Magnetic and radio-labeled bio-hybrid scaffolds to promote and track in vivo the progress of bone regeneration.

This work describes the preparation, characterization and functionalization with magnetic nanoparticles of a bone tissue-mimetic scaffold composed of collagen and hydroxyapatite obtained through a biomineralization process. Bone remodeling takes place over several weeks and the possibility to follow it in vivo in a quick and reliable way is still an outstanding issue. Therefore, this work aims to produce an implantable material that can be followed in vivo during bone regeneration by using the existing non-invasive imaging techniques (MRI). To this aim, suitably designed biocompatible SPIONs were linked to the hybrid scaffold using two different strategies, one involving naked SPIONs (nMNPs) and the other using coated and activated SPIONs (MNPs) exposing carboxylic acid functions allowing a covalent attachment between MNPs and collagen molecules. Physico-chemical characterization was carried out to investigate the morphology, crystallinity and stability of the functionalized materials followed by MRI analyses and evaluation of a radiotracer uptake ([99mTc]Tc-MDP). Cell proliferation assays in vitro were carried out to check the cytotoxicity and demonstrated no side effects due to the SPIONs. The achieved results demonstrated that the naked and coated SPIONs are more homogeneously distributed in the scaffold when incorporated during the synthesis process. This work demonstrated a suitable approach to develop a biomaterial for bone regeneration that allows the monitoring of the healing progress even for long-term follow-up studies.

Optimization of In Vivo Studies by Combining Planar Dynamic and Tomographic Imaging: Workflow Evaluation on a Superparamagnetic Nanoparticles System.

Molecular imaging holds great promise in the noninvasive monitoring of several diseases with nanoparticles (NPs) being considered an efficient imaging tool for cancer, central nervous system, and heart- or bone-related diseases and for disorders of the mononuclear phagocytic system (MPS). In the present study, we used an iron-based nanoformulation, already established as an MRI/SPECT probe, as well as to load different biomolecules, to investigate its potential for nuclear planar and tomographic imaging of several target tissues following its distribution via different administration routes. Iron-doped hydroxyapatite NPs (FeHA) were radiolabeled with the single photon γ-emitting imaging agent [99mTc]TcMDP. Administration of the radioactive NPs was performed via the following four delivery methods: (1) standard intravenous (iv) tail vein, (2) iv retro-orbital injection, (3) intratracheal (it) instillation, and (4) intrarectal installation (pr). Real-time, live, fast dynamic screening studies were performed on a dedicated bench top, mouse-sized, planar SPECT system from t = 0 to 1 hour postinjection (p.i.), and consequently, tomographic SPECT/CT imaging was performed, for up to 24 hours p.i. The administration routes that have been studied provide a wide range of possible target tissues, for various diseases. Studies can be optimized following this workflow, as it is possible to quickly assess more parameters in a small number of animals (injection route, dosage, and fasting conditions). Thus, such an imaging protocol combines the strengths of both dynamic planar and tomographic imaging, and by using iron-based NPs of high biocompatibility along with the appropriate administration route, a potential diagnostic or therapeutic effect could be attained.

In vivo imaging techniques for bone tissue engineering.

Bone is a dynamic tissue that constantly undergoes modeling and remodeling. Bone tissue engineering relying on the development of novel implant scaffolds for the treatment of pre-clinical bone defects has been extensively evaluated by histological techniques. The study of bone remodeling, that takes place over several weeks, is limited by the requirement of a large number of animals and time-consuming and labor-intensive procedures. X-ray-based imaging methods that can non-invasively detect the newly formed bone tissue have therefore been extensively applied in pre-clinical research and in clinical practice. The use of other imaging techniques at a pre-clinical level that act as supportive tools is convenient. This review mainly focuses on nuclear imaging methods (single photon emission computed tomography and positron emission tomography), either alone or used in combination with computed tomography. It addresses their application to small animal models with bone defects, both untreated and filled with substitute materials, to boost the knowledge on bone regenerative processes.

Iron Oxide Colloidal Nanoclusters as Theranostic Vehicles and Their Interactions at the Cellular Level.

Advances in surfactant-assisted chemical approaches have led the way for the exploitation of nanoscale inorganic particles in medical diagnosis and treatment. In this field, magnetically-driven multimodal nanotools that perform both detection and therapy, well-designed in size, shape and composition, are highly advantageous. Such a theranostic material—which entails the controlled assembly of smaller (maghemite) nanocrystals in a secondary motif that is highly dispersible in aqueous media—is discussed here. These surface functionalized, pomegranate-like ferrimagnetic nanoclusters (40⁻85 nm) are made of nanocrystal subunits that show a remarkable magnetic resonance imaging contrast efficiency, which is better than that of the superparamagnetic contrast agent Endorem©. Going beyond this attribute and with their demonstrated low cytotoxicity in hand, we examine the critical interaction of such nanoprobes with cells at different physiological environments. The time-dependent in vivo scintigraphic imaging of mice experimental models, combined with a biodistribution study, revealed the accumulation of nanoclusters in the spleen and liver. Moreover, the in vitro proliferation of spleen cells and cytokine production witnessed a size-selective regulation of immune system cells, inferring that smaller clusters induce mainly inflammatory activities, while larger ones induce anti-inflammatory actions. The preliminary findings corroborate that the modular chemistry of magnetic iron oxide nanoclusters stimulates unexplored pathways that could be driven to alter their function in favor of healthcare.

On the use of superparamagnetic hydroxyapatite nanoparticles as an agent for magnetic and nuclear in vivo imaging.

The identification of alternative biocompatible magnetic NPs for advanced clinical application is becoming an important need due to raising concerns about iron accumulation in soft issues associated to the administration of superparamagnetic iron oxide nanoparticles (NPs). Here, we report on the performance of previously synthetized iron-doped hydroxyapatite (FeHA) NPs as contrast agent for magnetic resonance imaging (MRI). The MRI contrast abilities of FeHA and Endorem® (dextran coated iron oxide NPs) were assessed by 1H nuclear magnetic resonance relaxometry and their performance in healthy mice was monitored by a 7 Tesla scanner. FeHA applied a higher contrast enhancement, and had a longer endurance in the liver with respect to Endorem® at iron equality. Additionally, a proof of concept of FeHA use as scintigraphy imaging agent for positron emission tomography (PET) and single photon emission computed tomography (SPECT) was given labeling FeHA with 99mTc-MDP by a straightforward surface functionalization process. Scintigraphy/x-ray fused imaging and ex vivo studies confirmed its dominant accumulation in the liver, and secondarily in other organs of the mononuclear phagocyte system. FeHA efficiency as MRI-T2 and PET-SPECT imaging agent combined to its already reported intrinsic biocompatibility qualifies it as a promising material for innovative nanomedical applications. STATEMENT OF SIGNIFICANCE: The ability of iron-doped hydroxyapatite nanoaprticles (FeHA) to work in vivo as imaging agents for magnetic resonance (MR) and nuclear imaging is demonstrated. FeHA applied an higher MR contrast in the liver, spleen and kidneys of mice with respect to Endorem®. The successful radiolabeling of FeHA allowed for scintigraphy/X-ray and ex vivo biodistribution studies, confirming MR results and envisioning FeHA application for dual-imaging.

A prototype PET/SPECT/X-rays scanner dedicated for whole body small animal studies.

OBJECTIVE: To present a prototype tri-modal imaging system, consisting of a single photon emission computed tomography (SPET), a positron emission tomography (PET), and a computed tomography (CT) subsystem, evaluated in planar mode. MATERIALS AND METHODS: The subsystems are mounted on a rotating gantry, so as to be able to allow tomographic imaging in the future. The system, designed and constructed by our group, allows whole body mouse imaging of competent performance and is currently, to the best of our knowledge, unequaled in a national and regional level. The SPET camera is based on two Position Sensitive Photomultiplier Tubes (PSPMT), coupled to a pixilated Sodium Iodide activated with Thallium (NaI(Tl)) scintillator, having an active area of 5x10cm2. The dual head PET camera is also based on two pairs of PSPMT, coupled to pixelated berillium germanium oxide (BGO) scintillators, having an active area of 5x10cm2. The X-rays system consists of a micro focus X-rays tube and a complementary metal-oxide-semiconductor (CMOS) detector, having an active area of 12x12cm2. RESULTS: The scintigraphic mode has a spatial resolution of 1.88mm full width at half maximum (FWHM) and a sensitivity of 107.5cpm/0.037MBq at the collimator surface. The coincidence PET mode has an average spatial resolution of 3.5mm (FWHM) and a peak sensitivity of 29.9cpm/0.037MBq. The X-rays spatial resolution is 3.5lp/mm and the contrast discrimination function value is lower than 2%. CONCLUSION: A compact tri-modal system was successfully built and evaluated for planar mode operation. The system has an efficient performance, allowing accurate and informative anatomical and functional imaging, as well as semi-quantitative results. Compared to other available systems, it provides a moderate but comparable performance, at a fraction of the cost and complexity. It is fully open, scalable and its main purpose is to support groups on a national and regional level and provide an open technological platform to study different detector components and acquisition strategies.

Versatile quarto stimuli nanostructure based on Trojan Horse approach for cancer therapy: Synthesis, characterization, in vitro and in vivo studies.

Nanostructured delivery and diagnostic systems that induces specific targeting properties by exploiting the local physicochemical tumour characteristics will be evaluated is the present work. It is well known that cancer cells have specific physicochemical characteristics, which can be taken into consideration for the design of a broad spectrum of drug delivery systems (DDS). Some of those characteristics including the different temperature environment their susceptibility when temperature ranges between 40 and 43°C where cell apoptosis is induced, the intra- and extra-cellular pH which varies from 6.0 to 6.8, for cancer cells, and 6.5 to 7.4 for normal cells respectively, (lysosomes acidic pH ranges 4-5). Additional significant factors are the overexpressed receptors on the tumour surface. Loading and release studies were carried out by using the anthracycline drug Doxorubicin and their cytotoxicity was evaluated by using the MTT assay in healthy and diseased cell lines. The highlight of this work is the in vitro and in vivo studies which were performed in order to evaluate different nanostructures as for their biodistribution, pharmacokinetic and toxicity per se.

Characterization of "γ-Eye": a Low-Cost Benchtop Mouse-Sized Gamma Camera for Dynamic and Static Imaging Studies.

PURPOSE: Several preclinical imaging systems are commercially available, but their purchase and maintenance costs make them unaffordable for the majority of small- and medium-sized groups. Taking into account the needs of average users, we developed "γ-eye", a mouse-sized, benchtop γ-camera suitable for in vivo scintigraphic imaging. PROCEDURES: The γ-eye is based on two position-sensitive photomultiplier tubes, coupled to a CsI(Na) pixelated scintillator and a low-energy lead collimator with parallel hexagonal holes. RESULTS: The spatial resolution of the system is 2 mm at 0 mm. The energy resolution is 26 % at 140 keV and the maximum recorded sensitivity 210 cps/MBq. The system was evaluated in a proof-of-concept animal study, using three different clinical Tc-99m-labeled radiopharmaceuticals. Phantom and animal studies demonstrate its ability to provide semiquantitative results even for short scans. CONCLUSIONS: Systems' performance, dimensions, and cost make γ-eye a unique solution for efficient whole-body mouse nuclear imaging.

Evaluation of ανß3-mediated tumor expression with a 99mTc-labeled ornithine-modified RGD derivative during glioblastoma growth in vivo.

In this study, a novel way of distinguishing the intrinsic relationship between ανß3 integrin targeting and detection of tumor growth by using a radiolabeled tracer based on a cyclic Arg-Gly-Asp (RGD) peptide was provided. The potential of the in vivo scintigraphic imaging of the developing vasculature from the early stage of tumor growth was evaluated. Alongside with the scintigraphic images, biodistribution studies were performed at distinct time points to validate this noninvasive imaging approach. The ability to noninvasively assess the tumor growth of ανß3 integrin-positive glioblastoma tumors provides a method to better understand tumor angiogenesis in vivo and allows for a direct assessment of anti-integrin treatment efficacy.

Hollow microspheres based on - Folic acid modified - Hydroxypropyl Cellulose and synthetic multi-responsive bio-copolymer for targeted cancer therapy: controlled release of daunorubicin, in vitro and in vivo studies.

HYPOTHESIS: Conventional chemotherapy drugs such as anthracyclines show no specific activity. They destroy cancer cells but also and the healthy ones, and for that reason exhibit high toxicity. In order to alleviate the toxic effects of chemotherapeutic drugs, the administration dose is being minimized, while their reactivity against tumor cells is lessened. This problem can be overcome or at least reduced by using nanoscale drug delivery systems to target the pathogenic area. The present work deals with the synthesis, characterization and biological evaluation of multi-responsive hollow microspheres coated with Hydroxypropyl Cellulose (HPC)-a biocompatible and thermosensitive polysaccharide-conjugated with folic acid as well promising drug vehicles for targeted cancer therapy. EXPERIMENTS: The synthetic route consists of two steps. In the first step, a single layer of sensitive copolymers is ((Methacrylic acid (MAA), N-(2-Hydroxypropyl) methacrylamide (HPMA) and N,N'-(disulfanediylbis(ethane-2,1-diyl))bis(2-methylacrylamide) (DSBMA)) fabricated on a sacrificial template of SiO2 and in the second step, an additional layer of the folic acid modified HPC coat the microspheres' surface. The layers fabrication is performed through a combination of distillation precipitation co-polymerization and chemical deposition method. The loading capacity (% LC) and encapsulation efficiency (% EE) percentages of the chemotherapeutic agent daunorubicin (DNR) in the fabricated microspheres were calculated through the standard curve methodology. In addition, the releasing properties of the resulting spheres are investigated, using the above mentioned methodology. It is worth mentioning that, spheres release the entrapped drug under combined conditions such acidic and reductive environment along with conventional hyperthermia. Cytotoxic activity of the synthesized spheres was investigated by using the well-established method of MTT assay in MCF-7 (breast cancer), HeLa (cervical cancer) and HEK 293 (Human Embryonic Kidney healthy cells) cell lines. Confocal and fluorescence microscopy were used to confirm the in vitro targeted ability of folic acid modified drug loaded microspheres in HeLa, to that overexpress folate receptors, MCF-7 and 3T3 cells, as negative folate cell substrate. Finally, radiolabelling of the spheres is performed, with a gamma emitting radionuclide ((99m)Tc), to assess their in vivo profile by means of scintigraphic imaging and biodistribution studies. FINDINGS: Hollow spheres release the encapsulated drug under acidic environment, conventional hyperthermia or in the presence of glutathione (reductive environment). The ability of modified drug carriers to target the HeLa cells, was confirmed by confocal and fluorescence microscopy. The resulting spheres are observed to be promising drug-carriers for cancer treatment due to their releasing properties under tumor's environment and high concentration in HeLa cells via endocytosis. In addition, the empty vehicles have no toxicity in healthy cells and present antimicrobial activity.

(99m)Tc-labeled aminosilane-coated iron oxide nanoparticles for molecular imaging of ανß3-mediated tumor expression and feasibility for hyperthermia treatment.

HYPOTHESIS: Dual-modality imaging agents, such as radiolabeled iron oxide nanoparticles (IO-NPs), are promising candidates for cancer diagnosis and therapy. We developed and evaluated aminosilane coated Fe3O4 (10±2nm) as a tumor imaging agent in nuclear medicine through 3-aminopropyltriethoxysilane (APTES) functionalization. We evaluated this multimeric system of targeted (99m)Tc-labeled nanoparticles (NPs) conjugated with a new RGD derivate (cRGDfK-Orn3-CGG), characterized as NPs-RGD as a potential thermal therapy delivery vehicle. EXPERIMENTS: Transmission Electron Microscopy (TEM) and spectroscopy techniques were used to characterize the IO-NPs indicating their functionalization with peptides. Radiolabeled IO-NPs (targeted, non-targeted) were evaluated with regard to their radiochemical, radiobiological and imaging characteristics. In vivo studies were performed in normal and ανß3-positive tumor (U87MG glioblastoma) bearing mice. We also demonstrated that this system could reach ablative temperatures in vivo. FINDINGS: Both radiolabeled IO-NPs were obtained in high radiochemical yield (>98%) and proved stable in vitro. The in vivo studies for both IO-NPs have shown significant liver and spleen uptake at all examined time points in normal and U87MG glioblastoma tumor-bearing mice, due to their colloidal nature. We have confirmed through in vivo biodistribution studies that the non-targeted (99m)Tc-NPs poorly internalized in the tumor, while the targeted (99m)Tc-NPs-RGD, present 9-fold higher tumor accumulation at 1h p.i. Accumulation of both IO-NPs in other organs was negligible. Blocking experiments indicated target specificity for integrin receptors in U87MG glioblastoma cells. The preliminary in vivo study of applied alternating magnetic field showed that the induced hyperthermia is feasible due to the aid of IO-NPs.

In vivo anticancer evaluation of the hyperthermic efficacy of anti-human epidermal growth factor receptor-targeted PEG-based nanocarrier containing magnetic nanoparticles.

Polymeric nanoparticles with targeting moieties containing magnetic nanoparticles as theranostic agents have considerable potential for the treatment of cancer. Here we report the chemical synthesis and characterization of a poly(D,L-lactide-co-glycolide)-b-poly(ethylene glycol)-based nanocarrier containing iron oxide nanoparticles and human epithelial growth factor receptor on the outer shell. The nanocarrier was also radiolabeled with (99m)Tc and tested as a theranostic nanomedicine, ie, it was investigated for both its diagnostic ability in vivo and its therapeutic hyperthermic effects in a standard A431 human tumor cell line. Following radiolabeling with (99m)Tc, the biodistribution and therapeutic hyperthermic effects of the nanosystem were studied noninvasively in vivo in tumor-bearing mice. A substantial decrease in tumor size correlated with an increase in both nanoparticle concentration and local temperature was achieved, confirming the possibility of using this multifunctional nanosystem as a therapeutic tool for epidermoid carcinoma.

Comparative in vitro stability and scintigraphic imaging for trafficking and tumor targeting of a directly and a novel 99mTc(I)(CO)3 labeled liposome.

Liposomes radiolabelling with diagnostic radionuclides is an excellent tool for studying pharmacokinetics with the view of developing liposome-based drug delivery agents. The aim of the present study is to evaluate the in vitro and in vivo behavior of a (99m)Tc-labeled liposome by applying either a direct labeling strategy via a carboxyl group, LP-COOH, or a surface chelating method via pyridyl ethyl cysteine compound (with the intermediate [99mTc(I)(CO)3(H2O)(3)](+)), LP-PEC. 99mTc-LP-COOH was obtained in high radiolabelling yield and radiochemical purity, while 99mTc(I)(CO)3-LP-PEC was initially purified before being in vitro and in vivo evaluated. 99mTc-LP-COOH was less stable in the presence of competitive for 99mTc ligands than 99mTc(I)(CO)3-LP-PEC. According to DLS measurements, the presence of serum as well as the applied radiolabelling conditions did not affect the liposomes' size. The different radiolabelling methods seemed to exert an influence on the biodistribution pattern of the liposomes with the 99mTc(I)(CO)3-LP-PEC showing slow blood clearance, which was also confirmed by in vivo scintigraphic imaging. Nevertheless, passive tumor targeting was attained at a similar extent no matter which radiolabelling technique was followed.

Targeted delivery of silver nanoparticles and alisertib: in vitro and in vivo synergistic effect against glioblastoma.

AIM: Targeted biocompatible nanoplatforms presenting multiple therapeutic functions have great potential for the treatment of cancer. MATERIALS & METHODS: Multifunctional nanocomposites formed by polymeric nanoparticles (PNPs) containing two cytotoxic agents - the drug alisertib and silver nanoparticles - were synthesized. These PNPs have been conjugated with a chlorotoxin, an active targeting 36-amino acid-long peptide that specifically binds to MMP-2, a receptor overexpressed by brain cancer cells. RESULTS: The individual and synergistic activity of these two cytotoxic agents against glioblastoma multiforme was tested both in vitro and in vivo. The induced cytotoxicity in a human glioblastoma-astrocytoma epithelial-like cell line (U87MG) was studied in vitro through a trypan blue exclusion test after 48 and 72 h of exposure. Subsequently, the PNPs' biodistribution in healthy animals and their effect on tumor reduction in tumor-bearing mice were studied using PNPs radiolabeled with (99m)Tc. CONCLUSION: Tumor reduction was achieved in vivo when using silver/alisertib@PNPs-chlorotoxin.

Dynamic in vivo imaging of dual-triggered microspheres for sustained release applications: synthesis, characterization and cytotoxicity study.

This paper deals with the synthesis, characterization and property evaluation of drug-loaded magnetic microspheres with pH-responsive cross-linked polymer shell. The synthetic procedure consists of 3 steps, of which the first two comprise the synthesis of a poly methyl methacrylate (PMMA) template and the synthesis of a shell by using acrylic acid (AA) and methyl methacrylate (MMA) as monomers, and divinyl benzene (DVB) as cross-linker. The third step of the procedure refers to the formation of magnetic nanoparticles on the microsphere's surface. AA that attaches pH-sensitivity in the microspheres and magnetic nanoparticles in the inner and the outer surface of the microspheres, enhance the efficacy of this intelligent drug delivery system (DDS), which constitutes a promising approach toward cancer therapy. A number of experimental techniques were used to characterize the resulting microspheres. In order to investigate the in vitro controlled release behavior of the synthesized microspheres, we studied the Dox release percentage under different pH conditions and under external magnetic field. Hyperthermia caused by an alternating magnetic field (AFM) is used in order to study the doxorubicin (Dox) release behavior from microspheres with pH functionality. The in vivo fate of these hybrid-microspheres was tracked by labeling them with the γ-emitting radioisotope (99m)Tc after being intravenously injected in normal mice. According to our results, microsphere present a pH depending and a magnetic heating, release behavior. As expected, labeled microspheres were mainly found in the mononuclear phagocyte system (MPS). The highlights of the current research are: (i) to illustrate the advantages of controlled release by combining hyperthermia and pH-sensitivity and (ii) to provide noninvasive, in vivo information on the spatiotemporal biodistribution of these microsphere by dynamic γ-imaging.

Biological evaluation of an ornithine-modified (99m)Tc-labeled RGD peptide as an angiogenesis imaging agent.

INTRODUCTION: Radiolabeled RGD peptides that specifically target integrin α(ν)ß(3) have great potential in early tumor detection through noninvasive monitoring of tumor angiogenesis. Based on previous findings of our group on radiopeptides containing positively charged aminoacids, we developed a new cyclic cRGDfK derivative, c(RGDfK)-(Orn)(3)-CGG. This new peptide availing the polar linker (Orn)(3) and the (99m)Tc-chelating moiety CGG (Cys-Gly-Gly) is appropriately designed for (99m)Tc-labeling, as well as consequent conjugation onto nanoparticles. METHODS: A tumor imaging agent, c(RGDfK)-(Orn)(3)-[CGG-(99m)Tc], is evaluated with regard to its radiochemical, radiobiological and imaging characteristics. RESULTS: The complex c(RGDfK)-(Orn)(3)-[CGG-(99m)Tc] was obtained in high radiochemical yield (>98%) and was stable in vitro and ex vivo. It presented identical to the respective, fully analytically characterized (185/187)Re complex retention time in RP-HPLC. In contrary to other RGD derivatives, we showed that the new radiopeptide exhibits kidney uptake and urine excretion due to the ornithine linker. High tumor uptake (3.87±0.48% ID/g at 60 min p.i.) was observed and was maintained relatively high even at 24 h p.i. (1.83±0.05 % ID/g), thus providing well-defined scintigraphic imaging. Accumulation in other organs was negligible. Blocking experiments indicated target specificity for integrin receptors in U87MG glioblastoma cells. CONCLUSION: Due to its relatively high tumor uptake, renal elimination and negligible abdominal localization, the new (99m)Tc-RGD peptide is considered promising in the field of imaging α(ν)ß(3)-positive tumors. However, the preparation of multifunctional SPECT/MRI contrast agents (RGD-conjugated nanoparticles) for dual modality imaging of integrin expressing tumors should be further investigated.

Menstrual and reproductive factors in women, genetic variation in CYP17A1, and pancreatic cancer risk in the European prospective investigation into cancer and nutrition (EPIC) cohort.

Menstrual and reproductive factors and exogenous hormone use have been investigated as pancreatic cancer risk factors in case-control and cohort studies, but results have been inconsistent. We conducted a prospective examination of menstrual and reproductive factors, exogenous hormone use and pancreatic cancer risk (based on 304 cases) in 328,610 women from the EPIC cohort. Then, in a case-control study nested within the EPIC cohort, we examined 12 single nucleotide polymorphisms (SNPs) in CYP17A1 (an essential gene in sex steroid metabolism) for association with pancreatic cancer in women and men (324 cases and 353 controls). Of all factors analyzed, only younger age at menarche (<12 vs. 13 years) was moderately associated with an increased risk of pancreatic cancer in the full cohort; however, this result was marginally significant (HR = 1.44; 95% CI = 0.99-2.10). CYP17A1 rs619824 was associated with HRT use (p value = 0.037) in control women; however, none of the SNPs alone, in combination, or as haplotypes were associated with pancreatic cancer risk. In conclusion, with the possible exception of an early age of menarche, none of the menstrual and reproductive factors, and none of the 12 common genetic variants we evaluated at the CYP17A1 locus makes a substantial contribution to pancreatic cancer susceptibility in the EPIC cohort.

Structural modifications of 99mTc-labelled bombesin-like peptides for optimizing pharmacokinetics in prostate tumor targeting.

PURPOSE: The main goal of the present study was to investigate the importance of the addition of a positively charged aa in the naturally occurring bombesin (BN) peptide for its utilization as radiodiagnostic agent, taking into consideration the biodistribution profile, the pharmacokinetic characteristics and the tumor targeting ability. METHODS: Two BN-derivatives of the general structure [M-chelator]-(spacer)-BN(2-14)-NH(2), where M: (99m)Tc or (185/187)Re, chelator: Gly-Gly-Cys-, spacer: -(arginine)(3)-, M-BN-A; spacer: -(ornithine)(3)-, M-BN-O; have been prepared and evaluated as tumor imaging agents. RESULTS: The peptides under study presented high radiolabelling efficiency (>98%), significant stability in human plasma (>60% intact radiolabelled peptide after 1h incubation) and comparable receptor binding affinity with the standard [(125)I-Tyr(4)]-BN. Their internalization rates in the prostate cancer PC-3 cells differed, although the amount of internalized peptide was the same. The biodistribution and the dynamic γ-camera imaging studies in normal and PC-3 tumor-bearing SCID mice have shown significant tumor uptake, combined with fast blood clearance, through the urinary pathway. CONCLUSION: The addition of the charged aa spacer in the BN structure was advantageous for biodistribution, pharmacokinetics and tumor targeting ability, because it reduced the upper abdominal radioactivity levels and increased tumor/normal tissue contrast ratios.

Spacer site modifications for the improvement of the in vitro and in vivo binding properties of (99m)Tc-N(3)S-X-bombesin[2-14] derivatives.

It has been shown that gastrin releasing peptide receptors (GRPRs) are overexpressed in various types of cancer cells. Bombesin is an analogue of the mammalian GRP that binds with high specificity and affinity to GRPRs. Significant research efforts have been lately devoted to the design of radiolabeled 8 or 14 aminoacid bombesin (BN) peptides for the detection (either with gamma or positron emitting radionuclides) and therapy (with beta(-) emitting radionuclides) of cancer. The specific aim of the present study was to further investigate the radiolabeled peptide structure and to determine whether the total absence of a linker or the use of a basic diverse amino acid linker could influence the biodistribution profile of the new compounds for specific targeting of human prostate cancer. Thus, two new derivatives with the structure Gly-Gly-Cys-X-BN[2-14], where linker X is either zero (I) or Orn-Orn-Orn (Orn: ornithine) (II) were designed and synthesized. The corresponding (99m)Tc-BN derivatives were obtained with high radiochemical yield (>98%) and had almost identical retention times in RP-HPLC with the (185/187)Re complexes, which were also characterized by ESI-MS. Metabolic stability was found to be high in human plasma, moderate in PC-3 cells, and rather low in mouse liver and kidney homogenates for both BN derivatives studied. The BN derivative without the spacer was less stable in cell culture and liver homogenates. A satisfactory binding affinity to GRPRs, in the nanomolar range, was obtained for both BN derivatives as well as for their Re complexes, with BN (II) demonstrating the highest one. In vitro internalization/externalization assays indicated that approximately 6% of BN (I) and approximately 25% of BN (II) were internalized into PC-3 cells. In vivo evaluation in normal Swiss mice and in tumor bearing SCID mice showed that BN (II) presented higher tumor and pancreas uptake than BN (I). Small animal SPECT dynamic imaging, carried out after an injection of BN (II) in mice bearing PC-3 tumors, resulted in PC-3 tumor delineation with low background activity. Overall, this study performed for two new N(3)S-X-BN[2-14] derivatives indicated that hydrophilicity and charge strongly affected the in vitro and in vivo binding properties and the biodistribution pattern. This finding is confirmed by SPECT imaging of BN (II), which is under further in vivo evaluation for detecting cancer-positive GRPRs.