Zita Pöstényi

99mTc-labelled nanosystem as tumour imaging agent for SPECT and SPECT/CT modalities

We report the synthesis, in vitro and in vivo investigation of folate-targeted, biocompatible, biodegradable self-assembled nanoparticles radiolabelled with (99m)Tc, as potential new SPECT or SPECT/CT imaging agent. Nanoparticles with hydrodynamic size in the range of 75-200 nm were prepared by self-assembly of chitosan and folated poly-γ-glutamic acid, and then radiolabelled with (99m)Tc. The nanoparticles target tumour cells overexpressing folate receptors and internalize specifically into them to realize early tumour diagnosis detected by SPECT and SPECT/CT modalities. Rat hepatocellular carcinoma cells were used as model system. Cell specificity and tumour targeting efficacy of these nanosystems were investigated in vitro, and in vivo using SPECT and fusion nanoSPECT/CT imaging. In vitro results showed that the radiolabeled nanosystem was efficiently internalized by tumour cells. Whole-body biodistribution of the new radiolabelled, folate-targeted nanoparticles revealed higher uptake in the tumorous kidney compared to the non-tumorous contralateral side. Uptake by the lungs and thyroids was negligible, which confirmed the stability of the nanoparticles in vivo. In vivo SPECT and SPECT/CT imaging visually reinforced the uptake results and were in accordance with the biodistribution data: the new nanoparticles as a targeted contrast agent improve tumour targeting and are able to detect folate-receptor-overexpressing tumours in animal models with enhanced contrast.

In vitro and biodistribution examinations of Tc-99m-labelled doxorubicin-loaded nanoparticles

BACKGROUND Nanoparticles represent promising drug carrier systems. In the case of cytostatics such as doxorubicin, carrier colloid systems as human serum albumin (HSA) nanoparticles, may increase their therapeutic efficiency and decrease their side-effects (toxicity) and any potential multidrug resistance. In the present study, doxorubicin, as a widely used antineoplastic agent, was incorporated into the matrix of human serum albumin and three different particle-sized doxorubicin-loaded HSA nanoparticles were prepared, using a previously described desolvation method. Our objective was to find out if different particle sizes of colloid carriers can allow regarding the given cytostatic agent. MATERIAL AND METHODS The three prepared nanoparticles were labelled using technetium (Tc-99m) and were tested for their physicochemical colloidal quality, fluctuations, and radiochemical stability. Biodistribution of different-sized radiolabelled colloids were determined by means of scintigraphic imaging studies in healthy male Wistar rats. Images were taken by gamma camera at several times and organ uptakes were estimated by quantitative ROI analysis. RESULTS In vitro measurements showed that more than 95% of doxorubicin proportion was permanently adsorbed to human serum albumin. Radiolabelled doxorubicin-loaded particles had high-degree and durable labelling efficiency and particle size stability. Biodistribution results had a close correlation to earlier described results of radiocolloids in similar particle size ranges. In vivo examinations verified that colloid carriers have insignificant size fluctuations after an intravenous application and they show the proper distribution according to their particle size. CONCLUSIONS Our investigations verified that different and stable particle sizes make drug carrier HSA nanoparticles possible to apply different drug targeting in a potential clinical use.

Lu-177-Labeled Zirconia Particles for Radiation Synovectomy

The present article describes the preparation of β-emitter lutetium-177-labeled zirconia colloid and its preliminary physicochemical and biological evaluation of suitability for local radionuclide therapy. The new (177)Lu-labeled therapeutic radiopharmaceutical candidate was based on the synthesis mode of a previously described zirconia nanoparticle system. The size and shape of the developed radiopharmaceutical compound were observed through a scanning electron microscope and dynamic light scattering methods. The radiocolloid had a 1.7 μm mean diameter and showed high in vitro radiochemical and colloid size stability at room temperature and during the blood sera stability test. After the in vitro characterizations, the product was investigated in the course of the treatment of a spontaneously diseased dog veterinary patients hock joint completed with single-photon emission computed tomography (SPECT) imaging follow-up measurements and a dual-isotope SPECT imaging tests with conventional (99m)Tc-methanediphosphonic acid bone scintigraphy. In the treated dog, no clinical side-effects or signs of histopathological changes of the joints were recorded during the treatment. SPECT follow-up studies clearly and conspicuously showed the localization of the (177)Lu-labeled colloid in the hock joint as well as detectable but negligible leakages of the radiocolloid in the nearest lymph node. On the basis of biological follow-up tests, the orthopedic team assumed that the (177)Lu-labeled zirconia colloid-based local radionuclide therapy resulted in a significant and long-term improvement in clinical signs of the patient without any remarkable side-effects.

HPLC analysis of blood-brain barrier penetration of 4-fluorodeprenyl

Validated HPLC analysis was developed in order to monitor the level of 4-fluorodeprenyl in rats. Male Wistar rats were intraperitoneally treated with 30 mg/kg of (-)-4-fluorodeprenyl. The rats were sacrificed after 5, 15, 30 and 60 min of treatment, and various tissues were isolated, such as serum, brain, CSF, liver, testis and lacrimal gland. Perchloric acid was given to aliquots, which were then homogenized, centrifuged and the supernatants were taken. The 4-fluorodeprenyl content was determined using reversed-phase HPLC, based on the comparison of the calibration line of the spiked samples. The level of 4-fluorodeprenyl was between 0.5 and 24 μg/g, showing maximum concentration in the brain and the liver after 5 min following its administration and in serum, CSF, testis, eyes and lacrimal gland after 15 min following its administration, while a relatively high concentration was found in the liver and the lacrimal gland.

Temperature increase induced by modulated electrohyperthermia (oncothermia®) in the anesthetized pig liver

AIM OF STUDY Is to show the intrahepatic temperature development in anesthetized pig. MATERIALS AND METHODS Temperature development in the liver of anesthetized pig is measured to study the thermal effects of capacitive coupled energy transfer. The treatment was made by modulated electrohyperthermia (mEHT, trade name: oncothermia ®), controlled by a fluoroptical temperature sensing positioned by the ultrasound-guided process. Various fits of coupling were studied. RESULTS The intrahepatic temperature at the end of the treatment ranged 40.5-44.8°C, while the skin temperature ranged 36.8-41.8°C depending on the coupling arrangement. CONCLUSION mEHT is a feasible method to deliver deep heat to the liver of an anesthetized pig.

Monitoring the level of 14C-labelled selegiline following oral administration

Background: Selegiline [(-)-deprenyl] is widely used for the treatment of Parkinson’s disease in humans. Objective: Time-dependence of tissue distribution of selegiline following per os administration to rats. Method: Oral administration of radiolabeled selegiline to rats resulted in a pattern of tissue distribution similar to that following intraperitoneal injection. Analyses were done using both reversed-phase HPLC and also by counting radioactivity in various body compartments of rats. Results: As a consequence of oral administration of 30 mg/kg of selegiline, its level in the stomach was extremely high (179.57 µg/g tissue through 54.67 µg/g at 15 min to 120 min), that is one magnitude higher than that in the serum level. High selegiline concentrations were also detected in the lacrimal glands (7.45 µg/g), kidneys (6.87 µg/g), livers (6.01 µg/g) and lungs (3.47 µg/g) after 30 minutes of application, which were higher than after intraperitoneal injections. Conclusion: The relatively high tissue levels remained for 120 min monitoring. Selegiline levels in the brain (1.69 µg/g) and in the testes (1.88 µg/g) were also considerably higher than following intraperitoneal administration during the entire period of observation (15 to 120 min).

177Lu Labeled Cyclic Minigastrin Analogues with Therapeutic Activity in CCK2R Expressing Tumors: Preclinical Evaluation of a Kit Formulation

Minigastrin (MG) analogues specifically target cholecystokinin-2 receptors (CCK2R) expressed in different tumors and enable targeted radiotherapy of advanced and disseminated disease when radiolabeled with a beta emitter such as 177Lu. Especially truncated MG analogues missing the penta-Glu sequence are associated with low kidney retention and seem therefore most promising for therapeutic use. Based on [d-Glu1,desGlu2-6]MG (MG11) we have designed the two cyclic MG analogues cyclo1,9[γ-d-Glu1,desGlu2-6,d-Lys9]MG (cyclo-MG1) and cyclo1,9[γ-d-Glu1,desGlu2-6,d-Lys9,Nle11]MG (cyclo-MG2). In the present work we have developed and preclinically evaluated a pharmaceutical kit formulation for the labeling with 177Lu of the two DOTA-conjugated cyclic MG analogues. The stability of the kits during storage as well as the stability of the radiolabeled peptides was investigated. A cell line stably transfected with human CCK2R and a control cell line without receptor expression were used for in vitro and in vivo studies with the radioligands prepared from kit formulations. In terms of stability 177Lu-DOTA-cyclo-MG2 showed advantages over 177Lu-DOTA-cyclo-MG1. Still, for both radioligands a high receptor-mediated cell uptake and favorable pharmacokinetic profile combining receptor-specific tumor uptake with low unspecific tissue uptake and low kidney retention were confirmed. Investigating the therapy efficacy and treatment toxicity in xenografted BALB/c nude mice a receptor-specific and comparable therapeutic effect could be demonstrated for both radioligands. A 1.7- to 2.6-fold increase in tumor volume doubling time was observed for receptor-positive tumors in treated versus untreated animals, which was 39-73% higher when compared to receptor-negative tumors. The treatment was connected with transient bone marrow toxicity and minor signs of kidney toxicity. All together the obtained results support further studies for the clinical translation of this new therapeutic approach.

Pharmacokinetics of selegiline in a rabbit model

Background: Selegiline is one of the most widely used monoamine oxidase B inhibitors for the treatment of Parkinson’s disease in early stage. Objective: Time-dependent distribution of selegiline was monitored in various tissues of rabbits treated with a dose of 30 mg/kg intravenously. Method: Selegiline content was determined by validated HPLC method following 5,15, 30, 60 and 120 minutes of treatment. Results: A time-dependent decline of selegiline tissue levels was observed in the iris, the cornea and the intraocular lens, while the maximum level of selegiline in the serum, in the brain, in the testis and in the retina was found at 15 min following administration.

Distribution of N-methyl-14C-labeled selegiline in the rat

Tissue distribution of selegiline including N-methyl-(14)C-selegiline was studied with three different techniques. Whole body autoradiography of labeled selegiline in rats completed the former results obtained in mice. Counting radioactivity by liquid scintillation method in various body compartments gave an in-depth numerical estimation of distribution, while RP-HPLC determination of selegiline determined the fate of intact, non-metabolized parent compound. Whole body autoradiography following 15 and 60 min of intraperitoneal application of N-methyl-(14)C-selegiline verified definite and time-dependent blood-brain penetration of selegiline. Quantitative determination of tissue concentrations by liquid scintillation and RP-HPLC methods following 5, 15, 60 and 180 min of intraperitoneal administration of selegiline unanimously verified both blood-brain and blood-testis penetration of the compound through the barrier.