Plasma and urinary CP I and CP III concentrations in chimeric mice with human hepatocytes after rifampicin administration.

The interest in transporter-mediated drug interactions has been increasing in the field of drug development. In this study, we measured the plasma and urinary concentrations of coproporphyrin (CP) I and CP III as endogenous substrates for organic anion-transporting polypeptide (OATP) using chimeric mice with human hepatocytes (PXB mice) and examined the influence of an OATP inhibitor, rifampicin (RIF). CP I and CP III were actively taken up intracellularly, and RIF inhibited the uptake in a concentration-dependent manner for both CP I and CP III in human hepatocytes (PXB-cells). Single doses of RIF at 10 and 30 mg/kg were orally or intravenously administered to PXB mice and wild-type ICR mice. Plasma concentrations (AUC0-8h) of CP I increased in both mice. However, a marked increase in CP III was only observed in ICR mice, after intravenous administration of RIF at 30 mg/kg. The IC50 values of RIF for intracellular CP I/III uptake and the unbound plasma concentrations of RIF suggested that the increase in plasma CP I is associated with the exposure of RIF to OATPs. The 24-h cumulative urinary excretions of CP I and CP III increased in both mice, but more markedly in PXB mice. Thus, RIF increased the plasma and urinary concentrations of CP I and CP III in the mice, as reported in humans, and CP I may be a more sensitive biomarker of OATP-mediated drug interactions in PXB mice.

A 73-Year-Old Man With Refractory Hemoptysis.

CASE PRESENTATION: A 73-year-old man with fever and fatigue presented to his doctor. He had a history of smoking (52 pack-years) and COPD on home oxygen therapy. The patient had no significant family medical history, illicit drug-use history, or recent alcohol use. Chest CT scan showed a slight infiltrative shadow of the left lower lobe on a background of emphysema. Broad-spectrum antibiotics were administered for community-acquired pneumonia without any clinical or radiologic improvement after 2 weeks of therapy. Additional tests showed rapid deterioration of renal function (creatinine level, which was 0.68 mg/dL 2 years earlier, had worsened to 2.08 mg/dL), BUN level of 49.8 mg/dL (reference range, 8- to 20 mg/dL), myeloperoxidase-anti-neutrophil cytoplasmic antibodies 484.0 units/mL (range, 0.0 to 3.4 units/mL), C-reactive protein level of 11.1 mg/dL (range, 0.0 to 0.14 mg/dL), hemoglobin level of 9.0 g/dL (range, 13.7 to 16.8 g/dL), and urinalysis protein 1+ and occult blood 3+. On physical examination, multiple lesions of purpura were observed on the body surface, and hemoptysis was present. No sputum, urine, or blood cultures were done. Based on the examination, the previous doctors suspected microscopic polyangiitis (MPA) rather than an atypical/resistant infectious disease. The patient was treated with high-dose methylprednisolone (500 mg for 2 days and 125 mg for 13 days), but hemoptysis reappeared, and the patient was subsequently transported to our hospital.

Discovery and Characterization of a Novel Allosteric Small-Molecule Inhibitor of NADP+-Dependent Malic Enzyme 1.

NADP+-dependent malic enzyme 1 (ME1) decarboxylates malate to form pyruvate and NADPH in the cytoplasm, where it mediates diverse biological functions related to the generation of lipids and other cellular building blocks. As such, ME1 has been implicated in the progression of cancers and has received attention as a promising drug target. Here we report the identification of a novel small-molecule inhibitor of ME1, designated AS1134900. AS1134900 is highly selective for ME1 compared with ME2 and uncompetitively inhibits ME1 activity in the presence of its substrates NADP+ and malate. In addition, X-ray crystal structure analysis of the enzyme-inhibitor complex revealed that AS1134900 binds outside the ME1 active site in a novel allosteric site. Structural comparison of the ME1 quaternary complex with AS1134900, NADPH, and Mn2+, alongside known crystal structures of malic enzymes, indicated the determined crystal ME1-inhibitor complex is in the open form conformation. These results provide insights and a starting point for further discovery of drugs that inhibit ME1 activity in cancer cells.

Dunaimycin C3, a new GRP78 downregulator from Streptomyces sp. RAN389.

A new member of the dunaimycin family, dunaimycin C3 (2), was isolated from a fermented broth of Streptomyces sp. RAN389. The molecular formula of 2 was established as C42H70O10 by high-resolution FAB-MS, and the structure was elucidated by NMR spectroscopic analyses. Dunaimycin C3 inhibited the expression of the molecular chaperone GRP78 in HT1080 G-L cells in the presence of 10 mM of 2-deoxyglucose with an IC50 of 8.4 nM.

Therapeutic plasma exchange for clinically amyopathic dermatomyositis (CADM) associated with rapidly progressive interstitial pneumonia.

BACKGROUND: Patients with clinically amyopathic dermatomyositis (CADM) with anti-melanoma differentiation-associated gene 5 antibody (anti-MDA5 Ab) frequently develop rapidly progressive interstitial pneumonia (RPIP), often with fatal outcomes. Therapeutic plasma exchange (TPE) has been reported as effective against CADM-RPIP refractory to conventional immunosuppressive therapy. However, the detailed mechanisms by which TPE improves disease activity of CADM-RPIP remain unclear. AIM: To elucidate the clinical and demographic characteristics of patients with anti-MDA5 Ab-positive CADM-RPIP treated with TPE and to analyze changes in laboratory findings before, during, and after TPE. MATERIALS & METHODS: Patients hospitalized for CADM-RPIP and treated with TPE in 2017 and 2018 were analyzed retrospectively. RESULTS: Three patients were successfully treated with TPE, with good tolerance. Anti-MDA5 Ab titers decreased significantly over the course of TPE. CONCLUSION: We emphasize that TPE could represent an effective treatment option for CADM-RPIP refractory to traditional therapy. Removal of anti-MDA5 Ab and other pathogenic factors may facilitate favorable outcomes.

A covalent small molecule inhibitor of glutamate-oxaloacetate transaminase 1 impairs pancreatic cancer growth.

Metabolic programs are rewired in cancer cells to support survival and tumor growth. Among these, recent studies have demonstrated that glutamate-oxaloacetate transaminase 1 (GOT1) plays key roles in maintaining redox homeostasis and proliferation of pancreatic ductal adenocarcinomas (PDA). This suggests that small molecule inhibitors of GOT1 could have utility for the treatment of PDA. However, the development of GOT1 inhibitors has been challenging, and no compound has yet demonstrated selectivity for GOT1-dependent cell metabolism or selective growth inhibition of PDA cell lines. In contrast, potent inhibitors that covalently bind to the transaminase cofactor pyridoxal-5'-phosphate (PLP), within the active site of the enzyme, have been reported for kynurenine aminotransferase (KAT) and gamma-aminobutyric acid aminotransferase (GABA-AT). Given the drug discovery successes with these transaminases, we aimed to identify PLP-dependent suicide substrate-type GOT1 inhibitors. Here, we demonstrate that PF-04859989, a known KAT2 inhibitor, has PLP-dependent inhibitory activity against GOT1 and shows selective growth inhibition of PDA cell lines.

Exposure to an Extremely-Low-Frequency Magnetic Field Stimulates Adrenal Steroidogenesis via Inhibition of Phosphodiesterase Activity in a Mouse Adrenal Cell Line.

Extremely low-frequency magnetic fields (ELF-MFs) are generated by power lines and household electrical devices. In the last several decades, some evidence has shown an association between ELF-MF exposure and depression and/or anxiety in epidemiological and animal studies. The mechanism underlying ELF-MF-induced depression is considered to involve adrenal steroidogenesis, which is triggered by ELF-MF exposure. However, how ELF-MFs stimulate adrenal steroidogenesis is controversial. In the current study, we investigated the effect of ELF-MF exposure on the mouse adrenal cortex-derived Y-1 cell line and the human adrenal cortex-derived H295R cell line to clarify whether the ELF-MF stimulates adrenal steroidogenesis directly. ELF-MF exposure was found to significantly stimulate adrenal steroidogenesis (p < 0.01-0.05) and the expression of adrenal steroid synthetic enzymes (p < 0.05) in Y-1 cells, but the effect was weak in H295R cells. Y-1 cells exposed to an ELF-MF showed significant decreases in phosphodiesterase activity (p < 0.05) and intracellular Ca2+ concentration (p < 0.01) and significant increases in intracellular cyclic adenosine monophosphate (cAMP) concentration (p < 0.001-0.05) and cAMP response element-binding protein phosphorylation (p < 0.05). The increase in cAMP was not inhibited by treatment with NF449, an inhibitor of the Gs alpha subunit of G protein. Our results suggest that ELF-MF exposure stimulates adrenal steroidogenesis via an increase in intracellular cAMP caused by the inhibition of phosphodiesterase activity in Y-1 cells. The same mechanism may trigger the increase in adrenal steroid secretion in mice observed in our previous study.

Discovery and preclinical profile of teneligliptin (3-[(2S,4S)-4-[4-(3-methyl-1-phenyl-1H-pyrazol-5-yl)piperazin-1-yl]pyrrolidin-2-ylcarbonyl]thiazolidine): a highly potent, selective, long-lasting and orally active dipeptidyl peptidase IV inhibitor for the treatment of type 2 diabetes.

Dipeptidyl peptidase IV (DPP-4) inhibition is suitable mechanism for once daily oral dosing regimen because of its low risk of hypoglycemia. We explored linked bicyclic heteroarylpiperazines substituted at the γ-position of the proline structure in the course of the investigation of l-prolylthiazolidines. The efforts led to the discovery of a highly potent, selective, long-lasting and orally active DPP-4 inhibitor, 3-[(2S,4S)-4-[4-(3-methyl-1-phenyl-1H-pyrazol-5-yl)piperazin-1-yl]pyrrolidin-2-ylcarbonyl]thiazolidine (8 g), which has a unique structure characterized by five consecutive rings. An X-ray co-crystal structure of 8 g in DPP-4 demonstrated that the key interaction between the phenyl ring on the pyrazole and the S(2) extensive subsite of DPP-4 not only boosted potency, but also increased selectivity. Compound 8 g, at 0.03 mg/kg or higher doses, significantly inhibited the increase of plasma glucose levels after an oral glucose load in Zucker fatty rats. Compound 8 g (teneligliptin) has been approved for the treatment of type 2 diabetes in Japan.

Fused bicyclic heteroarylpiperazine-substituted L-prolylthiazolidines as highly potent DPP-4 inhibitors lacking the electrophilic nitrile group.

Hypoglycemic agents with a mechanism of depeptidyl peptidase IV (DPP-4) inhibition are suitable for once daily oral dosing. It is difficult to strike a balance between inhibitory activity and duration of action in plasma for inhibitors bearing an electrophilic nitrile group. We explored fused bicyclic heteroarylpiperazine substituted at the γ-position of the proline structure in the investigation of L-prolylthiazolidines lacking the electrophilic nitrile. Among them, 2-trifluoroquinolyl compound 8g is the most potent, long-lasting DPP-4 inhibitor (IC(50) = 0.37 nmol/L) with high selectivity against other related peptidases. X-ray crystal structure determination of 8g indicates that CH-π interactions generated between the quinolyl ring and the guanidinyl group of Arg358 enhances the DPP-4 inhibitory activity and selectivity.

Comparison of tissue characteristics between acute coronary syndrome and stable angina pectoris. An integrated backscatter intravascular ultrasound analysis of culprit and non-culprit lesions.

BACKGROUND: Patients with acute coronary syndrome (ACS) have multiple complex coronary plaques associated with plaque vulnerability. The present study assessed the tissue characteristics of coronary plaques between ACS and stable angina pectoris (SAP) of culprit and non-culprit lesions using integrated backscatter intravascular ultrasound (IB-IVUS). METHODS AND RESULTS: IVUS was performed in 165 patients (40 patients with ACS) with 225 culprit (65 lesions in ACS) and 171 non-culprit lesions (42 lesions in ACS). The percentage of fibrous area (fibrous area/plaque area, %FIB) and the percentage of lipid area (lipid area/plaque area, %LIP) at the segment with minimal luminal area were calculated using IB-IVUS system. Culprit and non-culprit lesions with ACS showed a significant increase in %LIP (38±18 vs. 30±15%, P=0.002, and 38±21 vs. 32±17%, P=0.03, respectively) and a significant decrease in %FIB (59±15 vs. 63±12 %, P=0.04, and 57±18 vs. 62±14%, P=0.04, respectively) compared to those with SAP. On logistic regression analysis, not only culprit lesions but also non-culprit lesions with ACS patients were significantly associated with the lipid-rich plaque. CONCLUSIONS: Non-culprit coronary lesions with ACS patients are associated with the lipid-rich plaque, suggesting the extensive development of plaques instability in these patients.

Suppression of APP-containing vesicle trafficking and production of beta-amyloid by AID/DHHC-12 protein.

The metabolism of amyloid beta-protein precursor (APP) is regulated by various cytoplasmic and/or membrane-associated proteins, some of which are involved in the regulation of intracellular membrane trafficking. We found that a protein containing Asp-His-His-Cys (DHHC) domain, alcadein and APP interacting DHHC protein (AID)/DHHC-12, strongly inhibited APP metabolism, including amyloid beta-protein (Abeta) generation. In cells expressing AID/DHHC-12, APP was tethered in the Golgi, and APP-containing vesicles disappeared from the cytoplasm. Although DHHC domain-containing proteins are involved in protein palmitoylation, a AID/DHHC-12 mutant of which the enzyme activity was impaired by replacing the DHHC sequence with Ala-Ala-His-Ser (AAHS) made no detectable difference in the generation and trafficking of APP-containing vesicles in the cytoplasm or the metabolism of APP. Furthermore, the mutant AID/DHHC-12 significantly increased non-amyloidogenic alpha-cleavage of APP along with activation of a disintegrin and metalloproteinase 17, a major alpha-secretase, suggesting that protein palmitoylation involved in the regulation of alpha-secretase activity. AID/DHHC-12 can modify APP metabolism, including Abeta generation in multiple ways by regulating the generation and/or trafficking of APP-containing vesicles from the Golgi and their entry into the late secretary pathway in an enzymatic activity-independent manner, and the alpha-cleavage of APP in the enzymatic activity-dependent manner.

Lead optimization of [(S)-gamma-(arylamino)prolyl]thiazolidine focused on gamma-substituent: Indoline compounds as potent DPP-IV inhibitors.

Dipeptidyl peptidase IV (DPP-IV) inhibitors are looked to as a potential new antidiabetic agent class. A series of [(S)-gamma-(arylamino)prolyl]thiazolidine compounds in which the electrophilic nitrile is removed are chemically stable DPP-IV inhibitors. To discover a structure for the gamma-substituent of the proline moiety more suitable for interacting with the S(2) pocket of DPP-IV, optimization focused on the gamma-substituent was carried out. The indoline compound 22e showed a DPP-IV-inhibitory activity 100-fold more potent than that of the prolylthiazolidine 10 and comparable to that of NVP-DPP728. It also displayed improved inhibitory selectivity for DPP-IV over DPP8 and DPP9 compared to compound 10. Indoline compounds such as 22e have a rigid conformation with double restriction of the aromatic moiety by proline and indoline structures to promote interaction with the binding site in the S(2) pocket of DPP-IV. The double restriction effect provides a potent inhibitory activity which compensates for the decrease in activity caused by removing the electrophilic nitrile.

Inverse correlation between soluble CD40 ligand and soluble CD40 is absent in patients with unstable angina.

The CD40/CD40 ligand (CD40L) system mediates inflammatory processes important in atherogenesis and plaque instability. The expression of CD40L on activated T cells was suppressed by soluble CD40 (sCD40) in vitro. However, the relationship between soluble CD40L (sCD40L) and sCD40 in unstable angina (UA) is still unknown. Thirty-seven consecutive patients with recent chest pain or discomfort were recruited. Patients with both Braunwald's class IB-IIIB and with coronary stenosis (or stenoses) of >75% were assigned to the UA group (n = 19, aged 67.2 +/- 8.2 years), and the rest to the control group (n = 18, aged 63.4 +/- 8.7 years). The serum levels of sCD40L and sCD40, and the plasma levels of matrix metalloproteinase (MMP)-9, were measured by enzyme-linked immunosorbent assays. A significantly inverse correlation between sCD40L and sCD40 was shown in the controls (r = -0.72, P = 0.0007), but was absent in the UA group (r = -0.16, P not significant), although there was no statistical significance between these groups in terms of serum levels of sCD40L or sCD40. The difference of the regression slopes of these regression lines was statistically significant (P < 0.01). Additionally, there was a significant correlation between sCD40 and plasma levels of MMP-9 in the patients with and without UA (r = 0.58, P = 0.0096), but no significant correlation between sCD40L and MMP-9 levels (r = 0.00, P not significant). The balance between CD40 and CD40L may be lost in patients with UA. Soluble CD40 expression may also be related to MMP-9 expression in atherosclerotic tissues.

Immunohistochemical localization of glial cell line-derived neurotrophic factor and its receptor Ret in the rat sweat gland.

Glial cell line-derived neurotrophic factor (GDNF) was initially identified as a neurotrophic factor for dopaminergic neurons but its effects are not restricted to nervous tissue. It is reported that GDNF-induced intercellular signaling is necessary for the normal development of a variety of non-neural tissues. In this study, immunoreactivities of both GDNF and its functional receptor Ret were evaluated in normal adult rat sweat gland by light and electron microscopy. Ret was found in the epithelial cells of the excretory duct and the coiled secretory terminal of the sweat gland. Electron microscopic observation of the secretory epithelium showed that Ret immunoreactivity is localized in the basal area of the secretory cells, facing myoepithelial cells, but it was not observed in the basal infolding or in the apical region of these cells. On the other hand, GDNF was observed in most myoepithelial cells of the coiled secretory portion of the sweat gland and in a small number of nerve fibers innervating the gland. Although the GDNF-containing nerve fibers cannot be negated as a source of the ligand for Ret in the secretory cells, the finding that GDNF and Ret are localized face to face in adjoining cells in the sweat gland implies another novel trophic role of GDNF in this tissue.