Downregulation of CYP1A2, CYP2B6, and CYP3A4 in Human Hepatocytes by Prolyl Hydroxylase Domain 2 Inhibitors via Hypoxia-Inducible Factor-α Stabilization.

Hypoxia-inducible factor (HIF) is associated with the expression of CYP, but the underlying mechanism remains uncertain. In this study, we investigated the effect of HIF-α stabilization caused by novel prolyl hydroxylase domain (PHD) 2 inhibitors, which are HIF-α stabilizers that mimic hypoxia, on the expressions of CYP1A2, CYP2B6, and CYP3A4 in human hepatocytes. An mRNA expression analysis of human hepatocytes treated with PHD2 inhibitors for 72 hours showed the downregulation of genes encoding CYP1A2, CYP2B6, and CYP3A4. The mRNA repressions were accompanied with an increase in erythropoietin protein, a marker of HIF-α stabilization, indicating that HIF-α stabilization was involved in the downregulation of the CYP isoforms. To understand the underlying mechanisms, we assessed the relationship between the expressions of the CYP isoforms and those of their regulating transcription factors [aryl hydrocarbon receptor (AhR), AhR nuclear translocator (ARNT), constitutive androstane receptor (CAR), pregnane X receptor (PXR), and retinoid X receptor (RXR)] in human hepatocytes treated with the HIF-α stabilizers. As a result, the mRNA level of AhR did not decrease, although ARNT expression was repressed. On the other hand, the mRNA expression levels of CAR, PXR, and RXR were repressed and closely associated with those of CYP2B6 and CYP3A4. Although the underlying mechanism of the downregulation for CYP1A2 remains unclear, the presently reported results suggest that the downregulation of CYP2B6 and CYP3A4 via HIF-α stabilization is caused by a decrease in the expressions of CAR, PXR, and RXR. SIGNIFICANCE STATEMENT: We showed that hypoxia-inducible factor (HIF)-α stabilization downregulates CYP1A2, CYP2B6, and CYP3A4 using prolyl hydroxylase domain 2 inhibitors, which are HIF-α stabilizers, as a new tool to mimic hypoxia in human hepatocytes. To understand the underlying mechanisms, we assessed the relationship between the expressions of the CYP isoforms and those of their regulating transcription factors. Our findings would contribute to a better understanding of the hypoxia-triggered regulatory mechanism of drug-metabolizing enzymes in human hepatocytes.

Synthesis and Structure-Activity Relationship of C-Phenyl D-Glucitol (TP0454614) Derivatives as Selective Sodium-Dependent Glucose Cotransporter 1 (SGLT1) Inhibitors.

Sodium-glucose cotransporter 1 (SGLT1) is the primary transporter for glucose absorption from the gastrointestinal tract. While C-phenyl D-glucitol derivative SGL5213 has been reported to be a potent intestinal SGLT1 inhibitor for use in the treatment of type 2 diabetes, no SGLT1 selectivity was found in vitro (IC50 29 nM for hSGLT1 and 20 nM for hSGLT2). In this study we found a new method of synthesizing key intermediates 12 by a one-pot three-component condensation reaction and discovered C-phenyl D-glucitol 41j (TP0454614), which has >40-fold SGLT1 selectivity in vitro (IC50 26 nM for hSGLT1 and 1101 nM for hSGLT2). The results of our study have provided new insights into the structure-activity relationships (SARs) of the SGLT1 selectivity of C-glucitol derivatives.

Discovery of novel 2-[(4-hydroxy-6-oxo-2,3-dihydro-1H-pyridine-5-carbonyl)amino]acetic acid derivatives as HIF prolyl hydroxylase inhibitors for treatment of renal anemia.

Prolyl hydroxylase domain-containing protein (PHD) inhibitors are useful as orally administered agents for the treatment of renal anemia. Based on the common structures of known PHD inhibitors, we found novel PHD inhibitor 1 with a 2-[(4-hydroxy-6-oxo-2,3-dihydro-1H-pyridine-5-carbonyl)amino]acetic acid motif. The PHD2-inhibitory activity, lipophilicity (CLogP), and PK profiles (hepatocyte metabolism, protein binding, and/or elimination half-life) of this inhibitor were used as the evaluation index to optimize the structure and eventually discovered clinical candidate 42 as the suitable compound. Compound 42 was demonstrated to promote the production of erythropoietin (EPO) following oral administration in mice and rats. The predicted half-life of this compound in humans was 1.3-5.6 h, therefore, this drug may be expected to administer once daily with few adverse effects caused by excessive EPO production.

Novel 3H-[1,2,3]triazolo[4,5-c]pyridine derivatives as GPR119 agonists: Synthesis and structure-activity/solubility relationships.

We previously reported a novel series of 1H-pyrazolo[3,4-c]pyridine derivatives and the identification of compound 4b as a highly potent GPR119 agonist. However, the advancement of preclinical evaluations of compound 4b is expected to be difficult because of the compound's significantly poor aqueous solubility (0.71µM at pH6.8). In this article, we describe the further optimization of compound 4b focusing on the improvement of its aqueous solubility. Optimization of the central spacer, left-hand aryl group and right-hand piperidine N-capping group led to the identification of a potent GPR119 agonist, 3H-[1,2,3]triazolo[4,5-c]pyridine derivative 32o, with improved solubility (15.9µM at pH6.8).

Structure-activity relationship study at C9 position of kaitocephalin.

Kaitocephalin (KCP) isolated from Eupenicillium shearii PF1191 is an unusual amino acid natural product in which serine, proline, and alanine moieties are liked with carbon-carbon bonds. KCP exhibits potent and selective binding affinity for one of the ionotropic glutamate receptor subtypes, NMDA receptors (Ki=7.8nM). In this study, new structure-activity relationship studies at C9 of KCP were implemented. Eleven new KCP analogs with different substituents at C9 were prepared and employed for binding affinity tests using native ionotropic glutamate receptors. Replacement of the 3,5-dichloro-4-hydroxybenzoyl group of KCP with a 3-phenylpropionyl group resulted in significant loss of binding affinity for NMDARs (Ki=1300nM), indicating an indispensable role of the aromatic ring of KCP in the potent and selective binding to NMDARs. Other analogs showed potent binding affinity in a range of 11-270nM. These findings would directly link to develop useful chemical tools toward imaging and labeling of NMDARs.

Design and synthesis of 1H-pyrazolo[3,4-c]pyridine derivatives as a novel structural class of potent GPR119 agonists.

Design and synthesis of a novel class of 1H-pyrazolo[3,4-c]pyridine GPR119 receptor agonists are described. Lead compound 4 was identified through the ligand-based drug design approach. Modification of the left-hand aryl group (R(1)) and right-hand piperidine N-capping group (R(2)) led to the identification of compound 24 as a single-digit nanomolar GPR119 agonist.

(7S)-Kaitocephalin as a potent NMDA receptor selective ligand.

A structure-activity relationship (SAR) study of kaitocephalin (KCP), known to be a potent naturally occurring NMDA receptor ligand, was performed. This study led us to the discovery of (7S)-kaitocephalin as a highly selective NMDA receptor ligand. It displayed a 22-fold higher degree of selectivity for the NMDA receptor over KCP, though the binding affinity of (7S)-KCP [Ki = 29 nM] was 3.7-fold less potent than that of KCP [Ki = 7.8 nM].

The structure of (-)-kaitocephalin bound to the ligand binding domain of the (S)-α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/glutamate receptor, GluA2.

Glutamate receptors mediate the majority of excitatory synaptic transmission in the central nervous system, and excessive stimulation of these receptors is involved in a variety of neurological disorders and neuronal damage from stroke. The development of new subtype-specific antagonists would be of considerable therapeutic interest. Natural products can provide important new lead compounds for drug discovery. The only natural product known to inhibit glutamate receptors competitively is (-)-kaitocephalin, which was isolated from the fungus Eupenicillium shearii and found to protect CNS neurons from excitotoxicity. Previous work has shown that it is a potent antagonist of some subtypes of glutamate receptors (AMPA and NMDA, but not kainate). The structure of kaitocephalin bound to the ligand binding domain of the AMPA receptor subtype, GluA2, is reported here. The structure suggests how kaitocephalin can be used as a scaffold to develop more selective and high affinity antagonists for glutamate receptors.

Discovery of Novel Pyrazolylpyridine Derivatives for 20-Hydroxyeicosatetraenoic Acid Synthase Inhibitors with Selective CYP4A11/4F2 Inhibition.

20-Hydroxyeicosatetraenoic acid (20-HETE) is one of the major oxidized arachidonic acid (AA) metabolites produced by cytochrome P450 (CYP) 4A11 and CYP4F2 isozymes in the human liver and kidney. Numerous studies have suggested the involvement of 20-HETE in the pathogenesis of renal diseases, and suppression of 20-HETE production by inhibition of CYP4A11 and CYP4F2 may be an attractive therapeutic strategy for renal diseases. At first, we identified methylthiazole derivative 2 as a potent dual inhibitor of CYP4A11 and CYP4F2. An optimization study of a series of derivatives with a molecular weight of around 300 to improve aqueous solubility and selectivity against drug-metabolizing CYPs while maintaining the CYP4A11- and CYP4F2-inhibitory activities led to the identification of acetylpiperidine compound 11c. Compound 11c inhibited 20-HETE production in both human and rat renal microsomes and exhibited a favorable pharmacokinetic profile. Furthermore, 11c also significantly inhibited renal 20-HETE production in Sprague-Dawley rats after oral dosing at 0.1 mg/kg.

Clinical features and treatment of epidermal growth factor inhibitor-related late-phase papulopustular rash.

Papulopustular rash, an acneiform rash, appears on the seborrheic region during the first to second week of treatment with an epidermal growth factor receptor inhibitor (EGFRi). The rash gradually disappears after the fourth week; however, it persists or newly develops in other regions during EGFRi treatment. Because Staphylococcus aureus is frequently isolated from late-phase papulopustular rash, we assessed the incidence of bacterial infection and treatment outcomes of patients with late-phase papulopustular rash. Sixty-four cases treated with an EGFRi over 4 weeks who presented with papulopustular rash were assessed retrospectively. The median duration of EGFR inhibitor treatment was 5 months. Grade 2 and 3 papulopustular rash was observed in 47 and eight cases, respectively. Bacterial culture was performed in 51 cases, 50 of which yielded positive results: methicillin-sensitive S. aureus in 29, methicillin-resistant S. aureus in 14, Staphylococcus species in five, Pseudomonas aeruginosa in three, and other in four cases. Of the S. aureus isolates, 42% were resistant to minocycline and 40% to levofloxacin. After treatment with topical and/or oral antibiotics without topical corticosteroids, the papulopustular rash rapidly improved by an average of 2.9 ± 3.4 weeks. However, use of a combination of antibiotics and a topical corticosteroid prolonged the recovery period to an average of 18.9 ± 11.4 weeks. In conclusion, folliculitis that develops over 4 weeks after the initiation of EGFRi treatment is typically caused by staphylococcal infection. Bacterial culture is necessary due to the high rate of antibiotic resistance. It is important to distinguish late- from early-phase papulopustular rash and to treat using different approaches.

Primary extranodal non-Hodgkin's lymphoma of the common bile duct manifesting as obstructive jaundice: report of a case.

Primary non-Hodgkin's lymphoma (NHL) of the common bile duct (CBD) manifesting as obstructive jaundice is extremely rare: to our knowledge, only 22 cases of primary NHL arising from the CBD have been reported. The patient in this case report was a 63-year-old man who presented with obstructive jaundice. Abdominal sonography, positron emission tomography, and computed tomography showed a mass with abnormal 18-fluorodeoxyglucose uptake in pancreatic head. Magnetic resonance cholangiopancreatography demonstrated a strictured segment of the CBD with proximal bile duct dilatation. We performed pancreaticoduodenectomy for a presumptive diagnosis of pancreatic head carcinoma or cholangiocarcinoma of the CBD. However, the histological diagnosis was a primary, diffuse, large B-cell lymphoma of the CBD. He received three courses of combination chemotherapy, including rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP). The patient remains well, without evidence of tumor recurrence, 8 months after surgery. In summary, primary NHL of the CBD, despite its rarity, should be considered in the differential diagnosis of obstructive jaundice. An accurate histopathologic diagnosis and complete surgical resection, followed by combination chemotherapy plus rituximab may be effective.

[Nomogram as predictive model in clinical practice].

A nomogram which is developed based on logistic regression analysis with multiple factors provides accurate prediction in various situations. The ability of the nomograms to predict diagnosis, staging and prognosis in prostate cancer and other disease has been confirmed to be better than other predictive models such as risk stratification and artificial neural network. Making a nomogram requires a fixed number of patients and multiple steps such as validations and calibrations. And when nomograms are developed at other institutions, validations are essential for physicians before use at the actual clinical level. We review the clinical significance of nomograms and introduce the process of making a nomogram.

Total synthesis of (-)-kaitocephalin.

[reaction: see text] Total synthesis of the potent AMPA/KA receptor antagonist (-)-kaitocephalin (1) and its three diastereomers has been accomplished. The synthesis features strictly substrate-controlled operations to alpha-formylglutamate 3 starting with alpha-hydroxymethylglutamate 4. The requisite 2R,3S,7R-stereocenters were efficiently constructed by manipulation of stereoselective reactions: dihydroxylation of 7 followed by azide substitution of the corresponding thionocarbonate 10 and Cu-mediated allylation of an acyliminium ion 21. All of the protecting groups in 26 were removed simultaneously by AlCl3/Me2S to give 1.

The expression of the aurora-A gene and its significance with tumorgenesis in non-Hodgkin's lymphoma.

Non-Hodgkin's lymphoma (NHL) has a wide biological heterogeneity with various cell origin and biological features. Recent WHO classification of lymphoid neoplasms gives importance to immunological and cytogenetic features in addition to morphologic aspects of tumors. Several investigators have performed NHL subclassification based on other biological features of tumor cells. It is extremely important to clarify the proliferation mechanism of tumor cells, and understanding this mechanism may provide insight not only into the biology of tumors but also into the treatment strategy for NHL. Therefore, research focused on the cell cycle is one of the major approaches to the biology and the oncogenesis of NHL. The Aurora kinase family recently identified from Drosophila melamogaster is believed to be an essential kinase involved in mitotic cell cycles. Several groups have reported that Aurora kinases are overexpressed in some solid tumors, suggesting that Aurora kinases may be involved in tumor survival and proliferation. Here, we focus on the role of Aurora-A kinase in the tumorgenesis of NHL using our recent research data, and discuss the possibility of Aurora-A as a new molecular target of NHL treatment.

Efficient total synthesis of (-)-kaitocephalin.

A highly diastereoselective total synthesis of (-)-kaitocephalin, a novel antagonist of ionotropic glutamate receptors, was accomplished in 12 steps starting from 5-substituted proline ester via the aldol reaction with OBO-serine aldehyde, (E)-selective alpha,beta-dehydroamino acid synthesis using a new HWE reagent, and catalytic hydrogenation.

Aurora2/BTAK/STK15 is involved in cell cycle checkpoint and cell survival of aggressive non-Hodgkin's lymphoma.

Non-Hodgkin's lymphoma (NHL) has a wide biological heterogeneity and shows extremely variable responses to therapeutic measures. However, markers that indicate disease activity and determine treatment strategies for this malignancy are little recognized. Using the differential display method, we have identified Aurora2/BTAK/STK15, a centrosome-associated serine/threonine kinase, whose overexpression leads to centrosome amplification, chromosomal instability and transformation of mammalian solid tumours. Northern analysis with mRNA from a single tumour cell suspension of NHL confirmed that Aurora2/BTAK/STK15 was highly expressed in histologically aggressive types. To elucidate the function of Aurora2/BTAK/STK15 in NHL, Aurora2/BTAK/STK15 sense or antisense genes were transfected to B-cell lymphoma cell lines to generate overexpressed or under-regulated tumour cells. Aurora2/BTAK/STK15 antisense transfectant was barely established compared with a sense or vector-only transfectant. Two clones were finally established that exhibited a low proliferation rate and significantly increased G1 arrest compared with vector-only transfectants. Moreover, antisense oligo treatment in vitro showed that restriction of cell growth appeared in proportion to antisense oligo concentration. These results suggest that Aurora2/BTAK/STK15 is an effective candidate to indicate not only disease activity but also tumorigenesis of non-Hodgkin's lymphoma. Retardation of tumour cell growth resulting from the restriction of this gene's functions may be a novel therapeutic approach for non-Hodgkin's lymphoma.

Role of CD21 antigen in diffuse large B-cell lymphoma and its clinical significance.

Recent advances in immunological and molecular technology have prompted proposals to change tumour classification and treatment strategies. Cell surface antigens are now easy to access, and tumour origins and clinical characteristics are now readily identifiable. However, in diffuse large B-cell lymphoma (DLBCL), one of the heterogeneous forms of haematological malignancy, the clinical significance of tumour surface antigens has not been well documented. We analysed the tumour surface antigens of 50 tumours from newly diagnosed DLBCL patients by flow cytometry in accordance with their clinical characteristics and followed the patients for a median 3.7 years. Statistical analysis showed that CD21 expression was significantly negatively associated with mortality in DLBCL (CD21 negative versus positive; relative risk = 2.36, P < 0.05). As a result of these clinical observations, we generated CD21-overexpressed (CD21(+)) lymphoma cell lines after gene transfection and analysed tumour cell growth in vivo in immunocompromised mice. Mice challenged with vector-only transfectants and parental cells as controls died within 50 d. In contrast, mice injected with CD21(+) transfectants exhibited significantly reduced tumour growth and 83% survived long term (versus control groups; P < 0.05). Interestingly, all established CD21(+) transfectants (six clones from different bulks) showed homotypic aggregation during in vitro cell culture, and anti-CD21 antibodies did not block this aggregation. Expression of CD21 is strongly associated with increased survival in DLBCL in vivo. CD21 expression may be indirectly concerned with the expression of additional cell adhesion molecules.