Biochemical and crystallographic assessments of the effect of 4,6-O-disulfated disaccharide moieties in chondroitin sulfate E on chondroitinase ABC I activity.

O-sulfated N-acetyl-d-galactosamine (GalNAc) residues in chondroitin sulfate (CS) play a crucial role in chondroitinase ABC I (cABC-I) activity. CSA containing mainly 4-O-monosulfated GalNAc was a good substrate for the enzyme, but not CSE containing mainly 4,6-O-disulfated GalNAc [GalNAc(4S,6S)]. Each CS isomer exhibits structural heterogeneity; CSE has di-sulfated disaccharide units and mono-sulfated disaccharide units. Disaccharide composition analysis of digested products revealed that mono-sulfated disaccharide units in CSE contributed to the enzyme reactivity. Although enough substrate (CSA) was present in mixtures of CSA and CSE for reaction, the reactivity was reduced depending on the amount of CSE in the mixture. These results suggested that CSE is not only resistant to enzyme digestion but also attenuates enzyme activity. To understand the mechanism of action, crystallography of cABC-I in complex with unsaturated CSE-disaccharide, ΔDi-(4,6)S, was performed. Both 4-O- and 6-O-sulfate groups in ΔDi-(4,6)S interact with Arg500, suggesting that there was a greater interaction between ΔDi-(4,6)S and Arg500 than between mono-sulfated disaccharides and Arg500. Besides, this interaction attenuated enzyme activity by interfering with a function of Arg500, which is the charge neutralization of the carboxy group of D-glucuronic acid (GlcA) residues in CS. When interacting with the CSE-disaccharide unit [GlcAß1-3GalNAc(4S,6S)] in CS, cABC-I cannot interact with other CS-disaccharide units until it has digested the CSE-disaccharide unit. The low reactivity of cABC-I with CSE is attributable to two suggested factors: (a) resistance of E-units in CSE molecules to digestion by cABC-I, and (b) tendency of E-units in CSE molecules to attenuate cABC-I activity.

High-Throughput In Vitro Assay using Patient-Derived Tumor Organoids.

Patient-derived tumor organoids (PDOs) are expected to be a preclinical cancer model with better reproducibility of disease than traditional cell culture models. PDOs have been successfully generated from a variety of human tumors to recapitulate the architecture and function of tumor tissue accurately and efficiently. However, PDOs are unsuitable for an in vitro high-throughput assay system (HTS) or cell analysis using 96-well or 384-well plates when evaluating anticancer drugs because they are heterogeneous in size and form large clusters in culture. These cultures and assays use extracellular matrices, such as Matrigel, to create tumor tissue scaffolds. Therefore, PDOs have a low throughput and high cost, and it has been difficult to develop a suitable assay system. To address this issue, a simpler and more accurate HTS was established using PDOs to evaluate the potency of anticancer drugs and immunotherapy. An in vitro HTS was created that uses PDOs established from solid tumors cultured in 384-well plates. An HTS was also developed for assessment of antibody-dependent cellular cytotoxicity activity to represent the immune response using PDOs cultured in 96-well plates.

Advances in Understanding the Mechanism of Ophthalmic Viscosurgical Device Retention in the Anterior Chamber or on the Corneal Surface during Ocular Surgery.

Retention durability, especially in the eye, is one of the most important properties of ophthalmic viscosurgical devices (OVDs) during ocular surgery. However, the information on the physical properties of OVDs is insufficient to explain their retention durability. The purpose of this study is to clarify the mechanism of OVD retention to improve understanding of the behavior of OVDs during ocular surgery. To elucidate the mechanism of OVD retention, we have developed a new test method for measuring repulsive force. As a result, the maximum repulsive force of OVDs was positively and well correlated with the retention durability of investigated OVDs. Consequently, we demonstrated that the repulsive force could be used as an index of retention durability on the ocular surface and in the eye. We directly compared the intraocular retention durability of three OVDs (Shellgan, Viscoat, and Opegan-Hi) in ex vivo porcine eyes. Opegan-Hi was immediately removed from the anterior chamber, but Shellgan and Viscoat remained largely in the anterior chamber as determined by fluorescence imaging. These results showed that the intraocular retention behavior of OVDs was similar to their ocular surface behavior in our previous report, suggesting that retention durability is dependent on the OVD itself. The retention durability of Shellgan seemed to be higher than that of Viscoat, and the maximum repulsive force of Shellgan was 1.35-fold higher than that of Viscoat. Therefore, the repulsive force might be a useful index for assessing the difference in the retention durability between OVDs such as Shellgan and Viscoat.

Construction of in vitro patient-derived tumor models to evaluate anticancer agents and cancer immunotherapy.

An in vitro assay system using patient-derived tumor models represents a promising preclinical cancer model that replicates the disease better than traditional cell culture models. Patient-derived tumor organoid (PDO) and patient-derived tumor xenograft (PDX) models have been previously established from different types of human tumors to recapitulate accurately and efficiently their tissue architecture and function. However, these models have low throughput and are challenging to construct. Thus, the present study aimed to establish a simple in vitro high-throughput assay system using PDO and PDX models. Furthermore, the current study aimed to evaluate different classes of anticancer drugs, including chemotherapeutic, molecular targeted and antibody drugs, using PDO and PDX models. First, an in vitro high-throughput assay system was constructed using PDO and PDX established from solid and hematopoietic tumors cultured in 384-well plates to evaluate anticancer agents. In addition, an in vitro evaluation system of the immune response was developed using PDO and PDX. Novel cancer immunotherapeutic agents with marked efficacy have been used against various types of tumor. Thus, there is an urgent need for in vitro functional potency assays that can simulate the complex interaction of immune cells with tumor cells and can rapidly test the efficacy of different immunotherapies or antibody drugs. An evaluation system for the antibody-dependent cellular cytotoxic activity of anti-epidermal growth factor receptor antibody and the cytotoxic activity of activated lymphocytes, such as cytotoxic T lymphocytes and natural killer cells, was constructed. Moreover, immune response assay systems with bispecific T-cell engagers were developed using effector cells. The present results demonstrated that in vitro assay systems using PDO and PDX may be suitable for evaluating anticancer agents and immunotherapy potency with high reproducibility and simplicity.

Quantitative Assessment of Ophthalmic Viscosurgical Devices on Visibility, Spreadability, and Durability as Corneal Wetting Agents for the Wet Shell Technique.

PURPOSE: To evaluate ophthalmic viscosurgical devices (OVDs) as corneal wetting agents for the wet shell technique, a common procedure in Japan to maintain the wettability of corneal surfaces. METHODS: We surveyed Japanese ophthalmologists to determine the current state of the wet shell technique. After developing three ex vivo testing methods, we evaluated the corneal wetting properties of OVDs including 3% hyaluronic acid (HA) solution and OVD products, Opegan, Opelead, Viscoat, Shellgan, Discovisc, and Opegan-Hi. RESULTS: Overall, 214 ophthalmologists (70%) had performed the wet shell technique, and 91% of ophthalmologists who performed vitreous surgery had performed this technique. Using a questionnaire, we evaluated the performance of OVD as corneal wetting agents as follows: (i) visibility, smoothness of OVD surface; (ii) spreadability, coverage of the cornea; and (iii) retention durability, residual ratio of OVD on the corneal surface. The smoothness and spreadability of Opegan, Opelead, and 3% HA were superior to other OVDs. Adding an appropriate amount of balanced salt solution to the other OVDs improved smoothness and spreadability similar to that of Opelead or 3% HA. Shellgan and Viscoat, combination OVDs consisting of 3% HA and 4% chondroitin sulfate, showed high retention durability, resulting in remaining longer on the cornea compared with other OVDs. CONCLUSIONS: Physical properties of OVDs tested in this study may provide useful information for ophthalmologists to select a suitable OVD when performing the wet shell technique.

D-sorbitol can keep the viscosity of dispersive ophthalmic viscosurgical device at room temperature for long term.

The combination of 3% sodium hyaluronate (HA) and 4% sodium chondroitin sulfate (CS) is used as a dispersive ophthalmic viscosurgical device (OVD) during cataract surgery. For most OVDs containing HA, storage at 2-8 °C is recommended to preserve product characteristics. In order to develop a dispersive OVD that can be stored at room temperature, in this study, we searched additives which can stably maintain the viscosity, a key parameter of OVD, under preservation stability testing at 60 °C. The addition of D-sorbitol to a combination OVD, 3% HA and 4% CS, suppressed the reduction in viscosity compared with other OVDs with or without additives. The addition of D-sorbitol was also effective in improving the residual viscosity of the combination OVD after thermal treatment and light irradiation. Moreover, the OVD containing D-sorbitol can be stored at 25 °C with stably maintaining the initial viscosity for at least 24 months. In conclusion, the new dispersive OVD, 3% HA, 4% CS, and 0.5% D-sorbitol, can be stored at room temperature instead of under cold conditions and may represent an attractive option for clinical use because it is not necessary to bring the product to room temperature prior to use.

An In Vitro System for Evaluating Molecular Targeted Drugs Using Lung Patient-Derived Tumor Organoids.

Patient-derived tumor organoids (PDOs) represent a promising preclinical cancer model that better replicates disease, compared with traditional cell culture models. We have established PDOs from various human tumors to accurately and efficiently recapitulate the tissue architecture and function. Molecular targeted therapies with remarkable efficacy are currently in use against various tumors. Thus, there is a need for in vitro functional-potency assays that can be used to test the efficacy of molecular targeted drugs and model complex interactions between immune cells and tumor cells to evaluate the potential for cancer immunotherapy. This study represents an in vitro evaluation of different classes of molecular targeted drugs, including small-molecule inhibitors, monoclonal antibodies, and an antibody-drug conjugate, using lung PDOs. We evaluated epidermal growth factor receptor and human epidermal growth factor receptor 2 (HER2) inhibitors using a suitable high-throughput assay system. Next, the antibody-dependent cellular cytotoxicity (ADCC) activity of an anti-HER2 monoclonal antibody was evaluated to visualize the interactions of immune cells with PDOs during ADCC responses. Moreover, an evaluation system was developed for the immune checkpoint inhibitors, nivolumab and pembrolizumab, using PDOs. Our results demonstrate that the in vitro assay systems using PDOs were suitable for evaluating molecular targeted drugs under conditions that better reflect pathological conditions.

Rheological and Adhesive Properties to Identify Cohesive and Dispersive Ophthalmic Viscosurgical Devices.

The purpose of this study is to evaluate the usefulness of the rheological properties and adhesive force of ophthalmic viscosurgical devices (OVDs) as parameters for understanding and identifying the surgical behavior of cohesive and dispersive OVDs. The apparent viscosity, and the storage and loss moduli (dynamic rheological parameters) of 50% chondroitin sulfate (CS), 3% sodium hyaluronate (HA), Shellgan (the combination of 3% HA and 4% CS), Opegan (1% HA with a low molecular mass) and Opegan-Hi (1% HA with a high molecular mass) were obtained with a rheometer. The adhesive force of each sample was measured by using a texture analyzer. Opegan-Hi showed a solid-like behavior, while 50% CS showed a fluid-like behavior from their apparent viscosity and dynamic rheological parameters. Shellgan, 3% HA, and Opegan exhibited similar rheological properties and intermediate characteristics between Opegan-Hi and 50% CS, although their respective values were slightly different. Among these OVD samples, the adhesive force was higher in the order of 50% CS > Shellgan, 3% HA > Opegan > Opegan-Hi. The adhesive force of dispersive OVDs tended to be higher than that of cohesive OVDs, which correlated well with the removal times of OVDs from the eye that have previously been reported. In conclusion, we demonstrated that cohesive OVDs and dispersive OVDs have particular rheological and adhesive properties that can be applied to identify both types. These parameters obtained in this study provide useful information for a greater understanding and prediction of the behavior of OVDs in the eye during surgery.

Evaluation of anticancer agents using patient-derived tumor organoids characteristically similar to source tissues.

Patient-derived tumor xenograft models represent a promising preclinical cancer model that better replicates disease, compared with traditional cell culture; however, their use is low-throughput and costly. To overcome this limitation, patient-derived tumor organoids (PDOs) were established from human lung, ovarian and uterine tumor tissues, among others, to accurately and efficiently recapitulate the tissue architecture and function. PDOs were able to be cultured for >6 months, and formed cell clusters with similar morphologies to their source tumors. Comparative histological and comprehensive gene expression analyses proved that the characteristics of PDOs were similar to those of their source tumors, even following long-term expansion in culture. At present, 53 PDOs have been established by the Fukushima Translational Research Project, and were designated as Fukushima PDOs (F­PDOs). In addition, the in vivo tumorigenesis of certain F­PDOs was confirmed using a xenograft model. The present study represents a detailed analysis of three F­PDOs (termed REME9, 11 and 16) established from endometrial cancer tissues. These were used for cell growth inhibition experiments using anticancer agents. A suitable high-throughput assay system, with 96- or 384­well plates, was designed for each F­PDO, and the efficacy of the anticancer agents was subsequently evaluated. REME9 and 11 exhibited distinct responses and increased resistance to the drugs, as compared with conventional cancer cell lines (AN3 CA and RL95-2). REME9 and 11, which were established from tumors that originated in patients who did not respond to paclitaxel and carboplatin (the standard chemotherapy for endometrial cancer), exhibited high resistance (half-maximal inhibitory concentration >10 µM) to the two agents. Therefore, assay systems using F­PDOs may be utilized to evaluate anticancer agents using conditions that better reflect clinical conditions, compared with conventional methods using cancer cell lines, and to discover markers that identify the pharmacological effects of anticancer agents.

Evaluation system for arrhythmogenic potential of drugs using human-induced pluripotent stem cell-derived cardiomyocytes and gene expression analysis.

In recent years, human-induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs) have been widely used to develop evaluation systems for drug cardiotoxicity, including the arrhythmia caused by QT prolongation. To accurately assess the arrhythmogenic potential of drugs, associated with QT prolongation, we developed an evaluation system using hiPS-CMs and gene expression analysis. hiPS-CMs were treated with 8 arrhythmogenic and 17 non-arrhythmogenic drugs at several concentrations for 24 hr to comprehensively analyze gene expression. The results showed that 19 genes were upregulated in the arrhythmogenic drug-treated cells compared with their expression levels in the non-treated and non-arrhythmogenic drug-treated cells. The arrhythmogenic risks of the drugs were evaluated by scoring gene expression levels. The results indicated that arrhythmogenic risks could be inferred when cells were treated at a concentration 100 times higher than the maximum blood concentration of the drug. Thus, we succeeded in developing a system for evaluation of the arrhythmogenic potential of drugs using gene expression analysis.

Construction of a novel cell-based assay for the evaluation of anti-EGFR drug efficacy against EGFR mutation.

Epidermal growth factor receptor (EGFR) overexpression and EGFR-mediated signaling pathway dysregulation have been observed in tumors from patients with various cancers, especially non-small cell lung cancer. Thus, several anti-EGFR drugs have been developed for cancer therapy. For patients with known EGFR activating mutations (EGFR exon 19 in-frame deletions and exon 21 L858R substitution), treatment with an EGFR tyrosine kinase inhibitor (EGFR TKI; gefitinib, erlotinib or afatinib) represents standard first-line therapy. However, the clinical efficacy of these TKIs is ultimately limited by the development of acquired drug resistance such as by mutation of the gatekeeper T790 residue (T790M). To overcome this acquired drug resistance and develop novel anti-EGFR drugs, a cell-based assay system for EGFR TKI resistance mutant-selective inhibitors is required. We constructed a novel cell-based assay for the evaluation of EGFR TKI efficacy against EGFR mutation. To this end, we established non-tumorigenic immortalized breast epithelial cells that proliferate dependent on EGF (MCF 10A cells), which stably overexpress mutant EGFR. We found that the cells expressing EGFR containing the T790M mutation showed higher resistance against gefitinib, erlotinib and afatinib compared with cells expressing wild-type EGFR. In contrast, L858R mutant-expressing cells exhibited higher TKI sensitivity. The effect of T790M-selective inhibitors (osimertinib and rociletinib) on T790M mutant-expressing cells was significantly higher than gefitinib and erlotinib. Finally, when compared with commercially available isogenic MCF 10A cell lines carrying introduced mutations in EGFR, our EGFR mutant-overexpressing cells exhibited obviously higher responsiveness to EGFR TKIs depending on the underlying mutations because of the higher levels of EGFR phosphorylation in the EGFR mutant-overexpressing cells than in the isogenic cell lines. In conclusion, we successfully developed a novel cell-based assay for evaluating the efficacy of anti-EGFR drugs against EGFR mutation.

MUC5AC protects pancreatic cancer cells from TRAIL-induced death pathways.

We have previously reported that a specific siRNA transfected MUC5AC could knockdown MUC5AC expression and suppress in vivo tumor growth and metastasis, although it had no effects on in vitro cell growth, cell survival, proliferation and morphology. In the present study, we investigated which host immune cells induced these effects and how the effects were induced using immunocyte-depleted animal models. The tumor growth of SW1990/si-MUC5AC cells, which show no tumor growth when implanted subcutaneously into a nude mouse, was recovered when neutrophils were removed by anti-Gr-1 mAb administration. This result suggests that MUC5AC may suppress the antitumor effects of neutrophils by allowing tumor cells to escape the host immune system. Subsequently, we investigated the effects of MUC5AC on apoptosis induction mediated by TNF-related apoptosis-inducing ligand (TRAIL), one of the antitumor mechanisms of neutrophils. SW1990/si-MUC5AC cells showed significantly increased active caspase 3 expression after the addition of TRAIL. On the other hand, SW1990/si-mock cells showed no such changes. Our results indicate that MUC5AC inhibits TRAIL­induced apoptosis in human pancreatic cancer and may serve as an important indicator in diagnosis and prognosis.

Tumor-associated MUC5AC stimulates in vivo tumorigenicity of human pancreatic cancer.

MUC5AC, a high molecular weight glycoprotein, is overexpressed in the ductal region of human pancreatic cancer but is not detectable in the normal pancreas, suggesting its association with disease development. In the present study, we investigated the in vitro and in vivo effects of MUC5AC knockdown by short interfering RNA (siRNA) in the MUC5AC-overexpressing SW1990 and BxPC3 human pancreatic cancer cell lines in order to clarify its function. Significant decreases in the expression levels of MUC5AC mRNA and protein were observed in SW1990 and BxPC3 cells that had been stably transfected with a MUC5AC siRNA expression vector (SW1990/si-MUC5AC and BxPC3/si-MUC5AC cells) compared to those in cells transfected with an si-mock vector (SW1990/si-mock and BxPC3/si-mock cells). In in vitro studies, neither type of MUC5AC-knockdown cell showed any difference in cell survival, proliferation, or morphology from the si-mock cells or parental cells. However, in vivo xenograft studies demonstrated that MUC5AC knockdown significantly reduced the tumorigenicity and suppressed the tumor growth of si-MUC5AC cells compared to those of the si-mock cells. Immunohistochemical analysis revealed that CD45R/B220+ and Gr-1+ cells had infiltrated into the tumor tissue of the SW1990/si-MUC5AC cells. Furthermore, cancer-associated antigen specific antibodies were detected at high levels in the sera from the SW1990/si-MUC5AC cell-bearing mice. These results suggest that tumor-associated MUC5AC expressed on the surface of pancreatic cancer cells supports the escape of pancreatic cancer cells from immunosurveillance. The present findings highlight a new dimension of MUC5AC as a functional immunosuppressive agent and its important role in pancreatic cancer progression.

Anti-protein-bound polysaccharide-K monoclonal antibody binds the active structure and neutralizes direct antitumor action of the compound.

Protein-bound polysaccharide K (PSK) is extracted and purified from Coriolus versicolor (CM101), and is used as an anti-cancer agent. In this study, focusing on the direct actions of PSK, we investigated whether PSK reaches tumor and immune tissues with its active structure remaining intact, and the direct action of PSK was evaluated by its antitumor effects against MethA fibrosarcomas implanted in immunodeficient NOD/SCID mice. The results obtained suggest that PSK reaches the tumor tissue in its active form and exhibits antitumor effects against MethA cells.

Absorption and tissue distribution of an immunomodulatory alpha-D-glucan after oral administration of Tricholoma matsutake.

Alpha-D-glucan (MPG-1) separated from Tricholoma matsutake (CM6271) has been reported to show immunomodulatory activities. In this study, the plasma concentration and tissue distribution of MPG-1 after CM6271 oral administration were investigated as part of the action mechanism analysis. When CM6271 was orally administered in a single dose to mice, MPG-1 was absorbed via the intestinal tract, appeared in plasma after 16 h, was gradually excreted from the blood, and fell to background level after 48 h. The time course analysis of MPG-1 in plasma showed the following pharmacokinetic parameters of MPG-1: tmax = 24 h; Cmax = 161.1 ng/mL; AUC(0-infinity) = 2559.7 ng x h/mL. Moreover, MPG-1 was confirmed to localize in Peyer's patches, mesenteric lymph nodes (MLN), and the spleen and to promote IL-12 p70 production and NK cell activity. These results suggest that MPG-1 stimulated the intestinal immune system through Peyer's patches; moreover, it was taken into the blood and stimulated the systemic immune system.

Development of an enzyme-linked immunosorbent assay to detect an immunomodulatory alpha-D-glucan-protein complex, MPG-1, in basidiomycete Tricholoma matsutake and related processed foods.

We previously isolated a novel immunomodulatory alpha-(1,4)(1,6)(1,2)- d-glucan-protein complex (MPG-1) from mycelia of Tricholoma matsutake in basidiomycetes. In the present study, we raised a polyclonal antibody by immunizing rabbits with MPG-1 and constructed a sandwich enzyme-linked immunosorbent assay (ELISA) system to examine the distribution of MPG-1 among edible mushrooms and related processed foods. The system detected MPG-1 quantitatively at concentrations of more than 10 ng/mL, with a coefficient of variation of less than 10% by intra-assay and interassay precision. Analysis with the system of chemically modified MPG-1 suggested that the sugar moiety was mainly involved in the detection. The system detected MPG-1 in the extracts of the fruiting bodies of T. matsutake but not in those of 34 other basidiomycete species. Moreover, a significant amount of MPG-1 was detected in the extracts of their cultured mycelia. The antigenic structure of MPG-1 was relatively stable in terms of pH and temperature. MPG-1 was detected in processed foods supplemented with T. matsutake. These results suggest that MPG-1 is distributed predominantly in T. matsutake species and that the ELISA system can detect it in processed foods supplemented with T. matsutake.

Isolation and characterization of a novel immunomodulatory alpha-glucan-protein complex from the mycelium of Tricholoma matsutake in basidiomycetes.

Tricholoma matsutake, a high-class edible mushroom in Japan, has been reported to have excellent biological activities, but difficulty in cultivating the fruit bodies and limited bulk availability have restricted detailed studies. We have developed a method of culturing in tanks, enabling the bulk supply of the mycelia. The preparation (CM6271) exerts modulative effects on the immune competence of mice and rats. In this study, a sodium hydroxide extract of CM6271 was defatted followed by fractionation with a combination of ion exchange chromatography and gel filtration in order to identify the components involved in the expression of the activity, and a single peak fraction (MPG-1) was obtained with reversed phase chromatography. MPG-1 was a glycoprotein (sugar:protein ratio, 94.3:5.7) with a relative molecular mass of 360 kDa, and the sugar moiety contained about 90% glucose. NMR spectra and methylation analysis revealed that the alpha-1,4-linkage was the predominant glucan linkage with alpha-1,6- and alpha-1,2-linkages in the minority. The amino acid composition in the protein moiety was rich in glutamine, alanine, asparagine, leucine, glycine, valine, serine, threonine, isoleucine, and proline. MPG-1 was resistant to degradation with amylase or protease. The oral administration of MPG-1 promoted, in a dose-dependent manner, the recovery of the mouse natural killer cell activity and serum IL-12 level that had been reduced by the loading of restraint stress. The dose of MPG-1 (25 mg/kg) required for the expression of the effect decreases to 1/12 of that of CM6271 (300 mg/kg). Furthermore, MPG-1 formed a complex with TGF-beta1 in vitro, modulating the biological activity of TGF-beta1 by binding to its active form. These results indicate that the mycelium of T. matsutake contains a novel alpha-glucan-protein complex with immunomodulatory activities.

Inhibition of decrease in natural killer cell activity in repeatedly restraint-stressed mice by a biological response modifier derived from cultured mycelia of the basidiomycete Tricholoma matsutake.

OBJECTIVE: To develop a method to cope with stress-induced reduction in immunocompetence, we evaluated the immunomodulatory activities of a biological response modifier derived from the mycelia of the basidiomycete Tricholoma matsutake (CM6271) in mice under repeated restraint stress. METHODS: C57BL/6 mice were inserted, one per tube, into 50-ml polypropylene tubes into which more than 30 ventilation holes had been drilled, and were restrained everyday for 20 days in this fashion for set periods of time. Natural killer (NK) cell activity and NK1.1-positive cell counts in the spleen, ACTH and corticosterone levels in the blood were determined. CM6271 was orally administered daily during the restraint stress period. RESULTS: (1) When the mice were restrained in a confined space for 6 h per day for 20 days, the NK cell activity and the NK1.1-positive cell counts in the spleen significantly decreased after day 5 with an increase in the blood ACTH and corticosterone levels. (2) Oral administration of CM6271 during the restraint stress period significantly prevented the stress-induced decrease in NK cell activity. The effect was dependent on the timing, duration, and doses administered. (3) CM6271 did not significantly affect the splenic NK1.1-positive cell counts or the levels of blood ACTH and corticosterone in restraint-stressed mice. CONCLUSION: The above findings suggest that CM6271 inhibits the restraint stress-induced decrease of NK cell activity in a timing of administration and dose-dependent manner.