Chapter 8
Design of Polysaccharide-5-Fluorouracil Conjugates Exhibiting Antitumor Activities
Downloaded by UNIV OF PITTSBURGH on July 1, 2013 | http://pubs.acs.org Publication Date: August 15, 1991 | doi: 10.1021/bk-1991-0469.ch008
T. Ouchi, T. Banba, T. Z. Huang, and Y. Ohya Department of Applied Chemistry, Faculty of Engineering, Kansai University, Suita, Osaka 564, Japan
In order to provide a macromolecular prodrug of 5-fluorouracil(5FU) with reduced side-effects, having affinity for tumor cells and exhibiting a high antitumor activity, the design of polysaccharide-5FU conjugates was investigated. Chitin-5FU, chitosan-5FU, α-1,4-polygalactosamine-5FU, partially N-acetylated α-1,4-polygalactosamine-5FU, hyaluronic acid– 5FU and dextran-5FU conjugates exhibited significant survival effects against p388 lymphocytic leukemia in mice by intraperitoneal(ip) transplantation/intraperitoneal(ip) injection. Chitosan-5FU, chitosamino– oligosaccharide-5FU, and galactosamino-oligosaccharide-5FU conjugates showed higher growth-inhibitory effects against MH134Y hepatoma and M e t h - A fibrosarcoma i n mice than 5 F U , c h i t i n , oligosaccharides and their blends by subcutaneous(sc) implantation/ intravenous(iv) injection. The obtained conjugates did not display an acute toxicity in the high dose ranges.
5-Fluorouracil(5FU) has a remarkable antitumor activity (7-5), which is accompanied, however, by undesirable side-effects (4,5). Polysaccharides such as chitin, chitosan, a-l,4-polygalactosamine, N-acetyl-ct-l,4-polygalactosamine, hyaluronic acid and dextran are noteworthy as low or nontoxic, nonimmunogenetic, compatible and biodegradable polymers. Partially N-acetylated chitosan and a-l,4-polygalactosamine have been further reported to be selectively collected into tumor cells (6) and to inhibit growth of tumor cells (7), respectively. Especially, water-soluble hexa-Nacetyl chito-hexaose and a-l,4-polygalactosamine were found to act as growthinhibitors of tumors through stimulation of the host immuno-system (S). So, in order to provide a macromolecular prodrug of 5FU with reduced sideeffects, having affinity for tumor cells and exhibiting a high antitumor activity, the present paper is concerned with the design of hybrid type conjugates of chitin0097-6156/91/0469-0071$06.00/0 © 1991 American Chemical Society
In Polymeric Drugs and Drug Delivery Systems; Dunn, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.
72
POLYMERIC DRUGS AND DRUG DELIVERY SYSTEMS
5FU(1), chitosan-5FU(2), a-l,4-polygalactosamine-5FU(3), partially N-acetylated ocl,4-polygalactosamine-5FU(4), hyaluronic acid-5FU(5), dextran-5FU(6), and 6-0carboxymethyl chitin(CM-chitin)-5FU(7).
Downloaded by UNIV OF PITTSBURGH on July 1, 2013 | http://pubs.acs.org Publication Date: August 15, 1991 | doi: 10.1021/bk-1991-0469.ch008
Experimental Syntheses of Polysaccharide-5FU Conjugates. The conjugates of 5FUs attached to chitosan, a-l,4-polygalactosamine, partially iV-acetylated a-l,4-polygalactosamine and dextran at the 2-positions through hexamethylene spacer groups via carbamoyl bonds were synthesized by coupling reactions of chitosan, oc-l,4-polygalactosamine, partially N-acetylated a-l,4-polygalactosamine and dextran with 6-(5-fluorouracil-lyl)-hexamethylene isocyanate(8) (9), respectively. Chitosaminohexaose(COS6)5FU(9), chitosaminotriose(COS3)-5FU(10), galactosaminopentaose (GOS5)-5FU(li) and galactosaminotriose(GOS3)-5FU(12) conjugates were prepared by the same methods described above. The desired conjugates of 5FU attached to chitin and hyaluronic acid at 6-positions through hexamethylene and pentamethylene spacer groups via carbamoyl bonds were synthesized through coupling reactions of chitin and hyaluronic acid with the isocyanate (8) and l-[(5-aminopentyl)carbamoyl]- 5fluorouracil (13) (10), respectively. CM-chitin-5FU conjugate (7) was prepared by coupling reaction of CM-chitin with 4-[(amino-n-pentyl)-ester]-methylene-5FU(14) (77). These fixation steps of 5FU onto polysaccharides are shown in Schemes I-IV. Release Behavior of 5FU from Polysaccharide-5FU Conjugates. In order to evaluate the release behavior of 5FU from the polysaccharide-5FU conjugates, the hydrolyses of these conjugates were studied in vitro at 37°C in physiological saline. The carbamoyl and ester bonds of the obtained polysaccharide-5FU conjugates were cleaved to give only free 5FU itself but not to afford any 5FU derivative. After the release of 5FU, the glycoside bonds of backbone polysaccharides might be degraded slowly for a very long time. Survival Effect of Polysaccharide-5FU Conjugates against p388 Leukemia in Mice ip/ip. The survival effect of the 5FU conjugates was evaluated against p388 lymphocytic leukemia in female C D F j mice (30 untreated mice/group and 6 treated mice/group) by ip transplantation/ip injection according to the protocol for preliminary screening of the antitumor activity performed at the Japanese Foundation for Cancer Research. A total of lxlO leukemia cells were injected ip on day 0. The water-insoluble chitin-5FU(l), chitosan-5FU(2), a-l,4-polygalactosamine- 5FU(3), partially N-acetylated a-l,4-polygalactosamine- 5FU(4) and dextran-5FU(6) conjugates were sonicated in 0.05% Sorbate 80 and sterile normal saline, and then the obtained suspensions were administered ip. Hyaluronic acid-5FU(5) and CM-chitin5FU(7) conjugates were dissolved in sterile normal saline, and then the obtained aqueous solutions were administered ip. The mice received doses of 200-800mg/kg on days 1 and 5. The ratio of prolongation of life, T/C(%), which means the ratio of median survival of treated mice (T) to that of control mice (C) was evaluated as the survival effect. 6
In Polymeric Drugs and Drug Delivery Systems; Dunn, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.
In Polymeric Drugs and Drug Delivery Systems; Dunn, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.
2
2
90-100°C
NH-HCM
n 2
OH
NH-HCI
CH^)H -C> D M S O , pyridine H O
Chitin-5FU(jJ
CH
BS >
g-^CHj^NCO
NH^HCI
CH^)H
ί
„
5
Chitosan-5FUt2;n n=30) COS6-5FU(jy(n=6) COS3-5FU(10)(n=3)
2
CH OH
conjugates.
Scheme I. Preparation route for chitosan-5FU, COS6-5FU and C O S 3 - 5 F U
Chitosan/HCI or COS/HCI
2
CH OH
DMAC, LiCI 90-100°C
8,, T E A
pyridine,
NH -HCI
CH OH
Chitin
5FU
H
Xr
OCN—
