Publications by authors named "Noriyasu Hirasawa"

112 Publications

Evaluation of the Utility of Vial Packaging to Reduce Occupational Exposure to Antineoplastic Drugs in Preventing Breakages and Scattering.

PDA J Pharm Sci Technol 2021 May 14. Epub 2021 May 14.

Tohoku University, Graduate School of Pharmaceutical Sciences.

The dropping of glass vials based on negligence or accidental events that occur during the preparation or mixing of injectable drugs are examples of instances of occupational exposures occurring in a clinical setting. To reduce such risks, several types of glass vial packaging have been developed. We herein compared the resistance of base- and cup-type packaged vials to breakage and scattering of contents during falls with control vials. The falling heights at which test products were dropped were set to 70, 135, and 180 cm. Compared with the control group, appearance changes were inhibited in the cup-type groups. Significant differences were found between the cup-type and control groups at heights of 135 and 180 cm. Next, resistance of packaging to spilling and scattering of solution from the vial was determined. There was no scattering in any types of vials at a height of 70 cm because they were not broken. However, at heights of 135 and 180 cm, the mean scattering distance in the control groups were 50 and 70.6 cm, respectively. At these heights, some vials in the base-type and cup-type group were also cracked, but the solution stayed completely inside the covering packaging, indicating an obvious antiscattering ability. Vials packed in cup- and base-type packaging would lower the risk of the exposure of hazardous drugs during vial breakages. Because the base-type packaging did not show significant antibreakage effects, the cup-type packaging is more suited for hazardous drug packaging. However, cup-type packaging requires equipment investments from pharmaceutical manufacturers. Thus, cost-effectiveness and the target drug profile should be evaluated, and the use of cup- and base-type packaging, as well as control, forms should be selected accordingly.
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http://dx.doi.org/10.5731/pdajpst.2020.012286DOI Listing
May 2021

Lactate released from human fibroblasts enhances Ni elution from Ni plate.

Toxicology 2021 04 14;453:152723. Epub 2021 Feb 14.

Laboratory of Pharmacotherapy of Life-Style Related Diseases, Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Miyagi, Japan. Electronic address:

Elution of Ni ions from medical devices induces inflammation and toxicity. We previously reported that elution of Ni ions from Ni wires induced COX-2 expression and increased lactate production, but whether lactate is involved in the further elution of Ni ions remains unclear. In this study, using KMST-6, a human fibroblast cell line, we examined the molecular mechanisms by which Ni ions increase lactate release and the role of lactate in enhancing the elution of Ni ions. When KMST-6 cells were incubated on a Ni plate or stimulated with NiCl (1 mM), the expression of glucose transporter 1 (GLUT1), hexokinase 2 (HK2), and lactate dehydrogenase A (LDHA), and the release of lactate were enhanced. The NiCl (1 mM)-induced expression of these genes was inhibited by a hypoxia-inducible factor-1α (HIF-1α) inhibitor, PX-478 (10-25 μM). Stimulation of cells with a prolyl hydroxylase domain (PHD) inhibitor, roxadustat, increased the expression of these genes, lactate release, and elution of Ni ions at 10 μM. A monocarboxylate transporter-4 (MCT4) inhibitor, syrosingopine, inhibited lactate release from roxadustat-treated cells and reduced the elution of Ni ions by the cells at 10 μM. Finally, syrosingopine (10 μM) reduced the elution of Ni ions by the cells from the Ni plate. These results suggest that elution of Ni ions from metals promotes the production of lactate via HIF-1α-mediated gene expression and causes further Ni elution. Thus, Ni ions show a positive feedback mechanism of Ni elution, and this step may be potentially targeted to protect against metal elution from metal devices.
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http://dx.doi.org/10.1016/j.tox.2021.152723DOI Listing
April 2021

Functional Assessment of 12 Rare Allelic Variants Identified in a Population of 4773 Japanese Individuals.

J Pers Med 2021 Feb 2;11(2). Epub 2021 Feb 2.

Laboratory of Pharmacotherapy of Life-Style Related Diseases, Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai 980-8578, Japan.

Cytochrome P450 2C9 (CYP2C9) is an important drug-metabolizing enzyme that contributes to the metabolism of approximately 15% of clinically used drugs, including warfarin, which is known for its narrow therapeutic window. Interindividual differences in CYP2C9 enzymatic activity caused by genetic polymorphisms lead to inconsistent treatment responses in patients. Thus, in this study, we characterized the functional differences in CYP2C9 wild-type (CYP2C9.1), CYP2C9.2, CYP2C9.3, and 12 rare novel variants identified in 4773 Japanese individuals. These variants were heterologously expressed in 293FT cells, and the kinetic parameters (, , , catalytic efficiency, and ) of ()-warfarin 7-hydroxylation and tolbutamide 4-hydroxylation were estimated. From this analysis, almost all novel CYP2C9 variants showed significantly reduced or null enzymatic activity compared with that of the CYP2C9 wild-type. A strong correlation was found in catalytic efficiencies between ()-warfarin 7-hydroxylation and tolbutamide 4-hydroxylation among all studied CYP2C9 variants. The causes of the observed perturbation in enzyme activity were evaluated by three-dimensional structural modeling. Our findings could clarify a part of discrepancies among genotype-phenotype associations based on the novel rare allelic variants and could, therefore, improve personalized medicine, including the selection of the appropriate warfarin dose.
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http://dx.doi.org/10.3390/jpm11020094DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7912942PMC
February 2021

[Analysis of the Distribution of Community Pharmacies and Pharmacists in Miyagi Prefecture].

Yakugaku Zasshi 2021 ;141(2):273-279

Laboratory of Social Pharmacy Management, Graduate School of Pharmaceutical Sciences, Tohoku University.

In Japan, the aging of the population is serious problem. The Ministry of Health, Labour and Welfare is constructing a new support system for elderly people called "Community-based integrated care system". In this system, community pharmacists are expected to play an important role as healthcare professionals for the whole community, including elderly people. Since pharmacists will be needed to manage community health in addition to their daily tasks, it is required to reassess the distribution of community pharmacies and pharmacists. In this study, we surveyed their distribution in Miyagi prefecture by using statistical data from public institutions and reevaluated the distribution to raise problems. Based on the numbers of community pharmacies and pharmacists per 1000 population in the whole Miyagi prefecture, each area was ranked to 2 categories and analyzed regarding population, aging rate and inhabitable land area. It was disclosed that the higher aging rate areas had the higher rate of category below the average of whole Miyagi prefecture, especially in the number of pharmacists. When the numbers of pharmacies and pharmacists per the inhabitable land area were used, the uneven distribution became clearer than when those per population were used. These findings suggested that it was important to characterize the areas by not only the ratios of community pharmacies and pharmacists to population numbers but also by the aging rates and inhabitable land area, which were related to the work efficiency of pharmacists and accessibility for resident to pharmacies.
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http://dx.doi.org/10.1248/yakushi.20-00191DOI Listing
May 2021

Functional Characterization of 40 CYP3A4 Variants by Assessing Midazolam 1'-Hydroxylation and Testosterone 6-Hydroxylation.

Drug Metab Dispos 2021 Mar 31;49(3):212-220. Epub 2020 Dec 31.

Laboratory of Pharmacotherapy of Life-Style Related Diseases, Graduate School of Pharmaceutical Sciences (M.K., E.M.G.R., N.H., M.H.), Tohoku Medical Megabank Organization (E.H., S.S., D.S., S.T., K.K., M.H.), Advanced Research Center for Innovations in Next-Generation Medicine (E.H., A.U., N.H., M.H.), and Laboratory of Clinical Pharmacy, Faculty of Pharmaceutical Sciences (A.A., M.M., N.M.), Tohoku University, Sendai, Japan; Faculty of Pharmacy, Meijo University, Nagoya, Japan (T.N., A.O.); and Department of Pharmaceutical Sciences, Tohoku University Hospital, Sendai, Japan (M.M., N.M., N.H., M.H.)

CYP3A4 is among the most abundant liver and intestinal drug-metabolizing cytochrome P450 enzymes, contributing to the metabolism of more than 30% of clinically used drugs. Therefore, interindividual variability in CYP3A4 activity is a frequent cause of reduced drug efficacy and adverse effects. In this study, we characterized wild-type CYP3A4 and 40 CYP3A4 variants, including 11 new variants, detected among 4773 Japanese individuals by assessing CYP3A4 enzymatic activities for two representative substrates (midazolam and testosterone). The reduced carbon monoxide-difference spectra of wild-type CYP3A4 and 31 CYP3A4 variants produced with our established mammalian cell expression system were determined by measuring the increase in maximum absorption at 450 nm after carbon monoxide treatment. The kinetic parameters of midazolam and testosterone hydroxylation by wild-type CYP3A4 and 29 CYP3A4 variants ( , , and catalytic efficiency) were determined, and the causes of their kinetic differences were evaluated by three-dimensional structural modeling. Our findings offer insight into the mechanism underlying interindividual differences in CYP3A4-dependent drug metabolism. Moreover, our results provide guidance for improving drug administration protocols by considering the information on genetic polymorphisms. SIGNIFICANCE STATEMENT: CYP3A4 metabolizes more than 30% of clinically used drugs. Interindividual differences in drug efficacy and adverse-effect rates have been linked to ethnicity-specific differences in gene variants in Asian populations, including Japanese individuals, indicating the presence of polymorphisms resulting in the increased expression of loss-of-function variants. This study detected alterations in CYP3A4 activity due to amino acid substitutions by assessing the enzymatic activities of coding variants for two representative CYP3A4 substrates.
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http://dx.doi.org/10.1124/dmd.120.000261DOI Listing
March 2021

Suprabasin-null mice retain skin barrier function and show high contact hypersensitivity to nickel upon oral nickel loading.

Sci Rep 2020 09 3;10(1):14559. Epub 2020 Sep 3.

Department of Dermatology, Hamamatsu University School of Medicine, 1-20-1, Handayama, Higashi-Ku, Hamamatsu, 431-3192, Japan.

Suprabasin (SBSN) is expressed not only in epidermis but also in epithelial cells of the upper digestive tract where metals such as nickel are absorbed. We have recently shown that SBSN level is decreased in the stratum corneum and serum of atopic dermatitis (AD) patients, especially in intrinsic AD, which is characterized by metal allergy. By using SBSN-null (Sbsn) mice, this study was conducted to investigate the outcome of SBSN deficiency in relation to AD. Sbsn mice exhibited skin barrier dysfunction on embryonic day 16.5, but after birth, their barrier function was not perturbed despite the presence of ultrastructural changes in stratum corneum and keratohyalin granules. Sbsn mice showed a comparable ovalbumin-specific skin immune response to wild type (WT) mice and rather lower contact hypersensitivity (CHS) responses to haptens than did WT mice. The blood nickel level after oral feeding of nickel was significantly higher in Sbsn mice than in WT mice, and CHS to nickel was elevated in Sbsn mice under nickel-loading condition. Our study suggests that the completely SBSN deficient mice retain normal barrier function, but harbor abnormal upper digestive tract epithelium that promotes nickel absorption and high CHS to nickel, sharing the features of intrinsic AD.
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http://dx.doi.org/10.1038/s41598-020-71536-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7471289PMC
September 2020

Heterologous expression of high-activity cytochrome P450 in mammalian cells.

Sci Rep 2020 08 25;10(1):14193. Epub 2020 Aug 25.

Laboratory of Pharmacotherapy of Life-Style Related Diseases, Graduate School of Pharmaceutical Sciences, Tohoku University, 6-3 Aoba Aramaki, Aoba-ku, Sendai, 980-8578, Japan.

The evaluation of Cytochrome P450 (CYP) enzymatic activity is essential to estimate drug pharmacokinetics. Numerous CYP allelic variants have been identified; the functional characterisation of these variants is required for their application in precision medicine. Results from heterologous expression systems using mammalian cells can be integrated in in vivo studies; however, other systems such as E. coli, bacteria, yeast, and baculoviruses are generally used owing to the difficulty in expressing high CYP levels in mammalian cells. Here, by optimising transfection and supplementing conditions, we developed a heterologous expression system using 293FT cells to evaluate the enzymatic activities of three CYP isoforms (CYP1A2, CYP2C9, and CYP3A4). Moreover, we established co-expression with cytochrome P450 oxidoreductase and cytochrome b. This expression system would be a potential complementary or beneficial alternative approach for the pharmacokinetic evaluation of clinically used and developing drugs in vitro.
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http://dx.doi.org/10.1038/s41598-020-71035-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7447777PMC
August 2020

ATP-Binding Cassette Transporter C4 is a Prostaglandin D2 Exporter in HMC-1 cells.

Prostaglandins Leukot Essent Fatty Acids 2020 08 30;159:102139. Epub 2020 May 30.

Department of Pharmaceutical Sciences, Tohoku University Hospital, Sendai, Miyagi, 980-8574, Japan; Department of Pharmacy, Yamagata University Hospital, Yamagata, 990-9585, Japan. Electronic address:

ATP-binding cassette transporter C4 (ABCC4) is associated with multidrug resistance and the regulation of cell signalling. Some prostaglandins (PGs), including: PGE, PGF, PGE, and PGF are known substrates of ABCC4, and are released from some types of cells to exert their biological effects. In the present study, we demonstrate that PGD is a novel substrate of ABCC4 using a transport assay based on inside-out membrane vesicles prepared from ABCC4-overexpressing cells. Then, we used two types of cell lines with confirmed ABCC4 mRNA and PGD release capacity (human mast cell lines HMC-1 cells and human rhabdomyosarcoma cell lines TE671 cells) to evaluate the contribution of ABCC4. The extracellular levels of PGD were unchanged following addition of a selective ABCC4 inhibitor in TE671 cells. Pharmacological inhibition and knockdown of ABCC4 significantly reduced the extracellular levels of PGD by at least 53% in HMC-1 cells. Moreover, the extracellular levels of PGD decreased by at least 20% using the selective ABCC4 inhibitor in the other mast cell line RBL-2H3 cells. Therefore, our results suggest that ABCC4 functions as a PGD exporter in HMC-1 cells.
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http://dx.doi.org/10.1016/j.plefa.2020.102139DOI Listing
August 2020

Potential synergistic effects of novel hematopoietic prostaglandin D synthase inhibitor TAS-205 and different types of anti-allergic medicine on nasal obstruction in a Guinea pig model of experimental allergic rhinitis.

Eur J Pharmacol 2020 May 19;875:173030. Epub 2020 Feb 19.

Laboratory of Pharmacotherapy of Life-Style Related Diseases, Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, 980-8578, Miyagi, Japan.

Nasal obstruction is one of the most bothersome symptoms of allergic rhinitis (AR) affecting sleep-related quality of life in AR patients. Although several treatments were tested to control nasal obstruction, some patients with moderate to severe AR do not respond to current treatments, including the combined administration of different types of anti-allergic medicine. Thus, new options for AR treatment are needed. This study aimed to evaluate the effects of combined treatment with a novel inhibitor of hematopoietic prostaglandin D synthase (HPGDS), TAS-205, and different types of anti-allergic medicine on nasal obstruction in AR. Firstly, we demonstrated that TAS-205 selectively inhibited prostaglandin D (PGD) synthesis in an enzymatic assay in a cell-based assay and in vivo models of AR. Moreover, treatment with TAS-205 alone suppressed eosinophil infiltration into the nasal cavity and late phase nasal obstruction. The combined administration of TAS-205 with montelukast, a cysteinyl leukotriene receptor 1 antagonist, showed significant additive inhibitory effects on eosinophil infiltration and late phase nasal obstruction compared to treatment with each agent alone. In contrast, concomitant treatment with TAS-205 and fexofenadine, a histamine H blocker, showed inhibitory effects on late phase and early phase nasal obstruction, although the magnitude of the inhibitory effects upon combined administration was comparable to that of each single treatment. These results suggest that combined treatment with an HPGDS inhibitor and different types of anti-allergic medicine may be a promising strategy to control nasal obstruction in AR patients.
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http://dx.doi.org/10.1016/j.ejphar.2020.173030DOI Listing
May 2020

CYP2D6 genotyping analysis and functional characterization of novel allelic variants in a Ni-Vanuatu and Kenyan population by assessing dextromethorphan O-demethylation activity.

Drug Metab Pharmacokinet 2020 Feb 20;35(1):89-101. Epub 2019 Jul 20.

Laboratory of Pharmacotherapy of Life-Style Related Diseases, Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai 980-8578, Japan; Advanced Research Center for Innovations in Next-Generation Medicine, Tohoku University, Sendai, 980-8575, Japan; Tohoku Medical Megabank Organization, Tohoku University, Sendai, 980-8575, Japan; Department of Pharmaceutical Sciences, Tohoku University Hospital, Sendai 980-8574, Japan. Electronic address:

While CYP2D6 allele and phenotype frequencies have been extensively studied, currently, very little ethnically specific data is available regarding the East African and South Pacific region, including Kenya and Vanuatu. The absence of information regarding gene polymorphisms and their resulting clinical effects in these populations may hinder treatment strategies and patient outcome. Given the scarceness of CYP2D6 related data in these populations, the purpose of this study was to perform a pharmacogenomic analysis of the Kenyan and Ni-Vanuatu population and ultimately characterize the enzymatic properties of eight novel CYP2D6 variant proteins expressed in 293FT cells in vitro using dextromethorphan as a substrate. Our study revealed a prevalence of functional alleles in both populations a low frequency for decreased function defining genotypes in the Ni-Vanuatu population, with approximately 36% of our Kenyan subjects presenting substrate-dependent decreased function alleles. Additionally, 6 variants (P171L, G306R, V402L, K1, K2, and K3) showed significantly reduced intrinsic clearance compared to wild-type CYP2D6.1. Our findings aid in efforts to bridge the gap between pharmacogenomic analysis and clinical application, by providing useful information in the development of ethnic-specific strategies as well as stressing the importance of population-specific genotyping when conducting multi-regional clinical trials and designing therapeutic strategies.
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http://dx.doi.org/10.1016/j.dmpk.2019.07.003DOI Listing
February 2020

Hypoxia inhibits TNF-α-induced TSLP expression in keratinocytes.

PLoS One 2019 4;14(11):e0224705. Epub 2019 Nov 4.

Laboratory of Pharmacotherapy of Life-Style Related Diseases, Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Miyagi, Japan.

The expression of thymic stromal lymphopoietin (TSLP), a cytokine which greatly contributes to the induction of type I allergy, is upregulated in chronic inflammation such as atopic dermatitis and psoriasis. As hypoxia in the epidermis is important for maintaining skin homeostasis, we examined the regulation of TSLP expression by hypoxic conditions in normal skin epithelial tissues. TNF-α-induced expression of TSLP in human keratinocyte HaCaT and in mouse keratinocyte PAM212 cell lines were inhibited under hypoxic condition (1% O2), although the mRNA expressions of TNF-α, IL-6, IL-8, MCP-1, and VEGF-A were not inhibited. Hypoxia-mimicking conditions, which include NiCl2, CoCl2, and DMOG, an inhibitor of 2-oxoglutarate-dependent enzymes, also selectively inhibited TNF-α-induced TSLP expression. These results suggested that inactivation of prolyl hydroxylase by hypoxia and hypoxia-mimicking conditions is involved in the repression of TNF-α-induced TSLP expression. Interestingly, the inhibition of TSLP production by hypoxic treatment was significantly reversed by treatment with the HIF-2α antagonist but not with the HIF-1α inhibitor. DMOG-induced inhibition of TSLP promoter activity was dependent on the -71 to +185 bp promoter region, suggesting that the binding of HIF-2 to hypoxia response element (HRE) in this region repressed the TSLP expression. These results indicated that hypoxia and hypoxia-mimicking conditions inhibited TSLP expression via HIF-2 and HRE-dependent mechanisms. Therefore, PHD and HIF-2α could be a new strategy for treatment of atopic dermatitis and psoriasis.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0224705PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6827910PMC
April 2020

COX-2 induces T cell accumulation and IFN-γ production during the development of chromium allergy.

Autoimmunity 2019 Aug - Sep;52(5-6):228-234. Epub 2019 Oct 6.

Department of Advanced Prosthetic Dentistry, Graduate School of Dentistry, Tohoku University, Sendai, Japan.

Chromium (Cr) is commonly added into various metal alloys to improve some mechanical properties such as corrosion resistance, strength, and workability. However, Cr is also known to be a metal allergen for some individuals. Metal allergy is a T cell-mediated disease with symptoms of inflammation and swelling that involve inflammatory cytokines and prostaglandins. Hence, suppressing these inflammation paths by using COX-2 inhibitor might be useful in treating Cr allergy. In this study, mice were used with Cr-induced allergy challenge model. The mice were injected with celecoxib once per day for 7 days one hour after the challenge. Footpad samples were stained with haematoxylin and eosin (H&E), and lymphocytes were isolated from popliteal lymph nodes for the flow cytometric analysis. The results show that both prostaglandin E (PGE), a known mediator of inflammation, and cyclooxygenases (COX)-2 have important roles in the development of Cr allergy. Further, COX-2 inhibitor, celecoxib, was effective in relieving swelling and inflammation in Cr-allergic mice concordant with suppression of IFN-γ production by CD8 T cells and T cell accumulation in the lymph nodes. Therefore, the inhibition of COX-2 may be a therapeutic target for Cr allergy, and additional molecules in the PGE signalling pathway may also be an effective therapeutic target for the treatment of metal allergy.
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http://dx.doi.org/10.1080/08916934.2019.1662404DOI Listing
July 2020

extract and ergosterol reduce allergic reactions in an allergy mouse model by suppressing the degranulation of mast cells.

Biosci Biotechnol Biochem 2019 Dec 14;83(12):2280-2287. Epub 2019 Aug 14.

Laboratory of Pathophysiological Biochemistry, Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Miyagi, Japan.

The increasing number of patients suffering from allergic diseases is a global health problem. is an edible mushroom consumed as a health food in Asia, and has recently been reported to have anti-allergic effects. We previously reported that extract (GFE) and its active components, ergosterol and its derivatives, inhibited the antigen-induced activation of RBL-2H3 cells. Here, we demonstrated that GFE and ergosterol also had an inhibitory effect on the degranulation of bone marrow-derived mast cells (BMMCs) and alleviated anaphylactic cutaneous responses in mice. Using an air pouch-type allergic inflammation mouse model, we confirmed that oral administration of GFE and ergosterol suppressed the degranulation of mast cells . Our findings suggest that , including ergosterol as its active component, reduces type I allergic reactions by suppressing mast cell degranulation in mice, and might be a novel functional food that prevents allergic diseases.
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http://dx.doi.org/10.1080/09168451.2019.1654360DOI Listing
December 2019

A chalcone derivative suppresses the induction of TSLP in mice and human keratinocytes and attenuates OVA-induced antibody production in mice.

Eur J Pharmacol 2019 May 10;851:52-62. Epub 2019 Feb 10.

Laboratory of Pharmacotherapy of Life-Style Related Diseases, Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai 980-8578, Miyagi, Japan. Electronic address:

Thymic stromal lymphopoietin (TSLP) is a key epithelial-derived factor that aggravates allergic diseases. Therefore, TSLP inhibitors are candidate compounds for the treatment of allergic diseases. Previously, we reported that KCMH-1, a mouse keratinocyte cell line, constitutively produces TSLP. In this study, we tried to identify inhibitors of TSLP by screening 2169 compounds in KCMH-1 cells and found one such chalcone derivative (code no. 16D10). 16D10 inhibited TSLP expression and TSLP promoter activation in HaCaT cells, a human keratinocyte cell line. Although nuclear factor kappa-B (NF-κB) is a key transcription factor for the induction of TSLP, 16D10 did not inhibit the activation pathway of NF-κB, such as degradation of inhibitor of κB (IκB) and p65 nuclear translocation. 16D10 activated the Kelch-like ECH-associated protein 1 (Keap1)-nuclear factor (erythroid-derived 2)-like 2 (Nrf2) system, although this system was not involved in the inhibitory effect of 16D10. 16D10 also inhibited TSLP production in a lipopolysaccharide (LPS)- or ovalbumin (OVA)-induced air-pouch-type inflammation model. Further, repeated 16D10 administration diminished serum immunoglobulin G1 (IgG1) and IgE concentration in an OVA-induced air-pouch-type sensitization model. Taken together, these results indicate that 16D10 is an inhibitor of TSLP production and has an anti-allergic effect. This inhibitory effect is independent of the activation of NF-κB and the Keap1-Nrf2 system. Therefore, 16D10 could be a new type of candidate drug for allergic diseases.
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http://dx.doi.org/10.1016/j.ejphar.2019.02.007DOI Listing
May 2019

A steroid alkaloid derivative 02F04 upregulates thymic stromal lymphopoietin expression slowly and continuously through a novel Gq/11-ROCK-ERK1/2 signaling pathway in mouse keratinocytes.

Cell Signal 2019 05 19;57:58-64. Epub 2019 Jan 19.

Laboratory of Pharmacotherapy of Life-Style Related Diseases, Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai 980-8578, Miyagi, Japan. Electronic address:

Thymic stromal lymphopoietin (TSLP), a master switch of allergic inflammation, plays an important role in the pathogenesis of allergic diseases. Although many compounds upregulate TSLP expression in vivo or in vitro, most of them are pollutants or toxicants. In the previous study, for the first time, we found that a steroid alkaloid derivative 02F04, which has a unique skeletal structure compared with other TSLP-inducing chemicals, significantly induced TSLP production in mouse keratinocytes. However, it is not investigated thoroughly that how 02F04 produces TSLP and why. In this study, we did a detailed investigation on the inducible effect and underlying molecular mechanism of 02F04 on TSLP production. We found that the peak time of TSLP mRNA level induced by 02F04 at 48 h led to a slow and continuous TSLP production in PAM212 cells. Besides, 02F04-induced TSLP production was significantly suppressed by inhibitors of Rho-associated protein kinase (ROCK), guanine nucleotide-binding protein subunit alpha q/11 (Gq/11) and extracellular signal-regulated kinase 1/2 (ERK1/2) at not only protein but also mRNA levels, and by siRNA-mediated knockdown of Gq or G11. This suggested that ROCK, Gq/11 and ERK1/2 signaling pathways were involved in 02F04-induced TSLP production. Increase in the level of p-ERK1/2 induced by 02F04 was suppressed by both inhibitors of ROCK and Gq/11, indicating that ROCK and Gq/11 molecules were located at the upstream of ERK1/2 to regulate 02F04-induced TSLP production. Gq/11 was located at the upstream of ROCK because the specific Gq/11 inhibitor of YM-254890 significantly reduced 02F04-induced actin stress fiber formation. Taken together, 02F04 upregulates a slow and continuous TSLP production through a novel Gq/11-ROCK-ERK1/2 signaling pathway. The thorough understanding the effect and mechanism of 02F04 on TSLP production is expected to supply it as a novel TSLP-regulating compound and a potential new tool for investigating the role of TSLP in allergic disorders.
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http://dx.doi.org/10.1016/j.cellsig.2019.01.005DOI Listing
May 2019

Expression of Histidine Decarboxylase and Its Roles in Inflammation.

Int J Mol Sci 2019 Jan 16;20(2). Epub 2019 Jan 16.

Graduate School of Pharmaceutical Sciences, Tohoku University, 6-3 Aoba Aramaki, Aoba-ku, Sendai 980-8578, Japan.

Histamine is a well-known mediator of inflammation that is released from mast cells and basophils. To date, many studies using histamine receptor antagonists have shown that histamine acts through four types of receptors: H1, H2, H3, and H4. Thus, histamine plays more roles in various diseases than had been predicted. However, our knowledge about histamine-producing cells and the molecular mechanisms underlying histamine production at inflammatory sites is still incomplete. The histamine producing enzyme, histidine decarboxylase (HDC), is commonly induced at inflammatory sites during the late and chronic phases of both allergic and non-allergic inflammation. Thus, histamine levels in tissues are maintained at effective concentrations for hours, enabling the regulation of various functions through the production of cytokines/chemokines/growth factors. Understanding the regulation of histamine production will allow the development of a new strategy of using histamine antagonists to treat inflammatory diseases.
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http://dx.doi.org/10.3390/ijms20020376DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6359378PMC
January 2019

Functional characterization of 50 CYP2D6 allelic variants by assessing primaquine 5-hydroxylation.

Drug Metab Pharmacokinet 2018 Dec 25;33(6):250-257. Epub 2018 Aug 25.

Laboratory of Pharmacotherapy of Life-Style Related Diseases, Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai 980-8578, Japan; Tohoku Medical Megabank Organization, Tohoku University, Sendai 980-8575, Japan; Department of Pharmaceutical Sciences, Tohoku University Hospital, Sendai 980-8574, Japan; Advanced Research Center for Innovations in Next-Generation Medicine, Tohoku University, Sendai, 980-8575, Japan. Electronic address:

Cytochrome P450 2D6 (CYP2D6) is responsible for the metabolic activation of primaquine, an antimalarial drug. CYP2D6 is genetically polymorphic, and these polymorphisms are associated with interindividual variations observed in the therapeutic efficacy of primaquine. To further understand this association, we performed in vitro enzymatic analyses of the wild-type CYP2D6.1 and 49 CYP2D6 allelic variants, which were expressed in 293FT cells, using primaquine as a substrate. The concentrations of CYP2D6 variant holoenzymes were measured by using carbon monoxide (CO)-reduced difference spectroscopy, and the wild type and 27 variants showed a peak at 450 nm. The kinetic parameters K, V, and intrinsic clearance (V/K) of primaquine 5-hydroxylation were characterized. The kinetic parameters of the wild type and 16 variants were measured, but the values for the remaining 33 variants could not be determined because of low metabolite concentrations. Among the variants, six (i.e., CYP2D6.17, .18, .35, .39, .53, and .70) showed significantly reduced intrinsic clearance compared with that of CYP2D6.1. Three-dimensional structural modeling analysis was performed to elucidate the mechanism of changes in the kinetics of CYP2D6 variants. Our findings provide insights into the allele-specific activity of CYP2D6 for primaquine, which could be clinically useful for malaria treatment and eradication efforts.
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http://dx.doi.org/10.1016/j.dmpk.2018.08.004DOI Listing
December 2018

Development and application of a rapid and sensitive genotyping method for pharmacogene variants using the single-stranded tag hybridization chromatographic printed-array strip (STH-PAS).

Drug Metab Pharmacokinet 2018 Dec 22;33(6):258-263. Epub 2018 Aug 22.

Laboratory of Pharmacotherapy of Life-Style Related Diseases, Graduate School of Pharmaceutical Sciences, Tohoku University, 6-3 Aoba Aramaki, Aoba-ku, Sendai, 980-8578, Japan; Advanced Research Center for Innovations in Next-Generation Medicine, Tohoku University, Sendai, 980-8575, Japan; Tohoku Medical Megabank Organization, Tohoku University, 2-1 Seiryo-machi, Aoba-ku, Sendai, 980-8573, Japan; Department of Pharmaceutical Sciences, Tohoku University Hospital, 1-1 Seiryo-machi, Aoba-ku, Sendai, 980-8574, Japan. Electronic address:

Genetic polymorphisms contribute to inter-individual variability in the metabolism of multiple clinical drugs, including warfarin, thiopurines, primaquine, and aminoglycosides. A rapid and sensitive clinical assessment of various genome biomarkers is, therefore, required to predict the individual responsiveness of each patient to these drugs. In this study, we developed a novel genotyping method for the detection of nine pharmacogene variants that are important in the prediction of drug efficiency and toxicity. This genotyping method uses competitive allele-specific PCR and a single-stranded tag hybridization chromatographic printed-array strip (STH-PAS) that can unambiguously determine the presence or absence of the gene variant by displaying visible blue lines on the chromatographic printed-array strip. Notably, the results of our STH-PAS method were in 100% agreement with those obtained using standard Sanger sequencing and KASP assay genotyping methods for CYP4F2 gene deletion. Moreover, the results were obtained within 90 min, including the PCR amplification and signal detection processes. The sensitive and rapid nature of this novel method make it ideal for clinical genetic testing to predict drug efficacy and toxicity, and in doing so will aid in the development of individualized medicine and better patient care.
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http://dx.doi.org/10.1016/j.dmpk.2018.08.003DOI Listing
December 2018

Functional characterization of 40 CYP2B6 allelic variants by assessing efavirenz 8-hydroxylation.

Biochem Pharmacol 2018 10 8;156:420-430. Epub 2018 Sep 8.

Laboratory of Pharmacotherapy of Life-Style Related Diseases, Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai 980-8578, Japan; Advanced Research Center for Innovations in Next-Generation Medicine, Tohoku University, Sendai 980-8575, Japan; Tohoku Medical Megabank Organization, Tohoku University, Sendai 980-8575, Japan; Department of Pharmaceutical Sciences, Tohoku University Hospital, Sendai 980-8574, Japan. Electronic address:

Genetic variations within cytochrome P450 2B6 (CYP2B6) contribute to inter-individual variation in the metabolism of clinically important drugs, including cyclophosphamide, bupropion, methadone and efavirenz (EFZ). In this study, we performed an in vitro analysis of 40 CYP2B6 allelic variant proteins including seven novel variants identified in 1070 Japanese individuals. Wild-type and 39 variant proteins were heterologously expressed in 293FT cells to estimate the kinetic parameters (K, V, and CL) of EFZ 8-hydroxylation and 7-ethoxy-4-trifluoromethylcoumarin (7-ETC) O-deethylation activities. The concentrations of CYP2B6 variant holo-enzymes were measured by using carbon monoxide (CO)-reduced difference spectroscopy, and the wild-type and 28 variants showed a peak at 450 nm. The kinetic parameters were measured for the wild-type and 24 variant proteins. The values for the remaining 15 variants could not be determined because the enzymatic activity was not detected at the highest substrate concentration used. Compared to wild-type, six variants showed significantly decreased EFZ 8-hydroxylation CL values, while these values were significantly increased in another six variants, including CYP2B6.6. Although 7-ETC O-deethylation CL values of CYP2B6 variants did not differ significantly from that of CYP2B6.1, the CL ratios obtained for 7-ETC O-deethylation were highly correlated with EFZ 8-hydroxylation. Furthermore, three-dimensional structural modeling analysis was performed to elucidate the mechanism of changes in the kinetics of CYP2B6 variants. Our findings could provide evidence of the specific metabolic activities of the CYP2B6 proteins encoded by these variant alleles.
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http://dx.doi.org/10.1016/j.bcp.2018.09.010DOI Listing
October 2018

Ergosterol and its derivatives from Grifola frondosa inhibit antigen-induced degranulation of RBL-2H3 cells by suppressing the aggregation of high affinity IgE receptors.

Biosci Biotechnol Biochem 2018 Oct 2;82(10):1803-1811. Epub 2018 Jul 2.

a Mushroom Research Laboratory , Hokuto Corporation , Nagano , Japan.

Grifola frondosa is an edible mushroom consumed as a health food and/or traditional medicine in Asia. However, the anti-allergic effects of G. frondosa are not yet understood. In this study, we demonstrated the effects of G. frondosa extract (GFE) on IgE-mediated allergic responses, using antigen-stimulated RBL-2H3 cells. Three active compounds: ergosterol, 6β-methoxyergosta-7,22-dien-3β,5α-diol (MEDD), and 6-oxoergosta-7,22-dien-3β-ol (6-OXO) were isolated from GFE and shown to inhibit the antigen-induced release of β-hexosaminidase and histamine. Among the three active components, we focused on ergosterol because of its high content in GFE. Ergosterol inhibited the aggregation of high-affinity IgE receptor (FcεRI), which is the first step in the activation of mast cells and antigen-induced tyrosine phosphorylation. Furthermore, ergosterol suppressed antigen-increased IL-4 and TNF-α mRNA. Taken together, our findings suggest that G. frondosa, including ergosterol and its derivatives as active components, has the potential to be a novel functional food that prevents type I allergies.
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http://dx.doi.org/10.1080/09168451.2018.1490169DOI Listing
October 2018

Functional Characterization of 21 Allelic Variants of Dihydropyrimidine Dehydrogenase Identified in 1070 Japanese Individuals.

Drug Metab Dispos 2018 08 16;46(8):1083-1090. Epub 2018 May 16.

Laboratory of Pharmacotherapy of Life-Style Related Diseases, Graduate School of Pharmaceutical Sciences (E.H., Yo.N., F.A., Yu.N., N.H., M.H.), and Tohoku Medical Megabank Organization (S.S., J.Y., M.N., M.Y., M.H.), and Department of Pharmaceutical Sciences, Tohoku University Hospital, Sendai, Japan (M.M., H.Y., N.M., M.H.)

Dihydropyrimidine dehydrogenase (DPD, EC 1.3.1.2), encoded by the gene, is the rate-limiting enzyme in the degradation pathway of endogenous pyrimidine and fluoropyrimidine drugs such as 5-fluorouracil (5-FU). DPD catalyzes the reduction of uracil, thymine, and 5-FU. In Caucasians, mutations, including , , c.2846A>T, and c.1129-5923C>G/hapB3, are known to contribute to interindividual variations in the toxicity of 5-FU; however, none of these polymorphisms has been identified in the Asian population. Recently, 21 allelic variants, including some novel single-nucleotide variants (SNVs), were identified in 1070 healthy Japanese individuals by analyzing their whole-genome sequences (WGSs), but the functional alterations caused by these variants remain unknown. In this study, in vitro analysis was performed on 22 DPD allelic variants by transiently expressing wild-type DPD and 21 DPD variants in 293FT cells and characterizing their enzymatic activities using 5-FU as a substrate. DPD expression levels and dimeric forms were determined using immunoblotting and blue-native PAGE, respectively. Additionally, the values of three kinetic parameters-the Michaelis constant ( ), maximum velocity ( ), and intrinsic clearance ( = )-were determined for the reduction of 5-FU. Eleven variants exhibited significantly decreased intrinsic clearance compared with wild-type DPD. Moreover, the band patterns observed in the immunoblots of blue-native gels indicated that DPD dimerization is required for enzymatic activity in DPD. Thus, the detection of rare variants might facilitate severe adverse effect prediction of 5-FU-based chemotherapy in the Japanese population.
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http://dx.doi.org/10.1124/dmd.118.081737DOI Listing
August 2018

Points-to-consider documents: Scientific information on the evaluation of genetic polymorphisms during non-clinical studies and phase I clinical trials in the Japanese population.

Drug Metab Pharmacokinet 2018 Jun 15;33(3):141-149. Epub 2018 Mar 15.

Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Japan; National Institute of Health Sciences (NIHS), Tokyo, Japan.

Pharmacotherapy shows striking individual differences in pharmacokinetics and pharmacodynamics, involving drug efficacy and adverse reactions. Recent genetic research has revealed that genetic polymorphisms are important intrinsic factors for these inter-individual differences. This pharmacogenomic information could help develop safer and more effective precision pharmacotherapies and thus, regulatory guidance/guidelines were developed in this area, especially in the EU and US. The Project for the Promotion of Progressive Medicine, Medical Devices, and Regenerative Medicine by the Ministry of Health, Labour and Welfare, performed by Tohoku University, reported scientific information on the evaluation of genetic polymorphisms, mainly on drug metabolizing enzymes and transporters, during non-clinical studies and phase I clinical trials in Japanese subjects/patients. We anticipate that this paper will be helpful in drug development for the regulatory usage of pharmacogenomic information, most notably pharmacokinetics.
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http://dx.doi.org/10.1016/j.dmpk.2018.01.005DOI Listing
June 2018

All- Retinoic Acid Enhances Antibody Production by Inducing the Expression of Thymic Stromal Lymphopoietin Protein.

J Immunol 2018 04 2;200(8):2670-2676. Epub 2018 Mar 2.

Graduate School of Pharmaceutical Science, Tohoku University, Miyagi 980-8578, Japan;

Many classical vaccines contain whole pathogens and, thus, may occasionally induce adverse effects, such as inflammation. Vaccines containing purified rAgs resolved this problem, but, owing to their low antigenicity, they require adjuvants. Recently, the use of several cytokines, including thymic stromal lymphopoietin (TSLP), has been proposed for this purpose. However, it is difficult to use cytokines as vaccine adjuvants in clinical practice. In this study, we examined the effects of all- retinoic acid (atRA) on TSLP production and Ag-induced Ab production. Application of atRA onto the ear lobes of mice selectively induced TSLP production without inducing apparent inflammation. The effects appeared to be regulated via retinoic acid receptors γ and α. Treatment with atRA was observed to enhance OVA-induced specific Ab production; however, this effect was completely absent in TSLP receptor-knockout mice. An enhancement in Ab production was also observed when recombinant hemagglutinin was used as the Ag. In conclusion, atRA was an effective adjuvant through induction of TSLP production. Therefore, we propose that TSLP-inducing low m.w. compounds, such as atRA, may serve as effective adjuvants for next-generation vaccines.
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http://dx.doi.org/10.4049/jimmunol.1701276DOI Listing
April 2018

Zinc ions have a potential to attenuate both Ni ion uptake and Ni ion-induced inflammation.

Sci Rep 2018 02 13;8(1):2911. Epub 2018 Feb 13.

Laboratory of Pharmacotherapy of Life-Style Related Diseases, Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Miyagi, 980-8578, Japan.

Nickel ions (Ni) are eluted from various metallic materials, such as medical devices implanted in human tissues. Previous studies have shown that Ni enters inflammatory cells inducing inflammation. However, the regulation of Ni uptake in cells has not yet been reported in detail. In the present study, we investigated the effects of various divalent cations on Ni uptake and Ni-induced interleukin (IL)-8 production in the human monocytic cell line, THP-1. We demonstrated that ZnCl MnCl, and CoCl inhibited the Ni uptake, while CuCl, FeCl, MgCl, and divalent metal transporter (DMT)-1 inhibitor, Chlorazol Black, did not. Furthermore, ZnCl inhibited Ni-induced IL-8 production, correlating with the inhibition of Ni uptake. These results suggested that Ni uptake occurred through Zn, Mn, and Co-sensitive transporters and that the inhibition of Ni uptake resulted in the inhibition of IL-8 production. Furthermore, using an Ni wire-implanted mouse model, we found that Ni wire-induced expression of mouse macrophage inflammatory protein-2 (MIP-2) and cyclooxygenase-2 (COX-2) mRNA in the skin tissue surrounding the wire were enhanced by low Zn conditions. These results suggested that the physiological concentration of Zn modulates Ni uptake by inflammatory cells, and a Zn deficient state might increase sensitivity to Ni.
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http://dx.doi.org/10.1038/s41598-018-21014-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5811449PMC
February 2018

Rapid and sensitive multiplex single-tube nested PCR for the identification of five human Plasmodium species.

Parasitol Int 2018 Jun 31;67(3):277-283. Epub 2018 Jan 31.

Laboratory of Pharmacotherapy of Life-Style Related Diseases, Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai 980-8578, Japan; Department of Pharmaceutical Sciences, Tohoku University Hospital, Sendai 980-8574, Japan; Tohoku Medical Megabank Organization, Tohoku University, Sendai 980-8575, Japan. Electronic address:

Malaria is caused by five species of Plasmodium in humans. Microscopy is currently used for pathogen detection, requiring considerable training and technical expertise as the parasites are often difficult to differentiate morphologically. Rapid diagnostic tests are as reliable as microscopy and offer faster diagnoses but possess lower detection limits and are incapable of distinguishing among the parasitic species. To improve global health efforts towards malaria control, a rapid, sensitive, species-specific, and economically viable diagnostic method is needed. In this study, we designed a malaria diagnostic method involving a multiplex single-tube nested PCR targeting Plasmodium mitochondrial cytochrome c oxidase III and single-stranded tag hybridization chromatographic printed-array strip. The detection sensitivity was found to be at least 40 times higher than that of agarose gel electrophoresis with ethidium bromide. This system also enables the identification of both single- and mixed-species malaria infections. The assay was validated with 152 Kenyan samples; using nested PCR as the standard, the assay's sensitivity and specificity were 88.7% and 100.0%, respectively. The turnaround time required, from PCR preparation to signal detection, is 90min. Our method should improve the diagnostic speed, treatment efficacy, and control of malaria, in addition to facilitating surveillance within global malaria eradication programs.
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http://dx.doi.org/10.1016/j.parint.2018.01.005DOI Listing
June 2018

Nickel ions bind to HSP90β and enhance HIF-1α-mediated IL-8 expression.

Toxicology 2018 02 31;395:45-53. Epub 2018 Jan 31.

Laboratory of Pharmacotherapy of Life-Style Related Diseases, Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Miyagi, Japan. Electronic address:

Nickel ions (Ni) eluted from biomedical devices cause inflammation and Ni allergy. Although Ni and Co elicit common effects, Ni induces a generally stronger inflammatory reaction. However, the molecular mechanism by which Ni and Co induce such different responses remains to be elucidated. In the present study, we compared the effects of Ni and Co on the expression of interleukin (IL)-8 in human monocyte THP-1 cells. We report that NiCl but not CoCl induced the expression of IL-8; in contrast, CoCl elicited a higher expression of hypoxia-inducible factor-1α (HIF-1α). The NiCl-induced expression of IL-8 in late phase was blocked by a HIF-1α inhibitor, PX-478, indicating that NiCl targets additional factors responsible for activating HIF-1α. To identify such targets, proteins that bound preferentially to Ni-NTA beads were analyzed by LC/MS/MS. The analysis yielded heat shock protein 90β (HSP90β) as a possible candidate. Furthermore, Ni reduced the interaction of HSP90β with HIF-1α, and instead promoted the interaction between HIF-1α and HIF-1β, as well as the nuclear localization of HIF-1α. Using various deletion variants, we showed that Ni could bind to the linker domain on HSP90β. These results suggest that HSP90β plays important roles in Ni-induced production of IL-8 and could be a potential target for the regulation of Ni-induced inflammation.
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http://dx.doi.org/10.1016/j.tox.2018.01.006DOI Listing
February 2018

Functional characterization of 9 CYP2A13 allelic variants by assessment of nicotine C-oxidation and coumarin 7-hydroxylation.

Drug Metab Pharmacokinet 2018 Feb 22;33(1):82-89. Epub 2017 Nov 22.

Laboratory of Pharmacotherapy of Life-Style Related Diseases, Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, 980-8578, Japan; Department of Pharmaceutical Sciences, Tohoku University Hospital, Sendai, 980-8574, Japan; Tohoku Medical Megabank Organization, Tohoku University, Sendai, 980-8575, Japan. Electronic address:

Cytochrome P450 2A13 (CYP2A13) is responsible for the metabolism of chemical compounds such as nicotine, coumarin, and tobacco-specific nitrosamine. Several of these compounds have been recognized as procarcinogens activated by CYP2A13. We recently showed that CYP2A13*2 contributes to inter-individual variations observed in bladder cancer susceptibility because CYP2A13*2 might cause a decrease in enzymatic activity. Other CYP2A13 allelic variants may also affect cancer susceptibility. In this study, we performed an in vitro analysis of the wild-type enzyme (CYP2A13.1) and 8 CYP2A13 allelic variants, using nicotine and coumarin as representative CYP2A13 substrates. These CYP2A13 variant proteins were heterologously expressed in 293FT cells, and the kinetic parameters of nicotine C-oxidation and coumarin 7-hydroxylation were estimated. The quantities of CYP2A13 holoenzymes in microsomal fractions extracted from 293FT cells were determined by measuring reduced carbon monoxide-difference spectra. The kinetic parameters for CYP2A13.3, CYP2A13.4, and CYP2A13.10 could not be determined because of low metabolite concentrations. Five other CYP2A13 variants (CYP2A13.2, CYP2A13.5, CYP2A13.6, CYP2A13.8, and CYP2A13.9) showed markedly reduced enzymatic activity toward both substrates. These findings provide insights into the mechanism underlying inter-individual differences observed in genotoxicity and cancer susceptibility.
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http://dx.doi.org/10.1016/j.dmpk.2017.11.004DOI Listing
February 2018

EGFR transactivation is involved in TNF-α-induced expression of thymic stromal lymphopoietin in human keratinocyte cell line.

J Dermatol Sci 2018 Mar 18;89(3):290-298. Epub 2017 Dec 18.

Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Miyagi, 980-8578, Japan. Electronic address:

Background: Thymic stromal lymphopoietin (TSLP) is an epithelial cell-derived cytokine involved in the pathology of inflammatory skin diseases, such as atopic dermatitis and psoriasis. Tumor necrosis factor (TNF)-α, a key cytokine in inflammatory skin diseases, is a known TSLP inducer. TNF-α activates NF-κB and induces transactivation of epidermal growth factor receptor (EGFR) in epithelial cells. However, the detailed mechanism of TSLP induction by TNF-α has remained unclear.

Objective: We investigated the involvement of TNF-α-induced EGFR transactivation in TSLP expression.

Methods: HaCaT cells were stimulated with TNF-α or EGF in the presence or absence of an EGFR kinase inhibitor or other signaling inhibitors. The expression of TSLP mRNA was analyzed by RT-PCR and the phosphorylation level of signal proteins was analyzed by western blot. TSLP promoter and NF-κB transcription activities were analyzed by luciferase assay.

Results: TNF-α-induced TSLP expression was inhibited by the EGFR kinase inhibitor AG1478. While TSLP expression was induced by EGF, it was inhibited by the MEK inhibitor, U0126. Inhibitors of p38 and ADAM proteases suppressed the TNF-α-induced TSLP expression and EGFR phosphorylation, but not the EGF-induced expression.

Conclusion: TNF-α-induced EGFR transactivation results in TSLP induction through ERK activation. The activation of p38 and ADAM proteases mediates TNF-α-induced EGFR phosphorylation. These findings suggested that the TNF-α-induced EGFR transactivation pathway could be a target for the treatment of inflammatory skin diseases.
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http://dx.doi.org/10.1016/j.jdermsci.2017.12.008DOI Listing
March 2018

Induction of thymic stromal lymphopoietin by a steroid alkaloid derivative in mouse keratinocytes.

Int Immunopharmacol 2018 Feb 22;55:28-37. Epub 2017 Dec 22.

Laboratory of Pharmacotherapy of Life-Style Related Diseases, Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai 980-8578, Miyagi, Japan. Electronic address:

Thymic stromal lymphopoietin (TSLP) plays critical roles in inducing and exacerbating allergic diseases. Chemical compounds that induce TSLP production can enhance sensitization to antigens and exacerbate allergic inflammation. Hence, identifying such chemicals will be important to prevent an increase in allergic diseases. In the present study, we found, for the first time, that a steroid alkaloid derivative, code no. 02F04, concentration and time dependently induced mRNA expression and production of TSLP in a mouse keratinocyte cell line, PAM212. In particular, the activity of 02F04 was selective to TSLP. As an analogue of the liver X receptor (LXR) endogenous ligand, 02F04 rapidly increased ATP-binding cassette transporter A1 (ABCA1) expression by regulating the nuclear receptor of LXR. However, instead of being inhibited by the LXR antagonist, 02F04-induced TSLP production was delayed and markedly suppressed by inhibitors of phospholipase C (PLC), pan-protein kinase C (PKC), PKCδ, Rho-associated protein kinase (ROCK), extracellular signal-regulated kinase (ERK) 1/2, and IκΒ kinase 2 (IKK2). Treatment with 02F04 caused the formation of F-actin filaments surrounding the nucleus of PAM212 cells, which then disappeared following addition of ROCK inhibitor. 02F04 also induced phosphorylation of ERK1/2 from 2h after treatment, with a maximum at 24h, and increased nuclear factor-κB (NF-κB) promoter activity by 1.3-fold. Taken together, these results indicate that 02F04-induced TSLP production is regulated via distinct signal transduction pathways, including PLC, PKC, ROCK, ERK1/2, and NF-κB but not nuclear receptors. 02F04, with a unique skeletal structure in inducing TSLP production, can represent a potential new tool for investigating the role of TSLP in allergic diseases.
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http://dx.doi.org/10.1016/j.intimp.2017.11.045DOI Listing
February 2018

LPS priming in early life decreases antigen uptake of dendritic cells via NO production.

Immunobiology 2018 01 9;223(1):25-31. Epub 2017 Oct 9.

Laboratory of Pharmacotherapy of Life-Style Related Diseases, Graduate School of Pharmaceutical Sciences, Tohoku University, Japan. Electronic address:

Immunological mechanisms of hygiene hypothesis are expected to develop a novel strategy for allergy prevention. Although a large number of studies has investigated the relation between allergies and infection, little is known about the influence of the exposure to infections on antigen uptake by dendritic cells (DCs). In this study, we examined the effect of lipopolysaccharide (LPS) priming in early life on the antigen uptake ability of DCs by using an original mouse model. LPS priming in juvenile mice decreased the migration of antigen-capturing CD11c cells in the lymph nodes, but not in aged mice. Besides, the bone marrow-derived DCs (BMDCs) from juvenile LPS-primed mice had the poor antigen uptake ability, and constitutively produced NO through the inducible nitric oxide synthase (iNOS). Interestingly, the LPS priming-induced poor antigen uptake of BMDCs was mimicked by the NO donor, and recovered by the iNOS inhibitor. Additionally, LPS priming in juvenile mice prevented the allergic reactions, but not in aged mice. Our results suggested that an exposure to infections in early life prevents allergy through the alteration of the BM cells fate that is to induce the differentiation of BM cells into inhibitory DCs such as NO-producing DCs.
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http://dx.doi.org/10.1016/j.imbio.2017.10.018DOI Listing
January 2018