Secondary prevention measures in anaphylaxis patients: Data from the anaphylaxis registry.

BACKGROUND: Patients with a history of anaphylaxis are at risk of future anaphylactic reactions. Thus, secondary prevention measures are recommended for these patients to prevent or attenuate the next reaction. METHODS: Data from the Anaphylaxis Registry were analyzed to identify secondary prevention measures offered to patients who experienced anaphylaxis. Our analysis included 7788 cases from 10 European countries and Brazil. RESULTS: The secondary prevention measures offered varied across the elicitors. A remarkable discrepancy was observed between prevention measures offered in specialized allergy centers (84% of patients were prescribed adrenaline autoinjectors following EAACI guidelines) and outside the centers: Here, EAACI guideline adherence was only 37%. In the multivariate analysis, the elicitor of the reaction, age of the patient, mastocytosis as comorbidity, severity of the reaction, and reimbursement/availability of the autoinjector influence physician's decision to prescribe one. CONCLUSIONS: Based on the low implementation of guidelines concerning secondary prevention measures outside of specialized allergy centers, our findings highlight the importance of these specialized centers and the requirement of better education for primary healthcare and emergency physicians.

Preparation and in vitro evaluation of radiolabeled HA-PLGA nanoparticles as novel MTX delivery system for local treatment of rheumatoid arthritis.

Radiosynovectomy is a technique used to decrease inflammation of the synovial tissue by intraarticular injection of a ß-emitting radionuclide, such as 177Lu, which is suitable for radiotherapy due to its decay characteristics. Drug-encapsulating nanoparticles based on poly lactic­co­glycolic acid (PLGA) polymer are a suitable option to treat several arthritic diseases, used as anti-inflammatory drugs transporters of such as methotrexate (MTX), which has been widely used in the arthritis treatment (RA), and hyaluronic acid (HA), which specifically binds the CD44 and hyaluronan receptors overexpressed on the inflamed synovial tissue cells. The 1,4,7,10­Tetraazacyclododecane­1,4,7,10­tetraacetic acid (DOTA) was used as complexing agent of Lutetium-177 for radiotherapy porpoises. The aim of this research was to synthesize 177Lu-DOTA-HA-PLGA(MTX) as a novel, smart drug delivery system with target-specific recognition, potentially useful in radiosynovectomy for local treatment of rheumatoid arthritis. The polymeric nanoparticle system was prepared and chemically characterized. The MTX encapsulation and radiolabelling were performed with suitable characteristics for its in vitro evaluation. The HA-PLGA(MTX) nanoparticle mean diameter was 167.6 nm ±â€¯57.4 with a monomodal and narrow distribution. Spectroscopic techniques demonstrated the effective conjugation of HA and chelating agent DOTA to the polymeric nanosystem. The MTX encapsulation was 95.2% and the loading efficiency was 6%. The radiochemical purity was 96 ±â€¯2%, determined by ITLC. Conclusion: 177Lu-DOTA-HA-PLGA(MTX) was prepared as a biocompatible polymeric PLGA nanoparticle conjugated to HA for specific targeting. The therapeutic nanosystem is based on bi-modal mechanisms using MTX as a disease-modifying antirheumatic drug (DMARD) and 177Lu as a radiotherapeutic component. The 177Lu-DOTA-HA-PLGA(MTX) nanoparticles showed properties suitable for radiosynovectomy and further specific targeted anti-rheumatic therapy.

[99mTc-HYNIC-N-dodecylamide]: a new hydrophobic tracer for labelling reconstituted high-density lipoproteins (rHDL) for radioimaging.

Despite the widespread use of nanotechnology in radio-imaging applications, lipoprotein based delivery systems have received only limited attention so far. These studies involve the synthesis of a novel hydrophobic radio-imaging tracer consisting of a hydrazinonicotinic acid (HYNIC)-N-dodecylamide and 99mTc conjugate that can be encapsulated into rHDL nanoparticles (NPs). These rHDL NPs can selectively target the Scavenger Receptor type B1 (SR-B1) that is overexpressed on most cancer cells due to excess demand for cholesterol for membrane biogenesis and thus can target tumors in vivo. We provide details of the tracer synthesis, characterization of the rHDL/tracer complex, in vitro uptake, stability studies and in vivo application of this new radio-imaging approach.

99mTc-exendin(9-39)/octreotide: biokinetics and radiation dosimetry in healthy individuals.

AIM: About 90% of insulinomas are benign and 5-15% are malignant. Benign insulinomas express the glucagon-like peptide-1 receptor (GLP-1R, which recognizes exendin-4 and low levels of the somatostatin receptor (SSTR, which recognizes octreotide), whereas malignant insulinomas overexpress SSTR and low levels of GLP-1R. Recently, Lys(Tc-EDDA/HYNIC)-exendin(9-39)/Tc-EDDA/HYNIC-Tyr-octreotide was formulated to detect 100% of insulinomas. The aim of this study was to estimate the biokinetics and dosimetry of Tc-exendin(9-39)/octreotide in four healthy individuals. PARTICIPANTS AND METHODS: Tc-exendin(9-39)/octreotide was obtained from a lyophilized formulation with radiochemical purities of more than 97%, determined by reversed-phase high-performance liquid chromatography. Whole-body images from four healthy individuals were acquired at 20 min, 2, 6, and 24 h after Tc-exendin(9-39)/octreotide administration. Regions of interest were drawn around the source organs on each time frame. Each region of interest was corrected by background, attenuation, scattered radiation, and physical decay. The image sequence was used to extrapolate the Tc-exendin(9-39)/octreotide time-activity curves of each organ to adjust the biokinetic model and calculate the total number of disintegrations (N) that occurred in the source regions. N data were the input for the OLINDA/EXM code to calculate internal radiation doses. Furthermore, in a patient suspicious of harboring an insulinoma, whole-body single-photon emission computed tomography/computed tomography images were obtained at 3 h. RESULTS: For four healthy individuals, the blood activity showed a half-life value of 1.20 min for the fast component (T1/2 α=ln 2/34.71), 8.7 min for the first slow component (T1/2 ß=ln 2/4.76), and 1.7 h for the second slow component (T1/2 γ=ln 2/0.401). The average equivalent doses calculated for a study using 555 MBq were 15.10, 4.13, 3.08, 2.61, and 1.90 mSv for the kidneys, upper large intestinal wall, lower large intestinal wall, small intestine, and liver, respectively, with an effective dose of 2.33±0.51 mSv. In addition, images from a patient showed an average tumor/heart (blood) ratio of 2.7 at 3 h. CONCLUSION: All the absorbed doses were comparable to those known for most of the Tc studies. Tc-exendin(9-39)/octreotide obtained from kit formulations showed high tumor uptake in a patient with a malignant lesion, making it a promising imaging radiopharmaceutical to target GLP-1R and SSTR.

Biodegradable poly(D,L-lactide-co-glycolide)/poly(L-γ-glutamic acid) nanoparticles conjugated to folic acid for targeted delivery of doxorubicin.

A novel targeted drug delivery nanoparticle system based on poly(D,L-lactide-co-glycolide) acid (PLGA) for delivery of doxorubicin (DOX) was developed. DOX-PLGA NPs were obtained by the emulsification-solvent evaporation technique. Then, their surface was modified with poly(L-γ-glutamic acid) (γ-PGA) and finally conjugated to modified folic acid (FA) as a targeting ligand. The surface modification and FA conjugation were followed by UV-Vis and FT-IR spectroscopies. Morphology was observed by TEM/SEM. Particle size, PDI and zeta potential were measured using DLS studies. Encapsulation and loading efficiencies, and DOX release kinetics were determined. Specific uptake and cell viability of DOX-PLGA/γ-PGA-FA NPs were tested in HeLa cells. Quasi-spherical nanoparticles with a particle size lower than 600nm (DLS) were obtained. Spectroscopic techniques demonstrated the successful surface modification with γ-PGA and FA conjugation. Release profile of DOX-PLGA/γ-PGA-FA NPs showed a release of 55.4±0.6% after seven days, in an acidic environment. HeLa cells exhibited a decrease in viability when treated with DOX-PLGA/γ-PGA-AF NPs, and cellular uptake was attributed to FA receptor-mediated endocytosis. These results suggest that DOX-PLGA/γ-PGA-FA NPs are a potential targeted drug carrier for further applications in cancer therapy.

Hydrogels based on poly(ethylene glycol) as scaffolds for tissue engineering application: biocompatibility assessment and effect of the sterilization process.

Hydrogels are suitable materials to promote cell proliferation and tissue support because of their hydrophilic nature, porous structure and sticky properties. However, hydrogel synthesis involves the addition of additives that can increase the risk of inducing cytotoxicity. Sterilization is a critical process for hydrogel clinical use as a proper scaffold for tissue engineering. In this study, poly(ethylene glycol) (PEG), poly(ethylene glycol)-chitosan (PEG-CH) and multi-arm PEG hydrogels were synthesized by free radical polymerization and sterilized by gamma irradiation or disinfected using 70 % ethanol. The biocompatibility assessment in human fibroblasts and hemocompatibility studies (hemolysis, platelet aggregation, morphology of mononuclear cells and viability) in peripheral blood from healthy volunteers (ex vivo), were performed. The sterilization or disinfection effect on hydrogel structures was evaluated by FT-IR spectroscopy. Results indicated that hydrogels do not induce any damage to fibroblasts, erythrocytes, platelets or mononuclear cells. Moreover, there was no significant difference in the biocompatibility after the sterilization or disinfection treatment. However, after gamma irradiation, several IR spectroscopic bands were shifted to higher or lower energies with different intensity in all hydrogels. In particular, several bands associated to carboxyl or hydroxyl groups were slightly shifted, possibly associated to scission reactions. The disinfection treatment (70 % ethanol) and γ-irradiation at 13.83 ± 0.7 kGy did not induce morphological damages and yielded sterile and biocompatible PEG hydrogels potentially useful for clinical applications.

A freeze-dried kit formulation for the preparation of Lys(27)(99mTc-EDDA/HYNIC)-Exendin(9-39)/99mTc-EDDA/HYNIC-Tyr3-Octreotide to detect benign and malignant insulinomas.

About 90% of insulinomas are benign and 5%-15% are malignant. Benign insulinomas express the glucagon-like peptide-1 receptor (GLP-1R) and low levels of somatostatin receptors (SSTR), while malignant insulinomas over-express SSTR or GLP-1R in low levels. A kit for the preparation of Lys(27)((99m)Tc-EDDA/HYNIC)-Exendin(9-39)/(99m)Tc-EDDA/HYNIC-Tyr(3)Octreotide was formulated to detect 100% of insulinomas. The formulation showed radiochemical purity of 97±1%, high stability in human serum, and GLP-1R and SSTR affinity. The biodistribution and imaging studies demonstrated properties suitable for its use as a target-specific agent for the simultaneous molecular imaging of GRP-1R- and/or SSTR-positive tumors.

Design and biological evaluation of 99mTc-N2S2-Tat(49-57)-c(RGDyK): a hybrid radiopharmaceutical for tumors expressing α(v)ß(3) integrins.

UNLABELLED: The α(ν)ß(3) integrin is over-expressed in the tumor neovasculature and the tumor cells of glioblastomas. The HIV Tat-derived peptide has been used to deliver various cargos into cells. The aim of this research was to synthesize and assess the in vitro and in vivo uptake of (99m)Tc-N2S2-Tat(49-57)-c(RGDyK) ((99m)Tc-Tat-RGD) in α(ν)ß(3) integrin positive cancer cells and compare it to that of a conventional (99m)Tc-RGD peptide ((99m)Tc-EDDA/HYNIC-E-[c(RGDfK)]2). METHODS: The c(RGDyK) peptide was conjugated to a maleimidopropionyl (MP) moiety through Lys, and the MP group was used as the branch position to form a thioether with the Cys(12) side chain of the Tat(49-57)-spacer-N2S2 peptide. (99m)Tc-Tat-RGD was prepared, and stability studies were carried out by size exclusion HPLC analyses in human serum. The in vitro affinity for α(v)ß(3) integrin was determined by a competitive binding assay. In vitro internalization was determined using glioblastoma C6 cells. Biodistribution studies were accomplished in athymic mice with C6 induced tumors that had blocked and unblocked receptors. Images were obtained using a micro-SPECT/CT. RESULTS: (99m)Tc-Tat-RGD was obtained with a radiochemical purity higher than 95%, as determined by radio-HPLC and ITLC-SG analyses. Protein binding was 15.7% for (99m)Tc-Tat-RGD and 5.6% for (99m)Tc-RGD. The IC50 values were 6.7 nM ((99m)Tc-Tat-RGD) and 4.6 nM ((99m)Tc-RGD). Internalization in C6 cells was higher in (99m)Tc-Tat-RGD (37.5%) than in (99m)Tc-RGD (10%). Biodistribution studies and in vivo micro-SPECT/CT images in mice showed higher tumor uptake for (99m)Tc-Tat-RGD (6.98% ± 1.34% ID/g at 3h) than that of (99m)Tc-RGD (3.72%±0.52% ID/g at 3h) with specific recognition for α(v)ß(3) integrins. CONCLUSIONS: Because of the significant cell internalization (Auger and internal conversion electrons) and specific recognition for α(v)ß(3) integrins, the hybrid (99m)Tc-N2S2-Tat(49-57)-c(RGDyK) radiopharmaceutical is potentially useful for the imaging and possible therapy of tumors expressing α(v)ß(3) integrins.

Engineered multifunctional RGD-gold nanoparticles for the detection of tumour-specific alpha(v)beta(3) expression: chemical characterisation and ecotoxicological risk assessment.

Radiolabelled nanoparticles conjugated to peptides that target specific molecules can be used as agents for diagnosis. For example, technetium-99m-labelled gold nanoparticles conjugated to the cyclic [Arg-Gly-Asp-Phe-Lys(Cys)] (c[RGDfK(C)]) peptide are used for the molecular imaging of tumour-specific alpha(epsilon)beta(3) expression. Gold nanoparticles (AuNPs, 18-20 nm) are assumed to be safe at the concentrations used in diagnostic studies, and several studies of human toxicology and health implications have been discussed in a wide number of reports. However, no data are available on the potential ecotoxicity of radio-AuNPs conjugated to thiol-peptides after complete radionuclide decay. The aim of this study was to determine the ecotoxicological risk, measured by oxidative stress (OS) induction in Hyalella azteca, of a well-characterised multifunctional RGD-AuNP system (Tc-HYNIC-GGC-AuNP-c[RGDfK(C)]). TEM, UV-Vis, Raman, XPS, FT-IR and Far-IR spectroscopy techniques demonstrated that AuNPs were functionalised with the conjugated peptides. An acute toxicity test predicted an LC50 (72 h) of 1.83 cm2 per millilitre of medium. There were no statistically significant increases in OS and antioxidant defences after sub-acute exposure to a surface area concentration of 1/10 of the LC50. Tc-HYNIC-GGC-AuNP-c[RGDfK(C)] (20 nm) was demonstrated to be a chemically stable and nontoxic system in biological media after complete radionuclide decay because it does not promote the expression of OS and antioxidant defence enzymes, such as catalase, glutathione peroxidase and superoxide dismutase in Hyalella azteca.

Development of specific radiopharmaceuticals for infection imaging by targeting infectious micro-organisms.

Infectious diseases remain a major health problem and cause of death worldwide. A variety of radiopharmaceuticals are used for the imaging of infections and inflammation in the practice of nuclear medicine. Long-term clinical use has shown that the majority of radiolabeled probes cannot distinguish between inflammation and infection. Gallium-67-citrate binds to bacteria, but also to proteins accumulating at both sterile inflammation and bacterial infection sites. Other agents are used to interact with receptors or domains on circulating and infiltrating leukocytes or to label them directly. However, these probes cannot distinguish between infection and inflammation because they are not specific to infectious micro-organisms. This review examines the recent developments and applications of radiolabeled specific agents, such as antiviral drugs, antifungal, antibiotics and antimicrobial peptides, to visualize infectious foci by targeting viruses, fungi or bacteria.

Multimeric system of 99mTc-labeled gold nanoparticles conjugated to c[RGDfK(C)] for molecular imaging of tumor α(v)ß(3) expression.

Integrin α(V)ß(3) plays a critical role in tumor angiogenesis and metastasis. Suitably radiolabeled cyclic RGD peptides can be used for noninvasive imaging of α(V)ß(3) expression. The aim of this research was to prepare a multimeric system of technetium-99m-labeled gold nanoparticles conjugated to c[RGDfK(C)] and to evaluate its biological behavior as a potential radiopharmaceutical for molecular imaging of tumor angiogenesis. Hydrazinonicotinamide-GGC (HYNIC-GGC) and c[RGDfK(C)] peptides were synthesized and conjugated to gold nanoparticles (AuNP, 20 nm) by means of spontaneous reaction of the thiol groups of cysteine. The nanoconjugate was characterized by TEM, FT-IR, UV-vis, XPS, and Raman spectroscopy. To obtain (99m)Tc-HYNIC-GGC-AuNP-c[RGDfK(C)] ((99m)Tc-AuNP-RGD), the (99m)Tc-HYNIC-GGC radiopeptide was first prepared and added to 1.5 mL of AuNP solution (1 nM) followed by c[RGDfK(C)] (10 µL, 50 µM) at 18 °C with stirring for 15 min. Radiochemical purity (RP) was determined by size-exclusion HPLC and ITLC-SG analyses. In vitro binding studies were carried out in α(V)ß(3) receptor-positive C6 glioma cancer cells. Biodistribution studies were accomplished in athymic mice with C6-induced tumors with blocked and nonblocked receptors, and images were obtained using a micro-SPECT/CT. TEM and spectroscopy techniques demonstrated that AuNPs were functionalized with peptides. RP was 96 ± 2% without postlabeling purification. (99m)Tc-AuNP-RGD showed specific recognition for α(V)ß(3) integrins expressed in C6 cells, and 3 h after i.p. administration in mice, the tumor uptake was 8.18 ± 0.57% ID/g. Micro-SPECT/CT images showed evident tumor uptake. (99m)Tc-AuNP-RGD demonstrates properties suitable for use as a target-specific agent for molecular imaging of tumor α(V)ß(3) expression.

(99m)Tc-labelled gold nanoparticles capped with HYNIC-peptide/mannose for sentinel lymph node detection.

UNLABELLED: The aim of this research was to prepare a multifunctional system of technetium-99m-labelled gold nanoparticles conjugated to HYNIC-GGC/mannose and to evaluate its biological behaviour as a potential radiopharmaceutical for sentinel lymph node detection (SLND). METHODS: Hydrazinonicotinamide-Gly-Gly-Cys-NH(2) (HYNIC-GGC) peptide and a thiol-triazole-mannose derivative were synthesized, characterized and conjugated to gold nanoparticles (AuNP, 20 nm) to prepare a multifunctional system of HYNIC-GGC-AuNP-mannose by means of spontaneous reaction of the thiol (Cys) present in HYNIC-GGC sequence and in the thiol-mannose derivative. The nanoconjugate was characterized by transmission electron microscopy (TEM), IR, UV-Vis, Raman, fluorescence and X-ray photoelectron spectroscopy (XPS). Technetium-99m labelling was carried out using EDDA/tricine as coligands and SnCl(2) as reducing agent with further size-exclusion chromatography purification. Radiochemical purity was determined by size-exclusion HPLC and ITLC-SG analyses. In vitro binding studies were carried out in rat liver homogenized tissue (mannose-receptor positive tissue). Biodistribution studies were accomplished in Wistar rats and images obtained using a micro-SPECT/CT system. RESULTS: TEM and spectroscopy techniques demonstrated that AuNPs were functionalized with HYNIC-GGC-NH(2) and thiol-mannose through interactions with thiol groups and the N-terminal amine of cysteine. Radio-chromatograms showed radiochemical purity higher than 95%. (99m)Tc-EDDA/HYNIC-GGC-AuNP-mannose ((99m)Tc-AuNP-mannose) showed specific recognition for mannose receptors in rat liver tissue. After subcutaneous administration of (99m)Tc-AuNP-mannose in rats (footpad), radioactivity levels in the popliteal and inguinal lymph nodes revealed that 99% of the activity was extracted by the first lymph node (popliteal extraction). Biodistribution studies and in vivo micro-SPECT/CT images in Wistar rats showed an evident lymph node uptake (11.58 ± 1.98 %ID at 1 h) which was retained during 24 h with minimal kidney accumulation (0.98 ± 0.10 %ID) and negligible uptake in all other tissues. CONCLUSIONS: This study demonstrated that (99m)Tc-AuNP-mannose remains within the first lymph node during 24 h and therefore might be useful as a target-specific radionanoconjugate for SLND using "1-day" or "2-day" conventional protocols.