Cyclopolymerization and Polymers with Chain-Ring Structures

7 Divinyl Ether-Maleic Anhydride Copolymer and Its Derivatives: Toxicity and Immunological and Endocytic Behavior Downloaded via UNIV OF SYDNEY on July 14, 2018 at 20:19:02 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

1

L. GROS, H . RINGSDORF, and R. SCHNEE —Institut für Organische Chemie, Universität Mainz, Johann-Joachim-Becher-Weg 20, D-6500 Mainz, Federal Republic of Germany J. B. LLOYD—Biochemistry Research Laboratory, Department of Biological Sciences, University of Keele, Staffs, United Kingdom 3

E . RÜDE, H . U. SCHORLEMMER2, and H . STÖTTER —Institut für Mikrobiologie und Immunologie, Universität Mainz, Federal Republic of Germany

DIVEMA and some DIVEMA derivatives were synthesized and tested for toxicity and immunological behaviour. Toxicity can be varied by introducing different side groups into parent DIVEMA molecules by polymer analogous reactions. The compounds tested showed mitogenic effects, i.e. they stimulated lymphocyte proliferation. Astonishingly, a DIVEMA-DNP conjugate did not induce the production of DNP-specific antibodies. Our results suggest that this is due to the formation of DNP-specific active suppressor cells. Using serum free culture media, we could show for the f i r s t time that DIVEMA and derivatives stimulate macrophages to release cytotoxic factors into the supernatant. However, no significant release of lytic enzymes was detected. DIVEMA and the derivatives tested failed to stimulate endocytosis by macrophages of 125I-PVP and 198Au (colloidal); higher mol. wt. DIVEMA inhibited pinocytosis. We conclude that the only effect of the compounds tested on macrophages is the release of cytotoxic factors into the supernatant. 1

Current address: Röhm GmbH, Kirschenallee, D-6100 Darmstadt, Federal Republic of Germany.

Current address: Behringwerke, Postfach 1140, D-3550 Marburg/Lahn 1, Federal Republic of Germany.

Current address: Zentrum f. Innere Medizin der Universität, Steinhövelstr. 9, D-7900 Ulm, Federal Republic of Germany. 2

3

0097-6156/82/0195-0083$06.00/0 © 1982 American Chemical Society

Butler and Kresta; Cyclopolymerization and Polymers with Chain-Ring Structures ACS Symposium Series; American Chemical Society: Washington, DC, 1982.

84

POLYMERS WITH CHAIN-RING STRUCTURES

P h a r m a c o l o g i c a l l y a c t i v e p o l y m e r s , and e s p e c i a l l y p o l y m e r i c a n t i t u m o u r a g e n t s , have b e e n w i d e l y d i s c u s s e d , s y n t h e s i z e d and t e s t e d d u r i n g t h e p a s t decade 2 3 ). Among t h e many a n i o n i c p o l y m e r s t e s t e d f o r p h a r m a c o l o g i c a l a c t i v i t y ( 4 ) , DIVEMA, o r p y r a n c o p o l y m e r , a 1:2 alternating c y c T o c o p o l y m e r o f d i v i n y l - e t h e r a n d mal e i c a n h y d r i d e i n i t s h y d r o l y z e d f o r m ( 5 ) , has b e e n one o f t h e m o s t i n t e n s i v e l y s t u d i e d c o m p o u n d s . DIVEMA has been shown t o h a v e immune s t i m u l a t i n g , a n t i v i r a l a n d a n t i t u m o u r a c t i v i t i e s ( 4 , 6^, 7). These p r o p e r t i e s s u g g e s t e d t h a t DIVEMA mTght be an a p p r o p r i a t e c a r r i e r c a n d i d a t e f o r drug m o l e c u l e s , e s p e c i a l l y f o r a n t i t u mour a g e n t s . T h u s , t h e immunosuppressive folateantagonistsmethotrexate (MTX) ( 8 ) a n d C y t o x a n (cyclop h o s p h a m i d e ) ( 9 , 10) were l i n k e d t o p y r a n c o p o l y m e r (cf. 3). ~ 9

R

—

T h e r e a r e , h o w e v e r , many u n s o l v e d p r o b l e m s c o n c e r n i n g t o x i c i t y , e n d o c y t i c u p t a k e and i m m u n o g e n i c i t y o f DIVEMA. In o r d e r t o l e a r n more a b o u t t h e s t r u c t u r e d e p e n d e n c y o f t h e s e p r o p e r t i e s , we s y t h e s i z e d some DIVEMA d e r i v a t i v e s a n d i n v e s t i g a t e d DIVEMA a n d t h e new d e r i v a t i v e s with appropriate routine b i o l o g i c a l test systems. Compounds

Tested

DIVEMA s a m p l e s ( 1 a - 1d) w e r e k i n d l y s u p p l i e d by D r . D . B r e s l o w , HERCULES I n c . The s y n t h e s i s o f t h e d e r i v a t i v e s ( 1 1 ) w i l l be p u b l i s h e d e l s e w h e r e . The c o m pounds i n v e s t i g a t e d a r e l i s t e d i n T a b l e I. Acute

Toxicity

of

DIVEMA

a n d two DIVEMA

Derivatives

One d i s a d v a n t a g e o f DIVEMA i s i t s r e l a t i v e l y h i g h t o x i c i t y w h i c h i s m a i n l y c a u s e d by h i g h m o l e c u l a r w e i g h t f r a c t i o n s (7). In an a t t e m p t t o l o w e r t h e t o x i c i t y o f DIVEMA, d e r i v a t i v e s ( 3 ) a n d ( 4 ) were s y n t h e sized. The p y r r o l i d o n e s i d e c h a i n i s known t o be n o n t o x i c e . g . in poly ( v i n y l p y r r o l idone). Incorporation of the d i m e t h y l a m i n o - m o i e t y l e a d s to z w i t t e r i o n i c s t r u c t u r e s ; s u c h s t r u c t u r e s a r e known t o l o w e r t h e t o x i c i t y of the p o l y c a t i o n i c p o l y ( e t h y l e n e i m i n e ) ( 1 2 ) . A c u t e t o x i c i t y o f compounds ( 1 d ) , ( 3 F 7 and ( 4 b ) , a l l o f m o l . w t . 2 4 , 0 0 0 , was t e s t e d i n v i v o . M a l e C 5 7 / B L . 6 m i c e w e r e g i v e n a s i n g l e i . p . d o s e . T a b l e II shows t h e r e s u l t s . T o x i c i t y i s e x p r e s s e d a s LD 5 0 / 2 1 , t h e d o s e l e t h a l f o r 50% o f t h e a n i m a l s by day 21 a f t e r i n j e c t i o n of the polymer.


7.

GROS E T A L .

Table I .

Behavior of DIVEMA

and DIVEMA

Derivatives

85

Compounds synthesized and t e s t e d w i t h R groups d i s t r i b u t e d between r i n g and chain c a r b o x y l s .

NR

-R

X

MW 4400 11000 16800 24000

la b c d

-OH

2

-ΝΗ-ΙΟΗ,ί,-^^-ΝΟ*

5,3

16 800

3a b

-0-(CH ) -^p

12

16800 24000

4a b

-0-(CH,),-N(CH,)

20

16800 24000

5

-CMCHj^-CHa

4

16 800

a

a

a

COOH


POLYMERS WITH CHAIN-RING

STRUCTURES

Table I I . Acute t o x i c i t y of DIVEMA. and some d e r i v a t i v e s

Substance Nr. 1d

Dose (mg/kg)

Survivors (day 21)

300

6/10

400

4/10

300

4/8

400

0/8

300

8/8

400

7/8

500

4/8

600

4/8

700

4/8

LD50/21*

300-400

3b

c a . 300

4b

500-600

*LD50/21 - l e t h a l dose f o r 50% of t e s t animals by day 21.


7.

GROS ET AL.

Behavior of DIVEMA

and DIVEMA

Derivatives

87

The i n t r o d u c t i o n o f t h e p o t e n t i a l l y biocompatible p y r r o l i d o n e u n i t (polymer (3b)) unexpectedly leads to a s l i g h t l y h i g h e r t o x i c i t y , as compared t o t h e s t a r t i n g material (1d). Decrease of the polyanion c h a r a c t e r in the z w i t t e r i o n i c polymer ( 4 b ) , however, substantially lowers toxicity. Effects System.

of

DIVEMA

a n d DIVEMA

Derivatives

on t h e Immune — — —

DIVEMA i s known t o a c t upon d i f f e r e n t t y p e s o f immune c e l l s (J_3) s u c h a s t h e a n t i b o d y p r o d u c i n g Β c e l l s ( b o n e marrow d e r i v e d l y m p h o c y t e s ) ; on T - c e l l s ( t h y m u s p r o c e s s e d l y m p h o c y t e s ) and on m a c r o p h a g e s ( c e l l s w h i c h p h a g o c y t o s e f o r e i g n m a t e r i a l ) . The m e c h a n i s m s o f i n t e r a c t i o n b e t w e e n p o l y m e r s a n d t h e immune s y s t e m a r e v e r y c o m p l e x . In o r d e r t o i n v e s t i g a t e them, many d i f f e r e n t e x p e r i m e n t a l a p p r o a c h e s c a n be u s e d . Our a p p r o a c h i s b a s e d on t h e f o l l o w i n g questions: - A r e t h e DIVEMA d e r i v a t i v e s m i t o g e n i c , i . e . do t h e y s t i m u l a t e l y m p h o c y t e p r o l i f e r a t i on? - A r e t h e DIVEMA d e r i v a t i v e s a n t i g e n i c , i . e . do t h e y induce production of s p e c i f i c antibodies? - Do DIVEMA d e r i v a t i v e s s t i m u l a t e m a c r o p h a g e s in v i t r o , i . e . do t h e y i n d u c e t h e r e l e a s e o f l y t i c e n z y m e s o r cytotoxic factors? M i t o g e n i c A c t i v i t y o f DIVEMA D e r i v a t i v e s . A f i r s t a p p r o a c h t o t h e e f f e c t s o f a compound on t h e immune system i s to t e s t i t s a b i l i t y to s t i m u l a t e lymphocyte p r o l i f e r a t i o n . An a p p r o p r i a t e t e s t i s t o m e a s u r e t h e i n c o r p o r a t i o n o f H - t h y m i d i n e i n t o DNA o f d i v i d i n g eel 1 s : S u b s t a n c e s ( 1 a ) - ( 1 d ) , ( 3 a ) and ( 4 a ) a r e i n c u bated with s p l e e n c e l l c u l t u r e s of u n t r e a t e d mice i n a s e r u m c o n t a i n i n g c u l t u r e m e d i u m . A d e f i n e d amount o f radioactive H-thymidine is added. A f t e r i n c u b a t i o n , t h e c u l t u r e s a r e deep f r o z e n i n o r d e r t o s t o p c e l l pro l i f e r a t i o n a n d DNA i s i s o l a t e d by f i l t r a t i o n through glass f i b r e f i l t e r s . R a d i o a c t i v i t y is counted in a l i q u i d s c i n t i l l a t o r . Control c u l t u r e s are incubated w i t h o u t a n t i g e n ( p o l y m e r ) a d d e d a n d w i t h a known potent mitogen ( C o n c a n a v a l i η A.) A l l substances showed s i g n i f i c a n t m i t o g e n i c acti v i t y . H i g h m o l e c u l a r w e i g h t DIVEMA s t i m u l a t e d lympho c y t e p r o l i f e r a t i o n a t lower c o n c e n t r a t i o n than the c o r r e s p o n d i n g low m o l e c u l a r w e i g h t s a m p l e s . Derivatives (3a) and ( 4 a ) were s l i g h t l y l e s s a c t i v e . I t seems that t h e more t h e p o l y a n i o n i c c h a r a c t e r i s r e d u c e d by s u b s t i t u t i o n , t h e more t h e m i t o g e n i c a c t i v i t y i s l o w e r e d . 3

3


88


A n t i g e n i c i t y o f a D I V E M A - H a p t e n - C o n j u g a t e ( 2 ) . The r e s u l t s of the H - t h y m i d i n e i n c o r p o r a t i o n experiments g i v e no i n f o r m a t i o n a b o u t t h e t y p e o f l y m p h o c y t e s sti m u l a t e d by DIVEMA. I t i s l i k e l y , t h o u g h n o t f o r m a l l y p r o v e n , t h a t t h e e n h a n c e d H - t h y m i d i n e u p t a k e i s due t o p r o l i f e r a t i o n of Β l y m p h o c y t e s . Whether the polymer f u r t h e r m o r e i s a n t i g e n i c a n d l e a d s t o an i n c r e a s e i n t h e numbyr o f a n t i b o d y f o r m i n g c e l l s c a n be t e s t e d u s i n g e i t h e r t h e p o l y m e r i t s e l f o r a p o l y m e r - h a p t e n con j u g a t e . In t h e l a t t e r c a s e a low m o l e c u l a r w e i g h t c o m pound ( h a p t e n , w h i c h i s r e c o g n i z e d by t h e immune s y s t e m o n l y when b o u n d t o an i m m u n o g e n i c c a r r i e r ) i s l i n k e d t o t h e p o l y m e r . T h i s has t h e a d v a n t a g e t h a t a s t a n d a r d i z e d t e s t - s y s t e m common f o r a l l s u b s t a n c e s is available. E x p e r i m e n t a l r e s u l t s known so f a r ( e . g . 14,15,16) i n d i c a t e t h a t t h e amount o f a n t i b o d i e s p r o d u c e " ? a g a i n s t the p o l y m e r - h a p t e n c o n j u g a t e i s c l o s e l y r e l a t e d to the a n t i g e n i c i t y o f t h e p o l y m e r i t s e l f ; t h e more a n t i g e n i c the p o l y m e r , the h i g h e r the a n t i b o d y p r o d u c t i o n a g a i n s t a hapten f i x e d to i t . P o l y m e r ( 2 ) was u s e d as a t e s t s u b s t a n c e . I t con t a i n s t h e d i n i t r o p h e n y l - m o i e t y , a c l a s s i c a l h a p t e n (DNP). P r o d u c t i o n o f D N P - s p e c i f i c a n t i b o d i e s was m e a s u r e d by a radio-immunoassay (antigen-binding test). F e m a l e CBA m i c e w e r e i m m u n i z e d w i t h d i f f e r e n t d o s e s o f ( 2 ) ( d a y 0) a n d r e i m m u n i z e d ( b o o s t e r e d ) w i t h t h e same d o s e ( d a y 2 1 ) . On day 3 1 , a b l o o d s a m p l e was t a k e n and t h e a n t i b o d i e s i n t h e s e r u m were p r e c i p i t a t e d by adding a I-labelled v i n y l polymer c o n t a i n i n g DNPmoieties (DNP-VINAM), 3

3

1 2 5

DNP-VINAM

a well

established

experimental

method

(_1_7). The


radio-

7.

GROS ET AL.

Behavior of DIVEMA

and DIVEMA

Derivatives

89

a c t i v i t y r e m a i n i n g i n t h e s u p e r n a t a n t was m e a s u r e d a n d t h e p e r c e n t b i n d i n g o f h a p t e n t o t h e a n t i b o d i e s was calculated. No s i g n i f i c a n t a n t i b o d y p r o d u c t i o n was f o u n d . R e i n v e s t i g a t i o n o f t h e number o f c e l l s p r o d u c i n g DNPs p e c i f i c a n t i b o d i e s with the plaque assay (which is much more s e n s i t i v e t h a n t h e a n t i g e n b i n d i n g t e s t ) conf i r m e d t h i s r e s u l t . T h e s e f i n d i n g s w o u l d n o t be t o o s u r p r i s i n g i f a weakly immunogenic c a r r i e r l i k e polyv i n y l p y r r o l i d o n e ) ( P V P ) was u s e d . In t h e c a s e o f DIVEMA, i t i s u n e x p e c t e d , s i n c e t h i s s u b s t a n c e - i n c o n t r a s t t o PVP - shows s t r o n g i n f l u e n c e s on t h e immune s y s t e m , e . g . m i t o g e n i c i t y f o r l y m p h o c y t e s . One p o s s i b l e e x p l a n a t i o n i s t h a t ( 2 ) i n d u c e s t o l e r a n c e a g a i n s t DNP. In o r d e r t o v e r i f y t h i s a s s u m p t i o n , f e m a l e CBA m i c e w e r e g i v e n a s t r o n g l y i m m u n o g e n i c DNP p r o t e i n c o n j u g a t e c a l l e d DNP-KLH ( k e y h o l e l i m p e t h e m o c y a n i n e ) ( J 8 ) . One g r o u p o f a n i m a l s had b e e n p r e - t r e a t e d ("vaccinated") w i t h d i f f e r e n t d o s e s o f ( 2 ) , a c o n t r o l g r o u p had o n l y been g i v e n p h o s p h a t e b u f f e r i n j e c t i o n s on d a y s 1 0 , 6 a n d 1 b e f o r e i n j e c t i o n o f D N P - K L H . T a b l e i n shows t h e r e s u l t s of the a n t i g e n - b i n d i n g t e s t of blood samples from t h e two g r o u p s of m i c e a n d o f two c o n t r o l groups. T h e s e m e a s u r e m e n t s show t h a t s m a l l d o s e s o f DNPDIVEMA ( 2 ) g i v e n t o t h e m i c e b e f o r e t h e immune s y s t e m i s c h a l l e n g e d w i t h t h e p o t e n t a n t i g e n DNP-KLH, signific a n t l y suppress the f o r m a t i o n of D N P - s p e c i f i c antib o d i e s : (2) i n d u c e s t o l e r a n c e against DNP-KLH. T a k i n g i n t o a c c o u n t t h a t (2) i s m i t o g e n i c but s t i l l has no a n t i g e n i c e f f e c t , we s u g g e s t t h a t i n d u c t i o n o f t o l e r a n c e by t h i s p o l y m e r i s due t o f o r m a t i o n o f DNPs p e c i f i c s u p p r e s s o r c e l l s . T h i s w o u l d e x p l a i n why DIVEMA i s immune s t i m u l a t i n g ( i n d u c e s p r o l i f e r a t i o n o f lymphocytes) without causing production of a n t i b o d i e s : (2) i n d u c e s a c t i v e s u p p r e s s i o n and t h u s l e a d s t o t h e apparent lack of immunogenicity. E f f e c t s o f DIVEMA and DIVEMA P e r i v a t i y e s on Mouse Macrophages i n V i t r o . P o l y a n i o n s act n o t o n l y upon l y m p h o c y t e s , b u t a l s o on m a c r o p h a g e s ( 1 3 , ^_9). M a c r o p h a g e s a r e immune c e l l s w h i c h t a k e up f o r e i g n m a t e r i a l , h e l p l y m p h o c y t e s r e c o g n i z e and a t t a c k a n t i g e n s and p l a y an i m p o r t a n t r o l e i n t h e d e f e n s e o f t h e b o d y a g a i n s t tumour c e l l s (20). S c h u l t z e T ~ a l . ( 2 1 , 22) f o u n d t h a t m a c r o p h a g e s of D I V E M A - t r e a t e d mice i n h i b i t e d tumour c e l l proliferation i n v i t r o ; t h i s means t h a t DIVEMA a c t i v a t e s macrophages. The a u t h o r s d i d n o t f i n d c y t o t o x i c f a c t o r s i n t h e s u p e r n a t a n t of these macrophage c u l t u r e s . As p o l y a n i o n s c a n s t i m u l a t e many d i f f e r e n t m a c r o p h a g e f u n c t i o n s , we t r i e d t o f i n d o u t w h i c h f u n c t i o n s s


90


Table

III

I n d u c t i o n o f t o l e r a n c e by DNP-DIVEMA ( 2 ) a g a i n s t DNPK L H . Mean v a l u e s o f 5 a n i m a l s . A n t i b o d y p r o d u c t i o n i s e x p r e s s e d as % h a p t e n ( j-DNP-VINAM) precipitated by t h e a n t i b o d i e s i n t h e s e r u m ( g r o u p s A a n d B) o r as t i t e r 3 3 % , t h a t i s t h e r e c i p r o c a l o f t h e serum d i l u t i o n a t w h i c h 33% o f I-DNP-VINAM a r e b o u n d a n d p r e c i p i t a t e d ( g r o u p s C , D ) . T i t e r 33% was r e a c h e d i n g r o u p s C and D o n l y . 1 2 5

1

PBS CFA

2

5

= phosphate b u f f e r e d s a l i n e = complete Freund's Adjuvant

Group

Treatment

Dose of (2),pg mouse

(background PBS days -10,-6,-1 control) CFA day 0 NaCl day 21

Β ( e f f e c t of DNP-DIVEMA itself)

C DNP-KLH alone

D (2)+DNPKLH

fc) days - 1 0 , - 6 , - 1 CFA day 0 NaCl day 21

-2.1

0,1 1 10 100

PBS days - 1 0 , - 6 , - 1 DNP-KLH (100 pg in CFA) day 0 DNP-KLH (10 pg i n NaCl) day 21

(2) days -10,-6,-1 DNP-KLH days 0 and 21

antibodies in serum day 31

8.6 7.7 6.4 2.2

+ 1.9

+ 11.1 + 6.0 Τ 5.0 + 4.6

12,340

0,1 1 10 100

2,380 6,880 7,220 12,400


7.

GROS E T A L .

Behavior of DIVEMA

and DIVEMA

Derivatives

91

a r e a c t i v a t e d . We c h o s e i n v i t r o t e s t s i n o r d e r t o a v o i d i n f l u e n c e o f m e t a b o l i s m . Two m a c r o p h a g e functions were t e s t e d : - s e c r e t i o n o f l y s o s o m a l enzymes and - r e l e a s e o f c y t o t o x i c f a c t o r s i n t o t h e s u p e r n a t a n t by D I V E M A - t r e a t e d macrophage c u l t u r e s . In o r d e r t o m e a s u r e s e c r e t i o n o f l y s o s o m a l enzymes, m a c r o p h a g e s o f n o r m a l , u n t r e a t e d NMRI m i c e were i n c u b a t e d i n s e r u m - f r e e c u l t u r e medium (RPMI) a n d t r e a t e d w i t h d i f f e r e n t a m o u n t s o f compounds ( 1 c ) , ( 3 a ) , ( 4 a ) (mol.wt. 16,800),and ( 1 d ) , ( 3 b ) , (3c) (mol.wt. 24,000). S e c r e t i o n o f t h e f o l l o w i n g e n z y m e s was m e a s u r e d a f t e r 24 h o f i n c u b a t i o n u s i n g e s t a b l i s h e d b i o c h e m i c a l test systems : N-Acetyl-B-D-glucosaminidase ( N - A c - G l u ) (23), β - G l u c u r o n i d a s e ( β - G l u ) J2j4)and l a c t a t e d e h y d r o g e n a s e (LDH) (251 The l a t t e r enzyme i s l o c a t e d i n t h e c y t o p l a s m a n d i s n o t a c t i v e l y s e c r e t e d ( a s t h e o t h e r two e n z y m e s a r e ) b u t o n l y s e t f r e e i f t h e c e l l membrane i s d e s t r o y e d : t h e LDH l i b e r a t e d i n f o r m s a b o u t v i a b i l i t y o f c e l l cultures o r c y t o t o x i c e f f e c t s on m a c r o p h a g e s o f t h e s u b s t a n c e tested. None o f t h e s u b s t a n c e s c a u s e d s i g n i f i c a n t enzyme s e c r e t i o n by m a c r o p h a g e s . O n l y t h e p y r r o l i d o n e d e r i v a t i v e ( 3 a ) c a u s e d a s l i g h t i n c r e a s e o f LDH w i t h i n c r e a s i n g dose o f t h e p o l y m e r . T h i s f i n d i n g corresponds t o t h e h i g h e r a c u t e t o x i c i t y o f ( 3 b ) as c o m p a r e d t o u n s u b s t i t u t e d DIVEMA d e r i v a t i v e ( 1 b ) . These r e s u l t s a r e u n e x p e c t e d , s i n c e d e x t r a n s u l f a t e w i t h s i m i l a r m o l . w t . does i n d u c e s e c r e t i o n o f enzymes ( 2 6 ) . Release of c y t o t o x i c f a c t o r s i n t o the supernatant was t e s t e d by a d d i n g t h e s u p e r n a t a n t o f D I V E M A - t r e a t e d m a c r o p h a g e s t o c u l t u r e s o f d e f i n e d a m o u n t s o f P815 t u m o u r c e l l s a n d m e a s u r i n g LDH f r o m l y s e d t u m o u r c e l l s a f t e r 24 hr. o f i n c u b a t i o n . F i g u r e 1 shows s c h e m a t i c a l l y how t h e e x p e r i m e n t was done. A l l substances t e s t e d , ( 1 c ) , ( 1 d ) , ( 3 a ) , ( 3 b ) , ( 4 a ) , (4b), c a u s e d i n c r e a s i n g c y t o t o x i c a c t i v i t y o f t h e s u p e r n a t a n t w i t h i n c r e a s i n g doses o f t h e p o l y m e r s . Though the a b s o l u t e values of L D H - 1 i b e r a t i o n d i f f e r g r e a t l y f r o m one e x p e r i m e n t t o a n o t h e r , t h i s q u a l i t a t i v e result i s t h e same i n a l l t e s t s ( J J _ ) . One t y p i c a l diagram showing t h e dose dependency o f s u p e r n a t a n t m e d i a t e d t o x i c i t y c a u s e d by p o l y m e r ( 3 b ) i s shown i n F i g . 2. R e l a t i v e l y l o w c o n c e n t r a t i o n s (50 - 100 p g / m l ) were s u f f i c i e n t to cause s u b s t a n t i a l c y t o t o x i c i t y . As f a r as we know, t h i s i s t h e f i r s t e x a m p l e o f s u p e r n a t a n t - m e d i a t e d t o x i c i t y c a u s e d by DIVEMA. I t i s c o n t r a d i c t o r y t o t h e r e s u l t s o f S c h u l t z e t al.


92


tumor

cells

tumor

cells

supermatant

DETERGENT

macrophages

eel I-lysis measured by enzyme release

ι

• DIVEMA

lysis relative to detergent-effect

Figure I.

ι

detergent-effect : 1007· lysis

Test system for measuring cytotoxicity in the supernatant of macrophage cultures.

t

Figure 2.

Supernatant-mediated cytotoxicity caused by DIVEMA derivative (3b in Table II) (three independent runs).


7.

GROS E T A L .

Behavior of DIVEMA

and DIVEMA

Derivatives

93

( 2 1 , 2j2) ; t h i s may be due t o t h e f a c t t h a t t h e s e a u T h o r s used s e r u m - c o n t a i n i n g c u l t u r e m e d i a . Our r e s u l t s d e m o n s t r a t e t h a t e v e n c h e m i c a l l y m o d i f i e d DIVEMA c a u s e s - a t l e a s t i n v i t r o - t h e same b i o l o g i c a l e f f e c t ( r e l e a s e o f c y t o t o x i c f a c t o r s ) as DIVEMA i t s e l f . T h i s a s p e c t i s e s p e c i a l l y i m p o r t a n t i f one u s e s DIVEMA as a c a r r i e r polymer f o r antitumour agents hoping to r e t a i n immune s t i m u l a t i n g p r o p e r t i e s . Endocytic

Behaviour

o f DIVEMA a n d

Derivatives

M a c r o p h a g e s h a v e one more i m p o r t a n t p r o p e r t y : t h e y i n g e s t f o r e i g n m a t e r i a l v i a e n d o c y t o s i s , a p r o c e s s which r e s e m b l e s t h e i n g e s t i o n o f m a t e r i a l by a m o e b a e . P o l y m e r s may e i t h e r be t a k e n up t h e m s e l v e s o r may s t i m u l a t e e n d o c y t o s i s o f o t h e r s u b s t a n c e s . We t e s t e d t h e l a t t e r p o s s i b i l i t y w i t h two e s t a b l i s h e d t e s t systems ( 2 7 ) , ( 2 8 ) : -

Uptake

of

I - l a b e l led poly(vinylpyrrolidone) by r a t m a c r o p h a g e s . ( T h i s p o l y m e r i s up by p i n o c y t o s i s i n t h e f l u i d p h a s e ) . 1 2 5

(125j'-pvp) taken -

Uptake o f A u ( c o l l o i d a l ) by mouse m a c r o p h a g e s . ( T h i s c o l l o i d i s m a i n l y t a k e n up a f t e r a d s o r p t i o n a t the c e l l membrane). P o l y m e r s ( 1 a ) , ( 1 b ) , ( 1 d ) , ( 3 a ) , ( 4 a ) a n d ( 5 ) were t e s t e d . F i g u r e 3 shows t h e E n d o c y t i c I n d e x ( 2 8 ) m e a s u r e d The DIVEMA d e r i v a t i v e s ( 1 a ) , ( 3 a ) a n d ( 4 a ) ïïâve no s i g n i f i c a n t e f f e c t on t h e p i n o c y t o s i s o f J-PVP (they do n o t i n f l u e n c e t h e r a t e o f f o r m a t i o n o f p i n o c y t i c v e s i c l e s ) . P o l y m e r ( 1 d ) shows m a r k e d i n h i b i t i o n o f p i n o c y t o s i s at the highest dose.(This l a t t e r f i n d i n g in v i t r o corresponds to in vivo r e s u l t s of Breslow et au (29)). They f o u n d t h a t t h e removal o f c o l l o i d a l c a r b o n f r o m t h e b l o o d o f t e s t a n i m a l s i s i n h i b i t e d by h i g h m o l e c u l a r weight DIVEMA). Polymer (5) causes a d e c r e a s e o f p i n o c y t o s i s w i t h i n c r e a s i n g dose. The f a i l u r e o f d e r i v a t i v e s ( 3 a ) a n d ( 4 a ) t o i n f l u e n c e p i n o c y t o s i s may be due t o t h e i r r e d u c e d p o l y a n i o n - c h a r a c t e r . The i n h i b i t o r y e f f e c t o f t h e h y d r o p h o b i c d e r i v a t i v e ( 5 ) may be a t t r i b u t e d t o i n t e r a c t i o n s o f a l k y l c h a i n s w i t h t h e l i p i d membrane* These r e s u l t s o b t a i n e d with the I-PVP-test s y s t e m , as w e l l as e x p e r i m e n t s u s i n g t h e c o l l o i d a l g o l d t e s t s y s t e m , a r e d i s c u s s e d i n more d e t a i l i n r e f . (27). T h i s work i s p a r t o f t h e P h . D . t h e s i s o f R . S c h n e e (11). 1 9 8

1 2 5

1 2 5


94

POLYMERS

WITH

CHAIN-RING

STRUCTURES

(5)

0,1 + φ ο

(Ια)

(lb)

(1c)

(3a)

(4a)

χ φ C

M

Ο

iftl

Ul

o°R8

°g

concentrations

o^gg

oosg

ooog

ooog

in( pg/ml)

Figure 3. Stimulation of endocytosis by DIVEMA and DIVEMA derivatives. Endocytic index in the macrophage test system and I-PVP used as standard sub stance. 125


7.

GROS E T A L .

Behavior of DIVEMA

and DIVEMA

Derivatives

95

Acknowledgements We are indebted to Prof. Dr.D. Breslow for the DIVEMA samples. This work was supported in part by the B r i t i s h Science Research Council and the Deutsche Forschungsgemeinschaft. R.S. thanks the Fonds der Chemie for a Ph.D. fellowship. Literature Cited 1. Kostelnik, R.J. (Ed.), "Polymeric Delivery Systems", Midland Macromolecular Monographs V o l . 5, Gordon & Breach, N.Y. 1975 2. Donaruma, G.L., Vogl, O. (Eds.), "Polymeric Drugs" Academic Press, N.Y. 1978 3. Gros, L., Ringsdorf, H., Schupp, H., Angew.Chem.Int Ed., 1981, 93, 305 - 325 4. Donaruma, L.G., Ottenbrite, R.M., Vogl, O. (Eds.), "Anionic Polymeric Drugs", John Wiley & Sons, N.Y. 1980 5. B u t l e r , G.B., see 4., p. 49 - 142 6. Breslow, D.S., Pure Appl.Chem. 1976, 46, 103 7. Regelson, W., Morahan, P., Kaplan, A. in "Polye l e c t r o l y t e s and Their Applications" (A. Rembaum, E. Sélégny, Eds.) D. Reidel Publ. Comp., Dordrecht+ Boston 1975, p. 131 - 144 8. P r z y b y l s k i , M., Zaharko, D.S., Chirigos, M.A., Adamson, R.H., Schultz, R.M., Ringsdorf, H. Cancer Treat.Rep. 1978, 62, 1837 9. Hirano, T . , Klesse, W., Ringsdorf, H., Makromol. Chem. 1979, 180, 1123 10. Hirano, R. Ringsdorf, H., Zaharko, D.S., Cancer Res. 1980, 40, 2263 11. Schnee, R., Ph.D. T h e s i s , Mainz 1980 12. Kobayashi, S., Ringsdorf, H., in preparation (unpubl. results) 13. Butler, G.B., see 4 . , p. 185 - 210 14. Snippe, H., Nab, J . , van Eyk, R.V.W., Immunology 1974, 27, 761 15. Golan, D.R., B o r e l , Y., J.Exp.Med. 1971, 134, 1046 16. Schmidtke, J . R . , Dixon, R . J . , J.Exp.Med. 1972, 136 392 17. Wrede, J., Rüde, E., Thumb, R., Meyer-Debus, M. Eur.J.Immunol. 1973, 3,798 18. M i s h e l l , B.B., S h i i g i , S.M.,"Selected Methods in C e l l u l a r Immunology", W.H. Freeman & Co. San Francisco 1980 19. see 4., p. 234 - 236 and p. 196 - 198


96


20. James, K., McBride, B. and Stuart, H. (Eds.) "The Macrophage and Cancer" Proceedings of the European Reticuloendothelial Society, Symposium Edinburgh Sept. 12 - 14 , 1977, Edinburgh 1977 21. Schultz, R.M., Papamatheakis, J.Β., Luetzler, J . , Chirigos, M.A., Cancer Res. 1977, 37, 3338 22. Schultz, R.M., Papamatheakis, J.P., Chirigos, M.A., Cell Immunol. 1977, 29, 403 23. Woolen, J.P., Heyworth, R., Walker, P.G., Biochem.J. 1961, 78, 111 24. Talalay, P., Fishman, W.H., Huggins, C., J . B i o l . Chem. 1946, 166, 757 25. Bergmeyer, H.U. (Ed.) "Methoden der enzymatischen Analyse" Verlag Chemie, Weinheim FRG, 1970, p. 533 26. Schorlemmer, H.U., Burger, R., Hylton, W., A l l i s o n , A.C., C l i n . Immunol. Immunopathol. 1977, 7, 88 27. Pratten, M.K., Duncan, R., Cable, H.C., Schnee, R., Ringsdorf, H., Lloyd, J.B., Chemico-Biological Interactions, 1981, 35, 319 28. Pratten, M.K., Lloyd, J.B., Biochem.J. 1979, 180, 567 29. Breslow, D.S., Edwards, E.F., Newburg, N.R., Nature 1973, 246, 160 th

RECEIVED February 1, 1982.


Cyclopolymerization and Polymers with Chain-Ring Structures

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