Transcranial alternating current stimulation improves quality of life in Parkinson's disease: study protocol for a randomized, double-blind, controlled trial.

BACKGROUND: The neural cells in the brains of patients with Parkinson's disease (PWP) display aberrant synchronized oscillatory activity within the beta frequency range. Additionally, enhanced gamma oscillations may serve as a compensatory mechanism for motor inhibition mediated by beta activity and also reinstate plasticity in the primary motor cortex affected by Parkinson's disease. Transcranial alternating current stimulation (tACS) can synchronize endogenous oscillations with exogenous rhythms, thereby modulating cortical activity. The objective of this study is to investigate whether the addition of tACS to multidisciplinary intensive rehabilitation treatment (MIRT) can improve symptoms of PWP so as to enhance the quality of life in individuals with Parkinson's disease based on the central-peripheral-central theory. METHODS: The present study was a randomized, double-blind trial that enrolled 60 individuals with Parkinson's disease aged between 45 and 70 years, who had Hoehn-Yahr scale scores ranging from 1 to 3. Participants were randomly assigned in a 1:1 ratio to either the tACS + MIRT group or the sham-tACS + MIRT group. The trial consisted of a two-week double-blind treatment period followed by a 24-week follow-up period, resulting in a total duration of twenty-six weeks. The primary outcome measured the change in PDQ-39 scores from baseline (T0) to 4 weeks (T2), 12 weeks (T3), and 24 weeks (T4) after completion of the intervention. The secondary outcome assessed changes in MDS-UPDRS III scores at T0, the end of intervention (T1), T2, T3, and T4. Additional clinical assessments and mechanistic studies were conducted as tertiary outcomes. DISCUSSION: The objective of this study is to demonstrate that tACS can enhance overall functionality and improve quality of life in PWP, based on the framework of MIRT. Additionally, it seeks to establish a potential correlation between these therapeutic effects and neuroplasticity alterations in relevant brain regions. The efficacy of tACS will be assessed during the follow-up period in order to optimize neuroplasticity and enhance its potential impact on rehabilitation efficiency for PWP. TRIAL REGISTRATION: Chinese Clinical Trial Registry ChiCTR2300071969. Registered on 30 May 2023.

Association between autonomic dysfunction with motor and non-motor symptoms in patients with Parkinson's disease.

To investigate the association between autonomic dysfunction (AutD) and motor as well as non-motor symptoms (NMS) in patients with Parkinson's disease (PD). Fifty-three PD patients were divided into two groups based on the number of domains affected by AutD: a multi-domain AutD group (AutD-M) and a single-domain AutD group (AutD-S), as evaluated using the Scale for Outcomes in Parkinson's disease-Autonomic (SCOPA-AUT), which assesses autonomic symptoms, one of the NMS. A comprehensive comparison was conducted between the two groups, including clinical measures such as clinical scales, quantitative evaluations of motor function and exercise capacity. Spearman correlation analysis was employed to investigate the relationship between AutD severity and PD symptoms. Additionally, we performed multiple linear regression model analysis to determine whether associations between SCOPA-AUT scores and clinical assessments remained significant after adjusting for Hoehn and Yahr stage, sex, and age. PD patients in the AutD-M group exhibited significantly more severe NMS and motor symptoms compared to those in the AutD-S group. In correlation analysis, SCOPA-AUT scores showed significant correlations with multiple clinical symptoms, such as most of the NMS, 10-MWT and CPET parameters. Furthermore, regression analysis also revealed that more pronounced fatigue, anxiety, depressive symptoms, worse walking speed and impaired exercise capacity were associated with higher SCOPA-AUT scores. The presence of AutD is correlated with emotional disturbances, decreased exercise endurance, and impaired gait function in patients with PD. Early management of AutD may prove beneficial in alleviating some NMS and motor symptoms in PD.

Intermittent theta-burst stimulation combined with physical therapy as an optimal rehabilitation in Parkinson's disease: study protocol for a randomised, double-blind, controlled trial.

BACKGROUND: First-line rehabilitative strategies to improve motor deficits are based on functional training (physical or occupational therapy), which has been demonstrated to facilitate neural reorganisation. Accumulating evidence suggests that non-invasive brain stimulation techniques, such as repetitive TMS (rTMS), may enhance neuroplasticity, thereby facilitating neural reorganisation and recovery from Parkinson's disease. Evidence also shows that intermittent theta-burst stimulation (iTBS) can improve motor function and quality of life in patients by promoting the excitability and neural remodelling of cerebral cortex. We aimed to combine iTBS stimulation with physiotherapy to improve the rehabilitation effect compared to physiotherapy alone in patients with Parkinson's disease. METHODS: This randomised, double-blind clinical trial will enrol 50 Parkinson's disease patients aged 45-70 years with Hoehn and Yahr scale scores of 1-3. Patients are randomly assigned to either the iTBS + physiotherapy or sham-iTBS + physiotherapy group. The trial consists of a 2-week double-blind treatment period and a 24-week follow-up period. iTBS and sham-iTBS will be administered twice daily for 10 days based on physiotherapy. The primary outcome will be the third part of Movement Disorders-Unified Parkinson's Disease Rating Scale (MDS-UPDRS III) from the baseline to the first 2 days following completion hospitalised intervention. The secondary outcome will be 39-item Parkinson's Disease Questionnaire (PDQ-39) at 4 weeks, 12 weeks and 24 weeks after intervention. Tertiary outcomes are clinical evaluations and mechanism study outcomes such as NMSS, 6MWD, 10MT, TUG, BBS, MRI, and EEG, the length of time between the drug needs to be adjusted when symptoms fluctuate. DISCUSSION: The aim of this study is to demonstrate that iTBS can promote overall function and quality of life in Parkinson's disease patients using physiotherapy and that this efficacy may be associated with altered neuroplasticity in exercise-related brain regions. The iTBS combined with physiotherapy training model will be evaluated during a 6-month follow-up period. With significant improvement in quality of life and motor function, iTBS combined with physiotherapy can be considered as a first-line rehabilitation option for Parkinson's disease. The potential of iTBS to enhance neuroplasticity in the brain should have a more positive impact in increasing the generality and efficiency of physiotherapy, improving the quality of life and overall functional status of patients with Parkinson's disease. TRIAL REGISTRATION: Chinese Clinical Trial Registry ChiCTR2200056581. Registered on 8 February 2022.

Predictive Value of Exercise Blood Pressure Changes for Orthostatic Hypotension in Patients With Parkinson's Disease.

BACKGROUND AND PURPOSE: Orthostatic hypotension (OH) is common in patients with Parkinson's disease (PD). Early recognition OH is required with sensitive assessments. The purpose of this study was to determine whether blood pressure (BP) changes during exercise can predict the occurrence of OH in PD. METHODS: This prospective cohort study included 80 consecutive patients with PD. All patients agreed to participate in a baseline evaluation and cardiopulmonary exercise test (CPET). According to the initial active standing test (AST), those without OH (PD-nonOH) at baseline had their AST results followed up for 6 months. The main outcome was defined as whether patients without OH at baseline would develop OH after 6 months. Logistic regression analysis was applied to identify the relevant variables. A nomogram was constructed based on clinical features and identified variables. The concordance index (C-index) and area under the receiver operating characteristic curve (AUC) were used to evaluate the accuracy and predictive ability of the nomogram, respectively. RESULTS: CPET results indicated that peak load, peak heart rate, heart rate recovery at 1 min, and systolic BP change (ΔSBP) were lower in those with OH than in the PD-nonOH group (p<0.05) at baseline. Logistic regression analysis indicated that peak load and ΔSBP during CPET had significant effects on OH (p<0.05). Age, sex, peak load, and ΔSBP were used to construct the nomogram model (C-index=0.761). The prediction model had an AUC of 0.782 (95% confidence interval=0.649-0.889) and a specificity and sensitivity of 70.0% and 81.8%, respectively. CONCLUSIONS: This study has identified predictive factors for OH development in patients with PD. CPET could be used as a complementary examination to identify patients at a high risk of OH.

Multiplexed Imaging Reveals the Spatial Relationship of the Extracellular Acidity-Targeting pHLIP with Necrosis, Hypoxia, and the Integrin-Targeting cRGD Peptide.

pH (low) insertion peptides (pHLIPs) have been developed for cancer imaging and therapy targeting the acidic extracellular microenvironment. However, the characteristics of intratumoral distribution (ITD) of pHLIPs are not yet fully understood. This study aimed to reveal the details of the ITD of pHLIPs and their spatial relationship with other tumor features of concern. The fluorescent dye-labeled pHLIPs were intravenously administered to subcutaneous xenograft mouse models of U87MG and IGR-OV1 expressing αVß3 integrins (using large necrotic tumors). The αVß3 integrin-targeting Cy5.5-RAFT-c(-RGDfK-)4 was used as a reference. In vivo and ex vivo fluorescence imaging, whole-tumor section imaging, fluorescence microscopy, and multiplexed fluorescence colocalization analysis were performed. The ITD of fluorescent dye-labeled pHLIPs was heterogeneous, having a high degree of colocalization with necrosis. A direct one-to-one comparison of highly magnified images revealed the cellular localization of pHLIP in pyknotic, karyorrhexis, and karyolytic necrotic cells. pHLIP and hypoxia were spatially contiguous but not overlapping cellularly. The hypoxic region was found between the ITDs of pHLIP and the cRGD peptide and the Ki-67 proliferative activity remained detectable in the pHLIP-accumulated regions. The results provide a better understanding of the characteristics of ITD of pHLIPs, leading to new insights into the theranostic applications of pHLIPs.

Synthesis of 1,8-Naphthyridines by the Ionic Liquid-Catalyzed Friedlander Reaction and Application in Corrosion Inhibition.

A several of basic ionic liquids (ILs) were synthesized as green solvents and catalysts for the preparation of 1,8-naphthyridyl derivatives via the Friedlander reaction. [Bmmim][Im] exhibited remarkable catalytic activity to achieve the synthetic targets, and the reaction conditions were optimized. The model product 2,3-diphenyl-1,8-naphthyridine (1,8-Nap), with carboxyethylthiosuccinic acid (CETSA) to form an IL corrosion inhibitor ([1,8-Nap][CETSA]), and its corrosion inhibition performance for Q235 steel in 1 M HCl were researched by weight loss measurements, and the results showed that the inhibition efficiency was 96.95% when the concentration of [1,8-Nap][CETSA] was 1 mM at 35 °C. The electrochemical test verified that [1,8-Nap][CETSA] acted as a mixed-type inhibitor but mainly exhibited cathodic behavior. The inhibitor adsorbed on the metal surface was further proved by surface topography analysis.

The Role of the Diaphragm in Postural Stability and Visceral Function in Parkinson's Disease.

Background: In normal subjects, the diaphragm plays a key functional role in postural stability, articulation, respiration, defecation, and urination. Objectives: The aim of this study was to investigate the role of the diaphragm in postural stability and visceral function in patients with Parkinson's disease (PD) and to compare the diaphragm function by gender, Hoehn and Yahr (H&Y) staging, and motor subtypes. Methods: In total, 79 patients were enrolled in this cross-sectional study. The severity of the disease was assessed by the Movement Disorder Society-Unified Parkinson's Disease Rating Scale III and by H&Y staging. Postural stability was quantitatively recorded, and respiratory function was evaluated by spirometry. Several scales were used to evaluate visceral function in patients with PD. In addition, diaphragm ultrasound was used to measure the excursion, contraction velocity, and thickness of the diaphragm during quiet breathing, deep breathing, and the sniff test. Significant features were selected by the least absolute shrinkage and selection operator (LASSO) regression and fitted in the multivariate linear regression and Pearson's correlation analysis. Results: Diaphragm thickness and excursion during quiet breathing were significantly different between men and women and between H&Y stage 1-2 and stage 2.5-3, whereas the diaphragm function was not influenced by motor subtypes. It was shown that the diaphragmatic function was significantly correlated with postural stability, voice function, respiratory function, constipation, and urological function to varying degrees in patients with PD. Conclusion: The diaphragmatic function is associated with dysfunction in PD although it remains unclear as to whether the observed changes in the diaphragm are primary or secondary.

Efficacy of short-term multidisciplinary intensive rehabilitation in patients with different Parkinson's disease motor subtypes: a prospective pilot study with 3-month follow-up.

Parkinson's disease (PD) can be classified into three motor-based subtypes: postural instability/gait difficulty (PIGD), tremor dominant (TD), and indeterminate. The neuropathophysiological mechanisms of the three motor subtypes are different, which may lead to different responses to therapy. Sixty-nine patients with idiopathic Parkinson's disease (Hoehn-Yahr stage ≤ 3) were screened from 436 patients with Parkinsonism recruited through outpatient services and the internet. According to the Movement Disorder Society Unified Parkinson's Disease Rating Scale (MDS-UPDRS) TD/PIGD ratio, the patients were divided into PIGD (TD/PIGD ≤ 0.09; n = 36), TD (TD/PIGD ≥1.15; n = 19), and indeterminate (TD/PIGD = 0.90-1.15; n = 14) groups. All patients received 2 weeks of multidisciplinary intensive rehabilitation treatment (MIRT) during hospitalization, as well as a remote home rehabilitation health education class. Compared with the scores at admission, all patients showed significant improvements in their MDS-UPDRS III score, walking ability, balance, and posture control at discharge. Moreover, the MDS-UPDRS III score improvement was greater in the PIGD group than in the TD group. The follow-up data, collected for 3 months after discharge, showed that overall symptom improvement in each group was maintained for 1-3 months. Furthermore, there were no significant differences in the duration or grade effects of symptom improvement among the three groups. These findings suggest that 2 weeks of MIRT is effective for improving motor performance in all three motor subtypes. Patients in the PIGD group had a better response after hospitalization than those in the TD group. This study was approved by the Institutional Ethics Committee of Beijing Rehabilitation Hospital of Capital Medical University of China (approval No. 2018bkky022) on May 7, 2018 and registered with the Chinese Clinical Trial Registry (registration No. ChiCTR1900020771) on January 19, 2019.

Radiotheranostic Agent 64Cu-cyclam-RAFT-c(-RGDfK-)4 for Management of Peritoneal Metastasis in Ovarian Cancer.

PURPOSE: Ovarian cancer peritoneal metastases (OCPMs) are a pathophysiologically heterogeneous group of tumors that are rarely curable. αVß3 integrin (αVß3) is overexpressed on tumoral neovessels and frequently on ovarian cancer cells. Here, using two clinically relevant αVß3-positive OCPM mouse models, we studied the theranostic potential of an αVß3-specific radiopeptide, 64Cu-cyclam-RAFT-c(-RGDfK-)4 (64Cu-RaftRGD), and its intra- and intertumoral distribution in relation to the tumor microenvironment. EXPERIMENTAL DESIGN: αVß3-expressing peritoneal and subcutaneous models of ovarian carcinoma (IGR-OV1 and NIH:OVCAR-3) were established in nude mice. 64Cu-RaftRGD was administered either intravenously or intraperitoneally. We performed intratumoral distribution (ITD) studies, PET/CT imaging and quantification, biodistribution assay and radiation dosimetry, and therapeutic efficacy and toxicity studies. RESULTS: Intraperitoneal administration was an efficient route for targeting 64Cu-RaftRGD to OCPMs with excellent tumor penetration. Using the fluorescence surrogate, Cy5.5-RaftRGD, in our unique high-resolution multifluorescence analysis, we found that the ITD of 64Cu-RaftRGD was spatially distinct from, but complementary to, that of hypoxia. 64Cu-RaftRGD-based PET enabled clear visualization of multiple OCPM deposits and ascites and biodistribution analysis demonstrated an inverse correlation between tumor uptake and tumor size (1.2-17.2 mm). 64Cu-RaftRGD at a radiotherapeutic dose (148 MBq/0.357 nmol) showed antitumor activities by inhibiting tumor cell proliferation and inducing apoptosis, with negligible toxicity. CONCLUSIONS: Collectively, these results demonstrate the all-in-one potential of 64Cu-RaftRGD for imaging guided radiotherapy of OCPM by targeting both tumoral neovessels and cancerous cells. On the basis of the ITD finding, we propose that pairing αVß3- and hypoxia-targeted radiotherapies could improve therapeutic efficacy by overcoming the heterogeneity of ITD encountered with single-agent treatments.

Composition of Ophiopogon Polysaccharide, Notoginseng Total Saponins and Rhizoma Coptidis Alkaloids Inhibits the Myocardial Apoptosis on Diabetic Atherosclerosis Rabbit.

OBJECTIVE: To investigate the effects of Composition of Ophiopogon polysaccharide, Notoginseng total saponins and Rhizoma Coptidis alkaloids (CONR) on myocardial apoptosis of diabetic atherosclerosis (DA) rabbits METHODS: Sixty male New Zealand white rabbits were randomly divided into 6 groups [control group, model group, CONR high-dose group (450 mg/kg), CONR medium-dose group (150 mg/kg), CONR low-dose group (50 mg/kg), and simvastatin group] by using a completely random method, 10 in each group. DA model was established by intravenously injected alloxan combined with high-fat diet and abdominal aortic balloon injury. After mediation for 10 weeks, fasting blood glucose (FBG), glycosylated hemoglobin (GHB), glycosylated serum protein (GSP), fructoseamine (FRA), aldose reductase (AR), advanced glycation end products (AGEs) in serum were measured by enzyme linked immunosorbent assay (ELISA) method; the expression of receptor of AGEs (RAGE) in myocardial tissue were observed by immunohistochemical method; and p-Jun N-terminal kinase (p-JNK), caspase-3, B-cell lymphoma-2 (bcl-2) protein expression in myocardial tissue were measured by Western blotting. The myocardial apoptosis was detected by TdT-mediated dUTPnick-end labeling (TUNEL) method, and apoptosis index (AI) was calculated. RESULTS: Compared with the control group, serum FBG, GHB, GSP, FRA, AR, AGEs and the expression of myocardium RAGE, p-JNK, caspase-3 proteins, as well as apoptosis index (AI) were significantly increased and bcl-2 protein was significantly decreased in the model group (P<0.01). Compared with the model group, the levels of serum FBG, GHB, GSP, FRA and AR showed a significant decline in CONR high- and medium-dose groups (P<0.01). FBG and GHB showed a significant decline in CONR low-dose group (P<0.01). Compared with the model group, the expression of serum AGEs and myocardium RAGE, p-JNK and caspase-3 protein as well as AI were significantly decreased and bcl-2 protein was significantly up-regulated in all treatment groups (P<0.01); high-dose CONR had the most significant effect on abovementioned indices compared with other treatment groups (P<0.01). Middle-dose CONR had better effect on serum AGEs compared with the low-dose group (P<0.01); middle-dose CONR and simvastatin groups had better effect on the expression of caspase-3, bcl-2 protein, myocardium apoptosis compared with the CONR low-dose group (P<0.01). CONCLUSION: CONR may effectively inhibit myocardial apoptosis on DA rabbits by intervening AGEs-RAGE and JNK, caspase-3, and bcl-2 protein expressions.

Molecular characteristics, pathogenicity and medication regimen of Aeromonas hydrophila isolated from common carp (Cyprinus carpio L.).

Aeromonas hydrophila causes disease in fish known as Motile Aeromonas Septicemia (MAS), also named as bacterial hemorrhagic septicemia. In this study, a pathogenic A. hydrophila strain was isolated from common carp Cyprinus carpio L., which were suffering from severe hemorrhagic septicemia. According to the phylogenetic analysis derived from 16S rDNA sequence, the isolate formed a single branch in the A. hydrophila group, named AhHN1. Artificial infection results indicated that AhHN1 showed strong pathogenicity in C. carpio and the LD50 was 1.38 × 106 CFU/fish, the clinical symptoms and pathological features of infected fish were similar to those observed in natural infections. The antimicrobial susceptibility testing revealed that AhHN1 resistance to more than 13 kinds of antimicrobial agents. However, the AhHN1 strain exhibited an extremely sensitivity to enrofloxacin, the in vitro activities of enrofloxacin were subsequently investigated and drug selection window (MSW) was 0.0016-0.0125 µg/ml. Pharmacokinetics data showed that plasma concentration of enrofloxacin was 0.0016, 0.0148 and 0.0282 µg/ml at 24 hr after orally administered with 2.5, 5 and 10 mg/kg enrofloxacin. Moreover, dosing once a day of 2.5, 5 and 10 mg/kg enrofloxacin, which the relative protection ratio (RPS) was amounted to 33.3, 66.7, and 83.3%, respectively. Therefore, 5 mg/kg enrofloxacin was considered to be the rational regimen for controlling AhHN1 infection in C. carpio in the countries where the use of enrofloxacin is permitted in aquaculture. The aim of this study was to establish a scientific medication regimen for the prevention and therapy of the mutidrug-resistant A. hydrophila infection.

Uniform intratumoral distribution of radioactivity produced using two different radioagents, 64Cu-cyclam-RAFT-c(-RGDfK-)4 and 64Cu-ATSM, improves therapeutic efficacy in a small animal tumor model.

BACKGROUND: The present study proposed a new concept for targeted radionuclide therapy (TRT) to improve the intratumoral distribution of radioactivity using two different radiopharmaceuticals. We examined the efficacy of a combination of a tetrameric cyclic Arg-Gly-Asp (cRGD) peptide-based radiopharmaceutical, 64Cu-cyclam-RAFT-c(-RGDfK-)4 (64Cu-RaftRGD, an αVß3 integrin [αVß3] tracer), and 64Cu-diacetyl-bis (N4-methylthiosemicarbazone) (64Cu-ATSM, a supposed tracer for hypoxic metabolism) in a small animal tumor model. RESULTS: Mice with subcutaneous αVß3-positive U87MG glioblastoma xenografts were used. The intratumoral distribution of a near-infrared dye, Cy5.5-labeled RAFT-c(-RGDfK-)4 (Cy5.5-RaftRGD), 64Cu-RaftRGD, and 64Cu-ATSM was visualized by fluorescence imaging and autoradiography of the co-injected Cy5.5-RaftRGD with 64Cu-RaftRGD or 64Cu-ATSM at 3 h postinjection. Mice were treated with a single intravenous dose of the vehicle solution (control), 18.5 or 37 MBq of 64Cu-RaftRGD or 64Cu-ATSM, or a combination (18.5 MBq of each agent). The tumor volume, tumor cell proliferation, body weight, survival, and tumor and organ uptake of radiopharmaceuticals were assessed. It was shown that Cy5.5-RaftRGD colocalized with 64Cu-RaftRGD and could be used as a surrogate for the radioactive agent. The intratumoral distribution of Cy5.5-RaftRGD and 64Cu-ATSM was discordant and nearly complementary, indicating a more uniform distribution of radioactivity achievable with the combined use of 64Cu-RaftRGD and 64Cu-ATSM. Neither 64Cu-RaftRGD nor 64Cu-ATSM showed significant effects on tumor growth at 18.5 MBq. The combination of both (18.5 MBq each) showed sustained inhibitory effects against tumor growth and tumor cell proliferation and prolonged the survival of the mice, compared to that by either single agent at 37 MBq. Interestingly, the uptake of the combination by the tumor was higher than that of 64Cu-RaftRGD alone, but lower than that of 64Cu-ATSM alone. The kidneys showed the highest uptake of 64Cu-RaftRGD, whereas the liver exhibited the highest uptake of 64Cu-ATSM. No obvious adverse effects were observed in all treated mice. CONCLUSIONS: The combination of 64Cu-RaftRGD and 64Cu-ATSM achieved an improved antitumor effect owing to the more uniform intratumoral distribution of radioactivity. Thus, combining different radiopharmaceuticals to improve the intratumoral distribution would be a promising concept for more effective and safer TRT.

Enhancement of oral bioavailability and immune response of Ginsenoside Rh2 by co-administration with piperine.

Ginsenoside Rh2 (Rh2) is one of the major bioactive ginsenosides in Panax ginseng. However, the oral bioavailability of Rh2 is low, with P-glycoprotein (P-gp) and CYP3A4 being reported to be the main factors. The purpose of the present study was to determine the enhancing effect of piperine on the oral bioavailability as well as bioactivity of Rh2. The inhibitory effect of piperine on P-gp and CYP3A4 was determined using a Caco-2 monolayer model and a recombinant CYP3A4 metabolic system, respectively. The pharmacokinetics of oral Rh2 (10 mg·kg-1) administered alone or in combination with piperine (10 and 20 mg·kg-1) was performed in rats. The immune boosting effect of Rh2 was assessed in rats by measuring IL-12 level after treated by Rh2 alone or co-administered with piperine. The results indicated that piperine significantly increased the permeability of Rh2 and inhibited the metabolism of Rh2. The pharmacokinetic study results showed that the AUC of Rh2 was significantly increased in combination with piperine at high dose (20 mg·kg-1) when compared to the control group, with relative bioavailability of 196.8%. The increase of Rh2 exposure led to increased serum levels of IL-12. In conclusion, piperine may be used as a bioenhancer to improve pharmacological effect of Rh2 when given orally.

Positron emission tomography of cerebral angiogenesis and TSPO expression in a mouse model of chronic hypoxia.

The present study aimed to examine whether positron emission tomography (PET) could evaluate cerebral angiogenesis. Mice were housed in a hypoxic chamber with 8-9% oxygen for 4, 7, and 14 days, and the angiogenic responses were evaluated with a radiotracer, 64Cu-cyclam-RAFT-c(-RGDfK-)4, which targeted αVß3 integrin and was imaged with PET. The PET imaging results showed little uptake during all of the hypoxic periods. Immunofluorescence staining of the ß3 integrin, CD61, revealed weak expression, while the microvessel density assessed by CD31 staining increased with the hypoxic duration. These observations suggest that the increased vascular density originated from other types of vascular remodeling, unlike angiogenic sprouting. We then searched for any signs of vascular remodeling that could be detected using PET. PET imaging of 11C-PK11195, a marker of the 18-kDa translocator protein (TSPO), revealed a transient increase at day 4 of hypoxia. Because the immunofluorescence of glial markers showed unchanged staining over the early phase of hypoxia, the observed upregulation of TSPO expression probably originated from non-glial cells (e.g. vascular cells). In conclusion, a transient increase in TSPO probe uptake was detected with PET at only the early phase of hypoxia, which indicates an early sign of vascular remodeling induced by hypoxia.

Multiple Administrations of 64Cu-ATSM as a Novel Therapeutic Option for Glioblastoma: a Translational Study Using Mice with Xenografts.

Glioblastoma is the most aggressive malignant brain tumor in humans and is difficult to cure using current treatment options. Hypoxic regions are frequently found in glioblastoma, and increased levels of hypoxia are associated with poor clinical outcomes of glioblastoma patients. Hypoxia plays important roles in the progression and recurrence of glioblastoma because of drug delivery deficiencies and induction of hypoxia-inducible factor-1α in tumor cells, which lead to poor prognosis. We focused on a promising hypoxia-targeted internal radiotherapy agent, 64Cu-diacetyl-bis (N4-methylthiosemicarbazone) (64Cu-ATSM), to address the need for additional treatment for glioblastoma. This compound can target the overreduced state under hypoxic conditions within tumors. Clinical positron emission tomography studies using radiolabeled Cu-ATSM have shown that Cu-ATSM accumulates in glioblastoma and its uptake is associated with high hypoxia-inducible factor-1α expression. To evaluate the therapeutic potential of this agent for glioblastoma, we examined the efficacy of 64Cu-ATSM in mice bearing U87MG glioblastoma tumors. Administration of single dosage (18.5, 37, 74, 111, and 148 MBq) and multiple dosages (37 MBq × 4) of 64Cu-ATSM was investigated. Single administration of 64Cu-ATSM in high-dose groups dose-dependently inhibited tumor growth and prolonged survival, with slight and reverse signs of adverse events. Multiple dosages of 64Cu-ATSM remarkably inhibited tumor growth and prolonged survival. By splitting the dose of 64Cu-ATSM, no adverse effects were observed. Our findings indicate that multiple administrations of 64Cu-ATSM have effective antitumor effects in glioblastoma without side effects, indicating its potential for treating this fatal disease.

67Cu-Radiolabeling of a multimeric RGD peptide for αVß3 integrin-targeted radionuclide therapy: stability, therapeutic efficacy, and safety studies in mice.

OBJECTIVE: Copper-67 (Cu) is one of the most promising radionuclides for internal radiation therapy. Globally, several projects have recently been initiated for developing innovative approaches for the large-scale production of Cu. Encouraged by these, we performed Cu-radiolabeling of a tetrameric cyclic Arg-Gly-Asp (cRGD) peptide conjugate, cyclam-RAFT-c(-RGDfK-)4, which selectively targets αVß3 integrin (αVß3), the transmembrane receptor involved in tumor invasion, angiogenesis, and metastasis. We also evaluated the therapeutic potential and safety of this radiocompound. MATERIALS AND METHODS: Cu, produced through the Ni(α, p)Cu reaction, was used for the radiolabeling of cyclam-RAFT-c(-RGDfK-)4 at 70°C for 10 min. The radiolabeling efficiency and product stability were assessed using reversed-phase high-performance liquid chromatography and/or thin-layer chromatography. Mice with subcutaneous αVß3-positive U87MG-glioblastoma xenografts received a single intravenous injection of one of the following: Cu-cyclam-RAFT-c(-RGDfK-)4 (11.1 MBq), peptide control, or vehicle solution. The tumor volumes were measured, side effects were assessed in terms of body weight, routine hematology, and hepatic and renal functions, and the mouse radiation dosimetry was estimated. RESULTS: Cu-cyclam-RAFT-c(-RGDfK-)4 was produced with a radiochemical purity of 97.9±2.4% and a specific activity of 2.7±0.6 MBq/nmol and showed high in-vitro and in-vivo plasma stability. The administration of a single dose of Cu-cyclam-RAFT-c(-RGDfK-)4 resulted in significant tumor growth delay in comparison with that observed upon vehicle or peptide control administration, with an estimated tumor-absorbed dose of 0.712 Gy. No significant toxicity was observed in Cu-cyclam-RAFT-c(-RGDfK-)4-treated mice. CONCLUSION: Cu-cyclam-RAFT-c(-RGDfK-)4 would be a promising therapeutic agent for αVß3 integrin-targeted internal radiotherapy.

αVß3 Integrin-Targeted Radionuclide Therapy with 64Cu-cyclam-RAFT-c(-RGDfK-)4.

The transmembrane cell adhesion receptor αVß3 integrin (αVß3) has been identified as an important molecular target for cancer imaging and therapy. We have developed a tetrameric cyclic RGD (Arg-Gly-Asp) peptide-based radiotracer (64)Cu-cyclam-RAFT-c(-RGDfK-)4, which successfully captured αVß3-positive tumors and angiogenesis by PET. Here, we subsequently evaluated its therapeutic potential and side effects using an established αVß3-positive tumor mouse model. Mice with subcutaneous U87MG glioblastoma xenografts received single administrations of 37 and 74 MBq of (64)Cu-cyclam-RAFT-c(-RGDfK-)4 (37 MBq/nmol), peptide control, or vehicle solution and underwent tumor growth evaluation. Side effects were assessed in tumor-bearing and tumor-free mice in terms of body weight, routine hematology, and hepatorenal functions. Biodistribution of (64)Cu-cyclam-RAFT-c(-RGDfK-)4 with ascending peptide doses (0.25-10 nmol) and with the therapeutic dose of 2 nmol were determined at 3 hours and at various time points (2 minutes-24 hours) postinjection, respectively, based on which radiation-absorbed doses were estimated. The results revealed that (64)Cu-cyclam-RAFT-c(-RGDfK-)4 dose dependently slowed down the tumor growth. The mean tumor doses were 1.28 and 1.81 Gy from 37 and 74 MBq of (64)Cu-cyclam-RAFT-c(-RGDfK-)4, respectively. Peptide dose study showed that the tumor uptake of (64)Cu-cyclam-RAFT-c(-RGDfK-)4 dose dependently decreased at doses ≥1 nmol, indicating a saturation of αVß3 with the administered therapeutic doses (1 and 2 nmol). Combined analysis of the data from tumor-bearing and tumor-free mice revealed no significant toxicity caused by 37-74 MBq of (64)Cu-cyclam-RAFT-c(-RGDfK-)4 Our study demonstrates the therapeutic efficacy and safety of (64)Cu-cyclam-RAFT-c(-RGDfK-)4 for αVß3-targeted radionuclide therapy. (64)Cu-cyclam-RAFT-c(-RGDfK-)4 would be a promising theranostic drug for cancer imaging and therapy. Mol Cancer Ther; 15(9); 2076-85. ©2016 AACR.

Immuno-PET Imaging of HER3 in a Model in which HER3 Signaling Plays a Critical Role.

HER3 is overexpressed in various carcinomas including colorectal cancer (CRC), which is associated with poor prognosis, and is involved in the development of therapy resistance. Thus, an in vivo imaging technique is needed to evaluate the expression of HER3, an important therapeutic and diagnostic target. Here, we report successful HER3 PET imaging using a newly generated anti-human HER3 monoclonal antibody, Mab#58, and a mouse model of a HER3-overexpressing xenograft tumor. Furthermore, we assessed the role of HER3 signaling in CRC cancer tissue-originated spheroid (CTOS) and applied HER3 imaging to detect endogenous HER3 in CTOS-derived xenografts. Cell binding assays of 89Zr-labeled Mab#58 using the HER3-overexpressing cell line HER3/RH7777 demonstrated that [89Zr]Mab#58 specifically bound to HER3/RH7777 cells (Kd = 2.7 nM). In vivo biodistribution study in mice bearing HER3/RH7777 and its parent cell xenografts showed that tumor accumulation of [89Zr]Mab#58 in HER3/RH7777 xenografts was significantly higher than that in the control from day 1 to day 4, tending to increase from day 1 to day 4 and reaching 12.2 ± 4.5%ID/g. Radioactivity in other tissues, including the control xenograft, decreased or remained unchanged from day 1 to day 6. Positron emission tomography (PET) in the same model enabled clear visualization of HER3/RH7777 xenografts but not of RH7777 xenografts. CTOS growth assay and signaling assay revealed that CRC CTOS were dependent on HER3 signaling for their growth. In PET studies of mice bearing a CRC CTOS xenograft, the tumor was clearly visualized with [89Zr]Mab#58 but not with the 89Zr-labeled control antibody. Thus, tumor expression of HER3 was successfully visualized by PET with 89Zr-labeled anti-HER3 antibody in CTOS xenograft-bearing mice, a model that retains the properties of the patient tumor. Non-invasive targeting of HER3 by antibodies is feasible, and it is expected to be useful for cancer diagnosis and treatment.

Development of the Fibronectin-Mimetic Peptide KSSPHSRN(SG)5RGDSP as a Novel Radioprobe for Molecular Imaging of the Cancer Biomarker α5ß1 Integrin.

α5ß1 Integrin, a fibronectin receptor, is becoming a pertinent therapeutic target and a promising prognostic biomarker for cancer patients. The aim of this study was to functionalize an α5ß1-specific fibronectin-mimetic peptide sequence KSSPHSRN(SG)5RGDSP (called PR_b) as a positron emission tomography (PET) probe. PR_b was modified by addition of a ß-alanine residue, conjugated with 2-S-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (p-SCN-Bn-NOTA), and radiolabeled with (18)F based on the chelation of (18)F-aluminum fluoride. A control probe was produced by glycine to alanine substitution in the RGD motif of PR_b. Cell binding and blocking assays, autoradiographic evaluation of tissue binding and blocking, dynamic PET scans, and a biodistribution study were conducted using cell lines and murine tumor models with determined expression levels of α5ß1 and other related integrins. (18)F-PR_b was produced with a labeling yield of 22.3±1.9% based on (18)F-F(-), a radiochemical purity of >99%, and a specific activity of 30-70 GBq/µmol; it exhibited α5ß1-binding activity and specificity in vitro, ex vivo, and in vivo, and had a rapid blood clearance and a predominant renal excretion pathway. In vivo α5ß1-positive tumors could be clearly visualized by (18)F-PR_b PET imaging. Both imaging and biodistribution studies suggested higher uptake of (18)F-PR_b in α5ß1-positive tumors than in α5ß1-negative tumors and higher α5ß1-positive tumor uptake of (18)F-PR_b than the control probe. In contrast, there was no significant difference seen in the contralateral muscle uptake. A PET radioprobe, (18)F-PR_b, was developed de novo and potentially can be used for noninvasive detection of α5ß1 expression in tumors.

A limited overlap between intratumoral distribution of 1-(5-fluoro-5-deoxy-α-D-arabinofuranosyl)-2-nitroimidazole and copper-diacetyl-bis[N(4)-methylthiosemicarbazone].

Positron emission tomography (PET) imaging of tumor hypoxia provides valuable information for cancer treatment planning. Two types of PET tracers, nitroimidazole compounds and [62,64Cu] copper-diacetyl-bis[N(4)-methylthio- semicarbazone] (Cu-ATSM), have been used for imaging hypoxic tumors. High accumulation of these tracers in tumors was shown to predict poor prognosis. Both similar and different intratumoral distributions of these PET tracers have been reported with some studies questioning the dependence of the Cu-ATSM accumulation on hypoxia. In the present study, we compared the intratumoral distribution and cellular uptake of 1-(5-fluoro-5-deoxy-α-D-arabinofuranosyl)-2-nitroimidazole (FAZA) and Cu-ATSM. Intratumoral distributions of FAZA and Cu-ATSM compared by double tracer autoradiography in xenografts of 8 cancer cell lines and 3 cancer tissue originated spheroids (CTOSs) showed that only a limited overlap was observed between the regions with high levels of FAZA and Cu-ATSM accumulation in all the xenografts. Immunohistochemistry in the regions enriched with FAZA and Cu-ATSM in xenografts demonstrated that pimonidazole adducts were in regions that accumulated high levels of FAZA, while HIF-1α was in areas enriched with either tracer. In addition, we examined the cellular uptake of FAZA and Cu-ATSM at different levels of oxygen concentration in 4 cell lines and revealed that cellular uptake of FAZA was increased with the decrease of oxygen concentration from 20 to 2 and from 2 to 1%, while the Cu-ATSM uptake increased with the decrease of oxygen concentration from 20 to 2%, but did not increase with the decrease from 2 to 1%. Our findings indicate that intratumoral distributions of FAZA and Cu-ATSM were essentially non-overlapping and although hypoxia affects the buildup of both tracers, the accumulation of Cu-ATSM occurred at milder hypoxia compared to the conditions required for the accumulation of FAZA. Therefore, accumulation levels of FAZA and Cu-ATSM may be considered as independent biomarkers.