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This study aimed to establish a second national standard for antithrombin (AT) concentrate that can be used for potency assays of AT products. A collaborative study was conducted involving four laboratories, including national control laboratories and manufacturers in Korea, and the suitability of a candidate material to serve as the second national standard for AT concentrate was evaluated. The candidate material was manufactured using a process approved for Good Manufacturing Practices. The potency of the candidate was determined using the heparin cofactor chromogenic method. The candidate was calibrated against the third International Standard for AT concentrate (code 06/166). Participants provided data from 58 independent assays. Combined potency estimates were calculated by determining the geometric means of the results obtained from all assays performed at individual laboratories. The overall potency assessments were subsequently established as the geometric means of all results collected from all laboratories. According to the collaborative study results, the intra- and inter-laboratory variability showed acceptable geometric coefficient of variation of 1.2 %-3.4 % and 2.4 % respectively, and it deemed to serve as the Korean national standard for AT concentrate with the assigned potency as follows: 32.1 IU/vial (95 % confidence interval; 31.7-32.5 IU/vial)., Competing Interests: Declarations of interest None., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Plasma-derived medicinal products (PDMPs) are essential in the treatment of acute and chronic life-threatening diseases. The Korea Ministry of Food and Drug Safety has conducted a national lot release (NLR) of PDMPs since 2012 based on a summary protocol review system and lot release testing. However, few studies have investigated the performance or characteristics of the NLR framework. Over the past decade, the NLR of PDMPs was approximately 1000 per year, including mainly albumins, immunoglobulins, fibrin sealant kits, and coagulation factors, among others. The NLR system for PDMPs is similar to that for vaccines, except that PDMPs are manufactured using human plasma, which requires strict safety management. This study describes the status of NLR procedures for PDMPs and outlines the regulatory requirements needed to safely manage plasma for fractionation in Korea. This study can aid national control laboratories and marketing authorization holders in developing regulatory systems that assure the availability of safe and effective PDMPs., Competing Interests: Declaration of competing interest None., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The Eighth Asian National Control Laboratory (NCL) Network meeting, entitled "Biological Products Quality Control and Self-Sufficiency Strategy focusing on plasma-derived medicinal products (PDMPs)" was held in Seoul on 31 August 2023. The participants were NCL experts from Indonesia, Japan, Malaysia, the Philippines, Vietnam, and the Republic of Korea. Special lectures included the PDMPs self-sufficiency strategies of the World Health Organization (WHO) and Indonesian Food and Drug Authority, and a case study on Global Benchmarking Tool (GBT) assessment for vaccines by the Korea Ministry of Food and Drug Safety. The NCL delegates shared their current experiences with national lot releases and biological standardisation. The meeting contributed to a mutual understanding of the progress of the PDMPs self-sufficiency among Asian countries, the WHO's support strategies, and the NCL's plan for the preparation of the WHO GBT assessment. In the panel discussion, all participants agreed that building capacity in blood safety in the Asian region and harmonisation of relevant international regulatory requirements will support appropriate emergency preparedness, particularly source materials in the region, and will build the foundation for resolving the PDMPs supply insecurity that has worsened after the COVID-19 pandemic in some countries., (Copyright © 2024. Published by Elsevier Ltd.)

This study aimed to establish a second national standard for hepatitis B immunoglobulin (HBIG) that can be used for potency assays of hepatitis B and normal immunoglobulin. The candidate material was manufactured using a process approved as Good Manufacturing Practice. The freeze-dried candidate preparation was tested for physicochemical and biological properties, including pH, residual moisture, molecular size distribution, and potency. A collaborative study was performed involving four laboratories, including the National Institute of Food and Drug Safety Evaluation, as an official national control laboratory in Korea and manufacturers. The potency was calibrated against the second international standard for HBIG using two enzyme immunoassays: enzyme-linked immunosorbent assay and electrochemiluminescence immunoassay. Results from 240 assays were obtained from four laboratories, and combined potency estimates were obtained by calculating the geometric means. Intra- and inter-laboratory variability showed acceptable geometric coefficients of variation of 1.3-6.0 and 3.2-3.6%, respectively. The candidate preparation showed satisfactory stability in accelerated thermal degradation and real-time stability tests. Based on these results, the potency value of 105 IU/vial was assigned (95% confidence intervals: 100.0-109.2 IU/vial), and it was deemed suitable to serve as the Korean national standard for HBIG., Competing Interests: Declarations of competing interest None., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The embryonic stem cell test (EST) is a validated in vitro embryotoxicity test; however, as the inhibition of cardiac differentiation alone is used as a differentiation endpoint in the EST, it may not be a useful test to screen embryotoxic chemicals that affect the differentiation of noncardiac tissues. Previously, methylmercury (MeHg), cadmium and arsenic compounds, which are heavy metals that induce developmental neurotoxicity in vivo, were misclassified as nonembryotoxic with the EST. The aim of this study was to improve the EST to correctly screen such developmental neurotoxicants. We developed a neuronal endpoint (Tuj-1 ID₅₀) using flow cytometry analysis of Tuj-1-positive cells to screen developmental neurotoxicants (MeHg, valproic acid, sodium arsenate and sodium arsenite) correctly using an adherent monoculture differentiation method. Using Tuj-1 ID₅₀ in the EST instead of cardiac ID₅₀, all of the tested chemicals were classified as embryotoxic, while the negative controls were correctly classified as nonembryotoxic. To support the validity of Tuj-1 ID₅₀) , we compared the results from two experimenters who independently tested MeHg using our modified EST. An additional neuronal endpoint (MAP2 ID₅₀), obtained by analyzing the relative quantity of MAP2 mRNA, was used to classify the same chemicals. There were no significant differences in the three endpoint values of the two experimenters or in the classification results, except for isoniazid. In conclusion, our results indicate that Tuj-1 ID₅₀ can be used as a surrogate endpoint of the traditional EST to screen developmental neurotoxicants correctly and it can also be applied to other chemicals., (Copyright © 2011 John Wiley & Sons, Ltd.)

The Asia-Pacific Economic Cooperation (APEC) Harmonization Center (AHC) was established in 2009 with the purpose of promoting harmonization of regulatory processes for drugs and medical devices. The AHC held three training workshops on multiregional clinical trials (MRCTs); these workshops provided forums for discussing the value and potential benefits of MRCTs. Participants from regulatory agencies, the pharmaceutical industry, and academia identified many issues and made recommendations for resolving major challenges with the aim of improving the capacity of the Asia-Pacific region to carry out MRCTs.

Embryonic stem cell (ESC) research gave rise to the possibility that stem cell therapy could be used in the treatment of incurable diseases such as neurodegenerative disorders. However, problems related to the tumorigenicity of undifferentiated ESCs must be resolved before such cells can be used in the application of cell replacement therapies. In the present study, we attempted to determine biomarkers that predicted tumor formation of undifferentiated ESCs in vivo. We differentiated mouse ESCs (R1 cell line) into neural lineage using a 5-step method, and evaluated the expression of oncogenes (p53, Bax, c-myc, Bcl2, K-ras), telomerase-related genes (TERT, TRF), and telomerase activity and telomere length during differentiation of ESCs. The expression of oncogenes did not show a significant change during differentiation steps, but the expression of telomerase reverse transcriptase (TERT) and telomerase activity correlated with mouse ESCs differentiation. To investigate the possibility of mouse TERT (mTERT) as a biomarker of tumorigenicity of undifferentiated ESCs, we established mTERT knockdown ESCs using the shRNA lentivirus vector and evaluated its tumorigenicity in vivo using nude mice. Tumor volumes significantly decreased, and appearances of tumor formation in mice were delayed in the TERT-knockdown ESC treated group compared with the undifferentiated ESC treated group. Altogether, these results suggested that mTERT might be potentially beneficial as a biomarker, rather than oncogenes of somatic cells, for the assessment of ESCs tumorigenicity.

β-Glucans have been known to exhibit antitumor activities by potentiating host immunity by an unknown mechanism. The C-type lectin dectin-1, a β-glucan receptor, is found on the macrophage and can recognize various β-glucans. Previously, we demonstrated the presence of β-glucan receptor, dectin-1, on the Raw 264.7 cells as well as on murine mucosal organs, such as the thymus, the lung, and the spleen. In order to investigate immunopotentiation of innate immunity by β-glucan, we stimulated a murine macrophage Raw 264.7 cell line with β-glucans from Pleurotus ostreatus, Saccharomyces cerevisiae, and Laminaria digitata. Then, we analyzed cytokines such as tumor necrosis factor (TNF)-α and interleukin (IL)-6 by reverse transcription-polymerase chain reaction (RT-PCR). In addition we analyzed gene expression patterns in β-glucan-treated Raw 264.7 cells by applying total mRNA to cDNA microarray to investigate the expression of 7,000 known genes. When stimulated with β-glucans, the macrophage cells increased TNF-α expression. When co-stimulation of the cells with β-glucan and lipopolysaccharide (LPS), a synergy effect was observed by increased TNF-α expression. In IL-6 expression, any of the β-glucans tested could not induce IL-6 expression by itself. However, when co-stimulation occurred with β-glucan and LPS, the cells showed strong synergistic effects by increased IL-6 expression. Chip analysis showed that β-glucan of P. ostreatus increased gene expressions of immunomodulating gene families such as kinases, lectin associated genes and TNF-related genes in the macrophage cell line. Induction of TNF receptor expression by FACS analysis was synergized only when co-stimulated with β-glucan and LPS, not with β-glucan alone. From these data, β-glucan increased expressions of immunomodulating genes and showed synergistic effect with LPS.

Human mesenchymal stem cells (hMSCs) have generated a great deal of interest in clinical applications. The reason is that they may have the plasticity needed to differentiate into multiple lineages and the ability to expand ex vivo. For the therapeutic applications of hMSCs to be of practical use, it is crucial to assess the efficacy and safety of hMSCs in long-term ex vivo expansion. In this study, we cultured hMSCs by population doubling (PD) 60, and investigated their growth, osteogenic and adipogenic differential abilities, change of surface markers, telomerase activity, telomere length, and gene expression related to tumorigenesis. An in vivo tumorigenesis assay was also carried out. In long-term expanded hMSCs, the cells became aged above PD 30 and their adipogenic and osteogenic differentiation potential decreased. Telomerase activity unchanged whereas telomere length decreased and karyotypes were not changed. Gene expressions related to tumorigenesis decreased in proportion as the PD of hMSCs increased. In vivo transplantation of long-term cultured hMSCs to nude mice did not result in tumor formation. These findings suggest that diverse tests for cellular therapy should be considered during the ex vivo culture of hMSCs, particularly when a prolonged and extended propagation period is required.

The molecular mechanisms that couple growth arrest and cell differentiation were examined during adipogenesis. Here, to understand the cyclin-dependent kinase inhibitor (CKI) genes involved in the progression of adipogenic differentiation, we examined changes in the protein and mRNA expression levels of CKI genes in vitro. During the onset of growth arrest associated with adipogenic differentiation, two independent families of CKI genes, p27Kip1 and p18INK4c, were significantly increased. The expressions of p27Kip1 and p18INK4c, regulated at the level of protein and mRNA accumulation, were directly coupled to adipogenic differentiation. This finding was supported by the inhibition of adipogenic differentiation caused by short interfering RNA (siRNA). In this study, we investigated the regulatory effects of transforming growth factor beta-1 (TGFbeta-1) on CKI genes involved in adipogenic differentiation of bone marrow-derived human mesenchymal stem cells (hMSCs). Only the up-regulation of p18INK4c during adipogenic differentiation, and not that of the p27Kip1 gene was prevented by treatment with TGFbeta-1, one of the factors that inhibit adipogenesis in vitro. This finding indicates a close correlation between adipogenic differentiation and p18INK4c induction in hMSCs. Thus, these data demonstrate a role for the differentiation-dependent cascade expression of cyclin-dependent kinase inhibitors in regulating adipogenic differentiation, thereby providing a molecular mechanism that couples growth arrest and differentiation.

Purpose: The aim of this study was to evaluate the therapeutic effect and morphological alterations resulting from (166)Ho-chitosan complex (DW-166HC) in an animal model of prostate cancer., Methods: First, in a subcutaneous tumor model, 80 rats were randomly divided into four groups (n=20 in each group), and intratumoral injections of 0.05 ml (normal saline in group 1,( 165)Ho-chitosan complex solution in group 2, DW-166HC solution (10 mCi) in group 3, and DW-166HC solution (20 mCi) in group 4) were performed when the tumor measured approximately 1 cm along its long axis in each group. Further, in an orthotopic tumor model, 40 rats were similarly randomly divided into four groups (n=10 in each group), and intraprostatic injections of 0.05 ml [PBS in group 1,( 165)Ho-chitosan complex solution in group 2, DW-166HC solution (0.5 mCi) in group 3 and DW-166HC solution (1 mCi) in group 4] were performed at 1 week after implantation of the AIT cell line in the ventral prostate., Results: In the subcutaneous tumor model, mean tumor weights of groups 3 and 4 were significantly lower than those of groups 1 and 2 at 2 and 4 weeks post injection (p<0.05). At 2 and 4 weeks after injection in the orthotopic tumor model, the mean weights of the prostate, including tumor, in groups 3 and 4 were also significantly lower than those in groups 1 and 2 (p<0.05). No adverse injury was seen in adjacent organs at histopathologic examination., Conclusion: Intratumoral injection of the beta-emitting radionuclide (166)Ho as a form of complex solution with chitosan appears to be a promising alternative therapeutic modality for the local control of prostate cancer.

The regional distributions and frequencies of argyrophil endocrine cells in gastrointestinal (GI) tract of Balb/c-nu/ nu mouse were studied using Grimelius silver stain after abdominal subcutaneous implantation of COLO205. The experimental animals were divided into two groups, one is non-implanted group (Sham) and the other is COLO205- implanted group. Samples were collected from GI tract (fundus, pylorus, duodenum, jejunum, ileum, cecum, colon and rectum) at 21 days after implantation of COLO205 cells (1 x 10(6) cell/mouse). In this study, argyrophil cells were detected throughout the entire GI tract with various frequencies regardless of implantation. Most of these argyrophil cells in the mucosa of GI tract were generally spherical or spindle in shape (open type cell) while cells showing round in shape (close type cell) were found occasionally in gastric and/or intestinal gland regions. The regional distributions of argyrophil cells in COLO205 were similar to those of Sham. However, significant decreases of argyrophil cells were detected in COLO205 compared to those of Sham except for the jejunum and ileum. In the jejunum and ileum, argyrophil cells in COLO205 showed similar frequencies compared to those of Sham. In the pylorus, the most dramatically decreasement of argyrophil cells were detected in COLO205 compared to that of Sham. Implantation of COLO205 tumor cell line induced severe quantitative changes of argyrophil cell density, and the abnormality in density of GI endocrine cells may contribute to the development of gastrointestinal symptoms such as anorexia and indigestion, frequently encountered in patients with cancer.

Aim: To study the distributions and frequencies of intestinal endocrine cells in the C57BL/6 mouse with immunohistochemical method using seven types of specific antisera against chromogranin A (CGA), serotonin, somatostatin, glucagons, gastrin, cholecystokinin (CCK)-8 and human pancreatic polypeptide (hPP) after abdominal subcutaneous implantation of murine lung carcinoma (3LL)., Methods: The experimental animals were divided into two groups, one is non-implanted Sham and the other is 3LL-implanted group. Samples were collected from six regions of intestinal tract at 28(th) d after implantation of 3LL cells (1X10(5) cell/mouse)., Results: In this study, five types of immunoreactive (IR) cells were identified except for gastrin and hPP. The regional distributions of the intestinal endocrine cells in the 3LL-implanted group were similar to those of the non-implanted Sham. However, significant decreases of IR cells were detected in 3LL-implanted group compared to those of non-implanted Sham. CGA- and serotonin-IR cells significantly decreased in 3LL-implanted groups compared to that of non-implanted Sham. Somatostatin-IR cells in the jejunum and ileum and CCK-8-IR cells in the jejunum of 3LL-implanted groups significantly decreased compared to that of non-implanted Sham. In addition, glucagon-IR cells were restricted to the ileum and colon of non-implanted Sham., Conclusion: Implantation of tumor cell mass (3LL) induced severe quantifiable changes of intestinal endocrine cell density and the abnormality in density of intestinal endocrine cells may contribute to the development of gastrointestinal symptoms such as anorexia and indigestion, frequently encountered in patients with cancer.

Purpose: The objective of this study was to determine the fate of the 166Ho-chitosan complex (DW-166HC) in rats by examining its absorption, distribution and excretion after administration into the prostate., Methods: About 100 microCi of DW-166HC [containing 0.1875 mg of Ho(NO3)3.5H2O and 0.25 mg of chitosan] was administered intraprostatically. The level of radioactivity in blood, urinary and faecal excretion, and radioactivity distribution were examined. To determine the effect of chitosan in DW-166HC, 166Ho nitrate alone [0.1875 mg of Ho(NO3)3.5H2O] was administered into the prostate of male rats, and radioactivity distribution was examined using whole-body autoradiography., Results: After administration of DW-166HC into the prostate, cumulative urinary and faecal excretion over the period 0-72 h was 0.35% and 0.11%, respectively. The radioactivity at the administration site was extremely high at all time points up to 144 h (>98% of injected dose). The small amount of radioactivity which did transfer from the administration site distributed mainly to the liver, spleen, kidney cortex and bone. Compared with the DW-166HC group, the group that received 166Ho nitrate alone displayed three- to fourfold higher levels of radioactivity in the main tissues, including liver, spleen, kidney cortex and bone, at 24 h after administration (P < 0.05)., Conclusion: The results of this study show clearly that most of the administered DW-166HC remained at the administration site. It is concluded that the chitosan complex may be used to retain 166Ho within a limited area in cancer of the prostate.

The novel 1-(1-benzoylindoline-5-sulfonyl)-4-phenyl-4,5-dihydroimidazolones 2 shows highly potent and broad cytotoxicities. Their cytotoxicities against human lung carcinoma A549, human chronic myelogenous leukemia K562, and human ovarian adenocarcinoma SK-OV-3 are compatible with doxorubicin. Compound 2p (1-[(4-aminobenzoyl)indoline-5-sulfonyl])-4-phenyl-4,5-dihydroimidazolone) exhibits a cytotoxicity that is far more potent than doxorubicin and also exhibits highly effective antitumour activities against murine (3LL, Colon 26) and human xenograft (NCI-H23, SW620) tumor models.

Background: In the preambles to the safety and health standards that it has issued since 1987, the Occupational Safety and Health Administration (OSHA) projected that new safety standards would prevent over 2,600 death each year. For six safety standards issued since 1990, we compare OSHA's projections of the impact of full compliance on fatalities with actual fatality changes and examine the reasons for the differences., Methods: We reviewed the preambles to OSHA standards and the Regulatory Impact Analyses (RIAs) prepared for them to identify the baseline and the prevention factor that the agency used to project the number of deaths that would be prevented. We used three data sources to track the relevant categories of fatalities: the Census of Fatal Occupational Injuries (CFOI), the National Traumatic Occupational Fatality program, and OSHAs Fatality/Catastrophe investigations., Results: For all six standards, OSHA appeared to overestimate the number of deaths prevented. The availability of CFOI led to better estimates of the fatality baseline, but the prevention factor was always overestimated, especially for standards which emphasized training., Conclusions: OSHA needs to develop better methods for projecting injury impacts. Research is needed to help OSHA predict the effects of behavioral requirements (e.g., training) on actual work practices and injury outcomes. For non-fatal injuries, new methods of data collection will be required., (Copyright 2004 Wiley-Liss, Inc.)

DW2282 [(S)-(+)-4-phenyl-1-[N-(4-aminobenzoyl) indoline-5-sulfonyl]-4,5-dihydro-2-imidazolone].hydrochloride] was derived from diarylsulfonylurea and was identified as a prominent new anticancer agent. We examined the characteristics of DW2282 activity on the proliferation of human lung carcinoma cells, A549 and human leukemic cells, K562. DW2282 effectively inhibited cancer cell proliferation in vitro. Colony forming assay and viability tests demonstrated that DW2282 is a cytotoxic agent rather than a cytostatic agent. The isotope uptake test exhibited that DW2282 inhibited or inactivated protein synthesis. Also, under conditions which cause RNA or protein synthesis inhibition, by co-treatment with actinomycin D or cycloheximide, reduced the anticancer effects of DW2282. This means that the cytotoxicity of DW2282 depends partially on RNA or protein synthesis and proteins affected by DW2282 may inactivate or alter the process of the synthesis of another protein. DW2282 activity was highly diminished in the presence of colcemid, a metaphase spindle blocker. This result suggests that DW2282 may be related to the cell cycle. After exposure to DW2282, morphologically apoptotic cells appeared in A549 cells and fragmented DNA was detected in K562 cells. It demonstrated that apoptosis is one of the mechanisms by which DW2282 inhibits the proliferation of A549 and K562 cells.

We examined the ability of sulfonylurea derivative, DW2143 (4-phenyl-1-[1-(4-aminobenzoyl)-indoline-5-sulfonyl]-4,5-dihydro-2-imida zolone hydrochloride), to inhibit the growth of tumor cells in vitro and in vivo. When its anti-proliferative activities were tested on five murine tumor (B 16, Colon26, E1-4, 3LL and P388) and nine human tumor (BxPC-3, HepG2, Lovo, MCF-7, NCI-H69, SW480, WiDR, KB and KBV20C) cells of diverse tissue origins, the in vitro antitumor activities of DW2143 were comparable to those of doxorubicin against all tumor cell lines. In addition, the anti-proliferative activities of DW2143 against KBV20C, a vincristine-resistant cell line, are similar or superior to those of doxorubicin. When the in vivo antitumor activities using three murine tumor cells were tested after oral administration of DW2143, a wide range of tumor growth inhibition was observed. Tumor growth inhibition against 3LL at doses of 50 and 100 mg/kg DW2143 was 84.3% and 47.2%, respectively, which was comparable or superior to those of doxorubicin (5 mg/kg). Tumor growth inhibition of B16 at a dose of 100 mg/kg in the DW2143-treated group was 42% as compared to 54% for doxorubicin (5 mg/kg). When mice implanted with Colon26 were tested, tumor growth inhibition at a dose of 80 mg/kg DW2143 was 36% as compared with 37% for doxorubicin (5 mg/kg). Taken together, these results indicate that the novel sulfonylurea derivative, DW2143, is an attractive candidate for further development as a useful oral anticancer drug.

Novel 1-(1-benzoylindoline-5-sulfonyl)-4-phenyl-4,5-dihydroimidazolon es 3 synthesized show highly potent and broad cytotoxicities. Among them compound 3b (DW2143) exhibits much more potent cytotoxicities than doxorubicin and highly effective antitumor activities against murine (3LL, Colon 26) and human xenograft (NCI-H23, SW620) tumor models.

The activation of Jun/Fos is a crucial factor in transmitting the tumor promoting signal from the extracellular environment to nuclear transcription machinery. One of the final steps in signal transduction is the binding of Jun/Fos to the AP-1 site in order to express gene transcription. Utilizing this concept, we screened about 100 extracts of natural plants to search for a Jun-Fos function inhibitor. The methanol extract of Ampelopsis radix reduced Jun/Foc retardation remarkably. The active principles of the extract were isolated and purified by repeated column chromatography and their structures were identified as oleanolic acid glycosides known as momordin I, Id, and Ie. These compounds reduced the Jun/Fos-DNA interaction and their activities were quantitated with liquid scintillation counting of corresponding bands. Among them, momordin I had the strongest inhibitory activity, with an IC50 value of 22.8 micrograms/ml. The methanol extract and momordin I, Id and Ie also showed cell cytotoxicity against human cancer cell lines. As expected from a gel shift assay, momordin I showed the strongest cytotoxicity and its IC50 value was from 7.280 micrograms/ml to 16.05 micrograms/ml depending on the cell line. With these data, it may be concluded that the mechanism of anticancer activity of momordin I comes from its inhibitory effect on the protein-DNA interaction. The in vivo test was done only with the methanol extract. The extract showed measurable anticancer activity against murine colon cancer. The wet tumor weight reduction rate was 17.73% at 90 mg/kg dose. We suggest that the Jun/Fos-DNA interaction results in cell cytotoxicity.