Specific activation of pro-Infliximab enhances selectivity and safety of rheumatoid arthritis therapy.

During rheumatoid arthritis (RA) treatment, long-term injection of antitumor necrosis factor α antibodies (anti-TNFα Abs) may induce on-target toxicities, including severe infections (tuberculosis [TB] or septic arthritis) and malignancy. Here, we used an immunoglobulin G1 (IgG1) hinge as an Ab lock to cover the TNFα-binding site of Infliximab by linking it with matrix metalloproteinase (MMP) -2/9 substrate to generate pro-Infliximab that can be specifically activated in the RA region to enhance the selectivity and safety of treatment. The Ab lock significantly inhibits the TNFα binding and reduces the anti-idiotypic (anti-Id) Ab binding to pro-Infliximab by 395-fold, 108-fold compared with Infliximab, respectively, and MMP-2/9 can completely restore the TNFα neutralizing ability of pro-Infliximab to block TNFα downstream signaling. Pro-Infliximab was only selectively activated in the disease site (mouse paws) and presented similar pharmacokinetics (PKs) and bio-distribution to Infliximab. Furthermore, pro-Infliximab not only provided equivalent therapeutic efficacy to Infliximab but also maintained mouse immunity against Listeria infection in the RA mouse model, leading to a significantly higher survival rate (71%) than that of the Infliximab treatment group (0%). The high-selectivity pro-Infliximab maintains host immunity and keeps the original therapeutic efficiency, providing a novel strategy for RA therapy.

Development of Radiofluorinated Nicotinamide/Picolinamide Derivatives as Diagnostic Probes for the Detection of Melanoma.

Regarding the increased incidence and high mortality rate of malignant melanoma, practical early-detection methods are essential to improve patients' clinical outcomes. In this study, we successfully prepared novel picolinamide-benzamide (18F-FPABZA) and nicotinamide-benzamide (18F-FNABZA) conjugates and determined their biological characteristics. The radiochemical yields of 18F-FPABZA and 18F-FNABZA were 26 ± 5% and 1 ± 0.5%, respectively. 18F-FPABZA was more lipophilic (log P = 1.48) than 18F-FNABZA (log P = 0.68). The cellular uptake of 18F-FPABZA in melanotic B16F10 cells was relatively higher than that of 18F-FNABZA at 15 min post-incubation. However, both radiotracers did not retain in amelanotic A375 cells. The tumor-to-muscle ratios of 18F-FPABZA-injected B16F10 tumor-bearing mice increased from 7.6 ± 0.4 at 15 min post-injection (p.i.) to 27.5 ± 16.6 at 3 h p.i., while those administered with 18F-FNABZA did not show a similarly dramatic increase throughout the experimental period. The results obtained from biodistribution studies were consistent with those derived from microPET imaging. This study demonstrated that 18F-FPABZA is a promising melanin-targeting positron emission tomography (PET) probe for melanotic melanoma.

Rapamycin and hydroxychloroquine combination alters macrophage polarization and sensitizes glioblastoma to immune checkpoint inhibitors.

INTRODUCTION: The failure of immune checkpoint inhibitor (ICPi) on glioblastoma (GBM) treatment underscores the need for improving therapeutic strategy. We aimed to change tumor associated macrophage (TAM) from M2 type (anti-inflammatory) to M1 (pro-inflammatory) type to increase the therapeutic response of ICPi. We proposed that combined rapamycin (R) and hydroxychloroquine (Q) preferentially induce M2 cells death, as fatty acid oxidation was their major source of energy. METHODS: Macrophage polarization was characterized on mice and human macrophage cell lines by specific cytokines stimulation with or without RQ treatment under single culture or co-culture with GBM cell lines. Tumor sizes were evaluated on subcutaneous and intracranial GL261 mice models with or without RQ, anti-PD1 mAb treatment. Tumor volumes assessed by MRI scan and proportions of tumor infiltrating immune cells analyzed by flow cytometry were compared. RESULTS: In vitro RQ treatment decreased the macrophages polarization of M2, increased the phagocytic ability, and increased the lipid droplets accumulation. RQ treatment decreased the expression levels of CD47 and SIRPα on tumor cells and macrophage cells in co-culture experiments. The combination of RQ and anti-PD1 treatment was synergistic in action. Enhanced the intra-tumoral M1/M2 ratio, the CD8/CD4 ratio in the intracranial GL261 tumor model after RQ treatment were evident. CONCLUSION: We provide a rationale for manipulating the macrophage phenotype and increased the therapeutic effect of ICPi. To re-educate and re-empower the TAM/microglia opens an interesting avenue for GBM treatment.

Simplified quantification of 13N-ammonia PET myocardial blood flow: A comparative study with the standard compartment model to facilitate clinical use.

BACKGROUND: Short imaging protocol to quantify myocardial blood flow (MBF) and myocardial flow reserve (MFR) may enhance the clinical application of 13N-ammonia cardiac PET. We assessed the flow quantitation of 13N-ammonia PET implementing simple retention model and two-compartment model. METHODS: Fourteen healthy volunteers (HVT) and twenty-three clinical patients received 13N-ammonia PET/CT. The simple retention model used the first 7-minute image to quantify MBF. Global and regional MBF and MFR of the two models were compared. RESULTS: Global and regional MBF and MFR of these two models were highly correlated with mildly inferior correlation in RCA territory (global R2: rest MBF = 0.79, stress MBF = 0.65, MFR = 0.77; regional R2: rest MBF ≥ 0.72, stress MBF ≥ 0.52, MFR ≥ 0.68). There were significant differences for MFR (4.04 ± 0.72, 3.66 ± 0.48, p = .02) and rest MBF (0.69 ± 0.12, 0.78 ± 0.12, p = .02) between the two models in the HVT group. CONCLUSIONS: 13N-ammonia global and regional MBF and MFR from the simple retention model demonstrate strong correlations with that from the two-compartment model. Significant differences of MFR and rest MBF are noted in the HVT group, with a proposed normal reference value for the 13N-ammonia short simple retention protocol.

Evaluation of Radioiodinated Fluoronicotinamide/Fluoropicolinamide-Benzamide Derivatives as Theranostic Agents for Melanoma.

Malignant melanoma is the most harmful type of skin cancer and its incidence has increased in this past decade. Early diagnosis and treatment are urgently desired. In this study, we conjugated picolinamide/nicotinamide with the pharmacophore of 131I-MIP-1145 to develop 131I-iodofluoropicolinamide benzamide (131I-IFPABZA) and 131I-iodofluoronicotiamide benzamide (131I-IFNABZA) with acceptable radiochemical yield (40 ± 5%) and high radiochemical purity (>98%). We also presented their biological characteristics in melanoma-bearing mouse models. 131I-IFPABZA (Log P = 2.01) was more lipophilic than 131I-IFNABZA (Log P = 1.49). B16F10-bearing mice injected with 131I-IFNABZA exhibited higher tumor-to-muscle ratio (T/M) than those administered with 131I-IFPABZA in planar γ-imaging and biodistribution studies. However, the imaging of 131I-IFNABZA- and 131I-IFPABZA-injected mice only showed marginal tumor uptake in A375 amelanotic melanoma-bearing mice throughout the experiment period, indicating the high binding affinity of these two radiotracers to melanin. Comparing the radiation-absorbed dose of 131I-IFNABZA with the melanin-targeted agents reported in the literature, 131I-IFNABZA exerts lower doses to normal tissues on the basis of similar tumor dose. Based on the in vitro and in vivo studies, we clearly demonstrated the potential of using 131I-IFNABZA as a theranostic agent against melanoma.

Efficient Prodrug Activator Gene Therapy by Retroviral Replicating Vectors Prolongs Survival in an Immune-Competent Intracerebral Glioma Model.

Prodrug activator gene therapy mediated by murine leukemia virus (MLV)-based retroviral replicating vectors (RRV) was previously shown to be highly effective in killing glioma cells both in culture and in vivo. To avoid receptor interference and enable dual vector co-infection with MLV-RRV, we have developed another RRV based on gibbon ape leukemia virus (GALV) that also shows robust replicative spread in a wide variety of tumor cells. We evaluated the potential of GALV-based RRV as a cancer therapeutic agent by incorporating yeast cytosine deaminase (CD) and E. coli nitroreductase (NTR) prodrug activator genes into the vector. The expression of CD and NTR genes from GALV-RRV achieved highly efficient delivery of these prodrug activator genes to RG-2 glioma cells, resulting in enhanced cytotoxicity after administering their respective prodrugs 5-fluorocytosine and CB1954 in vitro. In an immune-competent intracerebral RG-2 glioma model, GALV-mediated CD and NTR gene therapy both significantly suppressed tumor growth with CB1954 administration after a single injection of vector supernatant. However, NTR showed greater potency than CD, with control animals receiving GALV-NTR vector alone (i.e., without CB1954 prodrug) showing extensive tumor growth with a median survival time of 17.5 days, while animals receiving GALV-NTR and CB1954 showed significantly prolonged survival with a median survival time of 30 days. In conclusion, GALV-RRV enabled high-efficiency gene transfer and persistent expression of NTR, resulting in efficient cell killing, suppression of tumor growth, and prolonged survival upon CB1954 administration. This validates the use of therapeutic strategies employing this prodrug activator gene to arm GALV-RRV, and opens the door to the possibility of future combination gene therapy with CD-armed MLV-RRV, as the latter vector is currently being evaluated in clinical trials.

pH-Responsive PEG-Shedding and Targeting Peptide-Modified Nanoparticles for Dual-Delivery of Irinotecan and microRNA to Enhance Tumor-Specific Therapy.

Irinotecan is one of the main chemotherapeutic agents for colorectal cancer (CRC). MicroRNA-200 (miR-200) has been reported to inhibit metastasis in cancer cells. Herein, pH-sensitive and peptide-modified liposomes and solid lipid nanoparticles (SLN) are designed for encapsulation of irinotecan and miR-200, respectively. These peptides include one cell-penetrating peptide, one ligand targeted to tumor neovasculature undergoing angiogenesis, and one mitochondria-targeting peptide. The peptide-modified nanoparticles are further coated with a pH-sensitive PEG-lipid derivative with an imine bond. These specially-designed nanoparticles exhibit pH-responsive release, internalization, and intracellular distribution in acidic pH of colon cancer HCT116 cells. These nanoparticles display low toxicity to blood and noncancerous intestinal cells. Delivery of miR-200 by SLN further increases the cytotoxicity of irinotecan-loaded liposomes against CRC cells by triggering apoptosis and suppressing RAS/ß-catenin/ZEB/multiple drug resistance (MDR) pathways. Using CRC-bearing mice, the in vivo results further indicate that irinotecan and miR-200 in pH-responsive targeting nanoparticles exhibit positive therapeutic outcomes by inhibiting colorectal tumor growth and reducing systemic toxicity. Overall, successful delivery of miR and chemotherapy by multifunctional nanoparticles may modulate ß-catenin/MDR/apoptosis/metastasis signaling pathways and induce programmed cancer cell death. Thus, these pH-responsive targeting nanoparticles may provide a potential regimen for effective treatment of colorectal cancer.

Evaluation of the Biological Behavior of a Gold Nanocore-Encapsulated Human Serum Albumin Nanoparticle (Au@HSANP) in a CT-26 Tumor/Ascites Mouse Model after Intravenous/Intraperitoneal Administration.

Colorectal cancer is one of the major causes of cancer-related death in Taiwan and worldwide. Patients with peritoneal metastasis from colorectal cancer have reduced overall survival and poor prognosis. Hybrid protein-inorganic nanoparticle systems have displayed multifunctional applications in solid cancer theranostics. In this study, a gold nanocore-encapsulated human serum albumin nanoparticle (Au@HSANP), which is a hybrid protein-inorganic nanoparticle, and its radioactive surrogate 111In-labeled Au@HSANP (111In-Au@HSANP), were developed and their biological behaviors were investigated in a tumor/ascites mouse model. 111In-Au@HSANP was injected either intravenously (iv) or intraperitoneally (ip) in CT-26 tumor/ascites-bearing mice. After ip injection, a remarkable and sustained radioactivity retention in the abdomen was noticed, based on microSPECT images. After iv injection, however, most of the radioactivity was accumulated in the mononuclear phagocyte system. The results of biodistribution indicated that ip administration was significantly more effective in increasing intraperitoneal concentration and tumor accumulation than iv administration. The ratios of area under the curve (AUC) of the ascites and tumors in the ip-injected group to those in the iv-injected group was 93 and 20, respectively. This study demonstrated that the ip injection route would be a better approach than iv injections for applying gold-albumin nanoparticle in peritoneal metastasis treatment.

Synthesis and biological evaluation of 2-(3,4-dimethoxyphenyl)-6-(2-[18F]fluoroethoxy)benzothiazole ([18F]FEDBT) for PET imaging of breast cancer.

Given the ever-present demand for improved PET radiotracer in oncology imaging, we have synthesized 2-(3,4-dimethoxyphenyl)-6-(2-[18F]fluoroethoxy)benzothiazole ([18F]FEDBT), a fluorine-18-containing fluoroethylated benzothiazole to explore its utility as a PET imaging tracer. [18F]FEDBT was prepared via kryptofix-mediated nucleophilic substitution of the tosyl group precursor. Fractionated ethanol-based solid-phase (SPE cartridge-based) purification afforded [18F]FEDBT in 60% radiochemical yield (EOB), with radiochemical purity in excess of 98% and the specific activity was 35GBq/µmol. The radiotracer displayed clearly higher cellular uptake ratio in various breast cancer cell lines MCF7, MDA-MB-468 and MDA-MB-231. However, both biodistribution and microPET studies have showed an higher abdominal accumulation of [18F]FEDMBT and the tumor/muscle ratio of 1.8 was observed in the MDA-MB-231 xenograft tumors mice model. Further the lipophilic improvement is needed for the reducement of hepatobilliary accumulation and to promote the tumor uptake for PET imaging of breast cancer.

Over-expression of cofilin-1 suppressed growth and invasion of cancer cells is associated with up-regulation of let-7 microRNA.

Cofilin-1, a non-muscle isoform of actin regulatory protein that belongs to the actin-depolymerizing factor (ADF)/cofilin family is known to affect cancer development. Previously, we found that over-expression of cofilin-1 suppressed the growth and invasion of human non-small cell lung cancer (NSCLC) cells in vitro. In this study, we further investigated whether over-expression of cofilin-1 can suppress tumor growth in vivo, and performed a microRNA array analysis to better understand whether specific microRNA would be involved in this event. The results showed that over-expression of cofilin-1 suppressed NSCLC tumor growth using the xenograft tumor model with the non-invasive reporter gene imaging modalities. Additionally, cell motility and invasion were significantly suppressed by over-expressed cofilin-1, and down-regulation of matrix metalloproteinase (MMPs) -1 and -3 was concomitantly detected. According to the microRNA array analysis, the let-7 family, particularly let-7b and let-7e, were apparently up-regulated among 248 microRNAs that were affected after over-expression of cofilin-1 up to 7 days. Knockdown of let-7b or let-7e using chemical locked nucleic acid (LNA) could recover the growth rate and the invasion of cofilin-1 over-expressing cells. Next, the expression of c-myc, LIN28 and Twist-1 proteins known to regulate let-7 were analyzed in cofilin-1 over-expressing cells, and Twist-1 was significantly suppressed under this condition. Up-regulation of let-7 microRNA by over-expressed cofilin-1 could be eliminated by co-transfected Twist-1 cDNA. Taken together, current data suggest that let-7 microRNA would be involved in over-expression of cofilin-1 mediated tumor suppression in vitro and in vivo.

Targeted antitumor prodrug therapy using CNGRC-yCD fusion protein in combination with 5-fluorocytosine.

BACKGROUND: The enzyme-prodrug system is considered a promising tool for tumor treatment when conjugated with a targeting molecule. The asparagine-glycine-arginine (NGR) motif is a developing and interesting targeting peptide that could specifically bind to aminopeptidase N (APN), which is an NGR receptor expressed on the cell membrane of angiogenic endothelial cells and a number of tumor cells within the tumor tissues. The objective of this study was to develop a novel targeted enzyme-prodrug system using 5-fluorocytosine (5-FC) and an NGR-containing peptide fused with yeast cytosine deaminase (yCD), i.e. CNGRC-yCD fusion protein, to target APN-expressing cells within the tumor tissues and to convert 5-FC into 5-fluorouracil (5-FU) to kill tumors. RESULTS: Both yCD and CNGRC-yCD proteins were cloned into the pET28a vector and expressed by an Escherichia coli host. Both yCD and CNGRC-yCD proteins were purified and the yields were approximately 20 mg/L with over 95 % purity. The binding assay demonstrated that the CNGRC-yCD fusion protein had specific binding affinity toward purified APN recombinant protein and high-APN-expressing cells, including human endothelial cells (HUVECs) and various types of human tumor cell lines, but not low-APN-expressing tumor cell lines. Moreover, the enzyme activity and cell viability assay showed that the CNGRC-yCD fusion protein could effectively convert 5-FC into 5-FU and resulted in significant cell death in both high-APN-expressing tumor cells and HUVECs. CONCLUSIONS: This study successfully constructs a new targeting enzyme-prodrug system, CNGRC-yCD fusion protein/5-FC. Systematic experiments demonstrated that the CNGRC-yCD protein retained both the APN-binding affinity of NGR and the enzyme activity of yCD to convert 5-FC into 5-FU. The combined treatment of the CNGRC-yCD protein with 5-FC resulted in the significantly increased cell death of high-APN-expressing cells as compared to that of low-APN-expressing cells.

Preparation and characterization of a novel Al(18)F-NOTA-BZA conjugate for melanin-targeted imaging of malignant melanoma.

Melanin is an attractive target for the diagnosis and treatment of malignant melanoma. Previous studies have demonstrated the specific binding ability of benzamide moiety to melanin. In this study, we developed a novel (18)F-labeled NOTA-benzamide conjugate, Al(18)F-NOTA-BZA, which can be synthesized in 30min with a radiochemical yield of 20-35% and a radiochemical purity of >95%. Al(18)F-NOTA-BZA is highly hydrophilic (logP=-1.96) and shows good in vitro stability. Intravenous administration of Al(18)F-NOTA-BZA in two melanoma-bearing mouse models revealed highly specific uptake in B16F0 melanotic melanoma (6.67±0.91 and 1.50±0.26%ID/g at 15 and 120min p.i., respectively), but not in A375 amelanotic melanoma (0.87±0.21 and 0.24±0.09%ID/g at 15 and 120min p.i., respectively). The clearance from most normal tissues was fast. A microPET scan of Al(18)F-NOTA-BZA-injected mice also displayed high-contrast tumor images as compared with normal organs. Owing to the favorable in vivo distribution of Al(18)F-NOTA-BZA after intravenous administration, the estimated absorption dose was low in all normal organs and tissues. The melanin-specific binding ability, sustained tumor retention, fast normal tissues clearance and thelow projected human dosimetry supported that Al(18)F-NOTA-BZA is a very promising melanin-specific PET probe for melanin-positive melanoma.

Development and Characterization of the Recombinant Human VEGF-EGF Dual-Targeting Fusion Protein as a Drug Delivery System.

The design, preparation, as well as structural and functional characterizations of the recombinant fusion protein hVEGF-EGF as a dual-functional agent that may target both EGFR (R: receptor) and angiogenesis are reported. hVEGF-EGF was found to bind to EGFR more strongly than did EGF, and to bind to VEGFR similarly to VEGF. Mass spectrometry measurements showed that the sites of DTPA (diethylenetriaminepentaacetic acid) conjugated hVEGF-EGF (for radiolabeling) were the same as those of its parent hEGF and hVEGF proteins. All DTPA-conjugated proteins retained similar binding capacities to their respective receptors as compared to their respective parent proteins. In vitro cell binding studies using BAEC (a bovine aortic endothelial cell) and MDA-MB-231 (a human breast cancer) cells expressing both EGFR and VEGFR confirmed similar results. Treating BAEC cells with hVEGF-EGF induced remarkable phosphorylation of EGFR, VEGFR, and their downstream targets ERK1/2. Nevertheless, the radiolabeled (111)In-DTPA-hVEGF-EGF showed cytotoxicity against MDA-MB-231 cells. Pharmacokinetic studies using (111)In-DTPA-hVEGF-EGF in BALB/c nude mice showed that appreciable tracer activities were accumulated in liver and spleen. In all, this study demonstrated that the fusion protein hVEGF-EGF maintained the biological specificity toward both EGFR and VEGFR and may be a potential candidate as a dual-targeting moiety in developing anticancer drugs.

Synthesis and evaluation of ¹²³/¹³¹I-Iochlonicotinamide as a novel SPECT probe for malignant melanoma.

Malignant melanoma expresses a highly aggressive metastasis. Early diagnosis of malignant melanoma is important for patient survival. Radiolabeled benzamides and nicotinamides have been reported to be attractive candidates for malignant melanoma diagnosis as they bind to melanin, a characteristic substance that displays in malignant melanoma, and show high tumor accumulation and retention. Herein, we designed and synthesized a novel (123/131)I-labeled nicotinamide derivative that specifically binds to melanin. (123/131)I-Iochlonicotinamide was prepared with good radiochemical yield (50-70%, decay corrected) and high specific radioactivity (50-80 GBq/µmol). (131)I-Iochlonicotinamide exhibited good in vitro stability (radiochemical purity >95% after a 24-h incubation) in human serum. High uptake of (123/131)I-Iochlonicotinamide in B16F0 melanoma cells compared to that in A375 amelanotic cells demonstrated its selective binding to melanin. Intravenous administration of (123/131)I-Iochlonicotinamide in a melanoma-bearing mouse model revealed high uptake in melanotic melanoma and high tumor-to-muscle ratio. MicroSPECT scan of (123/131)I-Iochlonicotinamide injected mice also displayed high contrast tumor imaging as compared with normal organs. The radiation-absorbed dose projection for the administration of (131)I-Iochlonicotinamide to human was based on the results of biodistribution study. The effective dose appears to be approximately 0.44 mSv/MBq(-1). The specific binding of (123/131)I-Iochlonicotinamide to melanin along with a prolonged tumor retention and acceptable projected human dosimetry suggest that it may be a promising theranostic agent for treating malignant melanoma.

Biological characterization of cetuximab-conjugated gold nanoparticles in a tumor animal model.

Gold nanoparticles (AuNPs) are widely applied to the diagnosis and treatment of cancer and can be modified to contain target-specific ligands via gold-thiolate bonding. This study investigated the pharmacokinetics and microdistribution of antibody-mediated active targeting gold nanoparticles in mice with subcutaneous lung carcinoma. We conjugated AuNPs with cetuximab (C225), an antibody-targeting epidermal growth factor receptor (EGFR), and then labeled with In-111, which created EGFR-targeted AuNPs. In vitro studies showed that after a 2 h incubation, the uptake of C225-conjugated AuNPs in high EGFR-expression A549 cells was 14.9-fold higher than that of PEGylated AuNPs; furthermore, uptake was also higher at 3.8-fold when MCF7 cells with lower EGFR-expression were used. MicroSPECT/CT imaging and a biodistribution study conducted by using a A549 tumor xenograft mouse model provided evidence of elevated uptake of the C225-conjugated AuNPs into the tumor cells as a result of active targeting. Moreover, the microdistribution of PEGylated AuNPs revealed that a large portion of AuNPs remained in the tumor interstitium, whereas the C225-conjugated AuNPs displayed enhanced internalization via antibody-mediated endocytosis. Our findings suggest that the anti-EGFR antibody-conjugated AuNPs are likely to be a plausible nano-sized vehicle for drug delivery to EGFR-expressing tumors.

Sensitivity of PEGylated interferon detection by anti-polyethylene glycol (PEG) antibodies depends on PEG length.

Attachment of poly(ethylene glycol) to proteins can mask immune epitopes to increase serum half-life, reduce immunogenicity, and enhance in vivo biological efficacy. However, PEGylation mediated epitope-masking may also limit sensitivity and accuracy of traditional ELISA. We previously described an anti-PEG-based sandwich ELISA for universal assay of PEGylated molecules. Here, we compared the quantitative assessment of PEGylated interferons by anti-PEG and traditional anti-interferon sandwich ELISA. The detection limits for PEG-Intron (12k-PEG) and Pegasys (40k-PEG) were 1.9 and 0.03 ng/mL for anti-PEG ELISA compared to 0.18 and 0.42 ng/mL for traditional anti-interferon sandwich ELISA. These results indicate that the anti-PEG sandwich ELISA was insensitive to PEGylation mediated epitope-masking and the sensitivity increased in proportion to the length of PEG. By contrast, PEG-masking interfered with detection by traditional anti-interferon sandwich ELISA. Human and mouse serum did not affect the sensitivity of anti-PEG ELISA but impeded traditional anti-interferon sandwich ELISA. The anti-PEG sandwich ELISA was comparable to anti-interferon sandwich ELISA and radioassay of 131I-Pegasys in pharmacokinetic studies in mice. The anti-PEG sandwich ELISA provides a sensitive, accurate, and convenient quantitative measurement of PEGylated protein drugs.

Evaluation of EGFR-targeted radioimmuno-gold-nanoparticles as a theranostic agent in a tumor animal model.

This study evaluated the tumor targeting and therapeutic efficacy of a novel theranostic agent (131)I-labeled immuno-gold-nanoparticle ((131)I-C225-AuNPs-PEG) for high epidermal growth factor receptor (EGFR)-expressed A549 human lung cancer. Confocal microscopy demonstrated the specific uptake of C225-AuNPs-PEG in A549 cells. (131)I-C225-AuNPs-PEG induced a significant reduction in cell viability, which was not observed when incubated with AuNPs-PEG and C225-AuNPs-PEG. MicroSPECT/CT imaging of tumor-bearing mice after intravenous injection of (123)I-C225-AuNPs-PEG revealed significant radioactivity retention in tumor suggested that (131)I-labeled C225-conjugated radioimmuno-gold-nanoparticles may provide a new approach of targeted imaging and therapy towards high EGFR-expressed cancers.

Synthesis and biological evaluation of technetium-99m labeled galactose derivatives as potential asialoglycoprotein receptor probes in a hepatic fibrosis mouse model.

Two galactose derivatives, a monovalent (99m)Tc-MAMA-MGal galactoside and a divalent (99m)Tc-MAMA-DGal galactoside, were synthesized and radiolabeled in high radiochemical purity (>98%). Dynamic microSPECT imaging and biodistribution study of two traces in normal and liver fibrosis mice showed that the (99m)Tc-MAMA-DGal revealed higher specific binding to asialoglycoprotein receptors in liver and then rapidly excreted via both hepatobiliary system and renal clearance. The results suggest that (99m)Tc-MAMA-DGal may be used as SPECT probes for noninvasive evaluation of asialoglycoprotein receptor-related liver dysfunction.

(18)F-FBHGal for asialoglycoprotein receptor imaging in a hepatic fibrosis mouse model.

Quantification of the expression of asialoglycoprotein receptor (ASGPR), which is located on the hepatocyte membrane with high-affinity for galactose residues, can help assess ASGPR-related liver diseases. A hepatic fibrosis mouse model with lower asialoglycoprotein receptor expression was established by dimethylnitrosamine (DMN) administration. This study developed and demonstrated that 4-(18)F-fluoro-N-(6-((3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)hexyl)benzamide ((18)F-FBHGal), a new (18)F-labeled monovalent galactose derivative, is an asialoglycoprotein receptor (ASGPR)-specific PET probe in a normal and a hepatic fibrosis mouse models. Immunoassay exhibited a linear correlation between the accumulation of GalH-FITC, a fluorescent surrogate of FBHGal, and the amount of ASGPR. A significant reduction in HepG2 cellular uptake (P <0.0001) was observed using confocal microscopy when co-incubated with 0.5µM of asialofetuin, a well known ASGPR blocking agent. Animal studies showed the accumulation of (18)F-FBHGal in fibrosis liver (14.84±1.10 %ID/g) was appreciably decreased compared with that in normal liver (20.50±1.51 %ID/g, P <0.01) at 30min post-injection. The receptor indexes (liver/liver-plus-heart ratio at 30min post-injection) of hepatic fibrosis mice derived from both microPET imaging and biodistribution study were significantly lower (P <0.01) than those of normal mice. The pharmacokinetic parameters (T(1/2)α, T(1/2)ß, AUC and Cl) derived from microPET images revealed prolonged systemic circulation of (18)F-FBHGal in hepatic fibrosis mice compared to that in normal mice. The findings in biological characterizations suggest that (18)F-FBHGal is a feasible agent for PET imaging of hepatic fibrosis in mice and may provide new insights into ASGPR-related liver dysfunction.

Radiometal-Labeled Chitosan Microspheres as Transarterial Radioembolization Agents against Hepatocellular Carcinoma.

Transarterial radioembolization (TARE) is an emerging treatment for patients with unresectable hepatocellular carcinoma (HCC). This study successfully developed radiometal-labeled chitosan microspheres (111In/177Lu-DTPA-CMS) with a diameter of 36.5 ± 5.3 µm for TARE. The radiochemical yields of 111In/177Lu-DTPA-CMS were greater than 90% with high radiochemical purities (>98%). Most of the 111In/177Lu-DTPA-CMS were retained in the hepatoma and liver at 1 h after intraarterial (i.a.) administration. Except for liver accumulation, radioactivity in each normal organ was less than 1% of the injected radioactivity (%IA) at 72 h after injection. At 10 days after injection of 177Lu-DTPA-CMS (18.6 ± 1.3 MBq), the size of the hepatoma was significantly reduced by around 81%, while that of the rats in the control group continued to grow. This study demonstrated the effectiveness of 177Lu-DTPA-CMS in the treatment of N1-S1 hepatoma. 111In/177Lu-DTPA-CMS have the potential to be a superior theranostic pair for the treatment of clinical hepatoma.