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BACKGROUND: Targeted real-time imaging during robot-assisted radical prostatectomy provides information on the localisation and extent of prostate cancer. We assessed the safety and feasibility of the prostate-specific membrane antigen (PSMA)-targeted fluorescent tracer OTL78 in patients with prostate cancer. METHODS: In this single-arm, phase 2a, feasibility trial with an adaptive design was carried out in The Netherlands Cancer Institute, Netherlands. Male patients aged 18 years or older, with PSMA PET-avid prostate cancer with an International Society of Urological Pathology (ISUP) grade group of 2 or more, who were scheduled to undergo robot-assisted radical prostatectomy with or without extended pelvic lymph node dissection were eligible. All patients had a robot-assisted radical prostatectomy using OTL78. Based on timing and dose, patients received a single intravenous infusion of OTL78 (0·06 mg/kg 1-2 h before surgery [dose cohort 1], 0·03 mg/kg 1-2 h before surgery [dose cohort 2], or 0·03 mg/kg 24 h before surgery [dose cohort 3]). The primary outcomes, assessed in all enrolled patients, were safety and pharmacokinetics of OTL78. This study is completed and is registered in the European Trial Database, 2019-002393-31, and the International Clinical Trials Registry Platform, NL8552, and is completed. FINDINGS: Between June 29, 2020, and April 1, 2021, 19 patients were screened for eligibility, 18 of whom were enrolled. The median age was 69 years (IQR 64-70) and median prostate-specific antigen concentration was 15 ng/mL (IQR 9·3-22·0). In 16 (89%) of 18 patients, robot-assisted radical prostatectomy was accompanied by an extended pelvic lymph node dissection. Three serious adverse events occurred in one (6%) patient: an infected lymphocele, a urosepsis, and an intraperitoneal haemorrhage. These adverse events were considered unrelated to the administration of OTL78 or intraoperative fluorescence imaging. No patient died, required a dose reduction, or required discontinuation due to drug-related toxicity. The dose-normalised maximum serum concentration (Cmax/dose) in patients was 84·1 ng/mL/mg for the 0·03 mg/kg dose and 79·6 ng/mL/mg for the 0·06 mg/kg dose, the half-life was 5·1 h for the 0·03 mg/kg dose and 4·7 h for the 0·06 mg/kg dose, the volume of distribution was 22·9 L for the 0·03 mg/kg dose and 19·5 L for the 0·06 mg/kg dose, and the clearance was 3·1 L/h for the 0·03 mg/kg dose and 3·0 L/h for the 0·06 mg/kg dose. INTERPRETATION: This first-in-patient study showed that OTL78 was well tolerated and had the potential to improve prostate cancer detection. Optimal dosing was 0·03 mg/kg, 24 h preoperatively. PSMA-directed fluorescence imaging allowed real-time identification of visually occult prostate cancer and might help to achieve complete oncological resections. FUNDING: On Target Laboratories.
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Próstata , Neoplasias da Próstata , Humanos , Masculino , Idoso , Próstata/patologia , Estudos de Viabilidade , Neoplasias da Próstata/diagnóstico por imagem , Neoplasias da Próstata/cirurgia , Antígeno Prostático Específico , Prostatectomia/efeitos adversos , Imagem Óptica , Tomografia por Emissão de Pósitrons combinada à Tomografia ComputadorizadaRESUMO
Suspicious nodules detected by radiography are often investigated by biopsy, but the diagnostic yield of biopsies of small nodules is poor. Here we report a method-NIR-nCLE-to detect cancer at the cellular level in real-time during biopsy. This technology integrates a cancer-targeted near-infrared (NIR) tracer with a needle-based confocal laser endomicroscopy (nCLE) system modified to detect NIR signal. We develop and test NIR-nCLE in preclinical models of pulmonary nodule biopsy including human specimens. We find that the technology has the resolution to identify a single cancer cell among normal fibroblast cells when co-cultured at a ratio of 1:1000, and can detect cancer cells in human tumors less than 2 cm in diameter. The NIR-nCLE technology rapidly delivers images that permit accurate discrimination between tumor and normal tissue by non-experts. This proof-of-concept study analyzes pulmonary nodules as a test case, but the results may be generalizable to other malignancies.
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Neoplasias Pancreáticas , Biópsia , Endoscopia , Humanos , Lasers , Microscopia Confocal/métodos , Neoplasias Pancreáticas/patologiaRESUMO
Although immunotherapies of tumors have demonstrated promise for altering the progression of malignancies, immunotherapies have been limited by an immunosuppressive tumor microenvironment (TME) that prevents infiltrating immune cells from performing their anticancer functions. Prominent among immunosuppressive cells are myeloid-derived suppressor cells (MDSC) and tumor-associated macrophages (TAM) that inhibit T cells via release of immunosuppressive cytokines and engagement of checkpoint receptors. Here, we explore the properties of MDSCs and TAMs from freshly isolated mouse and human tumors and find that an immunosuppressive subset of these cells can be distinguished from the nonimmunosuppressive population by its upregulation of folate receptor beta (FRß) within the TME and its restriction to the TME. This FRß+ subpopulation could be selectively targeted with folate-linked drugs. Delivery of a folate-targeted TLR7 agonist to these cells (i) reduced their immunosuppressive function, (ii) increased CD8+ T-cell infiltration, (iii) enhanced M1/M2 macrophage ratios, (iv) inhibited tumor growth, (v) blocked tumor metastasis, and (vi) improved overall survival without demonstrable toxicity. These data reveal a broadly applicable strategy across tumor types for reprogramming MDSCs and TAMs into antitumorigenic immune cells using a drug that would otherwise be too toxic to administer systemically. The data also establish FRß as the first marker that distinguishes immunosuppressive from nonimmunosuppressive subsets of MDSCs and TAMs. Because all solid tumors accumulate MDSCs and TAMs, a general strategy to both identify and reprogram these cells should be broadly applied in the characterization and treatment of multiple tumors. SIGNIFICANCE: FRß serves as both a means to identify and target MDSCs and TAMs within the tumor, allowing for delivery of immunomodulatory compounds to tumor myeloid cells in a variety of cancers.
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Receptor 2 de Folato/metabolismo , Células Mieloides/efeitos dos fármacos , Células Supressoras Mieloides/imunologia , Microambiente Tumoral/imunologia , Macrófagos Associados a Tumor/imunologia , Adenocarcinoma/patologia , Adenocarcinoma/secundário , Animais , Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/patologia , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Linhagem Celular Tumoral , Polaridade Celular , Técnicas de Reprogramação Celular , Citocinas/metabolismo , Ácido Fólico/farmacologia , Humanos , Imunomodulação/efeitos dos fármacos , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/secundário , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Células Mieloides/patologia , Células Supressoras Mieloides/metabolismo , Macrófagos Associados a Tumor/metabolismo , Regulação para CimaRESUMO
PURPOSE: The ability to locate and remove all malignant lesions during radical prostatectomy leads not only to prevent biochemical recurrence (BCR) and possible side effects but also to improve the life expectancy of patients with prostate cancer. Fluorescence-guided surgery (FGS) has emerged as a technique that uses fluorescence to highlight cancerous cells and guide surgeons to resect tumors in real time. Thus, development of tumor-specific near-infrared (NIR) agents that target biomarkers solely expressed on prostate cancer cells will enable to assess negative tumor margins and affected lymph nodes. EXPERIMENTAL DESIGN: Because PSMA is overexpressed in prostate cancer cells in >90% of the prostate cancer patient population, a prostate-specific membrane antigen (PSMA)-targeted NIR agent (OTL78) was designed and synthesized. Optical properties, in vitro and in vivo specificity, tumor-to-background ratio (TBR), accomplishment of negative surgical tumor margins using FGS, pharmacokinetics (PKs) properties, and preclinical toxicology of OTL78 were then evaluated in requisite models. RESULTS: OTL78 binds to PSMA-expressing cells with high affinity, concentrates selectively to PSMA-positive cancer tissues, and clears rapidly from healthy tissues with a half-time of 17 minutes. It also exhibits an excellent TBR (5:1) as well as safety profile in animals. CONCLUSIONS: OTL78 is an excellent tumor-specific NIR agent for use in fluorescence-guided radical prostatectomy and FGS of other cancers.
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Antígenos de Superfície/genética , Corantes Fluorescentes/farmacologia , Glutamato Carboxipeptidase II/genética , Neoplasias da Próstata/diagnóstico por imagem , Cirurgia Assistida por Computador , Animais , Antígenos de Superfície/isolamento & purificação , Linhagem Celular Tumoral , Modelos Animais de Doenças , Fluorescência , Corantes Fluorescentes/química , Regulação Neoplásica da Expressão Gênica/genética , Glutamato Carboxipeptidase II/isolamento & purificação , Xenoenxertos , Humanos , Raios Infravermelhos , Masculino , Margens de Excisão , Imagem Óptica , Próstata/cirurgia , Prostatectomia/métodos , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Neoplasias da Próstata/cirurgia , Espectroscopia de Luz Próxima ao InfravermelhoRESUMO
Because the most reliable therapy for cancer involves quantitative resection of all diseased tissue, considerable effort has been devoted to improving a surgeon's ability to locate and remove all malignant lesions. With the aid of improved optical imaging equipment, we and others have focused on developing tumor-targeted fluorescent dyes to selectively illuminate cancer nodules during surgery. We describe here the design, synthesis, optical properties, in vitro and in vivo tumor specificity/affinity, pharmacokinetics, preclinical toxicology, and some clinical application of a folate receptor (FR)-targeted NIR dye (OTL38) that concentrates specifically in cancer tissues and clears rapidly from healthy tissues. We demonstrate that OTL38 binds FR-expressing cells with â¼1 nM affinity and eliminates from receptor negative tissues with a half-time of <30 min. We further show that OTL38 enables visualization of malignant lesions at concentrations less than 100-fold those required to elicit signs of toxicity. Since OTL38 also provides excellent tumor contrast in both murine tumor models and human cancer patients, we conclude that OTL38 constitutes an excellent NIR dye for fluorescence-guided resection of malignant lesions in cancer patients.
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Corantes Fluorescentes/química , Corantes Fluorescentes/metabolismo , Ácido Fólico/química , Ácido Fólico/metabolismo , Raios Infravermelhos , Neoplasias/cirurgia , Cirurgia Assistida por Computador , Células A549 , Animais , Desenho de Fármacos , Fluorescência , Corantes Fluorescentes/síntese química , Receptores de Folato com Âncoras de GPI/química , Receptores de Folato com Âncoras de GPI/metabolismo , Ácido Fólico/síntese química , Humanos , Células KB , Camundongos , Simulação de Acoplamento Molecular , Neoplasias/diagnóstico por imagem , Neoplasias/metabolismo , Neoplasias/patologia , Conformação ProteicaRESUMO
Carbonic anhydrase IX (CAIX) is a membrane-spanning zinc metalloenzyme that catalyzes the reversible consumption of CO2 and water to form H+ + HCO3-. Many human cancers upregulate CAIX to help control the pH in their hypoxic microenvironments. The consequent overexpression of CAIX on malignant cells and low expression on normal tissues render CAIX a particularly attractive target for small molecule inhibitors, antibody-drug conjugates, and ligand-targeted drugs. In this study, CAIX-targeted fluorescent reporter molecules were initially exploited to investigate CAIX-specific binding to multiple cancer cell lines, where they were shown to display potent and selective binding to CAIX positive cells. A small molecule CAIX-targeted tubulysin B conjugate was then synthesized and examined for its ability to kill CAIX-expressing tumor cells in vitro. Potent therapeutic conjugates were subsequently tested in vivo and demonstrated to eliminate solid human tumor xenografts in murine tumor models without exhibiting overt signs of toxicity. Because most solid tumors contain hypoxic regions where CAIX is overexpressed, development of a method to selectively deliver drugs to these hypoxic regions could aid in the therapy of otherwise difficult to treat tumors.
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Antineoplásicos/farmacologia , Anidrase Carbônica IX/antagonistas & inibidores , Inibidores da Anidrase Carbônica/farmacologia , Neoplasias/tratamento farmacológico , Oligopeptídeos/farmacologia , Ácidos Pipecólicos/farmacologia , Animais , Antígenos de Neoplasias/metabolismo , Antineoplásicos/síntese química , Antineoplásicos/uso terapêutico , Anidrase Carbônica IX/metabolismo , Inibidores da Anidrase Carbônica/síntese química , Inibidores da Anidrase Carbônica/uso terapêutico , Linhagem Celular Tumoral , Desenho de Fármacos , Feminino , Células HEK293 , Humanos , Camundongos , Camundongos Nus , Neoplasias/patologia , Oligopeptídeos/síntese química , Oligopeptídeos/uso terapêutico , Ácidos Pipecólicos/síntese química , Ácidos Pipecólicos/uso terapêutico , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: Clinical applicability of folate receptor-targeted intraoperative molecular imaging (FR-IMI) has been established for surgically resectable pulmonary adenocarcinoma. A role for FR-IMI in other lung cancer histologies has not been studied. In this study, we evaluate feasibility of FR-IMI in patients undergoing pulmonary resection for squamous cell carcinomas (SCCs). METHODS: In a human clinical trial (NCT02602119), twelve subjects with pulmonary SCCs underwent FR-IMI with a near-infrared contrast agent that targets the folate receptor-α (FRα), OTL38. Near-infrared signal from tumors and benign lung was quantified to calculate tumor-to-background ratios (TBR). Folate receptor-alpha expression was characterized, and histopathologic correlative analyses were performed to evaluate patterns of OTL38 accumulation. An exploratory analysis was performed to determine patient and histopathologic variables that predict tumor fluorescence. RESULTS: 9 of 13 SCCs (in 9 of 12 of subjects) displayed intraoperative fluorescence upon NIR evaluation (median TBR, 3.9). OTL38 accumulated within SCCs in a FRα-dependent manner. FR-IMI was reliable in localizing nodules as small as 1.1 cm, and prevented conversion to thoracotomy for nodule localization in three subjects. Upon evaluation of patient and histopathologic variables, in situ fluorescence was associated with distance from the pleural surface, and was independent of alternative variables including tumor size and metabolic activity. CONCLUSIONS: This work demonstrates that FR-IMI is potentially feasible in 70% of SCC patients, and that molecular imaging can improve localization during minimally invasive pulmonary resection. These findings complement previous data demonstrating that â¼98% of pulmonary adenocarcinomas are localized during FR-IMI and suggest broad applicability for NSCLC patients undergoing resection.
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Non-small cell lung cancer (NSCLC) is the number one cancer killer in the United States. Despite attempted curative surgical resection, nearly 40% of patients succumb to recurrent disease. High recurrence rates may be partially explained by data suggesting that 20% of NSCLC patients harbor synchronous disease that is missed during resection. In this report, we describe the use of a novel folate receptor-targeted near-infrared contrast agent (OTL38) to improve the intraoperative localization of NSCLC during pulmonary resection. Using optical phantoms, fluorescent imaging with OTL38 was associated with less autofluorescence and greater depth of detection compared to traditional optical contrast agents. Next, in in vitro and in vivo NSCLC models, OTL38 reliably localized NSCLC models in a folate receptor-dependent manner. Before testing intraoperative molecular imaging with OTL38 in humans, folate receptor-alpha expression was confirmed to be present in 86% of pulmonary adenocarcinomas upon histopathologic review of 100 human pulmonary resection specimens. Lastly, in a human feasibility study, intraoperative molecular imaging with OTL38 accurately identified 100% of pulmonary adenocarcinomas and allowed for identification of additional subcentimeter neoplastic processes in 30% of subjects. This technology may enhance the surgeon's ability to identify NSCLC during oncologic resection and potentially improve long-term outcomes.
Assuntos
Adenocarcinoma/diagnóstico por imagem , Adenocarcinoma/metabolismo , Meios de Contraste , Receptores de Folato com Âncoras de GPI/metabolismo , Neoplasias Pulmonares/diagnóstico por imagem , Neoplasias Pulmonares/metabolismo , Imagem Molecular , Adenocarcinoma/genética , Adenocarcinoma/cirurgia , Animais , Modelos Animais de Doenças , Feminino , Receptor 1 de Folato/genética , Receptor 1 de Folato/metabolismo , Receptores de Folato com Âncoras de GPI/genética , Expressão Gênica , Humanos , Imuno-Histoquímica , Cuidados Intraoperatórios , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/cirurgia , Masculino , Camundongos , Imagem Molecular/métodos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
OBJECTIVE: To determine if intraoperative molecular imaging (IMI) can improve detection of malignant pulmonary nodules. BACKGROUND: 18-Fluorodeoxyglucose positron emission tomography (PET) is commonly utilized in preoperative assessment of patients with solid malignancies; however, false negatives and false positives remain major limitations. Using patients with pulmonary nodules as a study model, we hypothesized that IMI with a folate receptor targeted near-infrared contrast agent (OTL38) can improve malignant pulmonary nodule identification when combined with PET. METHODS: Fifty patients with pulmonary nodules with imaging features suspicious for malignancy underwent preoperative PET. Patients then received OTL38 before pulmonary resection. During resection, IMI was utilized to evaluate known pulmonary nodules and identify synchronous lesions. Tumor size, PET standardized uptake value, and IMI tumor-to-background ratios were compared for known and synchronous nodules via paired and unpaired t tests, when appropriate. Test characteristics of PET and IMI with OTL38 were compared. RESULTS: IMI identified 56 of 59 (94.9%) malignant pulmonary nodules identified by preoperative imaging. IMI located an additional 9 malignant lesions not identified preoperatively. Nodules only detected by IMI were smaller than nodules detected preoperatively (0.5 vs 2.4âcm; P < 0.01), but displayed similar fluorescence (tumor-to-background ratio 3.3 and 3.1; P = 0.50). Sensitivity of IMI and PET were 95.6% and 73.5% (P = 0.001), respectively; and positive predictive values were 94.2% and 89.3%, respectively (P > 0.05). Additionally, utilization of IMI clinically upstaged 6 (12%) subjects and improved management of 15 (30%) subjects. CONCLUSIONS: These data suggest that combining IMI with PET may provide superior oncologic outcomes for patients with resectable lung cancer.
Assuntos
Adenocarcinoma/diagnóstico por imagem , Cuidados Intraoperatórios/métodos , Neoplasias Pulmonares/diagnóstico por imagem , Imagem Molecular/métodos , Pneumonectomia , Tomografia por Emissão de Pósitrons/métodos , Nódulo Pulmonar Solitário/diagnóstico por imagem , Adenocarcinoma/cirurgia , Adulto , Idoso , Meios de Contraste , Feminino , Fluordesoxiglucose F18 , Humanos , Neoplasias Pulmonares/cirurgia , Masculino , Pessoa de Meia-Idade , Projetos Piloto , Cuidados Pré-Operatórios , Compostos Radiofarmacêuticos , Sensibilidade e Especificidade , Nódulo Pulmonar Solitário/cirurgia , Espectroscopia de Luz Próxima ao InfravermelhoRESUMO
Background. Intraoperative imaging can identify cancer cells in order to improve resection; thus fluorescent contrast agents have emerged. Our objective was to do a preclinical comparison of two fluorescent dyes, EC17 and OTL38, which both target folate receptor but have different fluorochromes. Materials. HeLa and KB cells lines were used for in vitro and in vivo comparisons of EC17 and OTL38 brightness, sensitivity, pharmacokinetics, and biodistribution. In vivo experiments were then performed in mice. Results. The peak excitation and emission wavelengths of EC17 and OTL38 were 470/520 nm and 774/794 nm, respectively. In vitro, OTL38 required increased incubation time compared to EC17 for maximum fluorescence; however, peak signal-to-background ratio (SBR) was 1.4-fold higher compared to EC17 within 60 minutes (p < 0.001). Additionally, the SBR for detecting smaller quantity of cells was improved with OTL38. In vivo, the mean improvement in SBR of tumors visualized using OTL38 compared to EC17 was 3.3 fold (range 1.48-5.43). Neither dye caused noticeable toxicity in animal studies. Conclusions. In preclinical testing, OTL38 appears to have superior sensitivity and brightness compared to EC17. This coincides with the accepted belief that near infrared (NIR) dyes tend to have less autofluorescence and scattering issues than visible wavelength fluorochromes.
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A chemically defined anti-CXCR4-auristatin antibody-drug conjugate (ADC) was synthesized that selectively eliminates tumor cells overexpressing the CXCR4 receptor. The unnatural amino acid p-acetylphenylalanine (pAcF) was site-specifically incorporated into an anti-CXCR4 immunoglobulinâ G (IgG) and conjugated to an auristatin through a stable, non-cleavable oxime linkage to afford a chemically homogeneous ADC. The full-length anti-CXCR4 ADC was selectively cytotoxic to CXCR4(+) cancer cells inâ vitro (half maximal effective concentration (EC50 )≈80-100â pM). Moreover, the anti-CXCR4 ADC eliminated pulmonary lesions from human osteosarcoma cells in a lung-seeding tumor model in mice. No significant overt toxicity was observed but there was a modest decrease in the bone-marrow-derived CXCR4(+) cell population. Because CXCR4 is highly expressed in a majority of metastatic cancers, a CXCR4-auristatin ADC may be useful for the treatment of a variety of metastatic malignancies.
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Anticorpos Monoclonais/química , Antineoplásicos/química , Imunoconjugados/química , Imunoterapia/métodos , Receptores CXCR4/química , Linhagem Celular Tumoral , HumanosRESUMO
A folate targeted camptothecin small molecule drug conjugate (SMDC) was synthesized using a monodisperse PEG spacer linked to folate via a releasable disulfide carbonate linker. Cell cytotoxicity in human KB cells exhibited an IC50 of 6nM. Importantly, activity of the prodrug was blocked by excess folate, demonstrating receptor-mediated celluar uptake of the PEG conjugate.
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Antineoplásicos Fitogênicos/química , Camptotecina/análogos & derivados , Ácido Fólico/química , Polietilenoglicóis/química , Pró-Fármacos/química , Antineoplásicos Fitogênicos/administração & dosagem , Antineoplásicos Fitogênicos/farmacocinética , Antineoplásicos Fitogênicos/farmacologia , Camptotecina/administração & dosagem , Camptotecina/farmacocinética , Camptotecina/farmacologia , Sistemas de Liberação de Medicamentos , Receptores de Folato com Âncoras de GPI/metabolismo , Humanos , Células KB , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Pró-Fármacos/administração & dosagem , Pró-Fármacos/farmacocinética , Pró-Fármacos/farmacologiaRESUMO
Activated macrophages play a significant role in initiation and progression of inflammatory diseases and may serve as the basis for the development of targeted diagnostic methods for imaging sites of inflammation. Folate receptor beta (FR-ß) is differentially expressed on activated macrophages associated with inflammatory disease states yet is absent in either quiescent or resting macrophages. Because folate binds with high affinity to FR-ß, development of folate directed imaging agents has proceeded rapidly in the past decade. However, reports of PET based imaging agents for use in inflammatory conditions remain limited. To investigate whether FR-ß expressing macrophages could be exploited for PET based inflammatory imaging, two separate folate-targeted PET imaging agents were developed, 4-[(18)F]-fluorophenylfolate and [(68)Ga]-DOTA-folate, and their ability to target activated macrophages were examined in a rodent inflammatory paw model. We further compared inflamed tissue uptake with 2-[(18)F]fluoro-2-deoxy-d-glucose ([(18)F]-FDG). microPET analysis demonstrated that both folate-targeted PET tracers had higher uptake in the inflamed paw compared to the control paw. When these radiotracers were compared to [(18)F]-FDG, both folate PET tracers had a higher signal-to-noise ratio (SNR) than [(18)F]-FDG, suggesting that folate tracers may be superior to [(18)F]-FDG in detecting diseases with an inflammatory component. Moreover, both folate-PET imaging agents also bind to FR-α which is overexpressed on multiple human cancers. Therefore, these folate derived PET tracers may also find use for localizing and staging FR(+) cancers, monitoring response to therapy, and for selecting patients for tandem folate-targeted therapies.
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Fluordesoxiglucose F18 , Ácido Fólico , Inflamação/diagnóstico , Tomografia por Emissão de Pósitrons , AnimaisRESUMO
Complete surgical resection of malignant disease is the only reliable method to cure cancer. Unfortunately, quantitative tumor resection is often limited by a surgeon's ability to locate all malignant disease and distinguish it from healthy tissue. Fluorescence-guided surgery has emerged as a tool to aid surgeons in the identification and removal of malignant lesions. While nontargeted fluorescent dyes have been shown to passively accumulate in some tumors, the resulting tumor-to-background ratios are often poor, and the boundaries between malignant and healthy tissues can be difficult to define. To circumvent these problems, our laboratory has developed high affinity tumor targeting ligands that bind to receptors that are overexpressed on cancer cells and deliver attached molecules selectively into these cells. In this study, we explore the use of two tumor-specific targeting ligands (i.e., folic acid that targets the folate receptor (FR) and DUPA that targets prostate specific membrane antigen (PSMA)) to deliver near-infrared (NIR) fluorescent dyes specifically to FR and PSMA expressing cancers, thereby rendering only the malignant cells highly fluorescent. We report here that all FR- and PSMA-targeted NIR probes examined bind cultured cancer cells in the low nanomolar range. Moreover, upon intravenous injection into tumor-bearing mice with metastatic disease, these same ligand-NIR dye conjugates render receptor-expressing tumor tissues fluorescent, enabling their facile resection with minimal contamination from healthy tissues.
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Fluorescência , Corantes Fluorescentes , Neoplasias Mamárias Experimentais/diagnóstico , Neoplasias/diagnóstico , Neoplasias/cirurgia , Animais , Feminino , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/química , Ácido Fólico/química , Glutaratos/química , Humanos , Raios Infravermelhos , Células KB , Camundongos , Camundongos Endogâmicos DBA , Estrutura Molecular , Células Tumorais Cultivadas , Ureia/análogos & derivados , Ureia/químicaRESUMO
INTRODUCTION: Detection of (subclinical) synovitis is relevant for both early diagnosis and monitoring of therapy of rheumatoid arthritis (RA). Previously, the potential of imaging (sub)clinical arthritis was demonstrated by targeting the translocator protein in activated macrophages using (R)-[11C]PK11195 and positron emission tomography (PET). Images, however, also showed significant peri-articular background activity. The folate receptor (FR)-ß is a potential alternative target for imaging activated macrophages. Therefore, the PET tracer [18F]fluoro-PEG-folate was synthesized and evaluated in both in vitro and ex vivo studies using a methylated BSA induced arthritis model. METHODS: [18F]fluoro-PEG-folate was synthesized in a two-step procedure. Relative binding affinities of non-radioactive fluoro-PEG-folate, folic acid and naturally circulating 5-methyltetrahydrofolate (5-Me-THF) to FR were determined using KB cells with high expression of FR. Both in vivo [18F]fluoro-PEG-folate PET and ex vivo tissue distribution studies were performed in arthritic and normal rats and results were compared with those of the established macrophage tracer (R)-[11C]PK11195. RESULTS: [18F]fluoro-PEG-folate was synthesized with a purity >97%, a yield of 300 to 1,700 MBq and a specific activity between 40 and 70 GBq/µmol. Relative in vitro binding affinity for FR of F-PEG-folate was 1.8-fold lower than that of folic acid, but 3-fold higher than that of 5-Me-THF. In the rat model, [18F]fluoro-PEG-folate uptake in arthritic knees was increased compared with both contralateral knees and knees of normal rats. Uptake in arthritic knees could be blocked by an excess of glucosamine-folate, consistent with [18F]fluoro-PEG-folate being specifically bound to FR. Arthritic knee-to-bone and arthritic knee-to-blood ratios of [18F]fluoro-PEG-folate were increased compared with those of (R)-[11C]PK11195. Reduction of 5-Me-THF levels in rat plasma to those mimicking human levels increased absolute [18F]fluoro-PEG-folate uptake in arthritic joints, but without improving target-to-background ratios. CONCLUSIONS: The novel PET tracer [18F]fluoro-PEG-folate, designed to target FR on activated macrophages provided improved contrast in a rat model of arthritis compared with the accepted macrophage tracer (R)-[11C]PK11195. These results warrant further exploration of [18F]fluoro-PEG-folate as a putative PET tracer for imaging (sub)clinical arthritis in RA patients.
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Artrite Experimental/diagnóstico por imagem , Macrófagos/diagnóstico por imagem , Compostos Radiofarmacêuticos , Sinovite/diagnóstico por imagem , Animais , Fluordesoxiglucose F18 , Ácido Fólico/análogos & derivados , Humanos , Processamento de Imagem Assistida por Computador , Imuno-Histoquímica , Ligantes , Masculino , Polietilenoglicóis , Cintilografia , Compostos Radiofarmacêuticos/síntese química , Ratos , Ratos WistarAssuntos
Receptores de Folato com Âncoras de GPI/metabolismo , Linfócitos T Citotóxicos/imunologia , Animais , Complexo CD3/imunologia , Linhagem Celular Tumoral , Técnicas de Cocultura , Ácido Fólico/química , Humanos , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fab das Imunoglobulinas/imunologia , Fragmentos Fc das Imunoglobulinas/química , Fragmentos Fc das Imunoglobulinas/imunologia , Imunoglobulina G/química , Imunoglobulina G/imunologia , Imunoterapia , Leucócitos Mononucleares/citologia , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Neoplasias/metabolismo , Neoplasias/patologia , Neoplasias/terapia , Ratos , Ratos Sprague-Dawley , Linfócitos T Citotóxicos/citologiaRESUMO
UNLABELLED: Folic acid has been frequently exploited to target attached drugs to cells that overexpress a folate receptor (FR). Unfortunately, folic acid and folate-linked drugs bind equally well to both major isoforms of the FR-that is, FR-α, which is primarily expressed on malignant cells, and FR-ß, which is upregulated on activated monocytes and macrophages. Because both major isoforms of FR can be expressed simultaneously in the same organism, folic acid cannot enable selective targeting of therapeutic and imaging agents to either tumor masses or sites of inflammation. In an effort to develop a targeting ligand that can selectively deliver attached imaging and therapeutic agents to tumor cells, we constructed a reduced and alkylated form of folic acid, N(5), N(10)-dimethyl tetrahydrofolate (DMTHF) that exhibits selectivity for FR-α. METHODS: DMTHF-(99m)Tc was injected into mice bearing FR-α-expressing tumor xenografts and imaged by γ-scintigraphy. The selectivity for FR-α over FR-ß in vivo was examined by γ-scintigraphic images of animal models of various inflammatory diseases such as apolipoprotein E-deficient mice with atherosclerosis, DBA/1 LacJ mice with induced arthritis, C57BL/6J mice with muscle injury, and BALB/C mice with both FR-α tumor and ulcerative colitis, by administration of equal doses of DMTHF-(99m)Tc and EC20-(99m)Tc. The uptake of radiochelates in various organs was quantified by biodistribution studies. DMTHF-near-infrared dye conjugate and DMTHF-Oregon green dye conjugates were synthesized and evaluated for FR-α selectivity over FR-ß in rat peritoneal macrophages and human peripheral blood monocytes, respectively, by flow cytometry. Fluorescence-guided imaging was also performed using folate and DMTHF dye conjugates. RESULTS: The new targeting ligand was found to bind malignant cells in mice with solid tumor xenografts but not peripheral blood monocytes or inflammatory macrophages in animal models of atherosclerosis, rheumatoid arthritis, muscle injury, or ulcerative colitis. Results from optical and radioimaging studies and biodistribution experiments confirm the differential specificity of this new ligand for malignant masses. CONCLUSION: The new targeting ligand DMTHF enables selective noninvasive imaging and therapy of tumor tissues in the presence of inflammation.
Assuntos
Receptor 1 de Folato/efeitos dos fármacos , Inflamação/diagnóstico por imagem , Neoplasias/diagnóstico por imagem , Compostos de Organotecnécio , Compostos Radiofarmacêuticos , Tetra-Hidrofolatos , Animais , Artrite/induzido quimicamente , Artrite/diagnóstico por imagem , Aterosclerose/diagnóstico por imagem , Células CHO , Linhagem Celular Tumoral , Quelantes/química , Quelantes/farmacocinética , Cricetinae , Cricetulus , Feminino , Citometria de Fluxo , Humanos , Células KB , Macrófagos Peritoneais/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Nus , Músculo Esquelético/diagnóstico por imagem , Músculo Esquelético/lesões , Compostos de Organotecnécio/química , Compostos de Organotecnécio/farmacocinética , Técnicas de Patch-Clamp , Cintilografia , Compostos Radiofarmacêuticos/química , Compostos Radiofarmacêuticos/farmacocinética , Ratos , Especificidade por Substrato , Tecnécio/química , Tecnécio/farmacocinética , Tetra-Hidrofolatos/química , Tetra-Hidrofolatos/farmacocinética , Distribuição TecidualRESUMO
Tumor progenitor cells represent a population of drug-resistant cells that can survive conventional chemotherapy and lead to tumor relapse. However, little is known of the role of tumor progenitors in prostate cancer metastasis. The studies reported herein show that the CXCR4/CXCL12 axis, a key regulator of tumor dissemination, plays a role in the maintenance of prostate cancer stem-like cells. The CXCL4/CXCR12 pathway is activated in the CD44(+)/CD133(+) prostate progenitor population and affects differentiation potential, cell adhesion, clonal growth and tumorigenicity. Furthermore, prostate tumor xenograft studies in mice showed that a combination of the CXCR4 receptor antagonist AMD3100, which targets prostate cancer stem-like cells, and the conventional chemotherapeutic drug Taxotere, which targets the bulk tumor, is significantly more effective in eradicating tumors as compared to monotherapy.
Assuntos
Células-Tronco Neoplásicas/metabolismo , Neoplasias da Próstata/metabolismo , Receptores CXCR4/metabolismo , Animais , Protocolos de Quimioterapia Combinada Antineoplásica , Benzilaminas , Adesão Celular , Proliferação de Células , Quimiocina CXCL12/metabolismo , Ciclamos , Docetaxel , Compostos Heterocíclicos/farmacologia , Humanos , Masculino , Camundongos , Metástase Neoplásica , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/patologia , Taxoides/farmacologiaRESUMO
A folate receptor targeted didemnin B conjugate was synthesized using a hydrophilic peptide spacer linked to folate via a releasable disulfide carbonate linker. Cell cytotoxicity and TNF-α inhibition in RAW264.7 macrophage-like cells exhibited IC(50)s of 13 and 5 nM, respectively. Folate didemnin B was found to be â¼50-100 fold more potent than didemnin B itself. More importantly, activity of the prodrug was blocked by excess folic acid, demonstrating receptor-mediated cellular uptake of the conjugate.
Assuntos
Depsipeptídeos/síntese química , Depsipeptídeos/farmacologia , Ácido Fólico/química , Inflamação/tratamento farmacológico , Animais , Carbono/química , Relação Dose-Resposta a Droga , Interações Hidrofóbicas e Hidrofílicas , Concentração Inibidora 50 , Macrófagos/citologia , Camundongos , Modelos Biológicos , Modelos Químicos , Peptídeos/química , Pró-Fármacos/farmacologia , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Due to the overexpression of a folate receptor (FR) on many malignant cells, folate-targeted drugs have been developed to improve the cancer specificity of chemotherapeutic agents. Therapeutic index is further enhanced with the use of self-immolative linkers that efficiently release the attached drug upon cellular internalization of the folate-drug conjugate. Because FR is also abundant in normal kidney proximal tubule (PT) cells, we sought to examine in real time the trafficking and release of folate-targeted drugs in the kidney in vivo. Thus, we conducted two-photon kidney imaging studies in mice utilizing a Förster resonance energy transfer (FRET) based folate conjugate that undergoes a color shift from red to green upon reduction of the disulfide bond linking folate to a surrogate drug molecule. Following infusion via intravenous injection, folate-FRET reached the kidney in its intact unreduced form. The folate-FRET conjugate was then filtered into the lumen of PT, where it was efficiently captured by FR. As FR transcytosed across PT, some disulfide reduction occurred, with reduced folate-FRET detectable in PT vesicles 30 min postinjection. Prolonged monitoring of folate-FRET in mice showed modest progression of reduction in PT cells over time. Moreover, inhibition of FR trafficking in PT cells by colchicine did not significantly affect the rate or extent of folate-FRET reduction. Finally, the lack of cytosolic accumulation of released drug surrogate in the PT suggests that drug release via disulfide bond reduction should cause little kidney toxicity.