Fibroblast inhibition by tocilizumab enabled gemcitabine/nab-paclitaxel rechallenge for pancreatic cancer.

Interleukin-6 (IL-6)/signal transducer and activator of transcription 3 (STAT3) pathway inhibition may overcome chemoresistance of metastatic pancreatic cancer (MPC). We sought to determine the safety and recommended dose of tocilizumab (TCZ), an IL-6 receptor monoclonal antibody, and biological correlates of tumor shrinkage in patients with gemcitabine (GEM)/nanoparticle albumin-bound paclitaxel (nab-PTX)-refractory MPC. This phase 1 study enrolled 10 patients with MPC who had progressed after GEM/nab-PTX. Patients initially received TCZ 8 mg/kg on Day 1 and nab-PTX 100 mg/m2 + GEM 750 mg/m2 on Days 2, 9, and 16. Before and at the end of Cycle 1, biopsy of liver metastases was performed 3-5 h after levofloxacin (LVFX) administration to measure LVFX infiltration into tumor tissue. No dose-limited toxicities occurred, and the recommended dosage of TCZ was determined to be 8 mg/kg. Treatment-emergent adverse events occurred in 80% of patients, of which decreased neutrophil count was the most common. Tumor reduction during Cycle 1 was observed in four patients, who were defined as responders. In paired-biopsy samples, responder-related biological activities were an increase of cleaved PARP expression of tumor nuclei (p = 0.01), a decrease of proliferative cancer-associated fibroblasts (CAFs) (p = 0.08), and an increase of LVFX infiltration in the tumor (p = 0.04). A decrease of phosphorylated STAT3 expression (p = 0.02) favored an increase in LVFX infiltration. In conclusion, TCZ + GEM/nab-PTX-rechallenge had a manageable safety profile and showed preliminary activity via inhibition of CAF and improved intratumoral drug infiltration in MPC.

Validation of a method to analyze size distribution of crovalimab-complement C5-eculizumab complexes in human serum.

Background: Crovalimab is a humanized monoclonal antibody targeting human complement C5. Patients switching from eculizumab to crovalimab are expected to form drug-target-drug complexes (DTDCs), since these antibodies each bind to a different epitope on complement C5. An analytical method to evaluate the size distribution of these DTDCs was developed and validated. Methods: Human serum samples were separated by size-exclusion chromatography (SEC) into eight fractions, and the concentration of crovalimab in each fraction was measured by ELISA. We evaluated SEC, ELISA and the combination of both methods (SEC-ELISA). Results: Predetermined validation acceptance criteria were met. Conclusion: The DTDC assay method was successfully validated. It enables us to evaluate the impact of DTDCs on clinical outcomes.

Imaging Mass Spectrometry (IMS) for drug discovery and development survey: Results on methods, applications and regulatory compliance.

Imaging mass spectrometry (IMS) is increasingly used for drug discovery and development to understand target enagement, tissue distribution, drug toxicity, and disease mechanisms, etc. However, this is still a relatively new technique that requires further development validation before it will be an acceptable technique to support regulated development of new drugs. Thus, best practices will need to be established to build more confidence and gain wider acceptance by the scientific community, pharmaceutical industry, and regulatory authorities. The Imaging Mass Spectrometry Society (IMSS) and the Japan Association for Imaging Mass Spectrometry (JAIMS) have conducted a thorough survey to gather information on the current state of IMS and to identify key issues. The survey was sent to researchers or managers in the position who are currently using IMS techniques in support of their drug discovery and development efforts and/or who plan to use such tools as best practices are established. The survey probes questions related to details regarding technical aspects of IMS, which includes data acquisition, data analysis and quantitation, data integrity, reporting, applications, and regulatory concerns. This international survey was conducted online through the Survey Monkey (https://www.surveymonkey.com) in both English and Japanese from September 14 through September 30, 2020.

Applications of MALDI mass spectrometry imaging for pharmacokinetic studies during drug development.

The concentration and distribution of a drug or its metabolites in tissues are key factors for understanding drug efficacy or toxicity. Conventional pharmacokinetic studies show that the plasma concentration of a drug is often unrelated to the intra-tissue concentration. Moreover, it is difficult to predict the distribution of a drug in tissues, particularly those with complex structures, even though the overall tissue concentration is measured by using homogenizing procedures. Mass spectrometry imaging (MSI) enables visualization of the spatial distribution and quantities of drugs in tissue sections without labeling, which can significantly impact on the development of new drugs and translational research. Recent advances in instrument technology and the knowledge accumulated to date could further improve the sensitivity, spatial resolution, and reproducibility of MSI. Here we present current applications of matrix-assisted laser desorption/ionization (MALDI)-MSI in pharmacokinetic imaging (PK-imaging) studies, give an overview of MALDI-MSI procedures, highlight the importance of internal standards, and give details of quantitative approaches. We also point out the need for standardizing MALDI-MSI techniques. PK-imaging using standardized MALDI-MSI methods, independent of instrument or technician expertise, is expected to contribute to acquiring reliable data in drug development and translational research in the future.

Collaborative study using common samples to evaluate the performance of anti-drug antibody assays constructed by different companies.

This study was undertaken to evaluate the performance of anti-drug antibody (ADA) assays constructed by each participating company using common samples including ADA, drug and human serum. The ADA assays constructed by each company showed good sensitivity and precision for evaluation of ADA. Cut points for screening and confirmatory assays and assay selectivity were determined by various calculation methods. In evaluations of blind ADA samples, nearly similar results were obtained by the study companies in determinations of whether samples were positive or negative except at the lowest sample concentration (5 ng/mL). In measurement of drug tolerance, for almost samples containing ADA and drugs, more positive results were obtained in assays using acid dissociation compared to those without acid dissociation. Overall, the performance of ADA assays constructed by the 10 companies participating in this study was acceptable in terms of sensitivity and reproducibility for detection and evaluation of immunogenicity in both patients and healthy subjects. On the other hand, based on results for samples containing ADA and drugs, validity of results for ADA assays conducted without acid dissociation was less meaningful and more difficult to evaluate. Thus, acid dissociation was confirmed to be useful for improving drug tolerance.

MALDI mass spectrometry imaging of erlotinib administered in combination with bevacizumab in xenograft mice bearing B901L, EGFR-mutated NSCLC cells.

Combination therapy of erlotinib plus bevacizumab improves progression-free survival of patients with epidermal growth factor receptor-mutated (EGFR-mutated) advanced non-small-cell lung cancer (NSCLC) compared with erlotinib alone. Although improved delivery and distribution of erlotinib to tumours as a result of the normalization of microvessels by bevacizumab is thought to be one of the underlying mechanisms, there is insufficient supporting evidence. B901L cells derived from EGFR-mutated NSCLC were subcutaneously implanted into mice, and mice were treated with bevacizumab or human IgG followed by treatment with erlotinib. The distribution of erlotinib in their tumours at different times after erlotinib administration was analysed by matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI). We also analysed the distribution of erlotinib metabolites and the distribution of erlotinib in tumours refractory to erlotinib, which were established by long-term treatment with erlotinib. We found that erlotinib was broadly diffused in the tumours from B901L-implanted xenograft mice, independently of bevacizumab treatment. We also found that erlotinib metabolites were co-localized with erlotinib and that erlotinib in erlotinib-refractory tumours was broadly distributed throughout the tumour tissue. Multivariate imaging approaches using MALDI MSI as applied in this study are of great value for pharmacokinetic studies in drug development.

Visualizing spatial distribution of alectinib in murine brain using quantitative mass spectrometry imaging.

In the development of anticancer drugs, drug concentration measurements in the target tissue have been thought to be crucial for predicting drug efficacy and safety. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is commonly used for determination of average drug concentrations; however, complete loss of spatial information in the target tissue occurs. Mass spectrometry imaging (MSI) has been recently applied as an innovative tool for detection of molecular distribution of pharmacological agents in heterogeneous targets. This study examined the intra-brain transitivity of alectinib, a novel anaplastic lymphoma kinase inhibitor, using a combination of matrix-assisted laser desorption ionization-MSI and LC-MS/MS techniques. We first analyzed the pharmacokinetic profiles in FVB mice and then examined the effect of the multidrug resistance protein-1 (MDR1) using Mdr1a/b knockout mice including quantitative distribution of alectinib in the brain. While no differences were observed between the mice for the plasma alectinib concentrations, diffuse alectinib distributions were found in the brain of the Mdr1a/b knockout versus FVB mice. These results indicate the potential for using quantitative MSI for clarifying drug distribution in the brain on a microscopic level, in addition to suggesting a possible use in designing studies for anticancer drug development and translational research.

Migration of mesenchymal cell fated to blastema is necessary for fish fin regeneration.

Urodeles and fish have higher regeneration ability in a variety of tissues and organs than do other vertebrate species including mammals. Though many studies have aimed at identifying the cellular and molecular basis for regeneration, relatively little is known about the detailed cellular behaviors and involved molecular basis. In the present study, a small molecule inhibitor was used to analyzed the role of phosphoinositide 3-kinase (PI3K) signaling during regeneration. We showed that the inhibitor disrupted the formation of blastema including the expression of characteristic genes. The failure of blastema formation was due to the impaired migration of mesenchymal cells to the distal prospective blastema region, although it had a little affect on cell cycle activation in mesenchymal cells. Moreover, we found that the epidermal remodeling including cell proliferation, distal cell migration and Akt phosphorylation was also affected by the inhibitor, implying a possible involvement of epidermis for proper formation of blastema. From these data, we propose a model in which distinct signals that direct the cell cycle activation, mesenchymal cell migration and epidermal remodeling coordinate together to accomplish the correct blastema formation and regeneration.

Identification of novel markers expressed during fin regeneration by microarray analysis in medaka fish.

Urodeles and fish have a remarkable ability to regenerate lost body parts, whereas many higher vertebrates, including mammals, retain only a limited capacity. It is known that the formation of specialized cell populations such as the wound epidermis or blastema is crucial for regeneration; however, the molecular basis for their formation has not been elucidated. Recently, approaches using differential display and microarray have been done in zebrafish for searching molecules involved in regeneration. Here, we used the medaka fish, a distantly diverged fish species, for microarray screening of transcripts up-regulated during regeneration. By setting criteria for selecting transcripts that are reliably and reproducibly up-regulated during regeneration, we identified 140 transcripts. Of them, localized in situ expression of 12 transcripts of 22 tested was detected either in differentiating cartilage, basal wound epidermis, or blastema. Our results provide useful molecular markers for dissecting the regeneration process at a fine cellular resolution.