Generation and in vivo characterization of a chimeric αvβ5-targeting antibody 14C5 and its derivatives

Background: Previous studies showed that radiolabeled murine monoclonal antibody (mAb) 14C5 and its Fab and F(ab')2 fragments, targeting αvβ5 integrin, have promising properties for diagnostic and therapeutic applications in cancer. To diminish the risk of generating a human anti-mouse antibody response in patients, chimeric variants were created. The purpose of this study was to recombinantly produce chimeric antibody (chAb) derivatives of the murine mAb 14C5 and to evaluate the in vitro and in vivo characteristics. Methods: In vitro stability, specificity, and affinity of radioiodinated chAb and fragments (Iodo-Gen method) were examined on high-expressing αvβ5 A549 lung tumor cells. In vivo biodistribution and pharmacokinetic characteristics were studied in A549 lung tumor-bearing Swiss Nu/Nu mice. Results: Saturation binding experiments revealed high in vitro affinity of radioiodinated chAb, F(ab')2, and Fab, with dissociation constants (KD) of 1.19 ± 0.19, 0.68 ± 0.10, and 2.11 ± 0.58 nM, respectively. ChAb 14C5 showed highest tumor uptake (approximately 10%ID/g) at 24 h post injection, corresponding with other high-affinity Abs. ChF(ab')2 and chFab fragments showed faster clearance from the blood compared to the intact Ab. Conclusions: The chimerization of mAb 14C5 and its fragments has no or negligible effect on the properties of the antibody. In vitro and in vivo properties show that the chAb 14C5 is promising for radioimmunotherapy, due to its high maximum tumor uptake and its long retention in the tumor. The chF(ab')2 fragment shows a similar receptor affinity and a faster blood clearance, causing less non-specific retention than the chAb. Due to their fast blood clearance, the fragments show high potential for radioimmunodiagnosis

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

Automated radiosynthesis of Al[18F]PSMA-11 for large scale routine use.

Objectives: We report a reproducible automated radiosynthesis for large scale batch production of clinical grade Al[F-18]PSMA-11. Methods: A SynthraFCHOL module was optimized to synthesize Al[F-18]PSMA-11 by Al[F-18]-chelation. Results Al[F-18]PSMA-11 was synthesized within 35 min in a yield of 21 +/- 3% (24.0 +/- 6.0 GBq) and a radiochemical purity > 95%. Batches were stable for 4 h and conform the European Pharmacopeia guidelines. Conclusions: The automated synthesis of Al[F-18]PSMA-11 allows for large scale production and distribution of Al [F-18]PSMA-11

Kinetic modeling and graphical analysis of 18F-fluoromethylcholine (FCho), 18F-fluoroethyltyrosine (FET) and 18F-fluorodeoxyglucose (FDG) PET for the discrimination between high-grade glioma and radiation necrosis in rats

Background : Discrimination between glioblastoma (GB) and radiation necrosis (RN) post-irradiation remains challenging but has a large impact on further treatment and prognosis. In this study, the uptake mechanisms of 18F-fluorodeoxyglucose (18F-FDG), 18F-fluoroethyltyrosine (18F-FET) and 18F-fluoromethylcholine (18F-FCho) positron emission tomography (PET) tracers were investigated in a F98 GB and RN rat model applying kinetic modeling (KM) and graphical analysis (GA) to clarify our previous results. Methods : Dynamic 18F-FDG (GB n = 6 and RN n = 5), 18F-FET (GB n = 5 and RN n = 5) and 18F-FCho PET (GB n = 5 and RN n = 5) were acquired with continuous arterial blood sampling. Arterial input function (AIF) corrections, KM and GA were performed. Results : The influx rate (Ki) of 18F-FDG uptake described by a 2-compartmental model (CM) or using Patlak GA, showed more trapping (k(3)) in GB (0.07 min(-1)) compared to RN (0.04 min(-1)) (p = 0.017). K-1 of 18F-FET was significantly higher in GB (0.06 ml/ccm/min) compared to RN (0.02 ml/ccm/min), quantified using a 1-CM and Logan GA (p = 0.036). 18F-FCho was rapidly oxidized complicating data interpretation. Using a 1-CM and Logan GA no clear differences were found to discriminate GB from RN. Conclusions : Based on our results we concluded that using KM and GA both 18F-FDG and 18F-FET were able to discriminate GB from RN. Using a 2-CM model more trapping of 18F-FDG was found in GB compared to RN. Secondly, the influx of 18F-FET was higher in GB compared to RN using a 1-CM model. Important correlations were found between SUV and kinetic or graphical measures for 18F-FDG and 18F-FET. 18F-FCho PET did not allow discrimination between GB and RN

The role of efflux transporters in refractory epilepsy: evaluation with PET tracers

Epilepsy is a neurological disorder characterized by seizures. Epilepsy is usually controlled, but not cured, with antiepileptic medication. However, 30% of epileptic patients do not have seizure control even with the best available antiepileptic drugs. A possible explanation of this form of epilepsy, also called refractory epilepsy, is postulated as the transporter hypothesis. This theory proposes that refractory epilepsy may be the consequence of an overexpression of efflux transport proteins that prevents antiepileptic drugs from penetrating the blood–brain barrier in sufficient concentration. Why patients are resistant to multiple AEDs with distinct mechanisms of action can thereby be explained by this hypothesis. Although several studies already investigated the role of the efflux transporters, P-gp and MRP, the aim of this thesis was visualizing the P-gp and their interaction with antiepileptic drugs with the use of a new PET tracer, 11C-desmethylloperamide. In chapter 1, an overview of the current findings in epilepsy is mentioned, with some greater attention to refractory epilepsy and the recovery strategies. In this chapter, the role of animal models in epilepsy research is also described. The structure of the brain, as well as the transport across the BBB is discussed in chapter 2. Moreover, the structure, localization in tissues, and the known substrates and inhibitors of the efflux transporters are cited, whereby the characteristics of P-gp are highlighted. The role of PET and SPECT in molecular imaging and the practical facts about kinetic modeling is discussed in chapter 3. Chapter 5 gives information about general experimental procedures, while chapter 6 deals with the radiosynthesis and in vivo evaluation of a novel PET tracer, 11C-desmethylloperamide, to visualize P-gp. The tracer was synthesized with a good radiochemical yield and specific activity. Although low brain uptake in wild-type mice was observed, P-gp inhibition with cyclosporine A resulted in significantly increased tracer uptake. Moreover, in P-gp knock-out mice an eight fold higher uptake was observed, suggesting that 11C-desmethylloperamide is an avid tracer to investigated P-gp. Moreover, no metabolisation in the brain occurs, while only one polar metabolite is observed in plasma. Nevertheless, the depletion of P-gp has no effect on the tracer metabolisation pattern. In chapter 7, the first acquirement, which has to be fulfilled in the transporter hypothesis, is investigated. By an indirect method the interaction of phenytoin, levetiracetam, topiramate, and sodium valproate on the P-gp at the BBB are observed. Therapeutic doses of phenytoin, levetiracetam, and topiramate in combination with 11C-desmethylloperamide resulted in brain uptake, which is statistically increased compared to baseline tracer uptake. Higher doses of antiepileptic drugs reduced the brain uptake again to baseline levels. These results confirm the fact that these AED shows interaction with P-gp, while P-gp probably plays no role in the transport of sodium valproate across the BBB, since no different tracer brain uptake is observed with and without antiepileptic drug administrations. Kinetic modeling with 11C-desmethylloperamide to visualize the P-gp mediated transport in the brain is the key topic of chapter 8. First, a kinetic model was accompliced in wild-type, with or without cyclosporine A pretreatment and in P-gp knock-out mice to identify a typical rate constant for P-gp transport. We proposed and evaluated an alternative method to determine the input function, essential for kinetic modeling, by using an 18F-FDG scan to delineate the left heart ventricle. Subsequently, a model, which best fitted the brain data, was determined as well as the rate constants. K1 in wild-type mice is statistical smaller than in P-gp knock-out mice and in cyclosporine A pretreated mice, while the k2 is in the same range in all mice. Since it was expected that K1, representing influx in the brain, should not change between the different groups tested, while k2, indicating efflux out of the brain, was supposed to be lower in P-gp knock-out mice and in the wild-type mice pretreated with cyclosporine A, we postulated a new theory to explain the results. . So, we propose K1 as a pseudo value in mice, representing a combination of passive influx of 11C-dLop through the BBB and a rapid energy dependent output by P-gp, while k2 corresponds to slow passive efflux out of the brain. Since 11C-desmethylloperamide kinetic modeling is useful to predict the presence of absence of functional P-gp, it was postulated that overexpression of P-gp in epileptic rats could be predicted by this non invasive method of kinetic modeling. The kinetic parameters (K1 and k2) obtained from a two-tissue compartment model were statistical different between non epileptic Sprague-Dawley and epileptic rats, when P-gp was partially blocked by cyclosporine A. Higher expression of P-gp, as indicated by immunohistochemical staining, can thus be indicated by the increase of k2 observed in the epileptic rat group. Thereby, kinetic modeling with 11C-desmethylloperamide is a useful non invasive method to evaluate the P-gp functionality in the brain. Chapter 9 focussed on the influence of specific activity of PET tracers on the brain tracer uptake. Our results clearly demonstrate statistical higher brain uptake of 11C-laniquidar when administered as low specific activity solutions, while high specific activity 11C-laniquidar solutions showed lower brain uptake. In comparison, no different 11C-dLop brain uptake was observed after high or low S.A. solutions. These results confirm the important role of specific activity, and subsequently the mass amount of PET tracers when used to investigate P-gp mediated efflux at the BBB

Effect of cyclosporin A administration on the biodistribution and multipinhole μSPECT imaging of [123-I]R91150 in rodent brain

Purpose P-glycoprotein (Pgp) is an efflux protein found amongst other locations in the blood–brain barrier. It is important to investigate the effect of Pgp modulation on clinically used brain tracers, because brain uptake of the tracer can be altered by blocking of the Pgp efflux transporter. The function of Pgp can be blocked with cyclosporin A. Methods We investigated the effect of cyclosporin A administration on the biodistribution of [123I]R91150 in rodents, and the effect of Pgp blocking on the quality of multipinhole μSPECT imaging with [123I]R91150. The influence of increasing doses of cyclosporin A on the brain uptake of [123I]R91150 was investigated in NMRI mice. A biodistribution study with [123I]R91150 was performed in male Sprague-Dawley rats pretreated with cyclosporin A and not pretreated. Brain uptake of [123I]R91150 after cyclosporin A injection was compared to the brain uptake in untreated animals, and a displacement study with ketanserin was performed in both groups. A multipinhole μSPECT brain imaging study was also performed using a Milabs U-SPECT-II camera in male Sprague-Dawley rats. To exclude the effect of possible metabolites, a metabolite study was also performed. Results At the highest cyclosporin A dose (50 mg/kg), a sevenfold increase in brain radioactivity concentration was observed in NMRI mice. Also, a dose-response relationship was established between the dose of cyclosporin A and the brain uptake of [123I]R91150 in mice. Compared to the control group, a five-fold increase in [123I]R91150 radioactivity concentration was observed in the brain of Sprague-Dawley rats after cyclosporin A treatment (50 mg/kg). Radioactivity concentration in the frontal cortex increased from 0.24±0.0092 to 1.58±0.097% injected dose per gram of tissue after treatment with cyclosporin A (at the 1-h time-point). Blood radioactivity concentrations did not increase to the same extent. The cortical activity was displaced by administration of ketanserin. A metabolite study confirmed that there was no increased metabolism of [123I]R91150 due to cyclosporin A. The visual quality of multipinhole μSPECT images with [123I]R91150 in Sprague-Dawley rats improved markedly after cyclosporin A pretreatment. Conclusion From the results obtained in the biodistribution studies, it can be concluded that [123I]R91150 is a substrate for Pgp in rodents. A relationship between the administered dose of cyclosporin A and the increase in [123I]R91150 brain radioactivity concentration was established. The overall quality of our multipinhole μSPECT images with [123I]R91150 in rats improved markedly after pretreatment of the animals with cyclosporin A

Synthesis, in vitro and in vivo evaluation, and radiolabeling of aryl anandamide analogues as candidate radioligands for in vivo imaging of fatty acid amide hydrolase in the brain

Fatty acid amide hydrolyase (FAAH) is one of the main enzymes responsible for terminating the signaling of endocannabinoids in the brain. Imaging FAAH in vivo using PET or SPECT is important to deeper understanding of its role in neuropsychiatric disorders. However, at present, no radioligand is available for mapping the enzyme in vivo. Here, we synthesized 18 aryl analogues of anandamide, FAAH's endogenous substrate, and in vitro evaluated their potential as metabolic trapping tracers. Interaction studies with recombinant FAAH revealed good to very good interaction of the methoxy substituted aryl anandamide analogues 17, 18, 19, and 20 with FAAH and they were identified as competing substrates, Compounds 17 and 18 did not display significant binding to CB1 and CB2 cannabinoid receptors and stand out as potential candidate metabolic trapping tracers. They were successfully labeled with C-11 in good yields and high radiochemical purity and displayed brain uptake in C57BL/6J mice. Radioligands [C-11]-17 and [C-11]-18 merit further investigation in vivo