Preparation and PET/CT imaging of implant directed 68Ga-labeled magnetic nanoporous silica nanoparticles.

BACKGROUND: Implant infections caused by biofilm forming bacteria are a major threat in orthopedic surgery. Delivering antibiotics directly to an implant affected by a bacterial biofilm via superparamagnetic nanoporous silica nanoparticles could present a promising approach. Nevertheless, short blood circulation half-life because of rapid interactions of nanoparticles with the host's immune system hinder them from being clinically used. The aim of this study was to determine the temporal in vivo resolution of magnetic nanoporous silica nanoparticle (MNPSNP) distribution and the effect of PEGylation and clodronate application using PET/CT imaging and gamma counting in an implant mouse model. METHODS: PEGylated and non-PEGylated MNPSNPs were radiolabeled with gallium-68 (68Ga), implementing the chelator tris(hydroxypyridinone). 36 mice were included in the study, 24 mice received a magnetic implant subcutaneously on the left and a titanium implant on the right hind leg. MNPSNP pharmacokinetics and implant accumulation was analyzed in dependence on PEGylation and additional clodronate application. Subsequently gamma counting was performed for further final analysis. RESULTS: The pharmacokinetics and biodistribution of all radiolabeled nanoparticles could clearly be visualized and followed by dynamic PET/CT imaging. Both variants of 68Ga-labeled MNPSNP accumulated mainly in liver and spleen. PEGylation of the nanoparticles already resulted in lower liver uptakes. Combination with macrophage depletion led to a highly significant effect whereas macrophage depletion alone could not reveal significant differences. Although MNPSNP accumulation around implants was low in comparison to the inner organs in PET/CT imaging, gamma counting displayed a significantly higher %I.D./g for the tissue surrounding the magnetic implants compared to the titanium control. Additional PEGylation and/or macrophage depletion revealed no significant differences regarding nanoparticle accumulation at the implantation site. CONCLUSION: Tracking of 68Ga-labeled nanoparticles in a mouse model in the first critical hours post-injection by PET/CT imaging provided a better understanding of MNPSNP distribution, elimination and accumulation. Although PEGylation increases circulation time, nanoparticle accumulation at the implantation site was still insufficient for infection treatment and additional efforts are needed to increase local accumulation.

Dissecting the target leukocyte subpopulations of clinically relevant inflammation radiopharmaceuticals.

BACKGROUND: Leukocyte subtypes bear distinct pro-inflammatory, reparative, and regulatory functions. Imaging inflammation provides information on disease prognosis and may guide therapy, but the cellular basis of the signal remains equivocal. We evaluated leukocyte subtype specificity of characterized clinically relevant inflammation-targeted radiotracers. METHODS AND RESULTS: Leukocyte populations were purified from blood- and THP-1-derived macrophages were polarized into M1-, reparative M2a-, or M2c-macrophages. In vitro uptake assays were conducted using tracers of enhanced glucose or amino acid metabolism and molecular markers of inflammatory cells. Both 18F-deoxyglucose (18F-FDG) and the labeled amino acid 11C-methionine (11C-MET) displayed higher uptake in neutrophils and monocytes compared to other leukocytes (P = 0.005), and markedly higher accumulation in pro-inflammatory M1-macrophages compared to reparative M2a-macrophages (P < 0.001). Molecular tracers 68Ga-DOTATATE targeting the somatostatin receptor type 2 and 68Ga-pentixafor targeting the chemokine receptor type 4 (CXCR4) exhibited broad uptake by leukocyte subpopulations and polarized macrophages with highest uptake in T-cells/natural killer cells and B-cells compared to neutrophils. Mitochondrial translocator protein (TSPO)-targeted 18F-flutriciclamide selectively accumulated in monocytes and pro-inflammatory M1 macrophages (P < 0.001). Uptake by myocytes and fibroblasts tended to be higher for metabolic radiotracers. CONCLUSIONS: The different in vitro cellular uptake profiles may allow isolation of distinct phases of the inflammatory pathway with specific inflammation-targeted radiotracers. The pathogenetic cell population in specific inflammatory diseases should be considered in the selection of an appropriate imaging agent.

Impact of DOTA-Chelators on the Antitumor Activity of 177Lu-DOTA-Rituximab Preparations in Lymphoma Tumor-Bearing Mice.

Background: This work aimed to evaluate the influence of two chelators: DOTA(SCN) and DOTA(NHS) on radioimmunotherapy using 177Lu-DOTA-Rituximab preparations in murine lymphoma xenograft models. Subsequently, based on animal data, the organ radiation-absorbed doses were extrapolated to humans (adult male). Materials and Methods: Therapeutic efficacy of 177Lu-DOTA-Rituximab was evaluated in male nude mice bearing either Raji (B lymphocyte, CD20+) and Jurkat (T lymphocyte, CD20) xenografts, utilizing an anti-CD20 antibody-Rituximab conjugate with either DOTA(SCN) or DOTA(NHS). The DOTA-Rituximab conjugates were prepared in the form of freeze-dried kits. Results: All radioimmunoconjugates were obtained with high radiolabeling yield (radiochemical purity, RCP > 95%) and specific activity of ca. 0.5 GBq/mg. Therapeutic effects of 177Lu-DOTA-Rituximab were observed in animals regardless whether DOTA(SCN) or DOTA(NHS) were used for conjugation. Importantly, therapy involving 177Lu-DOTA-Rituximab was more effective than use of Rituximab alone. Conclusions: The degree of antitumor efficacy was dependent on the type of applied bifunctional chelators conjugated to mAb. However, this difference was not statistically significant. Dosimetry calculations showed that the absorbed radiation doses extrapolated to humans were very low for osteogenic cells regardless of the conjugates. Organs like the liver and spleen, treated with 177Lu-DOTA(SCN)-Rituximab, showed similar radiation absorbed doses when compared with 177Lu-DOTA(NHS)-Rituximab.

Nanoparticles for SPECT and PET Imaging: Towards Personalized Medicine and Theranostics.

BACKGROUND: PET and SPECT imaging methods can be of excellent assistance for the development of new nanoparticle drug delivery systems, and at the same time, these investigations also offer the opportunity to produce exceptional new diagnostic and therapeutic radiopharmaceuticals, as well. With a multifunctional, nano-scaled drug delivery system, the diagnostic (imaging) methods and the therapy (delivering drugs or beta-emitter radionuclides) can be carried out using the same biological and pharmacological mechanisms. By combining therapy and diagnostics in one method or in one specifically targeted nanoparticle system, we can product theranostic pharmaceuticals, and its applications are important elements of personalized medicine. OBJECTIVES: This review takes a short historical look back to the radiocolloids, the great ancestors of (radiolabeled) nanoparticles and then describes the general features of current types of PET and SPECT imaging associated nanoparticle-based products and key radiolabeling methods; entering into details of potential prospective challenges related to radiotheranostic approaches and imaging guided therapy.

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.

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.

Evaluation of ¹77Lu-EDTMP in Dogs with Spontaneous Tumor Involving Bone: Pharmacokinetics, Dosimetry and Therapeutic Efficacy.

177Lu-EDTMP is currently being investigated as a potential agent for providing palliative care to the patients suffering from bone pain due to metastatic skeletal carcinoma. The present article describes the evaluation of 177Lu-EDTMP complex in four different canine patients with different types of primary and metastatic skeletal lesions with respect to its pharmacokinetic properties, dosimetry and therapeutic efficacy. The dogs were treated with a dose of ~44.4 MBq (1.2 mCi) per kg body weight of 177Lu-EDTMP, synthesized in-house with high radiochemical purity (98.8 ± 0.4 %) and excellent in vitro stability. The radiopharmaceutical showed favourable pharmacokinetic properties, such as, preferential accumulation at skeletal lesion sites and fast clearance from blood and other non-target organs through urinary route. The administered dose of the radiopharmacutical showed excellent therapeutic efficacy in case of a dog suffering from skeletal metastasis originating from primary tumor elsewhere. On the other hand, two of the remaining three patients with primary bone cancer showed stable disease intially with palliative effect. The fourth patient having metal implant induced osteosarcoma with severe limb oedema did not show any response to the treatment.

Lu-177-Labeled Zirconia Particles for Radiation Synovectomy.

The present article describes the preparation of ß-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 patient's 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.

[SPECT radiopharmaceuticals -- novelties and new possibilities]. / SPECT-radiofarmakonok -- újdonságok és új lehetoségek.

Actual state of affairs and future perspectives of SPECT radiopharmaceuticals regarding local and international data were summarized. Beyond conventional gamma-emitting radioisotopes, localization studies with beta emitting therapeutic radiopharmaceuticals hold increasing importance. Extension of hybrid (SPECT/CT) equipments has modified conventional scintigraphic and SPECT methods as well but more important changes come into the world through novel ligands for specific diagnoses and therapy.

Thulium-170-labeled microparticles for local radiotherapy: preliminary studies.

The present article describes the preparation, characterization, and biological evaluation of Thulium-170 ((170)Tm) [T1/2 = 128.4 days; Eßmax = 968 keV; Eγ = 84 keV (3.26%)] labeled tin oxide microparticles for its possible use in radiation synovectomy (RSV) of medium-sized joints. (170)Tm was produced by irradiation of natural thulium oxide target. 170Tm-labeled microparticles were synthesized with high yield and radionuclidic purity (> 99%) along with excellent in vitro stability by following a simple process. Particle sizes and morphology of the radiolabeled particles were examined by light microscope, dynamic light scattering, and transmission electron microscope and found to be of stable spherical morphology within the range of 1.4-3.2 µm. The preparation was injected into the knee joints of healthy Beagle dogs intraarticularly for biological studies. Serial whole-body and regional images were taken by single-photon-emission computed tomography (SPECT) and SPECT-CT cameras up to 9 months postadministration, which showed very low leakage (< 8% of I.D.) of the instilled particles. The majority of leaked radiocolloid particles were found in inguinal lymph nodes during the 9 months of follow-up. All the animals tolerated the treatment well; the compound did not show any possible radiotoxicological effect. These preliminary studies showed that 170Tm-labeled microparticles could be a promising nontoxic and effective radiopharmaceutical for RSV applications or later local antitumor therapy.

Folate receptor targeted self-assembled chitosan-based nanoparticles for SPECT/CT imaging: demonstrating a preclinical proof of concept.

A new biocompatible, biodegradable, self-assembled chitosan-based nanoparticulate product was successfully synthesized and radiolabeled with technetium-99m, and studied as a potential new SPECT or SPECT/CT imaging agent for diagnosis of folate receptor overexpressing tumors. In the present study we examined the conditions of a preclinical application of this labeled nanosystem in early diagnosis of spontaneously diseased veterinary patient using a human SPECT/CT device. The results confirmed that the nanoparticles accumulated in tumor cells overexpressing folate receptors, contrast agent revealed higher uptake in the tumor for a long time. Preclinical trials verified that the new nanoparticles are able to detect folate-receptor-overexpressing tumors in spontaneously diseased animal models with enhanced contrast.

(99m)Tc-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.

Multispecies animal investigation on biodistribution, pharmacokinetics and toxicity of 177Lu-EDTMP, a potential bone pain palliation agent.

INTRODUCTION: Radionuclide therapy (RNT) is an effective method for bone pain palliation in patients suffering from bone metastasis. Due to the long half-life, easy production and relatively low beta- energy, (177)Lu [T(1/2)=6.73 days, E(beta max)=497 keV, E(gamma)=113 keV (6.4%), 208 keV (11%)]-based radiopharmaceuticals offer logistical advantage for wider use. This paper reports the results of a multispecies biodistribution and toxicity studies of (177)Lu-EDTMP to collect preclinical data for starting human clinical trials. METHODS: (177)Lu-EDTMP with radiochemical purity greater than 99% was formulated by using a lyophilized kit of EDTMP (35 mg of EDTMP, 5.72 g of CaO and 14.1 mg of NaOH). Biodistribution studies were conducted in mice and rabbits. Small animal imaging was performed using NanoSPECT/CT (Mediso, Ltd., Hungary) and digital autoradiography. Gamma camera imaging was done in rabbits and dogs. Four levels of activity (9.25 through 37 MBq/kg body weight) of (177)Lu-EDTMP were injected in four groups of three dogs each to study the toxicological effects. RESULTS: (177)Lu-EDTMP accumulated almost exclusively in the skeletal system (peak ca. 41% of the injected activity in bone with terminal elimination half-life of 2130 and 1870 h in mice and rabbits, respectively) with a peak uptake during 1-3 h. Excretion of the radiopharmaceutical was through the urinary system. Imaging studies showed that all species (mouse, rat, rabbit and dog) take up the compound in regions of remodeling bone, while kidney retention is not visible after 1 day postinjection (pi). In dogs, the highest applied activity (37 MBq/kg body weight) led to a moderate decrease in platelet concentration (mean, 160 g/L) at 1 week pi with no toxicity. CONCLUSION: The protracted effective half-life of (177)Lu-EDTMP in bone supports that modifying the EDTMP molecule by introducing (177)Lu does not alter its biological behaviour as a specific bone-seeking tracer. Species-specific pharmacokinetic behavior differences were observed. Toxicity studies in dogs did not show any biological adverse effects. The studies demonstrate that (177)Lu-EDTMP is a promising radiopharmaceutical that can be further evaluated for establishing as a radiopharmaceutical for human use.

177Lu-EDTMP: a viable bone pain palliative in skeletal metastasis.

Designing ideal radiopharmaceuticals for use as bone pain palliatives require the use of a moderate energy beta() emitter as a radionuclide and a suitable polyaminophosphonic acid as a carrier molecule. Owing to its suitable decay characteristics [T(1/2) = 6.73 d, E((max)) = 497 keV, E() = 113 keV (6.4%), 208 keV (11%)] as well as the feasibility of large-scale production in adequate specific activity and radionuclidic purity using a moderate flux reactor, 177Lu could be considered as a promising radionuclide for palliative care in painful bone metastasis. The present study was therefore, oriented toward the preparation and biologic evaluation of 177Lu complex of ethylenediaminetetramethylene phosphonic acid (EDTMP) in various animal models, with an aim to prepare a viable radiopharmaceutical for bone pain palliation. 177Lu was produced with a specific activity of approximately 12 GBq/mg (approximately 324 mCi/mg) and radionuclidic purity of 99.98% by irradiation of natural Lu2O3 targeted at a thermal neutron flux of approximately 6 x 10(13) n/cm(2).s for 21 days. 177Lu-EDTMP complex was prepared in high-yield and excellent radiochemical purity (>99%), using EDTMP synthesized and characterized in-house. The complex exhibited excellent in vitro stability at room temperature. Biodistribution studies in Wistar rats showed a rapid skeletal accumulation of injected activity [(1.74 +/- 0.30)% per gram in femur at 3 hours postinjection] with a fast clearance from blood and minimal uptake in any of the major organs. Scintigraphic imaging studies carried out in normal Wistar rats, New Zealand white rabbits, as well as in Beagle dogs also demonstrated significant accumulation of the agent in the skeleton and almost no retention of activity in any other vital organs.

Preliminary studies with 188rhenium-tin colloid for radiation synovectomy: preparation, size determination, in-vivo distribution, effect and dosimetry studies.

BACKGROUND: Generator-produced beta-emitting radionuclides such as (188)Re are gaining in importance for radiosynoviorthesis because of their availability on a regular basis. MATERIAL AND METHODS: We prepared a (188)Re-tin colloid in a reaction carried out either at 100 degrees C or at room temperature (RT). The size of the colloid particles was measured with a laser lightscattering method, and their biodistribution, dosimetric aspects and therapeutic effects were studied in an antigen-induced arthritis (AIA) model in rabbits. (188)Re-tin colloid solution was injected intra-articularly into the knee joints of rabbits with AIA and imaging studies were performed. Blood samples were collected post injection for estimation of the blood residence time. We also injected 2 intact rabbits in the same manner with (188)Re perrhenate solution in order to observe its effects and distribution in the body. All the treated rabbit knees were subjected to histopathology. RESULTS: The colloid particle size distribution was different after preparation at the different reaction temperatures, with a more suitable mean of 4.53 micro m in the RT preparation. The dose delivered to the synovial surface was between 3.51 and 4.21 Gy and that to the bone surface was between 0.70 and 0.84 Gy. Histopathologic examination revealed the development of fibrous connective tissue in the AIA knees 4 weeks after treatment, but not in the control group. CONCLUSIONS: The (188)Re-tin colloid preparation used in this study was suitable for radiation synovectomy application. It requires modifications in the preparation protocol so as to increase the labeling efficiency in correlation with an appropriate particle size.

Sentinel lymph node detection in canine oncological patients.

BACKGROUND: Sentinel lymph node detection was investigated in dogs with spontaneously occurring tumours. MATERIAL AND METHODS: In this pilot study, 24 client-owned spontaneously tumorous dogs presented for sentinel lymph node detection. A multiple method was used with a nuclear medicine technique (injection of 99mTc human serum albumin colloid) with scintigraphy and intraoperative guidance, and blue dye injection. RESULTS: Of the 35 lymph nodes histologically demonstrated to contain metastases, 34 (97%) were found by radioguided surgery, which means that one would have been missed in the intraoperative localisation process; 31 nodes (89%) were clearly visualised in the gamma camera images; only 27 (77%) were blue-stained by vital dye; a mere 8 lymph nodes (23%) were enlarged and therefore easily detectable by palpation. CONCLUSIONS: Data obtained from the harmless application of the sentinel node concept are useful for the radiopharmaceutist. The sentinel lymph node concept is well applicable in the veterinary clinic.