Synthesis of two potential NK1-receptor ligands using [1-11C]ethyl iodide and [1-11C]propyl iodide and initial PET-imaging

BACKGROUND: The previously validated NK(1)-receptor ligand [O-methyl-(11)C]GR205171 binds with a high affinity to the NK(1)-receptor and displays a slow dissociation from the receptor. Hence, it cannot be used in vivo for detecting concentration changes in substance P, the endogenous ligand for the NK(1)-receptor. A radioligand used for monitoring these changes has to enable displacement by the endogenous ligand and thus bind reversibly to the receptor. Small changes in the structure of a receptor ligand can lead to changes in binding characteristics and also in the ability to penetrate the blood-brain barrier. The aim of this study was to use carbon-11 labelled ethyl and propyl iodide with high specific radioactivity in the synthesis of two new and potentially reversible NK(1)-receptor ligands with chemical structures based on [O-methyl-(11)C]GR205171. METHODS: [1-(11)C]Ethyl and [1-(11)C]propyl iodide with specific radioactivities of 90 GBq/μmol and 270 GBq/μmol, respectively, were used in the synthesis of [O-methyl-(11)C]GR205171 analogues by alkylation of O-desmethyl GR205171. The brain uptake of the obtained (2S,3S)-N-(1-(2- [1-(11)C]ethoxy-5-(3-(trifluoromethyl)-4H-1,2,4-triazol-4-yl)phenyl)ethyl)-2-phenylpiperidin-3-amine (I) and (2S,3S)-2-phenyl-N-(1-(2- [1-(11)C]propoxy-5-(3-(trifluoromethyl)-4H-1,2,4-triazol-4-yl)phenyl)ethyl)piperidin-3-amine (II) was studied with PET in guinea pigs and rhesus monkeys and compared to the uptake of [O-methyl-(11)C]GR205171. RESULTS: All ligands had similar uptake distribution in the guinea pig brain. The PET-studies in rhesus monkeys showed that (II) had no specific binding in striatum. Ligand (I) had moderate specific binding compared to the [O-methyl-(11)C]GR205171. The ethyl analogue (I) displayed reversible binding characteristics contrary to the slow dissociation rate shown by [O-methyl-(11)C]GR205171. CONCLUSION: The propyl-analogue (II) cannot be used for detecting changes in NK(1)-ligand levels, while further studies should be performed with the ethyl-analogue (I)

Blood-Brain Barrier Penetration of Zolmitriptan—Modelling of Positron Emission Tomography Data

Positron emissiontomography (PET) with the drug radiolabelled allows a direct measurement of brain or other organ kinetics, information which can be essential in drug development. Usually, however, a PET-tracer is administered intravenously (i.v.), whereas the therapeutic drug is mostly given orally or by a different route to the PET-tracer. In such cases, a recalculation is needed to make the PET data representative for the alternative administration route. To investigate the blood-brain barrier penetration of a drug (zolmitriptan) using dynamic PET and by PK modelling quantify the brain concentration of the drug after the nasal administration of a therapeutic dose. [11C]Zolmitriptan at tracer dose was administered as a short i.v. infusion and the brain tissue and venous blood kinetics of [11C]zolmitriptan was measured by PET in 7 healthy volunteers. One PET study was performed before and one 30min after the administration of 5mg zolmitriptan as nasal spray. At each of the instances, the brain radioactivity concentration after subtraction of the vascular component was determined up to 90min after administration and compared to venous plasma radioactivity concentration after correction for radiolabelled metabolites. Convolution methods were used to describe the relationship between arterial and venous tracer concentrations, respectively between brain and arterial tracer concentration. Finally, the impulse response functions derived from the PET studies were applied on plasma PK data to estimate the brain zolmitriptan concentration after a nasal administration of a therapeutic dose. The studies shows that the PET data on brain kinetics could well be described as the convolution of venous tracer kinetics with an impulse response including terms for arterial-to-venous plasma and arterial-to-brain impulse responses. Application of the PET derived impulse responses on the plasma PK from nasal administration demonstrated that brain PK of zolmitriptan increased with time, achieving about 0.5mg/ml at 30min and close to a maximum of 1.5mg/ml after 2hr. A significant brain concentration was observed already after 5min. The data support the notation of a rapid brain availability of zolmitriptan after nasal administratio

[11C]Flumazenil brain uptake is influenced by the blood-brain barrier efflux transporter P-glycoprotein

Background:

(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

Alteration in P-glycoprotein Functionality Affects Intrabrain Distribution of Quinidine More Than Brain Entry—A Study in Rats Subjected to Status Epilepticus by Kainate

This study aimed to investigate the use of quinidine microdialysis to study potential changes in brain P-glycoprotein functionality after induction of status epilepticus (SE) by kainate. Rats were infused with 10 or 20 mg/kg quinidine over 30 min or 4 h. Plasma, brain extracellular fluid (brain ECF), and end-of-experiment total brain concentrations of quinidine were determined during 7 h after the start of the infusion. Effect of pretreatment with tariquidar (15 mg/kg, administered 30 min before the start of the quinidine infusion) on the brain distribution of quinidine was assessed. This approach was repeated in kainate-treated rats. Quinidine kinetics were analyzed with population modeling (NONMEM). The quinidine microdialysis assay clearly revealed differences in brain distribution upon changes in P-glycoprotein functionality by pre-administration of tariquidar, which resulted in a 7.2-fold increase in brain ECF and a 40-fold increase in total brain quinidine concentration. After kainate treatment alone, however, no difference in quinidine transport across the blood–brain barrier was found, but kainate-treated rats tended to have a lower total brain concentration but a higher brain ECF concentration of quinidine than saline-treated rats. This study did not provide evidence for the hypothesis that P-glycoprotein function at the blood–brain barrier is altered at 1 week after SE induction, but rather suggests that P-glycoprotein function might be altered at the brain parenchymal level

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

Blood-Brain Barrier Transport : Investigation of Active Efflux using Positron Emission Tomography and Modelling Studies

This thesis examines the transport of exogenous molecules across the blood-brain barrier (BBB), focusing on active efflux, using positron emission tomography (PET), computer simulation and modelling. P-glycoprotein (P-gp) inhibition was studied using [11C]verapamil and [11C]hydroxyurea was investigated as a new marker for active efflux transport. Simulations were carried out to explore the importance of the efflux transporter location in the BBB. Brain concentrations of [11C]verapamil, [11C]GR205171 and [18F]altanserin were compared in various laboratory animal species and in humans. A central aspect of the studies has been the novel combination of dynamic PET imaging of the brain pharmacokinetics of a labelled drug, administered through an exponential infusion scheme allowing time-resolved consequence analysis of P-gp inhibition, and mathematical modelling of the obtained data. The methods are applicable to drugs under development and can be used not only in rodents but also in higher species, potentially even in humans, to investigate the effects of P-gp or other transporters on drug uptake in the brain. The inhibition of P-gp by cyclosporin A (CsA) and the subsequent change in brain concentrations of [11C]verapamil occurred rapidly in the sense that [11C]verapamil uptake increased rapidly after CsA administration but also in the sense that the increased uptake was rapidly reversible. The P-gp inhibition was best described by an inhibitory indirect effect model in which CsA decreased the transport of [11C]verapamil out of the brain. The model indicated that approximately 90% of the transport of [11C]verapamil was P-gp-mediated. The low brain concentrations of [11C]hydroxyurea appeared to be a result of slow transport across the BBB rather than active efflux. This exemplifies why the extent and the rate of brain uptake should be approached as two separate phenomena. The brain-to-plasma concentration ratios for the three studied radiotracers differed about 10-fold be-tween species, with lower concentrations in rodents than in humans, monkeys and pigs. The increase in brain concentrations after P-gp inhibition was somewhat greater in rats than in the other species. The findings demonstrate a need to include the dynamics of efflux inhibition in the experimental design and stress the importance of the choice of species in preclinical studies of new drug candidates

Engineered antibodies: new possibilities for brain PET?

Almost 50 million people worldwide are affected by Alzheimer's disease (AD), the most common neurodegenerative disorder. Development of disease-modifying therapies would benefit from reliable, non-invasive positron emission tomography (PET) biomarkers for early diagnosis, monitoring of disease progression, and assessment of therapeutic effects. Traditionally, PET ligands have been based on small molecules that, with the right properties, can penetrate the blood-brain barrier (BBB) and visualize targets in the brain. Recently a new class of PET ligands based on antibodies have emerged, mainly in applications related to cancer. While antibodies have advantages such as high specificity and affinity, their passage across the BBB is limited. Thus, to be used as brain PET ligands, antibodies need to be modified for active transport into the brain. Here, we review the development of radioligands based on antibodies for visualization of intrabrain targets. We focus on antibodies modified into a bispecific format, with the capacity to undergo transferrin receptor 1 (TfR1)-mediated transcytosis to enter the brain and access pathological proteins, e.g. amyloid-beta. A number of such antibody ligands have been developed, displaying differences in brain uptake, pharmacokinetics, and ability to bind and visualize the target in the brain of transgenic mice. Potential pathological changes related to neurodegeneration, e.g. misfolded proteins and neuroinflammation, are suggested as future targets for this novel type of radioligand. Challenges are also discussed, such as the temporal match of radionuclide half-life with the ligand's pharmacokinetic profile and translation to human use. In conclusion, brain PET imaging using bispecific antibodies, modified for receptor-mediated transcytosis across the BBB, is a promising method for specifically visualizing molecules in the brain that are difficult to target with traditional small molecule ligands

Cationization increases brain distribution of an amyloid-beta protofibril selective F(ab')2 fragment

Antibodies and fragments thereof are, because of high selectivity for their targets, considered as potential therapeutics and biomarkers for several neurological disorders. However, due to their large molecular size, antibodies/fragments do not easily penetrate into the brain. The aim of the present study was to improve the brain distribution via adsorptive-mediated transcytosis of an amyloid-beta (A beta) protofibril selective F(ab')2 fragment (F(ab')2-h158). F(ab')2-h158 was cationized to different extents and the specific and unspecific binding was studied in vitro. Next, cationized F(ab')2-h158 was labelled with iodine-125 and its brain distribution and pharmacokinetics was studied in mice. Cationization did not alter the in vitro affinity to A beta protofibrils, but increased the unspecific binding somewhat. Ex vivo experiments revealed a doubling of brain concentrations compared with unmodified F(ab')2-h158 and in vivo imaging with single photon emission computed tomography (SPECT) showed that the cationized F(ab')2-h158, but not the unmodified F(ab')2-h158 could be visualized in the brain. To conclude, cationization is a means to increase brain concentrations of therapeutic antibodies or fragments and may facilitate the use of antibodies/fragments as imaging biomarkers in the brain