Hiraku Onishi

Biodegradation and distribution of water-soluble chitosan in mice

Randomly 50% deacetylated chitin, called Chi, was examined on the biodegradability, body distribution and urinary excretion after the intraperitoneal (ip) administration to mice. These characteristics were investigated using fluorescein isothiocyanate (FITC)-labeled Chi (FTC-Chi). The in vitro biodegradability was investigated by incubation with lysozyme and murine plasma and urine. The degradation of Chi or FTC-Chi was accelerated by lysozyme, plasma and urine. The molecular weight was checked by gel-chromatography. The degradation product showed a fairly small molecular weight and contained no FTC-Chi of a large one. The body distribution and urinary excretion of FTC-Chi were investigated at 1, 14 and 24 h after the ip injection to mice. FTC-Chi moved fast to the kidney and urine, and was scarcely distributed to the liver, spleen, abdominal dropsy and plasma. Most of FTC-Chi was excreted into urine after 14 h, and the molecular weight of the excreted FTC-Chi was as small as that of the product obtained by the long in vitro incubation. Therefore, Chi is considered to be highly biodegradable and easily excreted in urine, and further it is suggested to have no problem on accumulation in the body; however, at the same time, Chi is found not to operate as a polymer support showing long retention in the body.

Application of chitin and chitosan derivatives in the pharmaceutical field

Chitin and chitosan derivatives are used as excipients and drug carriers in the pharmaceutical field. Their derivatization contributed to expansion of application and decrease toxicity. Chitosan is used as an excipient in oral dosage form. Chitosan tablet can exhibit a sustained drug release compared to commercial products. Films prepared using chitin or chitosan have been developed as wound dressings, oral mucoadhesive and water-resisting adhesive by virtue of their release characteristics and adhesion. Intratumoral administration of gadopentetic acid-chitosan complex nanoparticles (approximately 430 nm in diameter) has been more effective for gadolinium neutron-capture therapy compared with a group treated with the solution. Compared to intragastrical feeding with diphtheria toxoid (DT) in PBS, a strong enhancement of the systemic (IgG) and local (IgA) immune responses against DT has been observed in mice fed with DT loaded chitosan microparticles (approximately 4.7 microm in size). When DNA-loaded chitosan microspheres (1.15 - 1.28 microm) were intramuscularly administrated into mice, high beta-galactosidase and luciferase productions were obtained even after a long post-transfection period (12 weeks). N-Succinyl-chitosan (Suc-Chi) has been studied for cancer chemotherapy as a drug carrier and the conjugates of mitomycin C with Suc-Chi exhibited good antitumor activities against various tumors. Furthermore, trimethyl-chitosan and monocarboxymethyl-chitosan has been shown to be effective as intestinal absorption enhancers due to their physiological properties. Chitosan-thioglycolic acid conjugates has been found to be a promising candidate as scaffold material in tissue engineering due to their physicochemical properties. This review summarizes the application of chitin and chitosan derivatives for hospital preparations and drug carriers.

N-succinyl-chitosan as a drug carrier: water-insoluble and water-soluble conjugates.

N-succinyl-chitosan (Suc-Chi) has favourable properties as a drug carrier such as biocompatibility, low toxicity and long-term retention in the body. It was long retained in the systemic circulation after intravenous administration, and the plasma half-lives of Suc-Chi (MW: 3.4 x 10(5); succinylation degree: 0.81 mol/sugar unit; deacetylation degree: 1.0 mol/sugar unit) were ca. 100.3h in normal mice and 43 h in Sarcoma 180-bearing mice. The biodistribution of Suc-Chi into other tissues was trace apart from the prostate and lymph nodes. The maximum tolerable dose for the intraperitoneal injection of Suc-Chi to mice was greater than 2 g/kg. The water-insoluble and water-soluble conjugates could be prepared using a water-soluble carbodiimide and mitomycin C (MMC) or using an activated ester of glutaric MMC. In vitro release characteristics of these conjugates showed similar patterns, i.e. a pH-dependent manner, except that water-insoluble conjugates showed a slightly slower release of MMC than water-soluble ones. The conjugates of MMC with Suc-Chi showed good antitumour activities against various tumours such as murine leukaemias (L1210 and P388), B16 melanoma, Sarcoma 180 solid tumour, a murine liver metastatic tumour (M5076) and a murine hepatic cell carcinoma (MH134). This review summarizes the utilization of Suc-Chi as a drug carrier for macromolecular conjugates of MMC and the therapeutic efficacy of the conjugates against various tumours.

Biological characteristics of lactosaminated N-succinyl-chitosan as a liver-specific drug carrier in mice

Lactosaminated N-succinyl-chitosan (Lac-Suc) was prepared by reductive amination of N-succinyl-chitosan (Suc) and lactose using sodium cyanoborohydride. Six-day reaction using lactose (12.8-fold (w/w)) yielded Lac-Suc with lactosamination degree of 30% (mol/sugar unit). Fluorescein thiocarbamyl-Lac-Suc (Lac-Suc-FTC) was prepared by labeling Lac-Suc with fluorescein isothiocyanate. Lac-Suc-FTC was injected intravenously at a dose of either 1 (high dose) or 0.2 (low dose) mg/mouse. At both doses, Lac-Suc-FTC initially underwent fast hepatic clearance, showed maximum liver localization at 8 h, and the amounts localized there were maintained even at 48 h post-injection. Very slow excretion into feces and urine was observed. The ratio of liver AUC(0--48 h) to plasma AUC(0--48 h) at low dose was three times higher than that at high dose. On the other hand, the Suc derivative, Gal-Suc, obtained by reductive amination of Suc/galactose showed very little distribution to the liver similarly to Suc itself. Further, since the liver uptake of Lac-Suc-FTC was inhibited by asialofetuin, it was suggested that the liver distribution of Lac-Suc should be concerned with asialoglycoprotein receptor. Thus, Lac-Suc was found available as a carrier exhibiting a high affinity to and long retention in the liver.

Sustained release ketoprofen microparticles with ethylcellulose and carboxymethylethylcellulose.

Microparticulate systems for sustained release of ketoprofen were prepared and evaluated by monitoring drug release in the JP XIII second fluid, pH 6.8. All the microparticulate dosage forms were prepared using ketoprofen in the form of calcium salt (KP-Ca). Simple ethylcellulose microparticles of KP-Ca (EC-MP) exhibited the fairly rapid release in the first phase with slower release in the late period. Most of the drug was released from EC-MP showing high drug content. For polymer-coated microparticles of ketoprofen, Eudragit microparticles of KP-Ca (ER-MP) were first prepared, and then coated with ethylcellulose or with a mixture of carboxymethylethylcellulose and ethylcellulose to produce ethylcellulose-coated (EC-coat) and the mixture-coated microparticles (CMEC/EC-coat), respectively. Some polymer-coated microparticles showed drug release at nearly zero-order rate. Especially, CMEC/EC-coat prepared at a CMEC:EC ratio of 1:1 (w/w), named formation I, could supply the drug constantly and efficiently for about half a day except for an initial rapid release. When formation I was administered intraduodenally to rats, the plasma concentration of ketoprofen could be maintained at a nearly constant level. Kinetic analysis demonstrated that formation I showed a nearly zero-order release rate in vivo consistent with that observed in vitro.

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.

Development of oral acetaminophen chewable tablets with inhibited bitter taste

Various formulations with some matrix bases and corrigents were examined for development of oral chewable tablets which suppressed the bitter taste of acetaminophen, often used as an antipyretic for infants. Corn starch/lactose, cacao butter and hard fat (Witepsol H-15) were used for matrix bases, and sucrose, cocoa powder and commercial bitter-masking powder mixture made from lecithin (Benecoat BMI-40) were used for corrigents against bitter taste. The bitter taste intensity was evaluated using volunteers by comparison of test samples with standard solutions containing quinine at various concentrations. For the tablets made of matrix base and drug, Witepsol H-15 best inhibited the bitter taste of the drug, and the bitter strength tended to be suppressed with increase in the Witepsol H-15 amount. When the inhibitory effect on the bitter taste of acetaminophen solution was compared among the corrigents, each tended to suppress the bitter taste; especially, Benecoat BMI-40 exhibited a more inhibitory effect. Further, chewable tablets were made of one matrix base and one corrigent, and of one matrix base and two kinds of corrigents, their bitter taste intensities after chewing were compared. As a result, the tablets made of Witepsol H-15/Benecoat BMI-40/sucrose, of Witepsol H-15/cocoa powder/sucrose and of Witepsol H-15/sucrose best masked the bitter taste so that they were tolerable enough to chew and swallow. The dosage forms best masking bitter taste showed good release of the drug, indicating little change in bioavailability by masking.

Bioadhesive characteristics of chitosan microspheres to the mucosa of rat small intestine.

Chitosan (Chi) microspheres were examined in vitro and in vivo in terms of their bioadhesive characteristics to the mucosa of rat small intestine. Chi was labeled with fluorescein isothiocyanate (FITC), and the microspheres (FTC-MS) were prepared by the dry-in-oil method using the obtained fluorescein thiocarbamyl–chitosan (FTC-Chi). FTC-MS with a mean diameter of 27 μm and size distribution of a few micrometers to several tens of micrometers was used for the bioadhesion experiment. FTC-MS exhibited a tendency to adhere to each part of the small intestine to a greater extent than dissolved FTC-Chi, and the ratio of adhering FTC-MS increased as the amount of added FTC-MS decreased. FTC-MS showed slower transit following intraduodenal injection than oral administration. Following the intraduodenal injection of FTC-MS, more than half remained in the upper or middle part of the small intestine for over 8 h. Further, insulin-containing chitosan microspheres with a mean diameter of 20 μm and size distribution of 5 μm to 45 μm were checked in situ for drug absorption, but intraduodenal or intraileal application hardly gave any decrease in plasma glucose level at a very high dose. The present chitosan microsphere system showed good adhesion to the intestinal mucosa, but scarcely facilitated absorption of insulin.

Pharmacokinetics of prolonged-release CPT-11-loaded microspheres in rats.

CPT-11-containing microspheres composed of poly-D,L-lactic acid or poly (D,L-lactic acid-co-glycolic acid) copolymers were prepared by an oil-in-water evaporation method. The size and shape of the microspheres were examined, and the drug release rates were analyzed from the in vitro release profiles. CPT-11 aqueous solution was intravenously or intraperitoneally injected at 10 mg/kg, and microspheres were intraperitoneally administered at 50 mg eq CPT-11/kg in rats. The microspheres had an average diameter of around 10 microm and their shape was spherical. All the microspheres contained CPT-11 in a lactone form, and their drug contents and release profiles were basically similar to those of previous microspheres. After i.v. injection of CPT-11 solution, the CPT-11 plasma concentration decreased quickly, SN-38 decreased slowly at a much lower level, and SN-38 glucuronide (SN-38G) declined very slowly at a higher level than SN-38. The plasma concentration of CPT-11 reached a maximum at 30 min after i.p. administration of CPT-11 solution. The area under the plasma concentration-time curve (AUC) of CPT-11 after i.p. administration was somewhat lower compared with that after i.v. administration, but the plasma concentration-time profiles of SN-38 and SN-38G were nearly identical between i.v. and i.p. administration. An i.p. administration of the microspheres resulted in gradually increasing or almost constant CPT-11 levels. The levels of SN-38 were also stable during the observation period (4 days) except for the slowest releasing microsphere in which SN-38 was not detected after 24 h following administration. Intraperitoneal administration of any of the microspheres resulted in stable and similar levels of SN-38G after 24 h following administration. When judging from apparent simple pharmacokinetic analysis, an inconsistency was found between the in vitro drug release and the plasma level to a fair extent, but overall the in vivo drug release rate from microspheres was considered parallel to the in vitro one. The microspheres showing a faster release of CPT-11 exhibited higher plasma levels of CPT-11 and SN-38, explaining the previous results that efficacy was better when the in vitro release rate was higher. That the SN-38 level could be attained to a certain extent even at the range of modest or low plasma concentration of CPT-11 in each administration may be related to the non-linear metabolic conversion from CPT-11 to SN-38.

Potentiality of double liposomes containing salmon calcitonin as an oral dosage form

The hypocalcemic effects of salmon calcitonin (SCT) after oral administration in rats by means of SCT-loading double liposomes (DL) which consist of liposomes containing small liposomes were investigated. SCT-loading DL consisted of four types of the inner liposomes such as neutral liposomes (NL) and cationic charged liposomes (CL) prepared using Coatsome, and neutral (VET) and cationic charged (c-VET) liposomes prepared using a mechanochemical method and sizing to 100 nm by the extrusion procedure were prepared. DL could be prepared by a combination of mechanochemical and glass-beads methods at a high efficiency. DL produced the increase in bioavailability in all groups treated with SCT-loading liposomes except for c-VET. The bioavailability of VET-DL was not significantly different but the greatest among the samples used in this study regardless of the similar size of NL-DL and CL-DL, and was approx. 6.8-fold higher than that of SCT solution when taken orally. The group treated with c-VET showed the strongest hypocalcemic effects among the inner liposomes examined (P>0.05). Therefore, it is speculated that not only the size of liposomes but also the cationic charge plays an important role in the intestinal absorption of DL. These findings suggested the utility of DL as an oral dosage form of SCT.