Susceptibility to serious skin and subcutaneous tissue disorders and skin tissue distribution of sodium-dependent glucose co-transporter type 2 (SGLT2) inhibitors.

Objectives: In Japan, sodium-glucose co-transporter type 2 (SGLT2) inhibitors have been reported to be associated with serious skin and subcutaneous tissue disorders. A post-marketing surveillance (PMS) study suggested that the association was specific for ipragliflozin and, to a lesser extent for dapagliflozin. These studies were performed to confirm the association of 6 SGLT2 inhibitors with serious skin disorders in a clinical setting, to elucidate the role of melanin in serious skin disorders and to understand the underlying mechanisms. Methods: The latest PMS records were retrieved from the Japanese Adverse Drug Event Report (JADER) database, and the associations were analyzed by data mining techniques. In silico 3-D docking simulation of SGLT2 inhibitors with melanin was performed using the MOE software. The skin tissue distribution of SGLT2 inhibitors was evaluated using albino rats after oral administration at clinical doses. Results: The adjusted reporting odds ratio (95% confidential limit) was 1.667 (1.415, 1.963) for ipragliflozin, 0.514 (0.317, 0.835) for dapagliflozin, 0.149 (0.048, 0.465) for tofogliflozin, 0.624 (0.331, 1.177) for luseogliflozin, 0.590 (0.277, 1.257) for canagliflozin and 0.293 (0.073, 1.187) for empagliflozin, when drugs other than the SGLT2 inhibitors were referred, and the association was detected only for ipragliflozin in clinical use. In silico 3-D docking simulation suggested the influence of melanin in ipragliflozin-specific serious skin disorders. The skin tissue-to-plasma concentration ratio of ipragliflozin was 0.45 ± 0.20 (±SD) at 1 hr after administration and increased in a time-dependent manner to 5.82 ± 3.66 at 24 hr (p<0.05), but not in case of other SGLT2 inhibitors. Conclusions: Serious skin disorders were suggested to be specific for ipragliflozin. Interaction with melanin might be implicated in ipragliflozin-specific serious skin disorders. Ipragliflozin was retained in the skin tissue, which suggested its interaction with the skin tissue in serious skin disorders.

Semi-physiological pharmacokinetic-pharmacodynamic (PK-PD) modeling and simulation of 5-fluorouracil for thrombocytopenia in rats.

1. The aim of this study was to develop a simple pharmacokinetic-pharmacodynamic (PK-PD) model that could characterize the complete time-course of alterations in platelet counts to predict the onset and degree of thrombocytopenia, which severely limits the use of the anticancer agent 5-fluorouracil (5-FU), in rats. 2. Platelet counts were measured in rats following the intravenous administration of various doses of 5-FU for 4 days to obtain data for an analysis of the PK-PD model. Our PK-PD model consisted of a two-compartment PK model, with three compartments for the PD model and 10 structural PK-PD model parameters. 3. After the 5-FU treatment, platelet counts transiently decreased to a nadir level, showed a rebound to above the baseline level before recovering to baseline levels. Nadir platelet counts and rebounds varied with the AUC0-∞ level. The final PK-PD model effectively characterized platelet count data and final PD parameters were estimated with high certainty. 4. This PK-PD model and simulation may represent a valuable tool for quantifying and predicting the complete time-course of alterations in blood cell counts, and could contribute to the development of therapeutic strategies with 5-FU and assessments of various novel anticancer agents that are difficult to examine in humans.

Pharmacokinetic-pharmacodynamic (PK-PD) modeling and simulation of 5-fluorouracil for erythropenia in rats.

INTRODUCTION: The aim of the present study was to develop a simple pharmacokinetic-pharmacodynamic (PK-PD) model in rats that could predict the onset and degree of erythropenia, a severely toxic side effect that severely limits the use of the anticancer agent 5-fluorouracil (5-FU). METHODS: Total erythrocyte counts, hemoglobin (Hb) concentrations, and hematocrit (Hct) levels were measured in rats following the intravenous bolus administration of 5-FU for 4 days in order to obtain data for an analysis of the PK-PD model. Our PK-PD model consisted of a two-compartment PK model, with two compartments for the PD model and nine structural PK-PD model parameters. RESULTS: After the intravenous bolus administration of 5, 10, or 20 mg/kg of 5-FU to rats, absolute erythrocyte counts, Hb concentrations, and Hct levels transiently decreased, reached minimum levels on Days 7-14, and then returned to baseline levels. The nadir values (Cnadir) for rats treated with 5, 10, or 20 mg/kg of 5-FU were significantly decreased to approximately 79.4, 76.3, or 46.5% of the baseline value (Cbaseline) in erythrocyte counts, 86.3, 83.3, or 45.7% of Cbaseline in Hb concentrations, 88.6, 85.5, or 47.1% of Cbaseline in Hct levels, respectively. The PK-PD model effectively captured the features of erythropenia and Cnadir after 5-FU chemotherapy. This PK-PD model was successfully used to characterize the learner relationship between the area under the plasma 5-FU concentration-time curve (AUC0-∞) following the intravenous bolus administration of 5-FU and the Cnadir in erythrocyte counts, Hb concentrations, and Hct levels after the 5-FU treatment. DISCUSSION: The results of the present study suggest that the administration of a pharmacokinetically modified dose of 5-FU could minimize the Cnadir in erythrocyte counts, Hb concentrations, and Hct levels following the administration of 5-FU. The PK-PD model and simulation represent valuable approaches for quantifying and predicting erythropenia as well as determining individual doses and the time at which the subsequent course of the treatment should start.

Semi-physiological pharmacokinetic-pharmacodynamic modeling and simulation of 5-fluorouracil for the whole time course of alterations in leukocyte, neutrophil and lymphocyte counts in rats.

We aimed to develop a simple pharmacokinetic-pharmacodynamic (PK-PD) model to predict the onset and degree of severe toxic side effects that severely limit the use of many anticancer agents, such as myelosuppression, in rats. Our PK-PD model consisted of a two-compartment PK model, with one compartment representing proliferative cells and some transit compartments consisting of maturing cells, while the other compartment represented circulating blood cells for the PD model. The semi-physiological PK-PD model effectively captured the features of myelosuppression and the degree of the off-target toxicities observed after 5-fluorouracil (5-FU) chemotherapy, and helped simultaneously simulate the whole time course for alterations in leukocyte, neutrophil and lymphocyte counts after 5-FU treatment in rats. Interestingly, by plotting the nadir period of leukocyte, neutrophil and lymphocyte counts as determined by PK-PD analytical simulation curves against the area under the plasma 5-FU concentration-time curve (AUC0-∞) after intravenous administration of 5-FU, a linear relationship was inferred, with r2=0.989, 0.877 and 0.956, respectively. The semi-physiological PK-PD model is a valuable tool for evaluating a variety of novel cancer chemopreventive agents or emerging therapeutic strategies that are difficult to address in humans.

Controlled release of insulin from self-assembling nanofiber hydrogel, PuraMatrix™: application for the subcutaneous injection in rats.

The concept of this research is, using the acetyl-(Arg-Ala-Asp-Ala)4-CONH2 peptide hydrosol (PuraMatrix™, PM), to develop an new injectable formula of controlled insulin delivery for subcutaneous injection. PM has sol-gel phase transition behavior, and was developed as a scaffold in the field of tissue engineering. The aqueous media of the PM including insulin changed from a sol to a gel phase with increasing ion strength of phosphate ion and pH in working environments in vitro and in vivo. In this study, we examined the in vitro insulin dissolution behavior and the in vivo pharmacokinetics and pharmacodynamics after subcutaneous administration of PM-insulin sol (PM-Isol). In the in vitro release study, after PM-Isol was converted to a gel phase (PM-Igel), PM concentration-dependent and controlled release of insulin were observed at the final concentrations of PM between 0.1% and 2.0% (w/v). The PM-Isol is changed to gel form in vivo, and exhibited a sustained-release pharmacokinetics of insulin, where PM concentration-dependent prolongation of efficacy was found. The plasma glucose level markedly decreased, and the lowest plasma glucose level was maintained up to 24h when 2.0% (w/v) PM-Isol was administered subcutaneously to rats. The PM-Isol, we developed here, is applicable for the wild-type of insulin, and increased the bioavailability and hypoglycemic efficacy of insulin after subcutaneous injection. Hence, the PM is a useful inactive ingredient to produce various types of control-released system of insulin by making just a few changes in PM content of the formulation.

Replacement of neutral protamine Hagedorn insulin with the long-acting insulin analogue, detemir, improves glycemic control without weight gain in basal-bolus insulin therapy in Japanese patients with type 1 diabetes.

UNLABELLED: Aims/Introduction: The aim of the present study was to evaluate the efficacy of replacing neutral protamine Hagedorn insulin (NPH) with the long-acting insulin analogue, detemir, in clinical practice. MATERIALS AND METHODS: We carried out a retrospective study to compare the effects of replacing NPH with detemir in basal-bolus insulin therapy in Japanese patients with type 1 diabetes. A total of 19 patients were enrolled in the study, and changes in hemoglobin A1c (HbA1c), insulin dose, bodyweight, fasting blood glucose levels (FBG), within-patient variability in FBG and prevalence in hypoglycemia were monitored for 12 weeks before replacement and during three periods after replacement; 1-12 weeks (period 1), 13-24 weeks (period 2) and 25-36 weeks (period 3). RESULTS: HbA1c values improved significantly in periods 2 and 3. Despite the total insulin dose remaining unchanged throughout the study, the basal insulin dose increased from 0.24 to 0.27 IU/kg/day in period 2 and 0.28 IU/kg/day in period 3. Bodyweight decreased from 61.8 to 60.8 kg in period 1, whereas FBG improved throughout the study. Within-patient variability in FBG was lower with detemir treatment than with NPH, despite the number of hypoglycemic episodes increasing significantly after replacement. CONCLUSIONS: These findings show that the weight loss observed in patients was independent of the reduction in calorie intake resulting from less frequent hypoglycemic attacks. In Japanese patients with diabetes who received NPH, replacing NPH with detemir led to improvements in glycemic control without any weight gain. (J Diabetes Invest, doi: 10.1111/j.2040-1124.2010.00066.x, 2010).

Collagenolytic activity in sonic extracts of Tannerella forsythia.

OBJECTIVE: The purpose of the present study was to characterize the collagenolytic activity in a sonicated extract of Tannerella forsythia and to investigate the activation of proMMMP-2 and -9 by the T. forsythia extract. METHODS: The T. forsythia extract was incubated with type I collagen. The cleaved products were then analyzed by SDS-PAGE using the method of Laemmli. We studied the effects of cysteine, DTT, CaCl(2), and various proteinase inhibitors on collagenolytic activity. A HT1080 cell culture supernatant containing proMMP-2 and -9 was incubated with the T. forsythia extract and analyzed for the activation of proMMP-2 and -9 by gelatin zymography. RESULTS: The T. forsythia extract degraded type I collagen. Cysteine increased the collagenolytic activity of the extract, and 5mM CaCl(2) was required for this activity. The collagenolytic activity of T. forsythia was inhibited by N-ethylmaleimide, iodoacetamide, iodoacetic acid, EDTA, and leupeptin, but not by PMSF, E-64, TLCK, or TPCK. When proMMP-2 and -9 were incubated with the T. forsythia extract, gelatinases with the relative molecular masses of MMP-2 and -9 were produced. CONCLUSION: The present study suggests that T. forsythia extract is able to degrade type I collagen and activate proMMP-2 and -9.

Histomorphometric Evaluation of Cartilage Degradation using Rabbit Articular Chondrocytes Cultured in Alginate Beads - Effects of Hyaluronan.

OBJECTIVE: A 3-dimensional alginate bead culturing method using rabbit articular chondrocytes was studied for the screening of the effectiveness of drugs for articular diseases. DESIGN: The beads cultured with IL-1ß, TGF-ß, and Hyaluronan (HA) were evaluated histochemically with Alecian blue and immunohistochemically with CS-56 antibody. Chondrocytes in alginate beads were arbitrarily classified into four groups: 1) chodrocyte surrounded with cell-associated matrix (CAM) in which proteoglycan (PG) was positively stained (PG-possitive chondrocyte); 2) chondrocyte with PG-negative CAM; 3) PG-positive CAM alone, and 4) PG-negative CAM alone. Total sulfated GAG concentrations in the culture media were quantitated by dimethylmethylene blue (DMMB) assay. ProMMP-3, TIMP-1 and -2 concentrations in the culture media were determined by sandwich enzyme immunoassays. RESULTS: Significant increase of PG-nagative cells were immunohistochemically found by IL-1ß stimulation. The pretreatment with TGF-ß almost fully suppressed those increase of PG-negative cells by IL-1ß. Both GAG and proMMP-3 concentrations in the culture media were significantly increased after IL-1ß stimulation. There were no significant differences in both TIMP-1 and TIMP-2 concentrations in the culture media with or without IL-1ß stimulation. 800-kDa HA reduced significantly the number of PG-negative cells and proMMP-3 concentration in the culture media, but showed no effects on the concentrations of both TIMPs. CONCLUSIONS: Because this 3-dimensional chondrocyte culture in alginate beads is close to in vivo conditions, this method can be used for evaluation of the effectiveness of novel drugs for articular diseases.

The activation of matrix metalloproteinases by a whole-cell extract from Prevotella nigrescens.

Periradicular lesions are primarily evoked as a response to a bacterial challenge emanating from an infected root canal. Many bacteria such as those of the genera Porphyromonas, Prevotella, and others have been isolated from infected root canals. The cause of periradicular lesions is related to the destruction of the extracellular matrix (ECM). Matrix metalloproteinases (MMPs) such as interstitial collagenase (MMP-1), gelatinase A (MMP-2), gelatinase B (MMP-9), and so on are products of inflammatory cells and, once activated, are intimately involved in the degradation of the ECM. However, there are no reports regarding the destruction of the ECM by bacterial extracts from Prevotella nigrescens (P. nigrescens). The present study was conducted to evaluate the activating effect of a whole-cell extract (WCE) of P. nigrescens on proMMP-2 and proMMP-9. P. nigrescens WCE was mixed with proMMP-2 or proMMP-9 under many conditions, and the activation of these MMPs was determined by gelatin zymography. A band indicating a lower molecular weight of 66 kd or 84 kd, which migrated faster than the band of proMMP-2 (72 kd) or proMMP-9 (92 kd) respectively, was detected, which could be the active form of either MMP. The present study suggests that P. nigrescens might be able to activate proMMP-2 and proMMP-9 in vivo and that this activation might be related to the destruction of periapical tissues.

Cytotoxicity analysis of a novel titanium alloy in vitro: adhesion, spreading, and proliferation of human gingival fibroblasts.

A recently developed novel Ti-29Nb-13Ta-4.6Zr alloy (Ti-Ta) was investigated physically and chemically, and the results suggested it to be a possibly suitable dental material. In this study we analyzed the effects of the alloy, in comparison with those of other dental metals, on the adhesion, spreading, and proliferation of human gingival fibroblasts (Gin-1 cells) in vitro. The Gin-1 cells adhered and spread well on the Ti-Ta as well as on commercially pure titanium (Ti) and commercial Ti-6Al-7Nb alloy (Ti-Al), forming long processes showing a typical fibroblastic morphology that was close to that on glass. The proliferation of Gin-1 cells was significantly suppressed on Au-Pd-Ag alloy (Au-Pd) and commercially pure copper (Cu); however, the cells proliferated as well on Ti-Ta as they did on Ti, Ti-Al, and glass. Though most of the Gin-1 cells on Cu and about half of them on Au-Pd died after 1 day and 5 days of culture, respectively, the cells on Ti-Ta, Ti, Ti-Al and glass showed 100% viability even after 5 days of culture. These results suggest that the newly developed Ti-Ta alloy has biocompatibility as good as that of Ti and Ti-Al with respect to morphology and proliferation of Gin-1 cells in vitro.

Significance of 32-kDa cathepsin L secreted from cancer cells.

It is recognized that many cancer cells secrete cathepsin L to degrade the components of extracellular matrices and basement membranes, thus promoting tumor invasion and metastasis. However, very little information is available concerning the secreted forms of cathepsin L and their possible role in human cancer. We initially demonstrated that approximately 10-fold higher mature cathepsin L activity was secreted in a medium of human fibrosarcoma (HT 1080) cells, compared with their intracellular activity. A 32-kDa major-activity band, together with a 41-kDa faint-activity band, was detected in the medium by our newly developed gelatin zymography. The two forms were further confirmed to be cathepsin L by immunoblot analysis. Both were apparently secreted directly from the cells, as neither was affected when the cells were cultured in the presence of various kinds of proteinase inhibitors. Human tumor necrosis factor-alpha (TNF-alpha) stimulated not only the production of the 32-kDa cathepsin L, but also its secretion. Moreover, the 32-kDa cathepsin L activities in 3 colon and 2 lung cancer tissues were significantly higher than in normal tissues. Based on the foregoing, there are good reasons to speculate that the 32-kDa cathepsin L found in HT 1080 cell medium is involved in cancer invasion and metastasis.

Tissue inhibitors of metalloproteinases-1 (TIMP-1) and -2(TIMP-2) are major serum factors that stimulate the TIMP-1 gene in human gingival fibroblasts.

We demonstrate in this study that both TIMP-1 and TIMP-2 are major serum factors that stimulate the induction of TIMP-1 mRNA in quiescent human gingival fibroblasts (Gin-1 cells) at mid-G1 (6-9 h after serum stimulation) of the cell cycle, but not that of TIMP-2. When we chased the secretion of both TIMP proteins into culture medium containing 10% FCS freed of both TIMPs, TIMP-2 secretion rose to the level in 10% FCS after 24 h, but TIMP-1 secretion remained at a fairly low level even after 3 days, thus reflecting a contrastive difference in the induction of both TIMP mRNAs. The stimulating activity of TIMP-1 on the expression of the TIMP-1 gene switched over to inhibitory activity, when the TIMP-1 concentration in the culture medium exceeded about 30 ng/ml. The depletion of TIMP-1 and TIMP-2 from FCS affected remarkably the induction of c-jun and c-fos mRNAs, but not that of c-ets-1 mRNA. TIMP-1 and TIMP-2-dependent expression of AP-1 protein was further demonstrated by using nuclear extracts of Gin-1 cells in an electrophoretic mobility shift assay.

Inhibitory activity on matrix metalloproteinases and cell-proliferating activity of tissue inhibitor of metalloproteinases-1 (TIMP-1)-contrastive difference between human and bovine TIMP-1s on mouse cell proliferation.

Either human or bovine TIMP-1 inhibited matrix metalloproteinase (MMP) activities, such as collagenolytic, gelatinolytic and caseinolytic, expressed by mouse cells as well as those expressed by human cells. Bovine TIMP-1 stimulated the proliferation of both human and mouse cells, but human TIMP-1 only proliferate human cells and not mouse cells indicating that the cell-proliferating activity has more strict species specificity than MMP inhibitory activity. All the results from [(3)H]thymidine incorporation, the phosphorylation of tyrosine-containing cellular proteins and extracellular-signal-regulated protein kinases supported the cell-proliferating properties of both human and bovine TIMP-1s. Human TIMP-1 seems not to bind to TIMP-1 receptors on mouse cells.

Both tissue inhibitors of metalloproteinases-1 (TIMP-1) and TIMP-2 activate Ras but through different pathways.

Tissue inhibitors of metalloproteinases-1 (TIMP-1) and TIMP-2 have growth-stimulating activity for a wide range of cell types. Ras, which comprises a family of three members, i.e, Ha-Ras, Ki-Ras, and H-Ras, is known to participate in growth control in all its facets, including cell proliferation, transformation, differentiation, and apoptosis. In this study, we tested the hypothesis that Ras might be involved in the cell growth-promoting activity of TIMPs. Using MG-63 human osteosarcoma cells, we demonstrated that both TIMP-1 and TIMP-2 caused an increase in the Ras-GTP level in a dose-dependent manner. Our previous results indicated that TIMP-1 activity is mediated through the tyrosine kinase (TYK)/mitogen-activated protein kinase (MAPK) pathway. Here, we demonstrated that Ras activation by TIMP-1 was inhibited by a specific TYK inhibitor, herbimycin A, suggesting that the TYK/MAPK signaling pathway was involved in Ras activation by TIMP-1. However, the activation of Ras by TIMP-2 was inhibited by an inhibitor specific for cyclic AMP-dependent protein kinase (PKA), H89, suggesting the involvement of the PKA-mediated pathway. Furthermore, TIMP-2 promoted the formation of a complex between Ras-GTP and phosphoinositide 3-kinase.

Matrix metalloproteinase 9 in mucosally invasive gastric cancer.

BACKGROUND: Matrix metalloproteinase 9 (MMP-9) facilitates tumor invasion and metastasis via basement membrane degradation. Control of MMP-9 production by cancer-stromal cell interactions in these processes has been observed.METHODS: We measured plasma MMP-9 concentrations, using a one-step sandwich enzyme immunoassay, and also immunohistochemically localized MMP-9 in patients with small gastric cancers limited to the mucosa. The cancers were classified as intraepithelial tumor (Tis) and T1 disease according to the tumor-node-metastasis (TNM) classification of the International Union Against Cancer.RESULTS: Patients with T1 disease had a higher positivity rate for and mean value of MMP-9 than patients with Tis disease. In T1 tumors, MMP-9 expression determined immunohistochemically, was greater in cells in cancer stroma than in cells in noncancerous stroma, a situation found in only a few Tis tumors.CONCLUSIONS: These results suggest that MMP-9 is related to the initial step of gastric cancer invasion.