Polysaccharides as Drug Carriers - ACS Symposium Series (ACS

Chapter 14

Polysaccharides as Drug Carriers Activation Procedures and Biodégradation Studies 1

1

1

2

Etienne Schacht , Filip Vandoorne , Joan Vermeersch , and Ruth Duncan 1

Laboratory of Organic Chemistry, State University of Ghent, Ghent, Belgium Department of Biological Sciences, University of Keele, Keele, England

Downloaded by UNIV OF MICHIGAN ANN ARBOR on February 19, 2015 | http://pubs.acs.org Publication Date: September 4, 1987 | doi: 10.1021/bk-1987-0348.ch014

2

The activation of dextran and inulin by periodate oxidation, succinoylation and reaction with 4-nitrophenyl chloroformate was investigated. Hemiacetal formation during periodate oxidation of inulin caused only 50 percent of the aldehydes that were generated to be accessible for reaction with nucleophiles. Evidence was shown for the formation of different types of carbonates during the chloroformate activation of dextran. Model compounds of dextran were prepared using these activation procedures. Subsequent degradation by dextranase indicated a decrease of the rate of degradation for increasing degree of substitution. There i s a c u r r e n t i n t e r e s t i n i m p r o v i n g t h e p h a r m a c e u t i c a l p r o p e r t i e s o f e x i s t i n g drugs through c h e m i c a l t r a n s f o r m a t i o n o f t h e a c t i v e moiety i n t o low m o l e c u l a r weight as w e l l as macromolecular prodrugs. P o l y s a c c h a r i d e s a r e f r e q u e n t l y s e l e c t e d as c a r r i e r s f o r t h e p r e p a r a t i o n o f p o l y m e r i c prodrugs (J_, 2) . D i r e c t c o u p l i n g o f the drug w i t h the c a r r i e r i s o n l y p o s s i b l e p r o v i d e d t h e former has t h e a p p r o p r i a t e functionality. I n most c a s e s the p o l y s a c c h a r i d e o r t h e drug need t o be t r a n s f o r m e d i n t o a s u i t a b l e r e a c t i v e d e r i v a t i v e . A variety of a c t i v a t i o n p r o c e d u r e s a p p l i c a b l e t o p o l y s a c c h a r i d e s have been r e p o r ted. I n view o f the e v e n t u a l m e d i c a l a p p l i c a t i o n a p r o p e r c h a r a c t e r i z a t i o n o f t h e c h e m i c a l s t r u c t u r e o f t h e a c t i v a t e d polymer as w e l l as t h e polymer-drug c o n j u g a t e i s e s s e n t i a l . Recent r e i n v e s t i g a t i o n s have demonstrated t h a t t h e s e a c t i v a t i o n p r o c e d u r e s a r e o f t e n more complex than e x p e c t e d . F o r a number o f a c t i v a t i o n s side r e a c t i o n s occur i n v o l v i n g i n t e r a c t i o n o f the r e a c t i v e groups w i t h n e i g h b o u r i n g h y d r o x y l groups. T h i s w i l l be i l l u s t r a t e d i n t h e p r e s e n t paper f o r the a c t i v a t i o n o f d e x t r a n and i n u l i n u s i n g sodium p e r i o d a t e , s u c c i n i c a n h y d r i d e and 4 - n i t r o p h e n y l c h l o r o formate as a c t i v a t i n g a g e n t s . When u s i n g macromolecular prodrugs i t i s a d v i s a b l e t h a t t h e mac r o m o l e c u l a r c a r r i e r a f t e r f u l l f i l l i n g i t s f u n c t i o n s h o u l d be c l e a r e d

0097-6156/87/0348-0188S06.00/0 © 1987 American Chemical Society

In Controlled-Release Technology; Lee, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

14.

SCHACHT ET AL.

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Polysaccharides as Drug Carriers

from t h e body. Hence the b i o d e g r a d a b i l i t y o f the c a r r i e r and the e f f e c t o f c h e m i c a l m o d i f i c a t i o n on t h e b i o d e g r a d a b i l i t y a r e i m p o r t a n t a s p e c t s t o c o n s i d e r . . I n t h i s paper some d a t a w i l l be p r e s e n t e d d e a l i n g w i t h t h e b i o d e g r a d a b i l i t y o f d e x t r a n and some d e x t r a n d e r i v a tives .


A c t i v a t i o n o f d e x t r a n and i n u l i n P e r i o d a t e o x i d a t i o n . Aldehyde groups c a n be e a s i l y i n t r o d u c e d i n most p o l y s a c c h a r i d e s by r e a c t i o n w i t h sodium p e r i o d a t e . V i c i n a l d i o l s t r u c t u r e s g i v e r i s e to d i a l d e h y d e s . F o r d e x t r a n ( I ) h a v i n g t h r e e a d j a c e n t hydroxy1 groups i n each non-branched anhydro g l u c o p y r a n o s i d e r e p e a t u n i t the o x i d a t i o n i s a two s t e p r e a c t i o n : -CH

2

CH—0

/

\

CH

C H

l\ HO

-CH

/l C H C H II

II

0

0

L Q -

-ÇH

2

Î!H—Ο

/ CH

C H

C H

A

I\I HO

/, C H — ο \

\ O H

I X : H - C H OH

l

2

C H

ii 0 -

0

/[_ C H

L

o-

(I)

- C H

0

CH—0

/

\

CH 0

C H

/|_

C H — C H :H II I O H

L

O -

0

S i n c e t h e r a t e c o n s t a n t s f o r both o x i d a t i o n s t e p s a r e o f t h e same o r d e r o f magnitude (_3) d i f f e r e n t types o f aldehydes w i l l be p r e s e n t i n p a r t i a l o x i d i z e d dextran. Due to t h e occurrence of these c o n s e c u t i v e r e a c t i o n s the aldehyde content i n a p a r t i a l o x i d i z e d dextran can n o t be p r e d i c t e d from the amount o f p e r i o d a t e added. D e t a i l e d s t u d i e s o f the p e r i o d a t e o x i d a t i o n o f d e x t r a n and i n u l i n (4-6) have shown e v i d e n c e f o r t h e o c c u r r e n c e o f h e m i a c e t a l s t r u c t u r e s formed by r e a c t i o n o f aldehydes w i t h h y d r o x y l groups o f e i t h e r t h e same u n i t ( i n t r a - r e s i d u a l ) o r a n e i g h b o u r i n g u n i t ( i n t e r - r e s i d u a l ) . I n t e r - r e s i d u a l h e m i a c e t a l f o r m a t i o n reduces t h e number o f d i o l s t r u c -


190

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TECHNOLOGY


t u r e s s u s c e p t i b l e f o r o x i d a t i v e a t t a c k and hence slows down t h e r a t e of o x i d a t i o n . On t h e o t h e r hand a l d e h y d e s i n v o l v e d i n s t a b l e hemia c e t a l s t r u c t u r e s can be p r o t e c t e d f o r r e a c t i o n w i t h n u c l e o p h i l e s . T h i s i s t h e c a s e f o r i n u l i n where t h e C-3 a l d e h y d e i s c a p t u r e d i n a s t a b l e s i x membered h e m i a c e t a l s t r u c t u r e ( I I ) :

Despite these complications periodate o x i d a t i o n i s a convenient approach t o r e a c t i v e p o l y s a c c h a r i d e d e r i v a t i v e s . The p r o c e d u r e i s r a t h e r simple and i n c o n t r a s t w i t h most o t h e r a c t i v a t i o n methods t h e a c t i v a t e d polymer c a n be i s o l a t e d i n d r y form w i t h o u t i r r e v e r s i b l e crosslinkage taking place. S u c c i n i c a n h y d r i d e a c t i v a t i o n . C a r b o x y l i c groups c a n be e a s i l y i n t r o d u c e d i n p o l y s a c c h a r i d e s (P-OH) by r e a c t i o n w i t h s u c c i n i c anhydride (III). They c a n then be t r a n s f o r m e d i n t o r e a c t i v e e s t e r s and f i n a l l y coupled with drugs: 0 // CH2C V

P-OH +

I

0

CH C

X

9

—

P-0-C-CH CH -C-0H II II 0 0 o

1

o

— a c t i v a t i o n

— c o u p l i n g

1

0 (III) T h i s method was r e p o r t e d b e f o r e as a c o n v e n i e n t way t o c o u p l e drugs to d e x t r a n and s t a r c h ( 7 ) . F o r t h e attachment o f a l c o h o l s t h i s method i s o b v i o u s l y o n l y p r a c t i c a l when u s i n g the t r i - e s t e r d e r i v a t i v e s i n o r d e r t o a v o i d i n t r a - o r i n t e r - m o l e c u l a r r e a c t i o n s d u r i n g t h e subsequent c o u p l i n g p r o c e d u r e s . However a l s o w i t h amine type drugs s i m i l a r c o m p e t i t i v e s i d e r e a c t i o n may o c c u r as w i l l be i l l u s t r a t e d . We have i n v e s t i g a t e d t h e s u c c i n o y l a t i o n o f i n u l i n (In-OH) and the a c t i v a t i o n of t h e r e s u l t i n g p o l y a c i d u s i n g 1,1-carbonyl d i i m i d a zole (IV). Succinoylations of polysaccharides are frequently c a r r i e d out i n p y r i d i n e . Q u a n t i t a t i v e e s t e r i f i c a t i o n s g e n e r a l l y r e q u i r e l o n g r e a c t i o n times and e l e v a t e d temperature ( e . g . : 48h a t 70°C, r e f . 7 ) . However by u s i n g 1 - m e t h y l i m i d a z o l o r 4-dimethylamino p y r i d i n e as a c y l a t i o n c a t a l y s t we succeeded i n a c h i e v i n g f a s t and q u a n t i t a t i v e s u c c i n o y l a t i o n o f i n u l i n under r a t h e r m i l d r e a c t i o n c o n d i t i o n s as i s shown i n F i g u r e 1. The s u c c i n a t e d e r i v a t i v e o f i n u l i n was f u r t h e r t r a n s f o r m e d i n t o t h e c o r r e s p o n d i n g r e a c t i v e i m i d a z o l i d e by r e a c t i o n w i t h 1 , 1 - c a r b o n y l d i i m i d a z o l (CDI, I V ) : T


SCHACHT ET AL.

14.

In-0-C-CH CH -C-N H 2 2 „ ι

In-0-C-CH CH -C-0H il il 0 0 o

1

191


o

o

1

(CDI, IV)

1

(V)

+

C0

+

o

H-N

Ν

1

1

2


Ν

o

T h i s a c t i v a t i o n r e a c t i o n was f o l l o w e d by means o f p r o t o n NMR. This t e c h n i q u e a l l o w e d us t o m o n i t o r the c o n c e n t r a t i o n s o f t h e m o n o s u c c i n a t e groups, CDI, as w e l l as t h e i m i d a z o l l i b e r a t e d d u r i n g t h e r e a c tion. As can be seen from F i g u r e 2 t h e a c t i v a t i o n i s q u a n t i t a t i v e and completed w i t h i n 20 m i n u t e s . The C D I - a c t i v a t e d i n u l i n s u c c i n a t e (V) was s u b s e q u e n t l y c o u p l e d w i t h t h e N - g l y c y l d e r i v a t i v e o f p r o c a i n amide ( V I ) , t h e s y n t h e s i s o f which was r e p o r t e d b e f o r e ( 8 ) : V

+

H N-CH--C-NH-R . 2 H C l II

——In-0-C-CH CH -C-NH-CH -C-NH-R 0

D

M

0

F

9

9

II

II

0

0

II

0

(VI) w i t h H2N-R =

procainamide

T h i s r e a c t i o n o c c u r e d i n DMF and a t room temperature f o r 48 h o u r s . From t h e NMR spectrum o f t h e c o u p l i n g p r o d u c t ( i s o l a t e d v i a p r e p a r a t i v e g e l f i l t r a t i o n ) i t c o u l d be c a l c u l a t e d t h a t o v e r 95 p e r c e n t o f the c a r b o x y l groups had r e a c t e d w i t h t h e p r o c a i n a m i d e d e r i v a t i v e . T h i s d a t a demonstrate t h a t amino-type drugs c a n be e a s i l y l i n k e d i n h i g h y i e l d onto s u c c i n o y l a t e d p o l y s a c c h a r i d e s . However t h i s o n l y holds f o r polysaccharide d e r i v a t i v e s having a l l hydroxyls e s t e r i f i e d . When a s i m i l a r r e a c t i o n sequence as d e s c r i b e d b e f o r e was c a r r i e d o u t u s i n g a p a r t i a l l y s u c c i n o y l a t e d i n u l i n (approx. 50% e s t e r i f i e d ) a c o n s i d e r a b l e amount o f c r o s s l i n k e d p r o d u c t was o b t a i n e d . The r e m a i n i n g f r e e h y d r o x y l f u n c t i o n s a p p a r e n t l y compete w i t h t h e amino com pounds f o r r e a c t i o n w i t h t h e i m i d a z o l i d e d e r i v a t i v e . Hence t h e above d e s c r i b e d method i s o n l y a t t r a c t i v e p r o v i d e d t h e p o l y s a c c h a r i d e i s succinoylated quantitatively. 4-Nitrophenyl chloroformate a c t i v a t i o n . The r e a c t i o n o f p o l y s a c c h a r i d e s w i t h chloroformâtes f o r m i n g r e a c t i v e c a r b o n a t e d e r i v a t i v e s has o f t e n been used t o l i n k b i o a c t i v e compounds, such as a f f i n i t y l i g a n d s and enzymes, onto p o l y s a c c h a r i d e m a t r i x e s . I t was r e p o r t e d t h a t upon r e a c t i o n w i t h e t h y l c h l o r o f o r m a t e c y c l i c c a r b o n a t e as w e l l as e t h y l c a r b o n a t e s t r u c t u r e s a r e formed (9-13). A c t i v a t i o n with N-succinimido c h l o r o f o r m a t e (14), 2 , 4 , 5 - t r i c h l o r o f o r m a t e (14) and 4 - n i t r o p h e n y l c h l o r o f o r m a t e (14, 15) r e p o r t e d l y l e d to the i n t r o d u c t i o n o f p e n d i n g carbonate m o i e t i e s . R e c e n t l y we have r e i n v e s t i g a t e d (16) t h e a c t i v a t i o n o f d e x t r a n with 4-nitrophenyl chloroformate (VII). The c o n t e n t o f t h e 4 - n i t r o p h e n y l c a r b o n a t e groups i n a c t i v a t e d d e x t r a n c a n be e a s i l y determined


CONTROLLED-RELEASE TECHNOLOGY


192

F i g u r e 2. Time c o n v e r s i o n c u r v e f o r t h e r e a c t i o n o f i n u l i n monos u c c i n a t e w i t h CDI i n DMSO a t 25°C. [anhydrofructofuranoside u n i t s ] = 31 mM, [ C D I ] = 240 mM. Q

Q


14.

SCHACHT

ET

AL.


193


by h y d r o l y s i n g the r e a c t i o n p r o d u c t i n a l k a l i n e medium and a s s a y i n g f o r 4 - n i t r o p h e n o l a t e by means o f U.V.. The a c t i v a t i o n p e r c e n t a g e s thus determined (number o f 4 - n i t r o p h e n y l c a r b o n a t e u n i t s per hundred anhydro g l u c o p y r a n o s i d e u n i t s ) as a f u n c t i o n of time and f o r v a r y i n g amounts o f c h l o r o f o r m a t e a r e shown i n F i g u r e 3. In a l l cases the degree o f a c t i v a t i o n i n i t i a l l y i n c r e a s e s w i t h time, r e a c h e s a maximum and then s l o w l y d e c r e a s e s . A p l a u s i b l e e x p l a n a t i o n , o t h e r than hyd r o l y s i s o f the r e a c t i v e groups, i s r e a c t i o n o f the a r o m a t i c c a r b o nates with polymeric hydroxyls forming a l i p h a t i c i n t r a - or i n t e r m o l e c u l a r c a r b o n a t e s t r u c t u r e s ; e.g. i n t r a - m o l e c u l a r ( V I I I ) : 0

(VIII) S i n c e the r e a c t i o n p r o d u c t remains i n s o l u t i o n throughout the r e a c t i o n i n t e r - m o l e c u l a r rearrangements a r e n o t a major s i d e r e a c t i o n . The t o t a l c a r b o n a t e c o n t e n t i n the a c t i v a t e d polymer c o u l d be d e t e r mined by h y d r o l y s i n g the r e a c t i o n p r o d u c t w i t h excess barium h y d r o x i de (11). An example of the change o f b o t h the 4 - n i t r o p h e n y l c a r b o n a t e groups and the t o t a l c a r b o n a t e c o n t e n t d u r i n g the a c t i v a t i o n o f dext r a n w i t h 4 - n i t r o p h e n y l c h l o r o f o r m a t e i n DMSO/pyridine i s r e p r e s e n t e d i n F i g u r e 4. T h i s demonstrates t h a t i n c o n t r a d i c t i o n w i t h former l i t e r a t u r e r e p o r t s d i f f e r e n t types of c a r b o n a t e groups a r e formed d u r i n g the 4 - n i t r o p h e n y l c h l o r o f o r m a t e a c t i v a t i o n . A p p a r e n t l y the i n i t i a l l y formed a r o m a t i c c a r b o n a t e s f u r t h e r r e a c t w i t h n e i g h b o u r i n g p o l y m e r i c h y d r o x y l s . Moreover, i n p r e s e n c e of a s t r o n g e r base (e.g. t r i e t h y l a m i n e ) , c y c l i c c a r b o n a t e s a r e formed p r e d o m i n a n t l y . The 4n i t r o p h e n y l c a r b o n a t e groups form o n l y a minor f r a c t i o n o f the t o t a l c a r b o n a t e c o n t e n t i n d i c a t i n g the rearrangment t o be base c a t a l y s e d . F u r t h e r e v i d e n c e f o r the f o r m a t i o n of d i f f e r e n t c a r b o n a t e s p e c i e s i s p r e s e n t e d i n F i g u r e 5 showing the I.R. s p e c t r a o f a c t i v a t e d d e x t r a n p r e p a r e d (A) i n p r e s e n c e o f t r i e t h y l a m i n e , (B) i n p r e s e n c e o f p y r i dine. They show a b s o r p t i o n maxima f o r A: a t 1805 cnT^ (5-membered c y c l i c a l i p h a t i c c a r b o n a t e s ) and a weak band a t 1740 cm~^ (non s t r a i n e d a l i p h a t i c c a r b o n a t e s ) and f o r B: a t 1765 cm"l ( 4 - n i t r o p h e n y l c a r b o n a t e s ) w i t h a s h o u l d e r a t 1805 cm~"l ( c y c l i c c a r b o n a t e s ) . T h i s d a t a demonstrate t h a t d u r i n g the a c t i v a t i o n o f d e x t r a n w i t h 4 - n i t r o p h e n y l c h l o r o f o r m a t e d i f f e r e n t types o f c a r b o n a t e groups a r e formed and t h a t , depending on the r e a c t i o n c o n d i t i o n s , t h e degree o f a c t i v a t i o n might be much h i g h e r than what one would c o n c l u d e from the d a t a o b t a i n e d from the " c o n v e n t i o n a l " a l k a l i n e h y d r o l y s i s and U.V. a s s a y o f the 4 - n i t r o p h e n y l groups.



194

20


c ο

time

(h)

F i g u r e 3. 4 - N i t r o p h e n y l carbamate c o n t e n t d u r i n g the r e a c t i o n o f d e x t r a n w i t h 4 - n i t r o p h e n y l c h l o r o f o r m a t e i n DMSO/pyridine ( v o l . r a t i o 1/1) a t 0°C. [anhydro g l u c o s i d e s ] = 0.1 M; [chloroformate] = ( X ) : 90 mM, (Δ) : 50 mM, ( • ) : 12 mM, ( O ) : 8 mM. 0

c ο

30

+>

a >

S

20

10

0 0

10

20

30

40 time

50 (h)

F i g u r e 4. T o t a l carbamate c o n t e n t ( • ) and 4 - n i t r o p h e n y l c a r b a mate c o n t e n t (O) d u r i n g t h e a c t i v a t i o n o f d e x t r a n w i t h 4 - n i t r o p h e n y l c h l o r o f o r m a t e [anhydro g l u c o s i d e s ] = 0 . 1 M; [ c h l o r o f o r m a t e ^ = 50 mM. 0



SCHACHT ET AL.


F i g u r e 5. IR spectrum of a c t i v a t e d d e x t r a n p r e p a r e d of (A) t r i e t h y l a m i n e and (B) p y r i d i n e .

i n presence



196 Biodégradation of d e x t r a n

derivatives


One argument f o r the s e l e c t i o n o f d e x t r a n as a drug c a r r i e r has o f t e n been i t s s u s c e p t i b i l i t y to d e g r a d a t i o n . However c h e m i c a l m o d i f i c a t i o n o f the polymer may i m p a i r the b i o d e g r a d a b i l i t y . T h e r e f o r e we have s t u d i e d the biodégradation o f d e x t r a n and a number o f d e x t r a n d e r i v a t i v e s by d e x t r a n a s e . F o r t h i s study model d e r i v a t i v e s were p r e p a r e d u s i n g the a c t i v a t i o n p r o c e d u r e s d i s c u s s e d b e f o r e . P r e p a r a t i o n o f the d e x t r a n d e r i v a t i v e s Reduced d e x t r a n d i a l d e h y d e s . The r e a c t i o n o f d e x t r a n (D-OH) w i t h s o dium m e t a p e r i o d a t e i s a two s t e p r e a c t i o n l e a d i n g t o d i f f e r e n t k i n d s o f a l d e h y d e f u n c t i o n s . A l t h o u g h the a l d e h y d e c o n t e n t can n o t be p r e c i s e l y p r e d i c t e d from the amount o f p e r i o d a t e added, by approximat i o n 1.5 e q u i v a l e n t s o f p e r i o d a t e a r e r e q u i r e d p e r d i a l d e h y d e s t r u c ture. By v a r y i n g the amount o f p e r i o d a t e added to the p o l y s a c c h a r i d e d e x t r a n d i a l d e h y d e s w i t h v a r i a b l e degree of o x i d a t i o n were o b t a i n e d . I n o r d e r to a v o i d i n t e r a c t i o n o f the p o l y a l d e h y d e (D-CHO) w i t h the d e x t r a n a s e s the aldehyde groups were s u b s e q u e n t l y r e d u c e d by r e a c t i o n w i t h sodium b o r o h y d r i d e : + NalO,

+ NaBH.

D-OH

D-CH=0

D-CH -0H o

D e x t r a n carbamate d e r i v a t i v e s . A c t i v a t i o n of d e x t r a n w i t h 4 - n i t r o p h e n y l c h l o r o f o r m a t e and subsequent r e a c t i o n w i t h amines l e a d s t o the f o r m a t i o n of the c o r r e s p o n d i n g carbamate d e r i v a t i v e s : 2-Hydroxyprop y l a m i n e was s e l e c t e d as a model f o r amine type d r u g s . D-0-C-NH-CH -CH-CH 0

0

o

OH (IX)

By v a r y i n g the amount of c h l o r o f o r m a t e added p e r gram d e x t r a n d i f f e r e n t degrees o f a c t i v a t i o n and d e r i v a t i v e s o f v a r i a b l e carbamate c o n t e n t c o u l d be p r e p a r e d . I n the NMR spectrum o f the r e a c t i o n p r o d u c t (IX) the m e t h y l p r o t o n s o f the 2-hydroxy p r o p y l r a d i c a l s show up as a d o u b l e t a t δ = 1,3 ppm. From the i n t e g r a t i o n v a l u e s o f the NMR s i g n a l s the degree o f m o d i f i c a t i o n was c a l c u l a t e d . D e x t r a n monosuccinate e s t e r . A monosuccinate e s t e r o f d e x t r a n was p r e p a r e d as d e s c r i b e d b e f o r e by r e a c t i n g the p o l y s a c c h a r i d e w i t h s u c c i n i c a n h y d r i d e (30 m e q . / a n h y d r o g l u c o p y r a n o s i d e u n i t ) i n p r e s e n c e o f c a t a l y t i c amounts of 4-dimethylamino p y r i d i n e . Degradation

o f the d e x t r a n d e r i v a t i v e s by

dextranases

The d e x t r a n d e r i v a t i v e s were i n c u b a t e d w i t h d e x t r a n a s e (from Sigma Chem Comp., Grade I p r o d u c t ) i n a c i t r a t e b u f f e r pH=6 a t 37°C. The d e g r a d a t i o n o f the polymer was m o n i t o r e d by means o f g e l p e r m e a t i o n chromatography on an a n a l y t i c a l Sephadex G-25 column. F o r comparison d e g r a d a t i o n o f u n m o d i f i e d d e x t r a n was c a r r i e d out as w e l l . In order to d i f f e r e n t i a t e h y d r o l y t i c from d e x t r a n a s e - i n d u c e d p r o c e s s e s the d e g r a d a t i o n was a l s o i n v e s t i g a t e d i n absence of enzymes.


SCHACHT ET AL.

Hydrolytic

Enzymatic


197


— ι —

Time min

—1—

-ι— 60

30

60

F i g u r e 6. GPC chromatograms o f d e x t r a n T-70 and a 10% o x i d i z e d and s u b s e q u e n t l y reduced d e r i v a t i v e a f t e r i n c u b a t i o n a t pH 6 w i t h and w i t h o u t d e x t r a n a s e .

100

JO

80

0)

60

AO

20

20

AO

60

80 time

100

120

(m 1 η )

F i g u r e 7. Enzymatic d e g r a d a t i o n o f d e x t r a n and r e d u c e d d e x t r a n d i a l d e h y d e s by d e x t r a n a s e i n c i t r a t e b u f f e r pH 6 a t 37°C. Degree o f m o d i f i c a t i o n (%) : ( X ) : 50, ( Θ ) : 25, ( X ) : 10, (Δ) : 5, ( p ) : 2, ( O ) : 0.




198

0

4

'

0

20

1

1

40

' 60

' 80

time

100

(m 1 η )

F i g u r e 9. Enzymatic d e g r a d a t i o n o f d i f f e r e n t d e x t r a n d e r i v a t i v e s by d e x t r a n a s e . ( Δ ) : 5% o x . r e d . D e x t r a n , ( D ) : 3% s u c c i n o y l a t e d d e x t r a n , ( O ) : 4.5% c a r b a m o y l a t e d d e x t r a n .



14. SCHACHT ET AL.


199

The g e l p e r m e a t i o n chromatograms o b t a i n e d a f t e r d i f f e r e n t i n c u b a t i o n times f o r d e x t r a n and f o r a 10% o x i d i z e d and s u b s e q u e n t l y r e duced d e x t r a n i n p r e s e n c e , r e s p e c t i v e l y i n absence o f d e x t r a n a s e a r e shown on F i g u r e 6. I t i s c l e a r t h a t i n absence o f d e x t r a n a s e w i t h i n an i n c u b a t i o n p e r i o d o f 1 h no d e g r a d a t i o n o f n e i t h e r t h e p a r e n t d e x t r a n n o r t h e p e r i o d a t e a c t i v a t e d d e r i v a t i v e c a n be o b s e r v e d . However i n presence o f the dextranase d e g r a d a t i o n i s observed f o r both polymers, t h e u n m o d i f i e d polymer b e i n g degraded more r a p i d l y than the o x i d i z e d d e r i v a t i v e . The peak h e i g h t h o f t h e polymer s i g n a l was t a k e n as a measure f o r t h e polymer c o n t e n t i n t h e r e a c t i o n medium and measured a t d i f f e r e n t i n c u b a t i o n t i m e s . The r e s u l t s o b t a i n e d f o r t h e d e x t r a n a s e - i n d u c e d d e g r a d a t i o n o f p e r i o d a t e t r e a t e d as w e l l as c a r b a moylated d e x t r a n d e r i v a t i v e s o f v a r y i n g degree o f m o d i f i c a t i o n a r e shown i n F i g u r e s 7 and 8. I t i s c l e a r t h a t i n b o t h c a s e s t h e r a t e o f biodégradation d e c r e a s e s w i t h i n c r e a s i n g degree o f m o d i f i c a t i o n o f the polymer. F i g u r e 9 shows t h e r e s u l t s o b t a i n e d f o r t h r e e d i f f e r e n t types o f d e x t r a n d e r i v a t i v e s , m o d i f i e d t o a comparable degree. The o b s e r v e d d i f f e r e n c e s i n r a t e o f d e g r a d a t i o n might be caused by mutual d i f f e r e n c e s i n polymer c h a i n c o n f o r m a t i o n o r e l e c t r o s t a t i c c h a r g e s . The e f f e c t o f t h e n a t u r e o f t h e s u b s t i t u e n t s on t h e b i o d e g r a d a b i l i t y i s a t present being i n v e s t i g a t e d . Conclusion These d e g r a d a t i o n s t u d i e s o f d e x t r a n d e r i v a t i v e s , m u t u a l l y d i f f e r i n g i n t h e n a t u r e and t h e degree o f m o d i f i c a t i o n , c l e a r l y demonstrate t h a t t h e b i o d e g r a d a b i l i t y o f d e x t r a n i s s i g n i f i c a n t l y reduced upon c h e m i c a l m o d i f i c a t i o n o f t h e polymer backbone. T h i s phenomenon s h o u l d be beared i n mind when u s i n g d e x t r a n as a c a r r i e r m o l e c u l e f o r t h e p r e p a r a t i o n o f macromolecular drug d e r i v a t i v e s . Acknowledgments T h i s work was s u p p o r t e d by t h e B e l g i a n N a t i o n a l S c i e n c e F o u n d a t i o n (N.F.W.O.) and t h e B e l g i a n I n s t i t u t e f o r Encouragment o f R e s e a r c h i n A g r i c u l t u r e and I n d u s t r y (I.W.O.N.L.).

Literature Cited 1. Schacht, E.; "Polysaccharide Macromolecules as Drug Carriers"; In "Controlled Release of Drugs from Polymer Particles and Macromolecules"; Illum, L.; Davis, S.S.; Eds. John Wright; Bristol, in press. 2. Molteni, L.; In "Drug Carriers in Biology and Medicine"; Gregoriasis, G.; Ed.; Academic : New York, 1977; Chap. 6. 3. Ishak, M.F.; Painter, T.J. Carbohydr. Res. 1978, 64, 189-197. 4. Ruys, L.; Vermeersch, J . ; Schacht, E.; Goethals, E.; Gyselinck, P.; Braeckman, P.; Van Severen, R. Acta Pharm. Techn. 1983, 29(2), 105-112.


200


5. Schacht, E.; Ruys, L.; Vermeersch, J . ; Remon, J.P. J. Controlled Release 1978, 1(1), 33-46. 6. Vermeersch, J . ; Ph.D. Thesis, State University Ghent, Ghent, 1985. 7. Ferruti, P.; Tanzi, M.C.; Vaccaroni, F. Makromoleculare Chem. 1979, 180, 375-382. 8.

Ruys, L.; Schacht, E. Bull. Soc. Chim. Belg. 1984, 93(6), 483-


485.

9. Kennedy, J.F.; Barker, S.A.; Rosevear, A. J. Chem. Soc., Perkin Trans. 1. 1973, 2293-2299. 10. Kennedy, J.F.; Rosevear, A. J. Chem. Soc., Perkin Trans. 1, 1974, 757-762.

11. Kol'tsova, G.N.; Krylova, N.K.; Vasil'ev, A.E.; Ovsepyan, A.M.; Shlimak, V.M.; Rozenberg, G.Ya. Zh. Obsch. Khim. 1977, 44, 111182. 12.

Kennedy, J.F.; Tun, H.C. Carbohydr. Res. 1973, 26, 401-408.

13.

Chaves, M.S.; Arranz, F. Makromol. Chem. 1985, 184, 17-25.

14.

Wilcheck, M.; Miron, T. Biochem. Inst. 1982, 4, 629-635.

15. Vasil'ev, A.E.; Kol'tsova, G.N.; Krylova, N.K.; Ovsepyan, A.M.; Shlimak, V.M.; Rozenberg, G.Ya. Zh. Obshch. Khim. 1977, 47, 1641-1648. RECEIVED January 27, 1987


Polysaccharides as Drug Carriers - ACS Symposium Series (ACS

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