Engineered Cells as a Test Platform for Radiohaptens in Pretargeted Imaging and Radioimmunotherapy Applications.

Pretargeted imaging and radioimmunotherapy approaches are designed to have superior targeting properties over directly targeted antibodies but impose more complex pharmacology, which hinders efforts to optimize the ligands prior to human applications. Human embryonic kidney 293T cells expressing the humanized single-chain variable fragment (scFv) C825 (huC825) with high-affinity for DOTA-haptens (293T-huC825) in a transmembrane-anchored format eliminated the requirement to use other pretargeting reagents and provided a simplified, accelerated assay of radiohapten capture while offering normalized cell surface expression of the molecular target of interest. Using binding assays, ex vivo biodistribution, and in vivo imaging, we demonstrated that radiohaptens based on benzyl-DOTA and a second generation "Proteus" DOTA-platform effectively and specifically engaged membrane-bound huC825, achieving favorable tumor-to-normal tissue uptake ratios in mice. Furthermore, [86Y]Y-DOTA-Bn predicted absorbed dose to critical organs with reasonable accuracy for both [177Lu]Lu-DOTA-Bn and [225Ac]Ac-Pr, which highlights the benefit of a dosimetry-based treatment approach.

An N-Acetylgalactosamino Dendron-Clearing Agent for High-Therapeutic-Index DOTA-Hapten Pretargeted Radioimmunotherapy.

Clearing agents (CAs) can rapidly remove nonlocalized targeting biomolecules from circulation for hepatic catabolism, thereby enhancing the therapeutic index (TI), especially for blood (marrow), of the subsequently administered radioisotope in any multistep pretargeting strategy. Herein we describe the synthesis and in vivo evaluation of a fully synthetic glycodendrimer-based CA for DOTA-based pretargeted radioimmunotherapy (DOTA-PRIT). The novel dendron-CA consists of a nonradioactive yttrium-DOTA-Bn molecule attached via a linker to a glycodendron displaying 16 terminal α-thio-N-acetylgalactosamine (α-SGalNAc) units (CCA α-16-DOTA-Y3+; molecular weight: 9059 Da). Pretargeting [177Lu]LuDOTA-Bn with CCA α-16-DOTA-Y3+ to GPA33-expressing SW1222 human colorectal xenografts was highly effective, leading to absorbed doses of [177Lu]LuDOTA-Bn for blood, tumor, liver, spleen, and kidneys of 11.7, 468, 9.97, 5.49, and 13.3 cGy/MBq, respectively. Tumor-to-normal tissues absorbed-dose ratios (i.e., TIs) ranged from 40 (e.g., for blood and kidney) to about 550 for stomach.

Evaluation of glycodendron and synthetically modified dextran clearing agents for multistep targeting of radioisotopes for molecular imaging and radioimmunotherapy.

A series of N-acetylgalactosamine-dendrons (NAG-dendrons) and dextrans bearing biotin moieties were compared for their ability to complex with and sequester circulating bispecific antitumor antibody streptavidin fusion protein (scFv4-SA) in vivo, to improve tumor-to-normal tissue concentration ratios for multistep targeted (MST) radioimmunotherapy and diagnosis. Specifically, a total of five NAG-dendrons employing a common synthetic scaffold structure containing 4, 8, 16, or 32 carbohydrate residues and a single biotin moiety were prepared (NAGB), and for comparative purposes, a biotinylated-dextran with an average molecular weight of 500 kD was synthesized from amino-dextran (DEXB). One of the NAGB compounds, CA16, has been investigated in humans; our aim was to determine if other NAGB analogues (e.g., CA8 or CA4) were bioequivalent to CA16 and/or better suited as MST reagents. In vivo studies included dynamic positron-emission tomography (PET) imaging of (124)I-labeled-scFv4-SA clearance and dual-label biodistribution studies following MST directed at subcutaneous (s.c.) human colon adenocarcinoma xenografts in mice. The MST protocol consists of three injections: first, a scFv4-SA specific for an antitumor-associated glycoprotein (TAG-72); second, CA16 or other clearing agent; and third, radiolabeled biotin. We observed using PET imaging of the (124)I-labeled-scFv4-SA clearance that the spatial arrangement of ligands conjugated to NAG (i.e., biotin linked with an extended spacer, referred to herein as long-chain (LC)) can impact the binding to the antibody in circulation and subsequent liver uptake of the NAG-antibody complex. Also, NAGB CA32-LC or CA16-LC can be utilized during MST to achieve comparable tumor-to-blood ratios and absolute tumor uptake seen previously with CA16. Finally, DEXB was equally effective as NAGB CA32-LC at lowering scFv4-SA in circulation, but at the expense of reducing absolute tumor uptake of radiolabeled biotin.

Microfabricating Mirror-like Surface Precision Micro-Sized Amorphous Alloy Structures Using Jet-ECM Process.

Amorphous alloy (AA) is a high-performance metal material generally with significantly excellent mechanical and corrosion resistance properties and thus is considered as a desirable material selection for micro-scale articles. However, the microfabrication of AA still faces a variety of technical challenges mainly because the materials are too hard to process and easily lose their original properties, although at moderately high temperatures. In this study, jet-electrolyte electrochemical machining (Jet-ECM) was proposed to microfabricate the Zr-based AA because it is a low-temperature material-removal process based on the anode dissolution mechanism. The electrochemical dissolution characteristics and material removal mechanism of AA were investigated, and then the optimal process parameters were achieved based on the evaluation of the surface morphologies, surface roughness, geometrical profile, and machining accuracy of the machined micro-dimples. Finally, the feasibility was further studied by using Jet-ECM to fabricate arrayed micro-dimples using the optimized parameters. It was found that Jet-ECM can successfully microfabricate mirror-like surface AA arrayed precision micro-dimples with significantly high dimensional accuracy and geometrical consistency. Jet-ECM is a promisingly advantageous microfabrication process for the hard-to-machine AA.

Association between left ventricular remodeling and lipid profiles in obese children: an observational study.

Objective: Childhood obesity has become a prominent issue in the society, which can lead to left ventricular remodeling and severe cardiovascular complications in adulthood. It is beneficial to identify the causes of left ventricular remodeling so that targeted measures can be taken to prevent the cardiovascular disease. Therefore, this study aimed to explore the relationship between left ventricular remodeling and changes in blood lipid indexes in obese children. Methods: This study was conducted on 40 healthy non-obese children and 140 obese children diagnosed in the pediatric health department of our hospital. Clinical data collected from the two groups were compared. Echocardiography was performed to examine left ventricular configuration and cardiac function. Multiple linear regression analysis was conducted to assess the independent effects of blood lipid levels on echocardiographic parameters. Blood lipid indicators among different left ventricular structural patterns which were classified according to left ventricular mass indexes and relative wall thickness were compared. Results: Obese children exhibited significantly increased height, weight, body mass index (BMI), body fat percentage (BFP), blood pressure, triglycerides, total cholesterol, left ventricular internal diameter (LVIDd), interventricular septum (IVSd), left ventricular posterior wall diastolic thickness (LVPWd), myocardial mass (LVM) and relative wall thickness (RWT), as well as lower high-density lipoprotein cholesterol (HDL-C) and left ventricular ejection fraction (LVEF) compared to the non-obese children (P < 0.05). Multiple linear correlation analysis showed LVM had a significantly positive correlation with BMI (r = 3.21, P = 0.002) and SBP (r = 2.61, P = 0.01); LVMI had a significantly negative correlation with HDL-C (r = -2.45, P = 0.015); RWT had a significantly positive correlation with SBP (r = 2.50, P = 0.013) but a significantly negative correlation with HDL-C (r = -2.35, P = 0.02). Furthermore, there were significant differences in HDL-C values among children with different ventricular configurations (P < 0.05), with the lowest HDL-C value recorded in the concentric hypertrophy group. Conclusion: Obese children will develop left ventricular remodeling. The left ventricular configuration indexes are most significantly associated with serum HDL-C. Lower HDL-C level contributes to severer left ventricular hypertrophy, indicating a concentric hypertrophy pattern.

Fabricating Precise and Smooth Microgroove Structures on Zr-Based Metallic Glass Using Jet-ECM.

Zr-based metallic glasses (MGs) are promising materials for mold manufacturing due to their unique mechanical and chemical properties. However, the high hardness of metallic glasses and their tendency to crystallize at high temperatures make it challenging to fabricate precise and smooth microscale structures on metallic glasses. This limitation hampers the development of metallic glasses as molds. Jet electrochemical machining (jet-ECM) is a non-contact subtractive manufacturing technology that utilizes a high-speed electrolyte to partially remove material from workpieces, making it highly suitable for processing difficult-to-machine materials. Nevertheless, few studies have explored microgroove structures on Zr-based MGs using sodium nitrate electrolytes by jet-ECM. Therefore, this paper advocates the utilization of the jet-ECM technique to fabricate precise and smooth microgroove structures using a sodium nitrate electrolyte. The electrochemical characteristics were studied in sodium nitrate solution. Then, the effects of the applied voltages and nozzle travel rates on machining performance were investigated. Finally, micro-helical and micro-S structures with high geometric dimensional consistency and low surface roughness were successfully fabricated, with widths and depths measuring 433.7 ± 2.4 µm and 101.4 ± 1.6 µm, respectively. Their surface roughness was determined to be 0.118 ± 0.002 µm. Compared to non-aqueous-based methods for jet-ECM of Zr-based MGs, the depth of the microgrooves was increased from 20 µm to 101 µm. Furthermore, the processed microstructures had no uneven edges in the peripheral areas and no visible flow marks on the bottom.

An Investigation of the Efficient-Precise Continuous Electrochemical Grinding Process of Ti-6Al-4V.

Titanium alloys have many excellent characteristics, and they are widely used in aerospace, biomedicine, and precision engineering. Meanwhile, titanium alloys are difficult to machine and passivate readily. Electrochemical grinding (ECG) is an ideal technology for the efficient-precise machining of titanium alloys. In the ECG process of titanium alloys, the common approach of applying high voltage and active electrolytes to achieve high efficiency of material removal will lead to serious stray corrosion, and the time utilized for the subsequent finishing will be extended greatly. Therefore, the application of ECG in the field of high efficiency and precision machining of titanium alloys is limited. In order to address the aforementioned issues, the present study proposed an efficient-precise continuous ECG (E-P-C-ECG) process for Ti-6Al-4V applying high-pulsed voltage with an optimized duty cycle and low DC voltage in the efficient ECG stage and precise ECG stage, respectively, without changing the grinding wheel. According to the result of the passivation properties tests, the ideal electrolyte was selected. Optimization of the process parameters was implemented experimentally to improve the processing efficiency and precision of ECG of Ti-6Al-4V. Utilizing the process advantages of the proposed process, a thin-walled structure of Ti-6Al-4V was obtained with high efficiency and precision. Compared to the conventional mechanical grinding process, the compressive residual stress of the machined surface and the processing time were reduced by 90.5% and 63.3% respectively, and both the surface roughness and tool wear were obviously improved.

N-acetylgalactosamino dendrons as clearing agents to enhance liver targeting of model antibody-fusion protein.

Dendrimer clearing agents represent a unique class of compounds for use in multistep targeting (MST) in radioimmunotherapy and imaging. These compounds were developed to facilitate the removal of excess tumor-targeting monoclonal antibody (mAb) prior to administration of the radionuclide to minimize exposure of normal tissue to radiation. Clearing agents are designed to capture the circulating mAb, and target it to the liver for metabolism. Glycodendrons are ideally suited for MST applications as these highly branched compounds are chemically well-defined, thus advantageous over heterogeneous macromolecules. Previous studies have described glycodendron 3 as a clearing agent for use in three-step MST protocols, and early in vivo assessment of 3 showed promise. However, synthetic challenges have hampered its availability for further development. In this report we describe a new sequence of chemical steps which enables the straightforward synthesis and analytical characterization of this class of dendrons. With accessibility and analytical identification solved, we sought to evaluate both lower and higher generation dendrons for hepatocyte targeting as well as clearance of a model protein. We prepared a series of clearing agents where a single biotin is connected to glycodendrons displaying four, eight, sixteen or thirty-two α-thio-N-acetylgalactosamine (α-SGalNAc) units, resulting in compounds with molecular weights ranging from 2 to 17 kDa, respectively. These compounds were fully characterized by LCMS and NMR. We then evaluated the capacity of these agents to clear a model (131)I-labeled single chain variable fragment antibody-streptavidin ((131)I-scFv-SAv) fusion protein from blood and tissue in mice, and compared their clearing efficiencies to that of a 500 kDa dextran-biotin conjugate. Glycodendrons and dextran-biotin exhibited enhanced blood clearance of the scFv-SAv construct. Biodistribution analysis showed liver targeting/uptake of the scFv-SAv construct to be 2-fold higher for compounds 1 to 4, as well as for the 500 kDa dextran, over saline. Additionally, the data suggest the glycodendrons clear through the liver, whereas the dextran through reticuloendothelial system (RES) metabolism.

Variations of Habitat Quality and Ecological Risk and Their Correlations with Landscape Metrics in a Robust Human Disturbed Coastal Region-Case Study: Xinggang Town in Southern China.

This paper explores how landscape risk and habitat quality vary in coastal areas with strong anthropogenic disturbance based on a case study. We analyze the temporal-spatial variations of habitat quality and ecological risk in the coastal region by adopting the methods of theInVEST model and the ecological risk index. The correlations of habitat quality and ecological risk with landscape metrics are subsequently quantified. The results indicated the presence of obvious distance gradients in relation to the deterioration of habitat quality and the increase in ecological risk. Moreover, the gradient area close to the coastline exhibits significant habitat quality and ecological risk changes. The majority of landscape metrics show positive correlations with habitat quality and ecological risk, and these correlations vary with the distance gradients. Since the rapid urbanization of the coastal region, the marked expansion of built-up land and decrease in natural landscapes has significantly impacted the landscape pattern index and, consequently, changed the habitat quality and ecological risk level.

Efficacy of HER2-Targeted Intraperitoneal 225Ac α-Pretargeted Radioimmunotherapy for Small-Volume Ovarian Peritoneal Carcinomatosis.

Epithelial ovarian cancer (EOC) is often asymptomatic and presents clinically in an advanced stage as widespread peritoneal microscopic disease that is generally considered to be surgically incurable. Targeted α-therapy with the α-particle-emitting radionuclide 225Ac (half-life, 9.92 d) is a high-linear-energy-transfer treatment approach effective for small-volume disease and even single cells. Here, we report the use of human epidermal growth factor receptor 2 (HER2) 225Ac-pretargeted radioimmunotherapy (PRIT) to treat a mouse model of human EOC SKOV3 xenografts growing as peritoneal carcinomatosis (PC). Methods: On day 0, 105 SKOV3 cells transduced with a luciferase reporter gene were implanted intraperitoneally in nude mice, and tumor engraftment was verified by bioluminescent imaging (BLI). On day 15, treatment was started using 1 or 2 cycles of 3-step anti-HER2 225Ac-PRIT (37 kBq/cycle as 225Ac-Proteus DOTA), separated by a 1-wk interval. Efficacy and toxicity were monitored for up to 154 d. Results: Untreated PC-tumor-bearing nude mice showed a median survival of 112 d. We used 2 independent measures of response to evaluate the efficacy of 225Ac-PRIT. First, a greater proportion of the treated mice (9/10 1-cycle and 8/10 2-cycle; total, 17/20; 85%) survived long-term compared with controls (9/27, 33%), and significantly prolonged survival was documented (log-rank [Mantel-Cox] P = 0.0042). Second, using BLI, a significant difference in the integrated BLI signal area to 98 d was noted between controls and treated groups (P = 0.0354). Of a total of 8 mice from the 2-cycle treatment group (74 kBq total) that were evaluated by necropsy, kidney radiotoxicity was mild and did not manifest itself clinically (normal serum blood urea nitrogen and creatinine). Dosimetry estimates (relative biological effectiveness-weighted dose, where relative biological effectiveness = 5) per 37 kBq administered for tumors and kidneys were 56.9 and 16.1 Gy, respectively. One-cycle and 2-cycle treatments were equally effective. With immunohistology, mild tubular changes attributable to α-toxicity were observed in both therapeutic groups. Conclusion: Treatment of EOC PC-tumor-bearing mice with anti-HER2 225Ac-PRIT resulted in histologic cures and prolonged survival with minimal toxicity. Targeted α-therapy using the anti-HER2 225Ac-PRIT system is a potential treatment for otherwise incurable EOC.

Genome-Wide Characterization and Anthocyanin-Related Expression Analysis of the B-BOX Gene Family in Capsicum annuum L.

The transcription factors, B-box (BBX), belong to a subfamily of the zinc finger family of proteins and exhibit multiple biological functions in plant growth, development, and abiotic stress response pathways. In this study, a total of 23 CaBBX members were identified using the pepper reference genome database. According to the gene structure, conserved domains, and the phylogenetic tree, 23 CaBBX genes were divided into four groups, wherein the analysis of the promoter region indicated the presence of cis-acting elements related to plant development, hormones, and stress response. Interspecies collinearity analysis showed that the CaBBXs had three duplicated gene pairs, and the highest gene density was found on chromosomes 2 and 7. Transcriptome RNA-seq data and quantitative polymerase chain reaction (qRT-PCR) analysis of pepper plants spanning the entire period showed that more than half of the CaBBX genes were widely expressed in diversity tissues of pepper. Co-expression network analysis indicated that the CaBBXs and the anthocyanin structural genes had a close co-expression relationship. Thus, it was reasonably speculated that the CaBBX genes may be involved in the regulation of anthocyanin biosynthesis. Overall, this study involved the genome-wide characterization of the CaBBX family and may serve as a solid foundation for further investigations on CaBBX genes involved in the anthocyanin synthesis mechanisms and development in pepper.

Analysis of the response regulatory network of pepper genes under hydrogen peroxide stress.

Hydrogen peroxide (H2O2) is a regulatory component related to plant signal transduction. To better understand the genome-wide gene expression response to H2O2 stress in pepper plants, a regulatory network of H2O2 stress-gene expression in pepper leaves and roots was constructed in the present study. We collected the normal tissues of leaves and roots of pepper plants after 40 days of H2O2 treatment and obtained the RNA-seq data of leaves and roots exposed to H2O2 for 0.5-24 h. By comparing the gene responses of pepper leaves and roots exposed to H2O2 stress for different time periods, we found that the response in roots reached the peak at 3 h, whereas the response in leaves reached the peak at 24 h after treatment, and the response degree in the roots was higher than that in the leaves. We used all datasets for K-means analysis and network analysis identified the clusters related to stress response and related genes. In addition, CaEBS1, CaRAP2, and CabHLH029 were identified through a co-expression analysis and were found to be strongly related to several reactive oxygen species-scavenging enzyme genes; their homologous genes in Arabidopsis showed important functions in response to hypoxia or iron uptake. This study provides a theoretical basis for determining the dynamic response process of pepper plants to H2O2 stress in leaves and roots, as well as for determining the critical time and the molecular mechanism of H2O2 stress response in leaves and roots. The candidate transcription factors identified in this study can be used as a reference for further experimental verification.

Radiosynthesis of the tumor hypoxia marker [18F]TFMISO via O-[18F]trifluoroethylation reveals a striking difference between trifluoroethyl tosylate and iodide in regiochemical reactivity toward oxygen nucleophiles.

The MRI hypoxia marker trifluoromisonidazole (TFMISO) [1-(2-nitro-1H-imidazol-1-yl)-3-(2,2,2-trifluoroethoxy)propan-2-ol] was successfully labeled with (18)F to expand its role into a bimodal PET/MRI probe. (18)F-Labeling was achieved via a three-step procedure in which 2,2,2-[(18)F]trifluoroethyl p-toluenesulfonate prepared by (18)F-(19)F exchange served as the [(18)F]trifluoroethylating agent. The O-[(18)F]trifluoroethylation reaction proceeded efficiently to give the intermediate 1,2-epoxy-3-(2,2,2-[(18)F]trifluoroethoxy)propane, with approximately 60% of (18)F incorporated from the tosylate precursor, which was condensed with 2-nitroimidazole to yield [(18)F]TFMISO. Approximately 40% of the [(18)F]trifluoroethyl tosylate precursor was converted into the final product. In stark contrast, 2,2,2-[(18)F]trifluoroethyl iodide failed to produce [(18)F]TFMISO, giving instead 1,1-[(18)F]difluoro-2-iodoethoxy and 1-[(18)F]fluoro-2-iodovinyloxy analogs of [(18)F]TFMISO. Thus, this investigation has identified 2,2,2-[(18)F]trifluoroethyl tosylate as an excellent [(18)F]trifluoroethylating agent, which can convert efficiently an alcohol into the corresponding [(18)F]trifluoroethyl ether.

A Self-Assembling and Disassembling (SADA) Bispecific Antibody (BsAb) Platform for Curative Two-step Pretargeted Radioimmunotherapy.

PURPOSE: Many cancer treatments suffer from dose-limiting toxicities to vital organs due to poor therapeutic indices. To overcome these challenges we developed a novel multimerization platform that rapidly removes tumor-targeting proteins from the blood to substantially improve therapeutic index. EXPERIMENTAL DESIGN: The platform was designed as a fusion of a self-assembling and disassembling (SADA) domain to a tandem single-chain bispecific antibody (BsAb, anti-ganglioside GD2 × anti-DOTA). SADA-BsAbs were assessed with multiple in vivo tumor models using two-step pretargeted radioimmunotherapy (PRIT) to evaluate tumor uptake, dosimetry, and antitumor responses. RESULTS: SADA-BsAbs self-assembled into stable tetramers (220 kDa), but could also disassemble into dimers or monomers (55 kDa) that rapidly cleared via renal filtration and substantially reduced immunogenicity in mice. When used with rapidly clearing DOTA-caged PET isotopes, SADA-BsAbs demonstrated accurate tumor localization, dosimetry, and improved imaging contrast by PET/CT. When combined with therapeutic isotopes, two-step SADA-PRIT safely delivered massive doses of alpha-emitting (225Ac, 1.48 MBq/kg) or beta-emitting (177Lu, 6,660 MBq/kg) S-2-(4-aminobenzyl)-1,4,7,10-tetraazacyclododecane tetraacetic acid (DOTA) payloads to tumors, ablating them without any short-term or long-term toxicities to the bone marrow, kidneys, or liver. CONCLUSIONS: The SADA-BsAb platform safely delivered large doses of radioisotopes to tumors and demonstrated no toxicities to the bone marrow, kidneys, or liver. Because of its modularity, SADA-BsAbs can be easily adapted to most tumor antigens, tumor types, or drug delivery approaches to improve therapeutic index and maximize the delivered dose.See related commentary by Capala and Kunos, p. 377.

Synthesis of the PET Tracer 124I-Trametinib for MAPK/ERK Kinase Distribution and Resistance Monitoring.

Trametinib is an extremely potent allosteric inhibitor of mitogen-activated protein kinase (MAPK)/extracellular-signal-regulated kinase (ERK) (MEK) 1/2, which has been approved for treatment of metastatic melanoma and anaplastic thyroid cancer in patients with confirmed BRAFV600E/K mutations. Though trametinib is highly efficacious, adverse side effects, including skin, gastrointestinal, and hepatic toxicity, are dose-limiting and can lead to treatment termination. Development of a noninvasive tool to visualize and quantify the delivery and distribution of trametinib (either as a single agent or in combination with other therapeutics) to tumors and organs would be helpful in assessing therapeutic index, personalizing individual dose, and potentially predicting resistance to therapy. Methods: To address these issues, we have developed a radiolabeled trametinib and evaluated the in vitro and in vivo properties. 123I-, 124I-, and 131I-trametinib, pure tracer analogs to trametinib, were synthesized in more than 95% purity, with an average yield of 69.7% and more than 100 GBq/µmol specific activity. Results: Overall, 124I-trametinib uptake in a panel of cancer cell lines can be blocked with cold trametinib, confirming specificity of the radiotracer in vitro and in vivo. 124I-trametinib was taken up at higher rates in KRAS and BRAF mutant cell lines than in wild-type KRAS cancer cell lines. In vivo, biodistribution revealed high uptake in the liver 2 h after injection, followed by clearance through the gastrointestinal tract over 4 d. Importantly, uptake higher than expected was observed in the lung and heart for up to 24 h. Peak uptake in the skin and gastrointestinal tract was observed between 6 and 24 h, whereas in B16F10 melanoma-bearing mice peak tumor concentrations were achieved between 24 and 48 h. Tumor uptake relative to muscle and skin was relatively low, peaking at 3.4- to 8.1-fold by 72 h, respectively. The biodistribution of 124I-trametinib was significantly reduced in mice on trametinib therapy, providing a quantitative method to observe MEK inhibition in vivo. Conclusion:124I-trametinib serves as an in vivo tool to personalize the dose instead of using the current single-fixed-dose scheme and, when combined with radiomic data, to monitor the emergence of therapy resistance. In addition, the production of iodinated trametinib affords researchers the ability to measure drug distribution for improved drug delivery studies.

Alpha radioimmunotherapy using 225Ac-proteus-DOTA for solid tumors - safety at curative doses.

This is the initial report of an α-based pre-targeted radioimmunotherapy (PRIT) using 225Ac and its theranostic pair, 111In. We call our novel tumor-targeting DOTA-hapten PRIT system "proteus-DOTA" or "Pr." Herein we report the first results of radiochemistry development, radiopharmacology, and stoichiometry of tumor antigen binding, including the role of specific activity, anti-tumor efficacy, and normal tissue toxicity with the Pr-PRIT approach (as α-DOTA-PRIT). A series of α-DOTA-PRIT therapy studies were performed in three solid human cancer xenograft models of colorectal cancer (GPA33), breast cancer (HER2), and neuroblastoma (GD2), including evaluation of chronic toxicity at ~20 weeks of select survivors. Methods: Preliminary biodistribution experiments in SW1222 tumor-bearing mice revealed that 225Ac could not be efficiently pretargeted with current DOTA-Bn hapten utilized for 177Lu or 90Y, leading to poor tumor uptake in vivo. Therefore, we synthesized Pr consisting of an empty DOTA-chelate for 225Ac, tethered via a short polyethylene glycol linker to a lutetium-complexed DOTA for picomolar anti-DOTA chelate single-chain variable fragment (scFv) binding. Pr was radiolabeled with 225Ac and its imaging surrogate, 111In. In vitro studies verified anti-DOTA scFv recognition of [225Ac]Pr, and in vivo biodistribution and clearance studies were performed to evaluate hapten suitability and in vivo targeting efficiency. Results: Intravenously (i.v.) administered 225Ac- or 111In-radiolabeled Pr in mice showed rapid renal clearance and minimal normal tissue retention. In vivo pretargeting studies show high tumor accumulation of Pr (16.71 ± 5.11 %IA/g or 13.19 ± 3.88 %IA/g at 24 h p.i. for [225Ac]Pr and [111In]Pr, respectively) and relatively low uptake in normal tissues (all average ≤ 1.4 %IA/g at 24 h p.i.). Maximum tolerated dose (MTD) was not reached for either [225Ac]Pr alone or pretargeted [225Ac]Pr at administered activities up to 296 kBq/mouse. Single-cycle treatment consisting of α-DOTA-PRIT with either huA33-C825 bispecific anti-tumor/anti-DOTA-hapten antibody (BsAb), anti-HER2-C825 BsAb, or hu3F8-C825 BsAb for targeting GPA33, HER2, or GD2, respectively, was highly effective. In the GPA33 model, no complete responses (CRs) were observed but prolonged overall survival of treated animals was 42 d for α-DOTA-PRIT vs. 25 d for [225Ac]Pr only (P < 0.0001); for GD2, CRs (7/7, 100%) and histologic cures (4/7, 57%); and for HER2, CRs (7/19, 37%) and histologic cures (10/19, 56%) with no acute or chronic toxicity. Conclusions: [225Ac]Pr and its imaging biomarker [111In]Pr demonstrate optimal radiopharmacologic behavior for theranostic applications of α-DOTA-PRIT. For this initial evaluation of efficacy and toxicity, single-cycle treatment regimens were performed in all three systems. Histologic toxicity was not observed, so MTD was not observed. Prolonged overall survival, CRs, and histologic cures were observed in treated animals. In comparison to RIT with anti-tumor IgG antibodies, [225Ac]Pr has a much improved safety profile. Ultimately, these data will be used to guide clinical development of toxicity and efficacy studies of [225Ac]Pr, with the goal of delivering massive lethal doses of radiation to achieve a high probability of cure without toxicity.

From synthesis to biologics: preclinical data on a chemistry derived anticancer vaccine.

A fully synthetic anticancer vaccine 2 has been prepared via bioconjugation of unimolecular pentavalent construct 1-containing five prostate and breast cancer associated carbohydrate antigens, Globo-H, GM2, STn, TF and Tn-to maleimide-modified carrier protein KLH. An improved conjugation protocol has been developed, which allowed us to obtain a higher epitope ratio of the unimolecular pentavalent glycopeptide antigen to the carrier protein (505/1 versus 228/1 for the previous version). KLH conjugate 2 has been subsequently submitted to preclinical immunogenic evaluation in mice in the presence of QS-21 as an adjuvant. Through standard ELISA assay, this vaccine candidate showed high promise in inducing IgG and IgM antibodies against each of the five individual carbohydrate antigens. In addition, FACS analysis indicated that these antibodies were able to react with MCF-7 breast cancer cell lines expressing these five carbohydrate antigens.

Synthesis of Human Cancer Associated Globo-H (MBr1) Glycosylamino Acid: Some Mechanistic and Conformational Reinvestigations.

The synthesis of an extended globo-H (MBr1 antigen) in the form of a glycosylamino acid is reported. By careful NMR analysis, we found an interesting conformational "flip" on the E ring of some synthetic intermediates. An explanation offered for the successful [3+3] coupling of ABC acceptor 11 and DEF donor 10 possessing a C4 free hydroxyl to produce ß-galactoside in azaglycosidations is reinforced.

[Efficacy and adverse effects of film coated praziquantel for treatment of schistosomiasis japonica].

OBJECTIVE: To understand the adverse and curative effects of film coated praziquantel for treatment of schistosomiasis japonica. METHODS: A questionnaire survey was conducted among 6 to 65 years old inhabitants from epidemic areas of schistosomiasis japonica in Jiangxi, Anhui and Hubei provinces, while indirect haemagglutination assay (IHA) was also administrated. The Serological positives were tested by Kato-Katz technique. A total of 509 habitats of serological positives and healthy persons were given praziquantel. 460 targets with no relative symptoms were followed up for adverse effect of drugs in one month and efficacy evaluation of praziquantel was carried out in 104 parasitological positives 3 months post-treatment. RESULTS: The 84.7% (144/170) individuals thought film coated tablets were no smell or only with little smell while 92.9%(315/339) targets in control group fell that tablets smelled bad. The total side effect rates of film coated praziquantel group and control group in 1-2 hours post-treatment were 20.30% (27/133) and 83.18% (272/327) respectively. There was significantly different between two groups (chi(2) = 164.316, P < 0.05). The adverse effect rates of film coated praziquantel group in neuromuscular system, digestive system and cardiovascular system were 15.79% (21/133), 9.77% (13/133), 2.26% (3/133) individually which were significantly lower than those of control groups with 81.65% (267/327), 49.24% (161/327), 12.84% (42/327) in corresponding systems (chi(2)(neuromuscular system) = 175.188, chi(2)(digestive system) = 62.601, chi(2)(cardiovascular system) = 12.010, P < 0.05). And the adverse effect rate of allergic reaction of film coated group was no significant difference with control group (2.26%(3/133) versus 0.92% (3/327), chi(2) = 1.315, P = 0.235). One day after treatment, the adverse effect rate of film coated tablets of praziquantel decreased to 3.01% (4/133), significantly lower than that of control group (P < 0.05). There was no significant difference after 2 weeks treatment while the adverse effect rates decreased to 0.75%(1/133), 0.61% (2/327) in film coated praziquantel group and control group respectively (chi(2) = 0.029, P = 0.642). The curative rates 3 months post treatment were 84.91% (45/51) in film coated tablets of praziquantel group and 82.35% (42/53) in control group without significant difference (chi(2) = 1.536, P = 0.215). CONCLUSION: The smell and adverse effects of film coated tablets of praziquantel were decreased significantly and its efficacy for treatment of schistosomiasis japonica was equal to the control tablets. The film coated tablets of praziquantel might be applied in field widely after a further verification.