Final Report Summary - EURIPIDES (European research initiative to develop imaging probes for early in-vivo diagnosis and evaluation of response to therapeutic substances)
Executive summary:
Drug resistance is an important clinical problem. Globally, 50,000,000 people have epilepsy: current treatments are ineffective in 30%. Drug resistance is associated with higher rates of: unemployment, disadvantage, injury, somatic co-morbidity (affecting many body systems), psychiatric disorder, cognitive/memory decline, sudden unexplained death and other premature mortality, making chronic epilepsy pervasive and pernicious, with seizures being just one 'tip-of-the-iceberg' manifestation. Availability of several new AEDs has not solved drug resistance. We do not understand drug resistance.
One concept that has arisen for drug resistance is the transporter hypothesis, which maintains that broad resistance to antiepileptic drugs might relate to over-activity of non-specific transporter mechanisms that reduce the local concentration of AEDs within the brain tissue below a therapeutic level. The aims of EURIPIDES were to develop in-vivo imaging biomarker of multidrug transporter function as a generic tool for the prediction, diagnosis, monitoring and prognosis of major CNS diseases. EURIPIDES involved a multidisciplinary approach, based on integration of the clinical and basic science domains and on the synergistic combination of neurophysiological and biomolecular imaging methods.
In a comprehensive study of P-glycoprotein (Pgp) inhibitor based positron emission tomography (PET) tracers, we clarified that [11C]laniquidar, [11C]elacridar and [11C]tariquidar in tracer concentrations are recognised by Pgp and/or BCRP as substrates. We developed and validated, at least in epileptic animal models, eight [11C]-and three [18F]-PET radiotracers aimed to be either Pgp substrates or inhibitors, with further development of longer-lived PET radionuclides aimed at novel ways of imaging Pgp functionality. Biodistribution studies were performed in animal models of naïve, transporter knock-out and epileptic rodents. Even though these highly potent and selective radiolabelled Pgp inhibitors afforded only very low PET signals, three PET tracers ([11C]laniquidar, [11C]tariquidar, [11C]phenytoin) entered trials in humans. Using the established PET radiotracer [11C]verapmail as a model Pgp substrate and the PET receptor ligands and Pgp substrates [18F]MPPF and [11C]flumazenil, we could show that partial inhibition of the blood-brain-barrier (BBB) by administration of half-maximum inhibitor dose allows detection of regional differences in Pgp functionality at the rat BBB.
We successfully translated our findings in animal models to the human condition. Studies with [11C]VPM PET in healthy controls showed that TQD-induced Pgp modulation at the human (BBB) appeared to be transient and its magnitude directly proportionate to serum drug exposure. The optimal analytical model for baseline studies was less suitable for blocking studies, where an additional compartment improves the quality of the fits. Analysis with additional models showed small, but significant differences between patients with Alzheimer's Disease and healthy controls. This is the first direct evidence that Pgp transporter at the BBB are dysfunctional in sporadic AD and suggests that decreased Pgp function may be involved in the pathogenesis of AD. In patients with temporal lobe epilepsy (TLE), the model derived K1 (influx rate constant) values from drug-sensitive patients were higher than those from drug-resistant patients falling, which suggests more efficient Pgp function in drug-refractory patients resulting in lower drug concentrations in the brain. This difference was restricted to the temporal lobes both on the ipsilateral and contralatral sides. After tariquidar administration, we observed smaller K1 increases in the drug-resistant patients compared to healthy volunteers supporting over expression of Pgp function, with the ipsilateral epileptogenic hippocampus being most significantly affected. Functional in-vivo evidence of over active transporters in drug-resistant compared to drug-sensitive epileptic patients was complimented by histological assays of post-mortem brains and surgically resected epileptic tissue from drug-resistant patients. The change in VPM uptake after Pgp inhibition with TQD in the hippocampus correlated inversely with Pgp-immunopositive labeling in pharmaco-resistant TLE patients who underwent anterior temporal lobe resection for surgical treatment.
In summary, our findings provide evidence supporting the hypothesis of multidrug transporter overexpression as an important mechanism for developing pharmacoresistance in epilepsy. The availability of such imaging biomarkers will support the development of new treatment strategies targeted at multidrug transporter and aimed at reversing pharmacoresistance with selection of optimal patients and assessment of molecular targets. This will lead to improved health care through individualised treatment strategies, and at the same time to a reduction of health care costs by discontinuing ineffective therapies. The stage is thus set for direct translation to clinical trials. We have a tool to identify patients in whom P-glycoprotein overactivity could conceivably contribute to drug resistant epilepsy; the same tool can act to determine whether P-glycoprotein activity has been inhibited in practice as might be intended with treatment with a P-glycoprotein inhibitor such as TQD. Thus EURIPIDES has a lasting legacy, with potential for further research and clinical application.
Project Context and Objectives:
1.2 Summary description of project context and objectives:
Background and Aims
Resistance to drug treatment is an important hurdle in the therapy of many diseases of the central nervous system (CNS). Consequently, there is a pressing need to develop new and more effective treatment strategies. Whilst drug resistance in CNS diseases is multifactorial, there are two broad likely mechanisms:
(1) resistance to the desired pharmacological effects of CNS drugs occurs from alteration in the postulated target(s) the 'target hypothesis';
(2) inadequate access of CNS drugs to their targets across the blood-brain barrier (BBB) due to the overexpression or overactivity of multdrug transporters that efflux foreign chemicals from the brain the 'transporter hypothesis'.
Although the brain is among the best perfused organs in the body, drug entry into the brain is limited by the BBB, which is formed by brain endothelial cells that are closely linked by tight junctions (Reichel 2006). Substances can enter the brain either by passive diffusion across the endothelial cell membranes or by selective carrier transport. High lipophilicity is an essential characteristic of a substance to cross the BBB by diffusion. However, there are several examples of lipophilic drugs (e.g. anticancer drugs, antiepileptic drugs, anti-HIV drugs) that have poor access to brain tissue. The presence of multidrug efflux transporters, such as P-glycoprotein (Pgp), breast cancer resistance protein (BCRP) and multidrug resistance proteins (MRPs), at the luminal side of the endothelial cells of the blood capillaries is believed to be the basis for these observations (De Lange 2004, Loscher and Potschka 2005). These transporters are mainly, but not exclusively, members of the ABC (ATP-Binding Cassette) superfamily, including ABCG2 (BCRP), ABCB1 (Pgp), ABCC1 (MRP1), ABCC2 (MRP2).
Multidrug efflux transporters can contribute to drug resistance in brain diseases in two ways:
- by their constitutive expression in the BBB and blood-CSF barrier, they restrict the brain access of many drugs by enhancing drug extrusion from the brain, so that the levels of drugs in the brain cannot become sufficiently high for therapeutic efficacy.
- by intrinsic or acquired over-expression of multidrug transporters in the BBB or brain target tissue limits drug penetration into that tissue. This can result from alterations (such as polymorphisms) in genes that encode multidrug transporters, or from the effects of disease or drug treatment on expression of these transporters. The consequences of intrinsic or acquired over-expression of multidrug transporters depend on the affinity of respective substrates for the efflux transporters.
Over-expression of multidrug transporters at the BBB and beyond will prevent CNS drugs from reaching sufficiently high concentrations in critical cerebral regions despite substantial plasma levels (Sisodya 2003). This may, therefore, account for the failure of multiple diverse CNS drugs in some patients.
Several lines of evidence suggest that changes in Pgp expression and/or function might also play a key role in the causation and pathogenesis of certain neurological disorders, such as AD (Vogelsang et al. 2004, Lee et al. 2004) and epilepsy (Loscher 2002). For instance, regional overexpression of Pgp in the brains of epilepsy patients might contribute to drug resistance by impeding the access of antiepileptic drugs (AED) to the seizure focus (Loscher and Potschka 2002). These results suggest that Pgp is an attractive pharmacologic target to increase intracerebral penetration of certain drugs, such as anticancer and antiepileptic drugs (Bates 2002, Sisodiya and Bates 2006).
It has not been investigated whether expression of other transporters in the BBB is affected by Pgp inhibition, but the conformation of Pgp may be affected by substrate inhibitors, possibly in a genotype-dependent fashion (Kimchi-Sarfaty et al, 2007). A chronic study in a model of pharmacoresistant epilepsy with a combination of phenobarbital and the Pgp inhibitor TQD did not indicate that tolerance development may be an issue (Brandt et al., 2006). In contrast, development of tolerance to a combination with phenytoin and TQD was described in another chronic epilepsy model (Van Vliet et al., 2006). At present, it cannot be excluded that this is due to an effect on expression of other transporters, or genotype-dependent dynamic alterations in Pgp itself (Kimchi-Safarty et al, 2007).
In this project, we focused initially on Pgp, which is the most-widely studied multidrug transporter, is found in and contributes to the normal BBB, and as efflux transporter, actively transports substrates (including multiple CNS drugs) against concentration gradients from the brain to blood and cerebrospinal fluid. This hampers the build-up of adequate tissue levels of these drugs in the brain, greatly limiting their therapeutic efficacy. As such, the 'transporter hypothesis' of drug resistance is applicable to a broad range of CNS drugs and patients with a variety of CNS diseases who critically depend on these drugs accessing the brain tissue.
Our aim was to develop a generic tool for the prediction, diagnosis, monitoring and prognosis of major CNS diseases, as well as to provide support and guidance for therapeutic interventions.
The project aims were twofold:
(1) to develop radiotracers to image Pgp function and distribution to modulation of Pgp expression,
(2) to validate the use of Pgp radiotracers as in-vivo imaging biomarker by determining the contribution of Pgp overexpression to impaired brain uptake of drugs (pharmacoresistance) for the prediction of therapeutic response, or the contribution of impaired Pgp function to reduced clearance of toxic substances (e.g. β-amyloid) for the early in-vivo diagnosis of Alzheimer's Disease (AD).
Work strategy and general description
Circumvention of pharmacoresistance, or increasing clearance of drugs from the brain tissues, may involve inhibitors of Pgp transporters or sophisticated alternative therapies, but demonstration of overexpression or underactivity of transporter function is an essential and necessary first step. An in-vivo imaging biomarker of Pgp function is essential for identifying altered transporter activity in individual patients. If a relation between overexpression and therapy resistance, or in the case of AD underactivity, can be demonstrated, such a biomarker will provide unique and new pathophysiological information, the means for predicting treatment response, or early diagnosis, in individual patients. The availability of a radiotracer for imaging Pgp function in healthy volunteers does not make it a biomarker for drug resistance. In order to validate the usefulness of a biomarker, studies have to be designed for relevant and sensitive clinical populations, e.g. comparative studies in patients with refractory vs responsive CNS diseases.
Management structure and procedures
The Project Coordinator ensured the smooth operation of the project and guaranteed that all efforts were focused towards the objectives. He submitted all required progress reports, deliverables, financial statements to the European Commission, and, with the assistance of GABO he was responsible for the proper use of funds and their transfers to participants. The EURIPIDES office was established by and based at the coordinator in London and at GABO in Munich. The Project Office at the Coordinator was concerned with the scientific management and the co-ordination of all research activities. The Project Office at GABO was responsible for administrative, financial and contractual management and the organisational co-ordination of the project activities.
Objectives of EURIPIDES:
In line with this call's main aim 'to develop highly sensitive agent(s) for established in-vivo imaging modalities able to report and quantify cellular and/or molecular events in several major disease states or disease processes for early in-vivo diagnosis and/or evaluation of response to therapy', using positron emission tomography (PET) and single-photon emission tomography (SPET) in a multidisciplinary approach integrating radiochemistry, molecular and cellular biology, physics, pharmacology and pathology, our main objectives are:
- to discover and characterise novel PET and SPET radiopharmacological tracers, which are markers for the function and the expression of cerebral multidrug transporters that form part of the BBB (WP 01 Key milestones (KM) 1.5,14,15,22,23).
- to characterise and identify compounds / potential radiotracers in-vitro that are substrates for Pgp and acts as inhibitors of drug efflux across the BBB (WP03 KM3.3,4)
- to evaluate, determine kinetics and develop quantitative methodology to measure transporter expression and/or functionality of existing radiotracers known to be either Pgp substrates ([11C]-VPM) or Pgp inhibitors ([11C]-laniquidar) in naïve animals (WP02 KM2.3).and in healthy human volunteers (WP05 KM5.4,5) and to later apply these methods to novel radiotracers emerging from WP01, in naïve animals (WP02 KM2,5,7,8,12,15) and in healthy human volunteers (WP05 KM5.10-12).
- to carry out proof-of-concept studies with existing radiotracers known to be either Pgp substrates ([11C]-VPM, [11C]-flumazenil, [18F]-MPPF) or Pgp inhibitors ([11C]-laniquidar) to image Pgp function, and determine the efficacy of existing and novel Pgp modulators to enhance cerebral penetration of substrate tracers in CNS disease models, comparing drug-resistant and drug-responsive animals (WP02 KM2.4; WP04, KM4.3,4) and humans (WP 04 KM4.3,4; WP05 KM5.4,8; WP06 KM6.4,6-8) and similarly with novel radiotracers emerging from WP01 (WP02 KM2.9-11,13,14,16-18)
- to carry out proof-of-concept studies with [11C]-VPM to image Pgp function for early diagnosis of Alzheimer's Disease (AD) (WP07 KM7.3,4)
- to determine the anatomical distribution of transporter proteins in brain tissue from patients with epilepsy and AD (WP08 KM8.5-7)
Project Results:
1.3 Description of the main S&T results/foregrounds of EURIPIDES
WP01: Development of new radiotracers
Objectives
The main objective of this WP01 was to develop new radiotracers for in-vivo imaging of cerebral multidrug transporters (P-glycoprotein, Pgp; multidrug resistance proteins, MRPs; breast cancer resistance protein, BCRP) with PET and SPET. We initially followed two different approaches. First, we developed radiotracers based on known third-generation Pgp inhibitors (laniquidar, elacridar and TQD). These compounds were expected to bind with nanomolar affinity to Pgp at the blood-brain barrier (BBB) and provide higher PET signals than available Pgp substrate radiotracers such as (R)-[11C]VPM. Moreover, radiolabelled Pgp inhibitors were expected to allow for mapping of Pgp expression levels at the BBB and to afford signal increases rather than signal decreases (as radiolabelled substrates) in epileptic brain regions overexpressing Pgp.
As a second approach, we developed two PET tracers based on antiepileptic drugs (AEDs), i.e. [11C]phenytoin and [11C]mephobarbital. The rationale behind this approach was our expectation that radiolabelled AEDs would be only weakly transported by Pgp at the BBB and thereby afford higher PET signals than the high-affinity Pgp substrate (R)-[11C]VPM. This should enable an improved visualisation of regional Pgp activity in the epileptic brain as compared with (R)-[11C]VPM. In addition, it was expected that mapping the regional brain distribution of a radiolabelled AED should be clinically more meaningful in predicting drug resistance than using a surrogate marker of Pgp activity such as (R)-[11C]VPM.
Results
[11C]Radiolabelling
After the synthesis of appropriate radiolabelling percursors, [11C]laniquidar, [11C]elacridar, [11C]TQD and [11C]mephobarbital were synthesised by [11C]methylation of phenolic OH functions ([11C]elacridar and [11C]TQD), a carboxylic acid function ([11C]laniquidar) or an imide function ([11C]mephobarbital) using [11C]methyl triflate or [11C]methyl iodide. Moreover, a presumably BCRP-selective TQD analogue (Kuhnle et al, 2009)) was labelled by [11C]methylation of a carboxylic acid function.
[11C]Phenytoin was synthesised by reaction of 2,2-azido-diphenylamide with [11C]carbon monoxide. All radiotracers were obtained in good radiochemical yield, with acceptable radiochemical purity (greater than98%) and specific activity (greater than50 GBq/µmol). In addition, 3H-labelled versions of laniquidar and elacridar were synthesised by [3H]methylation of the respective desmethyl-precursor molecules with [3H]methyl nosylate.
Radiopharmacological characterisation of [11C]radiotracers
[11C]Laniquidar was tested in biodistribution experiments in rats with and without pretreatment with cyclosporine A or valspodar and in PET experiments in rats with and without pretreatment with TQD. [11C]Elacridar and [11C]TQD were tested in paired small-animal PET scans in naïve rats, wild-type mice and three different transporter knockout mouse models (Mdr1a/b(-/-), Bcrp1(-/-) and Mdr1a/b(-/-)Bcrp1(-/-)), each before and after administration of unlabelled compound. Moreover, in-vitro autoradiography was performed on rat or mouse brain slices with [11C]elacridar and [11C]TQD, with and without co-incubation with an excess of unlabelled compound (1 µM). The 11C-labelled BCRP-selective TQD analogue was characterised in small-animal PET scans in wild-type, Mdr1a/b(-/-), Bcrp1(-/-) and Mdr1a/b(-/-)Bcrp1(-/-) mice. [11C]Phenytoin was tested in PET scans in rats before and after administration of either TQD as Pgp inhibitor or the MRP inhibitor probenecid. [11C]Mephobarbital was tested in paired PET scans in rats, wild-type, Mdr1a/b(-/-) and Mrp1(-/-) mice, before and after administration of TQD or the MRP inhibitor MK571. In-vivo metabolism of all [11C]radiotracers was assessed in naïve rats or mice by analysis of plasma samples with a combined solid-phase extraction (SPE) and high-performance liquid chromatography (HPLC) assay.
Development of radiotracers for SPET imaging
We synthesised a series of iodinated elacridar/TQD analogues in order to come up with a Pgp inhibitor based SPET tracer. Unfortunately, none of these maintained the biological activity of elacridar/TQD itself as shown by in-vitro transport inhibition assays. One derivative (iodoelacridar-urea) was found to be only approximately 6 times less potent than elacridar and therefore labelled with iodine-125. A biodistribution study was performed in wild-type mice with [125I]iodoelacridar urea. The compound was shown to possess low brain uptake (less than0.5 %ID/g) which was increased after pretreatment of animals with cold TQD. Due to the similar in-vivo behaviour of [125I]iodoelacridar urea and [11C]elacridar this compound was not further pursued.
WP02: Biological evaluation of new radiotracers
Objectives
The main objective was to evaluate the feasibility of new radiotracers developed in WP01 for in vivo imaging of cerebral multidrug transporters with PET and SPET. These radiotracers have been used to study in vivo the distribution and function of multidrug transporters in animals and this had been related to actual brain uptake and distribution of CNS active drugs.
PET studies
The (R)-[11C]-VPM PET scans have been performed in a microPET scanner (AIT/TIHO) as well as in an HRRT scanner (VUA/LACDR) in female (AIT/TIHO) and male (VUA/LACDR) Sprague-Dawley rats. Essentially these studies have demonstrated that the results were not critically dependent on methodological aspects. In fact, the results were very similar: [11C]VPM had very low brain uptake at baseline conditions. For comparison with human PET data, kinetic modelling was performed to estimate the rate constants of tracer transport across the rat BBB.
Intracerebral microdialysis
Parallel studies with PET and microdialysis were too complex to be performed in the same animal. QUIN pharmacokinetics was therefore determined separately following i.v. administration at 2 different dosages. The 8 hr study covered a 1 hr blank period, a 4 hr infusion period and a 3 hr elimination period. Plasma and microdialysate concentrations, as well as total brain concentrations at termination of the experiment, were analyzed by HPLC and fluorescence detection.
Additional studies
A range of studies have been performed to support the studies on Pgp functionality and expression. Population modelling with NONMEM has been introduced and covariate analysis has allowed to demonstrate which parameters of brain uptake and distribution were influenced by TQD, epilepsy and other factors. For example, this approach confirmed that TQD specifically and concentration-dependently affects efflux from the brain. Moreover modelling techniques have been exchanged between institutes. Also rat data were compared with values from the human dose-escalation study, showing that TQD-induced increase in [11C]VPM uptake is distinctly stronger in rats than in man, whereas EC50 values were nearly identical.
PET studies
MicroPET scans were performed with [11C]-VPM before and after administration of TQD (3 mg/kg and 15 mg/kg) in rats 48 h after status epilepticus (SE) and respective control rats. For analysis of µPET scans after TQD administration, different brain regions of interest were outlined on magnet resonance images which were co-registered with the PET images. For comparison with the present μPET data, computer-assisted quantification of Pgp expression in immunohistochemically stained rat brain sections was performed in respective brain regions. Striking changes in [11C]-VPM brain uptake were obvious after administration of 3 mg/kg TQD (ED50) in animals 48 h after pilocarpine-induced SE which vanished after maximal Pgp inhibition (15 mg/kg). In rats 48 h post SE, the DV (=Kp) of cerebellum was decreased by 37.5% whereas DV of frontal motor cortex displayed an increase by 18.9% compared to control rats. In thalamus, a tendency towards DV decrease was found. The decrease of [11C]-VPM uptake in the cerebellum as well as the tendency for decrease in thalamus indicate higher Pgp activity induced by SE which is reflected by increased Pgp-labelled areas in respective brain regions. In conclusion, a µPET protocol suitable to quantify SE-induced changes in Pgp activity in distinct rat brain regions was established.
Intracerebral microdialysis
Remarkably, kainate-treated rats tended to have a lower total brain concentration but a higher brain ECF concentration of quinidine than saline-treated rats. This suggests that the Pgp function in kainate-treated rats is altered at the parenchymal level rather than at the BBB. TQD pre-administration increased the brain ECF brain concentration 7.2-fold but the total brain concentration about 40-fold, supporting an important role for Pgp in intra-brain distribution. After kainate-treatment alone however there was no difference in transport compared to control rat
Biological evaluation of radiotracers in animal models of drug resistance (Task 5)
Rats were selected which did or did not respond to treatment with the antiepileptic drug phenobarbital in the pilocarpine model of TLE. Similar to the observations in the basolateral amygdala stimulation model, epileptic rats in the pilocarpine model showed a pronounced variability in individual response to treatment with phenobarbital. 33% of epileptic rats did not respond appropriately to treatment with the maximal tolerable dose of phenobarbital. As previously described part of the animals in the pilocarpine model did also not respond to treatment with the common antiepileptic drug levetiracetam. As a definition of pharmacoresistance in animal models asks for refractoriness to monotherapy at tolerable doses with at least two current antiepileptic drugs, these findings support the suitability of the pilocarpine model for the investigation of pharmacoresistant epilepsy. Selected responders and nonresponders have been used to evaluate if differences in Pgp activity between pharmacosensitive and resistant rats can be detected and quantified after TQD administration using small-animal positron emission tomography with [11C]VPM.
Additional studies
Some animals were repeatedly scanned in a (human) 3T MRI scanner during epileptogenesis. These data were used together with available brain region MRI atlases to create atlases that reflect specific developments in responder and non-responder rat brains.
A time course study on Pgp expression was undertaken in relation to time-after SE, covering a period of 5 weeks. Results showed considerable variability between animals but as a general trend there was a transient early increase peaking at 2 days after SE and a second broader peak between 7-14 days after SE.
SE induction by unilateral intrahippocampal kainate injection did not reveal clear ipsilateral to contralateral differences in Pgp expression. However correlating quinidine brain pharmacokinetics before and after TQD treatment, with Pgp expression and seizure severity during SE in individual animals, hinted to a clear and highly interesting uncoupling of Pgp expression and function strongly influenced by seizure severity.
WP03: In vitro characterisation
Objectives
The main objective is to determine the most specific and potent inhibitor of Pgp for use as in-vivo molecular imaging tracer in patients with refractory neurological and psychiatric conditions.
Task 1: In-vitro characterisation of inhibition of transport by Pgp to identify compounds with the best inhibitory profile to be used in-vivo
The objective of this task was to rank inhibitors on the basis of inhibitory potency in an effort to identify an optimal inhibitor for the subsequent in-vitro / in-vivo experiments. We have tested and determined IC50 values for seven Pgp inhibitors to validate the use of these compounds as in-vivo probes for PET imaging. The P12-DRC group has produced 5 batches of elacridar derivates labeled with iodine and we have determined the efficacies of the derivatives compared to the parent compound. Ten novel elacridar derivatives in total have been tested but most have markedly reduced efficacy compared to the parent compound which strongly suggests that they will not be suitable for use as in-vivo probes. A 5th batch of compounds was tested with the 3-iodo derivative showing an interaction with Pgp. This compound has now being taken forward for in-vivo imaging experiments.
An aim of WP03 is to test novel pro-drug ligands of Pgp generated from the lab group of E. Arstad, UCL utilising an in-vitro Pgp assay. Seventeen novel ligands have been tested in a validated assay and this has lead to the identification of an optimal ligand pair. This optimal ligand pair will now be further validated in-vivo as a novel imaging probe (E. Arstad, UCL).
Task 2: Comparison of inhibition of wild-type and variant forms of ABCB1 by model inhibitors
The primary aim of this task was to characterise the inhibition of transport of wild type and variant forms of Pgp (i.e. containing combinations of 3 single nucleotide polymorphisms [SNPs] in the ABCB1 gene) by inhibitors. To validate the Xenopus oocyte expression system, two model Pgp substrates, digoxin and imatinib were shown to be transported by human ABCB1 expressing oocytes. This Pgp mediated transport was shown to be dependent on ATPase activity and was inhibited by the Pgp inhibitor PSC-833. Site directed mutagenesis has been performed on human Pgp cDNA to generate the three SNP variant haplotype corresponding to C1236T, G2677T and C3435T. No differences in transport were observed between the wild type and triple SNP variant, indicating that the triple SNP variant does not have a direct functional effect for transport or inhibition for the two tested model substrates.
An additional sub-task was to characterise the transport of AEDs by Pgp. To address this question seven transport systems expressing Pgp have been developed and utilised in this work package to study the transport of three major AEDs. The Pgp mediated transport of phenytoin was observed utilising the equilibrium method in both Pgp stably over expressing cell lines. This suggests that phenytoin is a weak substrate of Pgp that requires non-physiological (or high) protein expression of Pgp for transport. The transport of carbamazepine and lamotrigine by Pgp was also investigated with no Pgp mediated transport observed in the in-vitro systems tested suggesting that lamotrigine and carbamazepine are not transported by Pgp. This adds to the evidence that there is no unifying mechanism of drug transporter resistance and emphasises the need for a systems approach of AED transport at the BBB.
Task 3: Characterisation of the mechanism of induction of ABCB1 by drugs
The induction properties of AEDs of ABCB1 have been investigated in an in-vitro model of the BBB. No induction was observed of ABCB1 over a timecourse and with different concentration of AED. This suggests that at least in the in-vitro model tested, that AEDs do not induce ABCB1 mRNA expression.
Task 4: Systems approach for studying drug transport at the BBB
The aim of this task was to investigate the overall transport, influx and efflux, at the blood-brain barrier (BBB), and not only focus on Pgp mediated drug transport. This systems approach is going to be essential in understanding the success of drugs used for CNS diseases, and the development of novel ligands. Identifying these mechanisms of transport at the BBB will lead to the identification of imaging probes. We have investigated lamotrigine as the mechanisms that underpin the passage of this important AED at the blood-brain barrier to its site of action in the brain is poorly understood. Lamotrigine has been postulated to be delivered to its site of action in the brain favourably despite its physicochemical properties.
WP04: Evaluation of existing radiotracers for imaging Pgp function
Objectives
The main objective is to determine the contribution of Pgp activity to uptake and binding of existing PET tracer [18F]-MPPF, and [11C]-FMZ that are Pgp substrates.
[18F]-MPPF:
Naïve and epileptic rats (Task 1-4, P06-LMU)
First PET scans, examining the impact of Pgp modulation on [18F]-MPPF brain kinetics, were performed in naïve Sprague Dawley rats. Following optimization of the quantification procedure, the initial uptake of [18F]-MPPF in brain was used as a surrogate marker of the unidirectional blood-brain clearance (K1). Based on the consortiums decision to focus on the Pgp inhibitor TQD (TQD), the effect of TQD on the K1-surrogate was determined. TQD had a significant and dose-dependent effect, with an IC50 close to 5 mg/kg, and a 2.5-fold increase at the highest TQD dose. Additional PET scans have been performed with the Pgp modulator cyclosporine A (CsA) resulting in an increase of 30-40% for the K1-surrogate. These data clearly substantiate transport of [18F]-MPPF by Pgp further supporting its suitability for non-invasive Pgp imaging.
Patients and healthy controls (Task 5, P07-HCL):
Cyclosporine A (CsA) was used throughout the project by partner P07-HCL. Fifteen patients with drug-resistant focal epilepsy of temporal lobe origin with or without hippocampal sclerosis were originally selected for this protocol. Twelve of the 15 scanned patients (mean age ± SD = 43.9 ± 7 years) had paired 60-minute dynamic [18F]MPPF PET scans with and without concurrent cyclosporine A (CsA) IV infusion. The three remaining patients underwent only one [18F]MPPF PET (without CsA) and were then excluded from the protocol for various reasons. A few patients reported mild central nervous system side effects, such as vertigo, during the 24 hours following CsA infusion, suggesting that the latter promoted the activity and adverse events of concurrent anti-epileptic drugs. For the PET associated with CsA infusion, the latter was started one hour prior to [18F]-MPPF injection at a dosage of 2.5 ml/kg/h over 2h resulting in a steady plasma concentration of CsA during the entire PET study at or above the target level (= 2980 μg/l). A simplified reference tissue model was used to generate parametric images of 5-HT1A receptor binding potential (BP=Bmax/Kd) and K2 values. The cerebellar white matter and the thalamic region were both used as reference regions. The same protocol was used in eight healthy subjects. All controls also gave their informed consent to participate in this study. Seven of the 8 scanned patients (mean age ± SD = 41.8 ± 7.2 years) had paired 60-minute dynamic [18F]MPPF PET scans with and without continuous cyclosporine A (CsA) IV infusion. The last control recruited had to be excluded from the study after completion of the first PET scan because the occurrence of a deep venous thrombosis in the leg.[18F]MPPF PET data obtained in the 12 patients were analyzed using a set of pre-defined regions of interests and paired non-parametric Wilcoxon test. At baseline, all patients showed a major reduction of the binding potential (BP) of [18F]MPPF for 5-HT1A receptors in their epileptogenic temporal lobe.
The concurrent infusion of CsA was associated with:
1) a significant reduction of [18F]MPPF k2 in the majority of brain regions. The average k2 reduction amounted to 14% using the thalamic reference region versus 23% using the cerebellar reference region
2) a significant increase of [18F]MPPF BP in selected regions. The average BP increase was 9% using the thalamic reference region versus 16% using the cerebellar reference region. However, we found no impact of CsA infusion on [18F]MPPF BPND asymmetry index (AI) whatever the reference region used.
[11C]-FMZ:
Naïve and epileptic rats (Task 1-4, (P02-VUmc, P10-LACDR, P11-SEIN, P06-LMU)
The quantification procedures have been successfully optimized in naïve rats. The data of the [11C]FMZ PET scans have been modelled according to the Lassen and the Liefaard approach and the estimated parameters were remarkably similar. The values for Bmax were 31 ±13 and 34 ±5 ng/mL, respectively, and for the KD 7.5 ±1.7 and 5.8 ±0.9 ng/mL. This validates the full saturation approach as a reliable and actually more precise method for the assessment of GABAA receptor density and affinity. Moreover, as the method requires only a single scan it is cheaper, more time-efficient and causes less inconvenience and discomfort. Therefore this method has been selected for further experiments.
Epilepsy patients (Task 5 P02-VUmc, P10-LACDR, P11-SEIN)
The study has been performed successfully in ten patients with unilateral MTLE. MRI and PET images were co-registered (Vinci), grey and white matter were segmented (SPM5) and 45 ROIs were automatically delineated (PVElab). Metabolite corrected plasma levels were used for the input function and the Pons was used as the reference tissue. Modelling was performed with ROI analysis. Treatment with 2 mg/kg TQD caused a reduction in Binding Potential, an increase in the brain to plasma ratio VT and an increase in the k1/k2 ratio (15-19%). The changes in [11C]flumazenil in response to TQD did not vary much between regions. There was no evidence for changes in cerebral blood flow after TQD, as assessed by PET scans with [15O]H2O. Taken together these data indicate that flumazenil is a weak substrate for Pgp in humans, consistent with the preclinical studies in rats and mice.
WP05: Quantitative imaging of Pgp function in healthy controls
Objectives
The main objective is to characterize Pgp tracers first in healthy controls and to develop tracer kinetic models for quantifying Pgp related pharmacokinetic parameters.
Task 1: Development of tracer kinetic models for (R)-[11C]-VPM and newly developed Pgp PET tracers
Just prior to the start of EURIPIDES, a tracer kinetic model for (R)-[11C]VPM had been published. This single tissue compartment model provides the volume of distribution as outcome measure and a pilot study in normal subjects had shown excellent test-retest variability. However, the model assumed that one of the labelled metabolites, [11C]D617, enters the brain with kinetics similar to that of (R)-[11C]VPM itself. To assess whether this assumption is correct, [11C]D617 was synthesized and preclinical studies were performed to study its kinetics in the brain. These preclinical studies involved in vivo PET scans and ex vivo biodistribution studies, both before and after pre-treatment with TQD. The main finding was that the volume of distribution of [11C]D617 was comparable to that of (R)-[11C]VPM, but the increase after TQD pre-treatment was substantially lower. Although above mentioned assumption underlying the (R)-[11C]VPM model is not entirely correct, effects on quantification of baseline (R)-[11C]VPM studies is expected to be very small. A secondary finding was that [11C]D617 itself could be used as a substrate tracer of Pgp, but given its lower sensitivity to TQD pre-treatment there appears to be no clear advantage over (R)-[11C]VPM itself.
Task 2: (R)-[11C]VPM and newly developed Pgp PET tracers test-retest studies in healthy controls
Just prior to the start of EURIPIDES, preliminary (R)-[11C]VPM test-retest variability for the single tissue compartment model had been published for a rather small series of healthy subjects. Although whole brain test-retest was excellent, the subject population was too small to address test-retest variability at a regional level, which is important, as it forms the basis for determining the significance of changes seen in (intervention) studies in patients (e.g. within the epileptic focus). In addition, during the course of EURIPIDES, it was discovered that a constrained two tissue compartment model is to be preferred, especially for studies in patients. To increase statistical power, further test-retest data were acquired in healthy subjects. For regional analysis, PVElab, an automatic atlas-based approach developed by one of the partners, was used and test-retest variability was assessed for various outcome measures derived from different models used in the literature.
Task 3: Effects of Pgp modulators on cerebral (R)-[11C]VPM
The optimal TQD dose for modulating (R)-[11C]VPM kinetics was determined using a TQD dose escalation study. First, in a pilot study, healthy subjects had undergone paired (R)-[11C]VPM scans before and after administration of 2 mg/kg TQD (given iv over 30 min). In this study only a small increase (+24%) in the volume of distribution of (R)-[11C]VPM in whole brain was observed. Next, in a second study, subjects were scanned with (R)-[11C]VPM after administration of 3, 4, 6 or 8 mg/kg TQD. Measured increases in the volume of distribution of (R)-[11C]VPM in whole brain were fitted to a sigmoidal dose-response curve, which showed that half-maximum effect concentrations (EC50) of TQD were similar in humans and rats (EC50 561±24 ng/ml in humans and 544±32 ng/ml in rats, respectively), corresponding to a TQD dose of 3 mg/kg. Based on this dose escalation study, the dose of 3 mg/kg was considered to be optimal for assessing Pgp function with (R)-[11C]VPM both in humans and in rats. Interestingly, the maximum increase in (R)-[11C]VPM after the highest TQD dose (8 mg/kg in humans and 15 mg/kg in rats) was different between humans and rats. In humans there was only a 3-fold increase, whereas in rats a 12-fold increase was observed, pointing to species differences in Pgp between humans and rats.
Task 4: Effects of age and gender on cerebral (R)-[11C]VPM
The known small global age effect was confirmed in a second, relatively small, series of young and elderly healthy subjects, showing that the volume of distribution of (R)-[11C]VPM increased in amygdala, insula and cerebellum by 30, 26 and 25%, respectively. To study age effects in more detail and to investigate possible gender effects, the original (male) data set was extended with a third (middle aged) group and additional studies in females. It was confirmed that the volume of distribution of (R)-[11C]VPM increases with age in several cortical brain regions, strongly suggesting a progressive decrease in blood-brain barrier Pgp function with age. Interestingly, this age effect was only seen in males. In contrast, Pgp function in younger females was similar to that in older women, which in turn was similar to older men. Findings were similar for partial volume corrected data. The different aging patterns between men and women highlight the need to include both males and females in aging studies.
Task 5: Effects of partial volume correction on pharmacokinetic parameters derived from (R)-[11C]VPM and newly developed Pgp PET tracer studies
Several partial volume correction methods were investigated and implemented. These included partial volume correction based on both MRI segmentation techniques and novel reconstruction algorithms that take into account the point spread function of the scanner. These methods should be generally applicable, i.e. independent of the actual tracer being used. Both methods were validated by scanning subjects on two different scanners with different spatial resolution.
Finally, the existing software package PVElab was extended by including the so-called Hammer's volume of interest (VOI) template. This template includes 67 VOIs, incorporating all brain voxels, and is now available within the software for partial volume correction. The software can be downloaded by registering at http://nru.dk/pveout.
WP06: Quantitative imaging of Pgp function in temporal lobe epilepsy
Objectives
The main objective is to validate radiotracers for Pgp activity as biomarkers for drug resistance using drug-resistant and drug-responsive epilepsy as a biological model. The required proof-of-concept of a non-invasive molecular imaging-based tool is the ability to differentiate drug-resistant from drug-responsive patients. Epilepsy is the ideal condition for testing suitability of these tracers, as either surgical specimens or whole brains are available for direct in-vivo / ex-vivo comparative analysis (WP08).
Methodology development
New methodology was developed to underpin the clinical studies of this workpackage. A comprehensive comparison of kinetic models was undertaken for VPM and the one-tissue compartment model using a total plasma input function was found to be the most robust to alteration in peripheral metabolism, which is more extensive in epilepsy patients taking AEDs than in healthy volunteers, and to increased brain uptake after Pgp inhibition. Considering reproducibility and consistency in effect size after Pgp inhibition, the transport rate constant from plasma to brain, K1, was selected over the total volume of distribution, VT, as the parameter of choice to quantify Pgp function measured with VPM. When fitting the first 10min of dynamic data, thereby limiting the contamination of brain crossing radiolabelled metabolites, using the VPM fraction in plasma or total plasma as the input function gave similar K1 estimates.
Task 1: Test-retest studies in patients with epilepsy (P01-UCL, P08-UNIMAN)
The test-retest reproducibility (calculated as mean absolute difference) of VPM K1 in temporal lobe of epilepsy patients was found to be 5.6% (n=6) in pharmaco-sensitive TLE patients who underwent paired baseline PET scans on the brain HRRT camera. Test-retest scans were performed on the same day, in the morning and in the afternoon, similarly to the paired baseline-blocking scans with TQD (in this fixed order). There is no order effect of the time of day when the scans were performed, as indicated by the mean difference between test-retest scans of -0.8% in the temporal lobe. The reproducibility compares favourably to that determined for the corresponding region in healthy volunteers using the same kinetic model and outcome parameter on the lower spatial resolution HR+ camera (8.6% see WP05).
Task 2: Effect of chronic active epilepsy (P01-UCL, P03-MUW, P08-UNIMAN)
In a voxel-based comparison of VPM K1 ratio images to global mean using statistical parametric mapping (SPM8), VPM uptake was found to be significantly reduced by 14% in pharmaco-resistant (n=14) compared to pharmaco-sensitive patients (n=8) in ipsilateral amygdala, and bilateral parahippocampal, fusiform, inferior and medial temporal gyrus, suggesting Pgp overexpression in pharmaco-resistant mTLE exclusively in temporal lobe regions, but both ipsi- and contralateral to the epileptogenic focus. There was no area of significant difference, neither reduced nor increased VPM uptake, between pharmaco-sensitive patients and healthy controls (n=13). Pharmaco-sensitive TLE patients, unlike healthy controls but like pharmaco-resistant TLE patients, were treated with AEDs. In contrast with pharmaco-resistant TLE patients, pharmaco-sensitive TLE patients did not have seizures for many years. There was a negative correlation globally and within the ipsilateral hippocampus (Spearman r = -.604 p= 0.029) of VPM uptake in pharmaco-resistant patients with seizure frequency, implying that pharmaco-resistant TLE patients with frequent seizures had lower VPM uptake and hence elevated Pgp function
Task 3: Effect of Pgp inhibitors on ligand uptake in patients with epilepsy (P01-UCL, P08-UNIMAN)
A voxel-based analysis of VPM K1 images compared the response to two doses of TQD in pharmaco-resistant TLE patients with healthy controls and found significantly lower increases of VPM uptake in the ipsilateral hippocampus in patients (n=14, 24.3%) compared to controls (n=13, 62.4%). After 2mg/kg TQD, the hippocampal increase was lower (n=7, 16.5%) compared to 3mg/kg (n=7, 32.4%) but still significantly attenuated compared to the increases observed in healthy controls, both after 2mg/kg (n=7, 63.0%) and 3mg/kg (n=6, 66.8%), suggesting functionally elevated Pgp function in pharmaco-resistant mTLE in the epileptogenic hippocampus above all regions. Five of the 14 pharmaco-resistant TLE patients who had paired PET scans with VPM underwent anterior temporal lobe resection for surgical treatment of their epilepsy. The surgical specimens were analysed quantitatively by immunohistochemistry with antibodies for measurement of Pgp expression. There was a negative correlation (Spearman r = -.900 p= 0.034) between presurgical increases in VPM uptake after TQD in the hippocampus compared to the ipsilateral inferior and medial temporal gyrus and the corresponding difference in the percentage area of Pgp-immunopositive labelling, demonstrating a correspondence between ex-vivo Pgp expression and in-vivo Pgp function measured by PET.
Old Task 4: Influence of AEDs and acute seizures on brain Pgp function (P03-MUW)
After scanning the first patient twice with a scan-to-scan interval of three hours, it was realised that it is not possible to keep patients off medication for long enough to achieve significant reduction of plasma AED levels without increasing the risk of seizures. It was not feasible to assess the influence of AEDs on Pgp function with this approach. The technical annexe was amended and the study was replaced by the new task 4. Twenty patients with refractory epilepsy due to FCD or tumours would be scanned with VPM before and after TQD administration. As these patients were expected to be difficult to recruit, it was originally envisaged that joint efforts would be undertaken by MUW and UniMan/UCL. In the end, scanning only took place at UniMan/UCL with the consequence that data in fewer patients than anticipated were acquired. Instead, MUW recalled the pharmaco-resistant TLE patients previously scanned with VPM prior to surgery to undergo PET scanning again after surgical treatment in order to assess the effects of seizures of Pgp function (see Task 2).
New Task 4: Effect of TQD on brain Pgp function in patients with FCD and tumours (P01-UCL, P08-UNIMAN)
FCD is a congenital maldevelopment of the cortical tissue and one of the most common causes of refractory epilepsy. Unlike in TLE where several studies have demonstrated the overexpression of Pgp in brain tissues, studies on the neuronal expression and function of Pgp in patients with FCD are scarce. Unlike TLE where the hippocampus is contaminated by spillover of radioactivity from the adjacent choroid plexus, FCD, where the seizure focus is located far away from the choroid plexus, may allow analysis of epileptogenic brain regions without the need for partial volume correction. Compared individually against the group of 13 healthy controls, all 3 pharmaco-resistant FCD patients had reduced VPM uptake in close proximity to the area of FCD identified by MRI and the reduction extended further to other ipsilateral regions. This pilot study confirms that in FCD patients there is Pgp overexpression, not only in the epileptogenic area but also the distribution of Pgp overexpression is spread across to other cortical regions.
WP07: Quantitative imaging of Pgp function in Alzheimer's Disease
Objectives
Alzheimer's disease (AD) is the most common form of dementia. A major pathological hallmark of AD is the deposition of amyloid-β plaques in the brain. The origin of amyloid-ß depositions in Alzheimer's disease is unclear. The amyloid hypothesis proposes that this may be caused by an imbalance between amyloid-β production and clearance. The hypothesis we set out to test at the beginning of WP07 was that P-glycoprotein function is reduced in patients with Alzheimer's disease, so that an increase in binding potential (BPND) of (R)-[11C]VPM is expected in patients with Alzheimer's disease compared with healthy controls.
WP08: Neuropathological foundations of Pgp radiotracer binding
Objectives
The main objective was to establish, using resected and post mortem brain tissue, the neuroanatomical parameters that are essential for the interpretation of the in-vivo imaging data, and to determine the role and mechanisms of transporter-encoding gene variation in these parameters and in the neuroimaging patterns observed.
We made use of opportunities provided by surgically-resected tissue from patients with acute insult caused by the placement of intracranial depth electrodes and post mortem brain tissue from patients with (drug-resistant or drug-sensitive) mesial temporal lobe epilepsy and unilateral hippocampal sclerosis (henceforth referred to as 'epilepsy') to examine: (i) inflammation and changes in bloodbrain barrier in acute, subacute and chronic insults in epilepsy; (ii) the expression of multidrug transporters in epileptogenic and non-epileptogenic brain regions of patients with drug-resistant or drug-sensitive epilepsy to formally test the inherent assumptions of the transporter hypothesis and (iii) the neuropathological consequences of the clinical use of invasive intracranial electrode recording, which have not been studied in detail using markers of vascular integrity, inflammation and multidrug transporters. We hypothesized that there would be localized bloodbrain barrier vascular and inflammatory changes in epilepsy with acute and chronic insults and that there would be a higher expression of multidrug transporters in the epileptogenic, sclerotic hippocampus than in other non-epileptogenic brain regions of the drug-resistant epilepsy brains, but not in the drug-sensitive epilepsy and control brain, in support of a role for multidrug transporters in drug resistance in epilepsy. Our results have recently been published (Liu JY, Thom M, Catarino CB, Martinian L, Figarella-Branger D, Bartolomei F, Koepp M, Sisodiya SM. Neuropathology of the blood-brain barrier and pharmaco-resistance in human epilepsy. Brain 2012 Jul 14. [Epub ahead of print] PMID: 22750659).
We showed that:
(i) there is a highly localized overexpression of P-glycoprotein in the epileptogenic hippocampus of patients with drug-resistant epilepsy, which is compatible with the current hypothesis of multidrug resistance;
(ii) this overexpression appears specific to P-glycoprotein and does not affect other transporters, such as BCRP and MRP1;
(iii) the 'double cuff' expression of P-glycoprotein is likely to be associated with ongoing, chronic seizures, being absent in sustained remission;
(iv) a single, acute insult from intracranial electrode recording causes localized inflammation, increased bloodbrain barrier permeability and structural changes to blood vessels (such as the irregular expression of claudins and caveolins), but not P-glycoprotein overexpression;
(v) exposure to P-glycoprotein inducers is associated with increased P-glycoprotein expression in human brain, though the number of cases studied is small; and
(vi) chronic epilepsy is associated with inflammation, enhanced bloodbrain barrier permeability and increased P-glycoprotein expression, particularly in perivascular glia of the epileptogenic hippocampus.
Potential Impact:
1.4 The potential impact
Socio-economic impact and the wider societal implications of the project
Contribution to Community and social objectives
Epilepsy is the most common serious chronic neurological condition, associated not only with major comorbidities including mood disorders and cognitive dysfunction, but also with a reduced life expectancy and a mortality rate from seizure-related death as high as 1 per 100 patients p.a. among patients with uncontrolled seizures. In Europe, epilepsy affects 6 million people of all ages, and the costs attributable to epilepsy exceed approximately 20 billion EUROS p.a. The lifetime risk for epilepsy is 2%, but as high as 10% for a single seizure. The majority of these costs are indirect, in relation with the socio-economic consequences of uncontrolled seizures and side-effects. Healthcare costs are likely to fall, and EU will benefit from avoidance of wastage on AEDs likely to be ineffective for individual patients. Economic development and scientific competitiveness will benefit from the potential for commercialisation of our findings. The societal context of research was established through regular connections with patient and support organisations, exemplified by the Epilepsy Society UK co-funding to a large extent the epilepsy research activity of the co-ordinator at UCL.
Main dissemination activities and exploitation of results
The main conclusion from WP01 is that the Pgp inhibitor based PET tracers [11C]laniquidar, [11C]elacridar and [11C]TQD are unexpectedly in tracer concentrations recognised by Pgp and/or Bcrp as substrates, which results in low brain uptake and renders them unsuitable to visualise Pgp expression levels at the BBB. For TQD and elacridar, in-vivo PET results were confirmed in a series of in-vitro experiments using the respective 3H-labelled molecules. For laniquidar, in-vitro data suggest, in contrast to the in-vivo PET data, absence of Pgp/Bcrp transport. Despite these apparent limitations of [11C]laniquidar, [11C]elacridar and [11C]TQD to visualise cerebral Pgp with PET, the experiments conducted in this work package and WP02 have provided important insight into the pharmacology of these widely used reference Pgp inhibitors. Due to the fact that [11C]laniquidar, [11C]elacridar and [11C]TQD have not provided sufficiently high PET signals in rodents for the imaging of transporter distribution, the preparation of longer-lived versions of these molecules for PET and SPET imaging was not further pursued. Nevertheless [11C]laniquidar, [11C]elacridar and [11C]TQD have all entered WP05 to characterise their behaviour in healthy human subjects. In the future, we plan to assess if any of these tracers might be suitable to visualise Pgp expression levels in peripheral organs. Apart from a thorough in-vivo/in-vitro characterisation of laniquidar, elacridar and TQD, we have obtained first promising results with a 18F-labelled fexofenadine ester prodrug, which might allow for visualising cerebral P gp activity and overcome the limitation of low brain uptake obtained with (R) [11C]VPM, [11C]laniquidar, [11C]elacridar and [11C]TQD.
The results from the clinical studies performed as part of EURIPIDES have provided answers to the questions initially raised on pharmaco-resistance in epilepsy and demonstrated the utility of PET with a radiolabelled Pgp substrate as to study Pgp activity in vivo in humans:
1) Compared to pharmaco-sensitive patients and healthy controls at baseline, pharmaco-resistant patients have significantly reduced VPM uptake exclusively in the temporal lobes but the reduction is not restricted to ipsilateral epileptogenic regions but also extends to contralateral temporal lobe regions. Similarly in patients with FCD, VPM uptake is reduced not only in close proximity to the area of FCD identified by MRI but also the reduction extends further to other ipsilateral regions, supplementing the findings in TLE patients that Pgp overexpression is not limited to the epileptogenic seizure-onset zone but also spreads across to other cortical regions.
2) After partial blockade of Pgp with TQD, pharmaco-resistant TLE patients have attenuated increases of VPM uptake in the whole brain and more so in the ipsilateral epileptogenic area of the hippocampus compared to healthy controls, implicating functionally elevated Pgp function primarily in the epileptogenic hippocampus in pharmaco-resistant epilepsy.
3) Pharmaco-resistant TLE patients with good surgery outcome have higher VPM increases in the post-surgery PET scan than patients with poor outcome, pointing to diminished Pgp activity which may be caused by reduced seizure frequency and lower antiepileptic drug load in patients with good outcome. Furthermore, pharmaco-resistant TLE patients who have frequent seizures have the lowest VPM uptake, substantiating the contribution of seizures to the induction of Pgp overexpression in epilepsy.
4) In pharmaco-resistant TLE patients who underwent anterior temporal lobe resection for surgical treatment, the difference in percentage change in VPM uptake after Pgp inhibition with TQD in the hippocampus compared to a reference temporal lobe region is inversely correlated with the corresponding difference in percentage area of Pgp-immunopositive labelling, demonstrating that imaging with PET, a radiolabelled Pgp substrate and a Pgp inhibitor at a half saturating dose is a suitable tool to assess Pgp function in vivo in humans.
5) In high grade glioma patients, Pgp inhibition minimally alters perfusion but differentially increases VPM uptake into brain tissue and glioma, and preferentially into grade III but not grade IV regions, supporting the use of PET with a radiolabelled Pgp substrate as a promising tool to select patients in whom Pgp inhibitors could potentially increase chemotherapeutic efficacy.
The major achievements of this WP, in concert with the Consortium overall, are:
1. Demonstration that robust studies of human brain tissue, both post mortem and post surgical, can be of enormous value in taking understanding forwards; identification of a new paradigm for the study of the response of human brain tissue to injury (using intracranial electrode tracks as a 'model' for injury).
2. Development of robust reproducible methodology for quantitative analyses in drug resistance-related studies of fixed human brain tissue. These methods can continue to be used in these and other studies
3. Development of robust reproducible methodology for quantitative analyses in drug resistance-related studies of fixed human brain tissue. These methods can continue to be used in these and other studies
4. Determination that some antigens cannot be studied in material that has been fixed for longer periods of time. This important quantitative observation sets new standards in quantifying and interpreting immunohistochemical data.
5. Determination of the spatial and temporal patterns of expression of a variety of proteins putatively involved in drug resistance, or maintenance of blood-brain barrier integrity in the human brain. Several proteins were studied.
6. Substantiation of major assumptions implicit in the drug transporter hypothesis of drug resistance: that overexpression of P-glycoprotein is spatially localised, focused on epileptogenic tissue, and correlates with active seizures, declining with seizure freedom and being absent from brain that is simply injured but not epileptogenic (i.e. intracranial electrode tracks)
7. Correlation of in vivo activity of P-glycoprotein with ex vivo demonstration of degree of expression of P-glycoprotein. This is the first such study and elegantly demonstrates that P-glycoprotein can, at structural and functional levels, indeed play a role in drug resistance in epilepsy.
8. Demonstration that P-glycoprotein, and not other transporters studied such as BCRP and MRP1, has the appropriate pattern of expression to potentially mediate resistance.
Outlook and future research
There is now considerable evidence in support of the 'transporter hypothesis':
(1) relevant multidrug transporters are overexpressed in animal models of drug-resistant epilepsy. Whilst there has been partial evidence in favour of overexpression in human drug-resistant epilepsy, we have recently finally confirmed appropriate regional patterns of overexpression across the human brain in drug-resistant epilepsy compared to drug-responsive epilepsy using a unique post mortem human brain resource
(2) there is evidence that relevant multidrug transporters are capable of transporting many AEDs, in both animal models and ex vivo models using human brain cells;
(3) in animal models of epilepsy and drug-resistant epilepsy, temporary inhibition of the multidrug transporter, P-glycoprotein, most strongly implicated in mediating drug resistance through the transporter mechanism, leads to a transient, but definite, reduction in seizure frequency. This is the current position of the field.
List of Websites:
http://www.euripides-europe.com
Drug resistance is an important clinical problem. Globally, 50,000,000 people have epilepsy: current treatments are ineffective in 30%. Drug resistance is associated with higher rates of: unemployment, disadvantage, injury, somatic co-morbidity (affecting many body systems), psychiatric disorder, cognitive/memory decline, sudden unexplained death and other premature mortality, making chronic epilepsy pervasive and pernicious, with seizures being just one 'tip-of-the-iceberg' manifestation. Availability of several new AEDs has not solved drug resistance. We do not understand drug resistance.
One concept that has arisen for drug resistance is the transporter hypothesis, which maintains that broad resistance to antiepileptic drugs might relate to over-activity of non-specific transporter mechanisms that reduce the local concentration of AEDs within the brain tissue below a therapeutic level. The aims of EURIPIDES were to develop in-vivo imaging biomarker of multidrug transporter function as a generic tool for the prediction, diagnosis, monitoring and prognosis of major CNS diseases. EURIPIDES involved a multidisciplinary approach, based on integration of the clinical and basic science domains and on the synergistic combination of neurophysiological and biomolecular imaging methods.
In a comprehensive study of P-glycoprotein (Pgp) inhibitor based positron emission tomography (PET) tracers, we clarified that [11C]laniquidar, [11C]elacridar and [11C]tariquidar in tracer concentrations are recognised by Pgp and/or BCRP as substrates. We developed and validated, at least in epileptic animal models, eight [11C]-and three [18F]-PET radiotracers aimed to be either Pgp substrates or inhibitors, with further development of longer-lived PET radionuclides aimed at novel ways of imaging Pgp functionality. Biodistribution studies were performed in animal models of naïve, transporter knock-out and epileptic rodents. Even though these highly potent and selective radiolabelled Pgp inhibitors afforded only very low PET signals, three PET tracers ([11C]laniquidar, [11C]tariquidar, [11C]phenytoin) entered trials in humans. Using the established PET radiotracer [11C]verapmail as a model Pgp substrate and the PET receptor ligands and Pgp substrates [18F]MPPF and [11C]flumazenil, we could show that partial inhibition of the blood-brain-barrier (BBB) by administration of half-maximum inhibitor dose allows detection of regional differences in Pgp functionality at the rat BBB.
We successfully translated our findings in animal models to the human condition. Studies with [11C]VPM PET in healthy controls showed that TQD-induced Pgp modulation at the human (BBB) appeared to be transient and its magnitude directly proportionate to serum drug exposure. The optimal analytical model for baseline studies was less suitable for blocking studies, where an additional compartment improves the quality of the fits. Analysis with additional models showed small, but significant differences between patients with Alzheimer's Disease and healthy controls. This is the first direct evidence that Pgp transporter at the BBB are dysfunctional in sporadic AD and suggests that decreased Pgp function may be involved in the pathogenesis of AD. In patients with temporal lobe epilepsy (TLE), the model derived K1 (influx rate constant) values from drug-sensitive patients were higher than those from drug-resistant patients falling, which suggests more efficient Pgp function in drug-refractory patients resulting in lower drug concentrations in the brain. This difference was restricted to the temporal lobes both on the ipsilateral and contralatral sides. After tariquidar administration, we observed smaller K1 increases in the drug-resistant patients compared to healthy volunteers supporting over expression of Pgp function, with the ipsilateral epileptogenic hippocampus being most significantly affected. Functional in-vivo evidence of over active transporters in drug-resistant compared to drug-sensitive epileptic patients was complimented by histological assays of post-mortem brains and surgically resected epileptic tissue from drug-resistant patients. The change in VPM uptake after Pgp inhibition with TQD in the hippocampus correlated inversely with Pgp-immunopositive labeling in pharmaco-resistant TLE patients who underwent anterior temporal lobe resection for surgical treatment.
In summary, our findings provide evidence supporting the hypothesis of multidrug transporter overexpression as an important mechanism for developing pharmacoresistance in epilepsy. The availability of such imaging biomarkers will support the development of new treatment strategies targeted at multidrug transporter and aimed at reversing pharmacoresistance with selection of optimal patients and assessment of molecular targets. This will lead to improved health care through individualised treatment strategies, and at the same time to a reduction of health care costs by discontinuing ineffective therapies. The stage is thus set for direct translation to clinical trials. We have a tool to identify patients in whom P-glycoprotein overactivity could conceivably contribute to drug resistant epilepsy; the same tool can act to determine whether P-glycoprotein activity has been inhibited in practice as might be intended with treatment with a P-glycoprotein inhibitor such as TQD. Thus EURIPIDES has a lasting legacy, with potential for further research and clinical application.
Project Context and Objectives:
1.2 Summary description of project context and objectives:
Background and Aims
Resistance to drug treatment is an important hurdle in the therapy of many diseases of the central nervous system (CNS). Consequently, there is a pressing need to develop new and more effective treatment strategies. Whilst drug resistance in CNS diseases is multifactorial, there are two broad likely mechanisms:
(1) resistance to the desired pharmacological effects of CNS drugs occurs from alteration in the postulated target(s) the 'target hypothesis';
(2) inadequate access of CNS drugs to their targets across the blood-brain barrier (BBB) due to the overexpression or overactivity of multdrug transporters that efflux foreign chemicals from the brain the 'transporter hypothesis'.
Although the brain is among the best perfused organs in the body, drug entry into the brain is limited by the BBB, which is formed by brain endothelial cells that are closely linked by tight junctions (Reichel 2006). Substances can enter the brain either by passive diffusion across the endothelial cell membranes or by selective carrier transport. High lipophilicity is an essential characteristic of a substance to cross the BBB by diffusion. However, there are several examples of lipophilic drugs (e.g. anticancer drugs, antiepileptic drugs, anti-HIV drugs) that have poor access to brain tissue. The presence of multidrug efflux transporters, such as P-glycoprotein (Pgp), breast cancer resistance protein (BCRP) and multidrug resistance proteins (MRPs), at the luminal side of the endothelial cells of the blood capillaries is believed to be the basis for these observations (De Lange 2004, Loscher and Potschka 2005). These transporters are mainly, but not exclusively, members of the ABC (ATP-Binding Cassette) superfamily, including ABCG2 (BCRP), ABCB1 (Pgp), ABCC1 (MRP1), ABCC2 (MRP2).
Multidrug efflux transporters can contribute to drug resistance in brain diseases in two ways:
- by their constitutive expression in the BBB and blood-CSF barrier, they restrict the brain access of many drugs by enhancing drug extrusion from the brain, so that the levels of drugs in the brain cannot become sufficiently high for therapeutic efficacy.
- by intrinsic or acquired over-expression of multidrug transporters in the BBB or brain target tissue limits drug penetration into that tissue. This can result from alterations (such as polymorphisms) in genes that encode multidrug transporters, or from the effects of disease or drug treatment on expression of these transporters. The consequences of intrinsic or acquired over-expression of multidrug transporters depend on the affinity of respective substrates for the efflux transporters.
Over-expression of multidrug transporters at the BBB and beyond will prevent CNS drugs from reaching sufficiently high concentrations in critical cerebral regions despite substantial plasma levels (Sisodya 2003). This may, therefore, account for the failure of multiple diverse CNS drugs in some patients.
Several lines of evidence suggest that changes in Pgp expression and/or function might also play a key role in the causation and pathogenesis of certain neurological disorders, such as AD (Vogelsang et al. 2004, Lee et al. 2004) and epilepsy (Loscher 2002). For instance, regional overexpression of Pgp in the brains of epilepsy patients might contribute to drug resistance by impeding the access of antiepileptic drugs (AED) to the seizure focus (Loscher and Potschka 2002). These results suggest that Pgp is an attractive pharmacologic target to increase intracerebral penetration of certain drugs, such as anticancer and antiepileptic drugs (Bates 2002, Sisodiya and Bates 2006).
It has not been investigated whether expression of other transporters in the BBB is affected by Pgp inhibition, but the conformation of Pgp may be affected by substrate inhibitors, possibly in a genotype-dependent fashion (Kimchi-Sarfaty et al, 2007). A chronic study in a model of pharmacoresistant epilepsy with a combination of phenobarbital and the Pgp inhibitor TQD did not indicate that tolerance development may be an issue (Brandt et al., 2006). In contrast, development of tolerance to a combination with phenytoin and TQD was described in another chronic epilepsy model (Van Vliet et al., 2006). At present, it cannot be excluded that this is due to an effect on expression of other transporters, or genotype-dependent dynamic alterations in Pgp itself (Kimchi-Safarty et al, 2007).
In this project, we focused initially on Pgp, which is the most-widely studied multidrug transporter, is found in and contributes to the normal BBB, and as efflux transporter, actively transports substrates (including multiple CNS drugs) against concentration gradients from the brain to blood and cerebrospinal fluid. This hampers the build-up of adequate tissue levels of these drugs in the brain, greatly limiting their therapeutic efficacy. As such, the 'transporter hypothesis' of drug resistance is applicable to a broad range of CNS drugs and patients with a variety of CNS diseases who critically depend on these drugs accessing the brain tissue.
Our aim was to develop a generic tool for the prediction, diagnosis, monitoring and prognosis of major CNS diseases, as well as to provide support and guidance for therapeutic interventions.
The project aims were twofold:
(1) to develop radiotracers to image Pgp function and distribution to modulation of Pgp expression,
(2) to validate the use of Pgp radiotracers as in-vivo imaging biomarker by determining the contribution of Pgp overexpression to impaired brain uptake of drugs (pharmacoresistance) for the prediction of therapeutic response, or the contribution of impaired Pgp function to reduced clearance of toxic substances (e.g. β-amyloid) for the early in-vivo diagnosis of Alzheimer's Disease (AD).
Work strategy and general description
Circumvention of pharmacoresistance, or increasing clearance of drugs from the brain tissues, may involve inhibitors of Pgp transporters or sophisticated alternative therapies, but demonstration of overexpression or underactivity of transporter function is an essential and necessary first step. An in-vivo imaging biomarker of Pgp function is essential for identifying altered transporter activity in individual patients. If a relation between overexpression and therapy resistance, or in the case of AD underactivity, can be demonstrated, such a biomarker will provide unique and new pathophysiological information, the means for predicting treatment response, or early diagnosis, in individual patients. The availability of a radiotracer for imaging Pgp function in healthy volunteers does not make it a biomarker for drug resistance. In order to validate the usefulness of a biomarker, studies have to be designed for relevant and sensitive clinical populations, e.g. comparative studies in patients with refractory vs responsive CNS diseases.
Management structure and procedures
The Project Coordinator ensured the smooth operation of the project and guaranteed that all efforts were focused towards the objectives. He submitted all required progress reports, deliverables, financial statements to the European Commission, and, with the assistance of GABO he was responsible for the proper use of funds and their transfers to participants. The EURIPIDES office was established by and based at the coordinator in London and at GABO in Munich. The Project Office at the Coordinator was concerned with the scientific management and the co-ordination of all research activities. The Project Office at GABO was responsible for administrative, financial and contractual management and the organisational co-ordination of the project activities.
Objectives of EURIPIDES:
In line with this call's main aim 'to develop highly sensitive agent(s) for established in-vivo imaging modalities able to report and quantify cellular and/or molecular events in several major disease states or disease processes for early in-vivo diagnosis and/or evaluation of response to therapy', using positron emission tomography (PET) and single-photon emission tomography (SPET) in a multidisciplinary approach integrating radiochemistry, molecular and cellular biology, physics, pharmacology and pathology, our main objectives are:
- to discover and characterise novel PET and SPET radiopharmacological tracers, which are markers for the function and the expression of cerebral multidrug transporters that form part of the BBB (WP 01 Key milestones (KM) 1.5,14,15,22,23).
- to characterise and identify compounds / potential radiotracers in-vitro that are substrates for Pgp and acts as inhibitors of drug efflux across the BBB (WP03 KM3.3,4)
- to evaluate, determine kinetics and develop quantitative methodology to measure transporter expression and/or functionality of existing radiotracers known to be either Pgp substrates ([11C]-VPM) or Pgp inhibitors ([11C]-laniquidar) in naïve animals (WP02 KM2.3).and in healthy human volunteers (WP05 KM5.4,5) and to later apply these methods to novel radiotracers emerging from WP01, in naïve animals (WP02 KM2,5,7,8,12,15) and in healthy human volunteers (WP05 KM5.10-12).
- to carry out proof-of-concept studies with existing radiotracers known to be either Pgp substrates ([11C]-VPM, [11C]-flumazenil, [18F]-MPPF) or Pgp inhibitors ([11C]-laniquidar) to image Pgp function, and determine the efficacy of existing and novel Pgp modulators to enhance cerebral penetration of substrate tracers in CNS disease models, comparing drug-resistant and drug-responsive animals (WP02 KM2.4; WP04, KM4.3,4) and humans (WP 04 KM4.3,4; WP05 KM5.4,8; WP06 KM6.4,6-8) and similarly with novel radiotracers emerging from WP01 (WP02 KM2.9-11,13,14,16-18)
- to carry out proof-of-concept studies with [11C]-VPM to image Pgp function for early diagnosis of Alzheimer's Disease (AD) (WP07 KM7.3,4)
- to determine the anatomical distribution of transporter proteins in brain tissue from patients with epilepsy and AD (WP08 KM8.5-7)
Project Results:
1.3 Description of the main S&T results/foregrounds of EURIPIDES
WP01: Development of new radiotracers
Objectives
The main objective of this WP01 was to develop new radiotracers for in-vivo imaging of cerebral multidrug transporters (P-glycoprotein, Pgp; multidrug resistance proteins, MRPs; breast cancer resistance protein, BCRP) with PET and SPET. We initially followed two different approaches. First, we developed radiotracers based on known third-generation Pgp inhibitors (laniquidar, elacridar and TQD). These compounds were expected to bind with nanomolar affinity to Pgp at the blood-brain barrier (BBB) and provide higher PET signals than available Pgp substrate radiotracers such as (R)-[11C]VPM. Moreover, radiolabelled Pgp inhibitors were expected to allow for mapping of Pgp expression levels at the BBB and to afford signal increases rather than signal decreases (as radiolabelled substrates) in epileptic brain regions overexpressing Pgp.
As a second approach, we developed two PET tracers based on antiepileptic drugs (AEDs), i.e. [11C]phenytoin and [11C]mephobarbital. The rationale behind this approach was our expectation that radiolabelled AEDs would be only weakly transported by Pgp at the BBB and thereby afford higher PET signals than the high-affinity Pgp substrate (R)-[11C]VPM. This should enable an improved visualisation of regional Pgp activity in the epileptic brain as compared with (R)-[11C]VPM. In addition, it was expected that mapping the regional brain distribution of a radiolabelled AED should be clinically more meaningful in predicting drug resistance than using a surrogate marker of Pgp activity such as (R)-[11C]VPM.
Results
[11C]Radiolabelling
After the synthesis of appropriate radiolabelling percursors, [11C]laniquidar, [11C]elacridar, [11C]TQD and [11C]mephobarbital were synthesised by [11C]methylation of phenolic OH functions ([11C]elacridar and [11C]TQD), a carboxylic acid function ([11C]laniquidar) or an imide function ([11C]mephobarbital) using [11C]methyl triflate or [11C]methyl iodide. Moreover, a presumably BCRP-selective TQD analogue (Kuhnle et al, 2009)) was labelled by [11C]methylation of a carboxylic acid function.
[11C]Phenytoin was synthesised by reaction of 2,2-azido-diphenylamide with [11C]carbon monoxide. All radiotracers were obtained in good radiochemical yield, with acceptable radiochemical purity (greater than98%) and specific activity (greater than50 GBq/µmol). In addition, 3H-labelled versions of laniquidar and elacridar were synthesised by [3H]methylation of the respective desmethyl-precursor molecules with [3H]methyl nosylate.
Radiopharmacological characterisation of [11C]radiotracers
[11C]Laniquidar was tested in biodistribution experiments in rats with and without pretreatment with cyclosporine A or valspodar and in PET experiments in rats with and without pretreatment with TQD. [11C]Elacridar and [11C]TQD were tested in paired small-animal PET scans in naïve rats, wild-type mice and three different transporter knockout mouse models (Mdr1a/b(-/-), Bcrp1(-/-) and Mdr1a/b(-/-)Bcrp1(-/-)), each before and after administration of unlabelled compound. Moreover, in-vitro autoradiography was performed on rat or mouse brain slices with [11C]elacridar and [11C]TQD, with and without co-incubation with an excess of unlabelled compound (1 µM). The 11C-labelled BCRP-selective TQD analogue was characterised in small-animal PET scans in wild-type, Mdr1a/b(-/-), Bcrp1(-/-) and Mdr1a/b(-/-)Bcrp1(-/-) mice. [11C]Phenytoin was tested in PET scans in rats before and after administration of either TQD as Pgp inhibitor or the MRP inhibitor probenecid. [11C]Mephobarbital was tested in paired PET scans in rats, wild-type, Mdr1a/b(-/-) and Mrp1(-/-) mice, before and after administration of TQD or the MRP inhibitor MK571. In-vivo metabolism of all [11C]radiotracers was assessed in naïve rats or mice by analysis of plasma samples with a combined solid-phase extraction (SPE) and high-performance liquid chromatography (HPLC) assay.
Development of radiotracers for SPET imaging
We synthesised a series of iodinated elacridar/TQD analogues in order to come up with a Pgp inhibitor based SPET tracer. Unfortunately, none of these maintained the biological activity of elacridar/TQD itself as shown by in-vitro transport inhibition assays. One derivative (iodoelacridar-urea) was found to be only approximately 6 times less potent than elacridar and therefore labelled with iodine-125. A biodistribution study was performed in wild-type mice with [125I]iodoelacridar urea. The compound was shown to possess low brain uptake (less than0.5 %ID/g) which was increased after pretreatment of animals with cold TQD. Due to the similar in-vivo behaviour of [125I]iodoelacridar urea and [11C]elacridar this compound was not further pursued.
WP02: Biological evaluation of new radiotracers
Objectives
The main objective was to evaluate the feasibility of new radiotracers developed in WP01 for in vivo imaging of cerebral multidrug transporters with PET and SPET. These radiotracers have been used to study in vivo the distribution and function of multidrug transporters in animals and this had been related to actual brain uptake and distribution of CNS active drugs.
PET studies
The (R)-[11C]-VPM PET scans have been performed in a microPET scanner (AIT/TIHO) as well as in an HRRT scanner (VUA/LACDR) in female (AIT/TIHO) and male (VUA/LACDR) Sprague-Dawley rats. Essentially these studies have demonstrated that the results were not critically dependent on methodological aspects. In fact, the results were very similar: [11C]VPM had very low brain uptake at baseline conditions. For comparison with human PET data, kinetic modelling was performed to estimate the rate constants of tracer transport across the rat BBB.
Intracerebral microdialysis
Parallel studies with PET and microdialysis were too complex to be performed in the same animal. QUIN pharmacokinetics was therefore determined separately following i.v. administration at 2 different dosages. The 8 hr study covered a 1 hr blank period, a 4 hr infusion period and a 3 hr elimination period. Plasma and microdialysate concentrations, as well as total brain concentrations at termination of the experiment, were analyzed by HPLC and fluorescence detection.
Additional studies
A range of studies have been performed to support the studies on Pgp functionality and expression. Population modelling with NONMEM has been introduced and covariate analysis has allowed to demonstrate which parameters of brain uptake and distribution were influenced by TQD, epilepsy and other factors. For example, this approach confirmed that TQD specifically and concentration-dependently affects efflux from the brain. Moreover modelling techniques have been exchanged between institutes. Also rat data were compared with values from the human dose-escalation study, showing that TQD-induced increase in [11C]VPM uptake is distinctly stronger in rats than in man, whereas EC50 values were nearly identical.
PET studies
MicroPET scans were performed with [11C]-VPM before and after administration of TQD (3 mg/kg and 15 mg/kg) in rats 48 h after status epilepticus (SE) and respective control rats. For analysis of µPET scans after TQD administration, different brain regions of interest were outlined on magnet resonance images which were co-registered with the PET images. For comparison with the present μPET data, computer-assisted quantification of Pgp expression in immunohistochemically stained rat brain sections was performed in respective brain regions. Striking changes in [11C]-VPM brain uptake were obvious after administration of 3 mg/kg TQD (ED50) in animals 48 h after pilocarpine-induced SE which vanished after maximal Pgp inhibition (15 mg/kg). In rats 48 h post SE, the DV (=Kp) of cerebellum was decreased by 37.5% whereas DV of frontal motor cortex displayed an increase by 18.9% compared to control rats. In thalamus, a tendency towards DV decrease was found. The decrease of [11C]-VPM uptake in the cerebellum as well as the tendency for decrease in thalamus indicate higher Pgp activity induced by SE which is reflected by increased Pgp-labelled areas in respective brain regions. In conclusion, a µPET protocol suitable to quantify SE-induced changes in Pgp activity in distinct rat brain regions was established.
Intracerebral microdialysis
Remarkably, kainate-treated rats tended to have a lower total brain concentration but a higher brain ECF concentration of quinidine than saline-treated rats. This suggests that the Pgp function in kainate-treated rats is altered at the parenchymal level rather than at the BBB. TQD pre-administration increased the brain ECF brain concentration 7.2-fold but the total brain concentration about 40-fold, supporting an important role for Pgp in intra-brain distribution. After kainate-treatment alone however there was no difference in transport compared to control rat
Biological evaluation of radiotracers in animal models of drug resistance (Task 5)
Rats were selected which did or did not respond to treatment with the antiepileptic drug phenobarbital in the pilocarpine model of TLE. Similar to the observations in the basolateral amygdala stimulation model, epileptic rats in the pilocarpine model showed a pronounced variability in individual response to treatment with phenobarbital. 33% of epileptic rats did not respond appropriately to treatment with the maximal tolerable dose of phenobarbital. As previously described part of the animals in the pilocarpine model did also not respond to treatment with the common antiepileptic drug levetiracetam. As a definition of pharmacoresistance in animal models asks for refractoriness to monotherapy at tolerable doses with at least two current antiepileptic drugs, these findings support the suitability of the pilocarpine model for the investigation of pharmacoresistant epilepsy. Selected responders and nonresponders have been used to evaluate if differences in Pgp activity between pharmacosensitive and resistant rats can be detected and quantified after TQD administration using small-animal positron emission tomography with [11C]VPM.
Additional studies
Some animals were repeatedly scanned in a (human) 3T MRI scanner during epileptogenesis. These data were used together with available brain region MRI atlases to create atlases that reflect specific developments in responder and non-responder rat brains.
A time course study on Pgp expression was undertaken in relation to time-after SE, covering a period of 5 weeks. Results showed considerable variability between animals but as a general trend there was a transient early increase peaking at 2 days after SE and a second broader peak between 7-14 days after SE.
SE induction by unilateral intrahippocampal kainate injection did not reveal clear ipsilateral to contralateral differences in Pgp expression. However correlating quinidine brain pharmacokinetics before and after TQD treatment, with Pgp expression and seizure severity during SE in individual animals, hinted to a clear and highly interesting uncoupling of Pgp expression and function strongly influenced by seizure severity.
WP03: In vitro characterisation
Objectives
The main objective is to determine the most specific and potent inhibitor of Pgp for use as in-vivo molecular imaging tracer in patients with refractory neurological and psychiatric conditions.
Task 1: In-vitro characterisation of inhibition of transport by Pgp to identify compounds with the best inhibitory profile to be used in-vivo
The objective of this task was to rank inhibitors on the basis of inhibitory potency in an effort to identify an optimal inhibitor for the subsequent in-vitro / in-vivo experiments. We have tested and determined IC50 values for seven Pgp inhibitors to validate the use of these compounds as in-vivo probes for PET imaging. The P12-DRC group has produced 5 batches of elacridar derivates labeled with iodine and we have determined the efficacies of the derivatives compared to the parent compound. Ten novel elacridar derivatives in total have been tested but most have markedly reduced efficacy compared to the parent compound which strongly suggests that they will not be suitable for use as in-vivo probes. A 5th batch of compounds was tested with the 3-iodo derivative showing an interaction with Pgp. This compound has now being taken forward for in-vivo imaging experiments.
An aim of WP03 is to test novel pro-drug ligands of Pgp generated from the lab group of E. Arstad, UCL utilising an in-vitro Pgp assay. Seventeen novel ligands have been tested in a validated assay and this has lead to the identification of an optimal ligand pair. This optimal ligand pair will now be further validated in-vivo as a novel imaging probe (E. Arstad, UCL).
Task 2: Comparison of inhibition of wild-type and variant forms of ABCB1 by model inhibitors
The primary aim of this task was to characterise the inhibition of transport of wild type and variant forms of Pgp (i.e. containing combinations of 3 single nucleotide polymorphisms [SNPs] in the ABCB1 gene) by inhibitors. To validate the Xenopus oocyte expression system, two model Pgp substrates, digoxin and imatinib were shown to be transported by human ABCB1 expressing oocytes. This Pgp mediated transport was shown to be dependent on ATPase activity and was inhibited by the Pgp inhibitor PSC-833. Site directed mutagenesis has been performed on human Pgp cDNA to generate the three SNP variant haplotype corresponding to C1236T, G2677T and C3435T. No differences in transport were observed between the wild type and triple SNP variant, indicating that the triple SNP variant does not have a direct functional effect for transport or inhibition for the two tested model substrates.
An additional sub-task was to characterise the transport of AEDs by Pgp. To address this question seven transport systems expressing Pgp have been developed and utilised in this work package to study the transport of three major AEDs. The Pgp mediated transport of phenytoin was observed utilising the equilibrium method in both Pgp stably over expressing cell lines. This suggests that phenytoin is a weak substrate of Pgp that requires non-physiological (or high) protein expression of Pgp for transport. The transport of carbamazepine and lamotrigine by Pgp was also investigated with no Pgp mediated transport observed in the in-vitro systems tested suggesting that lamotrigine and carbamazepine are not transported by Pgp. This adds to the evidence that there is no unifying mechanism of drug transporter resistance and emphasises the need for a systems approach of AED transport at the BBB.
Task 3: Characterisation of the mechanism of induction of ABCB1 by drugs
The induction properties of AEDs of ABCB1 have been investigated in an in-vitro model of the BBB. No induction was observed of ABCB1 over a timecourse and with different concentration of AED. This suggests that at least in the in-vitro model tested, that AEDs do not induce ABCB1 mRNA expression.
Task 4: Systems approach for studying drug transport at the BBB
The aim of this task was to investigate the overall transport, influx and efflux, at the blood-brain barrier (BBB), and not only focus on Pgp mediated drug transport. This systems approach is going to be essential in understanding the success of drugs used for CNS diseases, and the development of novel ligands. Identifying these mechanisms of transport at the BBB will lead to the identification of imaging probes. We have investigated lamotrigine as the mechanisms that underpin the passage of this important AED at the blood-brain barrier to its site of action in the brain is poorly understood. Lamotrigine has been postulated to be delivered to its site of action in the brain favourably despite its physicochemical properties.
WP04: Evaluation of existing radiotracers for imaging Pgp function
Objectives
The main objective is to determine the contribution of Pgp activity to uptake and binding of existing PET tracer [18F]-MPPF, and [11C]-FMZ that are Pgp substrates.
[18F]-MPPF:
Naïve and epileptic rats (Task 1-4, P06-LMU)
First PET scans, examining the impact of Pgp modulation on [18F]-MPPF brain kinetics, were performed in naïve Sprague Dawley rats. Following optimization of the quantification procedure, the initial uptake of [18F]-MPPF in brain was used as a surrogate marker of the unidirectional blood-brain clearance (K1). Based on the consortiums decision to focus on the Pgp inhibitor TQD (TQD), the effect of TQD on the K1-surrogate was determined. TQD had a significant and dose-dependent effect, with an IC50 close to 5 mg/kg, and a 2.5-fold increase at the highest TQD dose. Additional PET scans have been performed with the Pgp modulator cyclosporine A (CsA) resulting in an increase of 30-40% for the K1-surrogate. These data clearly substantiate transport of [18F]-MPPF by Pgp further supporting its suitability for non-invasive Pgp imaging.
Patients and healthy controls (Task 5, P07-HCL):
Cyclosporine A (CsA) was used throughout the project by partner P07-HCL. Fifteen patients with drug-resistant focal epilepsy of temporal lobe origin with or without hippocampal sclerosis were originally selected for this protocol. Twelve of the 15 scanned patients (mean age ± SD = 43.9 ± 7 years) had paired 60-minute dynamic [18F]MPPF PET scans with and without concurrent cyclosporine A (CsA) IV infusion. The three remaining patients underwent only one [18F]MPPF PET (without CsA) and were then excluded from the protocol for various reasons. A few patients reported mild central nervous system side effects, such as vertigo, during the 24 hours following CsA infusion, suggesting that the latter promoted the activity and adverse events of concurrent anti-epileptic drugs. For the PET associated with CsA infusion, the latter was started one hour prior to [18F]-MPPF injection at a dosage of 2.5 ml/kg/h over 2h resulting in a steady plasma concentration of CsA during the entire PET study at or above the target level (= 2980 μg/l). A simplified reference tissue model was used to generate parametric images of 5-HT1A receptor binding potential (BP=Bmax/Kd) and K2 values. The cerebellar white matter and the thalamic region were both used as reference regions. The same protocol was used in eight healthy subjects. All controls also gave their informed consent to participate in this study. Seven of the 8 scanned patients (mean age ± SD = 41.8 ± 7.2 years) had paired 60-minute dynamic [18F]MPPF PET scans with and without continuous cyclosporine A (CsA) IV infusion. The last control recruited had to be excluded from the study after completion of the first PET scan because the occurrence of a deep venous thrombosis in the leg.[18F]MPPF PET data obtained in the 12 patients were analyzed using a set of pre-defined regions of interests and paired non-parametric Wilcoxon test. At baseline, all patients showed a major reduction of the binding potential (BP) of [18F]MPPF for 5-HT1A receptors in their epileptogenic temporal lobe.
The concurrent infusion of CsA was associated with:
1) a significant reduction of [18F]MPPF k2 in the majority of brain regions. The average k2 reduction amounted to 14% using the thalamic reference region versus 23% using the cerebellar reference region
2) a significant increase of [18F]MPPF BP in selected regions. The average BP increase was 9% using the thalamic reference region versus 16% using the cerebellar reference region. However, we found no impact of CsA infusion on [18F]MPPF BPND asymmetry index (AI) whatever the reference region used.
[11C]-FMZ:
Naïve and epileptic rats (Task 1-4, (P02-VUmc, P10-LACDR, P11-SEIN, P06-LMU)
The quantification procedures have been successfully optimized in naïve rats. The data of the [11C]FMZ PET scans have been modelled according to the Lassen and the Liefaard approach and the estimated parameters were remarkably similar. The values for Bmax were 31 ±13 and 34 ±5 ng/mL, respectively, and for the KD 7.5 ±1.7 and 5.8 ±0.9 ng/mL. This validates the full saturation approach as a reliable and actually more precise method for the assessment of GABAA receptor density and affinity. Moreover, as the method requires only a single scan it is cheaper, more time-efficient and causes less inconvenience and discomfort. Therefore this method has been selected for further experiments.
Epilepsy patients (Task 5 P02-VUmc, P10-LACDR, P11-SEIN)
The study has been performed successfully in ten patients with unilateral MTLE. MRI and PET images were co-registered (Vinci), grey and white matter were segmented (SPM5) and 45 ROIs were automatically delineated (PVElab). Metabolite corrected plasma levels were used for the input function and the Pons was used as the reference tissue. Modelling was performed with ROI analysis. Treatment with 2 mg/kg TQD caused a reduction in Binding Potential, an increase in the brain to plasma ratio VT and an increase in the k1/k2 ratio (15-19%). The changes in [11C]flumazenil in response to TQD did not vary much between regions. There was no evidence for changes in cerebral blood flow after TQD, as assessed by PET scans with [15O]H2O. Taken together these data indicate that flumazenil is a weak substrate for Pgp in humans, consistent with the preclinical studies in rats and mice.
WP05: Quantitative imaging of Pgp function in healthy controls
Objectives
The main objective is to characterize Pgp tracers first in healthy controls and to develop tracer kinetic models for quantifying Pgp related pharmacokinetic parameters.
Task 1: Development of tracer kinetic models for (R)-[11C]-VPM and newly developed Pgp PET tracers
Just prior to the start of EURIPIDES, a tracer kinetic model for (R)-[11C]VPM had been published. This single tissue compartment model provides the volume of distribution as outcome measure and a pilot study in normal subjects had shown excellent test-retest variability. However, the model assumed that one of the labelled metabolites, [11C]D617, enters the brain with kinetics similar to that of (R)-[11C]VPM itself. To assess whether this assumption is correct, [11C]D617 was synthesized and preclinical studies were performed to study its kinetics in the brain. These preclinical studies involved in vivo PET scans and ex vivo biodistribution studies, both before and after pre-treatment with TQD. The main finding was that the volume of distribution of [11C]D617 was comparable to that of (R)-[11C]VPM, but the increase after TQD pre-treatment was substantially lower. Although above mentioned assumption underlying the (R)-[11C]VPM model is not entirely correct, effects on quantification of baseline (R)-[11C]VPM studies is expected to be very small. A secondary finding was that [11C]D617 itself could be used as a substrate tracer of Pgp, but given its lower sensitivity to TQD pre-treatment there appears to be no clear advantage over (R)-[11C]VPM itself.
Task 2: (R)-[11C]VPM and newly developed Pgp PET tracers test-retest studies in healthy controls
Just prior to the start of EURIPIDES, preliminary (R)-[11C]VPM test-retest variability for the single tissue compartment model had been published for a rather small series of healthy subjects. Although whole brain test-retest was excellent, the subject population was too small to address test-retest variability at a regional level, which is important, as it forms the basis for determining the significance of changes seen in (intervention) studies in patients (e.g. within the epileptic focus). In addition, during the course of EURIPIDES, it was discovered that a constrained two tissue compartment model is to be preferred, especially for studies in patients. To increase statistical power, further test-retest data were acquired in healthy subjects. For regional analysis, PVElab, an automatic atlas-based approach developed by one of the partners, was used and test-retest variability was assessed for various outcome measures derived from different models used in the literature.
Task 3: Effects of Pgp modulators on cerebral (R)-[11C]VPM
The optimal TQD dose for modulating (R)-[11C]VPM kinetics was determined using a TQD dose escalation study. First, in a pilot study, healthy subjects had undergone paired (R)-[11C]VPM scans before and after administration of 2 mg/kg TQD (given iv over 30 min). In this study only a small increase (+24%) in the volume of distribution of (R)-[11C]VPM in whole brain was observed. Next, in a second study, subjects were scanned with (R)-[11C]VPM after administration of 3, 4, 6 or 8 mg/kg TQD. Measured increases in the volume of distribution of (R)-[11C]VPM in whole brain were fitted to a sigmoidal dose-response curve, which showed that half-maximum effect concentrations (EC50) of TQD were similar in humans and rats (EC50 561±24 ng/ml in humans and 544±32 ng/ml in rats, respectively), corresponding to a TQD dose of 3 mg/kg. Based on this dose escalation study, the dose of 3 mg/kg was considered to be optimal for assessing Pgp function with (R)-[11C]VPM both in humans and in rats. Interestingly, the maximum increase in (R)-[11C]VPM after the highest TQD dose (8 mg/kg in humans and 15 mg/kg in rats) was different between humans and rats. In humans there was only a 3-fold increase, whereas in rats a 12-fold increase was observed, pointing to species differences in Pgp between humans and rats.
Task 4: Effects of age and gender on cerebral (R)-[11C]VPM
The known small global age effect was confirmed in a second, relatively small, series of young and elderly healthy subjects, showing that the volume of distribution of (R)-[11C]VPM increased in amygdala, insula and cerebellum by 30, 26 and 25%, respectively. To study age effects in more detail and to investigate possible gender effects, the original (male) data set was extended with a third (middle aged) group and additional studies in females. It was confirmed that the volume of distribution of (R)-[11C]VPM increases with age in several cortical brain regions, strongly suggesting a progressive decrease in blood-brain barrier Pgp function with age. Interestingly, this age effect was only seen in males. In contrast, Pgp function in younger females was similar to that in older women, which in turn was similar to older men. Findings were similar for partial volume corrected data. The different aging patterns between men and women highlight the need to include both males and females in aging studies.
Task 5: Effects of partial volume correction on pharmacokinetic parameters derived from (R)-[11C]VPM and newly developed Pgp PET tracer studies
Several partial volume correction methods were investigated and implemented. These included partial volume correction based on both MRI segmentation techniques and novel reconstruction algorithms that take into account the point spread function of the scanner. These methods should be generally applicable, i.e. independent of the actual tracer being used. Both methods were validated by scanning subjects on two different scanners with different spatial resolution.
Finally, the existing software package PVElab was extended by including the so-called Hammer's volume of interest (VOI) template. This template includes 67 VOIs, incorporating all brain voxels, and is now available within the software for partial volume correction. The software can be downloaded by registering at http://nru.dk/pveout.
WP06: Quantitative imaging of Pgp function in temporal lobe epilepsy
Objectives
The main objective is to validate radiotracers for Pgp activity as biomarkers for drug resistance using drug-resistant and drug-responsive epilepsy as a biological model. The required proof-of-concept of a non-invasive molecular imaging-based tool is the ability to differentiate drug-resistant from drug-responsive patients. Epilepsy is the ideal condition for testing suitability of these tracers, as either surgical specimens or whole brains are available for direct in-vivo / ex-vivo comparative analysis (WP08).
Methodology development
New methodology was developed to underpin the clinical studies of this workpackage. A comprehensive comparison of kinetic models was undertaken for VPM and the one-tissue compartment model using a total plasma input function was found to be the most robust to alteration in peripheral metabolism, which is more extensive in epilepsy patients taking AEDs than in healthy volunteers, and to increased brain uptake after Pgp inhibition. Considering reproducibility and consistency in effect size after Pgp inhibition, the transport rate constant from plasma to brain, K1, was selected over the total volume of distribution, VT, as the parameter of choice to quantify Pgp function measured with VPM. When fitting the first 10min of dynamic data, thereby limiting the contamination of brain crossing radiolabelled metabolites, using the VPM fraction in plasma or total plasma as the input function gave similar K1 estimates.
Task 1: Test-retest studies in patients with epilepsy (P01-UCL, P08-UNIMAN)
The test-retest reproducibility (calculated as mean absolute difference) of VPM K1 in temporal lobe of epilepsy patients was found to be 5.6% (n=6) in pharmaco-sensitive TLE patients who underwent paired baseline PET scans on the brain HRRT camera. Test-retest scans were performed on the same day, in the morning and in the afternoon, similarly to the paired baseline-blocking scans with TQD (in this fixed order). There is no order effect of the time of day when the scans were performed, as indicated by the mean difference between test-retest scans of -0.8% in the temporal lobe. The reproducibility compares favourably to that determined for the corresponding region in healthy volunteers using the same kinetic model and outcome parameter on the lower spatial resolution HR+ camera (8.6% see WP05).
Task 2: Effect of chronic active epilepsy (P01-UCL, P03-MUW, P08-UNIMAN)
In a voxel-based comparison of VPM K1 ratio images to global mean using statistical parametric mapping (SPM8), VPM uptake was found to be significantly reduced by 14% in pharmaco-resistant (n=14) compared to pharmaco-sensitive patients (n=8) in ipsilateral amygdala, and bilateral parahippocampal, fusiform, inferior and medial temporal gyrus, suggesting Pgp overexpression in pharmaco-resistant mTLE exclusively in temporal lobe regions, but both ipsi- and contralateral to the epileptogenic focus. There was no area of significant difference, neither reduced nor increased VPM uptake, between pharmaco-sensitive patients and healthy controls (n=13). Pharmaco-sensitive TLE patients, unlike healthy controls but like pharmaco-resistant TLE patients, were treated with AEDs. In contrast with pharmaco-resistant TLE patients, pharmaco-sensitive TLE patients did not have seizures for many years. There was a negative correlation globally and within the ipsilateral hippocampus (Spearman r = -.604 p= 0.029) of VPM uptake in pharmaco-resistant patients with seizure frequency, implying that pharmaco-resistant TLE patients with frequent seizures had lower VPM uptake and hence elevated Pgp function
Task 3: Effect of Pgp inhibitors on ligand uptake in patients with epilepsy (P01-UCL, P08-UNIMAN)
A voxel-based analysis of VPM K1 images compared the response to two doses of TQD in pharmaco-resistant TLE patients with healthy controls and found significantly lower increases of VPM uptake in the ipsilateral hippocampus in patients (n=14, 24.3%) compared to controls (n=13, 62.4%). After 2mg/kg TQD, the hippocampal increase was lower (n=7, 16.5%) compared to 3mg/kg (n=7, 32.4%) but still significantly attenuated compared to the increases observed in healthy controls, both after 2mg/kg (n=7, 63.0%) and 3mg/kg (n=6, 66.8%), suggesting functionally elevated Pgp function in pharmaco-resistant mTLE in the epileptogenic hippocampus above all regions. Five of the 14 pharmaco-resistant TLE patients who had paired PET scans with VPM underwent anterior temporal lobe resection for surgical treatment of their epilepsy. The surgical specimens were analysed quantitatively by immunohistochemistry with antibodies for measurement of Pgp expression. There was a negative correlation (Spearman r = -.900 p= 0.034) between presurgical increases in VPM uptake after TQD in the hippocampus compared to the ipsilateral inferior and medial temporal gyrus and the corresponding difference in the percentage area of Pgp-immunopositive labelling, demonstrating a correspondence between ex-vivo Pgp expression and in-vivo Pgp function measured by PET.
Old Task 4: Influence of AEDs and acute seizures on brain Pgp function (P03-MUW)
After scanning the first patient twice with a scan-to-scan interval of three hours, it was realised that it is not possible to keep patients off medication for long enough to achieve significant reduction of plasma AED levels without increasing the risk of seizures. It was not feasible to assess the influence of AEDs on Pgp function with this approach. The technical annexe was amended and the study was replaced by the new task 4. Twenty patients with refractory epilepsy due to FCD or tumours would be scanned with VPM before and after TQD administration. As these patients were expected to be difficult to recruit, it was originally envisaged that joint efforts would be undertaken by MUW and UniMan/UCL. In the end, scanning only took place at UniMan/UCL with the consequence that data in fewer patients than anticipated were acquired. Instead, MUW recalled the pharmaco-resistant TLE patients previously scanned with VPM prior to surgery to undergo PET scanning again after surgical treatment in order to assess the effects of seizures of Pgp function (see Task 2).
New Task 4: Effect of TQD on brain Pgp function in patients with FCD and tumours (P01-UCL, P08-UNIMAN)
FCD is a congenital maldevelopment of the cortical tissue and one of the most common causes of refractory epilepsy. Unlike in TLE where several studies have demonstrated the overexpression of Pgp in brain tissues, studies on the neuronal expression and function of Pgp in patients with FCD are scarce. Unlike TLE where the hippocampus is contaminated by spillover of radioactivity from the adjacent choroid plexus, FCD, where the seizure focus is located far away from the choroid plexus, may allow analysis of epileptogenic brain regions without the need for partial volume correction. Compared individually against the group of 13 healthy controls, all 3 pharmaco-resistant FCD patients had reduced VPM uptake in close proximity to the area of FCD identified by MRI and the reduction extended further to other ipsilateral regions. This pilot study confirms that in FCD patients there is Pgp overexpression, not only in the epileptogenic area but also the distribution of Pgp overexpression is spread across to other cortical regions.
WP07: Quantitative imaging of Pgp function in Alzheimer's Disease
Objectives
Alzheimer's disease (AD) is the most common form of dementia. A major pathological hallmark of AD is the deposition of amyloid-β plaques in the brain. The origin of amyloid-ß depositions in Alzheimer's disease is unclear. The amyloid hypothesis proposes that this may be caused by an imbalance between amyloid-β production and clearance. The hypothesis we set out to test at the beginning of WP07 was that P-glycoprotein function is reduced in patients with Alzheimer's disease, so that an increase in binding potential (BPND) of (R)-[11C]VPM is expected in patients with Alzheimer's disease compared with healthy controls.
WP08: Neuropathological foundations of Pgp radiotracer binding
Objectives
The main objective was to establish, using resected and post mortem brain tissue, the neuroanatomical parameters that are essential for the interpretation of the in-vivo imaging data, and to determine the role and mechanisms of transporter-encoding gene variation in these parameters and in the neuroimaging patterns observed.
We made use of opportunities provided by surgically-resected tissue from patients with acute insult caused by the placement of intracranial depth electrodes and post mortem brain tissue from patients with (drug-resistant or drug-sensitive) mesial temporal lobe epilepsy and unilateral hippocampal sclerosis (henceforth referred to as 'epilepsy') to examine: (i) inflammation and changes in bloodbrain barrier in acute, subacute and chronic insults in epilepsy; (ii) the expression of multidrug transporters in epileptogenic and non-epileptogenic brain regions of patients with drug-resistant or drug-sensitive epilepsy to formally test the inherent assumptions of the transporter hypothesis and (iii) the neuropathological consequences of the clinical use of invasive intracranial electrode recording, which have not been studied in detail using markers of vascular integrity, inflammation and multidrug transporters. We hypothesized that there would be localized bloodbrain barrier vascular and inflammatory changes in epilepsy with acute and chronic insults and that there would be a higher expression of multidrug transporters in the epileptogenic, sclerotic hippocampus than in other non-epileptogenic brain regions of the drug-resistant epilepsy brains, but not in the drug-sensitive epilepsy and control brain, in support of a role for multidrug transporters in drug resistance in epilepsy. Our results have recently been published (Liu JY, Thom M, Catarino CB, Martinian L, Figarella-Branger D, Bartolomei F, Koepp M, Sisodiya SM. Neuropathology of the blood-brain barrier and pharmaco-resistance in human epilepsy. Brain 2012 Jul 14. [Epub ahead of print] PMID: 22750659).
We showed that:
(i) there is a highly localized overexpression of P-glycoprotein in the epileptogenic hippocampus of patients with drug-resistant epilepsy, which is compatible with the current hypothesis of multidrug resistance;
(ii) this overexpression appears specific to P-glycoprotein and does not affect other transporters, such as BCRP and MRP1;
(iii) the 'double cuff' expression of P-glycoprotein is likely to be associated with ongoing, chronic seizures, being absent in sustained remission;
(iv) a single, acute insult from intracranial electrode recording causes localized inflammation, increased bloodbrain barrier permeability and structural changes to blood vessels (such as the irregular expression of claudins and caveolins), but not P-glycoprotein overexpression;
(v) exposure to P-glycoprotein inducers is associated with increased P-glycoprotein expression in human brain, though the number of cases studied is small; and
(vi) chronic epilepsy is associated with inflammation, enhanced bloodbrain barrier permeability and increased P-glycoprotein expression, particularly in perivascular glia of the epileptogenic hippocampus.
Potential Impact:
1.4 The potential impact
Socio-economic impact and the wider societal implications of the project
Contribution to Community and social objectives
Epilepsy is the most common serious chronic neurological condition, associated not only with major comorbidities including mood disorders and cognitive dysfunction, but also with a reduced life expectancy and a mortality rate from seizure-related death as high as 1 per 100 patients p.a. among patients with uncontrolled seizures. In Europe, epilepsy affects 6 million people of all ages, and the costs attributable to epilepsy exceed approximately 20 billion EUROS p.a. The lifetime risk for epilepsy is 2%, but as high as 10% for a single seizure. The majority of these costs are indirect, in relation with the socio-economic consequences of uncontrolled seizures and side-effects. Healthcare costs are likely to fall, and EU will benefit from avoidance of wastage on AEDs likely to be ineffective for individual patients. Economic development and scientific competitiveness will benefit from the potential for commercialisation of our findings. The societal context of research was established through regular connections with patient and support organisations, exemplified by the Epilepsy Society UK co-funding to a large extent the epilepsy research activity of the co-ordinator at UCL.
Main dissemination activities and exploitation of results
The main conclusion from WP01 is that the Pgp inhibitor based PET tracers [11C]laniquidar, [11C]elacridar and [11C]TQD are unexpectedly in tracer concentrations recognised by Pgp and/or Bcrp as substrates, which results in low brain uptake and renders them unsuitable to visualise Pgp expression levels at the BBB. For TQD and elacridar, in-vivo PET results were confirmed in a series of in-vitro experiments using the respective 3H-labelled molecules. For laniquidar, in-vitro data suggest, in contrast to the in-vivo PET data, absence of Pgp/Bcrp transport. Despite these apparent limitations of [11C]laniquidar, [11C]elacridar and [11C]TQD to visualise cerebral Pgp with PET, the experiments conducted in this work package and WP02 have provided important insight into the pharmacology of these widely used reference Pgp inhibitors. Due to the fact that [11C]laniquidar, [11C]elacridar and [11C]TQD have not provided sufficiently high PET signals in rodents for the imaging of transporter distribution, the preparation of longer-lived versions of these molecules for PET and SPET imaging was not further pursued. Nevertheless [11C]laniquidar, [11C]elacridar and [11C]TQD have all entered WP05 to characterise their behaviour in healthy human subjects. In the future, we plan to assess if any of these tracers might be suitable to visualise Pgp expression levels in peripheral organs. Apart from a thorough in-vivo/in-vitro characterisation of laniquidar, elacridar and TQD, we have obtained first promising results with a 18F-labelled fexofenadine ester prodrug, which might allow for visualising cerebral P gp activity and overcome the limitation of low brain uptake obtained with (R) [11C]VPM, [11C]laniquidar, [11C]elacridar and [11C]TQD.
The results from the clinical studies performed as part of EURIPIDES have provided answers to the questions initially raised on pharmaco-resistance in epilepsy and demonstrated the utility of PET with a radiolabelled Pgp substrate as to study Pgp activity in vivo in humans:
1) Compared to pharmaco-sensitive patients and healthy controls at baseline, pharmaco-resistant patients have significantly reduced VPM uptake exclusively in the temporal lobes but the reduction is not restricted to ipsilateral epileptogenic regions but also extends to contralateral temporal lobe regions. Similarly in patients with FCD, VPM uptake is reduced not only in close proximity to the area of FCD identified by MRI but also the reduction extends further to other ipsilateral regions, supplementing the findings in TLE patients that Pgp overexpression is not limited to the epileptogenic seizure-onset zone but also spreads across to other cortical regions.
2) After partial blockade of Pgp with TQD, pharmaco-resistant TLE patients have attenuated increases of VPM uptake in the whole brain and more so in the ipsilateral epileptogenic area of the hippocampus compared to healthy controls, implicating functionally elevated Pgp function primarily in the epileptogenic hippocampus in pharmaco-resistant epilepsy.
3) Pharmaco-resistant TLE patients with good surgery outcome have higher VPM increases in the post-surgery PET scan than patients with poor outcome, pointing to diminished Pgp activity which may be caused by reduced seizure frequency and lower antiepileptic drug load in patients with good outcome. Furthermore, pharmaco-resistant TLE patients who have frequent seizures have the lowest VPM uptake, substantiating the contribution of seizures to the induction of Pgp overexpression in epilepsy.
4) In pharmaco-resistant TLE patients who underwent anterior temporal lobe resection for surgical treatment, the difference in percentage change in VPM uptake after Pgp inhibition with TQD in the hippocampus compared to a reference temporal lobe region is inversely correlated with the corresponding difference in percentage area of Pgp-immunopositive labelling, demonstrating that imaging with PET, a radiolabelled Pgp substrate and a Pgp inhibitor at a half saturating dose is a suitable tool to assess Pgp function in vivo in humans.
5) In high grade glioma patients, Pgp inhibition minimally alters perfusion but differentially increases VPM uptake into brain tissue and glioma, and preferentially into grade III but not grade IV regions, supporting the use of PET with a radiolabelled Pgp substrate as a promising tool to select patients in whom Pgp inhibitors could potentially increase chemotherapeutic efficacy.
The major achievements of this WP, in concert with the Consortium overall, are:
1. Demonstration that robust studies of human brain tissue, both post mortem and post surgical, can be of enormous value in taking understanding forwards; identification of a new paradigm for the study of the response of human brain tissue to injury (using intracranial electrode tracks as a 'model' for injury).
2. Development of robust reproducible methodology for quantitative analyses in drug resistance-related studies of fixed human brain tissue. These methods can continue to be used in these and other studies
3. Development of robust reproducible methodology for quantitative analyses in drug resistance-related studies of fixed human brain tissue. These methods can continue to be used in these and other studies
4. Determination that some antigens cannot be studied in material that has been fixed for longer periods of time. This important quantitative observation sets new standards in quantifying and interpreting immunohistochemical data.
5. Determination of the spatial and temporal patterns of expression of a variety of proteins putatively involved in drug resistance, or maintenance of blood-brain barrier integrity in the human brain. Several proteins were studied.
6. Substantiation of major assumptions implicit in the drug transporter hypothesis of drug resistance: that overexpression of P-glycoprotein is spatially localised, focused on epileptogenic tissue, and correlates with active seizures, declining with seizure freedom and being absent from brain that is simply injured but not epileptogenic (i.e. intracranial electrode tracks)
7. Correlation of in vivo activity of P-glycoprotein with ex vivo demonstration of degree of expression of P-glycoprotein. This is the first such study and elegantly demonstrates that P-glycoprotein can, at structural and functional levels, indeed play a role in drug resistance in epilepsy.
8. Demonstration that P-glycoprotein, and not other transporters studied such as BCRP and MRP1, has the appropriate pattern of expression to potentially mediate resistance.
Outlook and future research
There is now considerable evidence in support of the 'transporter hypothesis':
(1) relevant multidrug transporters are overexpressed in animal models of drug-resistant epilepsy. Whilst there has been partial evidence in favour of overexpression in human drug-resistant epilepsy, we have recently finally confirmed appropriate regional patterns of overexpression across the human brain in drug-resistant epilepsy compared to drug-responsive epilepsy using a unique post mortem human brain resource
(2) there is evidence that relevant multidrug transporters are capable of transporting many AEDs, in both animal models and ex vivo models using human brain cells;
(3) in animal models of epilepsy and drug-resistant epilepsy, temporary inhibition of the multidrug transporter, P-glycoprotein, most strongly implicated in mediating drug resistance through the transporter mechanism, leads to a transient, but definite, reduction in seizure frequency. This is the current position of the field.
List of Websites:
http://www.euripides-europe.com
