Stereospecific recognition of a spirosuccinimide type aldose reductase inhibitor (AS-3201) by plasma proteins: a significant role of specific binding by serum albumin in the improved potency and stability

AS-3201 [(3R)-2'-(4-bromo-2-fluorobenzyl)spiro[pyrrolidine-3,4'(1'H)-pyrrolo[1,2-a]pyrazine]-1',2,3',5(2'H)-tetrone] is a structurally novel and stereospecifically potent aldose reductase (AKR1B; EC 1.1.1.21) inhibitor, which contains a succinimide ring that undergoes ring-opening at physiological pH levels. To delineate intermolecular interactions governing its favorable pharmacokinetic profile, the interaction of AS-3201 (R-isomer) with plasma proteins, especially human serum albumin (HSA), was examined in comparison with that of the optical antipode (S-isomer). Fluorescence, kinetic, and high-performance frontal analyses showed that the R-isomer is more strongly bound than the S-isomer to sites I and II on HSA, and the R-isomer is particularly protected from hydrolysis, suggesting that the stable HSA-R-isomer complex contributes to its prolonged activity. The thermodynamic parameters for the specific binding indicated that in addition to hydrophobic interactions, hydrogen bonds contribute significantly to the R-isomer complex formation. (13)C NMR observations of the succinimide ring (5-(13)C enriched), which are sensitive to its ionization state, suggested the presence of a hydrogen bond between the R-isomer and HSA, and (19)F NMR of the pendent benzyl ring (2-(19)F) evaluated the equilibrium exchange dynamics between the specific sites. Furthermore, fatty acid binding or glycation (both are site II-oriented perturbations) inhibited the binding to one of the specific sites and reduced the stereospecificity of HSA toward the isomers, although the clinical influence of these perturbations on the R-isomer binding ratio seemed to be minor. Thus, the difference in the interaction mode at site II might be a major cause of the stereospecificity; this is discussed on the basis of putative binding modes. The present results, together with preliminary absorption and distribution profiles, provide valuable information on the stereospecific pharmacokinetic and pharmacodynamic properties of the R-isomer relevant for the therapeutic treatment of diabetic complications.     

Participation of epoxygenase activation in saikogenin D-induced inhibition of prostaglandin E(2) synthesis

We examined the effect of saikogenin D on arachidonic acid metabolism in C6 rat glioma cells to clarify its anti-inflammatory mechanism. Incubation of C6 cells with saikogenin D for 20 min resulted in the inhibition of prostaglandin E(2) production and the accumulation of an arachidonic acid metabolite that was found to be 11,12-dihydroxyeicosatrienoic acid, a metabolite of 11,12-epoxyeicosatrienoic acid. C6 cells expressed rat epoxygenase mRNAs, CYP1A1, CYP2B1 and CYP2J3, which converted arachidonic acid to epoxyeicosatrienoic acids. 11,12-Epoxyeicosatrienoic acid inhibited A23187-induced prostaglandin E(2) production and SKF-525A, an inhibitor of epoxygenase, attenuated the saikogenin D-induced inhibition of prostaglandin E(2) production in C6 cells. Furthermore, 11,12-epoxyeicosatrienoic acid and 11,12-dihydroxyeicosatrienoic acid, but not saikogenin D, inhibited the activity of cyclooxygenase in a cell-free condition. These data suggest that saikogenin D activates epoxygenases that rapidly convert arachidonic acid to epoxyeicosanoids and dihydroxyeicosatrienoic acids, and then the metabolites secondarily inhibit prostaglandin E(2) production.     

Use of structure-based virtual screening in the investigation of novel human sialidase inhibitors

Recent studies have determined the molecular mechanisms underlying the pathological alterations of sialic acid or its conjugates in various human disease states, and the sialic acid modifying enzyme sialidase has been adopted as an attractive therapeutic option for diseases like cancer, diabetes, inflammation, and arteriosclerosis. Isoform human sialidase inhibitors are also a good chemical tool for exploring the differential functions of human sialidases. In the current study, structure-based virtual screening was attempted to identify the compounds containing novel structures for human sialidase inhibition. The best-hit compound, containing a furan core structure with a dicarboxylic acid group, was purchased from Maybridge Chemical Company and was evaluated for its inhibitory activity against all four types of sialidases. This is the first report on the structure-based virtual screening of human sialidases and provides some structural insights for further efforts in this area.     

Ameliorating effect of FK614, a novel nonthiazolidinedione peroxisome proliferator-activated receptor gamma agonist, on insulin resistance in Zucker fatty rat

Effect of 3-(2,4-dichlorobenzyl)-2-methyl-N-(pentylsulfonyl)-3H-benzimidazole-5-carboxamide (FK614), a novel nonthiazolidinedione peroxisome proliferator-activated receptor (PPAR) gamma agonist, on glucose tolerance and insulin resistance in peripheral tissues and in liver using Zucker fatty rats (genetically obese and insulin-resistant) was evaluated and compared to other insulin sensitizers. FK614 (0.32, 1 and 3.2 mg/kg), two thiazolidinedione PPAR gamma agonists, rosiglitazone (0.1, 0.32, 1 and 3.2 mg/kg) and pioglitazone (1, 3.2 and 10 mg/kg), and a biguanide, metformin (320 and 1000 mg/kg), were orally administered to Zucker fatty rats once a day for 14 days. Zucker fatty rats treated with FK614 and rosiglitazone were subjected to evaluation by oral glucose tolerance test. Ameliorating effect of each compound on peripheral and hepatic insulin resistance was evaluated using a euglycemic-hyperinsulineamic clamp procedure. FK614 and rosiglitazone dose-dependently improved impaired glucose tolerance in Zucker fatty rats. In addition, FK614 dose-dependently ameliorated peripheral and hepatic insulin resistance in Zucker fatty rats, with the degree of its effect in peripheral tissues almost equivalent to that in liver when compared at each dose tested. Similar data indicating ameliorating effects on insulin resistance was obtained for rosiglitazone and pioglitazone. Metformin showed less potent effects than other insulin sensitizers and its effect in liver tended to be greater than that in peripheral tissues. These findings suggest clinical potential for FK614 as a treatment of type 2 diabetes, acting by ameliorating insulin resistance both in peripheral tissues and liver.     

Comparative assessment of ocular tissue distribution of drug-related radioactivity after chronic oral administration of 14C-levofloxacin and 14C-chloroquine in pigmented rats

Fluoroquinolones have been reported to have a high affinity for melanin. The ocular tissue distribution and accumulation of radioactivity was compared after repeated oral administration of 14C-levofloxacin and 14C-chloroquine at daily doses of 20 mg (0.054 mmol) kg(-1) and 28 mg (0.054 mmol) kg(-1), respectively, in pigmented rats for 84 days. The mean serum level at 24 h following each dose of 14C-levofloxacin was almost constant in the range of 0.33-0.45 nmol equiv mL(-1) after the 14th dose and thereafter. The melanin-containing ocular tissues, such as iris ciliary body and stratum pigment chorioides sclera, showed a much higher concentration of radioactivity than other non-pigmented ocular tissues. The respective concentration in iris ciliary body and stratum pigment chorioides sclera after the 1st dose was 126.47 and 74.91 nmol equiv g(-1), and gradually increased with increasing dose number, reaching 1261.81 and 447.45 nmol equiv g(-1) after the 84th dose, which was ca. 10 and 6 times higher, respectively, than after the 1st dose. The mean serum level following each dose of 14C-chloroquine was almost constant in the range 0.51-0.87 nmol equiv mL(-1) after the 7th dose and thereafter. The respective concentration in iris ciliary body and stratum pigment chorioides sclera after the 1st dose was 572.10 and 709.41 nmol equiv g(-1), and gradually increased with increasing dose number, reaching 33 317.92 and 12 322.90 nmol equiv g(-1) after the 84th dose, which was ca. 58 and 17 times higher, respectively, than after the 1st dose. The concentration in aqueous humour, cornea, lens, vitreous body and retina after the 84th dose was 1.84, 6.33, 0.48, 5.60 and 11.42 nmol equiv g(-1) for 14C-levofloxacin and 18.84, 264.99, 27.26, 158.43 and 1020.89 nmol equiv g(-1) for 14C-chloroquine (ca. 10, 42, 57, 28 and 89 times higher, respectively, than for 14C-levofloxacin). Especially, the concentration in the retina was markedly higher after 14C-chloroquine administration than after 14C-levofloxacin administration. The concentration and the extent of accumulation of radioactivity not only in melanin-containing ocular tissues but also in other non-pigmented ocular tissues, such as retina, after chronic oral administration of 14C-levofloxacin once daily for 84 days were much lower than those after multiple dosing with 14C-chloroquine under the same conditions. These results indicate that levofloxacin would have a much lower risk for ocular toxicity than chloroquine after chronic dosing.     

Integrated FDG-PET/CT compared with intravenous contrast-enhanced CT for evaluation of metastatic regional lymph nodes in patients with resectable early stage esophageal cancer

Objective  To assess whether integrated fluorodeoxyglucose positron emission tomography/computed tomography (FDG-PET/CT) can improve the diagnostic accuracy of metastatic regional lymph nodes (LNs) in esophageal cancer compared with contrast enhanced CT (CECT). Methods  We examined 180 consecutive patients with esophageal cancer by integrated PET/CT between April 2006 and March 2007. Eighteen patients (M:F 14:4) underwent radical esophagectomy after evaluations by PET/CT and CECT of 5–7-mm-thick slices 70–80 s after injection. Regional LNs of esophageal cancer were retrospectively reviewed on CECT images by two blinded evaluators on the basis of the following cutoff sizes: 7 mm for all regional LNs (Protocol A), 10 mm for paratracheal LNs (Protocol B), and 7 mm for others. In addition, the maximum standardized uptake value (SUVmax) on PET/CT was evaluated for positive uptake by LNs. Results  Of 210 LNs excised at surgery, 25 were positive and 185 were negative for metastasis at pathology. The PET/CT images identified 15 true-positive and 184 truenegative LNs, whereas CECT identified 15 true positives and 176 true negatives in Protocol A, and 14 true positives and 180 true negative in Protocol B. The sensitivity, specificity, accuracy, positive, and negative predictive values of PET/CT were respectively 60.0%, 99.5%, 94.8%, 93.8%, and 94.8%, whereas those of CECT were 60.0%, 95.1%, 91.0%, 62.5%, and 94.6% (Protocol A) and 56.0%, 97.3%, 92.4%, 73.7%, and 94.2% (Protocol B). A comparison of the two CECT protocols revealed fewer false-positive LNs in Protocol B, but slightly lower sensitivity in Protocol B than in Protocol A. Substantial numbers of false-positive LNs were determined by CECT in the paratracheal regions (6 of 9, 66.7%) and CECT revealed central necrosis in 4 of 15 (26.7%) true-positive LNs > 1.8 cm. The mean SUVmax on PET/CT was 2.9 (range 1.7–5.5) in true-positive LNs. The smallest LN metastasis detectable by PET/CT was 6 mm. Conclusions  Integrated PET/CT improves the PPV of regional LNs when compared with CECT.     

Photocrosslinkable chitosan as a dressing for wound occlusion and accelerator in healing process.

Application of ultraviolet light (UV-) irradiation to a photocrosslinkable chitosan (Az-CH-LA) aqueous solution resulted in an insoluble, flexible hydrogel like soft rubber within 60 s. The chitosan hydrogel could completely stop bleeding from a cut mouse tail within 30 s of UV-irradiation and could firmly adhere two pieces of sliced skins of mouse to each other. In order to evaluate its accelerating effect on wound healing, full thickness-skin incisions were made on the back of mice and subsequently an Az-CH-LA aqueous solution was added into the wound and irradiated with UV light for 90 s. Application of the chitosan hydrogel significantly induced wound contraction and accelerated wound closure and healing. Histological examinations also have demonstrated an advanced granulation tissue formation and epithelialization in the chitosan hydrogel treated wounds. The chitosan hydrogel due to its accelerating healing ability is considered to become an excellent dressing for wound occlusion and tissue adhesive in urgent hemostasis situations.     

Imatinib mesylate inhibits cell invasion of malignant peripheral nerve sheath tumor induced by platelet-derived growth factor-BB

Malignant peripheral nerve sheath tumor (MPNST) is rare, highly aggressive, resistant to radiochemotherapy, and associated with poor prognosis. Basic research to develop new treatment regimes is critically needed. This study was designed to identify motogenic factor(s) involved in MPNST cell invasion and inhibitor(s) of such invasive activity. We profiled the invasion-inducing activities of eight motogenic growth factors on two human MPNST cell lines, FU-SFT8611 and 9817, using in vitro Matrigel invasion assays. Platelet-derived growth factor-BB (PDGF-BB) was identified as the most effective MPNST cell invasion-inducing factor. Epidermal growth factor (EGF) and hepatocyte growth factor (HGF) also stimulated invasion in one MPNST cell line. Expressions of PDGF-BB and EGF receptors (PDGFR-beta and EGFR) mRNAs were detected more frequently and their proteins were expressed at higher levels in MPNST tissues than benign peripheral nerve sheath tumors (schwannomas and neurofibromas). In both MPNST cell lines, PDGF-BB induced tyrosine phosphorylation of PDGFR-beta but not of PDGFR-alpha, and specific PDGFR-beta inhibition by small interfering RNA to the receptor inhibited PDGF-BB-stimulated MPNST cell invasion, suggesting the predominant role of PDGFR-beta. Inhibition of PDGFR-beta phosphorylation by pretreatment with herbimycin A and imatinib mesylate effectively suppressed basement membrane invasion and cell growth in vitro. No mutations were present in exons 12 and 18 of PDGFR-beta in both MPNST cell lines and 10 human MPNST tissues examined. Our results indicated that PDGF-BB enhanced the invasive activity of MPNST cells through PDGFR phosphorylation and that imatinib inhibited such activity. The results provide the ground for further assessment of the therapeutic potential of imatinib in suppressing the invasion and growth of MPNST.     

Analysis of mRNA with microsomal fractionation using a SAGE-based DNA microarray system facilitates identification of the genes encoding secretory proteins

In the regulation of host defense responses such as inflammation and immunity, the secretory proteins, including membrane proteins, play central roles. Although many secretory proteins have been identified by using methods such as differential display, random screening, or the signal sequence trap method, each method suffers from poor reproducibility, low sensitivity, or time-consuming or laborious work. Therefore, the strategy for facilitating the selection of the genes encoding the secretory proteins is desired. In this paper, we describe a system for isolating the genes encoding secretory proteins by analyzing mRNAs with microsomal fractionation on serial analysis of gene expression (SAGE)-based DNA microarray system. This system succeeded in discriminating the genes encoding secretory proteins from ones encoding nonsecretory proteins with 80% accuracy. We applied this system to human T lymphocytes. As a result, we were able to identify the genes that are not only encoding secretory proteins but also expressing selectively in a specific subset of T lymphocytes. The SAGE-based DNA microarray system is a promising system to identify the genes encoding specific secretory proteins.