusions: In FSHD, increased cyclin A1 RNA and protein expression might contribute to muscle hypertrophy in affected muscles. While systemic carbohydrate oxidation is decreased (and fat oxidation increased) in men and women, mitochondrial dysfunction is present rather in men than women, which is in line with clinical observations that men are more severely affected than women.

0025 - IS RELATIVE RECOVERY INFLUENCED BY DRUG COMBINATIONS? Daniela Burau1, Philipp Simon2, Lisa Ehmann1, David Petroff2, Hermann Wrigge2 1Freie Universitaet Berlin, Berlin, Berlin, Germany, 2University of Leipzig, Leipzig, Sachsen, Germany

Background: When using retrodialysis as calibration method for microdialysis catheters, performance conditions in microdialysis and retrodialysis periods should be equal to ensure identical diffusion characteristics through the semipermeable membrane in both directions. Achieving equal conditions in in vivo microdialysis trials may be challenging, when complex medical settings could influence microdialysis: Concomitant drugs may alter the relative recovery (RR) of the investigated drug. Hence, the effect of combined microdialysis of commonly used antibiotics and analgetics on their respective RR was investigated in vitro as preparation of a clinical microdialysis trial in obese and non-obese patients to evaluate the applicability of retrodialysis as catheter calibration method.

Methods: Investigations of single drug and drug combinations were performed in a standardised in vitro microdialysis system. CMA 102 pumps transported isotonic NaCl (perfusate) through three CMA 63 catheters (flow rate: 2 µL/min). RR was determined for single antibiotics and analgetics as well as for combinations of at maximum four drugs resembling their clinical use: (i) meropenem, linezolid, metamizole, paracetamol; (ii) cefazolin, metronidazole, metamizole, paracetamol; and (iii) tigecycline, metamizole, paracetamol. Five samples per catheter were collected in both, microdialysis and retrodialysis experiments, and analysed by a validated HPLC-UV assay.

Results: Within- and between-catheter variability were calculated for all investigated settings. All RR values of the drug combinations differed from -11.5% (meropenem in combination (i)) to +5.6% (tigecycline in combination (iii)) compared to the RR values of the single drug.

Conclusion: The observed small deviations in RR values were considered not relevant for the investigated drugs as they were in the range of imprecision due to bioanalysis and microdialysis sampling technique. The applicability of the catheter calibration by retrodialysis in microdialysis investigations should not be affected using these drug combinations; which reduces the efforts of in vivo catheter calibration in clinical trials.

0026 - CONTINUOUS MONITORING WITH SURFACE MICRODIALYSIS OF THE STOMACH AFTER GASTROESOPHAGEAL RESECTION Oscar Åkesson1, Pernilla Abrahamsson2, Rianne Lundqvist Waninge2, Per-Jonas Blind3 1Lund University Hospital, Lund, Sweden, 2Senzime AB, Uppsala, Sweden, 3Umeå University Hospital, Umeå, Sweden

Introduction: After resection of the Esofagus and Gastroesophageal junction, gastro-intestinal continuity is commonly restored by Gastric Tube reconstruction and intrathoracic anastomosis. Ischemia of the anastomotic region is considered to play a role in anastomotic leakage. Surface microdialysis (µD) has been used in laboratory to measure local biochemic parameters in a specific organ. Data of local changes of metabolism in surgical reconstruction after gastroesophageal resection will be presented.

Methods: In this study we have used a µD probe (OnZurf Probe, Senzime AB, Uppsala) developed for use on organ-surfaces to evaluate ischemia of the Gastric tube reconstruction. Anaesthetized normoventilated pigs were used. Surface µD probes and an intraparenchymal O2 saturation Licox CC1.2 probe (Integra, NeuroScience, Plainsboro, NJ) were placed on the stomach. A gastroesophageal resection was made to produce ischemia at the Gastric tube. Continuous on-line µD data (lactate) was monitored with CliniSenz Analyzer (Senzime AB, Uppsala) every 5 minutes and off-line data was collected in microvials (lactate, glucose) every 20 minutes during the experiment.

Results: Total ischemia was recorded at the cranial part of the Gastric reconstruction, measured by O2 saturation probe. µD data showed values indicating severe ischemia at the top of the Gastric tube reconstruction and partial ischemia at the level of transection of the Gastroepiploic artery. A Control probe in the abdominal cavity showed no change during the experiment.

Conclusions: Surface µD can detect and grade severity of local ischemia in real time with CliniSenz Analyzer and Onzurf Probe. The OnZurf Probe could possibly be used in a clinical setting for post-operative surveillance of the local metabolism in a surgical reconstruction.

0027 - NEUROCHEMICAL INVESTIGATION OF SEIZURE-INDUCED OXIDATIVE STRESS USING MICRODIALYSIS SAMPLING Amanda Furness, Hasitha Weerarathne, Susan Lunte, Craig Lunte University of Kansas, Lawrence, KS, USA

Introduction: Approximately 70% of epileptic patients experience seizures that occur in a specific localized region of the brain. Patients are diagnosed with epilepsy after experiencing two or more unprovoked seizures. These seizures result in the production of reactive oxygen species (ROS), leading to oxidative stress. The goal of this research is to develop a local epilepsy animal model where multiple seizures are induced within one experiment, while investigating the role of seizures in the production of ROS and neurotransmitter release. Oxidative stress was measured using malondialdehyde (MDA), which is a biomarker of lipid peroxidation

Methods: Microdialysis was used to administer an epileptic agent, 3-mercaptapropionic acid (3-MPA), locally to the hippocampus of a rat in two, 30 minutes intervals. The time between the two administrations of 3-MPA was varied to allow 40, 60, or 180 minutes of recovery before the next dose was given, while simultaneously collecting dialysate for analysis. Glutamate, GABA, norepinephrine (NE), and dopamine (DA) concentrations were determined in the dialysate samples using liquid chromatography with either fluorescence or electrochemical detection. Additionally, MDA was monitored fluorometrically using thiobarbituric acid.

Results: Depending on the length of recovery time between seizures, a 60-80% attenuation in glutamate concentration was observed in response to the second epileptic event. Dopamine was also attenuated by 12%. During each 3-MPA administration, decreases in GABA and increases in NE were observed, as expected. MDA concentrations also increased during each administration of 3-MPA, indicating that the seizure results in oxidative stress.

Conclusions: An animal model for multiple local seizures was developed, and significant attenuation in the excitatory response was observed with the induction of the second seizure. Future work will focus on understanding the cause of the attenuation of the excitatory response and the role of oxidative stress.

Acknowledgements Funding provided by NIH Grant: R01NS066466-03

0028 - PROTEIN ADSORPTION TO MICRODIALYSIS MEMBRANES STUDIED BY QUANTITATIVE MASS SPECTROMETRY Torgny Undin, Andreas Dahlin, Katarina Hörnaeus, Sara Lind, Jonas Bergquist Dept of Chem-BMC, Uppsala University, Uppsala, Sweden

Introduction: Formation of adsorbed protein layers onto biomaterial in contact with biological matrices such as microdialysis membranes can be problematic due to reduced sampling efficiency, encapsulation and rejection of implants. Novel approaches to study the adsorption and to reduce the protein adsorption to surfaces are needed.

Methods: Microdialysis membranes were incubated with either cerebrospinal fluid or an artificial protein solution of mimicking this body fluid. Adsorbed proteins were desorbed with on-surface enzymatic digestion (oSED). The corresponding peptides were analyzed with reversed phase nano liquid chromatography (LC) coupled to electrospray ionization and high-resolving mass spectrometry (MS). Specific experiments were designed to determine the mechanism of the oSED digestion, specific time-resolved protein adsorption and suitable surface modifications to decrease protein adsorption.

Results: A non-destructive way for investigating protein adsorption using oSED and MS detection was developed. The oSED is an important step in the procedure and our results confirm that the digestion is the key step for protein desorption [Undin et al 2015]. Most proteins stay adsorbed onto the surface during the entire digestion process, while peptides seem to desorb rather easily from the surface. The approach allows for collection of time-resolved adsorption profiles of proteins and heat maps facilitate clustering of proteins depending on adsorption behavior [Undin et al 2016]. In on-going studies, the effect on pluronic surface deactivation of the microdialysis membrane to reduce protein adsorption is investigated.

Conclusions: Our data proves that MS analysis of proteins adsorbed and digested on microdialysis membranes is a versatile tool for surface characterization. The developed methods are promising tool for future studies of protein-biomaterial, protein-polymer, protein-protein interaction.

References: Undin, T., Dahlin, A., Hörnaeus, K., Bergquist, J., Bergström Lind, S. 2016, Analyst, DOI: 10.1039/C5AN02091C.

Undin, T., Bergström Lind, S., Dahlin, A. 2015 Future Science OA, DOI: 10.4155/FSO.15.32(2015)

0029 - DEVELOPMENT OF AN ONLINE MICRODIALYSIS-MICROCHIP ELECTROPHORESIS WITH LED-INDUCED FLUORESCENCE DETECTION SYSTEM FOR MONITORING EXCITATORY AMINO ACID NEUROTRANSMITTERS IN TRAUMATIC BRAIN INJURY Michael Hogard1,2, Nathan Oborny3,2, Susan Lunte1,2, Craig Lunte1,2 1Department of Chemistry, University of Kansas, Lawrence, KS, USA, 2Ralph N. Adams Institute for Bioanalytical Chemistry, Lawrence, KS, USA, 3Department of Bioengineering, University of Kansas, Lawrence, KS, USA

Introduction: Excitatory amino acid neurotransmitters (EAAs) are associated with a wide number of neurodegenerative diseases. One such condition is traumatic brain injury (TBI), a leading cause of death and disability worldwide. Consequently, there is a large interest in the development of analytical techniques that can monitor EAAs in vivo to assist in medical treatment for TBI patients. Traditional benchtop equipment is too cumbersome for use in a medical setting, so a mobile system that can be used on-site is preferable.

Methods: The EAAs glutamate and aspartate were separated from other amino acids and detected using microchip electrophoresis (ME) with a borate background electrolyte solution. The analytes were derivatized with naphthalene-2,3-decarboxaldehyde (NDA) for fluorescence detection. The separation was optimized using a benchtop laser-induced fluorescence system. A mobile system with integrated light-emitting diode-induced fluorescence (LED-IF) detection was designed and built in-house for use with the separation.

Results: The ME-LED-IF system has limits of detection are 250 nM - 1.3 µM for derivatized amino acids.1 Glutamate and aspartate were detected in rat brain MD samples and separated from any interfering species. L-cysteic acid was used as an internal standard and introduced in an integrated online derivatization in order to monitor the state of the system. The MD-ME-LED-IF system with integrated online derivatization will be used to monitor in vivo changes in EAA concentration in near-real time in a rat epilepsy model.

Conclusions: The developed separation and analytical system are capable of monitoring EAAs in MD samples in near-real time. Future work will use the system to study an animal model of TBI. Additionally, research is being done to improve NDA derivatization on-chip for lower limits of detection with the system.

References 1. Oborny, N., Lunte, S., et al. 2016. Analytical Sciences, Vol. 32, 35-40.

Acknowledgments Funding provided by NIH grant R01 NS042929

0030 - IN VITRO MICRODIALYSIS OF A THERAPEUTIC MONOCLONAL ANTIBODY Satyawan B. Jadhav1, Vipada Khaowroongrueng1, Matthias Fueth2, Wolfgang Richter2, Michael B. Otteneder2, Hartmut Derendorf1 1Department of Pharmaceutics, College of Pharmacy, University of Florida, Gainesville, FL, USA, 2Roche Pharma Research and Early Development, Pharmaceutical Science, Roche Innovation Center, Basel, Switzerland

Introduction: Determination of tissue interstitial concentrations is an important aspect in PK/PD of therapeutic monoclonal antibodies (mAbs) 1. Microdialysis (MD) is a well-established technique for the continuous sampling of small and few large molecules within the extracellular fluid spaces; however, there are no reports of its application in case of therapeutic mAbs. In the present work we are exploring the utility of MD in determination of interstitial concentrations of a mAb targeting insulin like growth factor-1 receptor (IGF-1R). The data from in vitro MD experiment using a radiolabeled antibody is presented.

Methods: In vitro microdialysis and retrodialysis experiments were performed at 1µl/min perfusion flow rate using a polyethylene membrane probe (1000 kDa MW cutoff) coupled with push pull perfusion technique 2. The dialysate samples were collected every 30 minutes upto 2 hours after equilibration. The % recovery of radioactivity was evaluated at different concentrations of mAb as 0.1, 1, 10, 100 and 250 µg/ml.

Results: The mean % recovery of anti-IGF-1R antibody in both in vitro microdialysis and retrodialysis experiments was found to be in the range of 9-27% across different concentrations. A similar recovery was observed for Secukinumab (IL-17 antibody) in dermal interstitial fluid using open flow microperfusion technique 3.

Conclusions: MD using large MW cutoff membrane coupled with push-pull perfusion mechanism appears to be a promising approach for interstitial sampling of anti IGF-1R antibody. Further evaluation of MD recovery using unlabeled antibody and in vivo microdialysis is ongoing.

0031 - AGREEMENT OF DOUBLE MEASUREMENTS WHEN DETERMINING SOFT TISSUE CONCENTRATIONS OF LINEZOLID IN NORMAL WEIGHT AND MORBIDLY OBESE PATIENTS BY MICRODIALYSIS Philipp Simon1,2, David Petroff3, Daniela Burau4, Lisa Ehmann4, Christian Nestler1, Arne Dietrich2,5, Andreas W. Reske1, Charlotte Kloft4, Markus Zeitlinger6, Frieder Kees7, Hermann Wrigge1,2 1University of Leipzig, Department of Anaesthesiology and Intensive Care Medicine, Leipzig, Germany, 2University of Leipzig, Integrated Research and Treatment Center (IFB) Adiposity Diseases, Leipzig, Germany, 3University of Leipzig, Clinical Trial Centre, Leipzig, Germany, 4Free University of Berlin, Institute of Pharmacy, Department of Clinical Pharmacy and Biochemistry, Berlin, Germany, 5University of Leipzig, Department of Visceral, Transplantation, Vascular and Thoracic Surgery, Leipzig, Germany, 6Medical University of Vienna, Department of Clinical Pharmacology, Vienna, Austria, 7University of Regensburg, Department of Pharmacology, Regensburg, Germany

Background: In morbidly obese patients, pharmacokinetic data for antibiotic treatment are lacking. We are currently performing a prospective microdialysis study in 120 morbidly obese and normal weight patients to determine different antibiotic concentrations in subcutaneous interstitial fluid (ISF). Typically, microdialysis measurements in vivo are associated with high variability. To investigate the intraindividual variability of the microdialysis technique we performed parallel double measurements in both upper arms. The aim was to determine the repeatability of microdialysis in a clinical setting.

Material/methods: Following approval from the ethics committee and federal authorities, and after obtaining written informed consent, patients scheduled for abdominal surgery were included. A single dose of 600mg linezolid was infused intravenously for perioperative antibiotic prophylaxis. Linezolid concentrations were measured in subcutaneous ISF in both upper arms simultaneously with two catheters at 10 time points after injection (0.5h, 1h, 1.5h, followed every hour up to 8h). Calculation of concentrations in the ISF was enabled by calibrating the catheters using retrodialysis. Samples were analysed with an HPLC assay and data using Bland-Altman methods for multiple observations per individual on a logarithmic scale.

Results: We recruited 28 patients (15 obese) and performed successful measurements except in one arm of one patient. The mean ratio of concentrations between the arms was 1.06 and the limits of agreement ranged between a factor 0.44 and 2.57. The comparison between the arms was similar in morbidly obese and normal weight patients.

Conclusions: Differences in tissue concentrations were observed to be quite high at the two sites and further analyses of potential causes of variations are needed. In addition, future studies with this method should include sufficiently large patient samples and results should be interpreted with caution.

0032 - LOCAL DRUG DELIVERY IN NEUROBLASTOMA Carles Monterrubio, Jaume Mora, Angel M Carcaboso Preclinical Therapeutics and Drug Delivery Research Program and Department of Pediatric Hematology and Oncology, Hospital Sant Joan de Déu Barcelona, Esplugues de Llobregat, Spain

Introduction: Clinical translation of new local drug delivery systems (DDS) for pediatric cancer treatment is challenging. It is not totally understood whether an anticancer agent locally released in the proximity of a solid tumor penetrates deeply into the bulk of the tumor, or whether the penetration is restricted to the margins in direct contact with the DDS. Thus, it remains unclear which group of patients would benefit from such new DDS. To tackle this question from the preclinical perspective we have applied a microdialysis method in a xenograft model of neuroblastoma.

Methods: The DDS consisted in polymer nanofibers loaded with the potent and poorly soluble anticancer drug SN-38. Hydroxypropyl-beta-cyclodextrin was employed to avoid nonspecific binding of SN-38 to the microdialysis device. The microdialysis probes could be calibrated by the zero flow rate method in vivo. We studied drug delivery in the simmulated tumor resection bed (surgical bed) by inserting microdialysis probes in the virtual space between the DDS and the skin of athymic nude mice. In mice with subcutaneous neuroblastomas, we inserted the DDS peritumorally and placed the microdialysis probe intratumorally at different depths (i.e., distances DDS-probe) into the tumor bulk. We collected dialysates and blood samples and analyzed SN-38 by HPLC with fluorescence detection.

Results: We observed that the drug released from the local DDS achieved potentially active concentrations in the virtual space of the surgical bed and it penetrated a maximum distance of 2 mm within the bulk of the tumor.

Conclusions: Microdialysis helped characterize local drug distribution in tumors and surgical bed. The results of our study will help select the candidate patients that would likely benefit of the novel local DDS in a future clinical trial.

0033 - EARLY DETECTION OF PANCREATIC FISTULA AFTER PANCREATICODUODENECTOMY Gísli Björn Bergmann1,2, Søren Pischke1, Håkon Haugaa1, Tor Inge Tønnessen1,2 1Oslo University Hospital, Division of Emergencies and Critical Care, Oslo, Norway, 2University of Oslo, Faculty of medicine, Oslo, Norway

Introduction & Aim: Pancreatic fistula due to anastomosis insufficiency is a relatively common (12-30%) complication after pancreaticoduodenectomy. These are often discovered with considerable delay, causing severe peritonitis, erosion of vessels and even death. Microdialysis catheters have been shown to detect of both inflammation and ischemia in a number of postoperative conditions and organs. The aim of the study was to investigate if microdialysis catheter monitoring could facilitate earlier detection of pancreatic fistula than current standard of care.

Methods: Thirty-five patients, 20 women and 15 men, aged 44 to 88 years old were investigated. A microdialysis catheter was fixed to the pancreaticojejunal anastomosis at the end of the procedure. Samples for analysis of glucose, lactate, pyruvate and glycerol were acquired hourly during the first 24 hours, then every 2-4 hours to discharge. Postoperative pancreatic fistula (POPF) was defined as at least three times higher concentration of amylase in drain fluid than the upper normal serum concentration on postoperative day 3 or later, or by the discovery of anastomosis leakage during re-operation.

Results: Patients who developed POPF (N=9) had significantly higher glycerol levels (P<0.01) in microdialysate than did patients without POPF (N=26) during the first 24 hours after which the difference diminished. A glycerol concentration of more than 400 µmol/L during the first 12 postoperative hours detected patients who later developed POPF with a sensitivity of 89% and specificity of 92%. Lactate and lactate to pyruvate ratio were significantly higher (P<0.05) and glucose was significantly lower (P<0.05) in patients with POPF from about 24 hours postoperatively.

Conclusions: A high level of glycerol in microdialysate is an early (first 12 hours) indicator of pancreatic fistula. Glucose, lactate and lactate to pyruvate ratio are indicators of peritonitis caused by the leakage. Thus, microdialysis monitoring detects pancreatic fistula several days earlier than current methods.

0034 - POSTOPERATIVE MICRODIALYSIS CATHETER MONITORING SUCCESSFULLY DETECTS COMMON COMPLICATIONS AFTER PANCREATIC TRANSPLANTATION Gisle Kjøsen1, Rune Horneland2, Ole Øyen2, Einar Martin Aandahl2,6, Tom Eirik Mollnes3,5, Audun Elnæs Berstad4, Gaute Hagen4, Eric Dorenberg4, Kari Dahl2, Andreas Barratt-Due1,3, Sören Pischke1,3, Gisli Björn Bergmann1, Tor Inge Tønnessen1,5, Håkon Haugaa1 1Division of Emergencies and Critical Care, Dept. of Anaesthesiology, Oslo, Norway, 2Division of Cancer Medicine, Surgery and Transplantation, Dept. of Transplantation Surgery, Oslo University Hospital, Oslo, Norway, 3Division of Diagnostics and Intervention, Dept. of Immunology, Oslo University Hospital, Oslo, Norway, 4Division of Diagnostics and Intervention, Dept. of Radiology and Nuclear medicine, Oslo, Norway, 5Institute of Clinical Medicine, University of Oslo, Oslo, Norway, 6The Biotechnology Institute, University of Oslo, Oslo, Norway

Introduction: The complication rate after pancreas transplantation is very high. There is currently a lack of reliable methods to uncover complications at an early stage.

In an ongoing study, we investigate whether microdialysis catheters are able to detect complications post transplantation. In this case report we describe the successful detection of venous thrombi and anastomosis leakage by microdialysis catheters.

Method: Four diabetic patients underwent technically similar solitary pancreas transplantations. A common aortic patch with both the coeliac trunk and the superior mesenteric artery was anastomosed "end-to-side" to the common iliac artery on the right side and an elongated portal vein was anastomosed "end-to-side" to the inferior caval vein. At the end of surgery, two microdialysis catheters were placed on the pancreatic surface. We measured glucose, lactate, pyruvate and glycerol at the bedside, sampled every 1-2 hours postoperatively.

Results: All four patients had uncomplicated initial postoperative courses. On the third postoperative day however, two of the patients had elevations in lactate and lactate to pyruvate ratio in microdialysis measurements. Doppler ultrasounds showed well-circulated grafts, but supplemental CT scans revealed venous thrombi in both patients. The patients underwent immediate angiographic intervention, and the thrombi were successfully removed. Both patients remain insulin-free nine months post transplantation.

Another patient had a 36-fold increase in microdialysis glycerol 20 hours postoperatively, the last patient had elevated microdialysis glycerol in excess of 1000µm from the 1st measurement. They were discharged on the 9th postoperative day, but readmitted with abscesses in relation to the enteric anastomoses, as detected by CT scans. They were both successfully treated, one required IV antibiotics alone, the other needed surgical drainage and IV antibiotics.

Conclusion: Monitoring of pancreas transplants with microdialysis catheters appear to detect early complications like anastomosis leakage and ischemia at an early stage, and may potentially improve graft survival.

0035 - CHANGES IN METABOLIC MARKERS AND MICROVASCULAR BLOOD FLOW IN THE SKIN DURING LOCAL INSULIN DELIVERY AND AFTER AN ORAL GLUCOSE LOAD Fredrik Iredahl, Alexandra Högstedt, Joakim Henricson, Folke Sjöberg, Erik Tesselaar, Simon Farnebo Linköping university, Linköping, Sweden

Background: Insulin has vasoactive properties that augments its delivery to the cells. The knowledge of microvascular effects of insulin in the skin is limited. The aim of this study was to investigate the metabolic and vasoactive actions of insulin in the skin, when it is (1) endogeneously produced and delivered to the skin via the blood stream or (2) directly delivered to the insterstitium via a microdialysis catheter.

Methods: Two microdialysis catheters were inserted in the skin of the forearm in 8 healthy subjects.Insulin was delivered through one catheter, while a control substance was delivered through the other. After one hour, subjects received an oral glucose load. Changes in glucose, lactate, pyruvate and urea in the dialysate were analysed. Skin perfusion was measured using laser speckle contrast imaging.

Results: During local delivery of insulin, no change in glucose concentration (p>0.96) was observed. Lactate and pyruvate concentrations were increased significantly after local insulin delivery, compared to control (lactate: p<0.04, pyruvate: p<0.02). Skin blood flow was increased around the catheter through which insulin was delivered, compared to control.

Glucose, lactate, pyruvate and insulin concentrations in the skin all increased after the oral glucose load. A further increase in skin blood flow during systemic glucose intake independent of local insulin delivery was observed.

Locally delivered insulin and an oral glucose load cause metabolic changes in the skin. Skin perfusion was increased after local insulin delivery. While blood flow surrounding the catheters was further increased after an oral glucose load, this is likely the result of needle insertion trauma, since no increase in perfusion was observed at a control site.

Conclusion: Local and systemic insulin has metabolic and vasoactive actions in the skin. Microdialysis combined with perfusion measurement enables minimally-invasive studies of these metabolic and vascular effects.

0036 - MONITORING LIVER GRAFTS IN PEDIATRIC RECIPIENTS Gísli Björn Bergmann1,2, Søren Pischke1, Håkon Haugaa1, Tor Inge Tønnessen1,2 1Oslo University Hospital, Division of Emergencies and Critical Care, Oslo, Norway, 2University of Oslo, Faculty of medicine, Oslo, Norway

Introduction: We have previously shown that microdialysis catheters implanted in liver transplants detect post-operative complications such as vascular occlusion and acute cellular rejection (ACR) with high sensitivity and specificity.

Methods: The method is now a standard monitoring technique in our pediatric liver transplant recipients. We are now investigating the implementation of a clinical decision tree based on cutoff values for detecting ischemia (intrahepatic lactate >2.6 mM and lactate to pyruvate (L/P)-ratio >20) and rejection (intrahepatic lactate >2 mM with normal L/P ratio). We are also exploring if catheters placed in the abdominal cavity may detect postoperative complications such as biliary leakage and infections. At the end of surgery, one catheter is inserted in the liver graft and another is placed in the abdominal cavity. Lactate, pyruvate, glucose, and glycerol are analyzed every 30-60 minutes. Intrahepatic lactate >2.6 mM with an L/P-ratio >20 prompts an ultrasound Doppler evaluation of the graft circulation within the next 30 minutes. ACR is suspected and a liver biopsy is performed if lactate increases to >2.0 mM with an L/P-ratio <20 on 3 consecutive measurements.

Results: We have monitored 27 liver grafts so far. Thirteen grafts have had a vascular event (including hepatic artery thrombosis or stenosis, portal vein thrombosis or stenosis, hepatic vein stenosis or hemorrhage/hematoma in the hilus of the liver). ACR was suspected in 10 grafts of which 6 were confirmed by biopsy. Two patients had a perforated small bowel and peritonitis. Disturbances in the graft circulation are detected several hours before circulating lactate and transaminases increase. ACR is detected several days before circulating transaminases and/or bilirubin increase. The abdominal catheters detect infectious complications and cases suggest that they may be useful in distinguishing thrombosis of the hepatic artery from portal vein thrombosis when the intrahepatic metabolism is monitored simultaneously.

0037 - TISSUE METABOLISM IN VASOSPASM THERAPY WITH SPASMOLYTICS Aleš Hejčl1, Filip Cihlář2, Martin Bolcha1, Jan Procházka3, Martin Sameš1 1Masaryk Hospital, Department of Neurosurgery, Ústí nad Labem, Czech Republic, 2Masaryk Hospital, Department of Radiology, Ústí nad Labem, Czech Republic, 3Masaryk Hospital, Department of Anesthesiology and Intensive Care Medicine, Ústí nad Labem, Czech Republic

Introduction: Clinically symptomatic vasospasm causing a delayed ischemic neurological deficit occurs in up to 30% of patients with subarachnoid hemorrhage (SAH). In the last 7 years we have treated 27 female patients and 7 male patients with vasospasm by intra-arterial application of either nimodipine or milrinone.

Methods: After securing a ruptured aneurysm, the patients were monitored using clinical evaluation and repeated TCD monitoring. We implanted multimodal brain monitoring probes including microdialysis in 5 patients with severe SAH (GOS < 9). In two patients the probes were implanted before interventions, in one of them ipsilaterally and in the other patient it was implanted in the contralateral hemisphere. In the other 3 patients the probes were implanted after administration of spasmolytics.

Results: Increased TCD velocities indicated treatment of vasospasm with intra-arterial administration of spasmolytics. Elevation of glucose concentrations and stabilization of the LP ratio were observed after intra-arterial administratin of spasmolytics.

Conclusions: Monitoring tissue meetabolism may aid in indicating vasospasm treatment using spasmolytics after SAH.

0038 - A COMPARISON BETWEEN 1 MDA AND 100 KDA MICRODIALYSIS PROBES ON CEREBRAL CHEMOKINE AND CYTOKINE CAPTURE AFTER EXPERIMENTAL TRAUMATIC BRAIN INJURY Fredrik Clausen, Lars Hillered Uppsala University, Neuroscience, Neurosurgery, Uppsala, Sweden

Introduction:Capturing protein biomarkers using intracerebral microdialysis for prognostic and diagnostic purposes is an expanding field in neurotrauma research. To enhance the protein content in MD-samples development of novel probe membranes is highly interesting.

Objectives: Novel probe membranes with a molecular cut off of 1MDa were tested for their ability to capture protein biomarkers in an in vivo setting and were compared to the more established 100kDa membranes.

Material and methods: We induced diffuse TBI in rats using the midline fluid percussion injury (mFPI) model or sham surgery (n=6 per group). The surgeries were followed by six hours of hourly sampling using Pluronics® F-127 coated MD-probes (CMA) with either 1MDa or 100kDa cut off membranes. The 1MDa probes required a push-pull pump system to ensure stable fluid recovery. The samples were analyzed using a multiplex MAGPIX kit for 27 chemokines, cytokines and growth factors, all involved in the inflammatory response to TBI.

Results: For the 100kDa membranes, TBI induced an increase in 17 of the 27 protein concentrations within the first 6 h after injury compared to sham injury and for the 1MDa membranes 19 of the 27 protein concentrations were increased compared to sham. However, the samples from 1MDa membranes had dramatically higher concentrations compared to 100kDa membranes for 9 proteins in TBI animals and 6 proteins for sham animals. Surprisingly, the higher concentrations were almost exclusively observed for low molecular weight chemokines.

Conclusions: The study shows that the 1MDa membranes perform as well or better for capturing the protein biomarkers analyzed in this study compared to the 100kDa membranes. However, the 1MDa membranes require a more complicated pump system to ensure a stable performance over the semipermeable membrane, which would require the development of miniaturized push-pull pumps suitable for application in the NICU setting.

0039 - EXPLORING FACTORS CAUSING LOW BRAIN PENETRATION OF THE OPIOID PEPTIDE DAMGO Annika Lindqvist, Siv Jönsson, Margareta Hammarlund-Udenaes Uppsala University, Uppsala, Sweden

In order to improve the treatment of pain, many opioid peptides with improved pharmacological properties have been developed without successful analgesia when administered IV compared to IT. Our earlier findings showed that a low penetration into the brain, with an unbound brain to blood ratio, Kp,uu, of 0.08, is an important reason for the lack of effect of the enkephalin analog DAMGO (H-Tyr-D-Ala-Gly-MePhe-Gly-ol). The aim of this study was to investigate the role of efflux transporters, metabolism in the brain and/or elimination through interstitial fluid bulk flow for the brain exposure of DAMGO.

The in vivo brain distribution of DAMGO was evaluated using microdialysis in the rat. Data was analyzed with population modeling in order to estimate the rate and extent of brain distribution. In a second study, the Pgp/Bcrp transporter inhibitors cyclosporine A and elacridar were co-administered with DAMGO to evaluate the importance of efflux transporters on the blood-brain barrier transport. Caco-2 experiments were performed to estimate DAMGO permeability and efflux ratio. Modeling gave a clearance into the brain of 1.1 and an efflux clearance 14 µl/min/g_brain. The efflux clearance was thus much higher than the bulk flow known from the literature. Co-administration with cyclosporine A and elacridar in vivo did not affect Kp,uu, suggesting that DAMGO is not a substrate for transporters inhibited by these compounds. The Caco-2 assay showed that DAMGO has a very low permeability, of the same size as mannitol. The efflux ratio was < 2 and not influenced by the blockers.

DAMGO has a higher efflux than influx clearance from brain to blood, limiting the brain distribution of this opioid peptide. The well-known efflux transporters Pgp and Bcrp do not seem to be responsible for the higher efflux of DAMGO, which opens up for an important role of other transporters at the BBB.

0040 - COMBINED MICROPET IMAGING AND MICRODIALYSIS SAMPLING OF OXYCODONE AS A STEP IN TRANSLATION OF PET DATA TO UNBOUND CONCENTRATIONS Sofia Gustafsson1, Jonas Eriksson2, Stina Syvänen3, Olof Eriksson2, Margareta Hammarlund-Udenaes1, Gunnar Antoni2 1Department of Pharmaceutical Biosciences, Translational PKPD, Uppsala University, Uppsala, Sweden, 2Department of Medicinal Chemistry, Preclinical PET Platform, Uppsala University, Uppsala, Sweden, 3Department of Public Health and Caring Sciences, Molecular Geriatrics, Uppsala University, Uppsala, Sweden

In order to aid CNS drug development and to optimize clinical practice, it is desirable to evaluate unbound, pharmacologically active, drug concentrations within the brain. Positron emission tomography (PET) is used in both preclinical and clinical settings but measures only total drug concentrations, making it difficult to predict the concentration of unbound drug and hence effective concentrations at target site. Increasing failure rates in CNS drug development and the urgent need of new CNS drugs highlights the necessity of translational methods and more accurate predictions of unbound rather than total concentrations within the brain. Hence, the aim of the current project is to investigate oxycodone brain distribution and to elucidate whether total concentrations of oxycodone, measured by microPET, could be mathematically converted into unbound concentrations, similar to those observed with simultaneous microdialysis.

Rats received a 60 min constant rate intravenous infusion of a tracer dose of [N-methyl-11C]oxycodone in combination with oxycodone (0.3mg/kg/h). Discrete blood sampling, continuous microdialysis sampling and microPET imaging was carried out during the 2 h experiment. Plasma and microdialysis samples were analyzed using LC-MS/MS. ROI calculations were performed using the software AMIDE. Total brain concentrations, measured by PET, were converted into unbound concentrations using the unbound volume of distribution in brain, Vu,brain, for oxycodone. Results are presented as mean±SD.

Active uptake of oxycodone in brain was observed with both methods. The unbound brain to blood concentration ratio of oxycodone was calculated to 2.9±0.3. PET data converted to unbound concentrations showed a good congruence with microdialysis data. However, an apparent deviation was observed in the elimination phase, likely due to radioactive metabolite formation.

The results indicate that neuropharmacokinetic concepts can be applied in PET in order to predict unbound drug concentrations in brain, given that compensation is made for metabolite formation.

0041 - SPME, A COMPLEMENTARY TOOL FOR MICRODIALYSIS FOR IN-VIVO INVESTIGATIONS: RAT BRAIN AND PLANT SAMPLING Barbara Bojko, Janusz Pawliszyn University of Waterloo, Waterloo, Canada

The focus of the presentation will be on in-vivo targeted and global tissue metabolomics study with the use of single SPME probe compared to microdialysis (MD). The comparison has been done using in vivo SPME and MD in the brain of freely moving rats. It has been demonstrated that in many analytical aspects SPME providing complementary information compared to MD and therefore use of both approaches simultaneously results in more comprehensive metabolite coverage and better characterization of a living system. The in-vivo SPME technology involves of monitoring drugs and biomarkers directly in organ during operation without need of a separate sample collection step. The approach is based on the 0.1mm in diameter flexible microfibre coated with biocompatible extraction phase, which is inserted into tissue with minimum damage or disturbance of the organ as the fiber of the size of acupuncture needle. The SPME and MD approaches were used to global chemical profiling of the striata of freely moving rat. After the in vivo sampling, the dialysates and SPME samples were separated by LC and the analytes were detected using the orbitrap mass spectrometer in positive ion mode. The MD data, as anticipated, was biased toward polar/hydrophilic compounds with lower LogP values. The SPME data, on the contrary, was biased to hydrophobic/less polar compounds. Among the metabolites detected by SPME are compounds from groups (carnitines, gangliosides, fatty acids and lysophospholipids including lysophosphatidic acid and lysophosphatidylethanolamine) of particular interest to clinicians because of their involvement in various neurological diseases and disorders. To date, no effective in vivo analytical method, applicable to lipids analysis is available. Similar results were obtained when live plant tissue was monitored. More hydrophobic pesticides are adsorbed on the MD tubings leading to curry-over and lower results.

All abstracts associated with this Session are to follow. Abstracts are sorted by presentation type and then abstract number (D for oral presentation and P for poster). Serial no. Pres. type Code 0005 O 0007 P 0008 O 0009 P 0010 O 0011 P 0012 P 0013 P 0014 O 0015 Key 0016 O 0017 P 0018 O 0019 P 0020 P 0021 O 0022 P 0023 P 0024 P 0025 P 0026 P 0027 P 0028 O 0029 P 0030 P 0031 P 0032 O 0033 P 0034 P 0035 P 0036 O 0037 P 0038 O 0039 P 0040 P 0041 O

Microdialysis2016: Author index (May 12 2016) Aandahl, Einar Martin

0034

Abrahamsson, Pernilla 0026

Adam, Dieter 0018

Åkerlund, Cecilia 0016

Åkesson, Oscar 0026

Antoni, Gunnar 0040

Barratt-Due, Andreas 0034

Barth, Aline 0019

Bellander, Bo-Michael 0016

Bergmann, Gisli Björn 0034

Bergmann, Gísli Björn 0033, 0036

Bergquist, Jonas 0028

Berstad, Audun Elnæs 0034

Blind, Per-Jonas 0026

Bojko, Barbara 0041

Bolcha, Martin 0037

Boschmann, Michael 0024

Brassart-Pasco, Sylvie 0014

Bremholm Ellebæk, Mark 0011

Burau, Daniela 0018, 0025, 0031

Carcaboso, Angel M 0032

Carpenter, Adrian 0021

Carpenter, Keri 0013, 0021

Carter, Eleanor 0013

Cihlář, Filip 0037

Clausen, Fredrik 0038

Dahler Eriksen, Bjarne 0011

Dahlin, Andreas 0028

Dahl, Kari 0034

Dalla Costa, Teresa 0012, 0020

de Lange, Elizabeth 0010

Derendorf, Hartmut 0007, 0018, 0019, 0022, 0030

de Witte, Wilhelmus 0010

Dietrich, Arne 0031

Djerada, Zoubir 0014

Dorenberg, Eric 0034

Dukic, Sylvain 0014

E. Helfer, Victória 0012

Ehmann, Lisa 0025, 0031

Ellebæk, Mark Bremholm 0017

Erdö, Franciska 0009

Eriksson, Jonas 0040

Eriksson, Olof 0040

Farnebo, Simon 0035

Fueth, Matthias 0030

Fuhrmann-Selter, Tania 0023

Furness, Amanda 0027

Gaelzer Silva Torres, Bruna 0020

Gallagher, Clare 0021

Grice, Peter 0021

G. S. Torres, Bruna 0012

Gustafsson, Sofia 0040

Hagen, Gaute 0034

Hammarlund-Udenaes, Margareta 0039, 0040

Hartman, Robin 0010

Haugaa, Håkon 0033, 0034, 0036

Heinrichs, Tobias 0019

Hejčl, Aleš 0037

Helmy, Adel 0021

Henricson, Joakim 0035

Hillered, Lars 0038

Hogard, Michael 0029

Högstedt, Alexandra 0035

Hörnaeus, Katarina 0028

Horneland, Rune 0034

Howe, Duncan 0021

Husby, Steffen 0017

Hutchinson, Peter 0013, 0021

Iredahl, Fredrik 0035

Jadhav, Satyawan B 0030

Jakobsen, Marianne Skytte 0017

Jakobsson, Johan 0016

Jalloh, Ibrahim 0013, 0021

Jönsson, Siv 0039

Kees, Frieder 0031

Kempker, Russell Ryan 0019

Khaowroongrueng, Vipada 0030

Kima, Peter 0007

Kjøsen, Gisle 0034

Kloft, Charlotte 0018, 0023, 0031

Lagishetty, Chakriadhar 0020

Lindqvist, Annika 0039

Lind, Sara 0028

Lundqvist Waninge, Rianne 0026

Lunte, Craig 0027, 0029

Lunte, Susan 0027, 0029

Macedo, Alexandre 0012

Mähler, Anja 0024

Martins, Frederico 0022

Mason, Andrew 0021

M. Bernardi, Priscila 0012

Menon, David 0013, 0021

Mollnes, Tom Eirik 0034

Molnár, Petra 0009

Monterrubio, Carles 0032

Mora, Jaume 0032

Muench, Hans-Georg 0018

Murphy, Michael 0021

Natan, Oscar 0010

Nelson, David 0016

Néris dos Santos, Camila 0020

Nestler, Christian 0031

Nordström, Carl-Henrik 0005

Oborny, Nathan 0029

Otteneder, Michael B 0030

Øyen, Ole 0034

Pakula, Anna 0024

Pawliszyn, Janusz 0041

Peloquin, Charles Arthur 0019

Petroff, David 0025, 0031

Pickard, John 0021

Pischke, Sören 0034

Pischke, Søren 0033, 0036

Procházka, Jan 0037

Qvist, Niels 0011, 0017

Reiss Axelsen, Anne 0011

Reske, Andreas W 0031

Richter, Wolfgang 0030

Risby, Kirsten 0017

Sabroe, Jonas Emil 0011

Sameš, Martin 0037

Schmidt, Stephan 0020

Schneider, Joanna 0024

Schroepf, Sebastian 0018

Shannon, Richard 0013, 0021

Sike, Mirabella 0009

Simon, Philipp 0025, 0031

Sjöberg, Folke 0035

Slimano, Florian 0014

Sonego Zimmermann, Estevan 0020

Spuler, Simone 0024

Stovell, Matthew 0021

Svensson, Mikael 0016

Syvänen, Stina 0040

Sziráki, István 0009

Tesselaar, Erik 0035

Thelin, Eric 0016

Tønnessen, Tor Inge 0033, 0034, 0036

Trampus, Péter 0009

Tsai, Tung-Hu 0015

Undin, Torgny 0028

van den Berg, Dirk-Jan 0010

van den Brink, Willem 0010

van der Heide, Susan 0013, 0021

Van Gulick, Laurence 0014

van Wijk, Rob 0010

Voelkner, Alexander 0007, 0022

Voelkner, Nivea 0007, 0022

Weerarathne, Hasitha 0027

Weiser, Christine 0023

Wong, Yin Cheong 0010

Wrigge, Hermann 0025, 0031

Wu, Dong 0008

Wu, Juan 0008

Zeitlinger, Markus 0031

Zhang, Qi 0008

Zhang, Qunlin 0008