Effects of habitual chitosan intake on bone mass, bone-related metabolic markers and duodenum CaBP D9K mRNA in ovariectomized SHRSP rats.

We have demonstrated that the habitual intake of chitosan can decrease bone mass in ovariectomized (OVX) SHRSP rats fed a low-Ca diet (0.1%). In the present study, we examined both the etiology of bone loss induced by dietary chitosan and the preventive effect of vitamin C supplementation. Rats were OVX and maintained on one of the following diets for 6 wk: 10% cellulose (CE). 10% chitosan (CH) or 10% chitosan with sodium ascorbate (CHVC). CH caused a significant reduction in bone mineral density (BMD) and stiffness in femurs and the fourth lumbar vertebrae (L4). There was no significant difference in intestinal Ca absorption between CH and CE, whereas CH intake significantly reduced intestinal P absorption. The bone loss in CH rats was accompanied with an increase in urinary Ca excretion and a decrease in serum Ca as well as a significant increment In serum PTH and 1,25(OH)2D3. The vitamin D receptor and calcium binding protein D9K mRNAs were also significantly increased in the duodenum of CH rats. Vitamin C supplementation to CH caused an increase in the Ca and P contents of femurs as well as BMD of the L4, with a decrease in urinary Ca excretion. These results indicate that dietary chitosan with low Ca intake possibly induces the loss of bone mass by enhancing urinary Ca excretion rather than by inhibiting Ca absorption, and that vitamin C supplementation could prevent bone loss caused by chitosan through the increment of retained Ca followed by suppression of urinary Ca excretion.

Capillary electrophoresis of high-molecular chitosan: the natural carbohydrate biopolymer.

Due to its high resolving power and diverse application range, capillary electrophoresis (CE) has been successfully applied to the analysis of carbohydrates. In this paper, a method for the determination of high-molecular chitosan (Mr 200,000) using CE is presented. We studied the optimal condition of buffer pH and type, and column type for determination of chitosan. Optimal CE performance was found when employing 100 mM triethylamine (TEA)-phosphate buffer, pH 2.0 and untreated fused-silica capillary (50 microm x 27 cm) for the chitosan analysis. Under optimum conditions, excellent linear responses were obtained in the concentration range of 1.25-20 microM, with a linear correlation coefficient of 0.9983. The standard deviations of the migration time and peak area were found to be 2.5 and 6.4%, respectively. This method could be readily applied to chitosan determination in real biological samples and commercial products.

Evaluation of N-succinyl-chitosan as a systemic long-circulating polymer.

The water-soluble, low toxic and less biodegradable chitosan derivative N-succinyl-chitosan (Suc-chitosan) was investigated for body distribution and urinary excretion on a long-time scale (24-72 h after i.v. injection) using a fluorescein-labeling technique. Fluorescein isothiocyanate (FITC)-labeled Suc-chitosan (Suc-chitosan-FTC) was characterized for molecular weight, succinylation degree and FTC content. Systemic retention and tissue distribution of Suc-chitosan-FTC were examined after i.v. administration to normal and Sarcoma 180 tumor-bearing mice. Suc-chitosan-FTC was sustained at a high level in the circulation over 72 h; that is, the plasma half-life in normal mice was 100.3 h and that in tumor-bearing mice was 43.0 h, which was longer than those of other long-circulating macromolecules reported to date. As to the tissues except blood circulation, Suc-chitosan-FTC was distributed very little in tissues other than the tumor. Although the total amount of Suc-chitosan-FTC residing in tested tissues decreased gradually, urinary excretion did not increase from 24 h after injection. These observations suggested that Suc-chitosan-FTC may be eliminated by mechanisms other than in the urine or moved to tissues other than those tested. The ratio of tumor accumulation reached a plateau at 48 h after injection, and the accumulation level, approximately 10%, was similar to those observed for other reported long-circulating macromolecules.

Potential of low molecular mass chitosan as a DNA delivery system: biocompatibility, body distribution and ability to complex and protect DNA.

Cationic polymers have the potential for DNA complexation and it is recognised that they may be useful as non-viral vectors for gene delivery. Highly purified chitosan fractions of < 5000 Da (N1), 5000-10,000 Da (N2) and > 10,000 Daltons (N3) were prepared and characterised in respect of their cytotoxicity, ability to cause haemolysis, ability to complex DNA as well as to protect DNA from nuclease degradation. Also the biodistribution of 125I-labelled chitosans was followed at 5 and 60 min after intravenous injection into male Wistar rats. All chitosan fractions displayed little cytotoxicity against CCRF-CEM and L132 cells (IC50 > 1 mg/ml), and they were not haemolytic (< 15% lysis after 1 and 5 h). Chitosan-DNA interaction at a charge ration of 1:1 was much greater than seen for poly(L-lysine) and complexation resulted in inhibition of DNA degradation by DNase II: 99.9 +/- 0.1, 99.1 +/- 1.5 and 98.5 +/- 2.0% for N1, N2 and N3, respectively. After intravenous injection, all the chitosans showed rapid blood clearance, the plasma levels at 1 h being 32.2 +/- 10.5% of recovered dose for N1 and 2.6 +/- 0.5% of recovered dose for N3. Liver accumulation was molecular mass dependent, being 26.5 +/- 4.9% of the recovered dose for N1 and 82.7 +/- 1.9% of the recovered dose for N3. The observations that the highly purified chitosan fractions used were neither toxic nor haemolytic, that they have the ability to complex DNA and protect against nuclease degradation and that low molecular weight chitosan can be administered intravenously without liver accumulation suggest there is potential to investigate further low molecular weight chitosans as components of a synthetic gene delivery system.

Evaluation of poly(vinyl alcohol)-gel spheres containing chitosan as dosage form to control gastrointestinal transit time of drugs.

Two types of poly(vinyl alcohol)-gel spheres were prepared with chitosan (CS/PVA-GS) and without chitosan (PVA-GS), and comparative studies were performed using these gel spheres (GSs). No change in particle size was observed by the addition of chitosan: nearly 45% of both particles were in the 5-10 microm range. In an in vivo gastrointestinal transit test, CS/PVA-GS prolonged the small-intestinal transit time more than PVA-GS. In an in vitro intestinal perfusion study, the mean transit time of these GSs was markedly reduced by pretreatment of the intestinal surface with a mucolytic agent, N-acetyl-L-cysteine, suggesting that the mucous layer on the intestinal surface plays an important role in controlling the transit rate of these GSs. The oral administration of aminophylline (theophylline) and ampicillin as model drugs incorporated in PVA-GS and CS/PVA-GS was examined in rats. While theophylline absorption from PVA-GS was not affected by the addition of chitosan, the improvement of ampicillin absorption by PVA-GS was enhanced by the chitosan combination.

Biodistribution and kinetics of holmium-166-chitosan complex (DW-166HC) in rats and mice.

UNLABELLED: The fate of 166Ho-chitosan complex, a radiopharmaceutical drug for cancer therapy, was determined by studying its absorption, distribution and excretion in rats and mice. METHODS: Holmium-166-chitosan complex [0.75 mg of Ho(NO3)3 x 5H2O and 1 mg chitosan/ head] was administered intrahepatically to male rats. Radioactive concentrations in blood, urinary and fecal excretion and radioactive distribution in tissues were examined. To determine the effects of chitosan in 166Ho-chitosan complex, 166Ho alone [0.75 mg of Ho(NO3)3 x 5H2O/head] was intrahepatically administered to male rats, and radioactive concentrations in blood, urinary and fecal excretion and radioactive distribution were examined. In B16 melanoma-transplanted nude mice, radioactive distribution after intratumoral administration of 166Ho-chitosan complex [0.075 mg of Ho(NO3)3 x 5H2O and 0.10 mg chitosan/head] was investigated also. RESULTS: After administration of 166Ho-chitosan complex, the radioactive concentrations in blood were low, and cumulative urinary and fecal excretions over a period of 0-72 hr were 0.53% and 0.54%, respectively. The radioactive concentrations in tissues and the whole-body autoradiography images showed that most of the administered radioactivity was localized at the administration site, and only slight radioactivity was detected from the liver, spleen, lungs and bones. On the other hand, results of intrahepatic administration of 166Ho alone showed high radioactive concentrations in the blood, and the whole-body autoradiographs showed that the administered radioactivity was distributed in many organs and tissues. These results strongly suggest that 166Ho is retained at the administration site only when it forms a chelate complex with chitosan. Autoradiographs after intratumoral administration of 166Ho-chitosan complex showed that radioactivity was localized at the site of administration without distribution to the other organs and tissues. CONCLUSION: Administered 166Ho-chitosan complex is retained at the administration site after either intrahepatic or intratumoral administration to rats or tumor-transplanted nude mice.

In vitro degradation rates of partially N-acetylated chitosans in human serum.

The initial degradation rates (r) in human serum of three chitosans with FA = 0.42, 0.51, and 0.60 were determined by measuring the decrease in viscosity as a function of time. A strong increase in r with increasing FA of the chitosans was observed, with r increasing proportionally to FA4.5. With increasing concentrations of lysozyme added to the reaction mixtures of chitosan and serum, the relative increase in degradation rate of chitosans with increasing FA was almost the same as that without lysozyme added. Addition of the chitinase inhibitor allosamidin (50 microM) did not inhibit the degradation rate of chitosan (FA = 0.60) by human serum. The results suggest that chitosans are actually mainly depolymerized by lysozyme in human serum, and not by other enzymes or other depolymerization mechanisms.

Pharmacokinetic characteristics and antitumor activity of the N-succinyl-chitosan-mitomycin C conjugate and the carboxymethyl-chitin-mitomycin C conjugate.

The conjugate between N-succinyl-chitosan (Suc-chitosan) and mitomycin C (MMC), named Suc-chitosan-MMC, and that between carboxymethyl-chitin (CM-chitin) and MMC, named CM-chitin-MMC, were investigated in vivo. As for the intraperitoneal drug administration using rats, the order of the maximum blood concentration of MMC was unconjugated MMC > CM-chitin-MMC > Suc-chitosan-MMC. The plasma concentration of MMC for Suc-chitosan-MMC was maintained at an almost constant level over 24 h. Pharmacokinetic analysis of each plasma concentration indicated that for each conjugate, the in vitro drug release reported previously was useful for the approximate estimation of the in vivo regeneration of MMC. The chemotherapeutic effect of MMC and the conjugates was investigated using mice bearing L1210 leukemia or B16 melanoma. Concerning antitumor activity against L1210 leukemia, the conjugates exhibited a marked effect at higher doses, and their effect increased even in the dose range where the effect of MMC decreased. MMC and the conjugates exhibited a good growth-inhibitory effect against B16 melanoma. Complete inhibition of growth of B16 melanoma was observed at the dose of 10 mg eq MMC/kg for CM-chitin-MMC.

Effects of chitosan--a coagulating agent for food processing wastes--in the diets of rats on growth and liver and blood composition.

Effects of feeding free chitosan to rats at graded levels up to 15 percent of the diet for eight weeks was investigated. Animals receiving diets containing 5 percent or less of chitosan grew well at comparable rates. Progressive growth reduction occurred when chitosan was increased to 10 and 15 percent of the diet and enlargement of liver and kidneys was observed only in animals receiving the highest level of dietary chitosan. Liver moisture, protein, lipid, ash, and nucleic acids; blood hemoglobin and packed cell volume; and serum total protein, albumin, ceruloplasmin and transferrin were determined. Values for these components of liver and blood were altered significantly in the animals receiving the highest level of chitosan when compared to control animals. However, in animals receiving 5 percent or less of dietary chitosan none of these measures of tissue composition was different from controls, except for liver protein concentration of rats fed the 5 percent of chitosan diet. Animal feeds containing coagulated by-products are not expected to contain over 0.2% chitosan in the total diet. No adverse effects have been observed at this level in rat feeding studies. Therefore the tolerance level for dietary chitosan appears to be well above the levels expected to be in animal feeds containing by-products recovered from food processing wastes by coagulation with chitosan.