Metastatic and sentinel lymph node mapping using intravenously delivered Panitumumab-IRDye800CW.

Rationale: Sentinel lymph node biopsy (SLNB) is a well-established minimally invasive staging procedure that maps the spread of tumour metastases from their primary site to the regional lymphatics. Currently, the procedure requires the local peri-tumoural injection of radiolabelled and/or optical agents, and is therefore operator dependent, disruptive to surgical workflow and restricted largely to a small subset of malignancies that can be readily accessed externally for local tracer injection. The present study set out to determine whether intravenous (IV) infusion of a tumor-targeted tracer could identify sentinel and metastatic lymph nodes (LNs) in order to overcome these limitations. Methods: We examined 27 patients with oral squamous cell carcinoma (OSCC), 18 of whom were clinically node negative (cN0). Patients were infused intravenously with 50mg of Panitumumab-IRDye800CW prior to surgical resection of their primary tumour with neck dissection and/or SLNB. Lymphadenectomy specimens underwent fluorescence molecular imaging to evaluate tracer distribution to LNs. Results: A total of 960 LNs were analysed, of which 34 (3.5%) contained metastatic disease. Panitumumab-IRDye800CW preferentially localized to metastatic and sentinel LNs as evidenced by a higher fluorescent signal relative to other lymph nodes. The median MFI of metastatic LNs was significantly higher than the median MFI of benign LNs (0.06 versus 0.02, p < 0.05). Furthermore, selecting the highest five fluorescence intensity LNs from individual specimens resulted in 100% sensitivity, 85.8% specificity and 100% negative predictive value (NPV) for the detection of occult metastases and 100% accuracy for clinically staging the neck. In the cN+ cohort, assessment of the highest 5 fluorescence LNs per patient had 87.5% sensitivity, 93.2% specificity and 99.1% NPV for the detection of metastatic nodes. Conclusion: When intravenously infused, a tumour-targeted tracer localized to sentinel and metastatic lymph nodes. Further validation of an IV tumor-targeted tracer delivery approach for SLNB could dramatically change the practice of SLNB, allowing its application to other malignancies where the primary tumour is not accessible for local tracer injection.

Modulation of immune functions, inflammatory response, and cytokine production following long-term oral exposure to three food additives; thiabendazole, monosodium glutamate, and brilliant blue in rats.

The food additives thiabendazole (TBZ), monosodium glutamate (MSG), and brilliant blue (BB) are commonly used in many daily-consumed food products worldwide. They are widely used in major agricultural and industrial applications. Yet, many of its toxicological aspects are still unclear, especially immune modulation. This research was therefore intended to investigate the effects of male Wistar rats' daily oral exposure for 90 days to TBZ (10 mg/kg b.wt), MSG (20 mg/kg b.wt), or BB (1.2 mg/kg b.wt) on the blood cells, immunity, and inflammatory indicators. The three tested food additives showed varying degrees of hematological alterations. Initially, megaloblastic anemia and thrombocytopenia were evident with the three tested food additives. At the same time, TBZ showed no significant changes in the leukogram element except eosinopenia. MSG induced leukopenia, lymphocytopenia, neutrophilia, and eosinophilia. BB evoked neutrophilia and lymphopenia. The immunoglobins M (IgM) and IgG were significantly reduced with the three tested food additives. In contrast, lysozyme and nitric oxide levels were elevated. A reduced considerably lymphocyte proliferation was detected with TBZ and MSG exposure without affecting the phagocytic activity. Various pathologic disturbances in splenic tissues have been detected. An obvious increase in CD4+ but a lessening in CD8+ immunolabeling was evident in TBZ and MSG groups. The cytokines, including interferon-gamma, tumor necrosis factor-alpha, and interleukin 1ß, 6, 10, and 13, were significantly upregulated in the spleen of rats exposed to TBZ, MSG, and BB. These results concluded that TBZ, MSG, and BB negatively affect hematological parameters, innate and humoral immune functions together with inflammatory responses. TBZ achieved the maximal negative impacts followed by MSG and finally with BB. Given the prevalence of these food additives, TBZ and MSG should be limited to a minimal volume use, or natural food additives should be used instead.

Feed-forward activation of STAT3 signaling limits the efficacy of c-Met inhibitors in esophageal squamous cell carcinoma (ESCC) treatment.

c-Hepatocyte growth factor receptor (Met) inhibitors have demonstrated clinical benefits in some types of solid tumors. However, the efficacy of c-Met inhibitors in esophageal squamous cell carcinoma (ESCC) remains unclear. In this study, we discovered that c-Met inhibitors induced "Signal Transducer and Activator of Transcription (STAT3)-addiction" in ESCC cells, and the feedback activation of STAT3 in ESCC cells limits the tumor response to c-Met inhibition. Mechanistically, c-Met inhibition increased the autocrine of several cytokines, including CCL2, interleukin 8, or leukemia inhibitory factor, and facilitated the interactions between the receptors of these cytokines and Janus Kinase1/2 (JAK1/2) to resultantly activate JAKs/STAT3 signaling. Pharmacological inhibition of c-Met together with cytokines/JAKs/STAT3 axis enhanced cancer cells regression in vitro. Importantly, combined c-Met and STAT3 inhibitors synergistically suppressed tumor growth and promoted the apoptosis of tumor cells without producing systematic toxicity. These findings suggest that inhibition of the STAT3 feedback loop may augment the response to c-Met inhibitors via the STAT3-mediated oncogene addiction in ESCC cells.

Pre-clinical study of IRDye800CW-nimotuzumab formulation, stability, pharmacokinetics, and safety.

BACKGROUND: Epidermal growth factor receptor (EGFR) is a target for cancer therapy as it is overexpressed in a wide variety of cancers. Therapeutic antibodies that bind EGFR are being evaluated in clinical trials as imaging agents for positron emission tomography and image-guided surgery. However, some of these antibodies have safety concerns such as infusion reactions, limiting their use in imaging applications. Nimotuzumab is a therapeutic monoclonal antibody that is specific for EGFR and has been used as a therapy in a number of countries. METHODS: Formulation of IRDye800CW-nimotuzumab for a clinical trial application was prepared. The physical, chemical, and pharmaceutical properties were tested to develop the specifications to determine stability of the product. The acute and delayed toxicities were tested and IRDye800CW-nimotuzumab was determined to be non-toxic. Non-compartmental pharmacokinetics analysis was used to determine the half-life of IRDye800CW-nimotuzumab. RESULTS: IRDye800CW-nimotuzumab was determined to be non-toxic from the acute and delayed toxicity study. The half-life of IRDye800CW-nimotuzumab was determined to be 38 ± 1.5 h. A bi-exponential analysis was also used which gave a t1/2 alpha of 1.5 h and t1/2 beta of 40.8 h. CONCLUSIONS: Here, we show preclinical studies demonstrating that nimotuzumab conjugated to IRDye800CW is safe and does not exhibit toxicities commonly associated with EGFR targeting antibodies.

Effect of Formalin Fixation for Near-Infrared Fluorescence Imaging with an Antibody-Dye Conjugate in Head and Neck Cancer Patients.

PURPOSE: This study evaluated the effect of formalin fixation for near-infrared (NIR) fluorescence imaging of an antibody-dye complex (panitumumab-IRDye800CW) that was intravenously administered to patients with head and neck squamous cell carcinoma (HNSCC) scheduled to undergo surgery of curative intent. PROCEDURES: HNSCC patients were infused with 25 or 50 mg of panitumumab-IRDye800CW followed by surgery 1-5 days later. Following resection, primary tumor specimens were imaged in a closed-field fluorescence imaging device, before and after formalin fixation. The fluorescence images of formalin-fixed specimens were compared with images prior to formalin fixation. Regions of interest were drawn on the primary tumor and on the adjacent normal tissue on the fluorescence images. The mean fluorescence intensity (MFI) and tumor-to-background ratios (TBRs) of the fresh and formalin-fixed tissues were compared. RESULTS: Of the 30 enrolled patients, 20 tissue specimens were eligible for this study. Formalin fixation led to an average of 10 % shrinkage in tumor specimen size (p < 0.0001). Tumor MFI in formalin-fixed specimens was on average 10.9 % lower than that in the fresh specimens (p = 0.0002). However, no statistical difference was found between the TBRs of the fresh specimens and those of the formalin-fixed specimens (p = 0.85). CONCLUSIONS: Despite the 11 % decrease in MFI between fresh and formalin-fixed tissue specimens, the relative difference between tumor and normal tissue as measured in TBR remained unchanged. This data suggests that evaluation of formalin-fixed tissue for assessing the accuracy of fluorescence-guided surgery approaches could provide a valid, yet more flexible, alternative to fresh tissue analysis. TRIAL REGISTRATION: NCT02415881.

The Efficacy of Breast Implant Irrigant Solutions: A Comparative Analysis Using an In Vitro Model.

BACKGROUND: Infections are challenging complications of implant-based breast reconstruction and augmentation. They pose a clinical challenge, with significant economic implications. One proposed solution is implant irrigation at the time of placement. There is no consensus on the optimal irrigant solution. METHODS: The authors tested the relative efficacy of 10% povidone-iodine, Clorpactin, Prontosan, triple-antibiotic solution, or normal saline (negative control) against two strains each of methicillin-resistant Staphylococcus aureus and Staphylococcus epidermidis. Sterile, smooth silicone implant disks were immersed in irrigant solution, then incubated in suspensions of methicillin-resistant S. aureus or S. epidermidis overnight. The disks were rinsed and sonicated to displace adherent bacteria from the implant surface, and the displaced bacteria were quantified. Normalized values were calculated to characterize the relative efficacy of each irrigant. RESULTS: Povidone-iodine resulted in reductions of the bacterial load by a factor of 10 to 10 for all strains. Prontosan-treated smooth breast implant disks had a 10-fold reduction in bacterial counts for all but one methicillin-resistant S. aureus strain. In comparison to Prontosan, triple-antibiotic solution demonstrated a trend of greater reduction in methicillin-resistant S. aureus bacterial load and weaker activity against S. epidermidis strains. Clorpactin reduced the recovered colony-forming units for only a single strain of S. epidermidis. Povidone-iodine demonstrated the greatest efficacy against all four strains. However, Clorpactin, triple-antibiotic solution, and Prontosan demonstrated similar efficacies. CONCLUSIONS: Povidone-iodine was the most efficacious of the irrigants at reducing methicillin-resistant S. aureus and S. epidermidis contamination. Given the recent lifting of the U.S. Food and Drug Administration moratorium, larger clinical studies of povidone-iodine as a breast implant irrigant solution are warranted. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, V.

Multifunctional Molecular Therapeutic Agent for Targeted and Controlled Dual Chemo- and Photodynamic Therapy.

A novel molecular therapeutic agent was designed and synthesized, which contains three functional components, namely, a zinc(II) phthalocyanine substituted with a 2,4-dinitrobenzenesulfonate (DNBS) group as a glutathione (GSH)-activated photosensitizer, a chemo-prodrug based on combretastatin A-4 (CA4) with a singlet oxygen-cleavable aminoacrylate linker, and a biotin moiety as a tumor-targeting ligand. The conjugate showed preferential uptake toward the biotin-receptor-positive HepG2 cells compared with the low biotin-receptor-expressed HCT-116 cells used as the negative control, resulting in the restoration of the fluorescence emission and singlet oxygen generation upon removal of the DNBS group by intracellular GSH. The singlet oxygen produced not only induced a significant photodynamic effect against HepG2 cells but also triggered the cascaded release of the chemocytotoxic CA4, leading to synergistic cytotoxicity as shown by the less-than-unity combination index.

Tumour-specific fluorescence-guided surgery for pancreatic cancer using panitumumab-IRDye800CW: a phase 1 single-centre, open-label, single-arm, dose-escalation study.

BACKGROUND: Complete surgical resection remains the primary curative option for pancreatic ductal adenocarcinoma, with positive margins in 30-70% of patients. In this study, we aimed to evaluate the use of intraoperative tumour-specific imaging to enhance a surgeon's ability to detect visually occult cancer in real time. METHODS: In this single-centre, open-label, single-arm study, done in the USA, we enrolled patients who had clinically suspicious or biopsy-confirmed pancreatic ductal adenocarcinomas and were scheduled for curative surgery. Eligible patients were 19 years of age or older with a life expectancy of more than 12 weeks and a Karnofsky performance status of at least 70% or an Eastern Cooperative Oncology Group or Zubrod level of one or lower, who were scheduled to undergo curative surgery. Patients were sequentially enrolled into each dosing group and 2-5 days before surgery, patients were intravenously infused with 100 mg of unlabelled panitumumab followed by 25 mg, 50 mg, or 75 mg of the near-infrared fluorescently labelled antibody (panitumumab-IRDye800CW). The primary endpoint was to determine the optimal dose of panitumumab-IRDye800CW in identifying pancreatic ductal adenocarcinomas as measured by tumour-to-background ratio in all patients. The tumour-to-background ratio was defined as the fluorescence signal of the tumour divided by the fluorescence signal of the surrounding healthy tissue. The dose-finding part of this study has been completed. This study is registered with ClinicalTrials.gov, NCT03384238. FINDINGS: Between April, 2018, and July, 2019, 16 patients were screened for enrolment onto the study. Of the 16 screened patients, two (12%) patients withdrew from the study and three (19%) were not eligible; 11 (69%) patients completed the trial, all of whom were clinically diagnosed with pancreatic ductal adenocarcinoma. The mean tumour-to-background ratio of primary tumours was 3·0 (SD 0·5) in the 25 mg group, 4·0 (SD 0·6) in the 50 mg group, and 3·7 (SD 0·4) in the 75 mg group; the optimal dose was identified as 50 mg. Intraoperatively, near-infrared fluorescence imaging provided enhanced visualisation of the primary tumours, metastatic lymph nodes, and small (<2 mm) peritoneal metastasis. Intravenous administration of panitumumab-IRDye800CW at the doses of 25 mg, 50 mg, and 75 mg did not result in any grade 3 or higher adverse events. There were no serious adverse events attributed to panitumumab-IRDye800CW, although four possibly related adverse events (grade 1 and 2) were reported in four patients. INTERPRETATION: To our knowledge, this study presents the first clinical use of panitumumab-IRDye800CW for detecting pancreatic ductal adenocarcinomas and shows that panitumumab-IRDye800CW is safe and feasible to use during pancreatic cancer surgery. Tumour-specific intraoperative imaging might have added value for treatment of patients with pancreatic ductal adenocarcinomas through improved patient selection and enhanced visualisation of surgical margins, metastatic lymph nodes, and distant metastasis. FUNDING: National Institutes of Health and the Netherlands Organization for Scientific Research.

Fluorescence-guided imaging for resection margin evaluation in head and neck cancer patients using cetuximab-800CW: A quantitative dose-escalation study.

Tumor-positive resection margins are present in up to 23% of head and neck cancer (HNC) surgeries, as intraoperative techniques for real-time evaluation of the resection margins are lacking. In this study, we investigated the safety and potential clinical value of fluorescence-guided imaging (FGI) for resection margin evaluation in HNC patients. We determined the optimal cetuximab-800CW dose by quantification of intrinsic fluorescence values using multi-diameter single-fiber reflectance, single-fiber fluorescence (MDSFR/SFF) spectroscopy. Methods: Five cohorts of three HNC patients received cetuximab-800CW systemically: three single dose cohorts (10, 25, 50 mg) and two cohorts pre-dosed with 75 mg unlabeled cetuximab (15 or 25 mg). Fluorescence visualization and MDSFR/SFF spectroscopy quantification was performed and were correlated to histopathology. Results: There were no study-related adverse events higher than Common Terminology Criteria for Adverse Events grade-II. Quantification of intrinsic fluorescence values showed a dose-dependent increase in background fluorescence in the single dose cohorts (p<0.001, p<0.001), which remained consistently low in the pre-dosed cohorts (p=0.6808). Resection margin status was evaluated with a sensitivity of 100% (4/4 tumor-positive margins) and specificity of 91% (10/11 tumor-negative margins). Conclusion: A pre-dose of 75 mg unlabeled cetuximab followed by 15 mg cetuximab-800CW was considered the optimal dose based on safety, fluorescence visualization and quantification of intrinsic fluorescence values. We were able to use a lower dose cetuximab-800CW than previously described, while remaining a high sensitivity for tumor detection due to application of equipment optimized for IRDye800CW detection, which was validated by quantification of intrinsic fluorescence values.

Optimal Dosing Strategy for Fluorescence-Guided Surgery with Panitumumab-IRDye800CW in Head and Neck Cancer.

PURPOSE: To identify the optimal dosing strategy for fluorescence-guided surgery in patients with head and neck squamous cell carcinoma, we conducted a dose-ranging study evaluating the anti-epidermal growth factor receptor (EGFR) therapeutic antibody, panitumumab, that was fluorescently labeled with the near-infrared dye IRDye800CW. PROCEDURES: Patients (n = 24) received either 0.5 or 1.0 mg/kg panitumumab-IRDye800CW in the weight-based dosing group or 25 or 50 mg panitumumab-IRDye800CW in the fixed dosing group. Following surgery, whole primary specimens were imaged in a closed-field device and the mean fluorescence intensity (MFI) and tumor-to-background ratio (TBR) were assessed. Clinical variables, including dose, time of infusion-to-surgery, age, unlabeled dose, gender, primary tumor site, and tumor size, were analyzed to evaluate the factors affecting the fluorescence intensity in order to identify the optimal dose for intraoperative fluorescence imaging. RESULTS: A total of 24 primary tumor specimens were imaged and analyzed in this study. Although no correlations between TBR and dose of panitumumab-IRDye800CW were found, there were moderate-strong correlations between the primary tumor MFI and panitumumab-IRDye800CW dose for fixed dose (mg) (R2 = 0.42) and for dose/weight (mg/kg) (R2 = 0.54). Results indicated that the optimal MFI was at approximately 50 mg for fixed dose and 0.75 mg/kg for dose/weight. No significant differences were found for the primary tumor MFI and TBRs between the weight-based dosing and the fixed dosing groups. MFIs significantly increased when the infusion-to-surgery window was reduced to within 2 days (vs. 3 days or more, p < 0.05). CONCLUSIONS: Antibody-based imaging for surgical resection is under investigation in multiple clinical trials. Our data suggests that a fixed dose of 50 mg is an appropriate diagnostic dose for successful surgical fluorescence imaging.

Evaluation of the time-dependent antiproliferative activity and liver microsome stability of 3 phenyl 4-(2-oxo-3-alkylimidazolidin-1-yl)benzenesulfonates as promising CYP1A1-dependent antimicrotubule prodrugs.

OBJECTIVES: In this study, the antiproliferative activity of 3 phenyl 4-(2-oxo-3-alkylimidazolidin-1-yl)benzenesulfonates (PAIB-SOs) was assessed in a time-dependent manner together with their hepatic stability and metabolism using human, mouse and rat liver microsomes. METHODS: CEU-818, -820 and -913 were selected as promising hit compounds. Their antiproliferative activity on human breast carcinoma MCF-7 cells was evaluated using escalating concentrations of drugs at 24, 36 and 48 h and the sulforhodamine B assay. Their hepatic stability was evaluated by HPLC-UV of extracts obtained from human, mouse and rat liver microsomes. KEY FINDINGS: The antiproliferative activity of PAIB-SOs is concentration and time-dependent and requires between 24 and 36 h of contact with MCF-7 cells to detect a significant antiproliferative activity. PAIB-SOs stability in microsomes usually decreases following this order: human ≈ (rat > mouse). The CEU-913 exhibits the longest half-life in rat and human liver microsomes while the CEU-820 exhibits the longest half-life in mouse liver microsomes. CONCLUSIONS: Our in vitro results suggest that PAIB-SOs should have a minimum contact time of 24 h with the tumour to trigger significant antitumoural activity. The activity of mouse liver microsomes towards PAIB-SOs is higher than rat microsomes and tends to be higher than human liver microsomes.

The Sentinel Margin: Intraoperative Ex Vivo Specimen Mapping Using Relative Fluorescence Intensity.

PURPOSE: Despite major advancements in surgical oncology, the positive margin rate for primary head and neck cancer resection remains around 15%-30%. In particular, the deep surface margin is the most challenging to adequately assess. Inadequate margins are directly correlated to poor survival, and as such, mitigation of these rates is critical to improve patient outcomes. We have developed an ex vivo imaging strategy that utilizes fluorescence intensity peaks (relative to background signal) of an injected anti-EGFR antibody conjugated to a fluorescent probe to locate potential close or positive margins on the deep surface of the resected tumor specimen. EXPERIMENTAL DESIGN: Twelve patients with head and neck cancer scheduled for surgery received systemic administration of a tumor-specific contrast-agent (panitumumab-IRDye800CW). After surgical resection, the tumor specimen was imaged using a fluorescence imager. The three highest fluorescence intensity-peaks on the deep surface of the specimen were isolated and correlated to histology to determine the margin distance at these regions. RESULTS: Relative fluorescence peak intensities identified the closest margin on the deep surface of the specimen within 2.5 minutes. The highest intensity peak consistently (100%) detected the closest margin to the tumor. The difference in tumor margin distance between the first and second highest fluorescence intensity peak averaged 2.1 ± 1.4 mm. The tumor-margin difference between the second and third highest peak averaged 1.6 ± 0.6 mm. CONCLUSIONS: Fluorescence intensity peaks can identify the region on the specimen where tumor is closest to specimen's edge on the deep surface. This technique could have broad applications in obtaining adequate margins in oncological surgery.

Anti-Claudin-1 Conjugated to a Near-Infrared Fluorophore Targets Colon Cancer in PDOX Mouse Models.

INTRODUCTION: Claudins are tight-junction proteins, which maintain an epithelial barrier in normal colon cells. Overexpression of Claudin-1 has been implicated for development of colon cancer. We postulated that Claudin-1 may be a useful target in near-infrared imaging and fluorescence-guided surgery. METHODS: We conjugated Claudin-1 antibody to LI-COR IR800DyeCW (Claudin-1-IRDye800CW). Western blotting of 9 human colon cancer cell lysates was performed. Animal imaging was performed with the LI-COR Pearl Trilogy Fluorescence Imaging System. A dose-response study was carried out with subcutaneous LS174T colon cancer cell line models. Increasing doses of Claudin-1-IRDye800CW via tail vein injection were administered to three groups of mice. Two groups of mice were used as controls (antibody alone, and dye alone). In vivo imaging was performed at 24, 48, and 72 h after administration of the conjugated dye. Orthotopic implantation of patient-derived tumors and cell lines was performed and peritoneal carcinomatosis models were created. After tumor growth, mice were administered Claudin-1-IRDye800CW and imaged in vivo 48 h later. The mice were euthanized and laparotomy was performed to assess internal organs and toxicity. RESULTS: Western blotting revealed that all colon cancer cell lysates expressed varying amounts of Claudin-1. All tumors demonstrated strong and specific fluorescence labeling at 800 nm, even with the lowest dose of 12.5 µg of Claudin-1-IRDye800CW. CONCLUSIONS: Claudin-1 is a useful target for near-infrared antibody-based imaging for visualization of colorectal tumors for future use in fluorescence-guided surgery.

Panitumumab-IRDye800CW for Fluorescence-Guided Surgical Resection of Colorectal Cancer.

BACKGROUND: Fluorescence-guided surgery (FGS) is a rapidly advancing field that may improve outcomes in several cancer types. Although screening has decreased colorectal cancer (CRC) mortality, it remains a common and often fatal malignancy. In this study, we sought to identify an optical imaging agent for the application of FGS technology to CRC. METHODS: We compared a panitumumab-IRDye800CW conjugate to an IgG-IRDye800CW isotype control. Mice were implanted with one of three CRC cell lines (LS174T, Colo205, and SW948) and imaged with open- and closed-filed fluorescence imaging systems. Fluorescent contrast was quantified by calculating the ratio between tumor and background fluorescence. After 10 d, the mice were sacrificed, and their tumors stained for microscopic imaging. RESULTS: Panitumumab-IRDye800CW produced significantly greater (P < 0.05) fluorescent contrast in all three cell lines. Average tumor to background ratio was 6.00 versus 2.60 for LS174T, 5.78 versus 2.52 for Colo205, and 4.31 versus 1.70 for SW948. A 1-mg tumor fragment produced significantly greater fluorescent contrast in the Colo205 and SW948 cell lines in the panitumumab-IRDye800CW group. Western blotting for epidermal growth factor receptor (EGFR) and a semiquantitative analysis of EGFR expression noted strong expression in all three cell lines; however, EGFR expression did not directly correlate to tumor to background ratio. CONCLUSIONS: Panitumumab-IRDye800CW produces significantly greater fluorescent contrast than IgG-IRDye800CW in a murine model of CRC and is a suitable agent for the application of FGS technology to CRC.

GABA, γ-Aminobutyric Acid, Protects Against Severe Liver Injury.

BACKGROUND: Acute liver failure (ALF) from severe acute liver injury is a critical condition associated with high mortality. The purpose of this study was to investigate the impact of preemptive administration of γ-aminobutyric acid (GABA) on hepatic injury and survival outcomes in mice with experimentally induced ALF. MATERIALS AND METHODS: To induce ALF, C57BL/6NHsd mice were administered GABA, saline, or nothing for 7 d, followed by intraperitoneal administration of 500 µg of tumor necrosis factor α and 20 mg of D-galactosamine. The study mice were humanely euthanized 4-5 h after ALF was induced or observed for survival. Proteins present in the blood samples and liver tissue from the euthanized mice were analyzed using Western blot and immunohistochemical and histopathologic analyses. For inhibition studies, we administered the STAT3-specific inhibitor, NSC74859, 90 min before ALF induction. RESULTS: We found that GABA-treated mice had substantial attenuation of terminal deoxynucleotidyl transferase dUTP nick end labeling-positive hepatocytes and hepatocellular necrosis, decreased caspase-3, H2AX, and p38 MAPK protein levels and increased expressions of Jak2, STAT3, Bcl-2, and Mn-SOD, with improved mitochondrial integrity. The reduced apoptotic proteins led to a significantly prolonged survival after ALF induction in GABA-treated mice. The STAT3-specific inhibitor NSC74859 eliminated the survival advantage in GABA-treated mice with ALF, indicating the involvement of the STAT3 pathway in GABA-induced reduction in apoptosis. CONCLUSIONS: Our results showed that preemptive treatment with GABA protected against severe acute liver injury in mice via GABA-mediated STAT3 signaling. Preemptive administration of GABA may be a useful approach to optimize marginal donor livers before transplantation.

STAT3 is involved in IL-6-Mediated Downregulation of Hepatic Transporters in Mice.

Interleukin (IL)-6 decreases hepatic expression of numerous transporters. Although IL-6 signaling occurs through STAT3, the extent of the involvement of the STAT3 signaling pathway has not been elucidated. PURPOSE: Our objective was to investigate whether IL-6-mediated effects occur through STAT3, and whether PXR plays a role in this regulation. METHOD: PXR null (-/-) or wild-type (+/+) male mice were pre-dosed with a selective STAT3 inhibitor S3I-201 (7.5 mg/kg ip) or vehicle (n=5-8/group) 30 minutes before receiving a single dose of IL-6 (1 µg ip) or saline. Animals were sacrificed after 6 hours and liver samples were analyzed using qRT-PCR and western blotting. RESULTS: As compared to saline controls, IL-6 decreased the expression of Cyp3a, Abcb1a, Abcc3, and Slco1a4 20-70% similarly in PXR (+/+) and (-/-) mice at 6 hr, while downregulation of Abcb11, Abcc2, Slc10a1and Slco2b1 was only seen in PXR (+/+). Pre-administration of S3I-201 attenuated IL-6-mediated changes of most transporters in PXR (+/+) and PXR (-/-) mice. At early times after IL-6 administration (10-120 minutes), transcript levels of Socs3, PXR, Abcb1a, Abcc3, Abcb11, Slco1a4 and Slco2b1were increased in PXR (+/+) mice. CONCLUSIONS: Our findings demonstrate that IL-6 imposes a significant downregulation of numerous ABC and SLC transporters in liver primarily through activation of the STAT3 signaling pathway. Based on time-dependent changes in transporter expression, downregulation likely occurs downstream of STAT3 activation.  As IL-6 is elevated in many diseases, understanding the underlying mechanism(s) involved in transporter dysregulation will allow us to predict potential drug-disease interactions.

Evaluation of antibody fragment properties for near-infrared fluorescence imaging of HER3-positive cancer xenografts.

In vivo imaging is influenced by the half-life, tissue penetration, biodistribution, and affinity of the imaging probe. Immunoglobulin G (IgG) is composed of discrete domains with known functions, providing a template for engineering antibody fragments with desired imaging properties. Here, we engineered antibody-based imaging probes, consisting of different combinations of antibody domains, labeled them with the near-infrared fluorescent dye IRDye800CW, and evaluated their in vivo imaging properties. Antibody-based imaging probes were based on an anti-HER3 antigen binding fragment (Fab) isolated using phage display. Methods: We constructed six anti-HER3 antibody-based imaging probes: a single chain variable fragment (scFv), Fab, diabody, scFv-CH3, scFv-Fc, and IgG. IRDye800CW-labeled, antibody-based probes were injected into nude mice bearing FaDu xenografts and their distribution to the xenograft, liver, and kidneys was evaluated. Results: These imaging probes bound to recombinant HER3 and to the HER3-positive cell line, FaDu. Small antibody fragments with molecular weight <60 kDa (scFv, diabody, and Fab) accumulated rapidly in the xenograft (maximum accumulation between 2-4 h post injection (hpi)) and cleared primarily through the kidneys. scFv-CH3 (80 kDa) had fast clearance and peaked in the xenograft between 2-3 hpi and cleared from xenograft in a rate comparable to Fab and diabody. IgG and scFv-Fc persisted in the xenografts for up to 72 hpi and distributed mainly to the xenograft and liver. The highest xenograft fluorescence signals were observed with IgG and scFv-Fc imaging probes and persisted for 2-3 days. Conclusion: These results highlight the utility of using antibody fragments to optimize clearance, tumor labeling, and biodistribution properties for developing anti-HER3 probes for image-guided surgery or PET imaging.

Tumor-targeted Dual-modality Imaging to Improve Intraoperative Visualization of Clear Cell Renal Cell Carcinoma: A First in Man Study.

Intraoperative imaging with antibodies labeled with both a radionuclide for initial guidance and a near-infrared dye for adequate tumor delineation may overcome the main limitation of fluorescence imaging: the limited penetration depth of light in biological tissue. In this study, we demonstrate the feasibility and safety of intraoperative dual-modality imaging with the carbonic anhydrase IX (CAIX)-targeting antibody 111In-DOTA-girentuximab-IRDye800CW in clear cell renal cell carcinoma (ccRCC) patients. Methods: A phase I protein dose escalation study was performed in patients with a primary renal mass who were scheduled for surgery. 111In-DOTA-girentuximab-IRDye800CW (5, 10, 30, or 50 mg, n=3 ccRCC patients per dose level) was administered intravenously and after 4 days SPECT/CT imaging was performed. Seven days after antibody injection, surgery was performed with the use of a gamma probe and near-infrared fluorescence camera. Results: In total, fifteen patients were included (12 ccRCC, 3 CAIX-negative tumors). No study-related serious adverse events were observed. All ccRCC were visualized by SPECT/CT and localized by intraoperative gamma probe detection (mean tumor-to-normal kidney (T:N) ratio 2.5 ± 0.8), while the T:N ratio was 1.0 ± 0.1 in CAIX-negative tumors. ccRCC were hyperfluorescent at all protein doses and fluorescence imaging could be used for intraoperative tumor delineation, assessment of the surgical cavity and detection of (positive) surgical margins. The radiosignal was crucial for tumor localization in case of overlying fat tissue. Conclusion: This first in man study shows that tumor-targeted dual-modality imaging using 111In-DOTA-girentuximab-IRDye800CW is safe and can be used for intraoperative guidance of ccRCC resection.

Safety of panitumumab-IRDye800CW and cetuximab-IRDye800CW for fluorescence-guided surgical navigation in head and neck cancers.

Purpose: To demonstrate the safety and feasibility of leveraging therapeutic antibodies for surgical imaging. Procedures: We conducted two phase I trials for anti-epidermal growth factor receptor antibodies cetuximab-IRDye800CW (n=12) and panitumumab-IRDye800CW (n=15). Adults with biopsy-confirmed head and neck squamous cell carcinoma scheduled for standard-of-care surgery were eligible. For cetuximab-IRDye800CW, cohort 1 was intravenously infused with 2.5 mg/m2, cohort 2 received 25 mg/m2, and cohort 3 received 62.5 mg/m2. For panitumumab-IRDye800CW, cohorts received 0.06 mg/kg, 0.5 mg/kg, and 1 mg/kg, respectively. Electrocardiograms and blood samples were obtained, and patients were followed for 30 days post-study drug infusion. Results: Both fluorescently labeled antibodies had similar pharmacodynamic properties and minimal toxicities. Two infusion reactions occurred with cetuximab and none with panitumumab. There were no grade 2 or higher toxicities attributable to cetuximab-IRDye800CW or panitumumab-IRDye800CW; fifteen grade 1 adverse events occurred with cetuximab-IRDye800CW, and one grade 1 occurred with panitumumab-IRDye800CW. There were no significant differences in QTc prolongation between the two trials (p=0.8). Conclusions: Panitumumab-IRDye800CW and cetuximab-IRDye800CW have toxicity and pharmacodynamic profiles that match the parent compound, suggesting that other therapeutic antibodies may be repurposed as imaging agents with limited preclinical toxicology data.