ABSTRACT
Emotional distress (ED), commonly characterized by symptoms of depression and/or anxiety, is prevalent in patients with cancer. Preclinical studies suggest that ED can impair antitumor immune responses, but few clinical studies have explored its relationship with response to immune checkpoint inhibitors (ICIs). Here we report results from cohort 1 of the prospective observational STRESS-LUNG study, which investigated the association between ED and clinical efficacy of first-line treatment of ICIs in patients with advanced non-small-cell lung cancer. ED was assessed by Patient Health Questionnaire-9 and Generalized Anxiety Disorder 7-item scale. The study included 227 patients with 111 (48.9%) exhibiting ED who presented depression (Patient Health Questionnaire-9 score ≥5) and/or anxiety (Generalized Anxiety Disorder 7-item score ≥5) symptoms at baseline. On the primary endpoint analysis, patients with baseline ED exhibited a significantly shorter median progression-free survival compared with those without ED (7.9 months versus 15.5 months, hazard ratio 1.73, 95% confidence interval 1.23 to 2.43, P = 0.002). On the secondary endpoint analysis, ED was associated with lower objective response rate (46.8% versus 62.1%, odds ratio 0.54, P = 0.022), reduced 2-year overall survival rate of 46.5% versus 64.9% (hazard ratio for death 1.82, 95% confidence interval 1.12 to 2.97, P = 0.016) and detriments in quality of life. The exploratory analysis indicated that the ED group showed elevated blood cortisol levels, which was associated with adverse survival outcomes. This study suggests that there is an association between ED and worse clinical outcomes in patients with advanced non-small-cell lung cancer treated with ICIs, highlighting the potential significance of addressing ED in cancer management. ClinicalTrials.gov registration: NCT05477979 .
Subject(s)
Carcinoma, Non-Small-Cell Lung , Immune Checkpoint Inhibitors , Lung Neoplasms , Psychological Distress , Humans , Carcinoma, Non-Small-Cell Lung/drug therapy , Carcinoma, Non-Small-Cell Lung/immunology , Carcinoma, Non-Small-Cell Lung/pathology , Immune Checkpoint Inhibitors/therapeutic use , Immune Checkpoint Inhibitors/adverse effects , Female , Male , Lung Neoplasms/drug therapy , Lung Neoplasms/immunology , Middle Aged , Aged , Prospective Studies , Depression/drug therapy , Anxiety/drug therapy , Treatment Outcome , Progression-Free Survival , Adult , Aged, 80 and overABSTRACT
OBJECTIVE: This study aimed to identify a predictive marker of response to epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) in patients with EGFR-mutant advanced lung adenocarcinoma. METHODS: A cohort of 190 patients with EGFR-mutant advanced lung adenocarcinoma was analyzed. Receiver operating characteristic curve analysis was used to evaluate the optimal cutoffs for fibrinogen levels, the neutrophil-to-lymphocyte ratio (NLR), and the platelet-to-lymphocyte ratio (PLR) for predicting progression-free survival (PFS). Univariate and multivariate survival analyses were performed to identify factors correlated with PFS and overall survival (OS). RESULTS: High NLR was associated with worse performance status. In univariate analysis, fibrinogen levels, NLR, and PLR were correlated with OS and PFS. In multivariate analysis, all three variables remained predictive of OS, whereas only fibrinogen levels and PLR were independent prognostic factors for PFS. Furthermore, the combination of fibrinogen levels and PLR (F-PLR score) could stratify patients into three groups with significantly different prognoses, and the score was independently predictive of survival. CONCLUSION: The F-PLR score predicted the prognosis of patients with EGFR-mutant advanced lung adenocarcinoma who received EGFR-TKIs, and this score may serve as a convenient blood-based marker for identifying high-risk patients.
Subject(s)
Adenocarcinoma of Lung , Carcinoma, Non-Small-Cell Lung , Lung Neoplasms , Adenocarcinoma of Lung/drug therapy , Adenocarcinoma of Lung/genetics , ErbB Receptors/genetics , Fibrinogen , Humans , Lung Neoplasms/drug therapy , Lung Neoplasms/genetics , Lymphocytes , Neutrophils , Prognosis , Protein Kinase Inhibitors/therapeutic use , Retrospective StudiesABSTRACT
The synthetic house-tree-person (S-HTP) drawing test is a projective measure primarily designed to assess specific complex personality traits. It is widely used in general psychological problems and mental illness such as psychological crisis intervention. Applicability and validity of S-HTP drawing test in cancer patients suffering from anxiety are still unclear and there are no reports on such research. The aim of this study was to explore the prevalence of anxiety in cancer patients and to investigate the applicability of S-HTP drawing test in such patients. Self-rating anxiety scale (SAS) and the S-HTP drawing test were applied to 167 cancer patients (58.7% male; 41.3% female), 52.92±10.43 years old. On SAS, anxiety rate was found in 16.17% cancer patients. Using the evaluation results from SAS as the dependent variable and the anxiety drawing characteristics as the independent variables, the logistic regression equation was established, and 9 drawing features were employed in the regression equation (χ2=56.982, P≤0.001, Nagelkerke R2=0.492). It is concluded that there is a positive correlation between S-HTP drawing test and SAS for anxiety state of cancer patients (p<0.01). S-HTP drawing test and SAS have interrater reliability and test-retest reliability. Our findings indicate that the S-HTP drawing test could help in screening anxiety in cancer patients.
ABSTRACT
In Alzheimer's disease, the density and spread of aggregated tau protein track well with neurodegeneration and cognitive decline, making the imaging of aggregated tau a compelling biomarker. A structure-activity relationship exploration around an isoquinoline hit, followed by an exploration of tolerated fluorination positions, allowed us to identify 9 (JNJ-64326067), a potent and selective binder to aggregated tau with a favorable pharmacokinetic profile and no apparent off-target binding. This was confirmed in rat and monkey positron emission tomography studies using [18F]9.
Subject(s)
Drug Discovery , Fluorine Radioisotopes/metabolism , Isoquinolines/pharmacokinetics , Positron-Emission Tomography , Pyridines/pharmacokinetics , tau Proteins/metabolism , Alzheimer Disease/diagnostic imaging , Alzheimer Disease/metabolism , Animals , Cells, Cultured , Female , Fluorine Radioisotopes/chemistry , Fluorine Radioisotopes/pharmacokinetics , Hepatocytes/metabolism , Humans , Isoquinolines/chemistry , Macaca mulatta , Male , Pyridines/chemistry , Rats , Rats, Wistar , Structure-Activity RelationshipABSTRACT
The P2X7 receptor is an adenosine triphosphate-gated ion channel, which is abundantly expressed in glial cells within the central nervous system and in the periphery. P2X7 receptor activation leads to the release of the proinflammatory cytokine IL-1ß in the brain, and antagonism of the P2X7 receptor is a novel therapeutic strategy to dampen adenosine triphosphate-dependent IL-1ß signaling. PET ligands for the P2X7 receptor will not only be valuable to assess central target engagement of drug candidates but also hold promise as surrogate markers of central neuroinflammation. Herein we describe the in vitro and in vivo evaluation of 18F-JNJ-64413739, an 18F-labeled PET ligand for imaging the P2X7 receptor in the brain. Methods: P2X7 receptor affinity and specificity, pharmacokinetics, metabolic stability, blood-brain barrier permeability, and off-target binding of JNJ-64413739 were evaluated in a series of in vitro, ex vivo, and in vivo assays. 18F-JNJ-64413739 was radiolabeled via a one-step nucleophilic aromatic substitution. The tracer was also studied in rhesus macaques, and PET images were analyzed with an arterial plasma input function-based Logan graphical analysis. Results: The potency (half-maximal inhibitory concentration) of the P2X7 receptor antagonist JNJ-64413739 is 1.0 ± 0.2 nM and 2.0 ± 0.6 nM at the recombinant human and rat P2X7 receptor, respectively, and the binding affinity is 2.7 nM (rat cortex binding assay) and 15.9 nM (human P2X7 receptor). In nonhuman primate PET imaging studies, dose-dependent receptor occupancy of JNJ-54175446 was observed in 2 rhesus monkeys. At a 0.1 mg/kg dose (intravenous) of JNJ-54175446, the receptor occupancy was calculated to be 17% by Logan graphical analysis, whereas a dose of 2.5 mg/kg yielded a receptor occupancy of 60%. Conclusion: The preclinical evaluation of 18F-JNJ-64413739 demonstrates that the tracer engages the P2X7 receptor. Reproducible and dose-dependent receptor occupancy studies with the P2X7 receptor antagonist JNJ-54175446 were obtained in rhesus monkeys. This novel PET tracer exhibits in vitro and in vivo characteristics suitable for imaging the P2X7 receptor in the brain and warrants further studies in humans.
Subject(s)
Blood-Brain Barrier/drug effects , Peptides/pharmacology , Pyrimidines/pharmacology , Radiopharmaceuticals/pharmacology , Receptors, Purinergic P2X7/metabolism , Animals , Brain/drug effects , Female , Humans , In Vitro Techniques , Inflammation/diagnostic imaging , Kinetics , Ligands , Macaca mulatta , Male , Mice, Knockout , Permeability/drug effects , Positron-Emission Tomography , Protein Binding , Radiochemistry , Rats , Tissue Distribution , Treatment OutcomeABSTRACT
PURPOSE: The P2X7 receptor, an adenosine triphosphate (ATP)-gated purinoreceptor, has emerged as one of the key players in neuroinflammatory processes. Therefore, developing a positron emission tomography (PET) tracer for imaging of P2X7 receptors in vivo presents a promising approach to diagnose, monitor, and study neuroinflammation in a variety of brain disorders. To fulfill the goal of developing a P2X7 PET ligand as a biomarker of neuroinflammation, [18F]JNJ-64413739 has been recently disclosed. PROCEDURES: We evaluated [18F]JNJ-64413739 in a rat model of neuroinflammation induced by an intracerebral injection of lipopolysaccharide (LPS). In vivo brain uptake was determined by PET imaging. Upregulation of neuroinflammatory biomarkers was determined by quantitative polymerase chain reaction (qPCR). Distribution of the tracer in the brain was determined by ex vivo autoradiography (ARG). The specificity of [18F]JNJ-64413739 was confirmed by performing blocking experiments with the P2X7 antagonist JNJ-54175446. RESULTS: Brain regions of rats injected with LPS had a significantly increased uptake (34 % ± 3 % s.e.m., p = 0.036, t test, standardized uptake value measured over the entire scanning period) of [18F]JNJ-64413739 relative to the corresponding brain regions of control animals injected with phosphate-buffered saline (PBS). The uptake in the contralateral regions and cerebellum was not significantly different between the groups of animals. The increase in uptake of [18F]JNJ-64413739 at the LPS-injected site observed by PET imaging was concordant with ex vivo ARG, upregulation of neuroinflammatory biomarkers, and elevated P2X7 expression levels. CONCLUSIONS: While further work is needed to study [18F]JNJ-64413739 in other types of neuroinflammation, the current results favorably characterize [18F]JNJ-64413739 as a potential PET tracer of central neuroinflammation.
Subject(s)
Brain/diagnostic imaging , Inflammation/diagnostic imaging , Inflammation/pathology , Peptides/chemistry , Positron-Emission Tomography , Radiopharmaceuticals/chemistry , Receptors, Purinergic P2X7/metabolism , Animals , Biomarkers/metabolism , Brain/pathology , Disease Models, Animal , Lipopolysaccharides , Peptides/pharmacokinetics , Pilot Projects , Positron Emission Tomography Computed Tomography , Pyridines/chemistry , Rats , Triazoles/chemistryABSTRACT
OBJECTIVE: This study aimed to examine the influence of oral acetylsalicylic acid on blood fluidity and infusion speed in the cancer patients with Peripherally Inserted Central Catheter (PICC). BACKGROUND: PICC is placed for prolonged chemotherapy of cancer patients. The fibrin sheaths, which consist of cellular substance and non-cellular substance, generate at the place of insertion and grow down all over the catheter. Finally they cover the vent of the catheter and lead to catheter dysfunctions such as the decrease of infusion speed. In addition, the high viscosity status of cancer patients could lead to acute embolization, which adds to the high risk of death. DESIGN: Randomized controlled trial. METHODS: This research was carried out between April 2013 and January 2014 in the second hospital of Xiangya, Central South University in Changsha, China. Initially 96 cancer participants with PICC were chosen and randomly allocated to experimental and control group. The participants of the experimental group were conducted route PICC maintain technique and took acetylsalicylic acid 100âmg per day after dinner, while the control group received route PICC maintain technique only. The infusion speed and hemorheology indexes of the two groups were tested before our study and at the end of the 2nd and 4th months with several instruments. RESULTS: Repeated measures analysis of variance indicated that taking acetylsalicylic acid orally had significant main effect on high shear blood viscosity and red blood cell deformability index (Pâ<â0.05), and it also had significant main effect as well as time effect on plasma viscosity (Pâ<â0.05); and time had significant main effect as well as interaction effect with oral acetylsalicylic acid on low shear blood viscosity and red blood cell aggregation index (Pâ<â0.05). Repeated measures ANOVA also showed that taking acetylsalicylic acid orally had significant main effect, time effect and interaction effect on infusion speed (Pâ<â0.01). CONCLUSION: Oral acetylsalicylic acid could improve hemorheology condition and the infusion speed related to fibrin sheath in the cancer patients.
Subject(s)
Aspirin/therapeutic use , Catheterization, Peripheral/instrumentation , Neoplasms/therapy , Administration, Oral , Catheterization, Peripheral/methods , Female , Hemorheology , Humans , Male , Middle AgedABSTRACT
BACKGROUND: The present study aimed to explore the association between RDW and CAS in patients with ischemic stroke, expecting to find a new and significant diagnosis index for clinical practice. METHODS: This cross-sectional study involves 432 consecutive patients with primary ischemic stroke (within 72 h). All subjects were confirmed by magnetic resonance imaging, and underwent physical examination, laboratory tests and carotid ultrasonography check. Finally, 392 patients were included according to the exclusion criteria. The odds ratios of independent variables were calculated using stepwise multiple logistic regression. RESULTS: Carotid intimal-medial thickness (IMT) and RDW are both significantly different between CAS group and control group. Univariate analyses show that high-sensitive C-reactive protein (Hs-CRP) and RDW (r=0.436) are both in significantly positive association with IMT. Stepwise multiple logistic regression shows that RDW is an independent protective factor of CAS in patients with ischemic stroke. Compared with the lowest quartile, the second to fourth quartiles are 1.13 (95% CI: 1.13-3.05), 2.02 (95% CI: 1.66-4.67), and 3.10 (95% CI: 2.46-7.65), respectively. CONCLUSION: The present study suggested that RDW level were higher than non-CAS in patients with primary ischemic stroke. Our results facilitated a bridge to connect RDW with ischemic stroke and further confirmed the role of RDW in the progression of the ischemic stroke.
Subject(s)
Brain Ischemia/blood , Carotid Artery Diseases/blood , Aged , Brain Ischemia/epidemiology , Brain Ischemia/etiology , Carotid Arteries/pathology , Carotid Artery Diseases/complications , Carotid Artery Diseases/diagnosis , China/epidemiology , Cross-Sectional Studies , Erythrocyte Indices , Female , Humans , Incidence , Magnetic Resonance Imaging , Male , Middle Aged , Odds Ratio , Risk FactorsABSTRACT
Aggregates of hyperphosphorylated tau (PHF-tau), such as neurofibrillary tangles, are linked to the degree of cognitive impairment in Alzheimer's disease. We have recently reported early clinical results of a novel PHF-tau targeting PET imaging agent, [F18]-T807. Since then, we have investigated a second novel PHF-tau targeting PET imaging agent, [F18]-T808, with different pharmacokinetic characteristics, which may be favorable for imaging Alzheimer's disease and other tauopathies. Here, we describe the first human brain images with [F18]-T808.
Subject(s)
Alzheimer Disease/diagnostic imaging , Benzimidazoles , Brain/diagnostic imaging , Brain/metabolism , Fluorodeoxyglucose F18 , Pyrimidines , tau Proteins/metabolism , Aged , Aged, 80 and over , Benzimidazoles/pharmacokinetics , Brain/drug effects , Brain Mapping , Female , Humans , Male , Mental Status Schedule , Middle Aged , Positron-Emission Tomography , Pyrimidines/pharmacokinetics , Time FactorsABSTRACT
The aim of this study is to explore the tissue distribution of PEGylated puerarin in acute myocardial ischemia model rats. Healthy male SD rats were randomly divided into two groups (30 each). Both were given PEGylated puerarin at a dose of 488 mg x kg(-1). After 5 min of medication, one group was normal rats, another group with acute myocardial ischemia was established by peritoneal injection of 50 mg x kg(-1) isoprenaline. After administration, the animals were executed at 30, 60, 90, 120, 150 and 180 min, then heart, liver, spleen, lung, kidney were extracted. The content of puerarin in organ tissue was determined by HPLC. The results showed that the AUC of tissue distribution of PEGylated puerarin in normal rats was liver > kidney > heart ≈ spleen > lung > brain. While the AUC of tissue distribution of PEGylated puerarin in acute myocardial ischemia model rats was liver ≈ heart > kidney > lung ≈ spleen > brain. AUC(heart) of PEGylated puerarin in acute myocardial ischemia model rats was 1.7 times than that of the normal rats, and there was significant difference (P < 0.05). Thus, PEGylated puerarin had a good heart-targeting property in early myocardial infarction area, drugs could accumulate in the ischemic myocardium. It provided important information for further study and clinic use of PEGylated puerarin.
Subject(s)
Isoflavones/pharmacokinetics , Myocardial Ischemia/metabolism , Animals , Brain/metabolism , Kidney/metabolism , Liver/metabolism , Lung/metabolism , Male , Myocardium/metabolism , Rats , Rats, Sprague-Dawley , Spleen/metabolism , Tissue DistributionABSTRACT
PURPOSE: We identified and validated [(18)F]-CP18, a DEVD (the caspase 3 substrate recognition motif) containing substrate-based compound as an imaging tracer for caspase-3 activity in apoptotic cells. PROCEDURES: CP18 was radiolabeled with fluorine-18 using click chemistry. The affinity and selectivity of CP18 for caspase-3 were evaluated in vitro. The biodistribution and metabolism pattern of [(18)F]-CP18 were assessed in vivo. [(18)F]-CP18 positron emission tomography (PET) scans were performed in a dexamethasone-induced thymic apoptosis mouse model. After imaging, the mice were sacrificed, and individual organs were collected, measured in a gamma counter, and tested for caspase-3 activity. RESULTS: In vitro enzymatic caspase-3 assay demonstrated specific cleavage of CP18. In vivo, [(18)F]-CP18 is predominantly cleared through the kidneys and urine, and is rapidly eliminated from the bloodstream. There was a sixfold increase in caspase activity and a fourfold increase of [(18)F]-CP18 retention in the dexamethasone-induced thymus of treated versus control mice. CONCLUSIONS: We report the use [(18)F]-CP18 as a PET tracer for imaging apoptosis. Our data support further development of this tracer for clinical PET applications.
Subject(s)
Apoptosis , Glycopeptides/pharmacokinetics , Molecular Imaging/methods , Positron-Emission Tomography/methods , Analysis of Variance , Animals , Caspase 3/metabolism , Cell Line, Tumor , Dexamethasone/adverse effects , Glycopeptides/chemistry , Humans , Linear Models , Mice , Radiopharmaceuticals/chemistry , Radiopharmaceuticals/pharmacokinetics , Thymus Gland/chemistry , Thymus Gland/drug effects , Tissue DistributionABSTRACT
PURPOSE: A novel caspase-3 substrate-based probe [(18)F]-CP18 was evaluated as an in vivo positron emission tomography (PET) imaging agent for monitoring apoptosis in tumors. METHODS: Uptake of [(18)F]-CP18 in cell assays and tumors was measured. Caspase-3/7 activities in cell lysates and tumor homogenates were determined. Autoradiography,Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), and cleaved caspase-3 immunostaining were performed on adjacent tumor sections to identify areas of apoptosis. RESULTS: The in vitro cell assays showed caspase-3-dependent uptake of [(18)F]-CP18 in tumor cells when treated with an apoptosis inducer. The in vivo microPET imaging signal of [(18)F]-CP18 in xenograft tumors correlated with the ex vivo caspase-3/7 activities in these tumors. Furthermore, tumor autoradiographies of [(18)F]-CP18 in tumor sections matched adjacent sections stained by TUNEL and caspase-3 immunohistochemistry (IHC). CONCLUSIONS: [(18)F]-CP18 demonstrated high affinity and selectivity for activated caspase-3 both in vitro and in vivo, and the results support [(18)F]-CP18 as a promising new PET imaging agent for apoptosis.
Subject(s)
Apoptosis , Glycopeptides/pharmacokinetics , Molecular Imaging/methods , Neoplasms/diagnostic imaging , Positron-Emission Tomography/methods , Radiopharmaceuticals/pharmacokinetics , Animals , Caspase 3/analysis , Caspase 3/metabolism , Caspase 7/analysis , Caspase 7/metabolism , Cell Line, Tumor , Glycopeptides/chemistry , Humans , Linear Models , Mice , Radiopharmaceuticals/chemistry , Tissue Distribution , Xenograft Model Antitumor AssaysABSTRACT
OBJECTIVE: We wished to develop a highly selective positron emission tomography (PET) imaging agent targeting PHF-tau in human Alzheimer's disease (AD) brains. METHODS: To screen potential tau binders, human AD brain sections were used as a source of native paired helical filament (PHF)-tau and Aß rather than synthetic tau aggregates or Aß fibrils generated in vitro to measure the affinity and selectivity of [(18)F]T807 to tau and Aß. Brain uptake and biodistribution of [(18)F]T807 in mice were also tested. RESULTS: In vitro autoradiography results show that [(18)F]T807 exhibits strong binding to PHF-tau-positive human brain sections. A dissociation constant (Kd) of [(18)F]T807 (14.6 nM) was measured using brain sections from the frontal lobe of AD patients. A comparison of autoradiography and double immunohistochemical staining of PHF-tau and Aß on adjacent sections demonstrated that [(18)F]T807 binding colocalized with immunoreactive PHF-tau pathology, but did not highlight Aß plaques. In vivo studies in mice demonstrated that [(18)F]T807 was able to cross the blood-brain barrier and washed out quickly. CONCLUSIONS: [(18)F]T807 demonstrates high affinity and selectivity to PHF-tau as well as favorable in vivo properties, making this a promising candidate as an imaging agent for AD.
Subject(s)
Alzheimer Disease/diagnosis , Brain/diagnostic imaging , Fluorine Radioisotopes , tau Proteins/chemistry , tau Proteins/drug effects , Aged , Aged, 80 and over , Amyloid beta-Peptides/metabolism , Animals , Autoradiography , Brain/pathology , Case-Control Studies , Female , Humans , Male , Mice , Mice, Transgenic , Middle Aged , Positron-Emission Tomography , Protein Binding/drug effects , Protein Binding/genetics , Tissue Distribution , tau Proteins/geneticsABSTRACT
Senile plaques and neurofibrillary tangles are prominent neuropathological hallmarks in Alzheimer's disease and are considered to be targets for therapeutic intervention as well as biomarkers for diagnostic in vivo imaging agents. While there are a number of amyloid-ß positron emission tomography (PET) tracers currently in different stages of clinical development and commercialization, there have been very few reports on imaging agents selectively targeting tau aggregates. In search of [18F]-PET tracers that possess great binding affinity and selectivity toward tau tangles, we tested more than 900 compounds utilizing a unique screening process. A competitive autoradiography assay was set up to test compounds for binding to native tau tangles and amyloid-ß plaques on human brain tissue sections. In our in vitro assays, the 18F labeled compound [18F]-T808 displayed a high level of binding affinity and good selectivity for tau aggregates over amyloid-ß plaques. [18F]-T808 showed rapid uptake and washout in rodent brains. Our in vitro and preclinical in vivo studies suggest that [18F]-T808 possesses suitable properties and characteristics to be a specific and selective PET probe for imaging of paired helical filament tau in human brains.
Subject(s)
Fluorine Radioisotopes , Positron-Emission Tomography/methods , Radiopharmaceuticals , tau Proteins , Amyloid beta-Peptides/metabolism , Animals , Autoradiography/methods , Fluorine Radioisotopes/metabolism , Humans , Mice , Mice, Inbred ICR , Plaque, Amyloid/diagnostic imaging , Plaque, Amyloid/metabolism , Protein Binding/physiology , Radiopharmaceuticals/chemistry , Radiopharmaceuticals/metabolism , Rats , Rats, Sprague-Dawley , tau Proteins/metabolismABSTRACT
Tissue kallikrein (KLK1) processes low-molecular weight kininogen to produce vasoactive kinins, which exert biological functions via kinin receptor signaling. Using various delivery approaches, we have demonstrated that tissue kallikrein through kinin B2 receptor signaling exhibits a wide spectrum of beneficial effects by reducing cardiac and renal injuries, restenosis and ischemic stroke, and by promoting angiogenesis and skin wound healing, independent of blood pressure reduction. Protection by tissue kallikrein in oxidative organ damage is attributed to the inhibition of apoptosis, inflammation, hypertrophy and fibrosis. Tissue kallikrein also enhances neovascularization in ischemic heart and limb. Moreover, tissue kallikrein/kinin infusion not only prevents but also reverses kidney injury, inflammation and fibrosis in salt-induced hypertensive rats. Furthermore, there is a wide time window for kallikrein administration in protection against ischemic brain infarction, as delayed kallikrein infusion for 24 h after cerebral ischemia in rats is effective in reducing neurological deficits, infarct size, apoptosis and inflammation. Importantly, in the clinical setting, human tissue kallikrein has been proven to be effective in the treatment of patients with acute brain infarction when injected within 48 h after stroke onset. Finally, kallikrein promotes skin wound healing and keratinocyte migration by direct activation of protease-activated receptor 1.
Subject(s)
Cardiovascular Diseases/metabolism , Cerebrovascular Disorders/metabolism , Kidney Diseases/metabolism , Skin/injuries , Skin/metabolism , Tissue Kallikreins/metabolism , Wound Healing , Animals , Cardiovascular Diseases/physiopathology , Cardiovascular Diseases/prevention & control , Cerebrovascular Disorders/physiopathology , Cerebrovascular Disorders/prevention & control , Humans , Kidney Diseases/prevention & control , Skin/pathology , Tissue Kallikreins/antagonists & inhibitors , Tissue Kallikreins/chemistry , Tissue Kallikreins/therapeutic useABSTRACT
Delivery of short interfering RNA (siRNA) to cells in culture, and in vivo, is possible with combined use of a receptor-specific monoclonal antibody (mAb) and avidin-biotin technology. In the present studies, the luciferase gene is transiently expressed in human 293 epithelial cells. The siRNA delivery system is composed of the siRNA, monobiotinylated on the 3'-terminus of the sense strand, and a conjugate of streptavidin (SA) and a mAb to the human insulin receptor (HIR). Exposure of cells to 3'-biotinyl-siRNA bound to the HIRMAb/SA conjugate, but not to unconjugated SA, avidin, or the HIRMAb, causes a >90% reduction in luciferase gene expression. The receptor-targeted siRNA effect is maximal at 48 h after delivery of the siRNA to the cells, and the effect is lost by 7 days after a single application of the targeted siRNA in culture. The KI of the receptor-targeted siRNA inhibition of gene expression is 30.5 +/- 11.7 nM, and significant inhibition is observed with siRNA concentrations as low as 3 nM. In conclusion, the combination of a receptor-specific targeting ligand, such as the HIRMAb, and avidin-biotin technology allows for high affinity capture of the monobiotinylated siRNA by the targeting mAb. The siRNA is effectively delivered to the cytosol of cells, and knockdown of gene expression with the HIRMAb/SA delivery system is comparable to RNA interference effects obtained with cationic polyplexes. Whereas the use of cationic polyplexes in vivo is problematic, the bond between the targeting mAb and the siRNA is stable with avidin-biotin technology, and RNAi effects at distant sites such as brain are observed in vivo following an intravenous administration of the targeted siRNA.
Subject(s)
Antibodies, Monoclonal/chemistry , Antigens, CD/immunology , Avidin/chemistry , Biotin/chemistry , RNA, Small Interfering/chemistry , RNA, Small Interfering/physiology , Receptor, Insulin/immunology , Animals , Antibodies, Monoclonal/immunology , Cell Line , Humans , Luciferases/genetics , Mice , RNA Interference/physiology , Streptavidin/chemistry , Time FactorsABSTRACT
The genetic engineering, expression, and validation of a fusion protein of avidin (AV) and a chimeric monoclonal antibody (mAb) to the human insulin receptor (HIR) is described. The 15 kDa avidin monomer was fused to the carboxyl terminus of the heavy chain of the HIRMAb. The fusion protein heavy chain reacted with antibodies specific for human IgG and avidin, and had the same affinity for binding to the HIR extracellular domain as the original chimeric HIRMAb. The fusion protein qualitatively bound biotinylated ligands, but was secreted fully saturated with biotin by COS cells, owing to the high level of biotin in tissue culture medium. Chinese hamster ovary (CHO) cells were permanently transfected with a tandem vector expressing the fusion protein genes, and high expressing cell lines were isolated by methotrexate amplification and dilutional cloning. The product expressed by CHO cells had high binding to the HIR, and migrated as a homogeneous species in size exclusion HPLC and native polyacrylamide gel electrophoresis. The CHO cells were adapted to a 4 week culture in biotin depleted medium, and the HIRMAb-AV fusion protein expressed under these conditions had 1 unoccupied biotin binding site per molecule, based on a [3H]-biotin ultrafiltration assay. The HIRMAb-AV increased biotin uptake by human cells >15-fold, and mediated the endocytosis of fluorescein-biotin, as demonstrated by confocal microscopy. In summary, the HIRMAb-AV fusion protein is a new drug targeting system for humans that can be adapted to monobiotinylated drugs or nucleic acids.
Subject(s)
Antibodies, Monoclonal/genetics , Antibodies, Monoclonal/pharmacology , Biotin/chemistry , Mutant Chimeric Proteins/genetics , Mutant Chimeric Proteins/pharmacology , Receptors, Drug/drug effects , Animals , Antibodies, Monoclonal/biosynthesis , Avidin/chemistry , Biotin/pharmacokinetics , Blotting, Western , CHO Cells , COS Cells , Cell Line, Tumor , Chlorocebus aethiops , Chromatography, High Pressure Liquid , Cricetinae , Cricetulus , DNA, Complementary/biosynthesis , DNA, Complementary/genetics , Dialysis , Drug Delivery Systems , Electrophoresis, Polyacrylamide Gel , Enzyme-Linked Immunosorbent Assay , Genetic Engineering , Humans , Microscopy, Confocal , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/pharmacology , Serum Albumin, Bovine/chemistry , UltrafiltrationABSTRACT
Mucopolysaccharidosis Type I, Hurler's Syndrome, is a lysosomal storage disorder that affects the brain. The missing enzyme, alpha-L-iduronidase (IDUA), does not cross the blood-brain barrier (BBB). To enable BBB transport of the enzyme, human IDUA was fused to the carboxyl terminus of the heavy chain of a chimeric monoclonal antibody (MAb) to the human insulin receptor (HIR). The HIRMAb crosses the BBB on the endogenous insulin receptor, and acts as a molecular Trojan horse to ferry into brain the IDUA. Transfection of COS cells resulted in high levels of IDUA enzyme activity both in the medium and in the intracellular space. The size of the fusion heavy chain, as measured with Western blotting and antibodies to either human IDUA or human IgG, was increased about 80 kDa, relative to the size of the heavy chain of the parent HIRMAb. The IDUA enzyme specific activity of the affinity purified HIRMAb-IDUA fusion protein was 363 +/- 37 U/microg protein, which is comparable to specific activity of recombinant IDUA. The accumulation of glycosoaminoglycans in Hurler fibroblasts was decreased 70% by treatment with the HIRMAb-IDUA fusion protein. Confocal microscopy showed targeting of the fusion protein to the lysosome. The HIRMAb-IDUA fusion protein bound with high affinity to the HIR, and was rapidly transported into the brain of the adult Rhesus monkey following intravenous administration. The HIRMAb-IDUA fusion protein is a new treatment for Hurler's syndrome, which has been specifically engineered to cross the human BBB.
Subject(s)
Antibodies, Monoclonal/pharmacokinetics , Blood-Brain Barrier/physiology , Iduronidase/pharmacokinetics , Mucopolysaccharidosis I/therapy , Protein Engineering/methods , Receptor, Insulin/chemistry , Recombinant Fusion Proteins/pharmacokinetics , Animals , Antibodies, Monoclonal/administration & dosage , Blood-Brain Barrier/drug effects , COS Cells , Chlorocebus aethiops , Disease Models, Animal , Fibroblasts , Humans , Iduronidase/administration & dosage , Iduronidase/chemistry , Mucopolysaccharidosis I/genetics , Recombinant Fusion Proteins/administration & dosage , Recombinant Fusion Proteins/chemistry , Transfection/methodsABSTRACT
BACKGROUND: Rats with experimental Parkinson's disease (PD) are treated with intravenous glial-derived neurotrophic factor (GDNF) plasmid DNA, non-viral gene therapy using Trojan horse liposomes (THLs) targeted with a monoclonal antibody (MAb) to the rat transferrin receptor (TfR). Expression of the transgene is confined to catecholaminergic cells by placement of the GDNF gene under the influence of the rat tyrosine hydroxylase (TH) promoter. METHODS: A 13-kb eukaryotic expression plasmid, designated pTHpro-GDNF, is engineered in which the human prepro GDNF cDNA is driven by 8 kb of the 5'-flanking sequence of the rat TH promoter (pro), and is 3'-flanked by the bovine growth hormone transcription termination sequence. The pTHpro-GDNF plasmid DNA is encapsulated in THLs targeted with a TfRMAb, and a single intravenous injection is given to rats at 2 weeks after experimental PD is induced by intra-cerebral 6-hydroxydopamine. RESULTS: Expression of the GDNF gene, under the influence of the TH promoter, is restricted compared to GDNF expression under the influence of the cytomegalovirus promoter. GDNF is elevated only in organs of the rat where TH gene expression is observed, including the substantia nigra, liver and adrenal gland. The single, delayed intravenous administration of the GDNF gene therapy causes a lasting reduction in apormorphine-induced rotation, which is correlated with a 19-fold increase in striatal TH enzyme activity. Both dose-response and time-responses are observed. CONCLUSIONS: Sustained therapeutic effects are achieved in experimental PD with a delayed single intravenous dosing of GDNF plasmid DNA gene therapy, using receptor-targeted THLs and a region-specific promoter.