2011.10.04.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 1 Development of Complex Curricula for Molecular Bionics and Infobionics Programs within a consortial* framework** Consortium leader PETER PAZMANY CATHOLIC UNIVERSITY Consortium members SEMMELWEIS UNIVERSITY, DIALOG CAMPUS PUBLISHER The Project has been realised with the support of the European Union and has been co-financed by the European Social Fund *** **Molekuláris bionika és Infobionika Szakok tananyagának komplex fejlesztése konzorciumi keretben ***A projekt az Európai Unió támogatásával, az Európai Szociális Alap társfinanszírozásával valósul meg. PETER PAZMANY CATHOLIC UNIVERSITY SEMMELWEIS UNIVERSITY sote_logo.jpg dk_fejlec.gif INFOBLOKK TÁMOP –4.1.2-08/2/A/KMR-2009-0006 2 Peter Pazmany Catholic University Faculty of Information Technology Biomedical imaging PHARMACOLOGICAL FMRI www.itk.ppke.hu (Orvosbiológiai képalkotás) (fMRI alkalmazása a gyógyszerkutatásban) VIKTOR GÁL, ZOLTÁN VIDNYÁNSZKY 2010.10.23 Biomedical Imaging: Pharmacological fMRI 2010.10.23 TÁMOP –4.1.2-08/2/A/KMR-2009-0006 3 The clinical challenges in drug discovery • Chronic diseases are increasing: Alzheimer's disease, psychiatric diseases, diabetes, atherosclerosis, arthritis, ...• Early onset • Slow progression • Poor prognosis • Clinical trials extremely difficult and costly:• Long duration (> 3 years) • Many co-morbidities, huge group sizes (> 1'000 patients / arm) • Low chance of success (8% entering phase 1 will reach market) Solution • Search for early indicators (biomarkers)• stratify patient population • monitor therapy efficacy • Imaging www.itk.ppke.hu Biomedical Imaging: Pharmacological fMRI 2010.10.23 TÁMOP –4.1.2-08/2/A/KMR-2009-0006 4 www.itk.ppke.hu TÁMOP –4.1.2-08/2/A/KMR-2009-0006 4 Stages of CNS drug discovery, candidate role of phMRI www.itk.ppke.hu Preclinicalresearch Drugdiscovery • Targetvalidation • Diseasemodels • Transgenics • Lead optimization • Earlyproofof CNS target • Biomarkerdevelopment • Sideeffects •Effectiveand safedosage • Animalmodels • Proofof target • Sideeffects •Effectiveand safedosage II PhaseI III Faileddrugs Success New potentials(phMRI) Biomedical Imaging: Pharmacological fMRI 2010.10.23 TÁMOP –4.1.2-08/2/A/KMR-2009-0006 5 www.itk.ppke.hu Role of Neuroimaging in drug discovery and development Four interrelated categories: Neuroreceptor mapping• PET tracers • SPECT tracers Structural imaging to examine morphological changes and theirconsequences. Metabolic mapping • 18FDG • magnetic resonance spectroscopy Functional mapping (fMRIand FDG PET ) to examine disease-drug interactions Biomedical Imaging: Pharmacological fMRI 2010.10.23 TÁMOP –4.1.2-08/2/A/KMR-2009-0006 6 www.itk.ppke.hu Role of human and animal fMRI in drug discovery fMRI is of most value at two distinct stages in the process of drug discovery: neuroscientific investigation of mechanisms of drug action providing quantitative markers of drug action, or endpoints, in candidate compounds for the clinic fMRI also providesameans of comparing the potential mechanisms of drug action, at the systems level, between the animal models and humans, as the compound is transferred from animals to humans. This approach offers two benefits: the potential for verification of the similarity between the animal model and the human and hence the value of the animal model in future testing. the potential for reduction of animal use for investigating mechanisms of drug action and their replacement with comparatively small cohorts of human volunteers. Biomedical Imaging: Pharmacological fMRI 2010.10.23 TÁMOP –4.1.2-08/2/A/KMR-2009-0006 7 www.itk.ppke.hu Applications of phMRI Measuring • Pharmaco-dynamic response • Pharmaco-kinetic characteristics Patient categorization• Stratification, subgroup definition Target identification:• proof of mechanism Early phase outcome study Alternative/surrogate marker of outcome Biomedical Imaging: Pharmacological fMRI 2010.10.23 TÁMOP –4.1.2-08/2/A/KMR-2009-0006 8 www.itk.ppke.hu Advantages of phMRI High information content• novel information • faster than conventional analyses Multi-modal• from anatomy to function and molecular information Non-invasive• minimal interference with physiology • repeated assessments, intrinsic controls, chronic treatment studies • increased statistical power • reduced group sizes Bridging the gap: translational research• mouse to man • identical readouts in pre-clinical and clinical studies Biomedical Imaging: Pharmacological fMRI 2010.10.23 TÁMOP –4.1.2-08/2/A/KMR-2009-0006 9 www.itk.ppke.hu Biomarkers Definition:Acharacteristicthatisobjectivelymeasuredandevaluatedasanindicatorofnormalbiologicalprocesses,pathogenicprocessesorpharmacologicresponsetoatherapeuticintervention (Lesko&Atkinson,AnnuRevPharmacolToxicol2001) Biomarkers and the Pharmaceutical Industry Imaging biomarkers enable: characterization of patient populations quantification of the extent to which new drugs reach intended targets, alter proposed pathophysiological mechanisms, achieve clinical outcomes as well as predict drug response. Biomedical Imaging: Pharmacological fMRI 2010.10.23 TÁMOP –4.1.2-08/2/A/KMR-2009-0006 10 www.itk.ppke.hu Neuralactivity Synapses Metabolic Communication Vascularresponse BOLD signal -Oxygene level -CBF (cerebral blood flow) -CBV (cerebral blood volume Drugtargets Glia Is the drug affecting neuronal activity or just the haemodynamic response? FMRI for investigating regional neurovascular coupling mechanisms through pharmacological challenges Biomedical Imaging: Pharmacological fMRI 2010.10.23 TÁMOP –4.1.2-08/2/A/KMR-2009-0006 11 www.itk.ppke.hu BOLD/CBF changes in baseline activity Norepetitive specific sensory stimulus(Single evoked activity) Cocaine: Breiter HC, et al,Neuron, 1997; 19:591-611 Nicotine: Bloom AS, et al,Human Brain Mapping, 1999; 8:235-244 Methamphetamine Völlm et al. Neuropsychopharmacology2004, 29,1715–1722 MDMA: Brevard et al. / Magnetic Resonance Imaging 24(2006) 707–714 Modulation of stimulus induced activity More treatment/disease specific Remifentanil: Tracey I (2001). Prospects for human pharmacological functional magnetic resonance imaging (phMRI). J Clin Pharmacol 41: 21S–28S. Modelling drug-induced responses Time Drug cc Time fMRI signal Time Drug cc Time fMRI signal Biomedical Imaging: Pharmacological fMRI 2010.10.23 TÁMOP –4.1.2-08/2/A/KMR-2009-0006 12 www.itk.ppke.hu Drug-induced responses: example Effects of MDMA (3,4-methylenedioxymethamphetamine) on monkey brain Brevard et al. / Magnetic Resonance Imaging 24 (2006) 707–714 Repetitive Visual stimulation 15min 5min 5min 35min 15min Repetitive Visual stimulation MDMA administration water vehicle control period baseline period mdma effects period Biomedical Imaging: Pharmacological fMRI 2010.10.23 TÁMOP –4.1.2-08/2/A/KMR-2009-0006 13 www.itk.ppke.hu BOLD changes in baseline activity Raphe nucleus, hypothalamus, hippocampus, amygdala, striatal and visual areas followed the same tonic activation pattern Modulation of stimulus induced activity VSB: average amplitude of BOLD response to visual stimulation, before MDMA VSB_MDMA: average amplitude of BOLD response to visual stimulation (after MDMA administration) Drug-induced responses: example Time Time fMRI signal MDMA administration fMRI signal VSB VSB_MDMA Biomedical Imaging: Pharmacological fMRI 2010.10.23 TÁMOP –4.1.2-08/2/A/KMR-2009-0006 14 www.itk.ppke.hu Classification of patients: defining subgroups in range disorders Intermediate phenotype of schizophrenia : (Mac Donald et al am J Psychiatry, 2005) Expectancy AX Context Processing Task Schizophrenia patients Nonschizophrenia psychosis patients Healthy subjects Biomedical Imaging: Pharmacological fMRI 2010.10.23 TÁMOP –4.1.2-08/2/A/KMR-2009-0006 15 www.itk.ppke.hu Early phase outcome measure: proof of concept (target) Rigth Inferior frontal cortex ACC Basal ganglia Left medial frontal region(middle frontal gyrus) Stroop task brain activation: basal ganglia, ACC, inferior frontal cortex (with right hemisphere dominance) Abnormal brain activation (left dominance over right hemisphere in the frontal cortex) in MS patients transiently normalizes after rivastigmineadministration Biomedical Imaging: Pharmacological fMRI 2010.10.23 TÁMOP –4.1.2-08/2/A/KMR-2009-0006 16 www.itk.ppke.hu A., C fibers harmful input painful004_cinguliant CONTEXT MOOD COGNITIVE STATE Molecularand anatomic STRUCTURE NOCICEPTIVE modulation Factors influencing pain experience SPINAL CORD Biomedical Imaging: Pharmacological fMRI 2010.10.23 TÁMOP –4.1.2-08/2/A/KMR-2009-0006 17 www.itk.ppke.hu www.itk.ppke.hu Perceived pain intensity depends on: Painis highly subjective experience as illustrated by the definition given from the International Association for the Study of Pain (Merksey and Bogduk, 1994) ‘‘an unpleasant sensory and emotional experience associated with actual or potential tissue damage, or described in terms of such damage.’’ Neuropathic Pain: caused by damage to or malfunction of the nervous system (no impending tissue damage in the background) Chronic Pain: pain that persists for more than three months one of largest medical health problems in the developed world, affecting ~ 20% of the adult population, particularly women and the elderly (Breivik et al., 2006). to improve the ability to diagnose chronic pain and develop new treatments we need robust and objective ‘‘readouts’’ of the pain experience. Biomedical Imaging: Pharmacological fMRI 2010.10.23 TÁMOP –4.1.2-08/2/A/KMR-2009-0006 18 www.itk.ppke.hu fMRI biomarker for chronic pain should provide an opportunity to: assess and correlate pain signals at varying times in either pre-intervention or post-intervention settings. generate a unique brain processing “fingerprint” in response to a specific task or stimulus correlate behavioral pain scores with most important and relevant brain regions generate more specific and relevant definition of pain in early clinical studies (Phase I and II); smaller studies could assess most promising endpoints Biomedical Imaging: Pharmacological fMRI 2010.10.23 TÁMOP –4.1.2-08/2/A/KMR-2009-0006 19 www.itk.ppke.hu Painmatrixmain components: Thalamus S1/S2 Insula(several divisions) ACC (several divisions) Prefrontal painful004_cinguliant painful003insula painful005SII SPM images: Gál et al. Unpublished investigation Biomedical Imaging: Pharmacological fMRI 2010.10.23 TÁMOP –4.1.2-08/2/A/KMR-2009-0006 20 www.itk.ppke.hu Modulation of the pain system via Remifentanil levelinsular Modulation of the pain system •Subjective pain experience controlled by „objective” FMRI vs. • Identifying regions associated with analgesia • Novel therapeutic strategies FMRI dose-response relationship• Finding effective dosage Phasicthermal pain Remifentanil(peripheraland CNS painkiller) 0, 0.5, 1.0, 2.0 ng/ml • computer controlled infusion Tracey I (2001). Prospects for human pharmacological functional magnetic resonance imaging (phMRI). J Clin Pharmacol 41: 21S–28S. Biomedical Imaging: Pharmacological fMRI 2010.10.23 TÁMOP –4.1.2-08/2/A/KMR-2009-0006 21 www.itk.ppke.hu Remifentanil drug dosage fMRIresponse painful005SII painful004_cinguliant levelinsular Insula Anterior cingulate cortex SII Dose dependent suppression of pain related activity within the pain matrix drug dosage Perceived Pain SPM images: Gál et al. Unpublished investigation Biomedical Imaging: Pharmacological fMRI 2010.10.23 TÁMOP –4.1.2-08/2/A/KMR-2009-0006 22 www.itk.ppke.hu Before administration fMRIresponse levelinsular Insula Right insula Pain evoked BOLD signal Dynamicmodulationof painmatrixactivity During remifentanyl administration Afterremifentanyladministration(eliminationphase) SPM images: Gál et al. Unpublishedinvestigation: (Wiseet al., 2003 Neuropsychopharmacology29, 626-635. ) Biomedical Imaging: Pharmacological fMRI 2010.10.23 TÁMOP –4.1.2-08/2/A/KMR-2009-0006 23 www.itk.ppke.hu Chronic pain model: Central sensitisation by topical capsaicin treatment (Petersen and Rowbotham, 1999, Zambreanu et al. Pain 2005) stimarea sensitization kronogramm Computer controlledMR-compatible mechanical stimulus presentation equipment -Topicalapplication of capsaicin, a vanilloidreceptor agonist, which elicits ongoing discharge in C-nociceptorsand induces an area of hyperalgesia. Biomedical Imaging: Pharmacological fMRI 2010.10.23 TÁMOP –4.1.2-08/2/A/KMR-2009-0006 24 www.itk.ppke.hu Central sensitisation by topical capsaicin treatment BOLD response BOLD response Gál et al. Unpublishedinvestigation Effects of central sensitiszation: talamus, insulaanterior, –BOLD responses in the different brain areas in the conditions of untreated(left column) and central sensitization (right column) when subjects categorized painful and non-painful stimuli Biomedical Imaging: Pharmacological fMRI 2010.10.23 TÁMOP –4.1.2-08/2/A/KMR-2009-0006 25 www.itk.ppke.hu Central sensitisation by topical capsaicin treatment BOLD response BOLD response Gál et al. Unpublishedinvestigation Effects of central sensitiszation: S2 cortex (left, right) –BOLD responses in the different brain areas in the conditions of untreated(left column) and central sensitization (right column) when subjects categorized painful and non-painful stimuli Biomedical Imaging: Pharmacological fMRI 2010.10.23 TÁMOP –4.1.2-08/2/A/KMR-2009-0006 26 www.itk.ppke.hu Animal fMRI D:\vision_projects\_presentations\tamop\ratimages\IMG_3604.JPG Comparing to human fMRI: Larger number of samples Testing of potentially noxius/lethal/less known• Intervention •stimulation (e.g. intracranial microstimulation) • chemical agents • genetic manipulations Translation of small animal models to human models)•May validate other drug development methods Biomedical Imaging: Pharmacological fMRI 2010.10.23 TÁMOP –4.1.2-08/2/A/KMR-2009-0006 27 www.itk.ppke.hu Preclinical imaging Bridging the gap: fMRI in translational studies Better match than behaviour? Basic neuroscience Biomarkers Drug discovery Animal models Transgenic approach Clinical trials Biomedical Imaging: Pharmacological fMRI 2010.10.23 TÁMOP –4.1.2-08/2/A/KMR-2009-0006 28 www.itk.ppke.hu Is it predictive: Do we learn anything about drugs in the human situation? • Major differences in receptors, circuits and function • Experiments normally carried out on anesthetized animals Is it cost and time efficient: How does it compare to conventional methods? Is it relevant: What can we learn about new compounds? • Difficulties at detecting tonic activation via BOLD methods • Limited stimulus delivery and behavioral response Is it ethical: May animals be "used" for research? Animal pharmacological MRI: issues D:\vision_projects\_presentations\tamop\ratimages\IMG_3604.JPG Biomedical Imaging: Pharmacological fMRI 2010.10.23 TÁMOP –4.1.2-08/2/A/KMR-2009-0006 29 www.itk.ppke.hu Ultra High field MRI Typical strengths: 4.5T, 7T, 9.4 T Bruker, Varian Pros: • high SNR • Higher chemical shift (also disadvantage) • 100µm or lower spatial resolution • T1 higher • Shorter imaging sessions (due to high SNR)- High susceptibility effects (even 15% signal change in BOLD), lower stimuli repetition required - Spin echo also gives BOLD contrast! Cons• High susceptibility effects • Poor field homogeneity • Variable signal loss • Higher TR for T1 D:\vision_projects\_presentations\tamop\ratimages\IMG_3602.JPG Biomedical Imaging: Pharmacological fMRI 2010.10.23 TÁMOP –4.1.2-08/2/A/KMR-2009-0006 30 www.itk.ppke.hu Resolution Spatial Typical – 0.1x0.1x0.3mm , 4-6 slices, 5min – 0.015x0.015x0.3mm , 20 slices, 1 day – Cytoarchitecturecan be visualized Time– Normally acquisition of a volume is not faster than at lower fields, but: – even single events (stimulus) can be detected by gradient or spin echo EPI – percent signal change can be 10 times higher than at 3T – Spectroscopy is accelerated substantially Biomedical Imaging: Pharmacological fMRI 2010.10.23 TÁMOP –4.1.2-08/2/A/KMR-2009-0006 31 www.itk.ppke.hu Preparation Intubation Catheterization (through the tail vein) Placement of the monitors: ECG heart rate Respiration (piezo-electric transducer ) Rectal temperature probe Mechanical stabilization acrylic stereotactic head holder(incisor bar and blunt earplugs,) Insertion of heating tube D:\vision_projects\_presentations\tamop\ratimages\IMG_3603.JPG Biomedical Imaging: Pharmacological fMRI 2010.10.23 TÁMOP –4.1.2-08/2/A/KMR-2009-0006 32 www.itk.ppke.hu Immobilization Training Mechanical restraining Anesthesia: • .-chloralose • Propofol • Medetomidine • Isoflurane Paralysis • mivacurium (curarization)• Enable awake, conscious experiments • Serious ethical issues D:\vision_projects\_presentations\tamop\ratimages\IMG_3598.JPG Biomedical Imaging: Pharmacological fMRI 2010.10.23 TÁMOP –4.1.2-08/2/A/KMR-2009-0006 33 www.itk.ppke.hu Direct effect of anesthesia on BOLD signal Elevating isofluran concentration decreases baseline level D:\vision_projects\_presentations\tamop\ratimages\IMG_3597.JPG D:\vision_projects\_presentations\tamop\ratimages\2010_06_15_isoflurane_timecourse.jpg D:\vision_projects\_presentations\tamop\ratimages\2010_06_15_isoflurane_axial_Tmap_masked.jpg Biomedical Imaging: Pharmacological fMRI 2010.10.23 TÁMOP –4.1.2-08/2/A/KMR-2009-0006 34 www.itk.ppke.hu Paw stimulation (electrical) One of the most frequently used sensory stimulation in small animal fMRI Needle electrodes are inserted under the skin/fixed around fingers Basic research in somatosensory system Indirect effect of drugs, anesthesia on the sensory system (deprivation) Scanning parameter optimization SPM images: Gál et al. Unpublished investigation Biomedical Imaging: Pharmacological fMRI 2010.10.23 TÁMOP –4.1.2-08/2/A/KMR-2009-0006 35 www.itk.ppke.hu Normalization of small animal brains Why normalize? Multiple subject experiments To report anatomical localization of fMRI effects Coregister with other modalities (MRI, autoradiography) How normalize/coregister? 3D digital atlases (Schweinhardt et al., 2003 Schwarz et al., Neuroimage 2006) derived from• Rat: Paxinos and Watson, 2005 • Mouse Paxinos and Franklin, 2001; Automation of coregistration•Tissue probability maps, brain templates coregistered with known atlases • In-house brain templates • Via finding anatomical landmarks Biomedical Imaging: Pharmacological fMRI 2010.10.23 TÁMOP –4.1.2-08/2/A/KMR-2009-0006 36 www.itk.ppke.hu MRI Contrast agents Animal MRI has the advantage to use potentially noxious contrast agents more freely than in human studies. Types of contrast materials used in clinical practice: Oral Intravascular – Gadolinium (and complexes): Paramagnetic – Manganese (and complexes): Paramagnetic – Iron oxide: Superparamagnetic • SPIO : Superparamagnetic Iron Oxide (SPIO) and UltraSPIO • Reduces T2 and T2* • Intravascular time: depending on particles size and coating – With long iv time they can be used as fMRI contrast agent (next slide) Biomedical Imaging: Pharmacological fMRI 2010.10.23 TÁMOP –4.1.2-08/2/A/KMR-2009-0006 37 www.itk.ppke.hu Potential Functional MRI Contrast agents Indicators of change in local blood flow MION-47, USPIO with long blood half-life Indicators for Ca2+ and other metal ions BAPTA-based Gd3+ complex Mn2+ as Ca2+ mimetic pH indicators Phosphonated Gd3+ complex, Endogenous amide protons Probes for metabolic activity Exogenous hemoglobin Genetically controlled contrast agents Ferritin Transferrin (Tf)-conjugated SPIOs Artificial lysine-rich protein Biomedical Imaging: Pharmacological fMRI 2010.10.23 TÁMOP –4.1.2-08/2/A/KMR-2009-0006 38 www.itk.ppke.hu Electrical microstimulation and fMRI Local and remote connections of a specific (stimulated) site can be mapped Method can detect selective modulatory effects of pharmacological agents on specific connections Methodological challenge: MR compatible electrode, MR signal is contaminated by electrical stimulation Pioneering work of Logothetis Lab: monkey V1 microstimulation (Tolias et al 2005, Neuron)• Significant BOLD signal change in V1, and extrastriate visual areas V1 V2/V3 MT/V5 Biomedical Imaging: Pharmacological fMRI 2010.10.23 TÁMOP –4.1.2-08/2/A/KMR-2009-0006 39 www.itk.ppke.hu Optogenetic fMRI Electrical stimulation is not selective: Afferents and efferents, passing axons Inhibitory and excitatory neurons are also activated Injection of viral vector (AAV5-CaMKIIa::ChR2(H134R)-EYFP)• into primary motor cortex • expression of channelrhodopsin (ChR2) • only in Ca2+/calmodulin-dependent protein kinase II (CaMKIIa)-expressing principal cortical neurons, (not in GABAergic or glial cells) • Activation/measurement 10 days after viral injection C:\Users\viktor\AppData\Local\Microsoft\Windows\Temporary Internet Files\Content.IE5\MZRIX1P8\MC900433897[1].png Lee et al 2010, Nature M1 Optical (laser diode) stimulation of motor cortex resulted in BOLD response shown in the site of stimulation and in relevant thalamic nuclei Biomedical Imaging: Pharmacological fMRI 2010.10.23 TÁMOP –4.1.2-08/2/A/KMR-2009-0006 40 www.itk.ppke.hu Summary Examples demonstrate promising capabilities of phMRI in: Measuring Pharmaco-dynamic response and pharmaco-kinetic characteristics Patient categorization, target identification: Early phase outcome or surrogate biomarker of outcome via BOLD/CBF changes in baseline activity or modulation of stimulus induced activity Animal fMRI broaden the potential of phMRI enabling: Larger number of samples Testing of potentially noxius/lethal/less knownchemical agents (drugs), intervention, stimulation (e.g. intracranial microstimulation)and genetic manipulations Translation of small animal models to human models(bridging the gap)• May validate other drug development methods