ABSTRACT
Immunotherapies are a promising advance in cancer treatment. However, because only a subset of cancer patients benefits from these treatments it is important to find mechanisms that will broaden the responding patient population. Generally, tumors with high mutational burdens have the potential to express greater numbers of mutant neoantigens. As neoantigens can be targets of protective adaptive immunity, highly mutated tumors are more responsive to immunotherapy. Given that external beam radiation 1) is a standard-of-care cancer therapy, 2) induces expression of mutant proteins and potentially mutant neoantigens in treated cells, and 3) has been shown to synergize clinically with immune checkpoint therapy (ICT), we hypothesized that at least one mechanism of this synergy was the generation of de novo mutant neoantigen targets in irradiated cells. Herein, we use KrasG12D x p53-/- sarcoma cell lines (KP sarcomas) that we and others have shown to be nearly devoid of mutations, are poorly antigenic, are not controlled by ICT, and do not induce a protective antitumor memory response. However, following one in vitro dose of 4- or 9-Gy irradiation, KP sarcoma cells acquire mutational neoantigens and become sensitive to ICT in vivo in a T cell-dependent manner. We further demonstrate that some of the radiation-induced mutations generate cytotoxic CD8+ T cell responses, are protective in a vaccine model, and are sufficient to make the parental KP sarcoma line susceptible to ICT. These results provide a proof of concept that induction of new antigenic targets in irradiated tumor cells represents an additional mechanism explaining the clinical findings of the synergy between radiation and immunotherapy.
Subject(s)
Antigens, Neoplasm/immunology , Immunotherapy , Mutation/genetics , Neoplasms/genetics , Neoplasms/immunology , Radiation , Animals , CD8-Positive T-Lymphocytes/immunology , Cell Line, Tumor , Clone Cells , Female , Histocompatibility Antigens Class II/metabolism , Immune Checkpoint Proteins/metabolism , Immunity , Mice , Proto-Oncogene Proteins p21(ras)/genetics , Tumor Suppressor Protein p53/genetics , VaccinationABSTRACT
Herein, we report a 64Cu positron emission tomography (PET) imaging agent that shows appreciable in vivo brain uptake and exhibits high specific affinity for beta-amyloid (Aß) aggregates, leading to the successful PET imaging of amyloid plaques in the brains of 5xFAD mice versus those of wild-type mice. The employed approach uses a bifunctional chelator with two Aß-interacting fragments that dramatically improves the Aß-binding affinity and lipophilicity for favorable blood-brain barrier penetration, while the use of optimized-length spacers between the Cu-chelating group and the Aß-interacting fragments further improves the in vivo Aß-binding specificity and brain uptake of the corresponding 64Cu PET imaging agent.
Subject(s)
Alzheimer Disease/diagnostic imaging , Alzheimer Disease/diagnosis , Chelating Agents/chemistry , Copper Radioisotopes/chemistry , Positron-Emission Tomography , Animals , Autoradiography , Chelating Agents/chemical synthesis , Mice, TransgenicABSTRACT
There is an unmet need for better therapeutic strategies for advanced prostate cancer. Poly (ADP-ribose) polymerase-1 (PARP-1) is a chromatin-binding DNA repair enzyme overexpressed in prostate cancer. This study evaluates whether PARP-1, on account of its proximity to the cell's DNA, would be a good target for delivering high-linear energy transfer Auger radiation to induce lethal DNA damage in prostate cancer cells. We analyzed the correlation between PARP-1 expression and Gleason score in a prostate cancer tissue microarray. A radio-brominated Auger emitting inhibitor ([77Br]Br-WC-DZ) targeting PARP-1 was synthesized. The ability of [77Br]Br-WC-DZ to induce cytotoxicity and DNA damage was assessed in vitro. The antitumor efficacy of [77Br]Br-WC-DZ was investigated in prostate cancer xenograft models. PARP-1 expression was found to be positively correlated with the Gleason score, thus making it an attractive target for Auger therapy in advanced diseases. The Auger emitter, [77Br]Br-WC-DZ, induced DNA damage, G2-M cell cycle phase arrest, and cytotoxicity in PC-3 and IGR-CaP1 prostate cancer cells. A single dose of [77Br]Br-WC-DZ inhibited the growth of prostate cancer xenografts and improved the survival of tumor-bearing mice. Our studies establish the fact that PARP-1 targeting Auger emitters could have therapeutic implications in advanced prostate cancer and provides a strong rationale for future clinical investigation.
Subject(s)
Poly(ADP-ribose) Polymerase Inhibitors , Prostatic Neoplasms , Animals , Humans , Male , Mice , Cell Line, Tumor , DNA Damage , Poly (ADP-Ribose) Polymerase-1/metabolism , Poly(ADP-ribose) Polymerase Inhibitors/pharmacology , Prostatic Neoplasms/metabolism , Radioisotopes/therapeutic useABSTRACT
Herein, we report a new series of divalent 2-(4-hydroxyphenyl)benzothiazole bifunctional chelators (BFCs) with high affinity for amyloid ß aggregates and favorable lipophilicity for blood-brain barrier penetration. The addition of an alkyl carboxylate ester pendant arm offers high binding affinity toward Cu(II). The novel BFCs form stable 64Cu-radiolabeled complexes and exhibit promising partition coefficient (logD) values of 1.05-1.85. Among the five compounds tested, the 64Cu-YW-15 complex exhibits significant staining of amyloid ß plaques in ex vivo autoradiography studies. In addition, biodistribution studies show that 64Cu-YW-15-Me exhibits moderate brain uptake (0.69 ± 0.08 %ID/g) in wild type mice.
Subject(s)
Alzheimer Disease , Chelating Agents , Positron-Emission Tomography , Animals , Mice , Alzheimer Disease/diagnostic imaging , Alzheimer Disease/metabolism , Amyloid beta-Peptides/chemistry , Benzothiazoles/chemistry , Benzothiazoles/pharmacokinetics , Chelating Agents/chemistry , Plaque, Amyloid/diagnostic imaging , Positron-Emission Tomography/methods , Tissue DistributionABSTRACT
Positron emission tomography (PET), which uses positron-emitting radionuclides to visualize and measure processes in the human body, is a useful noninvasive diagnostic tool for Alzheimer's disease (AD). The development of longer-lived radiolabeled compounds is essential for further expansion of the use of PET imaging in healthcare, and diagnostic agents employing longer-lived radionuclides such as 64Cu (t1/2 = 12.7 h, ß+ = 17%, ß- = 39%, electron capture EC = 43%, and Emax = 0.656 MeV) can accomplish this task. One limitation of 64Cu PET agents for neuroimaging applications is their limited lipophilicity due to the presence of several anionic groups needed to ensure strong Cu chelation. Herein, we evaluate a series of neutral chelators containing the 1,4,7-triazacyclononane or 2,11-diaza[3.3](2,6)pyridinophane macrocycles that have pyridyl-containing arms incorporating Aß-peptide-interacting fragments. The crystal structures of the corresponding Cu complexes confirm that the pyridyl N atoms are involved in binding to Cu. Radiolabeling and autoradiography studies show that the compounds efficiently chelate 64Cu, and the resulting complexes exhibit specific binding to the amyloid plaques in the AD mouse brain sections versus wild-type controls.
Subject(s)
Alzheimer Disease , Alzheimer Disease/diagnostic imaging , Alzheimer Disease/metabolism , Animals , Chelating Agents/chemistry , Ligands , Mice , Plaque, Amyloid , Positron-Emission Tomography/methods , Radiopharmaceuticals/chemistry , Radiopharmaceuticals/pharmacologyABSTRACT
Rationale: Idiopathic pulmonary fibrosis (IPF) is a progressive inflammatory lung disease without effective molecular markers of disease activity or treatment responses. Monocyte and interstitial macrophages that express the C-C motif CCR2 (chemokine receptor 2) are active in IPF and central to fibrosis.Objectives: To phenotype patients with IPF for potential targeted therapy, we developed 64Cu-DOTA-ECL1i, a radiotracer to noninvasively track CCR2+ monocytes and macrophages using positron emission tomography (PET).Methods: CCR2+ cells were investigated in mice with bleomycin- or radiation-induced fibrosis and in human subjects with IPF. The CCR2+ cell populations were localized relative to fibrotic regions in lung tissue and characterized using immunolocalization, single-cell mass cytometry, and Ccr2 RNA in situ hybridization and then correlated with parallel quantitation of lung uptake by 64Cu-DOTA-ECL1i PET.Measurements and Main Results: Mouse models established that increased 64Cu-DOTA-ECL1i PET uptake in the lung correlates with CCR2+ cell infiltration associated with fibrosis (n = 72). As therapeutic models, the inhibition of fibrosis by IL-1ß blockade (n = 19) or antifibrotic pirfenidone (n = 18) reduced CCR2+ macrophage accumulation and uptake of the radiotracer in mouse lungs. In lung tissues from patients with IPF, CCR2+ cells concentrated in perifibrotic regions and correlated with radiotracer localization (n = 21). Human imaging revealed little lung uptake in healthy volunteers (n = 7), whereas subjects with IPF (n = 4) exhibited intensive signals in fibrotic zones.Conclusions: These findings support a role for imaging CCR2+ cells within the fibrogenic niche in IPF to provide a molecular target for personalized therapy and monitoring.Clinical trial registered with www.clinicaltrials.gov (NCT03492762).
Subject(s)
Biomarkers/chemistry , Idiopathic Pulmonary Fibrosis/physiopathology , Lung/diagnostic imaging , Lung/physiopathology , Macrophages/physiology , Monocytes/physiology , Receptors, CCR2/chemistry , Adult , Aged , Aged, 80 and over , Animals , Disease Models, Animal , Female , Humans , Male , Mice , Middle Aged , Molecular Imaging , Positron-Emission TomographyABSTRACT
Alzheimer's Disease (AD) is the most common neurodegenerative disease, and efficient therapeutic and early diagnostic agents for AD are still lacking. Herein, we report the development of a novel amphiphilic compound, LS-4, generated by linking a hydrophobic amyloid-binding distyrylbenzene fragment with a hydrophilic triazamacrocycle, which dramatically increases the binding affinity toward various amyloid ß (Aß) peptide aggregates, especially for soluble Aß oligomers. Moreover, upon the administration of LS-4 to 5xFAD mice, fluorescence imaging of LS-4-treated brain sections reveals that LS-4 can penetrate the blood-brain barrier and bind to the Aß oligomers in vivo. In addition, the treatment of 5xFAD mice with LS-4 reduces the amount of both amyloid plaques and associated phosphorylated tau aggregates vs the vehicle-treated 5xFAD mice, while microglia activation is also reduced. Molecular dynamics simulations corroborate the observation that introducing a hydrophilic moiety into the molecular structure of LS-4 can enhance the electrostatic interactions with the polar residues of the Aß species. Finally, exploiting the Cu2+-chelating property of the triazamacrocycle, we performed a series of imaging and biodistribution studies that show the 64Cu-LS-4 complex binds to the amyloid plaques and can accumulate to a significantly larger extent in the 5xFAD mouse brains vs the wild-type controls. Overall, these results illustrate that the novel strategy, to employ an amphiphilic molecule containing a hydrophilic moiety attached to a hydrophobic amyloid-binding fragment, can increase the binding affinity for both soluble and insoluble Aß aggregates and can thus be used to detect and regulate various Aß species in AD.
Subject(s)
Alzheimer Disease/drug therapy , Amyloid beta-Peptides/chemistry , Drug Design , Neuroprotective Agents/chemical synthesis , Neuroprotective Agents/pharmacology , Styrenes/chemistry , Amyloid , Animals , Mice , Mice, Transgenic , Molecular Structure , Peptide Fragments , Plaque, Amyloid , Positron-Emission Tomography , Protein BindingABSTRACT
Herein, we report a new series of bifunctional chelators (BFCs) with a high affinity for amyloid aggregates, a strong binding affinity toward Cu(II), and favorable lipophilicity for potential blood-brain barrier penetration. The alkyl carboxylate ester pendant arms offer up to 3 orders of magnitude higher binding affinity toward Cu(II) and enable the BFCs to form stable 64Cu-radiolabeled complexes. Among the five compounds tested, the 64Cu-YW-7 and 64Cu-YW-10 complexes exhibit strong and specific staining of amyloid plaques in ex vivo autoradiography studies. Importantly, these BFCs have promising partition coefficient (log Doct) values of 0.91-1.26 and show some brain uptake in biodistribution studies using CD-1 mice. Overall, these BFCs could serve as lead compounds for the development of positron emission tomography imaging agents for AD diagnosis.
Subject(s)
Alzheimer Disease/diagnostic imaging , Amyloid beta-Peptides/metabolism , Chelating Agents/chemistry , Chelating Agents/metabolism , Copper Radioisotopes , Hydrophobic and Hydrophilic Interactions , Positron-Emission Tomography , Alzheimer Disease/metabolism , Blood-Brain Barrier/metabolismABSTRACT
MTH1 (MutT homolog 1) or NUDT1 (Nudix Hydrolase 1), also known as oxidized purine nucleoside triphosphatase, has potential as a biomarker for monitoring cancer progression and quantifying target engagement for relevant therapies. In this study, we validate one MTH1 inhibitor TH287 as a PET MTH1 radiotracer. TH287 was radiolabeled with tritium and the binding of [3H]TH287 to MTH1 was evaluated in live glioblastoma cells (U251MG) through saturation and competitive binding assays, together with in vitro enzymatic assays. Furthermore, TH287 was radiolabeled with carbon-11 for in vivo microPET studies. Saturation binding assays show that [3H]TH287 has a dissociation constant (Kd) of 1.97 ± 0.18 nM, Bmax of 2676 ± 122 fmol/mg protein for U251MG cells, and nH of 0.98 ± 0.02. Competitive binding assays show that TH287 (Ki: 3.04 ± 0.14 nM) has a higher affinity for MTH1 in U251MG cells compared to another well studied MTH1 inhibitor: (S)-crizotinib (Ki: 153.90 ± 20.48 nM). In vitro enzymatic assays show that TH287 has an IC50 of 2.2 nM in inhibiting MTH1 hydrolase activity and a Ki of 1.3 nM from kinetics assays, these results are consistent with our radioligand binding assays. Furthermore, MicroPET imaging shows that [11C]TH287 gets into the brain with rapid clearance from the brain, kidney, and heart. The results presented here indicate that radiolabeled TH287 has favorable properties to be a useful tool for measuring MTH1 in vitro and for further evaluation for in vivo PET imaging MTH1 of brain tumors and other central nervous system disorders.
Subject(s)
Biomarkers, Tumor/isolation & purification , DNA Repair Enzymes/genetics , Glioblastoma/diagnostic imaging , Phosphoric Monoester Hydrolases/genetics , Pyrimidines/pharmacology , Animals , Biomarkers, Tumor/metabolism , Brain/diagnostic imaging , Brain/metabolism , Cell Line, Tumor , Crizotinib/pharmacology , DNA Repair Enzymes/antagonists & inhibitors , DNA Repair Enzymes/isolation & purification , Glioblastoma/genetics , Glioblastoma/pathology , Heart/diagnostic imaging , Humans , Kidney/diagnostic imaging , Kidney/metabolism , Mice , Phosphoric Monoester Hydrolases/antagonists & inhibitors , Phosphoric Monoester Hydrolases/isolation & purification , Positron Emission Tomography Computed Tomography , Pyrimidines/chemistryABSTRACT
Copper plays a dual role as a nutrient and a toxin during bacterial infections. While uropathogenic Escherichia coli (UPEC) strains can use the copper-binding metallophore yersiniabactin (Ybt) to resist copper toxicity, Ybt also converts bioavailable copper to Cu(II)-Ybt in low-copper conditions. Although E. coli have long been considered to lack a copper import pathway, we observed Ybt-mediated copper import in UPEC using canonical Fe(III)-Ybt transport proteins. UPEC removed copper from Cu(II)-Ybt with subsequent re-export of metal-free Ybt to the extracellular space. Copper released through this process became available to an E. coli cuproenzyme (the amine oxidase TynA), linking this import pathway to a nutrient acquisition function. Ybt-expressing E. coli thus engage in nutritional passivation, a strategy of minimizing a metal ion's toxicity while preserving its nutritional availability. Copper acquisition through this process may contribute to the marked virulence defect of Ybt-transport-deficient UPEC.
Subject(s)
Copper/classification , Escherichia coli , Phenols/metabolism , Thiazoles/metabolism , Copper/metabolism , Copper/toxicity , Escherichia coli/drug effects , Escherichia coli/metabolismABSTRACT
PURPOSE: To evaluate whether tumor uptake of [89Zr]trastuzumab can distinguish HER2-positive from HER2-negative breast cancer. METHODS: Women with HER2-positive (n = 34) and HER2-negative (n = 16) breast cancer underwent PET/CT 5 ± 2 days following [89Zr]trastuzumab administration. HER2 status was determined based on immunohistochemistry and/or fluorescence in situ hybridization of primary or metastatic/recurrent tumor. Tumor [89Zr]trastuzumab uptake was assessed qualitatively and semiquantitatively as maximum standardized uptake value (SUVmax), and correlated with HER2 status. Additionally, intrapatient heterogeneity of [89Zr]trastuzumab uptake was evaluated. RESULTS: On a per-patient basis, [89Zr]trastuzumab-PET/CT was positive in 30/34 (88.2%) HER2-positive and negative in 15/16 (93.7%) HER2-negative patients. Considering all lesions, the SUVmax was not significantly different in patients with HER2-positive versus HER2-negative disease (p = 0.06). The same was true of when only hepatic lesions were evaluated (p = 0.42). However, after excluding hepatic lesions, tumor SUVmax was significantly higher in HER2-positive compared to HER2-negative patients (p = 0.003). A cutoff SUVmax of 3.2, determined by ROC analysis, demonstrated positive-predictive value of 83.3% (95% CI 65.3%, 94.4%), sensitivity of 75.8% (57.7%, 88.9%), negative-predictive value of 50% (24.7%, 75.3%), and specificity of 61.5% (95% 31.6%, 86.1%) for differentiating HER2-positive from HER2-negative lesions. There was intrapatient heterogeneity of [89Zr]trastuzumab uptake in 20% of patients with multiple lesions. CONCLUSIONS: [89Zr]trastuzumab has the potential to characterize the HER2 status of the complete tumor burden in patients with breast cancer, thus obviating repeat or multiple tissue sampling to assess intrapatient heterogeneity of HER2 status.
Subject(s)
Breast Neoplasms/diagnosis , Breast Neoplasms/metabolism , Positron Emission Tomography Computed Tomography , Radioisotopes , Radiopharmaceuticals , Receptor, ErbB-2/metabolism , Trastuzumab , Zirconium , Adult , Aged , Biomarkers, Tumor , Biopsy , Female , Humans , Image Processing, Computer-Assisted , Middle Aged , Multimodal Imaging/methods , Neoplasm Grading , Neoplasm Staging , Positron Emission Tomography Computed Tomography/methods , ROC Curve , Radioisotopes/administration & dosage , Radiopharmaceuticals/administration & dosage , Sensitivity and Specificity , Trastuzumab/administration & dosage , Zirconium/administration & dosageABSTRACT
The development of somastatin (SS) peptide analogues for the detection and treatment of neuroendocrine tumors has been successful with the recent FDA approval of 68Ga-DOTA-TATE and 177Lu-DOTA-TATE. The structure of these peptide constructs contains the peptide binding motif that binds to the receptor with high affinity, a chelator to complex the radioactive metal, and a linker between the peptide and chelator. However, these constructs suffer from rapid blood clearance, which limits their tumor uptake. In this study, this design has been further improved by incorporating a modification to control the in vivo pharmacokinetics. Adding a truncated Evans Blue (EB) dye molecule into the construct provides a prolonged half-life in blood as a result of its low micromolar affinity to albumin. We compared 177Lu-DOTA-TATE to the modified 177Lu Evans Blue compound (177Lu-DMEB-TATE), in vitro and in vivo in mice bearing A427-7 xenografts. The tumor uptake of 177Lu-DMEB-TATE was significantly greater than the uptake of 177Lu-DOTA-TATE in the biodistribution and SPECT-imaging studies. The therapeutic effect of the 177Lu-DMEB-TATE construct was superior to the that of the 177Lu-DOTA-TATE construct at the doses evaluated.
Subject(s)
Evans Blue/chemistry , Neuroendocrine Tumors/diagnostic imaging , Neuroendocrine Tumors/radiotherapy , Radiopharmaceuticals/pharmacokinetics , Theranostic Nanomedicine/methods , Animals , Antineoplastic Agents, Hormonal/therapeutic use , Cell Line, Tumor , Heterografts , Humans , Lutetium , Mice , Octreotide/therapeutic use , Radioisotopes , Radiopharmaceuticals/chemistry , Tissue Distribution , Tomography, Emission-Computed, Single-PhotonABSTRACT
Positron emission tomography (PET) imaging agents that detect amyloid plaques containing amyloid beta (Aß) peptide aggregates in the brain of Alzheimer's disease (AD) patients have been successfully developed and recently approved by the FDA for clinical use. However, the short half-lives of the currently used radionuclides 11C (20.4 min) and 18F (109.8 min) may limit the widespread use of these imaging agents. Therefore, we have begun to evaluate novel AD diagnostic agents that can be radiolabeled with 64Cu, a radionuclide with a half-life of 12.7 h, ideal for PET imaging. Described herein are a series of bifunctional chelators (BFCs), L1-L5, that were designed to tightly bind 64Cu and shown to interact with Aß aggregates both in vitro and in transgenic AD mouse brain sections. Importantly, biodistribution studies show that these compounds exhibit promising brain uptake and rapid clearance in wild-type mice, and initial microPET imaging studies of transgenic AD mice suggest that these compounds could serve as lead compounds for the development of improved diagnostic agents for AD.
Subject(s)
Alzheimer Disease/diagnosis , Amyloid beta-Peptides/chemistry , Copper Radioisotopes/chemistry , Radiopharmaceuticals/chemistry , Alzheimer Disease/diagnostic imaging , Animals , Autoradiography , Copper Radioisotopes/pharmacokinetics , Half-Life , Humans , Mice , Mice, Transgenic , Multimodal Imaging , Radiopharmaceuticals/pharmacokinetics , Spectrometry, Fluorescence , Tissue DistributionABSTRACT
Peptide receptor-based targeted molecular imaging and therapy of cancer is on the current forefront of nuclear medicine preclinical research and clinical practice. The frequent overexpression of gastrin-releasing peptide (GRP) receptors in prostate cancer stimulated the development of radiolabeled bombesin derivatives as high affinity peptide ligands for selective targeting of the GRP receptor. In this study, we have evaluated a novel (68)Ga-labeled bombesin derivative for PET imaging of prostate cancer in vivo. In addition, we were interested in testing the recently proposed "serve-and-protect" strategy to improve metabolic stability of radiolabeled peptides in vivo and to enhance tumor uptake. GRP receptor targeting peptides NOTA-BBN2 and (nat)Ga-NOTA-BBN2 demonstrated a characteristic antagonistic profile and high binding affinity toward the GRP receptor in PC3 cells (IC50 4.6-8.2 nM). Radiolabeled peptide (68)Ga-NOTA-BBN2 was obtained from NOTA-BBN2 in radiochemical yields greater than 62% (decay-corrected). Total synthesis time was 35 min, including purification using solid-phase extraction. (68)Ga-NOTA-BBN2 exhibited favorable resistance against metabolic degradation by peptidases in vivo within the investigated time frame of 60 min. Interestingly, metabolic stability was not further enhanced in the presence of protease inhibitor phosphoramidon. Dynamic PET studies showed high tumor uptake in both PC3- and LNCaP-bearing BALB/c nude mice (SUV5min > 0.6; SUV60min > 0.5). Radiotracer (68)Ga-NOTA-BBN2 represents a novel radiometal-based bombesin derivative suitable for GRP receptor targeting in PC3 and LNCaP mouse xenografts. Further increase of metabolic stability in vivo and enhanced tumor uptake were not observed upon administration of protease inhibitor phosphoramidon. This led to the conclusion that the recently proposed "serve-and-protect" strategy may not be valid for peptides exhibiting favorable intrinsic metabolic stability in vivo.
Subject(s)
Bombesin/chemistry , Gallium Radioisotopes/chemistry , Glycopeptides/chemistry , Positron-Emission Tomography/methods , Prostatic Neoplasms/diagnosis , Protease Inhibitors/chemistry , Animals , Cell Line, Tumor , Humans , Male , Mice , Mice, NudeABSTRACT
We previously showed that gastrin-releasing peptide receptor (GRPR) in the spinal cord is important for mediating nonhistaminergic itch. Neuromedin B receptor (NMBR), the second member of the mammalian bombesin receptor family, is expressed in a largely nonoverlapping pattern with GRPR in the superficial spinal cord, and its role in itch transmission remains unclear. Here, we report that Nmbr knock-out (KO) mice exhibited normal scratching behavior in response to intradermal injection of pruritogens. However, mice lacking both Nmbr and Grpr (DKO mice) showed significant deficits in histaminergic itch. In contrast, the chloroquine (CQ)-evoked scratching behavior of DKO mice is not further reduced compared with Grpr KO mice. These results suggest that NMBR and GRPR could compensate for the loss of each other to maintain normal histamine-evoked itch, whereas GRPR is exclusively required for CQ-evoked scratching behavior. Interestingly, GRPR activity is enhanced in Nmbr KO mice despite the lack of upregulation of Grpr expression; so is NMBR in Grpr KO mice. We found that NMB acts exclusively through NMBR for itch transmission, whereas GRP can signal through both receptors, albeit to NMBR to a much lesser extent. Although NMBR and NMBR(+) neurons are dispensable for histaminergic itch, GRPR(+) neurons are likely to act downstream of NMBR(+) neurons to integrate NMB-NMBR-encoded histaminergic itch information in normal physiological conditions. Together, we define the respective function of NMBR and GRPR in itch transmission, and reveal an unexpected relationship not only between the two receptors but also between the two populations of interneurons in itch signaling.
Subject(s)
Gastrin-Releasing Peptide/metabolism , Nociception/physiology , Posterior Horn Cells/physiology , Pruritus/physiopathology , Receptors, Bombesin/metabolism , Signal Transduction , Animals , Gastrin-Releasing Peptide/genetics , Histamine , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Neural Inhibition , Pruritus/chemically induced , Receptors, Bombesin/geneticsABSTRACT
Current translational cancer research is directed to the development of high affinity peptide ligands for targeting neuropeptide receptors overexpressed in different types of cancer. Besides their desired high binding affinity to the receptor, the suitability of radiolabeled peptides as targeting vectors for molecular imaging and therapy depends on additional aspects such as high tumor-to-background ratio, favorable clearance pattern from nontarget tissue, and sufficient metabolic stability in vivo. This study reports how a switch from the prosthetic group, N-succinimidyl-4-[(18)F]fluorobenzoate ([(18)F]SFB), to 2-deoxy-2-[(18)F]fluoro-d-glucose ([(18)F]FDG) effects the metabolic pathway of an (18)F-labeled bombesin derivative, QWAV-Sar-H-FA01010-Tle-NH2. (18)F-Labeled bombesin derivatives represent potent peptide ligands for selective targeting of gastrin-releasing peptide (GRP) receptor-expressing prostate cancer. Radiosynthesis of (18)F-labeled bombesin analogues [(18)F]FBz-Ava-BBN2 and [(18)F]FDG-AOAc-BBN2 was achieved in good radiochemical yields of ~50% at a specific activity exceeding 40 GBq/µmol. Both nonradioactive compounds FBz-Ava-BBN2 and FDG-AOAc-BBN2 inhibited binding of [(125)I]Tyr(4)-bombesin(1-14) in PC3 cells with IC50 values of 9 and 16 nM, respectively, indicating high inhibitory potency. Influence of each prosthetic group was further investigated in PC3 mouse xenografts using dynamic small animal PET imaging. In comparison to [(18)F]FBz-Ava-BBN2, total tumor uptake levels were doubled after injection of [(18)F]FDG-AOAc-BBN2 while renal elimination was increased. Blood clearance and in vivo metabolic stability were similar for both compounds. The switch from [(18)F]SFB to [(18)F]FDG as the prosthetic group led to a significant reduction in lipophilicity which resulted in more favorable renal clearance and increased tumor uptake. The presented single step radiolabeling-glycosylation approach represents an innovative strategy for site-directed peptide labeling with the short-lived positron emitter (18)F while providing a favorable pharmacokinetic profile of (18)F-labeled peptides.
Subject(s)
Bombesin/analogs & derivatives , Bombesin/metabolism , Fluorodeoxyglucose F18/analogs & derivatives , Fluorodeoxyglucose F18/metabolism , Prostatic Neoplasms/diagnosis , Animals , Bombesin/pharmacokinetics , Cell Line, Tumor , Fluorodeoxyglucose F18/pharmacokinetics , Humans , Male , Mice , Mice, Inbred BALB C , Positron-Emission Tomography/methods , Prostate/pathology , Prostatic Neoplasms/pathology , Tissue DistributionABSTRACT
Using adenovirus (Ad)-based vectors is a promising strategy for novel cancer treatments; however, current tracking approaches in vivo are limited. The C-terminus of the Ad minor capsid protein IX (pIX) can incorporate heterologous reporters to monitor biodistribution. We incorporated metallothionein (MT), a low-molecular-weight metal-binding protein, as a fusion to pIX. We previously demonstrated 99mTc binding in vitro to a pIX-MT fusion on the Ad capsid. We investigated different fusions of MT within pIX to optimize functional display. We identified a dimeric MT construct fused to pIX that showed significantly increased radiolabeling capacity. After Ad radiolabeling, we characterized metal binding in vitro. We explored biodistribution in vivo in control mice, mice pretreated with warfarin, mice preimmunized with wild-type Ad, and mice that received both warfarin pretreatment and Ad preimmunization. Localization of activity to liver and bladder was seen, with activity detected in spleen, intestine, and kidneys. Afterwards, the mice were euthanized and selected organs were dissected for further analysis. Similar to the imaging results, most of the radioactivity was found in the liver, spleen, kidneys, and bladder, with significant differences between the groups observed in the liver. These results demonstrate this platform application for following Ad dissemination in vivo.
Subject(s)
Metallothionein/chemistry , Metallothionein/metabolism , Radiopharmaceuticals/pharmacokinetics , Sodium Pertechnetate Tc 99m/pharmacokinetics , Animals , Cell Line, Tumor , Cloning, Molecular , Female , HEK293 Cells , Humans , Metallothionein/genetics , Mice , Mice, Inbred C57BL , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/metabolism , Tomography, Emission-Computed, Single-Photon/methods , Vaccination/methods , Warfarin/pharmacologyABSTRACT
A significant limiting factor to the human clinical application of conditionally replicative adenovirus (CRAd)-based virotherapy is the inability to noninvasively monitor these agents and their potential persistence. To address this issue, we proposed a novel imaging approach that combines transient expression of the human somatostatin receptor (SSTR) subtype 2 reporter gene with genetic labeling of the viral capsid with mCherry fluorescent protein. To test this dual modality system, we constructed the Ad5/3Δ24pIXcherry/SSTR CRAd and validated its capacity to generate fluorescent and nuclear signals in vitro and following intratumoral injection. Analysis of 64Cu-CB-TE2A-Y3-TATE biodistribution in mice revealed reduced uptake in tumors injected with the imaging CRAd relative to the replication-incompetent, Ad-expressing SSTR2 but significantly greater uptake compared to the negative CRAd control. Optical imaging demonstrated relative correlation of fluorescent signal with virus replication as determined by viral genome quantification in tumors. Positron emission tomography/computed tomography studies demonstrated that we can visualize radioactive uptake in tumors injected with imaging CRAd and the trend for greater uptake by standardized uptake value analysis compared to control CRAd. In the aggregate, the plasticity of our dual imaging approach should provide the technical basis for monitoring CRAd biodistribution and persistence in preclinical studies while offering potential utility for a range of clinical applications.
Subject(s)
Adenoviridae/physiology , Capsid/physiology , Luminescent Agents/metabolism , Luminescent Proteins/metabolism , Ovarian Neoplasms/virology , Receptors, Somatostatin/metabolism , Animals , Capsid/chemistry , Cell Line, Tumor , Coordination Complexes/pharmacokinetics , Female , HEK293 Cells , Humans , Mice , Neoplasm Transplantation , Oncolytic Virotherapy , Oncolytic Viruses/physiology , Peptides/pharmacokinetics , Receptors, Somatostatin/genetics , Virus Replication , Red Fluorescent ProteinABSTRACT
This study was aimed at developing a triazine-based modular platform for targeted PET imaging. We synthesized mono- or bis-cyclo(RGDfK) linked triazine-based conjugates specifically targeting integrin αvß3 receptors. The core molecules could be easily linked to targeting peptide and radiolabeled bifunctional chelator. The spacer core molecule was synthesized in 2 or 3 steps in 64-80% yield, and the following conjugation reactions with cyclo(RGDfK) peptide or bifunctional chelator were accomplished using "click" chemistry or amidation reactions. The DOTA-TZ-Bis-cyclo(RGDfK) 13 conjugate was radiolabeled successfully with (64)Cu(OAc)2 using a microfluidic method, resulting in higher specific activity with above 95% labeling yields compared to conventional radiolabeling (SA ca. 850 vs 600 Ci/mmol). The dimeric cyclo(RGDfK) peptide was found to display significant bivalency effect using I(125)-Echistatin binding assay with IC50 value as 178.5 ± 57.1 nM, which displayed a 3.6-fold enhancement of binding affinity compared to DOTA-TZ-cyclo(RGDfK) 14 conjugate on U87MG human glioblastoma cell. Biodistribution of all four conjugates in female athymic nude mice were evaluated. DOTA-"Click"-cyclo(RGDfK) 15 had the highest tumor uptake among these four at 4 h p.i. with 1.90 ± 0.65%ID/g, while there was no clear bivalency effect for DOTA-TZ-BisRGD in vivo, which needs further experiments to address the unexpected questions.
Subject(s)
Integrin alphaVbeta3/metabolism , Molecular Imaging/methods , Molecular Probes/chemistry , Peptides, Cyclic/chemistry , Positron-Emission Tomography , Triazines/chemistry , Animals , Click Chemistry , Copper Radioisotopes/chemistry , Female , Glioblastoma/metabolism , Humans , Iodine Radioisotopes/chemistry , Isotope Labeling , Mice , Mice, Nude , Microfluidic Analytical Techniques , Molecular Probes/metabolism , Molecular Probes/pharmacokinetics , Molecular Structure , Neoplasms, Experimental/metabolism , Peptides, Cyclic/metabolism , Peptides, Cyclic/pharmacokinetics , Rats , Structure-Activity Relationship , Tissue Distribution , Triazines/metabolism , Triazines/pharmacokinetics , Tumor Cells, CulturedABSTRACT
A series of new hexadentate and pentadentate chelators were designed and synthesized as chelators of (64)Cu. The new pentadentate and hexadentate chelators contain different types of donor groups and are expected to form neutral complexes with Cu(II). The new chelators were evaluated for complex kinetics and stability with (64)Cu. The new chelators instantly bound to (64)Cu with high labeling efficiency and maximum specific activity. All (64)Cu-radiolabeled complexes in human serum remained intact for 2 days. The (64)Cu-radiolabeled complexes were further challenged by EDTA in a 100-fold molar excess. Among the (64)Cu-radiolabeled complexes evaluated, (64)Cu-complex of the new chelator E was well tolerated with a minimal transfer of (64)Cu to EDTA. (64)Cu-radiolabeled complex of the new chelator E was further evaluated for biodistribution studies using mice and displayed rapid blood clearance and low organ uptake. (64)Cu-chelator E produced a favorable in vitro and in vivo complex stability profiles comparable to (64)Cu complex of the known hexadentate NOTA chelator. The in vitro and in vivo data highlight strong potential of the new chelator E for targeted PET imaging application.