Development of [177Lu]Lu-LNC1010 for peptide receptor radionuclide therapy of nasopharyngeal carcinoma.

PURPOSE: Somatostatin Receptor 2 (SSTR2)-targeted radiopharmaceutical [68Ga]Ga-DOTATATE has potential advantages in the diagnosis of nasopharyngeal carcinoma (NPC). This study introduces a novel long-lasting SSTR2 analogue, LNC1010, based on DOTATATE, a truncated Evans blue-binding moiety, and a polyethylene-glycol linker. We hypothesised that peptide receptor radionuclide therapy (PRRT) is more effective with [177Lu]Lu-LNC1010 than with [177Lu]Lu-DOTATATE in treating metastatic NPC. METHODS: We assessed binding characteristics of LNC1010 in vitro using C666-1 NPC cells and in-vivo pharmacokinetics of [68Ga]Ga/[177Lu]Lu-LNC1010 in C666-1 NPC xenografts via PET and SPECT imaging, biodistribution studies, and PRRT, and compared them with [68Ga]Ga/[177Lu] Lu-labelled DOTATATE. Furthermore, a proof-of-concept approach for imaging and therapy was conducted in a patient with metastatic NPC. RESULTS: LNC1010 exhibited strong uptake and specific affinity for SSTR2 in C666-1 NPC cells. PET and SPECT imaging demonstrated higher uptake and longer tumour retention of [68Ga]Ga/[177Lu]Lu-LNC1010 than [68Ga]Ga/[177Lu]Lu-DOTATATE in C666-1 NPC xenografts, indicating its suitability for PRRT applications in NPCs. Biodistribution studies confirmed the higher uptake and prolonged retention of [177Lu]Lu-LNC1010 than [177Lu]Lu-DOTATATE. In preclinical PRRT studies, [177Lu]Lu-LNC1010 showed greater inhibition of tumour growth in C666-1 NPC xenografts than [177Lu]Lu-DOTATATE. In a subsequent pilot clinical study, PRRT with [177Lu]Lu-LNC1010 achieved favourable therapeutic and negligible side effects in a patient with metastatic NPC. CONCLUSION: [177Lu]Lu-LNC1010 demonstrated increased tumour uptake and prolonged retention in SSTR2-positive NPCs, with superior anti-tumour efficacy to that of [177Lu]Lu-DOTATATE in preclinical studies. These findings suggest that PRRT with [177Lu]Lu-LNC1010 is a promising treatment for advanced NPC, extending the clinical scope of PRRT beyond neuroendocrine tumours.

A decade of blood-brain barrier permeability assays: Revisiting old traumatic brain injury rat data for new insights and experimental design.

Increased microvascular permeability at the level of the blood-brain barrier (BBB) often leads to vasogenic brain edema following traumatic brain injury (TBI). These pathologic conditions compromise the integrity of the neurovascular unit resulting in severe brain dysfunction. To quantify this permeability and assess ionic equillibrium, preclinical researchers have relied on the use of various molecular weight permeable dyes such as Evans Blue that normally cannot enter the brain parenchyma under homeostatic conditions. Evans Blue, the most cited of the molecular weight dyes, has reported reproducibility issues because of harsh extraction processes, suboptimal detection via absorbance, and wide excitation fluorescence spectra associated with the dye. Our laboratory group transitioned to Alexa Fluor 680, a far-red dye with improved sensitivity compared to Evans Blue and thus improved reproducibility to alleviate this issue. To evaluate our reproducibility and increase the rigor of our experimental design, we retrospectively analyzed our controlled cortical impact (CCI) experiments over the past 10 years to evaluate effect size with larger samples and potential sources of variability. All of our BBB permeability experiments were performed with Male, Sprague Dawley rats weighing between 225 and 300 g. Historically, Sprague Dawleys were randomly divided into treatment groups: SHAM, CCI, and a stem cell-based treatment from years 2007-2020. The assessment of microvascular hyperpermeability were evaluated by comparing the mean at minimum threshold, area at 1 k-2 k, and intensity density obtained from Alexa Fluor 680 permeability data. Studies utilizing Evans Blue were further compared by tip depth, diameter size, and the hemisphere of injury. Statistical evaluation utilizing the G Power software analysis did not yield a significant difference in sample size comparing experimental groups for Evans Blue and Alexa Fluor 680 analyzed brain tissue. Our analysis also demonstrated a trend in that recent studies (years 2018-2020) have yielded more compact sample sizes between experimental groups in Alexa Fluor 680 analyzed rats. This retrospective study further revealed that Alexa Fluor 680 image analysis provides greater sensitivity to BBB permeability following TBI in comparison to Evans Blue. Significant differences in sample size were not detected between Evans Blue and Alexa Fluor 680; there were significant differences found throughout year to year analysis at the lower range of thresholds. SUMMARY STATEMENT: This work provides a comparative analysis of BBB permeability assay techniques after CCI model of injury in rats.

Development of [177Lu]Lu-LNC1003 for radioligand therapy of prostate cancer with a moderate level of PSMA expression.

PURPOSE: Evans blue as an albumin binder has been widely used to improve pharmacokinetics and enhance tumor uptake of radioligands, including prostate-specific membrane antigen (PSMA) targeting agents. The goal of this study is to develop an optimal Evans blue-modified radiotherapeutic agent that could maximize the absolute tumor uptake and tumor absorbed dose thus the therapeutic efficacy to allow treatment of tumors even with moderate level of PSMA expression. METHODS: [177Lu]Lu-LNC1003 was synthesized based on PSMA-targeting agent and Evans blue. Binding affinity and PSMA targeting specificity were verified through cell uptake and competition binding assay in 22Rv1 tumor model that has moderate level of PSMA expression. SPECT/CT imaging and biodistribution studies in 22Rv1 tumor-bearing mice were performed to evaluate the preclinical pharmacokinetics. Radioligand therapy studies were conducted to systematically assess the therapeutic effect of [177Lu]Lu-LNC1003. RESULTS: LNC1003 showed high binding affinity (IC50 = 10.77 nM) to PSMA in vitro, which was comparable with that of PSMA-617 (IC50 = 27.49 nM) and EB-PSMA-617 (IC50 = 7.91 nM). SPECT imaging of [177Lu]Lu-LNC1003 demonstrated significantly improved tumor uptake and retention as compared with [177Lu]Lu-EB-PSMA and [177Lu]Lu-PSMA-617, making it suitable for prostate cancer therapy. Biodistribution studies further confirmed the remarkably higher tumor uptake of [177Lu]Lu-LNC1003 (138.87 ± 26.53%ID/g) over [177Lu]Lu-EB-PSMA-617 (29.89 ± 8.86%ID/g) and [177Lu]Lu-PSMA-617 (4.28 ± 0.25%ID/g) at 24 h post-injection. Targeted radioligand therapy results showed noteworthy inhibition of 22Rv1 tumor growth after administration of a single dose of 18.5 MBq [177Lu]Lu-LNC1003. There was no obvious antitumor effect after [177Lu]Lu-PSMA-617 treatment under the same condition. CONCLUSION: In this study, [177Lu]Lu-LNC1003 was successfully synthesized with high radiochemical purity and stability. High binding affinity and PSMA targeting specificity were identified in vitro and in vivo. With greatly enhanced tumor uptake and retention, [177Lu]Lu-LNC1003 has the potential to improve therapeutic efficacy using significantly lower dosages and less cycles of 177Lu that promises clinical translation to treat prostate cancer with various levels of PSMA expression.

Localized Increased Permeability of Blood-Brain Barrier for Antibody Conjugates in the Cuprizone Model of Demyelination.

The development of new neurotherapeutics depends on appropriate animal models being chosen in preclinical studies. The cuprizone model is an effective tool for studying demyelination and remyelination processes in the brain, but blood-brain barrier (BBB) integrity in the cuprizone model is still a topic for debate. Several publications claim that the BBB remains intact during cuprizone-induced demyelination; others demonstrate results that could explain the increased BBB permeability. In this study, we aim to analyze the permeability of the BBB for different macromolecules, particularly antibody conjugates, in a cuprizone-induced model of demyelination. We compared the traditional approach using Evans blue injection with subsequent dye extraction and detection of antibody conjugates using magnetic resonance imaging (MRI) and confocal microscopy to analyze BBB permeability in the cuprizone model. First, we validated our model of demyelination by performing T2-weighted MRI, diffusion tensor imaging, quantitative rt-PCR to detect changes in mRNA expression of myelin basic protein and proteolipid protein, and Luxol fast blue histological staining of myelin. Intraperitoneal injection of Evans blue did not result in any differences between the fluorescent signal in the brain of healthy and cuprizone-treated mice (IVIS analysis with subsequent dye extraction). In contrast, intravenous injection of antibody conjugates (anti-GFAP or non-specific IgG) after 4 weeks of a cuprizone diet demonstrated accumulation in the corpus callosum of cuprizone-treated mice both by contrast-enhanced MRI (for gadolinium-labeled antibodies) and by fluorescence microscopy (for Alexa488-labeled antibodies). Our results suggest that the methods with better sensitivity could detect the accumulation of macromolecules (such as fluorescent-labeled or gadolinium-labeled antibody conjugates) in the brain, suggesting a local BBB disruption in the demyelinating area. These findings support previous investigations that questioned BBB integrity in the cuprizone model and demonstrate the possibility of delivering antibody conjugates to the corpus callosum of cuprizone-treated mice.

Cognitive Impairments and blood-brain Barrier Damage in a Mouse Model of Chronic Cerebral Hypoperfusion.

Chronic cerebral hypoperfusion (CCH) is commonly involved in various brain diseases. Tight junction proteins (TJs) are key components constituting the anatomical substrate of the blood-brain barrier (BBB). Changes in cognitive function and BBB after CCH and their relationship need further exploration. To investigate the effect of CCH on cognition and BBB, we developed a bilateral common carotid artery stenosis (BCAS) model in Tie2-GFP mice. Mice manifested cognitive impairments accompanied with increased microglia after the BCAS operation. BCAS mice also exhibited increased BBB permeability at all time points set from D1 to D42. Furthermore, BCAS mice showed reduced expression of TJs 42 d after the operation. In addition, correct entrances of mice in radial arm maze test had a moderate negative correlation with EB extravasation. Our data suggested that BCAS could lead to cognitive deficits, microglia increase and BBB dysfunction characterized by increased BBB permeability and reduced TJs expression level. BBB permeability may be involved in the cognitive impairments induced by CCH.

CCM3 Loss-Induced Lymphatic Defect Is Mediated by the Augmented VEGFR3-ERK1/2 Signaling.

OBJECTIVE: Cerebral cavernous malformations (CCMs) can happen anywhere in the body, although they most commonly produce symptoms in the brain. The role of CCM genes in other vascular beds outside the brain and retina is not well-examined, although the 3 CCM-associated genes (CCM1, CCM2, and CCM3) are ubiquitously expressed in all tissues. We aimed to determine the role of CCM gene in lymphatics. Approach and Results: Mice with an inducible pan-endothelial cell (EC) or lymphatic EC deletion of Ccm3 (Pdcd10ECKO or Pdcd10LECKO) exhibit dilated lymphatic capillaries and collecting vessels with abnormal valve structure. Morphological alterations were correlated with lymphatic dysfunction in Pdcd10LECKO mice as determined by Evans blue dye and fluorescein isothiocyanate(FITC)-dextran transport assays. Pdcd10LECKO lymphatics had increased VEGFR3 (vascular endothelial growth factor receptor-3)-ERK1/2 (extracellular signal-regulated kinase 1/2) signaling with lymphatic hyperplasia. Mechanistic studies suggested that VEGFR3 is primarily regulated at a transcriptional level in Ccm3-deficient lymphatic ECs, in an NF-κB (nuclear factor κB)-dependent manner. CCM3 binds to importin alpha 2/KPNA2 (karyopherin subunit alpha 2), and a CCM3 deletion releases KPNA2 to activate NF-κB P65 by facilitating its nuclear translocation and P65-dependent VEGFR3 transcription. Moreover, increased VEGFR3 in lymphatic EC preferentially activates ERK1/2 signaling, which is critical for lymphatic EC proliferation. Importantly, inhibition of VEGFR3 or ERK1/2 rescued the lymphatic defects in structure and function. CONCLUSIONS: Our data demonstrate that CCM3 deletion augments the VEGFR3-ERK1/2 signaling in lymphatic EC that drives lymphatic hyperplasia and malformation and warrant further investigation on the potential clinical relevance of lymphatic dysfunction in patients with CCM.

Model Study for Interaction of Sublethal Doses of Zinc Oxide Nanoparticles with Environmentally Beneficial Bacteria Bacillus thuringiensis and Bacillus megaterium.

Zinc oxide nanoparticles (ZnO NPs), due to their antibacterial effects, are commonly used in various branches of the economy and can affect rhizobacteria that promote plant growth. We describe the effect of ZnO NPs on two model bacteria strains, B. thuringiensis and B. megaterium, that play an important role in the environment. The MIC (minimum inhibitory concentration) value determined after 48 h of incubation with ZnO NPs was more than 1.6 mg/mL for both strains tested, while the MBC (minimum bactericidal concentration) was above 1.8 mg/mL. We tested the effect of ZnO NPs at concentrations below the MIC (0.8 mg/mL, 0.4 mg/mL and 0.2 mg/mL (equal to 50%, 25% and 12,5% MIC, respectively) in order to identify the mechanisms activated by Bacillus species in the presence of these nanoparticles. ZnO NPs in sublethal concentrations inhibited planktonic cell growth, stimulated endospore formation and reduced decolorization of Evans blue. The addition of ZnO NPs caused oxidative stress, measured using nitroblue tetrazolium (NBT), and reduced the activity of catalase. It was confirmed that zinc oxide nanoparticles in sublethal concentrations change metabolic processes in Bacillus bacteria that are important for their effects on the environment. B. thuringiensis after treatment with ZnO NPs decreased indole acetic acid (IAA) production and increased biofilm formation, whereas B. megaterium decreased IAA production but, inversely, increased biofilm formation. Comparison of different Bacillus species in a single experiment made it possible to better understand the mechanisms of toxicity of zinc oxide nanoparticles and the individual reactions of closely related bacterial species.

Process Parameters Optimization, Characterization, and Application of KOH-Activated Norway Spruce Bark Graphitic Biochars for Efficient Azo Dye Adsorption.

In this work, Norway spruce bark was used as a precursor to prepare activated biochars (BCs) via chemical activation with potassium hydroxide (KOH) as a chemical activator. A Box-Behnken design (BBD) was conducted to evaluate and identify the optimal conditions to reach high specific surface area and high mass yield of BC samples. The studied BC preparation parameters and their levels were as follows: pyrolysis temperature (700, 800, and 900 °C), holding time (1, 2, and 3 h), and ratio of the biomass: chemical activator of 1: 1, 1.5, and 2. The planned BBD yielded BC with extremely high SSA values, up to 2209 m2·g-1. In addition, the BCs were physiochemically characterized, and the results indicated that the BCs exhibited disordered carbon structures and presented a high quantity of O-bearing functional groups on their surfaces, which might improve their adsorption performance towards organic pollutant removal. The BC with the highest SSA value was then employed as an adsorbent to remove Evans blue dye (EB) and colorful effluents. The kinetic study followed a general-order (GO) model, as the most suitable model to describe the experimental data, while the Redlich-Peterson model fitted the equilibrium data better. The EB adsorption capacity was 396.1 mg·g-1. The employment of the BC in the treatment of synthetic effluents, with several dyes and other organic and inorganic compounds, returned a high percentage of removal degree up to 87.7%. Desorption and cyclability tests showed that the biochar can be efficiently regenerated, maintaining an adsorption capacity of 75% after 4 adsorption-desorption cycles. The results of this work pointed out that Norway spruce bark indeed is a promising precursor for producing biochars with very promising properties.

Intraindividual comparison of [177Lu]Lu-DOTA-EB-TATE and [177Lu]Lu-DOTA-TOC.

PURPOSE: The radiolabelled somatostatin analogue [177Lu]Lu-DOTA-EB-TATE binds to albumin via Evans blue, thereby increasing the residence time in the blood and potentially allowing more therapeutic agent to be absorbed into the target tissue during peptide receptor radionuclide therapy. It was tested in selected patients whether the substance is superior to [177Lu]Lu-DOTA-TOC. METHODS: Activity kinetics in organs and tumours after [177Lu]Lu-DOTA-EB-TATE and [177Lu]Lu-DOTA-TOC were compared intraindividually in five patients with progressive somatostatin receptor-positive disease scheduled for radionuclide therapy. RESULTS: In comparison to [177Lu]Lu-DOTA-TOC, tumour doses per administered activity were higher for [177Lu]Lu-DOTA-EB-TATE in 4 of 5 patients (median ratio: 1.7; range: 0.9 to 3.9), kidney doses (median ratio: 3.2; range: 1.6 to 9.8) as well as spleen doses (median ratio: 4.7; range 1.2 to 6.2) in all patients, and liver doses in 3 of 4 evaluable patients (median ratio: 4.0; range: 0.7 to 4.9). The tumour to critical organs absorbed dose ratios were higher after [177Lu]Lu-DOTA-TOC in 4 of 5 patients. CONCLUSIONS: Prior to a treatment with [177Lu]Lu-DOTA-EB-TATE, it should be assessed individually whether the compound is superior to established substances.

Evans Blue Might Produce Pathologically Activated Neuroprotective Effects via the Inhibition of the P2X4R/p38 Signaling Pathway.

The main pathological features of ischemic stroke include neuronal damage and blood-brain barrier (BBB) dysfunction. Previous studies have shown that Evans Blue, a dye used to probe BBB integrity, could enter the brain only during the pathological status of ischemic stroke, indicating the potential pathologically activated therapeutic use of this chemical to treat ischemic stroke. In this study, we have reported that Evans Blue could produce in vitro neuroprotective effects against iodoacetic acid (IAA)-induced hypoxia neuronal death in HT22 cells. We further found that P2X purinoreceptor 4 (P2X4R), a subtype of ATP-gated cation channel, was expressed in HT22 cells. Evans Blue could prevent IAA-induced increase of P2X4R mRNA and protein expression. Interestingly, shRNA of P2X4R could protect against IAA-induced activation of p38, and SB203580, a specific inhibitor of p38, could reverse IAA-induced neurotoxicity, indicating that p38 is a downstream signaling molecule of P2X4R. Molecular docking analysis further demonstrated the possible interaction between Evans Blue and the ATP binding site of P2X4R. Most importantly, pre-treatment of Evans Blue could largely reduce neurological and behavioral abnormity, and decrease brain infarct volume in middle cerebral artery occlusion/reperfusion (MCAO) rats. All these results strongly suggested that Evans Blue could exert neuroprotective effects via inhibiting the P2X4R/p38 pathway, possibly by acting on the ATP binding site of P2X4R, indicating that Evans Blue might be further developed as a pathologically activated therapeutic drug against ischemic stroke.