The Bcr-Abl kinase inhibitor INNO-406 induces autophagy and different modes of cell death execution in Bcr-Abl-positive leukemias.

Bcr-Abl tyrosine kinase (TK) inhibitors are promising therapeutic agents for Bcr-Abl-positive (Bcr-Abl(+)) leukemias. Although they are known to promote caspase-mediated apoptosis, it remains unclear whether caspase-independent cell death-inducing mechanisms are also triggered. Here we demonstrated that INNO-406, a second-generation Bcr-Abl TK inhibitor, induces programmed cell death (PCD) in chronic myelogenous leukemia (CML) cell lines through both caspase-mediated and caspase-independent pathways. The latter pathways include caspase-independent apoptosis (CIA) and necrosis-like cell death (CIND), and the cell lines varied regarding which mechanism was elicited upon INNO-406 treatment. We also observed that the propensity toward CIA or CIND in cells was strongly associated with cellular dependency on apoptosome-mediated caspase activity. Cells that undergo CIND have a high apoptosome activity potential whereas cells that undergo CIA tend to have a lower potential. Moreover, we found that INNO-406 promotes autophagy. When autophagy was inhibited with chloroquine or gene knockdown of beclin1 by shRNA, INNO-406-induced cell death was enhanced, which indicates that the autophagic response of the tumor cells is protective. These findings suggest new insights into the biology and therapy of Bcr-Abl(+) leukemias.

The pharmacokinetics and effects of a long-acting preparation of superoxide dismutase (PC-SOD) in man.

AIM: To study the pharmacokinetics (PK), safety and tolerability of single rising doses up to 80 mg of superoxide dismutase covalently linked to lecithin (PC-SOD) in healthy White volunteers. METHODS: This double-blind, placebo-controlled, four-period cross-over study was performed in eight healthy volunteers (four male/four female). Three doses of PC-SOD (20, 40 and 80 mg) and placebo were administered intravenously in randomized order. Serum and urinary PC-SOD concentrations were measured predose and up to 96 h after dosing. In addition to standard safety measurements, the urinary excretion of N-acetyl-beta-glucosaminidase, alpha-glutathione S-transferase (alpha-GST) and pi-GST was measured to evaluate renal function. The PK of PC-SOD was analysed using noncompartmental and compartmental methods. RESULTS: All treatments were well tolerated, and no obvious relationship between adverse events and treatment was observed. No effects of PC-SOD on renal function could be detected. Dose normalized C(max) and AUC were not different between the different dosages, indicating linearity of plasma concentrations with dose. Estimated PC-SOD clearance was 2.54 ml min(-1)[95% confidence interval (CI) 2.07, 2.83]. The terminal half-life was estimated to be 1.54 days (95% CI 0.93, 2.15). SOD activity was elevated above baseline for 19 +/- 6 h after the 80-mg dose. CONCLUSIONS: Single intravenous administrations of PC-SOD in doses up to 80 mg were well tolerated in healthy White male and female volunteers. With the doses used, SOD activity was linearly related to the dose; after the 80-mg dose it was present for an appreciable period. These findings suggest that it is worthwhile to investigate PC-SOD in clinical conditions characterized by a high radical overload.

DDS for anti-aging and regenerative medicine (review).

In this paper we summarized, first the present status and history of the development of research in anti-aging and regenerative medicine in Japan, and secondly some of our research using DDS in the field of both medicine. The regenerative medicine has been developed in Japan by using the fund from the Government, particularly as the Millennium Project. While anti-aging medicine developed following the social interest on it in Japan and it was influenced by American Society (A4M). Next, we summarized our research on DDS for anti-aging and regenerative medicine. In most cases we used oily or solid nanoparticles as carriers of drug. Those particles have a property of both of targeting and slow release in the DDS technology. The two properties are important for anti-aging and regenerative medicine, since drugs have to be administered safely and for long time. We applied prostaglandin E1, granulocyte-colony stimulate factor (G-CSF), and retinoid into the systems.

Administration of optimum sustained-insulin release PLGA microcapsules to spontaneous diabetes-prone BB/Wor//Tky rats.

To show the possibility of sustained-release insulin formulation composed of PLGA, the optimum one was administered to BioBreeding rat, a model of spontaneous type I diabetes mellitus (IDDM). Every 2 weeks subcutaneous administration made their blood glucose level depend on the insulin release and food intake. However, all of them kept alive with little change or rather a little gain in body weight. Furthermore, some of pregnant rats with intermittent treatment bore fetuses, although additional insulin therapy seemed necessary. Therefore, the formulation could become a new tool as a provider of basal insulin for IDDM patients.

Drug-incorporating calcium carbonate nanoparticles for a new delivery system.

We devised a simple method for incorporating drugs into solid calcium carbonate nanoparticles (nano-CaCO3). The size of nano-CaCO3 was controlled by mixing speed. Washing the nanoparticles released little incorporated drug but much drug that was adsorbed on the surface. In an in vitro releasing test, granulocyte colony-stimulating factor incorporated in nano-CaCO3 was chemically stable and released very slowly. Subcutaneous injection of nano-CaCO3 incorporating betamethasone phosphate (BP) resulted in a smaller initial increase in plasma concentration and a subsequent sustained release in compared with betamethasone phosphate solution. Nano-CaCO3 may be useful to deliver hydrophilic drugs and bioactive proteins.

Treatment of experimental arthritis with poly(D, L-lactic/glycolic acid) nanoparticles encapsulating betamethasone sodium phosphate.

OBJECTIVE: To examine the therapeutic activity of hydrophilic glucocorticoid encapsulated in PLGA nanoparticles, which have shown slow release and are targeted to inflamed joints after intravenous administration, in experimental arthritis models. METHODS: Betamethasone sodium phosphate (BSP) encapsulated in PLGA nanoparticles with a size of 100-200 nm (PLGA-nanosteroid) was prepared using a modified oil in water emulsion solvent diffusion method with Zn ions and coated with lecithin. Rats with adjuvant arthritis (AA rats) and mice with anti-type II collagen antibody induced arthritis (AbIA mice) were treated intravenously with PLGA-nanosteroid after the initial sign of arthritis. RESULTS: In AA rats, a 30% decrease in paw inflammation was obtained in 1 day and maintained for 1 week with a single injection of 100 mug of PLGA-nanosteroid. Soft x ray examination 7 days after this treatment showed decreased soft tissue swelling. Moreover, the PLGA-nanosteroid was also highly effective in AbIA mice. A single injection of 30 mug of the PLGA-nanosteroid resulted in almost complete remission of the inflammatory response after 1 week. In contrast, the same dose of free BSP after three administrations only moderately reduced the severity of inflammation. In addition, a histological examination 7 days after the treatment showed a significant decrease of the inflammatory cells in the joints. CONCLUSION: The observed strong therapeutic benefit obtained with PLGA-nanosteroid may be due to the targeting of the inflamed joint and its prolonged release in situ. Targeted drug delivery using a sustained release PLGA-nanosteroid is a successful intervention in experimental arthritis.

Expression of neuronal growth inhibitory factor (metallothionein-III) in the salivary gland.

Metallothioneins (MTs) are metal-binding proteins that have been regarded as intrinsic factors for protecting cells and tissues from metal toxicity and oxidants. Among the three major classes of MTs, MT-III is different from other MTs because it has neuronal inhibitory activity and is only expressed in the central nervous system. Recent studies, however, have confirmed that MT-III is also expressed in organs other than the brain. These findings not only indicate that MT-III has a much wider tissue distribution than was originally thought, but also suggest that it might have other unknown activities. In the present study, we examined the human salivary and thyroid glands and demonstrated that the MT-III gene is also expressed in the salivary but not in the thyroid gland. While salivary ducts showed intense immuno-reactivity with anti-MT-III, weak immunoreactivity was observed in acinar cells. This, together with the findings that some neuromodulators (i.e. nerve growth factor, etc.) exist in the salivary gland and that MT-III may participate in the transport in renal tubules, suggest that MT-III may have other functions than cytoprotection in the salivary gland.

Comparison of various submucosal injection solutions for maintaining mucosal elevation during endoscopic mucosal resection.

BACKGROUND AND STUDY AIMS: One of the major complications of endoscopic mucosal resection (EMR) for gastrointestinal tumors is perforation, and the most effective way of preventing perforation is to elevate the lesion sufficiently by endoscopic injection of fluid into the submucosa. MATERIALS AND METHODS: In order to compare the lesion-lifting properties of several different solutions, 1 ml of each of the following solutions was injected into the submucosa of the resected porcine stomach: normal saline, 3.75 % NaCl, 20 % dextrose water, 10 % glycerin with 0.9 % NaCl plus 5 % fructose, and two sodium hyaluronate (SH) solutions. RESULTS: Significantly higher initial elevation was produced by both SH solutions, and it remained higher than that achieved by the other solutions at all times. Hypertonic solutions, especially 10 % glycerin with 0.9 % NaCl plus 5 % fructose, tended to produce and maintain greater mucosal elevation than normal saline, but the difference was not significant. CONCLUSIONS: SH solutions were the most suitable ones for producing and maintaining long-term mucosal elevation, while the superiority of hypertonic solutions over normal saline was not clearly demonstrated.

Different mixtures of sodium hyaluronate and their ability to create submucosal fluid cushions for endoscopic mucosal resection.

BACKGROUND AND STUDY AIMS: Sodium hyaluronate (SH) is a promising submucosal injection solution during endoscopic mucosal resection, but its high cost is an obstacle to more widespread use. The aim of this study was to identify an appropriate low-cost SH solution by varying the molecular weight of SH and mixing various solutions with it. MATERIALS AND METHODS: The viscoelasticity of various SH solutions was first measured. The concentrations of two 1 % SH preparations with different molecular weights (800 kDa and 1900 kDa) were adjusted to 0.5 %, 0.25 %, and 0.125 %, using 0.9 %/3.75 % normal saline (NS), 5 %/20 % dextrose water (DW), and a glycerin solution (Glyceol): 10 % glycerin with 0.9 % normal saline plus 5 % fructose. The ability of these SH solutions to create submucosal fluid cushions (SFCs) was then investigated in the stomachs of two live minipigs. RESULTS: The 0.25 % 1900 kDa SH/NS solution and the 0.125 % 1900 kDa SH/20 % DW solution created a similar viscoelasticity to that of the 0.5 % 800 kDa SH/NS solution. The ability of these solutions to create SFCs was also similar. In addition, the 0.125 % 1900 kDa SH/Glyceol solution created similar SFCs, with a synergistic effect of increased viscoelasticity and the hypertonic nature of glycerin. CONCLUSIONS: A mixture of higher molecular weight sodium hyaluronate with a sugar solution (particularly 20 % dextrose), with or without glycerin, should be regarded as a cost-effective option for creating SFCs instead of the conventional SH solution made with the same amount of a 1 % 800 kDa SH preparation and normal saline.

Oral tolerization with peptide 336-351 linked to cholera toxin B subunit in preventing relapses of uveitis in Behcet's disease.

Behcet's disease (BD) specific peptide (p336-351) was identified within the human 60 kD heat shock protein (HSP60). Oral p336-351 induced uveitis in rats which was prevented by oral tolerization with the peptide linked to recombinant cholera toxin B subunit (CTB). This strategy was adopted in a phase I/II clinical trial by oral administration of p336-351-CTB, 3 times weekly, followed by gradual withdrawal of all immunosuppressive drugs used to control the disease in 8 patients with BD. The patients were monitored by clinical and ophthalmological examination, as well as extensive immunological investigations. Oral administration of p336-351-CTB had no adverse effect and withdrawal of the immunosuppressive drugs showed no relapse of uveitis in 5 of 8 patients or 5 of 6 selected patients who were free of disease activity prior to initiating the tolerization regimen. After tolerization was discontinued, 3 of 5 patients remained free of relapsing uveitis for 10-18 months after cessation of all treatment. Control of uveitis and extra-ocular manifestations of BD was associated with a lack of peptide-specific CD4+ T cell proliferation, a decrease in expression of TH1 type cells (CCR5, CXCR3), IFN-gamma and TNF-alpha production, CCR7+ T cells and costimulatory molecules (CD40 and CD28), as compared with an increase in these parameters in patients in whom uveitis had relapsed. The efficacy of oral peptide-CTB tolerization will need to be confirmed in a phase III trial, but this novel strategy in humans might be applicable generally to autoimmune diseases in which specific antigens have been identified.

Insulin-loaded biodegradable PLGA microcapsules: initial burst release controlled by hydrophilic additives.

We investigated the controlled release of human insulin at an initial stage from poly(DL-lactic-co-glycolic acid) (PLGA, M(w) 6600) spherical matrices. PLGA microcapsules were prepared by the novel solvent evaporation multiple emulsion process. When the crystalline insulin was dispersed in dichloromethane as solid-in-oil (S/O) dispersion, it was found that most of insulin molecules were inlaid on the surface of PLGA microcapsules. Consequently, insulin-loaded PLGA microcapsules exhibited marked rapid release of insulin within several hours in both in vivo and in vitro experiments. On the other hand, the addition of glycerol or water in the primary dichloromethane dispersion results in drastically suppressed initial release. It was found by SEM observation that water- or glycerol-in-oil (W/O or G/O) type mini-emulsion droplets with a mean diameter of 300-500 nm were formed in this primary solution. This phenomenon can be theoretically presumed to occur because insulin and PLGA molecules, having amphiphilic properties, converge on the interface between the hydrophilic additive and dichloromethane. Hence, insulin molecules heterogeneously located in the inside of PLGA microcapsules, not on the surface, would be gradually released with PLGA hydrolytic decomposition. As an additional effect of glycerol, the initial burst was further suppressed due to the decrease of the glass transition temperature of PLGA from 42.5 to 36.7 degrees C. Since the annealing of PLGA molecules took place at around 37 degrees C, the porous structure of microspheres immediately disappeared after immersion in PBS or subcutaneous administration. The insulin diffusion through the water-filled pores would be effectively prevented. The strict controlled initial release of insulin from the PLGA microsphere suggested the possibility of utilization in insulin therapy for type I diabetic patients who need construction of a basal insulin profile.

Defining a T-cell epitope within HSP 65 in recurrent aphthous stomatitis.

The 65 kD heat shock protein (HSP) has been implicated in the aetiology of recurrent aphthous stomatitis (RAS). We have previously demonstrated that peptide 91-105 derived from the sequence of mycobacterial 65 kD HSP stimulates specifically lymphocytes from patients with RAS. In this investigation, we show that both CD4+ and CD8+ T cells were significantly stimulated with mycobacterial peptide 91-105. In contrast, the human homologous peptide 116-130 stimulated only CD4+ T cells. Inhibition studies showed that CD4+ T cells were class II restricted, whereas CD8+ T cells were class I restricted. We then used truncated or substituted peptides, and demonstrated that residues 95-105 appear to be important, and residue 104(Arg) critical, in stimulating the T cells. Thus, peptide 95- 105 may constitute a T-cell proliferative epitope in RAS. We postulate that the high load of micro-organisms that colonize the oral mucosa may initiate an immune response by the microbial HSP 65-derived peptide 95-105, stimulating the numerous Langerhans cells in the oral mucosa to activate a cross-reacting immune response to the homologous peptide 116-130 within the epithelial HSP 60, initiating the immunopathological changes that lead to RAS.

Lecithinized copper, zinc-superoxide dismutase ameliorates ischemia-induced myocardial damage.

We have reported that lecithin-conjugated recombinant human Cu, Zn-superoxide dismutase (lecithinized SOD) has greater pharmacological potency than unmodified SOD through an increase in cell membrane affinity and half-life in plasma. Recently, ischemia or hypoxia alone has been suggested to result in increased superoxide anions, which lead to apoptosis in cardiomyocytes. We tested the effect of lecithinized SOD in reducing the infarct size following prolonged myocardial ischemia without reperfusion. Rats were subjected to a 24-h left coronary occlusion. Lecithinized SOD, unmodified SOD, free lecithin derivative or PBS was administered intravenously 30 min before coronary occlusion. SOD concentration of the heart, measured by ELISA, was higher in the lecithinized SOD-treated group than in the other groups 24 h after administration. The infarct area ratio of the heart, assessed by TTC staining, in the lecithinized SOD-treated group was significantly smaller than those of the other groups. Both TUNEL-positive cardiomyocytes and DNA laddering were attenuated in the ischemic area of the heart treated with lecithinized SOD. Single bolus administration of lecithinized SOD had a cardioprotective effect against ischemia without reperfusion in the rat model of acute myocardial infarction, possibly due to its sustained high tissue concentration.

Osteoclast-like cells in an in vitro model of bone destruction by rheumatoid synovium.

OBJECTIVE: Osteoclasts may be involved in the process of rheumatoid bone destruction. To test this hypothesis, we developed an in vitro model of bone destruction by osteoclast-like cells derived from cultured rheumatoid synovial tissue without using any inducers. METHODS: Synovial tissues were obtained from rheumatoid arthritis and osteoarthritis patients and tissue pieces of about 2 mm(3) that contained synovial lining were cultured. Multinucleated cells derived from cultured synovial tissues were studied cytochemically and morphologically for osteoclast-specific markers. RESULTS: Fibroblast-like and macrophage-like cells from the tissue pieces proliferated in the coexistence of lymphocytes. After 14 days of culture, multinucleated cells with tartrate-resistant acid phosphatase activity appeared. These cells expressed vacuolar H(+)-ATPase, the vitronectin receptor and cathepsin K. Although binding of (125)I-labelled salmon calcitonin was very low, the cells contained ringed structures of F-actin and showed strong bone-resorbing activity on ivory slices. Proliferation of macrophage-like cells and formation of multinucleated cells continued during 6 months of culture in the presence of fibroblast-like cells. The bone-resorbing activity of multinucleated cells derived from rheumatoid synovial tissue was much higher than that of cells from osteoarthritis synovial tissue, and was related to the disease activity of rheumatoid arthritis. CONCLUSION: Our culture system reproduced in vitro the process of bone destruction by rheumatoid synovium, including the proliferation and fusion of precursor cells, polarization, activation and bone tissue resorption. This system may provide a tool for understanding the mechanisms of bone destruction in rheumatoid arthritis and for the development of new therapies to prevent bone destruction.

[Toxicity study of cefmatilen hydrochloride hydrate (S-1090) (6)--Six-month repeated oral dose toxicity study in dogs].

A six-month repeated oral dose toxicity study of Cefmatilen hydrochloride hydrate (S-1090) at dose levels of 40, 100 and 250 mg potency/kg/day was conducted in male and female beagle dogs. No toxicologically significant changes were observed in general conditions of all animals. Reddish-brown feces (due to chelated products of S-1090 or its decomposition products with Fe3+ in the diet) were observed in all treated groups. Plasma irons showed a tendency to increase in the males and females of the 250 mg potency/kg group. However, as no changes suggesting anemia or hepatic injury were observed in this group, the change of plasma iron was considered to have no toxicological significance. No toxicologically significant changes were observed in other examination items. The plasma S-1090 concentrations increased in a manner less than dose-proportional. Based on the above results, the NOAEL of S-1090 was assessed to be 250 mg potency/kg/day.

[Toxicity study of cefmatilen hydrochloride hydrate (S-1090) (7)--Three-month repeated oral dose toxicity study in juvenile dogs].

To evaluate the repeated oral dose toxicity of Cefmatilen hydrochloride hydrate (S-1090) in juvenile dogs, S-1090 was administered to juvenile beagle dogs at dose levels of 50, 100, 200 and 400 mg potency/kg/day for 3 months. No deaths occurred. Urinalysis in the 400 mg potency/kg group revealed positive reactions of occult blood and protein, and erythrocytes in sediments. Cystitis was observed in the 200 and 400 mg potency/kg groups. In the thyroids, an increased weight in some animals in the groups dosed at 100 mg potency/kg or more and an increased follicular colloid in the 400 mg potency/kg group were observed. However, no related changes were noted in other examination items. Red to dark-red feces (due to chelated products of S-1090 or its decomposition products with Fe3+ in the diet) were observed in all treated groups. Plasma S-1090 concentrations increased in a manner less than dose-proportional. The lesions of urinary bladder were judged as S-1090-induced toxic changes and the NOAEL of S-1090 in this study was assessed to be 100 mg potency/kg/day.

[Toxicity study of cefmatilen hydrochloride hydrate (S-1090) (5)--Six-month repeated oral dose toxicity study and supplement study in rats].

Cefmatilen hydrochloride hydrate (S-1090) was orally administered to rats at dose levels of 100, 300 and 1000 mg potency/kg once daily for 6 months. All the S-1090 treated groups showed soft feces, reddish-brown feces (due to chelated products of S-1090 or its decomposition products with Fe3+ in the diet), abdominal distention, increased food and water consumption, lower urine pH, and a decrease of white blood cells counts (except for males of the 100 mg potency/kg group). One male in the 300 mg potency/kg group showed mucous feces and marked decrease in body weight, and diet in the middle stage of the administration period. In necropsy of the survivors of all treated groups, marked cecal enlargement was noted. No remarkable changes were observed in the other examination items. From the early stage of the withdrawal period, animals in the 1000 mg potency/kg group showed again soft or mucous feces and a marked decrease in body weight. Of these animals, one male died and another male was sacrificed in a moribund state at about 2 weeks of the withdrawal period. Enterocolitis was observed in these cases. Almost all animals recovered within 3 weeks of withdrawal. A supplemental study of the 6-month toxicity study was conducted to examine the mechanisms of enterocolitis and the changes observable in the 100 or 300 mg potency/kg groups after drug withdrawal. As a reference, cefdinir (CFDN), an oral cephem antibiotic the same as S-1090, was added in the 1000 mg potency/kg group. No deaths occurred in any groups. Decreased intestinal flora were noted in all the groups treated with S-1090 or CFDN at the end of the dosing period. At 2 weeks of the withdrawal period, C. difficile and its D-1 toxin in the cecal contents were highly detected in the S-1090 300 and 1000 mg potency/kg groups and CFDN group. Inflammatory changes in the cecum and colon were observed in these groups. At 4 weeks of the withdrawal period, intestinal flora in the S-1090 groups almost returned to the condition before dosing, but those in the CFDN group were retained highly. Cecal D-1 toxin in the CFDN group was positive and higher than in the S-1090 groups. It was thus considered that the critical condition with enterocolitis resulted from C. difficile, which proliferated more rapidly than the other bacteria and D-1 toxin produced by this bacteria in the withdrawal period. Above changes were commonly observed in the CFDN group. The NOAEL of S-1090 was assessed to be 100 mg potency/kg/day which induced no enteritis.