CMIC Pharma Science Co.

Suita, Japan

CMIC Pharma Science Co.

Suita, Japan

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Goto R.,CMIC Pharma Science Co. | Miyakawa S.,RIBOMIC Inc. | Inomata E.,RIBOMIC Inc. | Takami T.,CMIC Inc. | And 3 more authors.
Journal of Mass Spectrometry | Year: 2017

Correct sequences are prerequisite for quality control of therapeutic oligonucleotides. However, there is no definitive method available for determining sequences of highly modified therapeutic RNAs, and thereby, most of the oligonucleotides have been used clinically without direct sequence determination. In this study, we developed a novel sequencing method called ‘hydrophobic tag sequencing’. Highly modified oligonucleotides are sequenced by partially digesting oligonucleotides conjugated with a 5′-hydrophobic tag, followed by liquid chromatography–mass spectrometry analysis. 5′-Hydrophobic tag-printed fragments (5′-tag degradates) can be separated in order of their molecular masses from tag-free oligonucleotides by reversed-phase liquid chromatography. As models for the sequencing, the anti-VEGF aptamer (Macugen) and the highly modified 38-mer RNA sequences were analyzed under blind conditions. Most nucleotides were identified from the molecular weight of hydrophobic 5′-tag degradates calculated from monoisotopic mass in simple full mass data. When monoisotopic mass could not be assigned, the nucleotide was estimated using the molecular weight of the most abundant mass. The sequences of Macugen and 38-mer RNA perfectly matched the theoretical sequences. The hydrophobic tag sequencing worked well to obtain simple full mass data, resulting in accurate and clear sequencing. The present study provides for the first time a de novo sequencing technology for highly modified RNAs and contributes to quality control of therapeutic oligonucleotides. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.


PubMed | University of California at San Francisco, CMIC Pharma Science Co., Louis Pasteur Center for Medical Research, Kyoto University and Tenri Hospital
Type: | Journal: Scientific reports | Year: 2016

Oxidative stress plays a pivotal role in the pathogenesis of asthma. Aquaporin-3 (AQP3) is a small transmembrane water/glycerol channel that may facilitate the membrane uptake of hydrogen peroxide (H2O2). Here we report that AQP3 potentiates ovalbumin (OVA)-induced murine asthma by mediating both chemokine production from alveolar macrophages and T cell trafficking. AQP3 deficient (AQP3(-/-)) mice exhibited significantly reduced airway inflammation compared to wild-type mice. Adoptive transfer experiments showed reduced airway eosinophilic inflammation in mice receiving OVA-sensitized splenocytes from AQP3(-/-) mice compared with wild-type mice after OVA challenge, consistently with fewer CD4(+) T cells from AQP3(-/-) mice migrating to the lung than from wild-type mice. Additionally, in vivo and vitro experiments indicated that AQP3 induced the production of some chemokines such as CCL24 and CCL22 through regulating the amount of cellular H2O2 in M2 polarized alveolar macrophages. These results imply a critical role of AQP3 in asthma, and AQP3 may be a novel therapeutic target.


PubMed | CMIC Pharma Science Co., Ribomic Inc. and CMIC Inc.
Type: | Journal: Journal of mass spectrometry : JMS | Year: 2016

Correct sequences are prerequisite for quality control of therapeutic oligonucleotides. However, there is no definitive method available for determining sequences of highly modified therapeutic RNAs, and thereby most of the oligonucleotides have been used clinically without direct sequence determination. In this study, we developed a novel sequencing method called hydrophobic tag sequencing. Highly modified oligonucleotides are sequenced by partially digesting oligonucleotides conjugated with a 5-hydrophobic tag, followed by liquid chromatography-mass spectrometry analysis. 5- hydrophobic tag-printed fragments (5-tag degradates) can be separated in order of their molecular masses from tag-free oligonucleotides by reversed-phase liquid chromatography. As models for the sequencing, the anti-VEGF aptamer (Macugen) and the highly modified 38-mer RNA sequences were analyzed under blind conditions. Most nucleotides were identified from the molecular weight of hydrophobic 5-tag degradates calculated from monoisotopic mass in simple full mass data. When monoisotopic mass could not be assigned, the nucleotide was estimated using the molecular weight of the most abundant mass. The sequences of Macugen and 38-mer RNA perfectly matched the theoretical sequences. The hydrophobic tag sequencing worked well to get simple full mass data, resulting in accurate and clear sequencing. The present study provides for the first time a de novo sequencing technology for highly modified RNAs, and contributes to quality control of therapeutic oligonucleotides.


PubMed | Chiyoda Corporation, CMIC Pharma Science Co., Tokyo Electron and National Cancer Center
Type: Journal Article | Journal: Drug metabolism and pharmacokinetics | Year: 2016

The chemistry of nano-surface and molecular-orientation limited (nSMOL) proteolysis is the Fab-selective limited proteolysis by making use the difference of protease nanoparticle diameter (200 nm) and antibody resin pore diameter (100 nm). In this report, we have demonstrated that the full validation for Bevacizumab bioanalysis in human plasma using nSMOL. The immunoglobulin fraction was collected by Protein A resin from plasma, then nSMOL reaction was performed using the FG nanoparticle-immobilized trypsin under the nondenaturing physiological condition at 50 C for 6 h. After removal of resin and nanoparticles, the signature peptide of Bevacizumab complementarity-determining region (CDR) and internal standard P14R were simultaneously quantified by LCMS multiple reaction monitoring (MRM). This nSMOL method quantification of Bevacizumab showed sensitivity of 0.146 g/ml and linearity of 0.146-300 g/ml. The intra- and inter-assay precision of lower limit of quantification (LLOQ), low quality control (LQC), middle quality control (MQC), and high quality control (HQC) was 7.94-15.2% and 14.6%, 7.15-13.5% and 11.7%, 2.63-6.47% and 5.83%, and 3.09-4.35% and 4.45%, respectively. These results indicate that nSMOL is also significant method for Bevacizumab bioanalysis in human plasma.


Tanaka Y.,Shiga University of Medical Science | Kume S.,Shiga University of Medical Science | Araki H.,Shiga University of Medical Science | Nakazawa J.,Shiga University of Medical Science | And 8 more authors.
Free Radical Biology and Medicine | Year: 2015

Free fatty acid-bound albumin (FFA-albumin)-related oxidative stress is involved in the pathogenesis of proximal tubular cell (PTC) damage and subsequent renal dysfunction in patients with refractory proteinuria. Nicotinamide adenine dinucleotide (NAD) metabolism has recently been focused on as a novel therapeutic target for several modern diseases, including diabetes. This study was designed to identify a novel molecule in NAD metabolism to protect PTCs from lipotoxicity-related oxidative stress. Among 19 candidate enzymes involved in mammalian NAD metabolism, the mRNA expression level of nicotinamide n-methyltransferase (NNMT) was significantly increased in both the kidneys of FFA-albumin-overloaded mice and cultured PTCs stimulated with palmitate-albumin. Knockdown of NNMT exacerbated palmitate-albumin-induced cell death in cultured PTCs, whereas overexpression of NNMT inhibited it. Intracellular concentration of 1-Methylnicotinamide (1-MNA), a metabolite of NNMT, increased and decreased in cultured NNMT-overexpressing and -knockdown PTCs, respectively. Treatment with 1-MNA inhibited palmitate-albumin-induced mitochondrial reactive oxygen species generation and cell death in cultured PTCs. Furthermore, oral administration of 1-MNA ameliorated oxidative stress, apoptosis, necrosis, inflammation, and fibrosis in the kidneys of FFA-albumin-overloaded mice. In conclusion, NNMT-derived 1-MNA can reduce lipotoxicity-mediated oxidative stress and cell damage in PTCs. Supplementation of 1-MNA may have potential as a new therapy in patients with refractory proteinuria. © 2015 Elsevier Inc.


Iwamoto N.,Chiyoda Corporation | Umino Y.,Chiyoda Corporation | Aoki C.,Chiyoda Corporation | Yamane N.,CMIC Pharma Science Co. | And 2 more authors.
Drug Metabolism and Pharmacokinetics | Year: 2016

The chemistry of nano-surface and molecular-orientation limited (nSMOL) proteolysis is the Fab-selective limited proteolysis by making use the difference of protease nanoparticle diameter (200 nm) and antibody resin pore diameter (100 nm). In this report, we have demonstrated that the full validation for Bevacizumab bioanalysis in human plasma using nSMOL. The immunoglobulin fraction was collected by Protein A resin from plasma, then nSMOL reaction was performed using the FG nanoparticle-immobilized trypsin under the nondenaturing physiological condition at 50°C for 6 h. After removal of resin and nanoparticles, the signature peptide of Bevacizumab complementarity-determining region (CDR) and internal standard P14R were simultaneously quantified by LCMS multiple reaction monitoring (MRM). This nSMOL method quantification of Bevacizumab showed sensitivity of 0.146 μg/ml and linearity of 0.146-300 μg/ml. The intra- and inter-assay precision of lower limit of quantification (LLOQ), low quality control (LQC), middle quality control (MQC), and high quality control (HQC) was 7.94-15.2% and 14.6%, 7.15-13.5% and 11.7%, 2.63-6.47% and 5.83%, and 3.09-4.35% and 4.45%, respectively. These results indicate that nSMOL is also significant method for Bevacizumab bioanalysis in human plasma. © 2015 The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.

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