P-glycoprotein at the blood-brain barrier: kinetic modeling of 11C-desmethylloperamide in mice using a 18F-FDG μPET scan to determine the input function

Purpose: The objective of this study is the implementation of a kinetic model for 11C-desmethylloperamide (11CdLop) and the determination of a typical parameter for P-glycoprotein (P-gp) functionality in mice. Since arterial blood sampling in mice is difficult, an alternative method to obtain the arterial plasma input curve used in the kinetic model is proposed. Methods: Wild-type (WT) mice (pre-injected with saline or cyclosporine) and P-gp knock-out (KO) mice were injected with 20 MBq of 11C-dLop, and a dynamic μPET scan was initiated. Afterwards, 18.5 MBq of 18F-FDG was injected, and a static μPET scan was started. An arterial input and brain tissue curve was obtained by delineation of an ROI on the left heart ventricle and the brain, respectively based on the 18F-FDG scan. Results: A comparison between the arterial input curves obtained by the alternative and the blood sampling method showed an acceptable agreement. The one-tissue compartment model gives the best results for the brain. In WT mice, the K1/k2 ratio was 0.4 ± 0.1, while in KO mice and cyclosporine-pretreated mice the ratio was much higher (2.0 ± 0.4 and 1.9 ± 0.2, respectively). K1 can be considered as a pseudo value K1, representing a combination of passive influx of 11C-desmethylloperamide and a rapid washout by P-glycoprotein, while k2 corresponds to slow passive efflux out of the brain. Conclusions: An easy to implement kinetic modeling for imaging P-glycoprotein function is presented in mice without arterial blood sampling. The ratio of K1/k2 obtained from a one-tissue compartment model can be considered as a good value for P-glycoprotein functionality

Reproducibility of quantitative (R)-[11C]verapamil studies

Background P-glycoprotein [Pgp] dysfunction may be involved in neurodegenerative diseases, such as Alzheimer's disease, and in drug resistant epilepsy. Positron emission tomography using the Pgp substrate tracer (R)-[11C]verapamil enables in vivo quantification of Pgp function at the human blood-brain barrier. Knowledge of test-retest variability is important for assessing changes over time or after treatment with disease-modifying drugs. The purpose of this study was to assess reproducibility of several tracer kinetic models used for analysis of (R)-[11C]verapamil data. Methods Dynamic (R)-[11C]verapamil scans with arterial sampling were performed twice on the same day in 13 healthy controls. Data were reconstructed using both filtered back projection [FBP] and partial volume corrected ordered subset expectation maximization [PVC OSEM]. All data were analysed using single-tissue and two-tissue compartment models. Global and regional test-retest variability was determined for various outcome measures. Results Analysis using the Akaike information criterion showed that a constrained two-tissue compartment model provided the best fits to the data. Global test-retest variability of the volume of distribution was comparable for single-tissue (6%) and constrained two-tissue (9%) compartment models. Using a single-tissue compartment model covering the first 10 min of data yielded acceptable global test-retest variability (9%) for the outcome measure K1. Test-retest variability of binding potential derived from the constrained two-tissue compartment model was less robust, but still acceptable (22%). Test-retest variability was comparable for PVC OSEM and FBP reconstructed data. Conclusion The model of choice for analysing (R)-[11C]verapamil data is a constrained two-tissue compartment model

Diurnal variation in P-glycoprotein-mediated transport and cerebrospinal fluid turnover in the brain.

Nearly all bodily processes exhibit circadian rhythmicity. As a consequence, the pharmacokinetic and pharmacodynamic properties of a drug may also vary with time of day. The objective of this study was to investigate diurnal variation in processes that regulate drug concentrations in the brain, focusing on P-glycoprotein (P-gp). This efflux transporter limits the distribution of many drugs in the brain. To this end, the exposure to the P-gp substrate quinidine was determined in the plasma and brain tissue after intravenous administration in rats at six different time points over the 24-h period. Our results indicate that time of administration significantly affects the exposure to quinidine in the brain. Upon inhibition of P-gp, exposure to quinidine in brain tissue is constant over the 24-h period. To gain more insight into processes regulating brain concentrations, we used intracerebral microdialysis to determine the concentration of quinidine in brain extracellular fluid (ECF) and cerebrospinal fluid (CSF) after intravenous administration at two different time points. The data were analyzed by physiologically based pharmacokinetic modeling using NONMEM. The model shows that the variation is due to higher activity of P-gp-mediated transport from the deep brain compartment to the plasma compartment during the active period. Furthermore, the analysis reveals that CSF flux is higher in the resting period compared to the active period. In conclusion, we show that the exposure to a P-gp substrate in the brain depends on time of administration, thereby providing a new strategy for drug targeting to the brain.Pharmacolog

ABC Transporters and Drug Resistance in Patients with Epilepsy

Resistance to antiepileptic drugs (AED) remains a major problem in clinical epileptology. This pharmacoresistance is independent of the choice of AEDs. Different hypotheses have been proposed to explain the neurobiological basis for pharmacoresistance in epilepsy. The transporter hypothesis is the mostly investigated theory. Hereby, overexpression of multidrug efflux transporters, such as P-glycoprotein (Pgp), at the blood-brain-barrier (BBB) is thought to be involved in pharmacoresistance in epilepsy by extruding AEDs from their target site. Accumulating evidence supports an overexpression of Pgp in pharmacoresistant epilepsy. Molecular Imaging studies provide unique opportunities for the in-vivo study of the transporter hypothesis in the central nervous system (CNS). Several studies demonstrated that positron emission tomography (PET) with [11C]-radiolabled Pgp substrates is a promising tool for in vivo investigation of Pgp function at the rat, monkey and human BBB. Quantification of Pgp over activity in epilepsy patients by in vivo imaging could be highly useful because altered treatment strategies or novel AED could then be applied

The role of ABC transporters and inflammation in drug-resistant epilepsy

This thesis explores two pathologies thought to be related to drug resistance in epilepsy that may themselves be causally related: 1) overexpression of drug transporters in capillaries and 2) inflammation. With regard to the first hypothesis, overexpression of the ATP-binding cassette (ABC) transporters, P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP), at the blood-brain barrier are thought to contribute to drug resistance in epilepsy. To measure ABC transporter activity in vivo, positron emission tomography (PET) imaging can be used, which requires a radiolabeled substrate and non-radiolabeled inhibitor. The second hypothesis suggests that inflammation increases P-gp expression and, conversely, that inhibiting inflammation decreases P-gp expression. Questions remain however, as to the interactions between ABC transporters and specific inflammatory proteins, as well as the cellular expression of the same inflammatory proteins in the human brain. In Paper I, we characterized the BCRP inhibitor Ko143 to determine if it was a candidate for use in PET imaging. P-gp activity has been measured using PET imaging with tracers such as [11C]Ndesmethyl-loperamide (a P-gp substrate) along with a P-gp inhibitor such as tariquidar, but a similar imaging paradigm has not yet been developed for BCRP. Therefore, we performed multiple in vitro assays to characterize Ko143 and to measure its interaction with P-gp, BCRP, and the multidrug resistance transporter 1 (MRP1). Data from the in vitro assays indicated that while Ko143 was a potent BCRP inhibitor (IC50 = 9.7 nM), at higher concentrations it was a substrate for P-gp (IC50 = 2.7 µM) and MRP1. In Paper II, because of reports questioning whether tariquidar is a P-gp inhibitor, we investigated tariquidar to determine the mechanism by which it interacts with P-gp. Using similar methods as outlined in Paper I, we found that tariquidar was a potent P-gp inhibitor at low concentrations (IC50 = 100 nM), but at higher micromolar concentrations it was a substrate and competitive inhibitor of BCRP. In Paper III, we sought to determine whether a relationship exists between ABC transporter expression and expression of the inflammatory enzymes cyclooxygenase (COX)-1 and -2, as well as the inflammation biomarker, translocator protein 18 kDa (TSPO). We used multiplex immunofluorescence to measure the expression of P-gp and BCRP as well as COX-1, COX-2, and TSPO in brain tissue samples from people with drug-resistant epilepsy. These tissue samples were classified as either having mesial temporal sclerosis (MTS) or not (non-MTS), in which the non-MTS samples acted as control tissue for MTS samples. When investigating the relationship between ABC transporters and the inflammatory proteins, the only correlation we observed was between BCRP and TSPO, in which increased BCRP density correlated linearly with increased TSPO density (P = 0.0003, r = 0.72131). No significant differences were found in the expression of any protein measured between MTS and nonMTS tissue samples. In Paper IV, we investigated the cellular expression of three inflammatory proteins COX-1, COX-2, and TSPO in brain tissue samples from people with drug-resistant epilepsy. To do so, we used multiplex immunofluorescence microscopy to measure the expression of these proteins in microglia, astrocytes, and neurons. We found that that COX-1 was predominately expressed in microglia, while COX-2 and TSPO were expressed in microglia and neurons. In summary, this thesis explored the mechanisms underlying drug resistance in epilepsy. We studied overexpression of ABC transporters and inflammation, two pathologies hypothesized to be involved in drug-resistant epilepsy that may themselves possibly be related. While Ko143 is specific for BCRP at nanomolar concentrations (similar to tariquidar for P-gp), its potential utility as a radiolabeled inhibitor was diminished by the fact that PET requires picomolar affinity—rather than the 9.7 nM we measured— to measure the low density of BCRP in the brain. With regard to inflammation, we found that COX-1 is primarily expressed in microglia, a trait that makes it, rather than COX-2, a better radioligand for studying neuroinflammation in patients with drug-resistant epilepsy, given that microglia produce the majority of pro-inflammatory cytokines in the brain

Imaging techniques to study drug transporter function in vivo

Transporter systems involved in the permeation of drugs and solutes across biological membranes are recognized as key determinants of pharmacokinetics. Typically, the action of membrane transporters on drug exposure to tissues in living organisms is inferred from invasive procedures, which cannot be applied in humans. In recent years, imaging methods have greatly progressed in terms of instruments, synthesis of novel imaging probes as well as tools for data analysis. Imaging allows pharmacokinetic parameters in different tissues and organs to be obtained in a non-invasive or minimally invasive way. The aim of this overview is to summarize the current status in the field of molecular imaging of drug transporters. The overview is focused on human studies, both for the characterization of transport systems for imaging agents as well as for the determination of drug pharmacokinetics, and makes reference to animal studies where necessary. We conclude that despite certain methodological limitations, imaging has a great potential to study transporters at work in humans and that imaging will become an important tool, not only in drug development but also in medicine. Imaging allows the mechanistic aspects of transport proteins to be studied, as well as elucidating the influence of genetic background, pathophysiological states and drug-drug interactions on the function of transporters involved in the disposition of drugs

PET imaging of P-glycoprotein at the blood-brain barrier with new 18F-tracers

Brain is protected from the molecules circulating in the bloodstream by the blood-brain barrier (BBB). Transporter proteins at the BBB, especially P-glycoprotein (P-gp), have been found to be important in regulating brain entry of several compounds by removing them out of the brain. Decreased or increased P-gp function is linked to several neurological diseases, such as epilepsy and Alzheimer’s disease. We developed novel radiotracers for measuring P-gp function by imaging using positron emission tomography (PET). PET scanner locates and measures radioactive tracers that have been injected into the body. We used fluorine-18 to label our P-gp imaging tracers. Fluorine-18 has a half-life of 110 min, optimal radionuclide properties for imaging, and permits transport to other imaging centers. These new radiotracers were evaluated in cells assays and in several animal models. A compound called [18F]MC225 was identified as a weak P-gp substrate. In the normal situation, it has a low uptake in the brain since it is transported out by P-gp but when P-gp function is inhibited, the brain uptake increases. In addition, [18F]MC225 had good selectivity to P-gp and moderate metabolic stability. Therefore, [18F]MC225 is a suitable tracer for measuring P-gp function with PET

Novel Treatment Strategies for Brain metastases of Breast Cancer

About 20-40% of advanced breast cancer patients will develop symptomatic brain metastases. Once the patients diagnosed with metastatic brain tumors, there is 80% mortality rate within one year. The presence of blood-brain barrier makes it difficult for drugs to reach the site of action in brain-related ailments. To overcome we came up with two strategies: First, we encapsulated the chemotherapy in a liposome and thereby significantly improving the plasma pharmacokinetics of chemotherapy. We also observed that tumor drug exposure significantly improved by liposomal formulation. This improvement in plasma drug pharmacokinetics and tumor drug accumulation after administration of the liposomal formulation decreased the tumor burden and significantly increased the median survival by 40% when compared to vehicle group in an experimental model of brain metastases. In another strategy, we want to modulate blood-brain barrier in brain metastases to increase permeation. Notch-4 signaling pathway plays an important role in angiogenesis and inhibition of Notch-4 by DAPT will increase the expression of vascular endothelial growth factor receptor-2 ultimately leading to leaky vasculature in metastatic brain tumor. In our studies, we found that inhibition of Notch-4 by DAPT increased the permeation 14C- Aminoisobutyric acid specifically in the brain metastases. We also observed that the progression of tumor burden was decreased when animals were administered both Notch-4 inhibitor and chemotherapy. We also found that median survival is increased by 20% in animals treated with chemotherapy with concurrent Notch-4 inhibition by DAPT. Finally, we evaluated the effect of chemotherapy on normal brain region adjacent to brain metastases. We found that the permeation of fluorescent tracers and 14C-paclitaxel increased in brain adjacent to the tumor. We also found that the expression of activated astrocytes increased in brain adjacent to tumors after chronic chemotherapy treatment in our brain metastases model. Together these results suggest that novel strategies improved survival in brain metastases of breast cancer. Future studies should aim at combining these individual strategies to further increase survival in a preclinical model. At the same time care should be taken not increase chemotherapy permeation into the normal brain as it may lead to unwanted effects like chemo-fog

Monitoring the Function of the P-glycoprotein Transporter at the Blood Brain Barrier

The P-glycoprotein (P-gp) transporter located at the blood-brain barrier (BBB) is an efflux transporter that pumps neurotoxic compounds out of the brain. Its main function is to protect the brain to ensure an appropriate neural function. Decreases in the P-gp function can result in increased accumulation of toxic compounds inside the brain such as beta-amyloid and this may cause the development of Alzheimer´s or other neurological disorders. By contrast, increases in the P-gp function can decrease the therapeutic drug concentration inside the brain and influence the efficacy of the treatment (drug resistance) as occurred in patients with intractable epilepsy. Thus, it is of interest to monitor the P-gp function in vivo to facilitate the early diagnosis of brain disorders and to monitor drug resistance. To this aim, we used Positron Emission Tomography (PET) imaging, a non-invasive technique that allows the quantification of biological processes in vivo, and the novel radiotracer [18F]MC225 which measures the P-gp function. The aim was to study the kinetic properties of the radiotracer in different species and prove its efficacy to measure increases and decrease in the P-gp function before its clinical evaluation. We conclude that the obtained results have broadened the knowledge of the P-gp function at the BBB. Moreover, the results highlight that [18F]MC225 may become the first radiofluorinated P-gp PET tracer able to measure both decreases and increases in the P-gp function in vivo. The radiolabeling with fluorine-18 would allow its distribution to other PET centers and improve the image quality