Chapter 28
Design of Poly(α-malic acid)—Antitumor Drug-Saccharide Conjugate Exhibiting CellSpecific Antitumor Activity
Downloaded by YORK UNIV on October 28, 2012 | http://pubs.acs.org Publication Date: March 5, 1993 | doi: 10.1021/bk-1993-0520.ch028
T. Ouchi, H . Kobayashi, Κ. Hirai, and Y. Ohya Department of Applied Chemistry, Faculty of Engineering, Kansai University, Suita, Osaka 564, Japan
Poly(α-malic acid) is of interest as a biodegradable and bioadsorbable poly(lactide) type drug carrier, which is able to covalently attach both drug and targeting moiety. The macromolecular prodrug of antitumor drug will reduce the side-effects, have the ability to target tumor cells and exhibit high antitumor activity. The design of poly(α-malic acid)/ 5-fluorouracil(5FU)/saccharide and poly(α-malic acid)/ adriamycin(ADR) /saccharide conjugates were investigated. Monosaccharides such as galactosamine, glucosamine and mannosamine were used as targeting moieties. Growth inhibition against various tumor cells in vitro and the survival effect against mice bearing tumor cells in vivo were tested. Poly(α-malic acid)/5FU/galactosamine and poly(α-malic acid)/ADR /galactosamine conjugates showed stronger growth-inhibitory effects than poly(α-malic acid)/5FU and poly(α-malic acid)/ADR conjugates against human hepatoma cells in vitro. These results could be explained by galactose receptor-mediated specific uptake into hepatocyte cells. Moreover, the conjugates of poly(α-malic acid) with 5FU exhibited significant survival effects against p388 lymphocytic leukemia in mice by intraperitoneal(ip) transplantation/ip injection. The obtained conjugates did not display an acute toxicity in the high dose ranges. Since poly(α-malic acid) has the application as a biodegradable and bioadsorbable poly lactide type drug carrier having reactive carboxyl groups, which is able to attach drugs and targeting moieties (1). In comparison with low molecular weight prodrug, the macromolecule/drug conjugate is expected to overcome the problem of the side-effects of the parent anticancer agent. We have already reported that poly(oc-malic acid) immobilizing 5-fluorouracil (5FU) exhibited an excellent survival effect against p388 lymphocytic leukemia cells in mice by intraperitoneal(ip) transplantation/ip injection (2). 5FU has a strong antitumor activity, which is accompanied, however, by undesirable side-effects (3,4). Doxorubicin (adriamycin; ADR) is one of the most prominent clinical antitumor agents, however its undesirable side-effects have also been cited. Additonally, some kinds of saccharides were recently reported to play important roles in biological recognition. So, in order to achieve the active targeting of macromolecule/drug conjugate, we have employed the saccharide residue as a targeting 0097-6156/93/0520-0382$06.00/0 © 1993 American Chemical Society
In Polymeric Delivery Systems; El-Nokaly, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1993.
28.
OUCHI E T AL.
Cell-Specific
Antitumor
Activity
383
moiety. This has the advantage of reducing the side-effects while exhibiting high antitumor activity. The present paper presents the design of poly(cc-malic acid)/ 5FU/saccharide and poly(oc-malic acid)/ADR/saccharide conjugates, which are expected to have affinity for tumor cells; the release behavior of parent drugs from the conjugates in aqueous solution, the survival effect of the conjugates against p388 lymphocytic leukemia in mice iplip and the growth-inhibitory effect of the conjugates against various tumor cells in vitro.
Downloaded by YORK UNIV on October 28, 2012 | http://pubs.acs.org Publication Date: March 5, 1993 | doi: 10.1021/bk-1993-0520.ch028
Experimental
S y n t h e s i s of Poly(o>malic acid)/Saccharide C o n j u g a t e ( l a - l c ) . Poly(ocmalic acid) (degree of polymerization: 11.3) was synthesized by ring-opening polymerization of malide benzyl ester and consequent H2/Pd reduction according to the method reported previously by our group (7). Three kinds of saccharide residues, such as galactosamine, glucosamine, mannosamine, were attached to poly(a-malic acid) via amide bonds using dicyclohexylcarbodiimide ( D C C ) to give poly(oc-malic acid)/saccharide conjugates l a , l b and l c , respectively (Scheme I). 0.534 g (2.59 mmol) of D D C was added to an ice-cooled solution of 1.50 g (12.9 mmol) of poly(oc-malic acid) and 0.298 g (2.59 mmol) of 1-hydroxybenzotriazole (HOBt) in 30 πύοίΝ,Ν -dimethylformamide ( D M F ) . The reaction mixture was stirred at 0°C for 1 h and at room temperature for 24 h. A solution of 0.558 g (2.59 mmol) of galactosamine hydroxychloride and 0.5 ml of triethylamine (TEA) i n D M F (15 ml) was added to the reaction mixture and stirred at room temperature for 24 h. The Ν ,N -dicyclohexylurea ( D C U ) formed was removed by filtration and washed with D M F . The filtrate was evaporated under reduced pressure to afford poly(cc-malic acid)/galactosamine conjugate la. Poly(oc-malic acid)/glucosamine 1 b and poly(oc-malic acid)/ mannosamine 1 c were prepared by the method described above. The degree of introduction of saccharide i n m o l % per carboxyl group of conjugates l a - 1 c was estimated by the 3,4-dinitrosalicylic acid (DNS) reduction method (5).
Synthesis of l-[(Amino-n-pentyl)-ester]-methylene-5FU Hydrochloride (4). The preparation of the 5 F U amino derivative, l-[(amino-n-pentyl)-ester]methylene-5FU hydrochloride 4, is shown i n Scheme II (6). 14.4 m l (100 mmol) of 37 v o l % formalin was added to 17.8 g (220 mmol) of 5 F U and the reaction mixture was stirred at 60°C for 45 m i n and then evaporated to give the oily l,3-dimethylol-5FU (2). 12.3 g (120 mmol) of D C C , 15.6 g (120 mmol) of t-butoxycarbonyl- amino-n-capronic acid and 0.7 g of 4-dimethylaminopyridine ( D M A P ) were added to the solution of 2 i n 300 m l of acetonitrile and then stirred for 4 h at room temperature. The D C U formed was filtered off and washed with dichloromethane. The resulting solution was washed three times with 1 Ν H C I and aq. satd. N a C l solution. The oil layer, dried with anhydrous sodium sulfate, was evaporated to give the oily residue. The crude product was purified by recrystallization from diethylether to afford the white solid of l-[(t-butoxycarbonyl-amino-n-pentyl)-ester]- methylene-5FU (3). Compound 3 was dissolved in dioxane containing 4 Ν H C I , stirred for 1 h at room temperature. The solution was evaporated to give the white powder of l-[(amino-n-pentyl)-ester]methylene-5FU hydrochloride (4). M.p. 188-189° C; yield 3 0 % . ER ( K B r ) : 3050 (N+H3), 3000, 2950 (CH2), 1720 (C=0 of 5 F U ) , 1740, (COO), 1270 (C-F), 1200 (COO), l l l O c m ' ^ C - N ) . H - N M R ( ( C D 3 ) 2 S O ) , δΐ.34 (m, 2 H , CH2), 1.6 (m, 4 H , C H 2 ) , 2.35 (t, 2 H , C H 2 ) , 2.73 (m, 2 H , C H 2 ) , 5,58 (s, 2 H , C H 2 ) , 8.05 (s, 3 H , N H 3 ) , 8.25 (d, 1H, H-6). C - N N M R ( ( C D 3 ) 2 S O ) ) , δ23.9, 25.4, 26.8, 33.2, 40.3, 7 0 . 8 ( C H 2 ) , 129.7, 138.6, 140.9, 157.7(5FU), 172.8 (C=0). U V ( M e O H ) : Xmax 262nm, emax 8800. 1
+
u
In Polymeric Delivery Systems; El-Nokaly, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1993.
384
POLYMERIC DELIVERY SYSTEMS
Synthesis of Poly(cc-malic acid)/5FU Saccharide (5) and Poly(a-malic acid)/5FU/Saccharide Conjugate (5a-5c). The conjugate of 5 F U s attached to poly(a-malic acid) via amide bonds through pentamethylene, monomethylene spacer groups and ester bonds (5) and the conjugate of poly(a-malic acid) fixing 5 F U via amide bonds through pentamethylene, monomethylene spacer groups and ester bonds, and fixing saccharide units via amide or ester bonds (5a-5c) were prepared by the reaction steps shown in Scheme III. Conjugate 5 has no saccharide unit. Conjugate 5 a, 5 b and 5 c have galactosamine, glucosamine, and mannosamine units as side saccharide residues, respectively. 1.05 g (5.09 mmol) of D D C was added to an ice-cooled solution of 0.590 g(5.09 mmol carboxyl unit) poly(a-malic acid) (7) and 1.58 g (5.09 mmol) of 4 i n acetonitrile (70 ml). 1 m l of T E A was added dropwise to the above solution at 0°C for 3 h and then the reaction mixture was stirred at room temperature for 20 h. After the D C U formed was removed by filtration, the acetonitrile-soluble part was evaporated under reduced pressure to afford crude 5. In order to completely remove D C U , crude 5 was subjected to gel chromatography on Sephadex L H - 2 0 . Elution with T H F and evaporation afforded a hygroscopic pale yellow powder. This powder was then reprecipitated using a THF/excess Et20-n-hexane (1:1, v/v) system to give the desired poly(a-malic acid)/5FU conjugate (5). 0.664 g (3.22 mmol) of D C C was added to an ice-cooled solution of 1.2 g (8.06 mmol carboxyl unit) of l a and 0.371 g (3.22 mmol) of H O S u in D M F (20 ml). The reaction mixture was stirred at OP C for 1 h and then at room temperature for 24 h. A solution of 1.25 g (4.03 mmol) of 4 and 0.7 m l of T E A was added to the reaction mixture and stirred at room temperature for 24 h. The D C U formed was removed by filtration and washed with D M F . The filtrate was evaporated under reduced pressure. In order to completely remove D C U , the residue was subjected to gel chromatography on Sephadex L H - 2 0 (Pharmacia Co.). Elution with D M F afforded poly(oc-malic acid)/5FU/galactosamine conjugate 5 a. Poly(oc-malic acid)/5FU/glucosamine conjugate 5 b and poly(a-malic acid)/5FU/mannosamine conjugate 5 c were prepared by the method described above. The values of the degree of saccharide on the polymer i n m o l % saccharide based on the number of substituted carboxylic acid groups (DS) for 5a, 5 b and 5 c were determined to be 24, 16 and 13 m o l % , respectively. Since only free 5 F U was obtained by hydrolysis of the conjugates under refluxing in aq. 3 Ν N a O H solution for 24 h, the degree of substitution of 5 F U i n m o l % per carboxyl group of conjugate (D5FU) of 5, 5a, 5b, 5 c were determined to be 20, 26, 24 and 25 m o l % , respectively. The D 5 F U value was obtained by G P C measurement of the released amount of 5 F U in the hydrolyzed solution (column; Shodex OHpak K B - 8 0 3 : eluent; 0.013 M K H 2 P 0 4 - N a 2 H P 0 4 buffer solution, p H 7.0: detector; U V 265 nm).
Downloaded by YORK UNIV on October 28, 2012 | http://pubs.acs.org Publication Date: March 5, 1993 | doi: 10.1021/bk-1993-0520.ch028
1
1
Synthesis of Poly(o>malic acid)/ADR Conjugate (6) and Poly(oc-malic acid)/ ADR/Saccharide (6a-6c). The poly(a-malic acid)/amide/ADR conjugate 6 and poly(oc-malic acid)/amide/ADR/saccharide conjugate 6a-6c were prepared by reacting A D R with poly(cc-malic acid) or poly(a-malic acid)/saccharide conjugates l a - l c , respectively (Scheme IV). Tlie obtained conjugates were purified by gel filtration chromatography. The degree of substitution of A D R in m o l % per carboxyl group of the conjugate(DADR) was estimated by U V measurement i n water. The synthetic results of 6 and 6a-6c are summarized in Table I.
Determination of Extent of Release of 5 F U and A D R from the Conjugates. The release behavior of 5 F U from poly(oc-malic acid) /5FU/saccharide
In Polymeric Delivery Systems; El-Nokaly, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1993.
Cell-Specific Antitumor Activity
OUCHI E T AL.
28.
H^OCHCO^OH
DCC/HOBt
385
H ^ O Ç H C O ^ Ç H C O ^ O H
6H d:=o
CH COOH
[ COOH
2
2
n=ll 3 Saccharide Poly (α-malic acid) (1)
è
2
la - lc
Scheme I. Preparation route for poly(a-malic acid)/amide/saccharide conjugates
Downloaded by YORK UNIV on October 28, 2012 | http://pubs.acs.org Publication Date: March 5, 1993 | doi: 10.1021/bk-1993-0520.ch028
l a - l c . l a ( ( ^ ) :galactosamine), l b ( ( § ) :glucosamine), lc(^):mannosamine).
Ν
|f
formalin
|
«>°0,45ΓηΓ
oK'
O ^ J
Η
CH OH 2
5FU
2 2
5
•
Boc-NH(CH ) CO-CH -N 2
Room Temp., 4h
0
Boc-:(CH ) COC3
Q
DCC, DMAP ^ j ^ j
2 + Brc-NH(CH ) COH
3
|f
2
3
3
4N HCl/Dioxan^
5
0
F
HCl.H N(CH ) CO-CH -N^=0 2
2
5
2
Scheme II. Preparation route for l-[(amino-n-pentyl)-ester]-methylene-5FU hydrochloride 4.
H4(OCHCO) ^OÇHCO) (OÇHCO) R
Ή
ι or +H N(CH ) CO—CH -N la-lc n \ _ 2
2
4»
5
2
N
I
VoHOSu Ç
2
R
CH
2
O — C H
2
n F
y •OH
CH COOH 2
0 0 0
F
J
(CH ) C 2
5
- N ^ ) c O
5 or 5a-5c
Scheme III. Preparation route for poly(oc-malic acid)/5FU/saccharide conjugates 5(y=0), 5a((5) igalactosamine), 5 b ( ( J ) :glucosamine), 5 c ( ( § ) rmannosamine).
In Polymeric Delivery Systems; El-Nokaly, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1993.
386
POLYMERIC DELIVERY SYSTEMS
H | ^OCHCO)
