Evaluation of a 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid-conjugated bombesin-based radioantagonist for the labeling with single-photon emission computed tomography, positron emission tomography, and therapeutic radionuclides

PURPOSE: G protein-coupled receptor agonists are being used as radiolabeled vectors for in vivo localization and therapy of tumors. Recently, somatostatin-based antagonists were shown to be superior to agonists. Here, we compare the new [111In/68Ga]-labeled bombesin-based antagonist RM1 with the agonist [111In]-AMBA for targeting the gastrin-releasing peptide receptor (GRPR). EXPERIMENTAL DESIGN: IC50, Kd values, and antagonist potency were determined using PC-3 and HEK-GRPR cells. Biodistribution and imaging studies were done in nude mice transplanted with the PC-3 tumor. The antagonist potency was assessed by evaluating the effects on calcium release and on receptor internalization monitored by immunofluorescence microscopy. RESULTS: The IC50 value of [(nat)In]-RM1 was 14 +/- 3.4 nmol/L. [(nat/111)In]-RM1 was found to bind to the GRPR with a Kd of 8.5 +/- 2.7 nmol/L compared with a Kd of 0.6 +/- 0.3 nmol/L of [111In]-AMBA. A higher maximum number of binding site value was observed for [111In]-RM1 (2.4 +/- 0.2 nmol/L) compared with [111In]-AMBA (0.7 +/- 0.1 nmol/L). [(nat)Lu]-AMBA is a potent agonist in the immunofluorescence-based internalization assay, whereas [(nat)In]-RM1 is inactive alone but efficiently antagonizes the bombesin effect. These data are confirmed by the calcium release assay. The pharmacokinetics showed a superiority of the radioantagonist with regard to the high tumor uptake (13.4 +/- 0.8% IA/g versus 3.69 +/- 0.75% IA/g at 4 hours after injection. as well as to all tumor-to-normal tissue ratios. CONCLUSION: Despite their relatively low GRPR affinity, the antagonists [111In/68Ga]-RM1 showed superior targeting properties compared with [111In]-AMBA. As found for somatostatin receptor-targeting radiopeptides, GRP-based radioantagonists seem to be superior to radioagonists for in vivo imaging and potentially also for targeted radiotherapy of GRPR-positive tumors.

Early gene expression analysis in 9L orthotopic tumor-bearing rats identifies immune modulation in molecular response to synchrotron microbeam radiation therapy

Synchrotron Microbeam Radiation Therapy (MRT) relies on the spatial fractionation of the synchrotron photon beam into parallel micro-beams applying several hundred of grays in their paths. Several works have reported the therapeutic interest of the radiotherapy modality at preclinical level, but biological mechanisms responsible for the described efficacy are not fully understood to date. The aim of this study was to identify the early transcriptomic responses of normal brain and glioma tissue in rats after MRT irradiation (400Gy). The transcriptomic analysis of similarly irradiated normal brain and tumor tissues was performed 6 hours after irradiation of 9 L orthotopically tumor-bearing rats. Pangenomic analysis revealed 1012 overexpressed and 497 repressed genes in the irradiated contralateral normal tissue and 344 induced and 210 repressed genes in tumor tissue. These genes were grouped in a total of 135 canonical pathways. More than half were common to both tissues with a predominance for immunity or inflammation (64 and 67% of genes for normal and tumor tissues, respectively). Several pathways involving HMGB1, toll-like receptors, C-type lectins and CD36 may serve as a link between biochemical changes triggered by irradiation and inflammation and immunological challenge. Most immune cell populations were involved: macrophages, dendritic cells, natural killer, T and B lymphocytes. Among them, our results highlighted the involvement of Th17 cell population, recently described in tumor. The immune response was regulated by a large network of mediators comprising growth factors, cytokines, lymphokines. In conclusion, early response to MRT is mainly based on inflammation and immunity which appear therefore as major contributors to MRT efficacy.

Implementation of an Anthropomorphic Phantom for the Evaluation of Proton Therapy Treatment Procedures

With an increasing number of institutions offering proton therapy, the number of multi-institutional clinical trials involving proton therapy will also increase in the coming years. The Radiological Physics Center monitors sites involved in clinical trials through the use of site visits and remote auditing with thermoluminescent dosimeters (TLD) and mailable anthropomorphic phantoms. Currently, there are no heterogeneous phantoms that have been commissioned to evaluate proton therapy. It was hypothesized that an anthropomorphic pelvis phantom can be designed to audit treatment procedures (patient simulation, treatment planning and treatment delivery) at proton facilities to confirm agreement between the measured dose and calculated dose within 5%/3mm with a reproducibility of 3%. A pelvis phantom originally designed for use with photon treatments was retrofitted for use in proton therapy. The relative stopping power (SP) of each phantom material was measured. Hounsfield Units (HU) for each phantom material were measured with a CT scanner and compared to the relative stopping power calibration curve. The tissue equivalency for each material was calculated. Two proton treatment plans were created; one which did not correct for material SP differences (Plan 1) and one plan which did correct for SP differences (Plan 2). Film and TLD were loaded into the phantom and the phantom was irradiated 3 times per plan. The measured values were compared to the HU-SP calibration curve and it was found that the stopping powers for the materials could be underestimated by 5-10%. Plan 1 passed the criteria for the TLD and film margins with reproducibility under 3% between the 3 trials. Plan 2 failed because the right-left film dose profile average displacement was -9.0 mm on the left side and 6.0 mm on the right side. Plan 2 was intended to improve the agreements and instead introduced large displacements along the path of the beam. Plan 2 more closely represented the actual phantom composition with corrected stopping powers and should have shown an agreement between the measured and calculated dose within 5%/3mm. The hypothesis was rejected and the pelvis phantom was found to be not suitable to evaluate proton therapy treatment procedures.

Risk of Second Malignant Neoplasms Following Proton Arc Therapy and Volumetric Modulated Arc Therapy for Prostate Cancer

The risk of second malignant neoplasms (SMNs) following prostate radiotherapy is a concern due to the large population of survivors and decreasing age at diagnosis. It is known that parallel-opposed beam proton therapy carries a lower risk than photon IMRT. However, a comparison of SMN risk following proton and photon arc therapies has not previously been reported. The purpose of this study was to predict the ratio of excess relative risk (RRR) of SMN incidence following proton arc therapy to that after volumetric modulated arc therapy (VMAT). Additionally, we investigated the impact of margin size and the effect of risk-minimized proton beam weighting on predicted RRR. Physician-approved treatment plans were created for both modalities for three patients. Therapeutic dose was obtained with differential dose-volume histograms from the treatment planning system, and stray dose was estimated from the literature or calculated with Monte Carlo simulations. Then, various risk models were applied to the total dose. Additional treatment plans were also investigated with varying margin size and risk-minimized proton beam weighting. The mean RRR ranged from 0.74 to 0.99, depending on risk model. The additional treatment plans revealed that the RRR remained approximately constant with varying margin size, and that the predicted RRR was reduced by 12% using a risk-minimized proton arc therapy planning technique. In conclusion, proton arc therapy was found to provide an advantage over VMAT in regard to predicted risk of SMN following prostate radiotherapy. This advantage was independent of margin size and was amplified with risk-optimized proton beam weighting.

Pencil Beam Scanning Proton Therapy for Pediatric Parameningeal Rhabdomyosarcomas: Clinical Outcome of Patients Treated at the Paul Scherrer Institute

BACKGROUND Parameningeal rhabdomyosarcomas (PM-RMSs) represent approximately 25% of all rhabdomyosarcoma (RMS) cases. These tumors are associated with early recurrence and poor prognosis. This study assessed the clinical outcome and late toxicity of pencil beam scanning (PBS) proton therapy (PT) in the treatment of children with PM-RMS. PROCEDURES Thirty-nine children with PM-RMS received neoadjuvant chemotherapy followed by PBS-PT at the Paul Scherrer Institute, with concomitant chemotherapy. The median age was 5.8 years (range, 1.2-16.1). Due to young age, 25 patients (64%) required general anesthesia during PT. The median time from the start of chemotherapy to PT was 13 weeks (range, 3-23 weeks). Median prescription dose was 54 Gy (relative biologic effectiveness, RBE). RESULTS With a mean follow-up of 41 months (range, 9-106 months), 10 patients failed. The actuarial 5-year progression-free survival (PFS) was 72% (95% CI, 67-94%) and the 5-year overall survival was 73% (95% CI, 69-96%). On univariate analysis, a delay in the initiation of PT (>13 weeks) was a significant detrimental factor for PFS. Three (8%) patients presented with grade 3 radiation-induced toxicity. The estimated actuarial 5-year toxicity ≥grade 3 free survival was 95% (95% CI, 94-96%). CONCLUSIONS Our data contribute to the growing body of evidence demonstrating the safety and effectiveness of PT for pediatric patients with PM-RMS. These preliminary results are encouraging and in line with other combined proton-photon and photons series; observed toxicity was acceptable.

Dose-painting intensity-modulated proton therapy for intermediate- and high-risk meningioma.

BACKGROUND Newly diagnosed WHO grade II-III or any WHO grade recurrent meningioma exhibit an aggressive behavior and thus are considered as high- or intermediate risk tumors. Given the unsatisfactory rates of disease control and survival after primary or adjuvant radiation therapy, optimization of treatment strategies is needed. We investigated the potential of dose-painting intensity-modulated proton beam-therapy (IMPT) for intermediate- and high-risk meningioma. MATERIAL AND METHODS Imaging data from five patients undergoing proton beam-therapy were used. The dose-painting target was defined using [68]Ga-[1,4,7,10-tetraazacyclododecane tetraacetic acid]- d-Phe(1),Tyr(3)-octreotate ([68]Ga-DOTATATE)-positron emission tomography (PET) in target delineation. IMPT and photon intensity-modulated radiation therapy (IMRT) treatment plans were generated for each patient using an in-house developed treatment planning system (TPS) supporting spot-scanning technology and a commercial TPS, respectively. Doses of 66 Gy (2.2 Gy/fraction) and 54 Gy (1.8 Gy/fraction) were prescribed to the PET-based planning target volume (PTVPET) and the union of PET- and anatomical imaging-based PTV, respectively, in 30 fractions, using simultaneous integrated boost. RESULTS Dose coverage of the PTVsPET was equally good or slightly better in IMPT plans: dose inhomogeneity was 10 ± 3% in the IMPT plans vs. 13 ± 1% in the IMRT plans (p = 0.33). The brain Dmean and brainstem D50 were small in the IMPT plans: 26.5 ± 1.5 Gy(RBE) and 0.002 ± 0.0 Gy(RBE), respectively, vs. 29.5 ± 1.5 Gy (p = 0.001) and 7.5 ± 11.1 Gy (p = 0.02) for the IMRT plans, respectively. The doses delivered to the optic structures were also decreased with IMPT. CONCLUSIONS Dose-painting IMPT is technically feasible using currently available planning tools and resulted in dose conformity of the dose-painted target comparable to IMRT with a significant reduction of radiation dose delivered to the brain, brainstem and optic apparatus. Dose escalation with IMPT may improve tumor control and decrease radiation-induced toxicity.

Understanding the Influence of Photon Energy on 6MV Non Reference Dosimetry and CT Dosimetry using TLD and OSLD

Measurement of the absorbed dose from ionizing radiation in medical applications is an essential component to providing safe and reproducible patient care. There are a wide variety of tools available for measuring radiation dose; this work focuses on the characterization of two common, solid-state dosimeters in medical applications: thermoluminescent dosimeters (TLD) and optically stimulated luminescent dosimeters (OSLD). There were two main objectives to this work. The first objective was to evaluate the energy dependence of TLD and OSLD for non-reference measurement conditions in a radiotherapy environment. The second objective was to fully characterize the OSLD nanoDot in a CT environment, and to provide validated calibration procedures for CT dose measurement using OSLD. Current protocols for dose measurement using TLD and OSLD generally assume a constant photon energy spectrum within a nominal beam energy regardless of measurement location, tissue composition, or changes in beam parameters. Variations in the energy spectrum of therapeutic photon beams may impact the response of TLD and OSLD and could thereby result in an incorrect measure of dose unless these differences are accounted for. In this work, we used a Monte Carlo based model to simulate variations in the photon energy spectra of a Varian 6MV beam; then evaluated the impact of the perturbations in energy spectra on the response of both TLD and OSLD using Burlin Cavity Theory. Energy response correction factors were determined for a range of conditions and compared to measured correction factors with good agreement. When using OSLD for dose measurement in a diagnostic imaging environment, photon energy spectra are often referenced to a therapy-energy or orthovoltage photon beam – commonly 250kVp, Co-60, or even 6MV, where the spectra are substantially different. Appropriate calibration techniques specifically for the OSLD nanoDot in a CT environment have not been presented in the literature; furthermore the dependence of the energy response of the calibration energy has not been emphasized. The results of this work include detailed calibration procedures for CT dosimetry using OSLD, and a full characterization of this dosimetry system in a low-dose, low-energy setting.

Testosterone Therapy Differently Regulates The Anti- And Pro-inflammatory Cytokines In The Plasma And Prostate Of Rats Submitted To Chronic Ethanol Consumption (uchb).

Ethanol consumption damages the prostate, and testosterone is known by anti-inflammatory role. The cytokines were investigated in the plasma and ventral prostate of UChB rats submitted or not to testosterone therapy by ELISA and Western blot, respectively. Additionally, inflammatory foci and mast cells were identified in the ventral prostate slides stained by hematoxylin and eosin and toluidine blue, respectively. Inflammatory foci were found in the ethanol-treated animals and absent after testosterone therapy. Plasma levels of IL-6 and IL-10 were not changed while TNFα and TFG-β1 were increased in the animals submitted testosterone therapy. Regarding to ventral prostate, IL-6 did not alter, while IL-10, TNFα, and TFG-β1 were increased after testosterone therapy. Ethanol increases NFR2 in addition to high number of intact and degranulated mast cell which were reduced after testosterone therapy. So, ethanol and testosterone differentially modulates the cytokines in the plasma and prostate.

Genetic Predictors Of Long-term Response To Growth Hormone (gh) Therapy In Children With Gh Deficiency And Turner Syndrome: The Influence Of A Socs2 Polymorphism.

There is great interindividual variability in the response to GH therapy. Ascertaining genetic factors can improve the accuracy of growth response predictions. Suppressor of cytokine signaling (SOCS)-2 is an intracellular negative regulator of GH receptor (GHR) signaling. The objective of the study was to assess the influence of a SOCS2 polymorphism (rs3782415) and its interactive effect with GHR exon 3 and -202 A/C IGFBP3 (rs2854744) polymorphisms on adult height of patients treated with recombinant human GH (rhGH). Genotypes were correlated with adult height data of 65 Turner syndrome (TS) and 47 GH deficiency (GHD) patients treated with rhGH, by multiple linear regressions. Generalized multifactor dimensionality reduction was used to evaluate gene-gene interactions. Baseline clinical data were indistinguishable among patients with different genotypes. Adult height SD scores of patients with at least one SOCS2 single-nucleotide polymorphism rs3782415-C were 0.7 higher than those homozygous for the T allele (P < .001). SOCS2 (P = .003), GHR-exon 3 (P= .016) and -202 A/C IGFBP3 (P = .013) polymorphisms, together with clinical factors accounted for 58% of the variability in adult height and 82% of the total height SD score gain. Patients harboring any two negative genotypes in these three different loci (homozygosity for SOCS2 T allele; the GHR exon 3 full-length allele and/or the -202C-IGFBP3 allele) were more likely to achieve an adult height at the lower quartile (odds ratio of 13.3; 95% confidence interval of 3.2-54.2, P = .0001). The SOCS2 polymorphism (rs3782415) has an influence on the adult height of children with TS and GHD after long-term rhGH therapy. Polymorphisms located in GHR, IGFBP3, and SOCS2 loci have an influence on the growth outcomes of TS and GHD patients treated with rhGH. The use of these genetic markers could identify among rhGH-treated patients those who are genetically predisposed to have less favorable outcomes.

Dyspareunia And Lubrication In Premature Ovarian Failure Using Hormonal Therapy And Vaginal Health.

To evaluate vaginal microbiological and functional aspects in women with and without premature ovarian failure (POF) and the relationship with sexual function. A cross-sectional study of 36 women with POF under hormonal therapy who were age-matched with 36 women with normal gonadal function. The vaginal tropism was assessed through hormonal vaginal cytology, vaginal pH and vaginal health index (VHI). Vaginal flora were assessed by the amine test, bacterioscopy and culture for fungi. Sexual function was evaluated through the questionnaire Female Sexual Function Index (FSFI). Women in both groups were of similar age and showed similar marital status. The two groups presented vaginal tropic scores according to the VHI but the tropism was worse among women in the POF group. No difference was observed with respect to hormonal cytology and pH. Vaginal flora was similar in both groups. Women with POF showed worse sexual performance with more pain and poorer lubrication than women in the control group. The VHI, the only parameter evaluated showing statistical difference between the groups, did not correlate with the domains of pain and lubrication in the FSFI questionnaire. These findings suggest that the use of systemic estrogen among women with POF is not enough to improve complaints of lubrication and pain despite conferring similar tropism and vaginal flora. Other therapeutic options need to be evaluated.

The Effect Of Soy Dietary Supplement And Low Dose Of Hormone Therapy On Main Cardiovascular Health Biomarkers: A Randomized Controlled Trial.

To assess the effects of a soy dietary supplement on the main biomarkers of cardiovascular health in postmenopausal women compared with the effects of low-dose hormone therapy (HT) and placebo. Double-blind, randomized and controlled intention-to-treat trial. Sixty healthy postmenopausal women, aged 40-60 years, 4.1 years mean time since menopause were recruited and randomly assigned to 3 groups: a soy dietary supplement group (isoflavone 90mg), a low-dose HT group (estradiol 1 mg plus noretisterone 0.5 mg) and a placebo group. Lipid profile, glucose level, body mass index, blood pressure and abdominal/hip ratio were evaluated in all the participants at baseline and after 16 weeks. Statistical analyses were performed using the χ2 test, Fisher's exact test, Kruskal-Wallis non-parametric test, analysis of variance (ANOVA), paired Student's t-test and Wilcoxon test. After a 16-week intervention period, total cholesterol decreased 11.3% and LDL-cholesterol decreased 18.6% in the HT group, but both did not change in the soy dietary supplement and placebo groups. Values for triglycerides, HDL-cholesterol, glucose level, body mass index, blood pressure and abdominal/hip ratio did not change over time in any of the three groups. The use of dietary soy supplement did not show any significant favorable effect on cardiovascular health biomarkers compared with HT. The trial is registered at the Brazilian Clinical Trials Registry (Registro Brasileiro de Ensaios Clínicos - ReBEC), number RBR-76mm75.

Influence Of Delivery Method On Neuroprotection By Bone Marrow Mononuclear Cell Therapy Following Ventral Root Reimplantation With Fibrin Sealant.

The present work compared the local injection of mononuclear cells to the spinal cord lateral funiculus with the alternative approach of local delivery with fibrin sealant after ventral root avulsion (VRA) and reimplantation. For that, female adult Lewis rats were divided into the following groups: avulsion only, reimplantation with fibrin sealant; root repair with fibrin sealant associated with mononuclear cells; and repair with fibrin sealant and injected mononuclear cells. Cell therapy resulted in greater survival of spinal motoneurons up to four weeks post-surgery, especially when mononuclear cells were added to the fibrin glue. Injection of mononuclear cells to the lateral funiculus yield similar results to the reimplantation alone. Additionally, mononuclear cells added to the fibrin glue increased neurotrophic factor gene transcript levels in the spinal cord ventral horn. Regarding the motor recovery, evaluated by the functional peroneal index, as well as the paw print pressure, cell treated rats performed equally well as compared to reimplanted only animals, and significantly better than the avulsion only subjects. The results herein demonstrate that mononuclear cells therapy is neuroprotective by increasing levels of brain derived neurotrophic factor (BDNF) and glial derived neurotrophic factor (GDNF). Moreover, the use of fibrin sealant mononuclear cells delivery approach gave the best and more long lasting results.

Mastication Movements And Sleep Quality Of Patients With Myofascial Pain: Occlusal Device Therapy Improvements.

Patients with myofascial pain experience impaired mastication, which might also interfere with their sleep quality. The purpose of this study was to evaluate the jaw motion and sleep quality of patients with myofascial pain and the impact of a stabilization device therapy on both parameters. Fifty women diagnosed with myofascial pain by the Research Diagnostic Criteria were enrolled. Pain levels (visual analog scale), jaw movements (kinesiography), and sleep quality (Epworth Sleepiness Scale; Pittsburgh Sleep Quality Index) were evaluated before (control) and after stabilization device use. Range of motion (maximum opening, right and left excursions, and protrusion) and masticatory movements during Optosil mastication (opening, closing, and total cycle time; opening and closing angles; and maximum velocity) also were evaluated. Repeated-measures analysis of variance in a generalized linear mixed models procedure was used for statistical analysis (α=.05). At baseline, participants with myofascial pain showed a reduced range of jaw motion and poorer sleep quality. Treatment with a stabilization device reduced pain (P<.001) and increased both mouth opening (P<.001) and anteroposterior movement (P=.01). Also, after treatment, the maximum opening (P<.001) and closing (P=.04) velocities during mastication increased, and improvements in sleep scores for the Pittsburgh Sleep Quality Index (P<.001) and Epworth Sleepiness Scale (P=.04) were found. Myofascial pain impairs jaw motion and quality of sleep; the reduction of pain after the use of a stabilization device improves the range of motion and sleep parameters.

Morphological Aspects And Cox-2 Expression After Exposure To 780-nm Laser Therapy In Injured Skeletal Muscle: An In Vivo Study.

The effectiveness of low-level laser therapy in muscle regeneration is still not well known. To investigate the effects of laser irradiation during muscle healing. For this purpose, 63 rats were distributed to 3 groups: non-irradiated control group (CG); group irradiated at 10 J/cm(2) (G10); and group irradiated at 50 J/cm(2) (G50). Each group was divided into 3 different subgroups (n=7), and on days 7, 14 and 21 post-injury the rats were sacrificed. Seven days post-surgery, the CG showed destroyed zones and extensive myofibrillar degeneration. For both treated groups, the necrosis area was smaller compared to the CG. On day 14 post-injury, treated groups demonstrated better tissue organization, with newly formed muscle fibers compared to the CG. On the 21(st) day, the irradiated groups showed similar patterns of tissue repair, with improved muscle structure at the site of the injury, resembling uninjured muscle tissue organization. Regarding collagen deposition, the G10 showed an increase in collagen synthesis. In the last period evaluated, both treated groups showed statistically higher values in comparison with the CG. Furthermore, laser irradiation at 10 J/cm(2) produced a down-regulation of cyclooxygenase 2 (Cox-2) immunoexpression on day 7 post-injury. Moreover, Cox-2 immunoexpression was decreased in both treated groups on day 14. Laser therapy at both fluencies stimulated muscle repair through the formation of new muscle fiber, increase in collagen synthesis, and down-regulation of Cox-2 expression.

Development And Characterization Of A Cationic Lipid Nanocarrier As Non-viral Vector For Gene Therapy.

The aim of the present work was to produce a cationic solid lipid nanoparticle (SLN) as non-viral vector for protein delivery. Cationic SLN were produced by double emulsion method, composed of softisan(®) 100, cetyltrimethylammonium bromide (CTAB), Tween(®) 80, Span(®) 80, glycerol and lipoid(®) S75 loading insulin as model protein. The formulation was characterized in terms of mean hydrodynamic diameter (z-ave), polydispersity index (PI), zeta potential (ZP), stability during storage time, stability after lyophilization, effect of toxicity and transfection ability in HeLa cells, in vitro release profile and morphology. SLN were stable for 30days and showed minimal changes in their physicochemical properties after lyophilization. The particles exhibited a relatively slow release, spherical morphology and were able to transfect HeLa cells, but toxicity remained an obstacle. Results suggest that SLN are nevertheless promising for delivery of proteins or nucleic acids for gene therapy.