Drug review process advancement and required manufacturer and contract research oraganization responses.

The United States Senate passed the "FDA Modernization Act 2.0." on September 29, 2022. Although the effectiveness of this Bill, which aims to eliminate the mandatory use of laboratory animals in new drug development, is limited, it represents a significant trend that will change the shape of drug applications in the United States and other countries. However, pharmaceutical companies have not taken major steps towards the complete elimination of animal testing from the standpoint of product safety, where they prioritize patient safety. Nonetheless, society is becoming increasingly opposed to animal testing, and efforts will be made to use fewer animals and conduct fewer animal tests as a natural and reasonable response. These changes eventually alter the shape of new drug applications. Based on the assumption that fewer animal tests will be conducted or fewer animals will be used in testing, this study explored bioinformatics and new technologies as alternatives to compensate for reduced information and provide a picture of how future new drug applications may look. The authors also discuss the directions that pharmaceutical companies and nonclinical contract research organizations should adopt to promote the replacement, reduction, and refinement of animals used in research, teaching, testing, and exhibitions.

Transparency-enhancing technology allows the three-dimensional assessment of esophageal carcinoma obtained by endoscopic submucosal dissection.

BACKGROUND: Although much progress has been made in diagnosis of carcinomas, no established methods have been confirmed to elucidate their morphological features. METHODS: Three-dimensional structure of esophageal carcinomas was assessed using transparency-enhancing technology. Endoscopically resected esophageal squamous cell carcinoma was fluorescently stained, optically cleared using a transparency-enhancing reagent called LUCID, and visualized using laser scanning microscopy. The resulting microscope images were converted to virtual HE images for observation using ImageJ software. RESULTS: Microscopic observation and image editing enabled three-dimensional image reconstruction and conversion to virtual HE images. The structure of abnormal blood vessels in esophageal carcinoma recognized by endoscopy could be observed in the 3 dimensions. Squamous cell carcinoma and normal squamous epithelium could be distinguished in the virtual HE images. CONCLUSIONS: The results suggested that transparency-enhancing technology and virtual HE images may be feasible for clinical application and represent a novel histopathological method for evaluating endoscopically resected specimens.

Anamorelin Induced Acute Hyperglycemia in a Patient with Advanced Pancreatic Cancer and Diabetes: A Case Report.

Anamorelin (ANAM) is a novel ghrelin receptor agonist for the treatment of cancer cachexia. In clinical trials of ANAM, glucose metabolism disorders as adverse effects were relatively frequent, however, when and how they occur remains unclear. Moreover, the safety in patients with pancreatic cancer and/or diabetes has not been clarified because most previous studies focused on patients with non-small cell lung cancer and had excluded patients with poorly controlled diabetes. Herein, a 66-year-old man with advanced pancreatic cancer and diabetes was administered ANAM, and acute hyperglycemia was developed and could be monitored by the self-monitoring of blood glucose (SMBG). Increasing the insulin dose failed to control hyperglycemia adequately, but the hyperglycemia ameliorated quickly after ANAM discontinuation. The continuous glucose monitoring (CGM) revealed that the sensor glucose levels had remained in the high range throughout the day during ANAM administration despite using 1.5 times more insulin. Our report is one of the few that describe the details of ANAM-induced hyperglycemia and provides important information for the safe and effective use of ANAM.

The Ki67 index evaluation of pancreatic neuroendocrine tumors using 3D immunohistochemistry in small tissue specimens.

BACKGROUND: /Objectives: This study aimed to evaluate the usefulness of three-dimensional (3D) immunohistochemistry for the Ki67 index of small tissue specimens of pancreatic neuroendocrine tumor (PanNET). METHODS: Clinicopathological materials from 17 patients with PanNET who underwent surgical resection at Jichi Medical University Hospital were analyzed. We compared the Ki67 index of endoscopic ultrasonography-fine-needle aspiration biopsy (EUS-FNAB) specimens, surgical specimens, and small tissue specimens hollowed from paraffin blocks of surgical specimens that were substituted for EUS-FNAB specimens ("sub-FNAB"). The sub-FNAB specimens were optically cleared using LUCID (IlLUmination of Cleared organs to IDentify target molecules) and analyzed using 3D immunohistochemistry. RESULTS: The median Ki67 index in FNAB, sub-FNAB, and surgical specimens with conventional immunohistochemistry were 1.2% (0.7-5.0), 2.0% (0.5-14.6), and 5.4% (1.0-19.4), respectively. The median Ki67 index in sub-FNAB specimens with tissue clearing was calculated separately using the total number of cells on multiple images ("multiple slice"), with the image of the fewest positive cells ("coldspot"), and with the image of most positive cells ("hotspot"), which were 2.7% (0.2-8.2), 0.8% (0-4.8), and 5.5% (2.3-12.4), respectively. PanNET grade evaluated for the hotspot of the surgical specimens was significantly more consistent with those of the hotspot than multiple images of sub-FNAB specimens (16/17 vs. 10/17, p = 0.015). Hotspot evaluation using 3D immunohistochemistry of the sub-FNAB specimens showed agreement with the assessment of the surgical specimens (Kappa coefficient: 0.82). CONCLUSIONS: Tissue clearing and 3D immunohistochemistry for the Ki67 index can potentially improve the preoperative evaluation of EUS-FNAB specimens of PanNET in routine clinical practice.

Addressing practical issues in the smooth implementation of revised guidelines for non-clinical studies of vaccines for infectious disease prevention.

Herein, we investigated possible practical issues for the smooth implementation of the revised Japanese Guidelines for Non-clinical Studies of Vaccines for the Prevention of Infectious Diseases, which were raised in response to public comments on the proposed guideline revision and a gap analysis of the World Health Organization and European Medicines Agency guidelines. We identified main issues such as the non-clinical safety studies of adjuvants and evaluation of local cumulative tolerance in toxicity studies. The revised Japanese Pharmaceuticals and Medical Devices Agency (PMDA)/Ministry of Health, Labour and Welfare (MHLW) guidelines require non-clinical safety studies for vaccines containing new adjuvants, but additional safety pharmacology studies or safety studies in two animal species may be required if non-clinical safety studies raise any concerns (i.e., systemic distribution). Adjuvant biodistribution studies may aid in understanding vaccine characteristics. The evaluation of local cumulative tolerance in non-clinical studies, which was the focus of the Japanese review, can be omitted by including a warning in the package insert to avoid injection to the same site. The study's findings will be reflected in a Q&A to be released by the Japanese MHLW. We hope that this study will contribute to the global and harmonized development of vaccines.

Proposal for the revision of guidelines for clinical trials of vaccines to prevent infectious diseases in Japan.

The development of vaccines against infectious diseases requires a different approach from that of therapeutics, because vaccines are inoculated into healthy individuals and have a preventive effect by activating the immunity of the inoculated human. In Japan, "The Guideline for Clinical Trials of Vaccines for the Prevention of Infectious Diseases" was published in 2010 before changes occurred in the vaccine development environment in Japan, such as the introductions of foreign vaccines and simultaneous global development. This study aimed to identify current challenges in vaccine development through a questionnaire-based survey of pharmaceutical companies in Japan and by comparing the domestic and international guidelines and surveying review reports of 35 vaccines approved in Japan between April 2010 and December 2020. Identified challenges included the requirement for protective efficacy trials, efficacy evaluation of combination vaccines, development of multiregional and foreign clinical trials, and immunization of older adults and immunocompromised patients. We propose that new vaccines against infectious diseases should be evaluated for the protective efficacy, preferably through multiregional clinical trials. Additionally, differences in the incidence of infectious diseases or in epidemic virus strains between regions may affect the trials, when multiregional clinical trials are conducted, but immunogenicity-based studies can be conducted if a correlation between protective efficacy and immunogenicity has been established. We suggest that licensed combination vaccines can be used as comparators when an antigen is added to a licensed combination vaccine. We also proposed that the efficacy of a vaccine in non-major subjects, such as older adults or immunocompromised patients could be evaluated by comparing immunogenicity in major subjects with the confirmed protective effects of the vaccine. It is expected that these revisions will lead to the rapid advancement of vaccine development, which should contribute to the improvement of public health.

On-skin paintable biogel for long-term high-fidelity electroencephalogram recording.

Long-term high-fidelity electroencephalogram (EEG) recordings are critical for clinical and brain science applications. Conductive liquid-like or solid-like wet interface materials have been conventionally used as reliable interfaces for EEG recording. However, because of their simplex liquid or solid phase, electrodes with them as interfaces confront inadequate dynamic adaptability to hairy scalp, which makes it challenging to maintain stable and efficient contact of electrodes with scalp for long-term EEG recording. Here, we develop an on-skin paintable conductive biogel that shows temperature-controlled reversible fluid-gel transition to address the abovementioned limitation. This phase transition endows the biogel with unique on-skin paintability and in situ gelatinization, establishing conformal contact and dynamic compliance of electrodes with hairy scalp. The biogel is demonstrated as an efficient interface for long-term high-quality EEG recording over several days and for the high-performance capture and classification of evoked potentials. The paintable biogel offers a biocompatible and long-term reliable interface for EEG-based systems.

Proposal for the revision of the guidelines for Non-clinical studies of vaccines for the prevention of infectious diseases in Japan.

The efficacy and safety of vaccines for the prevention of infectious diseases are mostly evaluated based on the induction of an immune response against antigens, and do not necessarily depend on the dose administered. Therefore, there are some specific aspects that need to be considered in the development of vaccines and have been described in "The Guidelines for the non-clinical studies of vaccines for the prevention of infectious disease" in Japan. Recent changes in the vaccine development field, such as the introduction of vaccines developed overseas in Japan and vaccine development on a global scale have increased the need for revision of these guidelines. In this study, we identified the current challenges in the development of vaccines through comparison of Japanese and international guidelines. We conducted a questionnaire-based survey of pharmaceutical industries in Japan, and found issues related to non-clinical studies, such as the necessity of safety pharmacology studies and repeated-dose toxicity studies for each route of administration. We examined international guidelines on these issues as well as review reports by regulatory authorities, and determined that the results of repeated-dose toxicity studies can be used to decide whether safety pharmacology studies are required, and that studies to evaluate toxicity due to systemic effects may not be necessary for both intramuscular and subcutaneous administration. We propose revision of the guidelines for the non-clinical studies of vaccines in Japan taking international harmonizaion into account. We expected that the revised guidelines will promote smooth and rational vaccine development.

Guidance on the need for contraception related to use of pharmaceuticals: the Japan Agency for Medical Research and Development Study Group for providing information on the proper use of pharmaceuticals in patients with reproductive potential.

BACKGROUND: The U.S. Food and Drug Administration (FDA) and European Medicines Agency (EMA) have published guidelines on the use of cancer treatments in young people of reproductive potential. However, no such guideline is available in Japan. Therefore, this project aimed to gather relevant data and draft a respective guidance paper. METHODS: From April 2019 to March 2021, the Study Group for Providing Information on the Proper Use of Pharmaceuticals in Patients with Reproductive Potential at the Japan Agency for Medical Research and Development gathered opinions from experts in reproductive medicine, toxicology, and drug safety measures. The group considered these opinions, the FDA and EMA guidelines, and relevant Japanese guidelines and prepared a guidance paper, which they sent to 19 related organizations for comment. RESULTS: By November 2020, the draft guidance paper was completed and sent to the related organizations, 17 of which provided a total of 156 comments. The study group finalized the guidance paper in March 2021. CONCLUSIONS: The "Guidance on the Need for Contraception Related to Use of Pharmaceuticals" (The report of the Study Group for Providing Information on the Proper Use of Pharmaceuticals in Patients with Reproductive Potential, Research on Regulatory Science of Pharmaceuticals and Medical Devices, Japan Agency for Medical Research and Development: JP20mk0101139) is expected to help Japanese healthcare professionals provide fertility-related care and advice to adolescents, and young adults with cancer and their families.

[Reproductive and Developmental Toxicity of Drugs-Background of the Birth of Thalidomide].

INTRODUCTION: Thalidomide was a terrible drug‒induced suffering that should not have occurred, but it was revived in the market after more than 40 years. When we understand the historical background that caused the tragedy, safety measures for drugs have been established based on the lessons learned. We learn the background of the birth of this world from"The God and the Devil's Drug Thalidomide"and make use of it to the present.

Considerations of the Japanese Research Working Group for the ICH S6 & Related Issues Regarding Nonclinical Safety Assessments of Oligonucleotide Therapeutics: Comparison with Those of Biopharmaceuticals.

This white paper summarizes the current consensus of the Japanese Research Working Group for the ICH S6 & Related Issues (WGS6) on strategies for the nonclinical safety assessment of oligonucleotide-based therapeutics (ONTs), specifically focused on the similarities and differences to biotechnology-derived pharmaceuticals (biopharmaceuticals). ONTs, like biopharmaceuticals, have high species and target specificities. However, ONTs have characteristic off-target effects that clearly differ from those of biopharmaceuticals. The product characteristics of ONTs necessitate specific considerations when planning nonclinical studies. Some ONTs have been approved for human use and many are currently undergoing nonclinical and/or clinical development. However, as ONTs are a rapidly evolving class of drugs, there is still much to learn to achieve optimal strategies for the development of ONTs. There are no formal specific guidelines, so safety assessments of ONTs are principally conducted by referring to published white papers and conventional guidelines for biopharmaceuticals and new chemical entities, and each ONT is assessed on a case-by-case basis. The WGS6 expects that this report will be useful in considering nonclinical safety assessments and developing appropriate guidelines specific for ONTs.

[Current Issues in Photosafety Evaluation].

Phototoxicity is a toxic response elicited by topically applied or systemically administered photoreactive chemicals after exposure to light and can be broadly categorized into photoirritation, photoallergy, photogenotoxicity, and photocarcinogenicity. The need in the 21st century for accurate evaluation of photosafety has led to the publication of a number of guidelines from government agencies in Europe and the U.S.A. as well as the Organisation for Economic Co-operation and Development (OECD). In this review, we first discuss the mechanisms of phototoxicity and how they can be evaluated. We then discuss the state of the art and challenges now faced in photosafety evaluation of pharmaceuticals and cosmetics. Additionally, we describe the latest developments in OECD test guidelines (TG) for assessing photosafety, including revisions to the in vitro 3T3 neutral red uptake (NRU) phototoxicity test (TG 432) and the newly adopted reactive oxigen species (ROS) assay (TG 495). We will emphasize the importance of selecting the most appropriate means of evaluation with reference to the latest guidelines and other legal criteria for conducting photosafety evaluation.

Artificial Intelligence-Assisted motion capture for medical applications: a comparative study between markerless and passive marker motion capture.

We aimed to determine whether artificial intelligence (AI)-assisted markerless motion capture software is useful in the clinical medicine and rehabilitation fields. Currently, it is unclear whether the AI-assisted markerless method can be applied to individuals with lower limb dysfunction, such as those using an ankle foot orthosis or a crutch. However, as many patients with lower limb paralysis and foot orthosis users lose metatarsophalangeal (MP) joint flexion during the stance phase, it is necessary to estimate the accuracy of foot recognition under fixed MP joint motion. The hip, knee, and ankle joint angles during treadmill walking were determined using OpenPose (a markerless method) and the conventional passive marker motion capture method; the results from both methods were compared. We also examined whether an ankle foot orthosis and a crutch could influence the recognition ability of OpenPose. The hip and knee joint data obtained by the passive marker method (MAC3D), OpenPose, and manual video analysis using Kinovea software showed significant correlation. Compared with the ankle joint data obtained by OpenPose and Kinovea, which were strongly correlated, those obtained by MAC3D presented a weaker correlation. OpenPose can be an adequate substitute for conventional passive marker motion capture for both normal gait and abnormal gait with an orthosis or a crutch. Furthermore, OpenPose is applicable to patients with impaired MP joint motion. The use of OpenPose can reduce the complexity and cost associated with conventional passive marker motion capture without compromising recognition accuracy.

Ultraflexible organic light-emitting diodes for optogenetic nerve stimulation.

Organic electronic devices implemented on flexible thin films are attracting increased attention for biomedical applications because they possess extraordinary conformity to curved surfaces. A neuronal device equipped with an organic light-emitting diode (OLED), used in combination with animals that are genetically engineered to include a light-gated ion channel, would enable cell type-specific stimulation to neurons as well as conformal contact to brain tissue and peripheral soft tissue. This potential application of the OLEDs requires strong luminescence, well over the neuronal excitation threshold in addition to flexibility. Compatibility with neuroimaging techniques such as MRI provides a method to investigate the evoked activities in the whole brain. Here, we developed an ultrathin, flexible, MRI-compatible OLED device and demonstrated the activation of channelrhodopsin-2-expressing neurons in animals. Optical stimulation from the OLED attached to nerve fibers induced contractions in the innervated muscles. Mechanical damage to the tissues was significantly reduced because of the flexibility. Owing to the MRI compatibility, neuronal activities induced by direct optical stimulation of the brain were visualized using MRI. The OLED provides an optical interface for modulating the activity of soft neuronal tissues.

Blockade of the fractalkine-CX3CR1 axis ameliorates experimental colitis by dislodging venous crawling monocytes.

Chemokine systems modulate inflammatory and immune responses in inflammatory bowel disease (IBD). The colons of IBD patients show increased levels of fractalkine (FKN) and high numbers of FKN receptor-positive (CX3CR1+) cells; however, the FKN-CX3CR1 axis's role in intestinal inflammation, especially in intravascular leukocyte behaviors, still remains unclear. Here, we show that interruption of the FKN-CX3CR1 axis by anti-FKN monoclonal antibody (mAb) ameliorates murine colitis through regulation of intravascular monocyte behaviors in murine colitis models. FKN expression was detectable in vascular endothelium and CX3CR1+ macrophages accumulated in the mucosal lamina propria and submucosa of the inflamed colons. CD115+ monocytes tethered to the venous endothelium and expressed pro-inflammatory mediators. The anti-FKN mAb improved colitis symptoms, markedly reduced pro-inflammatory factors in the colon, maintained blood vessel integrity and reduced tethered monocytes in the inflamed veins. Intravital imaging revealed that CD115+Gr-1low/- monocytes crawled on the apical surfaces of venous endothelium, and anti-FKN mAb rapidly dislodged the crawling monocytes and inhibited their patrolling behavior. These findings suggest that the FKN-CX3CR1 axis triggers the patrolling behavior of crawling monocytes on the venous endothelium of inflamed colons, and accelerates the subsequent leukocyte activation and infiltration by locally producing inflammatory cytokines and chemokines. The mAb also ameliorated symptoms in another IBD model, T-cell-transferred colitis. Blocking the FKN-CX3CR1 axis with an anti-FKN mAb considerably inhibits the colitis-triggered inflammatory cascades, which may be an alternative strategy to treat IBD.

A Simulation Study of Light Propagation in the Spinal Cord for Optogenetic Surface Stimulation.

For utilizing optogenetics in neuroscience research, a proper setup is necessary, which delivers sufficient light to target cells and minimizes unexpected side effects caused by light exposure. In this study, we were interested in the area of influence of optical surface stimulation on a spinal cord tissue. We built a 3D spinal cord structure of rat and utilized the Monte-Carlo methods to simulate the light transport in it. We first evaluated light propagation in homogeneous nervous tissue models. For a 10-mW, 470-nm light source, light intensity of 1 mW=mm2 was detected at depths of 1:14 and 1:77 mm in white and grey matter, respectively. This indicated a narrower spreading pattern of light in the white matter than in the grey matter. Since the grey matter, which contains the somatosensory pathways, is an important target of spinal cord stimulation, we focused on investigating how much light could reach this area in a multi-layered structure. The results showed that when an optical fiber was positioned in the center line of the spinal cord dorsal surface, most of the light energy was absorbed before reaching the grey matter. In contrast, when we put the fiber on a lateral position, 0:8 mm away from the central line, relatively sufficient light intensity could be detected deep into the lamina 5 area. The experimental results obtained herein suggest that tissue type and the position of stimulation could greatly affect the area of influence of light stimulation in a 3D spinal cord. It is important to consider the location of the interested neural pathways and plan a proper stimulation site before conducting optogenetic surface stimulation of the spinal cord.

Applying Multichannel Optogenetic System for Epidural Spinal Cord Stimulation in Rats.

This study reports on the technique of applying multichannel optogenetic system to spinal cord stimulation in rats. Epidural spinal cord stimulation has been shown to reactivate spinalized hind limb motion; however, the stimulating parameters and detailed mechanism remain unclear. In order to utilize the high spatial resolution and cell type selectivity of optogenetics for studying the mechanism behind epidural spinal cord stimulation, a multichannel optical fiber bundle was designed, composed of 720 optical fibers of 200 $\mu $m diameter arranged in a 48$\times $ textbf15 matrix cover the vertebral columns of rats from level T13 to L2. The stimulating location was controlled by changing the direction of projection of a laser diode, and the appropriate projecting angle to obtain the maximum optical power output of each fiber was determined by a hill-climbing algorithm. A spinal cord window was developed to fit the head of the optical fiber bundle onto the dorsal part of rat spinal cord. Preliminary test in a rat revealed different stimulating area distribution of the optogenetically induced tibialis anterior (TA) and medial gastrocnemius (MG) muscle reactions and demonstrated the capability of the system for in-vivo study.

Influence of Gd-EOB-DTPA on T1 dependence of the proton density fat fraction using magnetic resonance spectroscopy.

This phantom study assessed the effect of Gd-EOB-DTPA on T1 bias (difference in T1 between water and fat) of the proton density fat fraction when using magnetic resonance spectroscopy. Phantoms containing varying fat percentages, without and with Gd-EOB-DTPA (precontrast and postcontrast, respectively), were scanned with repetition times ranging from 1000 to 5000 ms. The relationship between the proton density fat fraction at a reference repetition time of 5000 ms and that using different repetition times, was evaluated in the precontrast and postcontrast phantoms using linear regression and Bland-Altman analyses. In the precontrast phantom, as the repetition time increased, the slope tended to approach one. In the postcontrast phantom, the slope and intercept were near one and zero, respectively. The mean difference was smaller in the postcontrast phantom (range - 0.24 to - 0.01%) than in the precontrast phantom (range 0.12 to 3.52%). We conclude that I1 bias is minimized by Gd-EOB-DTPA.