CO carbonylation and first evaluation as a P-gp tracer in rats

BACKGROUND: At present, several positron emission tomography (PET) tracers are in use for imaging P-glycoprotein (P-gp) function in man. At baseline, substrate tracers such as R-[(11)C]verapamil display low brain concentrations with a distribution volume of around 1. [(11)C]phenytoin is supposed to be a weaker P-gp substrate, which may lead to higher brain concentrations at baseline. This could facilitate assessment of P-gp function when P-gp is upregulated. The purpose of this study was to synthesize [(11)C]phenytoin and to characterize its properties as a P-gp tracer. METHODS: [(11)C]CO was used to synthesize [(11)C]phenytoin by rhodium-mediated carbonylation. Metabolism and, using PET, brain pharmacokinetics of [(11)C]phenytoin were studied in rats. Effects of P-gp function on [(11)C]phenytoin uptake were assessed using predosing with tariquidar. RESULTS: [(11)C]phenytoin was synthesized via [(11)C]CO in an overall decay-corrected yield of 22 ± 4%. At 45 min after administration, 19% and 83% of radioactivity represented intact [(11)C]phenytoin in the plasma and brain, respectively. Compared with baseline, tariquidar predosing resulted in a 45% increase in the cerebral distribution volume of [(11)C]phenytoin. CONCLUSIONS: Using [(11)C]CO, the radiosynthesis of [(11)C]phenytoin could be improved. [(11)C]phenytoin appeared to be a rather weak P-gp substrate

(R)-[11C]Verapamil PET studies to assess changes in P-glycoprotein expression and functionality in rat blood-brain barrier after exposure to kainate-induced status epilepticus

<p>Abstract</p> <p>Background</p> <p>Increased functionality of efflux transporters at the blood-brain barrier may contribute to decreased drug concentrations at the target site in CNS diseases like epilepsy. In the rat, pharmacoresistant epilepsy can be mimicked by inducing status epilepticus by intraperitoneal injection of kainate, which leads to development of spontaneous seizures after 3 weeks to 3 months. The aim of this study was to investigate potential changes in P-glycoprotein (P-gp) expression and functionality at an early stage after induction of status epilepticus by kainate.</p> <p>Methods</p> <p><it>(R)</it>-[¹¹C]verapamil, which is currently the most frequently used positron emission tomography (PET) ligand for determining P-gp functionality at the blood-brain barrier, was used in kainate and saline (control) treated rats, at 7 days after treatment. To investigate the effect of P-gp on <it>(R)</it>-[¹¹C]verapamil brain distribution, both groups were studied without or with co-administration of the P-gp inhibitor tariquidar. P-gp expression was determined using immunohistochemistry in post mortem brains. <it>(R)</it>-[¹¹C]verapamil kinetics were analyzed with approaches common in PET research (Logan analysis, and compartmental modelling of individual profiles) as well as by population mixed effects modelling (NONMEM).</p> <p>Results</p> <p>All data analysis approaches indicated only modest differences in brain distribution of <it>(R)</it>-[¹¹C]verapamil between saline and kainate treated rats, while tariquidar treatment in both groups resulted in a more than 10-fold increase. NONMEM provided most precise parameter estimates. P-gp expression was found to be similar for kainate and saline treated rats.</p> <p>Conclusions</p> <p>P-gp expression and functionality does not seem to change at early stage after induction of anticipated pharmacoresistant epilepsy by kainate.</p

On The Rate and Extent of Drug Delivery to the Brain

To define and differentiate relevant aspects of blood–brain barrier transport and distribution in order to aid research methodology in brain drug delivery. Pharmacokinetic parameters relative to the rate and extent of brain drug delivery are described and illustrated with relevant data, with special emphasis on the unbound, pharmacologically active drug molecule. Drug delivery to the brain can be comprehensively described using three parameters: Kp,uu (concentration ratio of unbound drug in brain to blood), CLin (permeability clearance into the brain), and Vu,brain (intra-brain distribution). The permeability of the blood–brain barrier is less relevant to drug action within the CNS than the extent of drug delivery, as most drugs are administered on a continuous (repeated) basis. Kp,uu can differ between CNS-active drugs by a factor of up to 150-fold. This range is much smaller than that for log BB ratios (Kp), which can differ by up to at least 2,000-fold, or for BBB permeabilities, which span an even larger range (up to at least 20,000-fold difference). Methods that measure the three parameters Kp,uu, CLin, and Vu,brain can give clinically valuable estimates of brain drug delivery in early drug discovery programmes

Synthesis and in vivo evaluation of [123I]-3-I-CO: a potential SPECT tracer for the serotonin 5-HT2A receptor

The aim of this doctoral dissertation was the precursor synthesis, radiosynthesis and in vivo evaluation of [123I]-3-I-CO as a possible new tracer for imaging of the central serotonin 5-HT2A receptor with SPECT. [123I]-(4-fluorophenyl)[1-(3-iodophenethyl)piperidin-4-yl] methanone ([123I]-3-I-CO) demonstrates good affinity for the 5-HT2A receptor (Ki = 0.51 nM) and good selectivity ratios over other receptor types and was therefore selected as the ligand. First, the in vivo behaviour of a currently used 5-HT2A SPECT tracer, [123I]-R91150, was evaluated (chapter 3), and brain uptake of the tracer was assessed in rodents, as a standard for comparison. The influence of P-glycoprotein blocking (with cyclosporin A) on the biodistribution and brain uptake of [123I]-R91150 was also evaluated in rodents, and these results were compared with the data obtained from the normal biodistribution studies with [123I]-R91150. Also, the influence of P-glycoprotein blocking on pinhole μSPECT imaging with [123I]-R91150 in rodents was investigated. In NMRI mice, a dose-dependent influence of cyclosporin A on the brain uptake of [123I]-R91150 was observed, indicating that the increased brain uptake is the result of a decreased efflux of tracer out of the brain after blocking of the P-glycoprotein efflux transporter with cyclosporin A. Pre-treatment of Sprague-Dawley rats with cyclosporin A resulted in a drastically increased brain uptake of [123I]-R91150 (brain uptake increased seven-fold after Pglycoprotein blocking) and a vastly improved pinhole μSPECT imaging quality. From these results it can be concluded that [123I]-R91150 is a substrate for P-glycoprotein efflux in vivo, and that it’s brain efflux can be blocked by administration of cyclosporin A. Organic synthesis of the tributylstannyl precursor for the radiosynthesis of [123I]-3-I-CO was performed in adequate yield. An average yield of about 85 % was obtained in the radiosynthesis reaction. The radioligand was purified with semi-preparative HPLC, and radiochemical purities of > 95 % were obtained consistently. The radioligand was stable at room temperature until 48 h after synthesis. A logP value of 3.10 ± 0.10 was obtained for [123I]-3-I-CO (chapter 4). In vivo evaluation of [123I]-3-I-CO in NMRI mice revealed high initial brain uptake (6.26 ± 1.36 % ID/g tissue at 10 min post injection), but radioactivity concentrations in brain decreased rapidly over time. No radiolabelled metabolites were observed in blood or brain of NMRI mice. Brain uptake of [123I]-3-I-CO was also investigated in Sprague-Dawley rats (chapter 5): highest brain radioactivity concentrations were obtained in the occipital (0.942 ± 0.034 % ID/g tissue at 20 min post injection) and frontal cortex (0.674 ± 0.074 % ID/g tissue at 20 min post injection). Blood radioactivity concentrations were consistently low (a maximum value of 0.062 ± 0.014 % ID/g tissue was obtained at 20 min post injection). An average frontal cortex-to-cerebellum ratio of about 1.7 was obtained. In the Sprague-Dawley rat biodistribution studies, a rapid washout of radioactivity from the brain was observed. [123I]-3-I-CO was displaced from the 5-HT2A receptor by ketanserin: radioactivity concentration in the 5-HT2A rich areas of the brain decreased by 50 % after ketanserin displacement. Nevertheless, the residual radioactivity levels in cerebellum after ketanserin displacement remained high, especially compared to the results obtained with [123I]-R91150, and are probably caused by aspecific binding of the radioligand to brain tissues. No radiolabelled metabolites could be detected in the blood or brain of Sprague-Dawley rats. The influence of P-glycoprotein modulation with cyclosporin A on the brain uptake of [123I]-3-I-CO was also investigated. On average, a 67 % increase in [123I]-3-I-CO radioactivity concentration was observed throughout the brain after treatment of the animals with cyclosporin A. We can conclude from these results that [123I]-3-I-CO is at least a partial substrate for P-glycoprotein efflux, but the increase in brain radioactivity concentration after cyclosporin A treatment was not as large as the increase observed with [123I]-R91150 (chapter 3). Although cortical tissues could be visualized using pinhole μSPECT imaging with [123I]-3-ICO, aspecific binding of the radioligand was observed in the cerebellum, probably limiting its application as a serotonin 5-HT2A receptor tracer in humans. μSPECT imaging quality also did not improve after cyclosporin A pre-treatment of the animals. Although the initial rodent studies demonstrated promising brain uptake of the radioligand, it can be concluded that [123I]-3-I-CO probably has very limited potential as a 5-HT2A tracer for SPECT, due to high aspecific binding and rapid washout of the radioligand out of the brain. Also, compared to other clinically used brain tracers (for example [123I]-R91150), the specific ‘signal’ of [123I]-3-I-CO in brain is too limited for application as a tracer in brain receptor imaging studies

Imaging p-glycoprotein function: prediction of treatment response in mesial temporal lobe epilepsy

Background: Overexpression of multidrug efflux transporters at the blood–brain barrier, such as P-glycoprotein (Pgp), might contribute to pharmacoresistance by reducing target-site concentrations of antiepileptic drugs (AEDs). We assessed Pgp activity in vivo in patients with mesial temporal lobe epilepsy (mTLE). / Methods: Fourteen pharmacoresistant mTLE patients with unilateral hippocampal sclerosis (HS), three patients with pharmacoresistant epilepsy due to focal cortical dysplasia (FCD), eight seizure-free mTLE patients and 13 healthy controls underwent baseline PET scans with the Pgp substrate (R)- [¹¹C]verapami (VPM). Pharmacoresistant mTLE patients and healthy controls underwent a second VPM PET scan following infusion of the Pgp-inhibitor tariquidar (TQD). The transfer rate constant from plasma to brain, K1, was estimated using a single-tissue compartment model with a VPM-in-plasma arterial input function. Analysis was performed on the first 10min of dynamic data containing limited radiolabeled metabolites. Regions were defined automatically using a brain atlas (ROI analysis), and ratios of VPM-K1 values were calculated between a reference region (parietal cortex) and target regions. Parametric maps of VPM-K1 were also generated using generalised linear least squares and was used for SPM voxel-based analysis. For the voxel-based analysis at baseline we created VPM PET images corrected for differences in whole brain radiotracer uptake. Furthermore, we compared VPM PET scans with epileptic tissues removed during epilepsy surgery and measured peripheral markers of Pgp function: PBMC ABCB1 mRNA, ABCB1 polymorphism and S100B. / Findings: The ROI analysis revealed differences in VPM metabolism between mTLE patients and healthy controls which is caused by AED-mediated hepatic cytochrome P450 enzyme induction in mTLE patients requiring images to be normalised for global brain differences. When using ROI analysis and normalised VPM ratios there was no difference in VPM-K1 ratios in pharmacoresistant compared to seizure-free mTLE patients or healthy controls. The ROI analysis after partial Pgp-inhibition with TQD showed attenuated global increases of VPM brain uptake in pharmacoresistant mTLE patients compared to healthy controls but there where no regional differences. The voxel-based analysis at baseline revealed that pharmacoresistant mTLE patients had reduced VPM uptake compared to seizure-free mTLE patients and healthy controls in ipsi- and contralateral temporal lobes. Higher Pgp activity was associated with higher seizure frequency. After Pgp-inhibition with TQD pharmacoresistant mTLE patients had reduced increases of VPM brain uptake in the whole brain and ipsilateral hippocampus, implicating Pgp overactivity in the epileptogenic hippocampus. The difference in percentage change in VPM brain uptake after Pgp-inhibition with TQD inversely correlated with the difference in percentage area of Pgp immunopositive labeling in pharmacoresistant mTLE patients who underwent epilepsy surgery. Pharmacoresistant epilepsy patients with FCD had reduced VPM brain uptake in close proximity to the area of FCD but also extending to other ipsilateral regions. There were no differences in peripheral markers of Pgp function between the three groups. Our results support the hypothesis of Pgp overactivity in pharmacoresistant epilepsy

P-glycoprotein inhibition as a strategy to increase drug delivery across the blood-brain barrier: focus on antidepressants

Depression is among the leading causes of disability worldwide. Currently available antidepressant drugs have unsatisfactory efficacy, with up to 60% of depressed patients failing to respond adequately to treatment. Emerging evidence has highlighted a potential role for the efflux transporter P-glycoprotein (P-gp), expressed at the blood-brain barrier (BBB), in the aetiology of treatment-resistant depression. In this thesis, the potential of P-gp inhibition as a strategy to enhance the brain distribution and pharmacodynamic effects of antidepressant drugs was investigated. Pharmacokinetic studies demonstrated that administration of the P-gp inhibitors verapamil or cyclosporin A (CsA) enhanced the BBB transport of the antidepressants imipramine and escitalopram in vivo. Furthermore, both imipramine and escitalopram were identified as transported substrates of human P-gp in vitro. Contrastingly, human P-gp exerted no effect on the transport of four other antidepressants (amitriptyline, duloxetine, fluoxetine and mirtazapine) in vitro. Pharmacodynamic studies revealed that pre-treatment with verapamil augmented the behavioural effects of escitalopram in the tail suspension test (TST) of antidepressant-like activity in mice. Moreover, pre-treatment with CsA exacerbated the behavioural manifestation of an escitalopram-induced mouse model of serotonin syndrome, a serious adverse reaction associated with serotonergic drugs. This finding highlights the potential for unwanted side-effects which may occur due to increasing brain levels of antidepressants by P-gp inhibition, although further studies are needed to fully elucidate the mechanism(s) at play. Taken together, the research outlined in this thesis indicates that P-gp may restrict brain concentrations of escitalopram and imipramine in patients. Moreover, we show that increasing the brain distribution of an antidepressant by P-gp inhibition can result in an augmentation of antidepressant-like activity in vivo. These findings raise the possibility that P-gp inhibition may represent a potentially beneficial strategy to augment antidepressant treatment in clinical practice. Further studies are now warranted to evaluate the safety and efficacy of this approach