ABSTRACT
Plasma-derived therapeutic proteins are produced through an industrial fractionation process where proteins are purified from individual intermediates, some of which remain unused and are discarded. Relatively few plasma-derived proteins are exploited clinically, with most of available plasma being directed towards the manufacture of immunoglobulin and albumin. Although the plasma proteome provides opportunities to develop novel protein replacement therapies, particularly for rare diseases, the high cost of plasma together with small patient populations impact negatively on the development of plasma-derived orphan drugs. Enabling therapeutics development from unused plasma fractionation intermediates would therefore constitute a substantial innovation. To this objective, we characterized the proteome of unused plasma fractionation intermediates and prioritized proteins for their potential as new candidate therapies for human disease. We selected ceruloplasmin, a plasma ferroxidase, as a potential therapy for aceruloplasminemia, an adult-onset ultra-rare neurological disease caused by iron accumulation as a result of ceruloplasmin mutations. Intraperitoneally administered ceruloplasmin, purified from an unused plasma fractionation intermediate, was able to prevent neurological, hepatic and hematological phenotypes in ceruloplasmin-deficient mice. These data demonstrate the feasibility of transforming industrial waste plasma fraction into a raw material for manufacturing of new candidate proteins for replacement therapies, optimizing plasma use and reducing waste generation.
Subject(s)
Ceruloplasmin , Iron Metabolism Disorders , Neurodegenerative Diseases , Proteome , Adult , Humans , Animals , Mice , Ceruloplasmin/genetics , Ceruloplasmin/metabolism , Proteome/metabolism , Rare Diseases , Industrial WasteABSTRACT
The clinical manifestation of Parkinson's disease (PD) appears when neurodegeneration is already advanced, compromising the efficacy of disease-modifying treatment approaches. Biomarkers to identify the early stages of PD are therefore of paramount importance for the advancement of the therapy of PD. In the present study, by using a mouse model of PD obtained by subchronic treatment with the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and the clearance inhibitor probenecid (MPTPp), we identified prodromal markers of PD by combining in vivo positron emission tomography (PET) imaging and ex vivo immunohistochemistry. Longitudinal PET imaging of the dopamine transporter (DAT) by [18F]-N-(3-fluoropropyl)-2ß-carboxymethoxy-3ß-(4-iodophenyl) nortropane ([18F]-FP-CIT), and brain glucose metabolism by 2-deoxy-2-[18F]-fluoroglucose ([18F]-FDG) were performed before MPTPp treatment and after 1, 3, and 10 MPTPp administrations, in order to assess relation between dopamine neuron integrity and brain connectivity. The results show that in vivo [18F]-FP-CIT in the dorsal striatum was not modified after the first administration of MPTPp, tended to decrease after 3 administrations, and significantly decreased after 10 MPTPp administrations. Post-mortem immunohistochemical analyses of DAT and tyrosine hydroxylase (TH) in the striatum showed a positive correlation with [18F]-FP-CIT, confirming the validity of repeated MPTPp-treated mice as a model that can reproduce the progressive pathological changes in the early phases of PD. Analysis of [18F]-FDG uptake in several brain areas connected to the striatum showed that metabolic connectivity was progressively disrupted, starting from the first MPTPp administration, and that significant connections between cortical and subcortical regions were lost after 10 MPTPp administrations, suggesting an association between dopamine neuron degeneration and connectivity disruption in this PD model. The results of this study provide a relevant model, where new drugs that can alleviate neurodegeneration in PD could be evaluated preclinically.
Subject(s)
Parkinson Disease , Tropanes , Humans , Parkinson Disease/diagnostic imaging , Parkinson Disease/drug therapy , Parkinson Disease/pathology , Dopamine/metabolism , Probenecid/pharmacology , Probenecid/therapeutic use , Dopaminergic Neurons/pathology , Fluorodeoxyglucose F18/therapeutic use , Dopamine Plasma Membrane Transport Proteins/metabolism , Corpus Striatum/diagnostic imaging , Corpus Striatum/metabolism , Nerve Degeneration/diagnostic imaging , Nerve Degeneration/pathologyABSTRACT
BACKGROUND: One intrastriatal administration of quinolinic acid (QA) in rats induces a lesion with features resembling those observed in Huntington's disease. Our aim is to evaluate the effects of the cysteinyl leukotriene receptor antagonist montelukast (MLK), which exhibited neuroprotection in different preclinical models of neurodegeneration, on QA-induced neuroinflammation and regional metabolic functions. METHODS: The right and left striatum of Sprague Dawley and athymic nude rats were injected with QA and vehicle (VEH), respectively. Starting from the day before QA injection, animals were treated with 1 or 10 mg/kg of MLK or VEH for 14 days. At 14 and 30 days post-lesion, animals were monitored with magnetic resonance imaging (MRI) and positron emission tomography (PET) using [18F]-VC701, a translocator protein (TSPO)-specific radiotracer. Striatal neuroinflammatory response was measured post-mortem in rats treated with 1 mg/kg of MLK by immunofluorescence. Rats treated with 10 mg/kg of MLK also underwent a [18F]-FDG PET study at baseline and 4 months after lesion. [18F]-FDG PET data were then used to assess metabolic connectivity between brain regions by applying a covariance analysis method. RESULTS: MLK treatment was not able to reduce the QA-induced increase in striatal TSPO PET signal and MRI lesion volume, where we only detected a trend towards reduction in animals treated with 10 mg/kg of MLK. Post-mortem immunofluorescence analysis revealed that MLK attenuated the increase in striatal markers of astrogliosis and activated microglia in the lesioned hemisphere. We also found a significant increase in a marker of anti-inflammatory activity (MannR) and a trend towards reduction in a marker of pro-inflammatory activity (iNOS) in the lesioned striatum of MLK-compared to VEH-treated rats. [18F]-FDG uptake was significantly reduced in the striatum and ipsilesional cortical regions of VEH-treated rats at 4 months after lesion. MLK administration preserved glucose metabolism in these cortical regions, but not in the striatum. Finally, MLK was able to counteract changes in metabolic connectivity and measures of network topology induced by QA, in both lesioned and non-lesioned hemispheres. CONCLUSIONS: Overall, MLK treatment produced a significant neuroprotective effect by reducing neuroinflammation assessed by immunofluorescence and preserving regional brain metabolism and metabolic connectivity from QA-induced neurotoxicity in cortical and subcortical regions.
Subject(s)
Encephalitis , Neuroprotective Agents , Neurotoxicity Syndromes , Rats , Animals , Neuroprotective Agents/pharmacology , Neuroprotective Agents/therapeutic use , Rats, Sprague-Dawley , Quinolinic Acid/toxicity , Quinolinic Acid/metabolism , Fluorodeoxyglucose F18/metabolism , Neuroinflammatory Diseases , Corpus Striatum/metabolism , Neurotoxicity Syndromes/pathology , Encephalitis/pathology , Disease Models, AnimalABSTRACT
Rationale: The αvß6- and αvß8-integrins, two cell-adhesion receptors upregulated in many tumors and involved in the activation of the latency associated peptide (LAP)/TGFß complex, represent potential targets for tumor imaging and therapy. We investigated the tumor-homing properties of a chromogranin A-derived peptide containing an RGDL motif followed by a chemically stapled alpha-helix (called "5a"), which selectively recognizes the LAP/TGFß complex-binding site of αvß6 and αvß8. Methods: Peptide 5a was labeled with IRDye 800CW (a near-infrared fluorescent dye) or with 18F-NOTA (a label for positron emission tomography (PET)); the integrin-binding properties of free peptide and conjugates were then investigated using purified αvß6/αvß8 integrins and various αvß6/αvß8 single - or double-positive cancer cells; tumor-homing, biodistribution and imaging properties of the conjugates were investigated in subcutaneous and orthotopic αvß6-positive carcinomas of the pancreas, and in mice bearing subcutaneous αvß8-positive prostate tumors. Results: In vitro studies showed that 5a can bind both integrins with high affinity and inhibits cell-mediated TGFß activation. The 5a-IRDye and 5a-NOTA conjugates could bind purified αvß6/αvß8 integrins with no loss of affinity compared to free peptide, and selectively recognized various αvß6/αvß8 single- or double-positive cancer cells, including cells from pancreatic carcinoma, melanoma, oral mucosa, bladder and prostate cancer. In vivo static and dynamic optical near-infrared and PET/CT imaging and biodistribution studies, performed in mice with subcutaneous and orthotopic αvß6-positive carcinomas of the pancreas, showed high target-specific uptake of fluorescence- and radio-labeled peptide by tumors and low non-specific uptake in other organs and tissues, except for excretory organs. Significant target-specific uptake of fluorescence-labeled peptide was also observed in mice bearing αvß8-positive prostate tumors. Conclusions: The results indicate that 5a can home to αvß6- and/or αvß8-positive tumors, suggesting that this peptide can be exploited as a ligand for delivering imaging or anticancer agents to αvß6/αvß8 single- or double-positive tumors, or as a tumor-homing inhibitor of these TGFß activators.
Subject(s)
Carcinoma , Pancreatic Neoplasms , Prostatic Neoplasms , Male , Animals , Mice , Humans , Chromogranin A/metabolism , Positron Emission Tomography Computed Tomography , Tissue Distribution , Peptides/chemistry , Integrins/metabolism , Transforming Growth Factor beta/metabolismABSTRACT
Glioblastoma (GBM) is a common and deadly form of brain tumor in adults. Dysregulated metabolism in GBM offers an opportunity to deploy metabolic interventions as precise therapeutic strategies. To identify the molecular drivers and the modalities by which different molecular subgroups of GBM exploit metabolic rewiring to sustain tumor progression, we interrogated the transcriptome, the metabolome, and the glycoproteome of human subgroup-specific GBM sphere-forming cells (GSC). L-fucose abundance and core fucosylation activation were elevated in mesenchymal (MES) compared with proneural GSCs; this pattern was retained in subgroup-specific xenografts and in subgroup-affiliated human patient samples. Genetic and pharmacological inhibition of core fucosylation significantly reduced tumor growth in MES GBM preclinical models. Liquid chromatography-mass spectrometry (LC-MS)-based glycoproteomic screening indicated that most MES-restricted core-fucosylated proteins are involved in therapeutically relevant GBM pathological processes, such as extracellular matrix interaction, cell adhesion, and integrin-mediated signaling. Selective L-fucose accumulation in MES GBMs was observed using preclinical minimally invasive PET, implicating this metabolite as a potential subgroup-restricted biomarker.Overall, these findings indicate that L-fucose pathway activation in MES GBM is a subgroup-specific dependency that could provide diagnostic markers and actionable therapeutic targets. SIGNIFICANCE: Metabolic characterization of subgroup-specific glioblastoma (GBM) sphere-forming cells identifies the L-fucose pathway as a vulnerability restricted to mesenchymal GBM, disclosing a potential precision medicine strategy for targeting cancer metabolism.
Subject(s)
Brain Neoplasms , Glioblastoma , Humans , Glioblastoma/pathology , Fucose/metabolism , Signal Transduction , Brain Neoplasms/pathology , Neoplastic Stem Cells/pathology , Cell Line, TumorABSTRACT
We examined effects of exposing female and male mice for 33 weeks to 45% or 60% high fat diet (HFD). Males fed with either diet were more vulnerable than females, displaying higher and faster increase in body weight and more elevated cholesterol and liver enzymes levels. Higher glucose metabolism was revealed by PET in the olfactory bulbs of both sexes. However, males also displayed altered anterior cortex and cerebellum metabolism, accompanied by a more prominent brain inflammation relative to females. Although both sexes displayed reduced transcripts of neuronal and synaptic genes in anterior cortex, only males had decreased protein levels of AMPA and NMDA receptors. Oppositely, to anterior cortex, cerebellum of HFD-exposed mice displayed hypometabolism and transcriptional up-regulation of neuronal and synaptic genes. These results indicate that male brain is more susceptible to metabolic changes induced by HFD and that the anterior cortex versus cerebellum display inverse susceptibility to HFD.
Subject(s)
Diet, High-Fat , Obesity , Animals , Mice , Male , Female , Diet, High-Fat/adverse effects , Obesity/metabolism , Brain/metabolism , Body Weight , Neurons/metabolismABSTRACT
The aim of the present study is to investigate the synergic role of 68Ga-PSMA PET/MRI and 68Ga-DOTA-RM2 PET/MRI in prostate cancer (PCa) staging. We present pilot data on twenty-two patients with biopsy-proven PCa that underwent 68Ga-PSMA PET/MRI for staging purposes, with 19/22 also undergoing 68Gaa-DOTA-RM2 PET/MRI. TNM classification based on image findings was performed and quantitative imaging parameters were collected for each scan. Furthermore, twelve patients underwent radical prostatectomy with the availability of histological data that were used as the gold standard to validate intraprostatic findings. A DICE score between regions of interest manually segmented on the primary tumour on 68Ga-PSMA PET, 68Ga-DOTA-RM2 PET and on T2 MRI was computed. All imaging modalities detected the primary PCa in 18/19 patients, with 68Ga-DOTA-RM2 PET not detecting any lesion in 1/19 patients. In the remaining patients, 68Ga-PSMA and MRI were concordant. Seven patients presented seminal vesicles involvement on MRI, with two of these being also detected by 68Ga-PSMA, and 68Ga-DOTA-RM2 PET being negative. Regarding extraprostatic disease, 68Ga-PSMA PET, 68Ga-DOTA-RM2 PET and MRI resulted positive in seven, four and five patients at lymph-nodal level, respectively, and at a bone level in three, zero and one patients, respectively. These preliminary results suggest the potential complementary role of 68Ga-PSMA PET, 68Ga-DOTA-RM2 PET and MRI in PCa characterization during the staging phase.
ABSTRACT
The high glycolytic activity of multiple myeloma (MM) cells is the rationale for use of Positron Emission Tomography (PET) with 18F-fluorodeoxyglucose ([18F]FDG) to detect both bone marrow (BM) and extramedullary disease. However, new tracers are actively searched because [18F]FDG-PET has some limitations and there is a portion of MM patients who are negative. Glutamine (Gln) addiction has been recently described as a typical metabolic feature of MM cells. Yet, the possible exploitation of Gln as a PET tracer in MM has never been assessed so far and is investigated in this study in preclinical models. Firstly, we have synthesized enantiopure (2S,4R)-4-fluoroglutamine (4-FGln) and validated it as a Gln transport analogue in human MM cell lines, comparing its uptake with that of 3H-labelled Gln. We then radiosynthesized [18F]4-FGln, tested its uptake in two different in vivo murine MM models, and checked the effect of Bortezomib, a proteasome inhibitor currently used in the treatment of MM. Both [18F]4-FGln and [18F]FDG clearly identified the spleen as site of MM cell colonization in C57BL/6 mice, challenged with syngeneic Vk12598 cells and assessed by PET. NOD.SCID mice, subcutaneously injected with human MM JJN3 cells, showed high values of both [18F]4-FGln and [18F]FDG uptake. Bortezomib significantly reduced the uptake of both radiopharmaceuticals in comparison with vehicle at post treatment PET. However, a reduction of glutaminolytic, but not of glycolytic, tumor volume was evident in mice showing the highest response to Bortezomib. Our data indicate that [18F](2S,4R)-4-FGln is a new PET tracer in preclinical MM models, yielding a rationale to design studies in MM patients.
ABSTRACT
Glioblastoma (GBM) is a highly aggressive tumor of the brain. Despite the efforts, response to current therapies is poor and 2-years survival rate ranging from 6-12%. Here, we evaluated the preclinical efficacy of Metformin (MET) as add-on therapy to Temozolomide (TMZ) and the ability of [18F]FLT (activity of thymidine kinase 1 related to cell proliferation) and [18F]VC701 (translocator protein, TSPO) Positron Emission Tomography (PET) radiotracers to predict tumor response to therapy. Indeed, TSPO is expressed on the outer mitochondrial membrane of activated microglia/macrophages, tumor cells, astrocytes and endothelial cells. TMZ-sensitive (Gli36ΔEGFR-1 and L0627) or -resistant (Gli36ΔEGFR-2) GBM cell lines representative of classical molecular subtype were tested in vitro and in vivo in orthotopic mouse models. Our results indicate that in vitro, MET increased the efficacy of TMZ on TMZ-sensitive and on TMZ-resistant cells by deregulating the balance between pro-survival (bcl2) and pro-apoptotic (bax/bad) Bcl-family members and promoting early apoptosis in both Gli36ΔEGFR-1 and Gli36ΔEGFR-2 cells. In vivo, MET add-on significantly extended the median survival of tumor-bearing mice compared to TMZ-treated ones and reduced the rate of recurrence in the TMZ-sensitive models. PET studies with the cell proliferation radiopharmaceutical [18F]FLT performed at early time during treatment were able to distinguish responder from non-responder to TMZ but not to predict the duration of the effect. On the contrary, [18F]VC701 uptake was reduced only in mice treated with MET plus TMZ and levels of uptake negatively correlated with animals' survival. Overall, our data showed that MET addition improved TMZ efficacy in GBM preclinical models representative of classical molecular subtype increasing survival time and reducing tumor relapsing rate. Finally, results from PET imaging suggest that the reduction of cell proliferation represents a common mechanism of TMZ and combined treatment, whereas only the last was able to reduce TSPO. This reduction was associated with the duration of treatment response. TSPO-ligand may be used as a complementary molecular imaging marker to predict tumor microenvironment related treatment effects.
ABSTRACT
OBJECTIVE: Neuroinflammation is considered a key driver for neurodegeneration in several neurological diseases, including amyotrophic lateral sclerosis (ALS). SOD1 mutations cause about 20% of familial ALS, and related pathology might generate microglial activation triggering neurodegeneration. 11 C-PK11195 is the prototypical and most validated PET radiotracer, targeting the 18-kDa translocator protein which is overexpressed in activated microglia. In this study, we investigated microglia activation in asymptomatic (ASYM) and symptomatic (SYM) SOD1 mutated carriers, by using 11 C-PK11195 and PET imaging. METHODS: We included 20 subjects: 4 ASYM-carriers, neurologically normal, 6 SYM-carriers with probable ALS, and 10 healthy controls. A receptor parametric mapping procedure estimated 11 C-PK11195 binding potentials and voxel-wise statistical comparisons were performed at group and single-subject levels. RESULTS: Both the SYM- and ASYM-carriers showed significant microglia activation in cortical and subcortical structures, with variable patterns at individual level. Clusters of activation were present in occipital and temporal regions, cerebellum, thalamus, and medulla oblongata. Notably, SYM-carriers showed microglia activation also in supplementary and primary motor cortices and in the somatosensory regions. INTERPRETATION: In vivo neuroinflammation occurred in all SOD1 mutated cases since the presymptomatic stages, as shown by a significant cortical and subcortical microglia activation. The involvement of sensorimotor cortex became evident at the symptomatic disease stage. Although our data indicate the role of in vivo PET imaging for assessing resident microglia in the investigation of SOD1-ALS pathophysiology, further studies are needed to clarify the temporal and spatial dynamics of microglia activation and its relationship with neurodegeneration.
Subject(s)
Amides , Amyotrophic Lateral Sclerosis , Brain , Inflammation , Isoquinolines , Microglia , Positron-Emission Tomography , Superoxide Dismutase-1/genetics , Adult , Aged , Amyotrophic Lateral Sclerosis/diagnostic imaging , Amyotrophic Lateral Sclerosis/genetics , Amyotrophic Lateral Sclerosis/immunology , Amyotrophic Lateral Sclerosis/metabolism , Brain/diagnostic imaging , Brain/immunology , Brain/metabolism , Female , Heterozygote , Humans , Inflammation/diagnostic imaging , Inflammation/immunology , Inflammation/metabolism , Male , Microglia/immunology , Microglia/metabolism , Middle Aged , Prodromal SymptomsABSTRACT
Cyclooxygenase-2 (COX-2) is involved in the inflammatory response, and its recurrent overexpression in cancers as well as in neurodegenerative disorders has made it an important target for therapy. For this reason, noninvasive imaging of COX-2 expression may represent an important diagnostic tool. In this work, a COX-2 inhibitor analogue, VA426 [1-(4-fluorophenyl)-3-(2-methoxyethyl)-2-methyl-5-(4-(methylsulfonil)phenyl)-1H-pyrrole], was synthesized and radiolabelled with the 11C radioisotope. The ex vivo biodistribution profile of 11C-VA426 was evaluated in the brain and periphery of healthy rats and mice and in brain and periphery of inflammation models, based on the administration of LPS. 11C-VA426 synthesis with the tBuOK base showed optimal radiochemical yield (15 ± 2%) based on triflate activity, molar activity (range 37-148 GBq/µmol), and radiochemical purity (>95%). Ex vivo biodistribution studies showed a fast uptake of radioactivity but a rapid washout, except in regions expressing COX-2 (lungs, liver, and kidney) both in rats and in mice, with maximum values at 30 and 10 minutes p.i., respectively. LPS administration did not show significant effect on radioactivity accumulation. Celecoxib competition experiments performed in rats and mice treated with LPS produced a general target unrelated reduction of radioactivity concentration in all peripheral tissues and brain areas examined. Finally, in agreement with the negative results obtained from biodistribution experiments, radiometabolites analysis revealed that 11C-VA426 is highly unstable in vivo. This study indicates that the compound 11C-VA426 is not currently suitable to be used as radiopharmaceutical for PET imaging. This family of compounds needs further implementation in order to improve in vivo stability.
Subject(s)
Carbon Radioisotopes , Cyclooxygenase 2/analysis , Cyclooxygenase Inhibitors , Isotope Labeling/methods , Radiopharmaceuticals/chemical synthesis , Animals , Biotransformation , Celecoxib/pharmacology , Corpus Striatum/diagnostic imaging , Corpus Striatum/metabolism , Cyclooxygenase 2/metabolism , Cyclooxygenase Inhibitors/chemical synthesis , Cyclooxygenase Inhibitors/chemistry , Cyclooxygenase Inhibitors/pharmacokinetics , Drug Evaluation, Preclinical , Drug Stability , Inflammation/chemically induced , Inflammation/diagnostic imaging , Ligands , Lipopolysaccharides/toxicity , Liver/metabolism , Male , Mice , Organ Specificity , Positron-Emission Tomography , Radiopharmaceuticals/chemistry , Radiopharmaceuticals/pharmacokinetics , Rats , Rats, Sprague-Dawley , Tissue DistributionABSTRACT
PURPOSE: To assess the capability of 18F-FAZA PET/CT in identifying intratumoral hypoxic areas in early and locally advanced non-small cell lung cancer (NSCLC) patients and to compare 18FFAZA PET/CT with 18F-FDG PET/CT and histopathological biomarkers and to investigate whether the assessment of tumour to blood (T/B) and tumour to muscle (T/M) ratios provide comparable information regarding the hypoxic fractions of the tumour. MATERIALS AND METHODS: Seven patients with NSCLC were prospectively enrolled (3 men, 4 women; median age: 71 years; range 63-80). All patients underwent to 18F-FDG PET/CT and 18F-FAZA PET/CT before surgery. Maximum standardized uptake value (SUVmax) was used to evaluate 18FFDG PET/CT images, while 18F-FAZA PET/CT images have been interpreted by using tumour-toblood (T/B) and tumour-to-muscle (T/M) ratio. Surgery was performed in all patients; immunohistochemical analysis for hypoxia biomarkers was performed on histologic tumor samples. RESULTS: All lung lesions showed intense 18F-FDG uptake (mean SUVmax: 7.35; range: 2.35-25.20). A faint 18F-FAZA uptake was observed in 6/7 patients (T/B < 1.2) while significant uptake was present in the remaining 1/7 (T/B and T/M=2.24). On both 2 and 4 h imaging after injection, no differences were observed between T/M and T/B (p=0.5), suggesting that both blood and muscle are equivalent in estimating the background activity for image analysis. Immunohisotchemical analysis showed low or absent staining for hypoxia biomarkers in 3 patients (CA-IX and GLUT-1: 0%; HIF-1α: mean 3.3%; range 0-10). Two patients showed staining for HIF-1α of 5%, with CA-IX being 60% and 30%, respectively and GLUT-1 being 30% and 80%, respectively; in 1/7 HIF-1α was 10%, CA-IX was 50% and GLUT-1 was 90%. In one patient a higher percentage of HIF-1α and CA-IX (20% and 70%, respectively) positive cells was present, with GLUT-1 being 30%. CONCLUSIONS: To the best of our knowledge, this is the first paper assessing hypoxia and glucose metabolism in comparison with immunohistochemistry in patients candidate to surgery for NSCLC. Although including a small number of patients, useful insight regarding correlation between imaging and immunohistochemistry are reported along with methodological suggestions for clinical practice.
Subject(s)
Carcinoma, Non-Small-Cell Lung/diagnostic imaging , Lung Neoplasms/diagnostic imaging , Positron Emission Tomography Computed Tomography , Aged , Aged, 80 and over , Biomarkers, Tumor , Carcinoma, Non-Small-Cell Lung/pathology , Carcinoma, Non-Small-Cell Lung/surgery , Female , Fluorodeoxyglucose F18 , Humans , Immunohistochemistry , Lung Neoplasms/pathology , Lung Neoplasms/surgery , Male , Middle Aged , Nitroimidazoles , Preoperative Care , Prospective Studies , RadiopharmaceuticalsABSTRACT
UNLABELLED: AFRA-DMF5.3 is a human antibody fragment that, as a dimer, specifically binds to the α-folate receptor (FR) on ovary cancer cells. Pharmacokinetic and biodistribution parameters of (131)I-AFRA-DFM5.3 after intravenous administration in animal models support its potential therapeutic use. We evaluated its preclinical specificity and therapeutic efficacy in tumor models. METHODS: A negative control, AFRA-DFM6.1, was obtained by protein engineering. The activity and specificity of (131)I-AFRA-DFMs were evaluated by systemic administration (intravenous) in subcutaneous tumor xenograft-bearing nude mice. Pharmacokinetics, biodistribution, and efficacy were assessed by intraperitoneal administration of (131)I-AFRA-DFM5.3 in nude mice bearing 2 different intraperitoneal ovarian carcinoma xenografts. Treatments were tested at different doses and as single or double administrations 1 wk apart. RESULTS: In subcutaneous models, (131)I-AFRA-DFM5.3, but not the negative control, was found to reside on FR-positive tumor masses and significantly reduced tumor growth. In intraperitoneal models, early accumulation on free-floating clumps of ovarian cancer cells and solid peritoneal masses was evident after 1 h, and tumor uptake was stable for up to 3 h. The high tumor uptake determined the efficacy of (131)I-AFRA-DFM5.3. The best antitumor activity, with more than 50% of treated animals cured, was achieved with 2 locoregional treatments of intraperitoneally growing tumors on days 2 and 9. CONCLUSION: These results suggest that radioimmunotherapy with (131)I-AFRA-DFM5.3 is feasible and leads to significantly prolonged survival. These preclinical data provide the basis for the rationale design of therapeutic treatments of ovarian cancer patients with a radiolabeled anti-FR antibody fragment.
Subject(s)
Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Immunoglobulin Fragments/chemistry , Immunoglobulin Fragments/pharmacology , Ovarian Neoplasms/radiotherapy , Protein Multimerization , Animals , Antineoplastic Agents/pharmacokinetics , Antineoplastic Agents/therapeutic use , Cell Line, Tumor , Disease Models, Animal , Drug Administration Schedule , Female , Folate Receptor 1/immunology , Humans , Immunoglobulin Fragments/metabolism , Immunoglobulin Fragments/therapeutic use , Mice , Ovarian Neoplasms/metabolism , Protein Structure, Quaternary , RadioimmunotherapyABSTRACT
The question of the serum HER2 extracellular domain (HER2/ECD) measurement for prediction of response to the anti-HER2 antibody Trastuzumab is still an open and current matter of clinical debate. To elucidate the involvement of shed HER2/ECD in HER2-driven tumor progression and in guiding therapy of individual patients, we examined biological effects exerted by elevated HER2/ECD in cancer growth and in response to Trastuzumab. To this purpose SKOV3 tumor cells were stably transfected to release a recombinant HER2/ECD molecule (rECD). Transfectants releasing high levels of 110-kDa rECD, identical in size to native HER2/ECD (nECD), grew significantly slower than did controls, which constitutively released only basal levels of nECD. While transmembrane HER2 and HER1 were expressed at equal levels by both controls and transfected cells, activation of these molecules and of downstream ERK2 and Akt was significantly reduced only in rECD transfectants. Surface plasmon resonance analysis revealed heterodimerization of the rECD with HER1, -2, and -3. In cell growth bioassays in vitro, shed HER2 significantly blocked HER2-driven tumor cell proliferation. In mice, high levels of circulating rECD significantly impaired HER2-driven SKOV3 tumor growth but not that of HER2-negative tumor cells. In vitro and in mice, Trastuzumab significantly inhibited tumor growth due to the rECD-facilitated accumulation of the antibody on tumor cells. Globally our findings sustain the biological relevance of elevated HER2/ECD levels in the outcome of HER2-disease and in the susceptibility to Trastuzumab-based therapy.
Subject(s)
Antibodies, Monoclonal/therapeutic use , Antineoplastic Agents/therapeutic use , Breast Neoplasms/drug therapy , Breast Neoplasms/pathology , Receptor, ErbB-2/chemistry , Receptor, ErbB-2/metabolism , Animals , Antibodies, Monoclonal, Humanized , Breast Neoplasms/genetics , Cell Line, Tumor , Cell Proliferation , Female , Humans , Mice , Mice, Nude , Protein Isoforms/chemistry , Protein Isoforms/genetics , Protein Isoforms/metabolism , Protein Structure, Tertiary , Receptor, ErbB-2/genetics , Signal Transduction/physiology , TrastuzumabABSTRACT
INTRODUCTION: The mouse monoclonal antibody MOv18, directed against the alpha-isoform of folate receptor (FR), was investigated to identify the optimal radioconjugate for radioimmunotherapy of minimal residual disease in ovarian cancer. METHODS: Pharmacokinetics, biodistribution, long-term therapeutic efficacy and toxicity of MOv18, labeled with the beta-emitters (131)I, (90)Y and (177)Lu, were compared in a xenografted mouse model, composed by two cell lines, A431FR and A431MK, differing only for FR expression. RESULTS: A shorter blood clearance and a higher tumor uptake were observed for (90)Y- and (177)Lu- compared to (131)I-MOv18, and a shorter blood pharmacokinetics was recorded in A431FR-bearing animals. At equitoxic maximum tolerable doses, the general irradiation by (131)I- and (90)Y-MOv18 gives rise to strong targeted effects on A431FR and nontargeted effects on A431MK tumors, while (177)Lu-MOv18 was able to eradicate small size tumor masses expressing the antigen of interest exerting only mild non-targeted effects. CONCLUSION: (177)Lu-MOv18 at the maximal tolerated dose is the immunoradioconjugate with the best therapeutic window in experimental conditions of small tumor volume.
Subject(s)
Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/therapeutic use , Carrier Proteins/metabolism , Gene Expression Regulation, Neoplastic , Neoplasms/radiotherapy , Radioimmunotherapy , Radioisotopes/chemistry , Receptors, Cell Surface/metabolism , Animals , Antibodies, Monoclonal/pharmacokinetics , Antibodies, Monoclonal/toxicity , Antibodies, Monoclonal, Murine-Derived , Cell Line, Tumor , Female , Folate Receptors, GPI-Anchored , Humans , Iodine Radioisotopes/chemistry , Isotope Labeling , Lutetium/chemistry , Maximum Tolerated Dose , Mice , Neoplasms/genetics , Neoplasms/metabolism , Neoplasms/pathology , Time Factors , Tissue Distribution , Transplantation, Heterologous , Tumor Burden , Yttrium Radioisotopes/chemistryABSTRACT
AIM: In our multicentric ongoing phase I activity escalation study, (90)Y-labeled ibritumomab tiuxetan (Ze-valin was administered in activity per kilo twice- and three times the maximum tolerable dose of 15 MBq/kg suggested for nonmyeloablative treatments by the U.S. registration study. The radioinduced myelodepression was overcome by stem cell autografting. The dosimetric aim was to correlate possible extramedullary toxicities to the organ-absorbed doses or to the biologic effective dose (BED). This is a conceptually more suitable parameter, as it takes into account not only the absorbed dose, but also the influence of the dose rate and of the tissue repair mechanism. METHODS: Pretreatment planar dosimetry was performed on 16 patients with a median 200 MBq of (111)In-Zevalin. Conjugate view technique, background, attenuation, and partial scatter correction were adopted. Blood samples and a planar whole body scintigram were collected at least at 0.5, 48, 96, and 120 hours. Individual organ mass correction was based on a computed tomography scan. Internal dose calculation was performed by the OLINDA/EXM software. One (1) week after dosimetry, 12 patients were treated with 30 MBq/kg and 4 patients with 45 MBq/kg of (90)Y-Zevalin. RESULTS: The absorbed dose per unit activity (Gy/GBq) were (median and range of 16 dosimetric studies): heart wall 3.8 [0.5, 9.7]; kidneys 4.9 [2.8, 10.5]; liver 5.5 [3.9, 8.9]; lungs 2.8 [0.4, 6.8]; red marrow 1.1 [0.8, 2.1]; spleen 6.3 [1.5, 10.9]; and testes 4.6 [3.0, 16.7]. The absorbed dose (Gy) for the 4 patients administered with 45 MBq/kg were (median and range): heart wall 17.6 [9.4, 25.1]; kidneys 16.3 [7.9, 20.3]; liver 20.9 [15.4, 24.3]; lungs 7.7 [5.6, 11.4]; red marrow 3.0 [2.4, 3.3]; spleen 28.4 [18.9, 30.8]; and testes 16.5 [12.2, 17.3]. No extramedullary toxicity was observed. CONCLUSIONS: The administration of 45 MBq/kg of (90)Y ibritumomab tiuxetan to 4 patients with stem cell autografting was free from extramedullary toxicity. This is in agreement with both organ doses and BEDs below the corresponding toxicity thresholds. For these clinical and dosimetric reasons, a further increase in injectable activity could have been conceivable. If the more appropriate BED parameter were chosen for toxicity limit calculations, a wider margin of increase would have been possible. Our theoretical investigation demonstrates that, in this particular case of (90)Y Zevalin therapy, the uncertainty about radiobiological parameters was not a limiting factor for a BED-based calculation of the maximum injectable activity.
Subject(s)
Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/therapeutic use , Lymphoma, Non-Hodgkin/immunology , Lymphoma, Non-Hodgkin/therapy , Myeloablative Agonists/immunology , Myeloablative Agonists/therapeutic use , Antibodies, Monoclonal/administration & dosage , Antibodies, Monoclonal/adverse effects , Dose-Response Relationship, Radiation , Humans , Lymphoma, Non-Hodgkin/pathology , Myeloablative Agonists/administration & dosage , Myeloablative Agonists/adverse effects , Radioimmunotherapy , Yttrium RadioisotopesABSTRACT
The monoclonal antibody (mAb) MOv18 binds the membrane alpha isoform of the folate receptor (FR) which is overexpressed in human ovarian carcinoma cells. Exploiting the targeting capacity of this mAb, we developed and preclinically validated a protocol for the stable labeling of the mAb with 90Y, an isotope which has shown promise in cancer radioimmunotherapy. MOv18 was derivatized with the stable macrocyclic ligand p-isothiocyanatobenzyl-1,4,7,10-tetraazacyclododecane-1,4,7,10- tetraacetic acid (Bz-DOTA). MOv18-Bz-DOTA conjugates were labeled with 90Y or 111In under metal-free and good laboratory practice conditions. At the optimal Bz-DOTA/mAb derivatization ratio of 4-5, conjugates maintained binding activity up to 6 months, were efficiently labeled with 90Y or 111In (mean labeling yield 85 and 64%, associated to a final mean specific activity of 74 and 37 MBq/mg) and displayed a mean immunoreactivity of 60 and 58%, respectively. The radiolabeled preparations were stable in human serum, with >97% radioactivity associated to mAb at 48 h after labeling. The ability of 90Y- and 111In-MOv18 to localize FR on tumors in vivo was analyzed in nude mice bearing tumors induced by isogenic cell lines differing only in the presence or absence of the relevant antigen [A431FR (FR-positive) and A431tMock (FR-negative)]. In vivo biodistribution in organs other than tumor was comparable in non-tumor-, A431tMock- and A431FR-bearing mice, whereas the median tumor uptake of the radiolabeled reagents, expressed as area under the curve (AUC) and maximum uptake (Umax), was significantly higher (sixfold to sevenfold) in A431FR than in A431tMock tumors (P=0.0465 and P=0.0332, respectively). Mean maximum uptake (% ID/g) for 90Y-MOv18 was 53.7 and 7.4 in A431FR and A431tMock respectively; corresponding values for 111In-Mov18 were 45.0 and 11.3. These data demonstrate the feasibility of 90Y-labeling of MOv18 without compromising antibody binding ability and the immunoreagent-specific localization in vivo on FR-expressing tumors, suggesting the suitability of 90Y-MOv18 for clinical studies.
Subject(s)
Antibodies, Monoclonal/pharmacokinetics , Carcinoma/metabolism , Organometallic Compounds/pharmacokinetics , Ovarian Neoplasms/metabolism , Antibodies, Monoclonal/therapeutic use , Carcinoma/radiotherapy , Female , Humans , Isotope Labeling , Organometallic Compounds/therapeutic use , Ovarian Neoplasms/radiotherapy , Radioimmunotherapy , Tissue Distribution , Yttrium RadioisotopesABSTRACT
BACKGROUND AND AIM: Calcitonin (hCT) is a useful serum marker for the diagnosis and monitoring of medullary thyroid carcinoma (MTC). However, hCT values provided by different methods may differ, leading to difficulties in the interpretation of hCT results. In this study we compared four immunoradiometric (IRMA) and radioimmunometric (RIA) assays for hCT determination. MATERIAL AND METHODS: hCT was measured in 35 patients by means of the following commercially available IRMA or RIA kits: CT US (Biosource), IRMA hCT (Schering-CIS bio international), ultra-sensitive calcitonin (DSL), and calcitonin assay (Scantibodies). A comparison of the distribution of the hCT values measured by the tested IRMA-RIAs and a correlation analysis were performed. RESULTS: The hCT values were widely dispersed and the classification of the patients according to the hCT cutoff value varied depending on the assay used. CONCLUSION: Despite efforts to develop new, highly specific antibodies, the evaluation of this marker is still flawed by analytical inaccuracy. hCT values are widely dispersed depending on the method used for marker measurement; as a consequence, patient classification according to the hCT cutoff value is still dependent on the assay used.