Thiamine hydrochloride has been suggested as a natural, safe yet effective alternative for chemical insect repellents. However, there is a demand for a reassessment of the minimum required dose that is sufficient to perform a topical repellency on the human skin. Therefore, the purpose of the current work is to establish a dose–response curve from which the effective dose (ED) is calculated. A series of increasing concentrations of thiamine hydrochloride were applied to the forearm of adult volunteers, the number of bites was counted and the percent repellency calculated accordingly. Data of percent repellency were converted to probit values which were plotted against log doses. A linear relation was obtained from the dose–response curve with an r² = 0.958. Statistical validation of the equation was tested through linear regression analysis, where the slope and intercept were found significant from zero. No significant difference was shown between observed and expected responses (p > 0.05). ED 50 and 99.9% were computed from the linear equation and found to be 4.57 and 344 mg, respectively. This finding can be supported by future works in which a proper formulation of thiamine hydrochloride in the respective doses would be presented. One can get prolonged safe protection against insect bites.

Colony stimulating factor 1 (CSF1) receptor (CSF1R) is a receptor protein-tyrosine kinase specifically expressed in monocyte-lineage cells, such as monocytes and macrophages. In this study, we characterized the pharmacological properties of an azetidine compound, JTE-952 ((2S)-3-{[2-({3-[4-(4-cyclopropylbenzyloxy)-3-methoxyphenyl]azetidine-1-yl}carbonyl)pyridin-4-yl]methoxy}propane-1,2-diol), which is a novel CSF1R tyrosine kinase inhibitor. JTE-952 potently inhibited human CSF1R kinase activity, with a half maximal inhibitory concentration of 11.1 nmol/L, and inhibited the phosphorylation of CSF1R in human macrophages and the CSF1-induced proliferation of human macrophages. It also inhibited human tropomyosin-related kinase A activity, but only at concentrations 200-fold higher than that required to inhibit the activity of CSF1R in inducing the proliferation of human macrophages. JTE-952 displayed no marked inhibitory activity against other kinases. JTE-952 potently inhibited lipopolysaccharide-induced proinflammatory cytokine production by human macrophages and in whole blood. JTE-952 (≥3 mg/kg given orally) also significantly attenuated the CSF1-induced priming of lipopolysaccharide-induced tumor necrosis factor-alpha production in mice and arthritis severity in a mouse model of collagen-induced arthritis. Taken together, these results indicate that JTE-952 is an orally available compound with potent and specific inhibitory activity against CSF1R, both in vitro and in vivo. JTE-952 is a potentially clinically useful agent for various human inflammatory diseases, including rheumatoid arthritis.

Oxaliplatin is a first-line clinical drug in cancer treatment and its side effects of peripheral neuropathic pain have also attracted much attention. Neuroinflammation induced by oxidative stress-mediated activation of nuclear factor-kappa B (NF-κB) plays an important role in the course. Current studies have shown that curcumin has various biological activities like antioxidant, anti-inflammatory, antitumor and so on, while few studies were conducted about its role in oxaliplatin-induced peripheral neuropathic pain. The aim of this study is to verify the mechanism of curcumin alleviating oxaliplatin-induced peripheral neuropathic pain. Intraperitoneal injection with oxaliplatin (4 mg/kg body weight) was given to the rats twice a week and last for four weeks to establish the model rats. Gavage administration of curcumin (12.5, 25, and 50 mg/kg body weight, respectively) was conducted for consecutive 28 d to explore the effects and potential mechanism. Our results showed that curcumin administration could increase mechanical withdrawal threshold and decrease the paw-withdrawal times of cold allodynia significantly; meanwhile, motor nerve conduction velocity (MNCV) and sense nerve conduction velocity (SNCV) were both increased and the injured neurons of the spinal cord were repaired. In addition, curcumin administration increased superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), catalase (CAT) and reduced malondialdehyde (MDA). Moreover, the curcumin operation inhibited the activated of NF-κB and level of inflammatory factors like tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6). In conclusion, these findings suggested that curcumin could alleviate oxaliplatin-induced peripheral neuropathic pain; the mechanism might be inhibiting oxidative stress-mediated activation of NF-κB and mitigating neuroinflammation.

The Hippo intracellular signaling pathway plays a pivotal role in cell fate determination. Although previous studies have identified many components of the Hippo pathway, the whole picture of the Hippo network is just beginning to be delineated. Recent discoveries in the past decade have shed light on a newly discovered signaling network where the Hippo pathway interplays with several types of non-coding RNAs, including microRNAs, long non-coding RNAs and circular RNAs, to mediate diverse biological processes. Those non-coding RNAs communicate with each other to maintain cellular homeostasis. In this review article, we summarize the current and emerging understanding of the roles of non-coding RNAs in the regulation of and by the Hippo pathway.

The aim of the present study was to investigate the “chronotoxicity” of streptomycin (SM) in relation to its circadian periodicity. Male ICR mice were injected intraperitoneally with SM (780 mg/kg, one shot) one of six time points throughout the day. Mortality was monitored until 14 d after the injection and clearly differed depending on the timing of the injection (i.e., mice were more sensitive to injection during the dark phase). Moreover, when mice were administered with non-lethal doses of SM (550 mg/kg, every 24 h for 3 d, in the light phase or dark phase), the levels of nephrotoxicity indicators (blood urea nitrogen and renal levels of malondialdehyde and cyclooxygenase-2) were significantly increased by the injection in the dark phase, but not in the light phase. These results suggested that SM showed clear chronotoxicity. Our current data indicated that chronotoxicology may provide valuable information on the importance of injection timings for evaluations of toxicity and undesirable side effects.

Allergic contact dermatitis (ACD) is one of the most common skin diseases caused by hapten-modified proteins. Metformin, a drug commonly prescribed for type II diabetes, has been demonstrated to have various biological functions beyond its antidiabetic effects. However, its role in ACD remains unknown. In the present study, we found that metformin reduced the production of nitric oxide (NO) and the level of proinflammatory cytokines such as tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6 in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. These anti-inflammatory effects were also demonstrated on bone marrow-derived macrophages (BMDMs). Furthermore, metformin also enhanced autophagic flux, inhibited the phosphorylation of the serine/threonine protein kinase (AKT)/mammalian target of rapamycin (mTOR), mitogen-activated protein kinases (MAPKs) related protein levels and the level of miR-221 in LPS-stimulated RAW264.7 cells. Besides, metformin attenuated 2,4-dinitrofluorobenzene (DNFB)-induced ACD and inhibited proinflammatory cytokines in the ear. In addition, metformin ameliorated ACD partly through the inhibition of macrophage activation and the induction of autophagic flux. Taken together, our data indicated that metformin ameliorates ACD through enhanced autophagic flux to inhibit macrophage activation and provides a potential contribution to ACD treatment.

We have recently found that the synthetic curcumin derivative CNB-001 suppresses lipopolysaccharide (LPS)-induced nitric oxide (NO) production in cultured microglia, demonstrating that it exerts anti-neuroinflammatory effects by regulating microglial activation. To explore the molecular mechanisms underlying the anti-inflammatory effect of CNB-001, the present study investigated whether CNB-001 is also effective for microglial NO production induced by other stimulants than LPS. Treatment of primary cultured rat microglia with thrombin, a serine protease that has been proposed as a mediator of cerebrovascular injuries, caused the expression of inducible NO synthase (iNOS) and the production of NO. The thrombin-induced NO production was completely blocked by the presence of SCH-79797, a selective protease-activated receptor 1 (PAR-1) antagonist, suggesting that the effect of thrombin is mediated by PAR-1. CNB-001 (1–10 µM) attenuated the thrombin-induced iNOS expression and NO production without affecting the PAR-1 expression. In addition, thrombin treatment caused rapid phosphorylation of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase (MAPK). The changes in ERK and p38 MAPK were significantly suppressed by the presence of CNB-001. These results demonstrate that CNB-001 suppresses thrombin-stimulated microglial activation by inhibiting the ERK and p38 MAPK pathways.

Pathogenesis-related (PR) proteins are inducible and accumulated in plants upon pathogen challenge for survival. Interest in these proteins has arisen in many fields of research, including areas of protein defense mechanisms and plant-derived allergens. In this study, we cloned a PR protein gene (OJPR) from Oenanthe javanica, which consisted of 465 bp with an approximate molecular mass of 16 kDa. The DNA and deduced amino acid sequences of OJPR were 87% similar to Pimpinella brachycarpa PR-1 together with a glycine-rich loop which is a signature motif of PR-10. In microarray analysis, OJPR-transfected Raw264.7 (OJPR⁺) upregulated high mobility group box 1 and protein kinase Cα, and downregulated chemokine ligand 3 and interleukin 1β which are all related to toll-like receptor 4 (TLR4) and inflammation. TAK-242 and PD98059 inhibited the activation by OJPR, suggesting that OJPR transduce TLR4-mediated signaling. Interestingly, OJPR increased anti-viral repertoires, including interferon (IFN)α, IFNγ, OAS1, and Mx1 in CD4⁺ primary T cells. Taken together, we concluded that OJPR may play a role in modulating host defense responses via TLR signal transduction and provide new insights into the therapeutic and diagnostic advantages as a potential bioactive protein.

Elevated intraocular pressure (IOP) is the major cause of glaucoma, which is the second leading cause of blindness. However, current glaucoma treatments cannot completely regulate IOP and progression of glaucoma. Our group recently found that autotaxin (ATX) activity in human aqueous humor (AH) was positively correlated with increased IOP in various subtypes of glaucoma. To develop new IOP-lowering treatments, we generated a novel ATX inhibitor as an ophthalmic drug by high-throughput screening, followed by inhibitor optimization. Administration of the optimized ATX inhibitor (Aiprenon) reduced IOP in laser-treated mice exhibiting elevated IOP and higher level of ATX activity in AH and normal mice in vivo. The stimulation of ATX induced outflow resistance in the trabecular pathway; however, administration of Aiprenon recovered the outflow resistance in vitro. The in vitro experiments implied that the IOP-lowering effect of Aiprenon could be correlated with the altered cellular behavior of trabecular meshwork (TM) and Schlemm’s canal endothelial (SC) cells. Overall, our findings showed that ATX had major impact in regulating IOP as a target molecule, and potent ATX inhibitors such as Aiprenon could be a promising therapeutic approach for lowering IOP.

Recent clinical studies indicate that sodium–glucose cotransporter 2 (SGLT2) inhibitors exhibit a renoprotective effect. While studies at the single nephron level suggest that direct effects of SGLT2 inhibitors on renal hemodynamics may be a possible mechanism underlying their renoprotective effect, few studies have focused on such direct effects at the whole-kidney level. In the present study, we investigated the acute and direct effect of SGLT2 inhibition on creatinine clearance, an index of whole-kidney glomerular filtration rate (GFR), in a rat model of type 2 diabetes. Twelve to fifteen-week-old male Spontaneously Diabetic Torii (SDT) fatty rats and Sprague-Dawley rats were used as diabetic animals and non-diabetic controls, respectively. Under general anesthesia, baseline urine samples were collected from the left and right ureters for 1 h. The selective SGLT2 inhibitor ipragliflozin or vehicle was subsequently administered intravenously and post-drug urine was collected for 1 h. Baseline and post-drug blood samples were collected immediately before baseline urine collection and immediately after post-drug urine collection, respectively. Plasma glucose, urine volume, urinary glucose and albumin excretion were measured, and creatinine clearance was calculated. Blood pressure and heart rate were monitored continuously throughout the experiment. A single intravenous injection of ipragliflozin increased both urine output and glucose excretion, but reduced creatinine clearance without affecting systemic blood pressure. These results suggest that SGLT2 inhibition directly reduced whole-kidney GFR, most likely due to a reduction in intraglomerular pressure, by altering local renal hemodynamics, which may contribute to the renoprotective effects demonstrated in clinical studies.

Glucose-stimulated insulin secretion is controlled by both exocytosis and endocytosis in pancreatic β-cells. Although endocytosis is a fundamental step to maintain cellular responses to the secretagogue, the molecular mechanism of endocytosis remains poorly defined. We have previously shown that in response to high concentrations of glucose, guanosine 5′-diphosphate (GDP)-bound Rab27a is recruited to the plasma membrane where IQ motif-containing guanosine 5′-triphosphatase (GTPase)-activating protein 1 (IQGAP1) is expressed, and that complex formation promotes endocytosis of secretory membranes after insulin secretion. In the present study, the regulatory mechanisms of dissociation of the complex were investigated. Phosphorylation of IQGAP1 on serine (Ser)-1443, a site recognized by protein kinase Cε (PKCε), inhibited the interaction of GDP-bound Rab27a with IQGAP1 in a Cdc42-independent manner. Glucose stimulation caused a translocation of PKCε from the cytosol to the plasma membrane. In addition, glucose-induced endocytosis was inhibited by the knockdown of IQGAP1 with small interfering RNA (siRNA). However, the expression of the non-phosphorylatable or phosphomimetic form of IQGAP1 could not rescue the inhibition, suggesting that a phosphorylation-dephosphorylation cycle of IQGAP1 is required for endocytosis. These results suggest that IQGAP1 phosphorylated by PKCε promotes the dissociation of the IQGAP1-GDP-bound Rab27a complex in pancreatic β-cells, thereby regulating endocytosis of secretory membranes following insulin secretion.

High-density lipoprotein (HDL) particles that are formed in vivo adopt a disk-shaped structure, in which the periphery of the discoidal phospholipid bilayer is surrounded by apolipoprotein. Such discoidal nanoparticles can be reconstituted with certain apolipoproteins and phospholipids and are commonly called lipid nanodisks. Apolipoprotein E (apoE), one of the HDL constituent proteins, serves as a ligand for the low-density lipoprotein (LDL) receptor. Thus, it is considered that biocompatible delivery vehicles targeting LDL receptors could be prepared by incorporating apoE as the protein component of lipid nanodisks. To enhance targeting efficiency, we designed lipid nanodisks with a large number of ligands using a peptide with the LDL receptor-binding region of apoE combined with a high lipid affinity sequence (LpA peptide). In our study, the LpA peptide spontaneously formed discoidal complexes (LpA nanodisks) of approximately 10 nm in size, equivalent to native HDL. LpA peptides on nanodisks adopted highly α-helical structures, a competent conformation capable of interacting with LDL receptors. As anticipated, the uptake of LpA nanodisks into LDL receptor-expressing cells (HepG2) was higher than that of apoE nanodisks, suggesting an enhanced targeting efficiency via the enrichment of LDL receptor-binding regions on the particle. Biodistribution studies using ¹¹¹In-labeled LpA nanodisks showed little splenic accumulation and prolonged retention in blood circulation, reflecting the biocompatibility of LpA nanodisks. High accumulation of ¹¹¹In-labeled LpA nanodisks was observed in the liver as well as in implanted tumors, which abundantly express LDL receptors. Thus, LpA nanodisks are potential biocompatible delivery vehicles targeting LDL receptors.

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is characterized by disabling fatigue of at least 6 months, in addition to symptoms such as muscle pain and muscle weakness. There is no treatment provides long-term benefits to most patients. Recently, clinical research suggested the involvement of pyruvate dehydrogenase (PDH) in ME/CFS. PDH is a crucial enzyme in the mitochondria matrix that links glycolysis to the tricarboxylic acid cycle and oxidative phosphorylation. However, it is little known whether PDH could be a therapeutic target. The purpose of this study was to establish ME/CFS in mice and to investigate the involvement of PDH in ME/CFS. To induce the chronic fatigue in mice, a repeated forced swimming test was conducted. To evaluate fatigue, we measured immobility time in forced swimming test and starting time of grooming. An open field test was conducted on day 8. After 25 d of the forced swimming test, the mitochondrial fraction in gastrocnemius muscle was isolated and PDH activity was measured. Moreover, we evaluated the effect of PDH activation by administering sodium dichloroacetate (DCA). In ME/CFS mice group, the immobility time and starting time of grooming increased time-dependently. In addition, the moved distance was decreased in ME/CFS mice. PDH activity was decreased in the mitochondrial fraction of the gastrocnemius muscle of the forced swimming group. DCA treatment may be beneficial in preventing fatigue-like behavior in ME/CFS. These findings indicate that ME/CFS model was established in mice and that a decrease in mitochondrial PDH activity is involved with the symptom of ME/CFS.

Leukocyte migration across the blood–brain barrier (BBB) is an important step in the progression of brain dysfunction in systemic inflammation. The purpose of this study was to identify the key regulatory molecule(s) at the BBB among the cluster of differentiation (CD) antigens involved in leucocyte migration in lipopolysaccharide (LPS)-induced systemic inflammation based on their absolute protein expressions. Here, we identified the absolute expression levels of 17 CD antigens in isolated brain capillaries (Bcap) of LPS-administered mice. Among them, the expression levels of CD54 and CD106 were dramatically increased in LPS-administered mice compared to the control by 6.21- and 3.67-fold, respectively. In peripheral blood mononuclear cells, the expression levels of CD11a/CD18, the counter-receptor for CD54, were similar to those of CD54 in Bcap of LPS-administered mice. On the other hand, the expression level of CD49d, part of CD29/CD49d complex, which is the counter-receptor for CD106, was under the limit of quantification. It is thus likely that CD54 at the BBB is predominantly involved in promoting leukocyte migration across the BBB in systemic inflammation. The expression levels of CD9, CD49c and CD97, which are thought to be involved in cell-to-cell interaction, were decreased by 40–60% in Bcap of LPS-administered mice. In contrast, the expression levels of 9 transporters, 2 receptors, and 1 tight junction-related protein in Bcap of LPS-administered mice were essentially unchanged compared to the control. These results suggest that enhancement of leucocyte migration in systemic inflammation involves dynamic changes of CD antigens without alterations of other major functional molecules.

Previously, we reported that coffee extract and its constituents, caffeic acid (CA) and p-coumaric acid, inhibit infection by the hepatitis C virus (HCV). In the present report, we identified another coffee-related compound, tannic acid (TA), which also inhibits HCV infection. We systematically evaluated which steps of the viral lifecycle were affected by CA and TA. TA substantially inhibits HCV RNA replication and egression, while CA does not. The infectivity of the HCV pretreated with CA or TA was almost lost. Cellular attachment of HCV particles and their interaction with apolipoprotein E, which is essential for HCV infectivity, were significantly reduced by CA. These results indicate that CA inhibits HCV entry via its direct effect on viral particles and TA inhibits HCV RNA replication and particle egression as well as entry into host cells. Taken together, our findings may provide insights into CA and TA as potential anti-HCV strategies.

Combination therapy is often an effective strategy to treat cancer. In this study, we examined the growth-inhibitory effects of Am80 (tamibarotene), a specific retinoic acid receptor (RAR) α/β agonist, in combination with a histone deacetylase (HDAC) inhibitor, suberoylanilide hydroxamic acid (SAHA), or a DNA methyl transferase (DNMT) inhibitor, 5-aza-2′-deoxycytidine, on androgen receptor (AR)-positive and AR-negative prostate cancer cell lines (LNCaP and PC-3, respectively). We found that the combination therapy of SAHA and Am80 showed an enhanced growth-inhibitory effect on LNCaP cells. Further studies with various HDAC isotype-selective inhibitors showed that SAHA and KD5170 (a selective class I and II HDAC inhibitor) each increased the RARα protein level in LNCaP cells. Our results indicate that the target of the enhancing effect belongs to the Class IIb HDACs, especially HDAC6. Dual targeting of Class IIb HDAC and RARα may be a candidate therapeutic strategy for prostate cancer.

Hydrogen sulfide (H₂S) is an endogenous gaseous transmitter known to play an important role in biological functions. For the hepatic and intrahepatic targeting of H₂S prodrug at the cellular level, we developed two types of sulfo-albumins, in which five sulfide groups (source of H₂S) were covalently bound to succinylated (Suc) or galactosylated (Gal) bovine serum albumin (BSA). Sulfo-BSA-Suc and polyethylene glycol (PEG)-Sulfo-BSA-Gal, both released H₂S in the 5 mM glutathione solution, but not in the plasma. Sulfo-BSA-Suc and PEG-Sulfo-BSA-Gal were taken up by RAW264.7 cells (mouse macrophage-like cells) and Hep G2 cells (human hepatocellular carcinoma cells), respectively, and H₂S was released. These results indicate that Sulfo-BSA-Suc and PEG -Sulfo-BSA-Gal selectively released H₂S intracellularly. In a biodistribution study, up to 80% of ¹¹¹In-labeled Sulfo-BSA-Suc and PEG-Sulfo-BSA-Gal rapidly accumulated in the liver, 30 min after intravenous injection in mice. Furthermore, ¹¹¹In-labeled Sulfo-BSA-Suc and PEG-Sulfo-BSA-Gal predominantly accumulated in liver nonparenchymal (endothelial cells and Kupffer cells) and parenchymal cells (hepatocytes), respectively. These findings suggest that targeted delivery of H₂S prodrug to a specific type of liver cells was successfully achieved by bioconjugation.

CXC chemokine ligand 10 (CXCL10) is a CXC chemokine family protein that transmits signals by binding to its specific receptor, CXCR3. CXCL10 is also known as an interferon-γ-inducible chemokine involved in various biological phenomena, including chemotaxis of natural killer (NK) cells and cytotoxic T lymphocytes, that suppress tumor growth and inhibition of angiogenesis. In this study, we examined the effects of forced expression of CXCL10 in a murine colon carcinoma cell line (CT26) on growth and metastasis in syngeneic mice. We first established CT26 cells that were stably expressing murine CXCL10 (CT26/CXCL10) and compared their growth with their parental CT26 cells in vitro and in vivo. The in vitro growth of the CT26/CXCL10 and CT26 cells was comparable, whereas the in vivo growth of the CT26/CXCL10 cells in the skin was strongly suppressed. Liver metastasis of the CT26/CXCL10 cells was also significantly suppressed after intra-splenic implantation. Removal of NK cells by the administration of anti-asialo GM1 antibody canceled the suppression of subcutaneous growth and liver metastasis of CT26/CXCL10 cells. Immunofluorescence clearly showed that abundant NKp46-positive NK cells were recruited into the liver metastatic lesions of the CT26/CXCL10 cells, consistent with specific NK cell migration towards the culture supernatant from the CT26/CXCL10 cells in the chemotaxis assay using transwells. These findings indicate that CXCL10 prevents in vivo growth and metastasis of colon carcinoma cells by recruiting NK cells, suggesting that forced expression of CXCL10 in the colon tumors by gene delivery should lead to a favorable clinical outcome.

Contact hypersensitivity (CHS) to preservatives is receiving increased attention. Parabens are widely used in foods, pharmaceutics and cosmetics as preservatives. The skin sensitizing activity of parabens remains controversial but a few investigations have been made as to whether parabens could facilitate sensitization to other chemicals. We have shown that di-n-butyl phthalate (DBP), a phthalate ester, has an adjuvant effect in a fluorescein isothiocyanate (FITC)-induced CHS mouse model. We have also demonstrated that DBP activates transient receptor potential ankyrin 1 (TRPA1) cation channels expressed on sensory neurons. Comparative studies of phthalate esters revealed that TRPA1 agonistic activity and the adjuvant effect on FITC-CHS coincide. Here we focused on two commonly used parabens, butyl paraben (BP) and ethyl paraben (EP), as to their adjuvant effects. BALB/c mice were epicutneously sensitized with FITC in acetone in the presence or absence of a paraben. Sensitization to FITC was evaluated as the ear-swelling response after FITC challenge. BP but not EP enhanced skin sensitization to FITC, but the effect of BP was much weaker than that of DBP. Mechanistically, BP enhanced the trafficking of FITC-presenting CD11c⁺ dendritic cells (DCs) from the skin to draining lymph nodes as well as cytokine production by draining lymph nodes. When the TRPA1 agonistic activity was measured with a cell line expressing TRPA1, BP exhibited higher activity than EP. The present study provides direct in vivo evidence that BP causes sensitization to other chemicals by means of a mouse FITC-CHS model.

Biosimilar products of therapeutic antibodies have been launched all over the world. They can relieve some of the economic burden of medicines. Although clinical trials have demonstrated the equivalency of biosimilar products with their reference product, biosimilar products are not commonly used in clinical practice. One reason is that the structural difference between the reference product and a biosimilar one remains unclear. We analyzed glycoforms and amino acids of an infliximab biosimilar product approved in Japan compared to that of the reference product (Remicade^®). By combination of papain digestion and LC/ time-of-flight (TOF)-MS, we established a valuable method to analyze these therapeutic antibodies. Nine glycoforms were detected in infliximab, and a difference in amino acids was observed. In the glycoforms of MMF, MGnF/GnMF, GnGn, GnGnF, AGnF/GnAF, and AAF, the relative intensities were significantly different between the reference and biosimilar product. Furthermore, we elucidated that the content rate of the C-terminal lysine was different among glycoforms. In conclusion, our analytical method can analyze not only amino acids but also carbohydrate chains of therapeutic antibodies, and will provide a useful strategy to evaluate bio-medicines including biosimilar antibodies.