Publications by authors named "Shinji Iwasaki"

59 Publications

Discovery of a novel series of GPR119 agonists: Design, synthesis, and biological evaluation of N-(Piperidin-4-yl)-N-(trifluoromethyl)pyrimidin-4-amine derivatives.

Bioorg Med Chem 2021 Jul 9;41:116208. Epub 2021 May 9.

Cardiovascular & Metabolic Drug Discovery Unit, Takeda Pharmaceutical Company Ltd, 26-1, Muraokahigashi 2-chome, Fujisawa, Kanagawa 251-8555, Japan.

We undertook an optimization effort involving propan-2-yl 4-({6-[5-(methanesulfonyl)-2,3-dihydro-1H-indol-1-yl]pyrimidin-4-yl}oxy)piperidine-1-carboxylate 1, which we had previously discovered as a novel G protein-coupled receptor 119 (GPR119) agonist. To occupy a presumed hydrophobic space between the pyrimidine and piperidine rings in interaction with GPR119, we replaced the linker oxygen with nitrogen. Subsequently, the introduction of a substituent at the bridging nitrogen atom was explored. We found that the installation of N-trifluoromethyl group 10 not only enhanced GPR119 agonist activity but also considerably improved the human ether-à-go-go-related gene (hERG) inhibition profile. These improvements were not observed for non-fluorinated substituents, such as ethyl analog 8b. The next optimization effort focused on the exploration of a new surrogate structure for the indoline ring and the isosteric replacements of the piperidine N-Boc group to improve solubility, metabolic stability, and oral bioavailability. As a result, N-{1-[3-(2-fluoropropan-2-yl)-1,2,4-oxadiazol-5-yl]piperidin-4-yl}-6-{[1-(methanesulfonyl)piperidin-4-yl]oxy}-N-(trifluoromethyl)pyrimidin-4-amine (27) was identified as a potent and orally bioavailable GPR119 agonist. This compound augmented insulin secretion and effectively lowered plasma glucose excursion in a diabetic animal model after oral administration. In this study, we discuss the designs, syntheses, and biological activities of a novel series of N-(piperidin-4-yl)-N-(trifluoromethyl)pyrimidin-4-amine derivatives as GPR119 agonists, and to determine the distinctive effect of the N-trifluoromethyl group on hERG inhibition, we also discuss the conformational preference of representative compounds.
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http://dx.doi.org/10.1016/j.bmc.2021.116208DOI Listing
July 2021

An Automated Multicycle Immunoaffinity Enrichment Approach Developed for Sensitive Mouse IgG1 Antibody Drug Analysis in Mouse Plasma Using LC/MS/MS.

Anal Chem 2021 04 13;93(16):6348-6354. Epub 2021 Apr 13.

Department of Drug Metabolism and Pharmacokinetics, Takeda Pharmaceuticals International, Inc., 35 Landsdowne Street, Cambridge, Massachusetts 02139, United States.

In the immuno-oncology field, surrogate mouse monoclonal antibodies are often preferred in establishing proper PK/PD/efficacy correlations as well as supporting anticipated mouse to human translation. Thus, a highly sensitive and specific bioanalytical method is needed in quantifying those surrogate mouse antibodies after dosing in mice. Unfortunately, when specific reagents, such as recombinant target antigen and anti-idiotypic antibody, are not available, measuring mouse surrogate antibody drugs in mice is very challenging for ligand binding assay (LBA) due to the severe cross reactivity potential. Different from LBA, if at least one unique surrogate peptide can be identified from the surrogate antibody sequence, the immunoaffinity enrichment based LC/MS/MS assay may be able to differentiate the analyte response from the high endogenous immunoglobulin background and provide adequate sensitivity. Herein, a new automated multicycle immunoaffinity enrichment method was recently developed to extract a surrogate mouse IgG1 (mIgG1) antibody drug from mouse plasma using a commercially available antimouse IgG1 secondary antibody. In the assay, reuse of the capture antibody up to six times mostly resolved the binding capacity issue caused by the abundant endogenous mIgG1 and made the immunoaffinity enrichment step more cost-effective. Combined with a unique surrogate peptide identified from the antibody, the LC/MS/MS assay achieved a limit of quantitation of 5 ng/mL with satisfactory assay precision, accuracy, and dynamic range. The successful implementation of this novel approach in discovery pharmacokinetic (PK) studies eliminates the dependence on specially generated immunoaffinity capturing reagents.
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http://dx.doi.org/10.1021/acs.analchem.1c00698DOI Listing
April 2021

Design, Synthesis, and Evaluation of (2-Aminocyclopropyl)phenyl Derivatives as Novel Positron Emission Tomography Imaging Agents for Lysine-Specific Demethylase 1 in the Brain.

J Med Chem 2021 04 17;64(7):3780-3793. Epub 2021 Mar 17.

Research, Takeda Pharmaceutical Company Limited, 26-1 Muraoka-Higashi 2-Chome, Fujisawa, Kanagawa 251-8555, Japan.

Dysregulation of histone H3 lysine 4 (H3K4) methylation is implicated in the pathogenesis of neurodevelopmental disorders. Lysine-specific demethylase 1 (LSD1) determines the methylation status of H3K4 through flavin adenine dinucleotide (FAD)-mediated histone demethylation. Therefore, LSD1 inhibition in the brain can be a novel therapeutic option for treating these disorders. Positron emission tomography (PET) imaging of LSD1 allows for investigating LSD1 expression levels under normal and disease conditions and validating target engagement of therapeutic LSD1 inhibitors. This study designed and synthesized (2-aminocyclopropyl)phenyl derivatives with irreversible binding to LSD1 as PET imaging agents for LSD1 in the brain. We optimized lipophilicity of the lead compound to minimize the risk of nonspecific binding and identified with high selectivity over monoamine oxidase A and B, which are a family of FAD-dependent enzymes homologous to LSD1. PET imaging in a monkey showed a high uptake of [F] to regions enriched with LSD1, indicating its specific binding to LSD1.
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http://dx.doi.org/10.1021/acs.jmedchem.0c01937DOI Listing
April 2021

Discovery of a novel series of indolinylpyrimidine-based GPR119 agonists: Elimination of ether-a-go-go-related gene liability using a hydrogen bond acceptor-focused approach.

Bioorg Med Chem 2021 Mar 23;34:116034. Epub 2021 Jan 23.

Cardiovascular & Metabolic Drug Discovery Unit, Takeda Pharmaceutical Company Ltd, 26-1, Muraokahigashi 2-chome, Fujisawa, Kanagawa 251-8555, Japan.

We previously identified a novel series of indolinylpyrimidine derivatives exemplified by 2 in Figure 1, which is an indoline based derivative, as potent GPR119 agonists. Despite the attractive potency of 2, this compound inhibited the human ether-a-go-go-related gene (hERG) K channel. We elucidated crucial roles of the methylsulfonyl group of 2 in its interaction with the hERG channel and the GPR119 receptor, presumably as a hydrogen bond acceptor (HBA). To remove the undesirable hERG inhibitory activity, a strategy was implemented to arrange an HBA on a less conformationally flexible framework at the indoline 5-position instead of the methylsulfonyl group. This successfully led to the discovery of a piperidinone ring as a desirable motif at the indoline 5-position, which could minimize hERG liability as shown by 24b. Further optimization focused on the reduction of lipophilicity in terms of more favorable drug-like properties. Consequently, the introduction of a hydroxy group at the 3-position of the piperidinone ring effectively reduced lipophilicity without compromising GPR119 potency, resulting in the identification of (3S)-3-hydroxy-1-{1-[6-({1-[3-(propan-2-yl)-1,2,4-oxadiazol-5-yl]piperidin-4-yl}oxy)pyrimidin-4-yl]- 2,3-dihydro-1H-indol-5-yl}piperidin-2-one ((S)-29) as a novel, potent, and orally bioavailable GPR119 agonist with a well-balanced profile. The pharmacological effects of this compound were also confirmed after single and chronic oral administration in diabetic animal models.
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http://dx.doi.org/10.1016/j.bmc.2021.116034DOI Listing
March 2021

Characterization of plasma protein binding in two mouse models of humanized liver, PXB mouse and humanized TK-NOG mouse.

Xenobiotica 2021 Jan 25;51(1):51-60. Epub 2020 Aug 25.

Drug Metabolism and Pharmacokinetics Research Laboratories, Research, Takeda Pharmaceutical Company Limited, Fujisawa city, Japan.

The unbound fractions in plasma ( ) in two mouse models of humanized liver mice, PXB and humanized TK-NOG mice, were compared with human values using equilibrium dialysis method. A good relationship between values obtained from PXB mice and humans was observed; the of 34/39 compounds (87.2%) in PXB mice were within 3-fold of human . In contrast, a weak correlation was observed between human and humanized TK-NOG mouse values; the of 15/24 compounds (62.5%) in humanized TK-NOG mice were within 3-fold of human . As different profiles of plasma protein binding (PPB) profiles were observed between PXB and humanized TK-NOG mice, evaluation is necessary in each mouse model to utilize these humanized liver mice for pharmacological, drug-drug interaction (DDI), and toxicity studies. The unbound fraction in the mixed plasma of human and SCID mouse plasma (85:15) was well correlated with in PXB mice (38/39 compounds within a 3-fold). Thus, this artificial PXB mouse plasma could be used to evaluate PPB.
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http://dx.doi.org/10.1080/00498254.2020.1808735DOI Listing
January 2021

A Translational Physiologically Based Pharmacokinetics/Pharmacodynamics Framework of Target-Mediated Disposition, Target Inhibition and Drug-Drug Interactions of Bortezomib.

AAPS J 2020 04 14;22(3):66. Epub 2020 Apr 14.

Drug Metabolism and Pharmacokinetics, Takeda Pharmaceuticals International, Co., 35 Landsdowne Street, Cambridge, Massachusetts, USA.

Bortezomib is a potent 20S proteasome inhibitor approved for the treatment of multiple myeloma and mantle cell lymphoma. Despite the extensive clinical use of bortezomib, the mechanism of the complex time-dependent pharmacokinetics of bortezomib has not been fully investigated in context of its pharmacodynamics (PD) and drug-drug interaction (DDI) profiles. Here, we aimed to develop a mechanistic physiologically based (PB) PK/PD model to project PK, blood target inhibition and DDI of bortezomib in patients. A minimal PBPK/PD model consisting of six compartments was constructed using a bottom-up approach with pre-clinical data and human physiological parameters. Specifically, the target-mediated drug disposition (TMDD) of bortezomib in red blood cells (RBC), which determines target inhibition in blood, was characterized by incorporating the proteasome binding affinity of bortezomib and the proteasome concentration in RBC. The hepatic clearance and fraction metabolized by different CYP isoforms were estimated from in vitro metabolism and phenotyping experiments. The established model adequately characterized the multi-exponential and time-dependent plasma pharmacokinetics, target binding and blood proteasome inhibition of bortezomib. Further, the model was able to accurately predict the impact of a strong CYP3A inducer (rifampicin) and inhibitor (ketoconazole) on bortezomib exposure. In conclusion, the mechanistic PBPK/PD model successfully described the complex pharmacokinetics, target inhibition and DDIs of bortezomib in patients. This study illustrates the importance of incorporating target biology, drug-target interactions and in vitro clearance parameters into mechanistic PBPK/PD models and the utility of such models for pharmacokinetic, pharmacodynamic and DDI predictions.
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http://dx.doi.org/10.1208/s12248-020-00448-xDOI Listing
April 2020

Direct Drug Delivery of Low-Permeable Compounds to the Central Nervous System Via Intranasal Administration in Rats and Monkeys.

Pharm Res 2019 Apr 1;36(5):76. Epub 2019 Apr 1.

Drug Metabolism and Pharmacokinetics Research Laboratories, Takeda Pharmaceutical Co., Ltd., 26-1, Muraoka-Higashi 2-chome, Fujisawa, Kanagawa, 251-8555, Japan.

Purpose: Intranasal administration enhances drug delivery to the brain by allowing targeted-drug delivery. Here, we investigated the properties that render a compound suitable for intranasal administration, and the differences between rodents and non-human primates in delivery to the brain.

Methods: The delivery of 10 low-permeable compounds to the brain, including substrates of efflux drug transporters expressed in the blood-brain barrier (didanosine, metformin, zolmitriptan, cimetidine, methotrexate, talinolol, ranitidine, atenolol, furosemide, and sulpiride) and two high-permeable compounds (ropinirole and midazolam) was evaluated following intranasal and intravenous administration in rats. Six of the 12 compounds (metformin, cimetidine, methotrexate, talinolol, sulpiride, and ropinirole) were also evaluated in monkeys, which have a similar nasal cavity anatomical structure to humans.

Results: In rats, most of the low-permeable compounds displayed an obvious increase in the brain/plasma concentration ratio (K) by intranasal administration (despite their substrate liability for efflux drug transporters); this was not observed with the high-permeable compounds. Similarly, intranasal administration increased K for all low-permeable compounds in monkeys.

Conclusions: Compound permeability is a key determinant of K increase by intranasal administration. This route of administration is more beneficial for low-permeable compounds and enhances their delivery to the brain in rodents and non-human primates.
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http://dx.doi.org/10.1007/s11095-019-2613-8DOI Listing
April 2019

Prediction of human pharmacokinetics of long half-life compounds using chimeric mice with humanised liver.

Xenobiotica 2019 Dec 18;49(12):1379-1387. Epub 2019 Mar 18.

Drug Metabolism and Pharmacokinetics Research Laboratories, Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited , Fujisawa , Kanagawa , Japan.

1. The prediction of human pharmacokinetic (PK) parameters is an important theme to select drug candidates from preclinical studies. It is essential to improve the prediction accuracy of compound half-life () in humans. In this study, the predictability of in humans using PXB mice®, chimeric mice with humanised liver, was assessed using 14 compounds showing long in humans. 2. After intravenous administration of the compounds to PXB mice, the plasma concentration-time profiles were fitted using one- or two-compartment models and the human clearance (CL) and distribution volume (Vd) were predicted from single-species scaling. Using the obtained parameters, the in humans was predicted. Using PXB mice, the predicted values of 71.4% of the compounds were within two-fold of the actual values. Meanwhile, based on predictions using SCID mice, the host strain of the PXB mice, only 7.1% of tested compounds were within two-fold. 3. In conclusion, we demonstrated the novel utility of PXB mice for human PK predictions of compounds having long in humans.
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http://dx.doi.org/10.1080/00498254.2019.1579394DOI Listing
December 2019

T-448, a specific inhibitor of LSD1 enzyme activity, improves learning function without causing thrombocytopenia in mice.

Neuropsychopharmacology 2019 07 22;44(8):1505-1512. Epub 2018 Dec 22.

Neuroscience Drug Discovery Unit, Research, Takeda Pharmaceutical Company Limited, 26-1, Muraoka-Higashi 2-chome, Fujisawa, Kanagawa, 251-8555, Japan.

Dysregulation of histone H3 lysine 4 (H3K4) methylation has been implicated in the pathogenesis of several neurodevelopmental disorders. Targeting lysine-specific demethylase 1 (LSD1), an H3K4 demethylase, is therefore a promising approach to treat these disorders. However, LSD1 forms complexes with cofactors including growth factor independent 1B (GFI1B), a critical regulator of hematopoietic differentiation. Known tranylcypromine-based irreversible LSD1 inhibitors bind to coenzyme flavin adenine dinucleotide (FAD) and disrupt the LSD1-GFI1B complex, which is associated with hematotoxicity such as thrombocytopenia, representing a major hurdle in the development of LSD1 inhibitors as therapeutic agents. To discover LSD1 inhibitors with potent epigenetic modulation and lower risk of hematotoxicity, we screened small molecules that enhance H3K4 methylation by the inhibition of LSD1 enzyme activity in primary cultured rat neurons but have little impact on LSD1-GFI1B complex in human TF-1a erythroblasts. Here we report the discovery of a specific inhibitor of LSD1 enzyme activity, T-448 (3-((1S,2R)-2-(cyclobutylamino)cyclopropyl)-N-(5-methyl-1,3,4-thiadiazol-2-yl)benzamide fumarate). T-448 has minimal impact on the LSD1-GFI1B complex and a superior hematological safety profile in mice via the generation of a compact formyl-FAD adduct. T-448 increased brain H3K4 methylation and partially restored learning function in mice with NMDA receptor hypofunction. T-448-type LSD1 inhibitors with improved safety profiles may provide unique therapeutic approaches for central nervous system disorders associated with epigenetic dysregulation.
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http://dx.doi.org/10.1038/s41386-018-0300-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6785089PMC
July 2019

Application of unbound liver-to-plasma concentration ratio to quantitative projection of cytochrome P450-mediated drug-drug interactions using physiologically based pharmacokinetic modelling approach.

Xenobiotica 2019 Nov 3;49(11):1251-1259. Epub 2019 Feb 3.

a Drug Metabolism and Pharmacokinetics Research Laboratories, Takeda Pharmaceutical Co., Ltd. , Fujisawa , Kanagawa , Japan.

1. This study evaluated the prediction accuracy of cytochrome P450 (CYP)-mediated drug-drug interaction (DDI) using minimal physiologically-based pharmacokinetic (PBPK) modelling incorporating the hepatic accumulation factor of an inhibitor (i.e. unbound liver/unbound plasma concentration ratio [K]) based on 22 clinical DDI studies. 2. K values were estimated using three methods: (1) ratio of cell-to-medium ratio in human cryopreserved hepatocytes (C/M) at 37 °C to that on ice (K), (2) multiplication of total liver/unbound plasma concentration ratio (K) estimated from C/M at 37 °C with unbound fraction in human liver homogenate (K) and (3) observed K in rats after intravenous infusion (K). 3. PBPK model using each K projected the area under the curve (AUC) increase of substrates more accurately than the model assuming a K of 1 for the average fold error and root mean square error did. Particularly, the model with a K of 1 underestimated the AUC increase of triazolam following co-administration with CYP3A4 inhibitor itraconazole by five-fold, whereas the AUC increase projected using the model incorporating the K, K, or K of itraconazole and hydroxyitraconazole was within approximately two-fold of the actual value. 4. The results indicated that incorporating K into the PBPK model improved the accuracy of DDI projection.
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http://dx.doi.org/10.1080/00498254.2018.1547461DOI Listing
November 2019

Quantitative prediction of the extent of drug-drug interaction using a physiologically based pharmacokinetic model that includes inhibition of drug metabolism determined in cryopreserved hepatocytes.

Xenobiotica 2018 Aug 4;48(8):770-780. Epub 2017 Sep 4.

b Drug Metabolism and Pharmacokinetics Research Laboratories, Takeda Pharmaceutical Co., Ltd , Fujisawa , Japan.

1. A physiologically based pharmacokinetic (PBPK) model that includes inhibition constant evaluated in cryopreserved hepatocytes was used to predict drug-drug interactions (DDIs) between orally administered nifedipine, a CYP substrate, and fluconazole or ketoconazole, CYP inhibitors, in rats. 2. The Kp,uu, ratio of unbound inhibitor concentration in liver ([I]liver,u) to that in plasma ([I]sys,u), of fluconazole and ketoconazole was 1.0 and 13.0, indicating that ketoconazole accumulates in liver. The ratios of inhibition constants in rat liver microsomes (Ki,mic,u) to that in rat cryopreserved hepatocytes (Ki,hep,u) for fluconazole and ketoconazole were 1.5 and 25.5, which were similar to the Kp,uu and suggested that cryopreserved hepatocytes could mimic the hepatic accumulation of inhibitors. 3. The increases in AUC of nifedipine predicted by the minimal PBPK model using [I]liver,u/Ki,mic,u and [I]sys,u/Ki,hep,u were within 1.5-fold of the observed values for both inhibitors, whereas the model using [I]sys,u/Ki,mic,u underestimated the AUC increase caused by ketoconazole 21-fold. 4. These results indicated that hepatic accumulation factor of an inhibitor is required for a precise DDI projection and that cryopreserved hepatocytes would be useful to obtain the Ki including hepatic accumulation factor. It was demonstrated that PBPK model using Ki,hep,u could be a valuable approach for quantitative DDI projection.
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http://dx.doi.org/10.1080/00498254.2017.1370744DOI Listing
August 2018

Mechanism-Based Pharmacokinetic/Pharmacodynamic Modeling of the Glucagon-Like Peptide-1 Receptor Agonist Exenatide to Characterize Its Antiobesity Effects in Diet-Induced Obese Mice.

J Pharmacol Exp Ther 2017 09 11;362(3):441-449. Epub 2017 Jul 11.

Drug Metabolism and Pharmacokinetics Research Laboratories (S.I., T.H., I.C., N.S., A.F., N.A.) and Integrated Technology Research Laboratories (T.A.), Takeda Pharmaceutical Co., Ltd., Kanagawa, Japan.

In addition to their potent antidiabetic effects, glucagon-like peptide-1 (GLP-1) analogs lower body weight in humans. Hence, agonistic targeting of the GLP-1 receptor could be a valid approach to target obesity. However, quantitative analyses of the pharmacokinetic/pharmacodynamic (PK/PD) relationship between GLP-1 analogs and their antiobesity effect have not been reported in either animals or humans. Therefore, the present study was performed to establish a mechanism-based PK/PD model of GLP-1 receptor agonists using the GLP-1 analog exenatide for the development of promising new antiobesity drugs. Exenatide was administered to high-fat diet-induced obese C57BL/6J mice via subcutaneous bolus and continuous infusion. Food intake and body-weight reductions were observed and depended on the plasma concentrations of exenatide. The homeostatic feedback model, in which food intake is assumed to be regulated by appetite control signals, described the relationship among the plasma concentration-time profile of exenatide, food intake, and body weight. The estimated IC of exenatide against food intake was 2.05 pM, which is similar to the reported K value of exenatide in rat brain and the estimated EC value for augmentation of insulin secretion in humans. The PK/PD model simulation indicated that subcutaneous infusion would show a stronger effect on body-weight reduction than bolus dosing would. This novel, quantitative PK/PD model could be used for antiobesity research and development of GLP-1 analogs, GLP-1 secretagogues, GLP-1 degradation inhibitors, and combinations thereof by allowing the estimation of appropriate pharmacokinetic profiles and dosing regimens.
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http://dx.doi.org/10.1124/jpet.117.242651DOI Listing
September 2017

LSD1 Inhibitor T-3775440 Inhibits SCLC Cell Proliferation by Disrupting LSD1 Interactions with SNAG Domain Proteins INSM1 and GFI1B.

Cancer Res 2017 09 30;77(17):4652-4662. Epub 2017 Jun 30.

Oncology Drug Discovery Unit, Pharmaceutical Research Division, Takeda Pharmaceutical Company Ltd., Fujisawa, Kanagawa, Japan.

T-3775440 is an irreversible inhibitor of the chromatin demethylase LSD1, which exerts antiproliferative effects by disrupting the interaction between LSD1 and GFI1B, a SNAG domain transcription factor, inducing leukemia cell transdifferentiation. Here, we describe the anticancer effects and mechanism of action of T-3775440 in small-cell lung cancer (SCLC). T-3775440 inhibited proliferation of SCLC cells and retarded SCLC tumor growth T-3775440 disrupted the interaction between LSD1 and the transcriptional repressor INSM1, thereby inhibiting expression of neuroendocrine-associated genes, such as INSM1 silencing phenocopied the effects of T-3775440 on gene expression and cell proliferation, consistent with the likelihood T-3775440 mediated its effects in SCLC by inhibiting INSM1. T-3775440 also inhibited proliferation of an SCLC cell line that overexpressed GFI1B, rather than INSM1, by disrupting the interaction between LSD1 and GFI1B. Taken together, our results argue that LSD1 plays an important role in neuroendocrine-associated transcription and cell proliferation of SCLC via interactions with the SNAG domain proteins INSM1 and GFI1B. Targeting these critical interactions with LSD1 inhibitors offers a novel rational strategy to therapeutically manage SCLC. .
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http://dx.doi.org/10.1158/0008-5472.CAN-16-3502DOI Listing
September 2017

Discovery of novel 5-oxa-2,6-diazaspiro[3.4]oct-6-ene derivatives as potent, selective, and orally available somatostatin receptor subtype 5 (SSTR5) antagonists for treatment of type 2 diabetes mellitus.

Bioorg Med Chem 2017 08 9;25(15):4175-4193. Epub 2017 Jun 9.

Research Division Medicinal Chemistry Laboratory, SCOHIA PHARMA, Inc., 26-1, Muraoka-Higashi 2-Chome, Fujisawa, Kanagawa 251-8555, Japan.

Somatostatin receptor subtype 5 (SSTR5) has emerged as a novel attractive drug target for type 2 diabetes mellitus. Starting from N-benzyl azetidine derivatives 1 and 2 as in-house hit compounds, we explored the introduction of a carboxyl group into the terminal benzene of 1 to enhance SSTR5 antagonistic activity by the combination of the substituents at the 3-position of the isoxazoline. Incorporation of a carboxyl group at the 4-position of the benzene ring resulted in a significant enhancement in potency, however, the 4-benzoic acid derivative 10c exhibited moderate human ether-a-go-go related gene (hERG) inhibitory activity. A subsequent optimization study revealed that replacement of the 4-benzoic acid with an isonipecotic acid dramatically reduced hERG inhibition (5.6% inhibition at 30μM) by eliminating π-related interaction with hERG K channel, which resulted in the identification of 1-(2-((2,6-diethoxy-4'-fluorobiphenyl-4-yl)methyl)-5-oxa-2,6-diazaspiro[3.4]oct-6-en-7-yl)piperidin-4-carboxylic acid 25a (hSSTR5/mSSTR5 IC=9.6/57nM). Oral administration of 25a in high-fat diet fed C57BL/6J mice augmented insulin secretion in a glucose-dependent manner and lowered blood glucose concentration.
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http://dx.doi.org/10.1016/j.bmc.2017.06.007DOI Listing
August 2017

Discovery of novel somatostatin receptor subtype 5 (SSTR5) antagonists: Pharmacological studies and design to improve pharmacokinetic profiles and human Ether-a-go-go-related gene (hERG) inhibition.

Bioorg Med Chem 2017 08 13;25(15):4153-4162. Epub 2017 Jun 13.

Research Division, Takeda Pharmaceutical Co., Ltd., 26-1, Muraoka-Higashi 2-Chome, Fujisawa, Kanagawa 251-8555, Japan.

Somatostatin (SST) is a peptide hormone comprising 14 or 28 amino acids that inhibits endocrine and exocrine secretion via five distinct G-protein-coupled receptors (SSTR1-5). SSTR5 has an important role in inhibiting the secretion of pancreatic and gastrointestinal hormones (e.g., insulin, GLP-1, PYY) through the binding of SSTs; hence, SSTR5 antagonists are expected to be novel anti-diabetic drugs. In the course of our lead generation program of SSTR5 antagonists, we have discovered a novel spiroazetidine derivative 3a. However, pharmacological evaluation of 3a revealed that it had to be administered at a high dose (100mg/kg) to show a persistent glucose-lowering effect in an oral glucose tolerance test (OGTT). We therefore initiated an optimization study based on 3a aimed at improving the antagonistic activity and mean residence time (MRT), resulting in the identification of 2-cyclopropyl-5-methoxybiphenyl derivative 3k. However, 3k did not show a sufficient persistent glucose-lowering effect in an OGTT; moreover, hERG inhibition was observed. Hence, further optimization study of the biphenyl moiety of compound 3k, focused on improving the pharmacokinetic (PK) profile and hERG inhibition, was conducted. Consequently, the introduction of a chlorine atom at the 6-position on the biphenyl moiety addressed a putative metabolic soft spot and increased the dihedral angle of the biphenyl moiety, leading to the discovery of 3p with an improved PK profile and hERG inhibition. Furthermore, 3p successfully exhibited a persistent glucose-lowering effect in an OGTT at a dose of 3mg/kg.
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http://dx.doi.org/10.1016/j.bmc.2017.06.003DOI Listing
August 2017

Use of proximal side-hole micro-balloon catheter in transcatheter hepatic arterial chemoembolization.

Minim Invasive Ther Allied Technol 2017 Dec 12;26(6):372-376. Epub 2017 May 12.

a Department of Radiology , Kochi University, Kochi Medical School , Kochi , Japan.

We report on a 70-year-old man with unresectable multiple hepatocellular carcinomas who underwent treatment with transcatheter hepatic arterial chemoembolization. In treating a tumor in segment 1 of the liver, the proximal side-hole micro-balloon catheter, which has been newly developed, was useful.
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http://dx.doi.org/10.1080/13645706.2017.1322614DOI Listing
December 2017

Comparison of predictability for human pharmacokinetics parameters among monkeys, rats, and chimeric mice with humanised liver.

Xenobiotica 2017 Dec 2;47(12):1052-1063. Epub 2017 Mar 2.

a Drug Metabolism and Pharmacokinetics Research Laboratories, Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited , Fujisawa , Japan.

1. The aim of the present study was to evaluate the usefulness of chimeric mice with humanised liver (PXB mice) for the prediction of clearance (CL) and volume of distribution at steady state (Vd), in comparison with monkeys, which have been reported as a reliable model for human pharmacokinetics (PK) prediction, and with rats, as a conventional PK model. 2. CL and Vd values in PXB mice, monkeys and rats were determined following intravenous administration of 30 compounds known to be mainly eliminated in humans via the hepatic metabolism by various drug-metabolising enzymes. Using single-species allometric scaling, human CL and Vd values were predicted from the three animal models. 3. Predicted CL values from PXB mice exhibited the highest predictability: 25 for PXB mice, 21 for monkeys and 14 for rats were predicted within a three-fold range of actual values among 30 compounds. For predicted human Vd values, the number of compounds falling within a three-fold range was 23 for PXB mice, 24 for monkeys, and 16 for rats among 29 compounds. PXB mice indicated a higher predictability for CL and Vd values than the other animal models. 4. These results demonstrate the utility of PXB mice in predicting human PK parameters.
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http://dx.doi.org/10.1080/00498254.2016.1265160DOI Listing
December 2017

Unbound liver concentration is the true inhibitor concentration that determines cytochrome P450-mediated drug-drug interactions in rat liver.

Xenobiotica 2017 Jun 20;47(6):488-497. Epub 2016 Jul 20.

a Pharmaceutical Research Division, Drug Metabolism and Pharmacokinetics Research Laboratories, Takeda Pharmaceutical Company Limited , Fujisawa , Japan.

1. In order to identify the best inhibitor concentration for the accurate prediction of magnitude of a hepatic cytochrome P450 (CYP)-mediated drug-drug interaction (DDI), the DDI between nifedipine, the CYP substrate probe, and fluconazole, ketoconazole, or ritonavir, the CYP inhibitors, in in situ rat liver perfusion system and rats were investigated. 2. In in situ system, the intrinsic clearance (CLint) of nifedipine was decreased after co-infusion of the CYP inhibitors. The decrease in in situ CLint of nifedipine was most comparable to that in in vitro CLint in rat liver microsomes calculated by using the unbound liver concentrations of inhibitors ([I]liver,u). The ratios of unbound liver concentration to unbound hepatic vein concentration (Kp,uu) of ketoconazole and ritonavir were 4.0-8.0 and 18.4-21.1, suggesting a concentrative uptake of them into liver. 3. In rats, the DDI effects of orally administered nifedipine with constant infusion of the inhibitors were investigated. The most accurate prediction of magnitude of DDI was achieved when [I]liver,u was applied as the inhibitor concentration. 4. These results indicated that [I]liver,u is the most reliable inhibitor concentration for CYP-mediated DDI and it is necessary to consider the concentrative uptake of inhibitors into liver for the quantitative prediction of DDI.
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http://dx.doi.org/10.1080/00498254.2016.1204485DOI Listing
June 2017

A successful case of deceased donor liver transplantation for a patient with intrahepatic arterioportal fistula.

Hepatol Res 2016 Dec 31;46(13):1409-1415. Epub 2016 May 31.

Department of Gastroenterological Surgery, Transplant Surgery and Surgical Oncology, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama, Japan.

Intrahepatic arterioportal fistula (IAPF) is a rare cause of portal hypertension that is often difficult to treat with interventional radiology or surgery. Liver transplantation for IAPF is extremely rare. We report a case of bilateral diffuse IAPF with severe portal hypertension requiring deceased donor liver transplantation (DDLT). A 51-year-old woman with no past medical history was admitted to another hospital complaining of abdominal distension and marasmus. A computed tomography scan and digital subtraction angiography indicated a massive pleural effusion, ascites, and a very large IAPF. Several attempts of interventional embolization of the feeding artery failed to ameliorate arterioportal shunt flow. As ruptures of the esophageal varices became more frequent, hepatic encephalopathy worsened. After repeated, uncontrollable attacks of hepatic coma, the patient was referred to our facility for further treatment. Surgical approaches to IAPF other than liver transplantation were challenging because of diffuse collateralization; therefore, we placed the patient on the national waiting list for DDLT. Although her Model for End-Stage Liver Disease score was relatively low, she received a DDLT 2 months after the waiting period. The postoperative course was uneventful, and the patient was discharged 44 days after her transplant. Liver transplantation may be a valid treatment option for uncontrollable IAPF with severe portal hypertension.
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http://dx.doi.org/10.1111/hepr.12701DOI Listing
December 2016

Peroxiredoxin 1 promotes pancreatic cancer cell invasion by modulating p38 MAPK activity.

Pancreas 2015 Mar;44(2):331-40

From the Departments of *Pharmacology, †Pathology, ‡Surgery, and §Gastroenterology and Hepatology, School of Medicine, Kochi University, Nankoku, Kochi, Japan.

Objective: The aim of this study was to investigate the role of peroxiredoxin 1 (Prdx1) in the invasiveness of pancreatic ductal adenocarcinoma (PDAC) cells.

Methods: Immunohistochemistry was used to determine overexpression of Prdx1 in human PDAC tissues. Immunoprecipitation and immunocytochemistry were used to determine the interaction and intracellular distribution of Prdx1 and a member of the mitogen-activated protein kinase (MAPK) family protein, p38 MAPK, in PDAC cells. Finally, immunocytochemistry and Matrigel invasion assay were used to examine the effects of Prdx1 and p38 MAPK on the formation of cell protrusions and PDAC cell invasion.

Results: Prdx1 is overexpressed in human PDAC tissues. Peroxiredoxin 1 interacts with active forms of p38 MAPK, and complexes of Prdx1 and phosphorylated p38 MAPK localize at the leading edges of migrating PDAC cells. Suppression of Prdx1 decreases active p38 MAPK localized in cell protrusions and inhibits the invasiveness of PDAC cells. Consequently, suppression of Prdx1 inhibits membrane ruffling and protrusions. The p38 MAPK inhibitor SB203580 also decreases the formation of membrane protrusions and inhibits invasiveness.

Conclusions: Prdx1 associates with the formation of membrane protrusions through modulation of the activity of p38 MAPK, which in turn promotes PDAC cell invasion.
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http://dx.doi.org/10.1097/MPA.0000000000000270DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4327392PMC
March 2015

RUVBL1 directly binds actin filaments and induces formation of cell protrusions to promote pancreatic cancer cell invasion.

Int J Oncol 2014 Jun 10;44(6):1945-54. Epub 2014 Apr 10.

Department of Gastroenterology and Hepatology, School of Medicine, Kochi University, Nankoku, Kochi 783-8505, Japan.

We report a novel function of RUVBL1 molecule in pancreatic cancer cells. Previous reports describe that RUVBL1 belongs to the family of AAA+ ATPases that associate with chromatin-remodelling complexes and have important roles in transcriptional regulation, the DNA damage response, telomerase activity and cellular transformation. We show that knockdown of RUVBL1 inhibited the motility and invasiveness of pancreatic cancer cells. RUVBL1 localized in the cytoplasm bound filamentous actin (F-actin) in cell protrusions, and increased concentration of monomeric globular-actin (G-actin) in cell protrusions of migrating pancreatic cancer cells. Cytoplasmic RUVBL1 functioned in additional formation of actin filaments in cell protrusions. Consequently, cytoplasmic RUVBL1 contributed to the formation of membrane protrusions by promoting peripheral actin polymerization. Our results imply that these RUVBL1-actin interactions could enhance the invasive properties of pancreatic cancer cells.
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http://dx.doi.org/10.3892/ijo.2014.2380DOI Listing
June 2014

Evaluation of Ki-67 index in EUS-FNA specimens for the assessment of malignancy risk in pancreatic neuroendocrine tumors.

Endoscopy 2014 Jan 11;46(1):32-8. Epub 2013 Nov 11.

Department of Pathology and Molecular Diagnostics, Aichi Cancer Center Hospital, Nagoya, Japan.

Background And Study Aim: Malignancy in pancreatic neuroendocrine tumors (PNETs) is graded by assessing the resected specimens according to the World Health Organization (WHO) 2010 criteria. The feasibility of such grading using endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) specimens remains unclear. The aim of this study was to ascertain the optimal method of measuring the Ki-67 index in EUS-FNA specimens, using resected specimens as the criterion standard.

Patients And Methods: A total of 58 consecutive patients diagnosed with PNETs between March 1998 and May 2011 were included. The study measured intratumoral Ki-67 index heterogeneity, concordance rates of PNET grading by EUS-FNA with grade of the resected tumor, optimal method of measuring the Ki-67 index in EUS-FNA specimens, and survival analysis based on EUS-FNA specimen grading.

Results: Intratumoral dispersion of Ki-67 index in resected specimens was 0.033 for Grade 1 and 0.782 for Grade 2 tumors (P<0.001). Concordance rates for WHO classification between EUS-FNA and resected specimens were 74.0% using the mean Ki-67 index in EUS-FNA specimens and 77.8% using the highest Ki-67 index. The concordance rate rose to 90% when EUS-FNA samples with less than 2000 tumor cells were excluded (26% of EUS-FNA cases). The Kaplan-Meier survival curves were significantly stratified by the EUS-FNA grading of PNETs with 5-year survival rates of 100%, 58.3%, and 0%, for Grade 1, Grade 2, and neuroendocrine carcinoma (NEC) tumors, respectively.

Conclusions: Grading of PNETs by the highest Ki-67 index in EUS-FNA specimens with adequate cellularity has a high concordance with grading of resected specimens, and can predict long term patient survival with high accuracy.
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http://dx.doi.org/10.1055/s-0033-1344958DOI Listing
January 2014

[Revision of the training curriculum of Japan Society of Gastroenterology: liver].

Nihon Shokakibyo Gakkai Zasshi 2013 05;110(5):812-9

Aichi Medical University Graduate School of Medicine, Japan.

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May 2013

BART inhibits pancreatic cancer cell invasion by inhibiting ARL2-mediated RhoA inactivation.

Int J Oncol 2011 Nov 10;39(5):1243-52. Epub 2011 Aug 10.

Department of Gastro-enterology and Hepatology, Kochi University Medical School, Nankoku, Kochi 783-8505, Japan.

We report that BART plays a role in inhibiting cell invasion by regulating the activity of the Rho GTPase protein RhoA in pancreatic cancer cells. BART was originally identified as a binding partner of ARL2, a small G-protein implicated as a regulator of microtubule dynamics and folding. We show that BART interacts with GTP-bound ARL2 and is required for the binding of GTP-bound ARL2 with active forms of RhoA at leading edges in migrating cancer cells. GTP-bound ARL2 inactivates RhoA and BART prevents ARL2 from regulating RhoA activity. Thus, BART binds to and functions as an inhibitor of ARL2 at leading edges of migrating cells, thereby increasing the amount of active RhoA. Treatment with the Rho inhibitor C3 exoenzyme induces cell invasion by pancreatic cancer cells to the same level as that of cells in which BART is stably knocked down by RNA interference. GTP-bound ARL2 acts as a RhoA inhibitor by a mechanism that involves the induction of actin-cytoskeleton rearrangements. We show that BART decreases actin-cytoskeleton rearrangements by inhi-biting ARL2 function and by increasing the amount of active RhoA in pancreatic cancer cells. Our results imply that BART increases active RhoA by inhibiting ARL2 function, which in turn inhibits invasiveness of cancer cells.
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http://dx.doi.org/10.3892/ijo.2011.1156DOI Listing
November 2011

Velamentous cord insertion caused by oblique implantation after in vitro fertilization and embryo transfer.

J Obstet Gynaecol Res 2011 Nov 9;37(11):1698-701. Epub 2011 Jun 9.

Department of Obstetrics and Gynecology, Showa University School of Medicine, Tokyo, Japan.

We present a case of a 36-year-old pregnant female after intracytoplasmic sperm injection. Ultrasonographic examination at 8 weeks' gestation revealed umbilical cord insertion with a viable fetus located on the septum membrane of dichorionic twin pregnancy near the anterior wall, while the other fetus was observed to have vanished. Next, this umbilical cord was seen to connect to the anterior wall and the placenta developed on the posterior wall later in the pregnancy. As a result, velamentous cord insertion with long membranous umbilical vessels developed at the time of delivery. The present case indicates that the assessment of the cord insertion site during the early gestation period is very important to predict any abnormality of the cord insertion site at the time of delivery. Furthermore, this case is valuable to understand the pathophysiological development of the placenta and velamentous cord insertion.
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http://dx.doi.org/10.1111/j.1447-0756.2011.01555.xDOI Listing
November 2011

A novel and comprehensive mouse model of human non-alcoholic steatohepatitis with the full range of dysmetabolic and histological abnormalities induced by gold thioglucose and a high-fat diet.

Liver Int 2011 Apr 19;31(4):542-51. Epub 2011 Jan 19.

Department of Gastroenterology and Hepatology, Kochi Medical School, Kochi, Japan.

Background: The search for effective treatments of non-alcoholic steatohepatitis (NASH), now the most common chronic liver disease in affluent countries, is hindered by a lack of animal models having the range of anthropometric and pathophysiological features as human NASH.

Aims: To examine if mice treated with gold thioglucose (GTG) - known to induce lesions in the ventromedial hypothalamus, leading to hyperphagia and obesity - and then fed a high-fat diet (HF) had a comprehensive histological and dysmetabolic phenotype resembling human NASH.

Methods: C57BL/6 mice were injected intraperitoneally with GTG and then fed HF for 12 weeks (GTG+HF). The extent of abdominal adiposity was assayed by CT scanning. A glucose tolerance test and an insulin tolerance test were performed to evaluate insulin resistance (IR). Histological, molecular and biochemical analyses were also performed.

Results: Gold thioglucose+HF induced dysmetabolism, with hyperphagia, obesity with increased abdominal adiposity, IR and consequent steatohepatitis, with hepatocyte ballooning, Mallory-Denk bodies, perivenular and pericellular fibrosis as seen in adult NASH, paralleled by an increased expression of the profibrogenic factors, transforming growth factor-β1 and TIMP-1. Plasma adiponectin and the expression of adiponectin receptor 1 and receptor 2 were decreased, while PPAR-γ and FAS were increased in the livers of GTG+HF mice. In addition, GTG+HF mice showed glucose intolerance and severe IR.

Conclusions: Treatment with GTG and HF diet induce, in mice, a comprehensive model of human NASH, with the full range of dysmetabolic and histological abnormalities.
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http://dx.doi.org/10.1111/j.1478-3231.2010.02443.xDOI Listing
April 2011

[Anti smooth muscle antibody].

Nihon Rinsho 2010 Jun;68 Suppl 6:596-8

Department of Gastroenterology and Hepatology, Kochi Medical School.

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June 2010

Matrix metalloproteinase-1 expression in splenic angiosarcoma metastasizing to the serous membrane.

Int J Clin Exp Pathol 2010 Jun 25;3(6):634-9. Epub 2010 Jun 25.

Department of Pathology, Kochi Medical School, Nankoku, Kochi, Japan.

Angiosarcoma involving the serous membrane may mimic mesothelioma; therefore, the term "pseudomesotheliomatous angiosarcoma" has been suggested for this entity. However, the pathogenesis of pseudomesotheliomatous angiosarcoma remains unclear. Here, we report an autopsy case of splenic angiosarcoma, which systemically metastasized to the serous membrane of both the peritoneum and pleura, closely resembling a mesothelioma. The spindle-shaped tumor cells exhibited marked invasion of the lymphatic vessels and invaded the serous membrane causing thickening of the fibrous tissues like desmoplastic mesothelioma. In the present case, immunohistochemical staining showed that the tumor expressed not only the endothelial cell markers, such as CD31, vascular endothelial growth factor receptor 3, and podoplanin (D2-40), but also matrix metalloproteinase-1 (also known as collagenase-1), which is known to increase the invasiveness of mesothelioma cells. MMP-1 expression was not observed in the other cases of angiosarcoma, examined. This tumor might systemically metastasize to the serous membrane via the lymphatic route and might generate the fibrous stroma aided by the matrix metalloproteinase-1.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2907126PMC
June 2010

A novel membrane fusion-mediated plant immunity against bacterial pathogens.

Genes Dev 2009 Nov 15;23(21):2496-506. Epub 2009 Oct 15.

Graduate School of Science, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan.

Plants have developed their own defense strategies because they have no immune cells. A common plant defense strategy involves programmed cell death (PCD) at the infection site, but how the PCD-associated cell-autonomous immunity is executed in plants is not fully understood. Here we provide a novel mechanism underlying cell-autonomous immunity, which involves the fusion of membranes of a large central vacuole with the plasma membrane, resulting in the discharge of vacuolar antibacterial proteins to the outside of the cells, where bacteria proliferate. The extracellular fluid that was discharged from the vacuoles of infected leaves had both antibacterial activity and cell death-inducing activity. We found that a defect in proteasome function abolished the membrane fusion associated with both disease resistance and PCD in response to avirulent bacterial strains but not to a virulent strain. Furthermore, RNAi plants with a defective proteasome subunit PBA1 have reduced DEVDase activity, which is an activity associated with caspase-3, one of the executors of animal apoptosis. The plant counterpart of caspase-3 has not yet been identified. Our results suggest that PBA1 acts as a plant caspase-3-like enzyme. Thus, this novel defense strategy through proteasome-regulating membrane fusion of the vacuolar and plasma membranes provides plants with a mechanism for attacking intercellular bacterial pathogens.
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http://dx.doi.org/10.1101/gad.1825209DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2779742PMC
November 2009

Traumatic bilateral testicular dislocation: a recovery of spermatogenesis by orchiopexy 15 years after the onset.

Fertil Steril 2008 Nov 9;90(5):2009.e9-11. Epub 2008 Jun 9.

Department of Urology, Showa University Hospital, Tokyo, Japan.

Objective: To report a patient with azoospermia who achieved an induction of spermatogenesis after undergoing orchiopexy of long-standing bilateral traumatic dislocated testes.

Design: Case report.

Setting: Clinical.

Patient(s): A 33-year-old man.

Intervention(s): Imaging studies, orchiopexy, testicular biopsy, and a review of similar cases.

Main Outcome Measure(s): Recovery of spermatogenesis after orchiopexy.

Result(s): A patient with azoospermia with bilateral traumatic dislocated testes due to a motorcycle accident 15 years previously. A bilateral testicular dislocation in the superficial inguinal pouch was diagnosed by palpation, imaging studies, and exploration. A recovery of spermatogenesis was obtained after performing orchiopexy of bilateral dislocated testes.

Conclusion(s): The recovery of spermatogenesis in patients with azoospermia with a bilateral testicular dislocation therefore may be successfully obtained after appropriately performing orchiopexy.
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http://dx.doi.org/10.1016/j.fertnstert.2008.01.105DOI Listing
November 2008
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