Inhibitory effect of high molecular weight water-soluble chitosan on hypoxia-induced inflammatory cytokine production

Chitosan is widely used to treat patients with hypoxia-induced diseases such as ischemia, neuronal death, cerebral stroke, and cerebral infarction. Using the ELISA method, we examined the effect of high molecular weight water-soluble chitosan (WSC) on inflammatory cytokine production in the desferrioxamine (DFX, known to mimic hypoxia)-stimulated human mast cell line HMC-1. DFX significantly increased interleukin (IL)-6, IL-8, and tumor necrosis factor (TNF)-alpha production compared with the control in a time-dependent manner (p<0.05), but did not affect IL-1alpha production and mRNA expression. The increase in IL-6, IL-8, and TNF-alpha levels was significantly inhibited by WSC in a dose-dependent manner with IC(50) values of 0.77, 0.88, and 2.5 microg/ml, respectively. The maximal inhibition rate of IL-6, IL-8, and TNF-alpha production by WSC was 64+/-9.7%, 80+/-9.4% and 54+/-4.5%, respectively. In addition, WSC inhibited DFX-induced activation of nuclear factor (NF)-kappaB. In conclusion, these results suggest that WSC is an inhibitor of NF-kappaB under hypoxic conditions, which might explain its beneficial effect in the treatment of hypoxia-induced inflammatory diseases.     

Low molecular weight quaternised chitosan (I): synthesis and characterisation

For a better understanding of the behaviour of macromolecules in vitro and in vivo, their structural and chemical properties that may be influential as experimental variables need to be characterised. N-Trimethyl chitosan chloride and N-triethyl chitosan chloride have been synthesised from chitosan to increase the solubility range of these polymers. However, little is known about the effect of the degree of quaternisation, molecular weight, viscosity and different substitution groups on the polymer's ability to enhance the transport of large hydrophilic compounds, such as peptide and protein drugs, across intestinal and nasal epithelia and on their toxicity profile. This study describes the synthesis of various quaternised chitosan polymers from low and medium molecular weight chitosan. These polymers were characterised to determine if any relationships between their degree of quaternisation, molecular weight and viscosity could be found which will determine their behaviour as absorption enhancers in future studies.     

Effects of high molecular weight water-soluble chitosan on in vitro fertilization and ovulation in mice fed a high-fat diet

A high molecular weight water-soluble chitosan (WSC) with an average molecular weight of 300 kD and a deacethylation level of over 90% was produced using a simple multi-step membrane separation process. It is known that WSC prevents obesity induced by a high-fat diet. Consequently, this study investigated whether or not WSC improved the ovarian dysfunction caused by obesity in mice. The mice were fed a high density protein and lipid diet for 4 weeks, followed by the administration of WSC at 480 mg/kg body weight per day for 4 days. Thereafter, the changes in body weight, ovulation rate, in vivo and in vitro fertilization and embryonic development were measured. WSC markedly reduced the body weight of obese mice fed with a high-fat diet, but not in mice fed with a normal diet. WSC had significant effects on the ovulation rate, both the in vivo and in vitro fertilization rates and embryonic development. These results indicate an improvement in the ovarian and oviduct dysfunction caused by obesity, and suggest an adjustment in the internal secretions and metabolic functions.     

Antidiabetic action of low molecular weight chitosan in genetically obese diabetic KK-Ay mice

Recently, we reported that low molecular weight (LMW) chitosan (chitosan lactate, average MW: 20,000) prevents the progression of low dose (100 mg/kg, i.p.) streptozotocin-induced slowly progressive diabetes mellitus in male ICR mice. The present study was designed to clarify the effects of LMW chitosan on hyperglycemia, hyperinsulinemia and hypertriglyceridemia in genetically obese diabetic male KK-Ay mice. LMW chitosan (0.05%, 0.2% or 0.8% water solution) was given daily as drinking water to male KK-Ay mice for 11 weeks, from 5 weeks of age. The non-fasting serum glucose levels of control mice continued to increase slowly throughout the experimental period. LMW chitosan lowered the serum glucose levels in a dose-dependent manner. In these diabetic mice, hyperinsulinemia and hypertriglyceridemia were observed, and LMW chitosan was dose-dependently effective in improving both serum biochemical parameters. LMW chitosan at three doses improved overdrinking and polyuria observed in these diabetic mice. It is concluded from these results that LMW chitosan may be useful for the treatment of obesity-related type 2 diabetes mellitus.     

The effect of high molecular weight kininogen on neutrophil adhesion to polymer surfaces

The adhesion of neutrophils on a biomaterial surface depends on the surface chemistry of the material and the cell, as well as the composition and conformation of adsorbed protein and the adherence of other cells when the biomaterial is exposed to circulating blood. In this study, HK and HKa were allowed to adsorb on three different polyurethanes: underivatized (PU-base), quaternized (PU-NR4), sulfonated (PU-SO3). The effect of kininogen adsorption on the degree of neutrophil adhesion was examined. The surface density of the adsorbed protein was also investigated. The PU-NR4 surface adsorbed the most HK and HKa and had the high degree of neutrophil adhesion. Although the surface density of adsorbed HK and HKa on the PU-SO3 surface, the degree of neutrophil on adhesion was significantly lower when compared to the PU-NR4 and PU-base surfaces. HK and HKa contain binding sites for both anionic surfaces and neutrophils in the same domain (D5H). When adsorbed to the anionic PU-SO3 surfaces, HK and HKa did not have the neutrophil binding sites available and therefore, exhibited an anti-adhesive effect. In contrast, the neutrophil binding domains D3 and DsH of adsorbed kininogens were available on the PU-NR4 and PU-base surfaces. Thus, adsorbed kininogens on these two surfaces lost their anti-adhesive property and this led to a high degree of neutrophil adhesion.     

Low molecular weight quaternised chitosan (11): in vitro assessment of absorption enhancing properties

N-Trimethyl chitosan chloride (TMC; high molecular weight) and N-trimethyl chitosan oligosaccharide (TMO; low molecular weight) with different degrees of quaternisation were synthesised and evaluated for their absorption enhancing properties across mucosal epithelia. These quaternised chitosan derivatives (0.0625% w/v-0.5% w/v) showed a significant decrease in the transepithelial electrical resistance (TEER) of cultured rabbit tracheal epithelial cell monolayers as compared to the control. The degree of quaternisation and concentration of the compounds influenced the extent of the reduction in TEER. Higher degrees of quaternisation and an increase in the concentration of the compound were associated with a more pronounced reduction in the TEER. The TMO derivatives seemed to be more effective in lowering the TEER of tracheal cell monolayers as compared to the TMC polymers. Ciliary beat frequency (CBF) is the main defence mechanism of the respiratory tract and is therefore a useful parameter in evaluating the toxicity of nasally administered drugs and additives. The effect of the synthesised chitosan derivatives on the CBF of human nasal epithelial cells at pH 7.4 was determined by a method based on an analogue contrast enhancement technique. The TMO oligomers exhibited lower inhibition of the CBF of human nasal epithelial cells compared to that of the TMC polymers. It was proposed that this reduced effect on the CBF is due to the lower viscosity and molecular weight of TMO. However, no acute toxicity was found with any of the synthesised chitosan derivatives by means of the CBF tests conducted in this study.     

Low molecular weight chitosan inhibits obesity induced by feeding a high-fat diet long-term in mice

Three low molecular weight chitosans (molecular weight: 21, 46 and 130 kDa) obtained by enzymatic hydrolysis of a high molecular weight chitosan (average molecular weight: 650 kDa) had low viscosity and were water-soluble. The effects of these water-soluble chitosans on pancreatic lipase (in-vitro) and the elevation of plasma triacylglycerol concentration after the oral lipid tolerance test were examined in mice. The water-soluble 46-kDa chitosan was the most effective at inhibiting pancreatic lipase activity (in-vitro) and plasma triacylglycerol elevation after the oral lipid tolerance test. Based on this result, the effects of the 46-kDa chitosan on increases in bodyweight, various white adipose tissue weights, and plasma and liver lipids were examined in mice fed a high-fat diet for 20 weeks. Water-soluble 46-kDa chitosan (300 mg kg(-1), twice daily) prevented increases in bodyweight, various white adipose tissue weights and liver lipids (cholesterol and triacylglycerol) in mice fed a high-fat diet, and further increased the faecal bile acid and fat. The results suggest that the lipid-lowering effects of the 46-kDa chitosan may be mediated by increases in faecal fat and/or bile acid excretion resulting from the binding of bile acids, and by a decrease in the absorption of dietary lipids (triacylglycerol and cholesterol) from the small intestine as a result of the inhibition of pancreatic lipase activity. Water-soluble 46-kDa chitosan (100 and 300 mg kg(-1), twice daily) did not cause liver damage with the elevation of glutamic oxaloacetic transaminase and glutamic pyruvic transaminase, or kidney damage with the elevation of blood nitrogen urea. It was concluded that water-soluble 46-kDa chitosan is a safe functional food.     

Low molecular weight chitosan nanoparticles as new carriers for nasal vaccine delivery in mice.

High molecular weight (Mw) chitosan (CS) solutions have already been proposed as vehicles for nasal immunization. The aim of the present work was to investigate the potential utility of low Mw CS in the form of nanoparticles as new long-term nasal vaccine delivery vehicles. For this purpose, CS of low Mws (23 and 38 kDa) was obtained previously by a depolymerization process of the commercially available CS (70 kDa). Tetanus toxoid (TT), used as a model antigen, was entrapped within CS nanoparticles by an ionic cross-linking technique. TT-loaded nanoparticles were first characterized for their size, electrical charge, loading efficiency and in vitro release of antigenically active toxoid. The nanoparticles were then administered intranasally to conscious mice in order to study their feasibility as vaccine carriers. CS nanoparticles were also labeled with FITC-BSA and their interaction with the rat nasal mucosa examined by confocal laser scanning microcopy (CLSM). Irrespective of the CS Mw, the nanoparticles were in the 350 nm size range, and exhibited a positive electrical charge (+40 mV) and associated TT quite efficiently (loading efficiency: 50-60%). In vitro release studies showed an initial burst followed by an extended release of antigenically active toxoid. Following intranasal administration, TT-loaded nanoparticles elicited an increasing and long-lasting humoral immune response (IgG concentrations) as compared to the fluid vaccine. Similarly, the mucosal response (IgA levels) at 6 months post-administration of TT-loaded CS nanoparticles was significantly higher than that obtained for the fluid vaccine. The CLSM images indicated that CS nanoparticles can cross the nasal epithelia and, hence, transport the associated antigen. Interestingly, the ability of these nanoparticles to provide improved access to the associated antigen to the immune system was not significantly affected by the CS Mw. Indeed, high and long-lasting responses could be obtained using low Mw CS molecules. Furthermore, the response was not influenced by the CS dose (70-200 microg), achieving a significant response for a very low CS dose. In conclusion, nanoparticles made of low Mw CS are promising carriers for nasal vaccine delivery.     

一种低分子壳聚糖硫酸酯铝的制备

本文采用正交试验设计方法对壳聚糖进行氧化降解,获得了一种低相对分子质量的水溶性壳聚糖,并在此基础上经硫酸化和盐交换制得了一种低相对分子质量壳聚糖硫酸酯式铝盐,壳聚糖氧化降解实验结果表明降解的最佳条件是温度为80度,过氧化氢浓度为5％,降解时间为1h,在此条件一产品收率可达52．68％,其粘均相对分子质量为3990,对制是的低相对分子质量壳聚糖硫酸酯铝进行了红外光谱分析和部分理化性质测定,经测定定样品中有机硫（S）含量为9．10％,铝（Al)含量为20．58％,样品制酸力为188．59mL.g^-1。     

Low molecular weight heparins: a developmental perspective

Low molecular weight heparins (LMWHs) are now universally accepted as drugs of choice for post-surgical prophylaxis of deep vein thrombosis (DVT). Currently these agents are also being developed for the treatment of thrombosis and various cardiovascular indications. Due to manufacturing differences, each of the LMWHs exhibits a distinct pharmacological and biochemical profile. The specific activity of these agents in the anticoagulant assays ranges from 35 - 45 anti-IIa U/mg, whereas the specific activity in terms of anti-Xa units is designated as 80 - 145 anti-Xa U/mg. These LMWHs are capable of producing product-specific dose- and time-dependent antithrombotic effects in animal models of thrombosis. While the ex vivo effects are initially present at doses that are antithrombotic, these agents have been found to produce sustained antithrombotic effects without any detectable ex vivo anticoagulant actions. In experimental animal models and in various clinical trials, these agents have also been found to release tissue factor pathway inhibitor (TFPI) after both iv. and sc. administration. Repeated administration of LMWHs produces progressively stronger antithrombotic effects; however, the haemorrhagic responses vary and are largely dependent on the product used. The release of TFPI following iv. and sc. administration in a primate model also demonstrates product individuality and the relevance of this inhibitor to the actions of LMWHs. Furthermore, repeated administration, mimicking the post-surgical prophylaxis of DVT, leads to product-based augmentation of the antithrombotic or haemorrhagic effects. Antithrombotic and haemorrhagic studies are discussed, comparing the pharmacological profile of some of the available LMWHs. Product individuality, in terms of relative potency in different assays and the failure of standardisation protocols to provide any guidelines for product substitution and prediction of the clinical effects, is also addressed.     

Low molecular weight chitosan prevents the progression of low dose streptozotocin-induced slowly progressive diabetes mellitus in mice

The present study was designed to clarify the effect of low molecular weight (LMW) chitosan (chitosan lactate, average MW: 20000) on the progression of slowly progressive non-insulin-dependent diabetes mellitus (NIDDM) induced by a single i.p. injection of low dose (100 mg/kg) streptozotocin (STZ) to 8-week-old male ICR mice. The non-fasting serum glucose levels of STZ-treated control mice continued to rise throughout the experimental period until 23 weeks after STZ treatment. The 0.2% or 0.8% chitosan (water solution), given as drinking water from prediabetic stage (2 weeks after STZ treatment), markedly prevented the time course-related rise of serum glucose levels of diabetic mice. In addition, the reduction of relative numbers of insulin-immunoreactive cells (beta-cells) in the islets of diabetic mice at 24 weeks after STZ treatment was markedly prevented by 0.2% or 0.8% chitosan administration. However, the progression of hyperglycemia in diabetic mice was not affected by 0.2% glucosamine, a monosaccharide of chitosan. The glucose levels of normal mice were not affected by 0.8% chitosan administration. When 0.2% chitosan administration was stopped at 20 weeks, these animals had still maintained significantly lower serum glucose levels, compared to control animals, even at 5 weeks after stopping the administration. These results indicate that LMW chitosan prevents the progression of low dose STZ-induced slowly progressive NIDDM.     

Inhibition of angiogenesis by cleaved high molecular weight kininogen (HKa) and HKa domain 5

High molecular weight kininogen (HK) is an abundant, multi-domain plasma protein that circulates in plasma primarily in its single chain form. Proteolytic cleavage of HK by plasma kallikrein releases the vasoactive nanopeptide bradykinin (BK), and converts HK into two-chain HK (HKa). BK appears to have pro-angiogenic activity, most likely mediated through binding to B1 and B2 receptors on endothelial cells. Conversely, HKa and its domain 5, but not (single chain) HK, have potent anti-angiogenic activity comparable to other endogenous angiogenesis inhibitors. The mechanism by which HKa exerts its anti-angiogenic activity remains controversial, but appears to involve binding to cell surface tropomyosin and induction of apoptosis of proliferating endothelial cells. A role for tropomyosin in mediating the anti-angiogenic signals of other anti-angiogenic proteins such as endostatin and histidine-proline-rich glycoprotein (HPRG) has also been reported. Here we review the physiological importance of high molecular weight kininogen in angiogenesis, with emphasis on the mechanism(s) by which this activity is mediated.     

Identification of high molecular weight serine-proteases in Loxosceles intermedia (brown spider) venom.

High molecular weight serine-proteases have been identified in Loxosceles intermedia (brown spider) venom. The mechanism by which Loxosceles spp venoms cause dermonecrotic injury (a hallmark of loxoscelism) is currently under investigation, but it seems to be molecularly complex and in some instance proteases might be expected to play a role in this skin lesion. In the present investigation, when we submitted L. intermedia venom to linear gradient 3-20% SDS-PAGE stained by a monochromatic silver method we detected a heterogeneous protein profile in molecular weight, ranging from 850- to 5-kDa. In an attempt to detect zymogen molecules of proteolytic enzymes, venom aliquots were treated with several exogenous proteases. Among them, trypsin activated two gelatinolytic molecules of 85- and 95-kDa in the venom. In experiments of hydrolysis inactivation using different protease inhibitors for four major class of proteases, we detected that only serine-type protease inhibitors were able to inactivate the 85- and 95-kDa enzymes in the venom. An examination of the 85- and 95-kDa gelatinolytic activities as a function of pH showed that these proteases had no apparent activities at pH below 5.0 and higher than 9.0 and displayed little activity at pH 6.0. with the optimal pH for their activities ranging from 7.0 to 8.0. Evaluation of the functional specificities of the 85- and 95-kDa venom proteases showed that these proteases efficiently degrade gelatin (denatured collagen) but have no proteolytic activity on hemoglobin, immunoglobulin, albumin, librinogen or laminin, suggesting specificity of their proteolytic actions. We describe here two serine-proteases activities in L. intermedia venom probably involved in the harmful effects of the venom.     

A comparative study on hypoglycemic and hypocholesterolemic effects of high and low molecular weight chitosan in streptozotocin-induced diabetic rats.

The hypoglycemic and hypocholesterolemic effects of high and low molecular weight chitosan were evaluated in streptozotocin (STZ)-induced diabetic rats. Rats were divided into three groups of normal rats (Experiment I) and three groups of diabetic rats (Experiment II). The first group received a cellulose (control) diet, the second group received a low MW (1.4 x 10(4)Da) chitosan diet and the third group received a high MW (1.0 x 10(6)Da) chitosan diet. All three diets were containing 0.5% cholesterol. Experiment I: rats fed with high MW or low MW chitosan diet had increased high density lipoprotein (HDL) cholesterol. However, chitosan did not affect plasma glucose in normal rats. Experiment II: significantly decreased plasma glucose and total cholesterol and increased HDL cholesterol and fecal cholesterol excretion were observed in diabetic rats fed with high MW chitosan diet than animals fed with cellulose diet. However, no statistical significant difference in plasma glucose and total cholesterol was observed in diabetic rats fed with low MW chitosan. The total content of SCFAs in cecum was significantly increased and the ratio of acetate to propionate was slight but significantly decreased in diabetic rats after consuming high MW chitosan diet. The activities of hepatic hexokinase were significantly increased and the intestinal disaccharidases including sucrase and maltase were significantly decreased in normal and diabetic rats fed with high MW chitosan diet. Results obtained from the present study demonstrated the potential of high MW chitosan in reducing hyperglycemia and hypercholesterolemia in STZ-induced diabetic rats.     

低分子肝素相对分子质量与抗凝活性关系研究*

目的:研究低分子肝素相对分子质量与抗凝活性之间的关系。方法:用生色底物法测定不同相对分子质量的低分子肝素6个分级和未分级样品的抗Ⅹa因子和抗Ⅱa因子效价,相对分子质量采用高效体积排阻色谱法(high performance size exclusion chromatography,HPSEC)测定。结果:抗Ⅹa因子与抗Ⅱa因子效价比随着相对分子质量的降低而增大。结论:在测定范围内,低分子肝素相对分子质量降低,抗凝活性降低,抗栓作用增强。     

甘草酸-低分子壳聚糖偶联物大鼠体内组织分布研究

目的：研究甘草酸-低分子壳聚糖（GA-LMWC）偶联物在大鼠体内组织分布特征,探讨GA-LMWC偶联物作为肾靶向药物的可行性。方法：将甘草酸溶液和GA-LMWC偶联物溶液分别经尾静脉注射按剂量10 mg·kg^-1给予SD大鼠,并于给药后1,4,8 h取各组织（心、肝、脾、肺、肾）,采用HPLC测定各组织（心、肝、脾、肺、肾）中甘草酸的含量。结果：大鼠尾静脉注射GA-LMWC偶联物后1,4,8 h在肾脏中的分布较甘草酸组显著提高,分别为甘草酸组的1.34倍（P<0.05）、1.46倍（P<0.001）和2.83倍（P<0.01）;在肝脏和脾脏的分布较甘草酸组显著降低（P<0.01）。结论：与游离甘草酸相比,GA-LMWC偶联物改变了甘草酸大鼠体内分布特征,显著增加了GA在肾脏的分布,延长了其肾脏滞留时间,增强了甘草酸的肾脏靶向性。     

Prenatal oral (gavage) developmental toxicity study of decabromodiphenyl oxide in rats

Decabromodiphenyl oxide (DBDPO) is a highly effective flame retardant that is primarily used in electrical and electronic equipment with a secondary, but important, application in upholstery textiles. DBDPO, the second largest volume brominated flame retardant in use today, has undergone a wide range of toxicology tests in mammalian species with the results indicating a no-adverse-effect level of approximately 1000 mg/kg/day in oral repeated-dose studies. An oral prenatal developmental toxicity study of the commercial DBDPO product (97% purity) was performed under current EPA OPPTS and OECD guidelines. Female Sprague-Dawley rats (25 mated females/group) received 0,100, 300 or 1000 mg DBPDO/kg/ day via gavage in corn oil during gestation days 0 through 19. All females survived until scheduled sacrifice. No clinical signs of toxicity were observed. Pregnancy rates in the control and treated groups ranged from 96% to 100% and provided 23 or more litters in each group for evaluation on gestation day 20. No effect of treatment was seen in maternal gestational parameters (body weight, body weight gain, and food consumption), uterine implantation data, liver weight, or necropsy findings. Likewise, no effect of treatment was seen in fetal body weights, fetal sex distribution, or during the fetal external, visceral, or skeletal examinations. The NOEL (no-observable-effect level) for maternal and developmental toxicity was 1000 mg DBPDO/kg/day, the highest dose level administered on gestation days 0 to 19.     

Interaction of indomethacin with low molecular weight chitosan,and improvements of some pharmaceutical properties of indomethacin by low molecular weight chitosans

The interaction of four kinds of low molecular weight chitosans, which were different in molecular weight and degree of deacetylation, were studied in solution and the solid state using an anti-inflammatory drug, indomethacin (IM), as an acidic model molecule. The solubility of IM enhanced with increasing concentration of low molecular weight chitosan, especially C-I which had the lowest molecular weight and least degree of deacetylation. The C-I crystal complex was obtained in a molar ratio of 16:1 (IM: C-I) from aqueous solution. The data suggested that the acetyl group and amino group of chitosan played an important role in the complexation. The IM dissolution rates from kneaded mixtures with low molecular weight chitosans were enhanced in the order of C-I >C-II >C-III >C-IV (molecular weight: C-IV >C-III >C-II >C-I). The oral absorption rate of IM from C-I kneaded mixture was improved compared with IM alone.