7 Heparin Derivatives of High Molecular Weight L . M E S T E R , A . A M I T A M A Y A , and M . M E S T E R
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Institut de Chimie des Substances Naturelles, C.N.R.S., 91190 Gif-Sur-Yvette, France
Many attempts have been made in the last two deca des (1-6) to modify the s t r u c t u r a l features of the hepa r i n molecule (7,8) in order to produce changes in i t s b i o l o g i c a l a c t i v i t i e s (9,12), but only very few, to mo dify the size of the molecule (10,11,13). High molecu lar weight heparin preparations are now obtained through methacrylation of heparin and polymerization of the he parin methacrylate monomer. Methacrylation and polymerization Heparin (0.1 mMol) is dissolved in 0.5 Ν sodium hydroxide solution and methacryl chloride (4 m M o l s ) is added with s t i r r i n g at room temperature and then heated to 80°C for 2 hours. The solution is neutralized with acetic acid and evaporated under reduced pressure. The heparin methacrylate ester (Figure 1) is poly merized by heating with a z o d i i s o b u t y r o n i t r i l e as cata lyst in 1,4-dioxane solution at pH = 5. The polymeriza tion is stopped by adding hydroquinone and the solution is evaporated under reduced pressure. Operating in this way, the main product is a water soluble heparin methacrylate polymer having a molecular weight of about 40.000, as shown by u l t r a c e n t r i f u g a t i o n . The molecular weight of the initial heparin being 18.000, the polymer is a dimer of heparin. The polymerized hepa r i n is isolated from unchanged heparin by passage through a Sephadex G-200 column, equilibrated with an aqueous ammonia solution to pH = 8. The heparin-rich fractions are detected by their methacromatic effect with toluene blue. The fractions containing the water soluble heparin polymer are l y o p h i l i z e d and investigated by u l t r a c e n t r i fugation.
0-8412-0426-8/78/47-077-113$05.00/0 ©
1978 A m e r i c a n C h e m i c a l Society
In Carbohydrate Sulfates; Schweiger, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
In Carbohydrate Sulfates; Schweiger, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978. Figure 1.
Methacrylated heparin segment
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Water i n s o l u b l e m e t h a c r y l a t e d h e p a r i n p o l y m e r s are o b t a i n e d when more m e t h a c r y l c h l o r i d e i s u s e d f o r e s t e r i f i c a t i o n and when t h e p o l y m e r i z a t i o n i s c a r r i e d o u t a t pH = 3. The i n s o l u b l e h e p a r i n p r e p a r a t i o n s a r e w a s h e d t h r e e t i m e s w i t h i c e c o l d w a t e r , c e n t r i f u g a t e d and l y o p h i l i z e d . The m o l e c u l a r w e i g h t o f t h e i n s o l u b l e h e p a r i n p o l y m e r s i s h i g h e r t h a n 2 0 0 . 0 0 0 . The w a t e r i n s o l u b l e p o l y m e r s may be u s e d f o r c o a t i n g m e t a l s u r f a c e s w i t h l a y e r s showing a n t i t h r o m b i c activity.
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Ultracentrifugation U l t r a c e n t r i f u g a t i o n o f h e p a r i n ( I ) and o f m e t h a c r y l a t e d h e p a r i n p o l y m e r ( I I ) was c a r r i e d out i n a s u c r o s e g r a d i e n t o f 5 % t o 20 %, i n SPINC0 C e n t r i f u g (41.000RPM, 30 h o u r s , 2 ° C ) , u s i n g s e r u m a l b u m i n (SA) and c y t o c h r o m (CY) as i n t e r n a l r e f e r e n c e s . On ' F i g u r e 2, f r a c t i o n I i s h e p a r i n w i t h a c o n s t a n t o f s e d i m e n t a t i o n S = 2 c o r r e s p o n d i n g to a m o l e c u l a r w e i g h t o f 18.000, f r a c t i o n I I i s a p o l y m e r o f m e t h a c r y l a t e d h e p a r i n , i s o l a t e d on S e p h a d e x G-200 c o l u m n and s h o w i n g a s e d i m e n t a t i o n c o n s t a n t S = 4, w h i c h c o r r e s ponds to a m o l e c u l a r w e i g h t of about 40.000. C o p o l y m e r i ζat i o n C o p o l y m e r i z a t i o n of m e t h a c r y l a t e d h e p a r i n with v i n y l o r m e t h a c r y l monomers i s an u n i q u e m e t h o d to c h a n g e t h e g e o m e t r y and p h y s i c o - c h e m i c a l p r o p e r t i e s o f t h e h e p a r i n m o l e c u l e . F i g u r e 3 shows : A. a f r a g m e n t o f t h e m e t h a c r y 1 a t e d h e p a r i n p o l y m e r and _B. a f r a g m e n t o f a c o polymer of m e t h a c r y l a t e d h e p a r i n w i t h b u t y l m e t h a c r y l a t e . C o p o l y m e r i z a t i o n of m e t h a c r y l a t e d h e p a r i n w i t h v i n y l laur a t e gave l i p o s o l u b l e p o l y m e r s . Copolymerization with r e t i c u l a n t s , like divinylbenzene or Ν , Ν - m e t h y 1 e n e b i s - a c r y l a m i d e i s e s p e c i a l l y e f f e c t i v e to change the geometry of the p o l y m e r i z e d he parin molecule. T
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One o f t h e most i m p o r t a n t c u r r e n t a i m s f o r c h e m i c a l m o d i f i c a t i o n of the s t r u c t u r e of h e p a r i n i s the d i s s o c i a t i o n o f i t s a n t i t h r o m b i c and a n t i l i p e m i c a c t i v i t i e s . A d e c r e a s e i n t h e f i r s t a n d / o r an i n c r e a s e i n t h e s e c o n d has b e e n o b s e r v e d e a r l i e r by p a r t i a l h y d r o l y s i s (4^) , p e r i o d a t e o x i d a t i o n (6^), i r r a d i a t i o n (.5) o r m o d i f i c a t i o n o f t h e N - s u l f ο g r o u p s (2^^3) . A s i m i l a r d i s s o c i a t i o n o f t h e two a c t i v i t i e s i s r e p o r t e d t h r o u g h p o l y m e r i z a t i o n and c o p o l y m e r i z a t i o n methacry1ated heparin.
In Carbohydrate Sulfates; Schweiger, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
now of
CARBOHYDRATE SULFATES
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116
Figure 2. IJltracentrifligation in sucrose gradient 5-20% (Spinco, 41.000 RPM, 30 hr, 2°C); heparin (I) and methacrylated heparin polymer (II).
CHI CH - C I CO - HEPARIN
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In Carbohydrate Sulfates; Schweiger, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
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Heparin Derivatives
117
Table I shows thrombin-time and anti1ipemic a c t i v i t y of the methacrylated heparin polymer (II) and of the copolymer of methacrylated heparin with butyl methacrylate ( I I I ) , compared with the corresponding values obtained with heparin (I) in rats by intravenous i n j e c tion of 1 mg/kg doses of the compounds. A l l samples were dissolved in physiological sodium chloride s o l u t i o n . Polymer (II) shows an increased antithrombic a c t i v i t y of short duration and a decreased antilipemic a c t i v i t y . The butyl methacrylate copolymer (III), distinguished i t s e l f through a considerably increased antilipemic a c t i v i t y with s l i g h t l y decreased antithrombic a c t i v i t y . Similar results were obtained through intravenous administration of 2 mg/kg doses of the compounds in rabb i t s , as shown in Table I I . In subcutaneous administration (5 mg/kg) most of the polymers and copolymers show a h e p a r i n - l i k e a c t i v i ty, however, their antilipemic a c t i v i t y was decreased or considerably delayed. C i r c u l a r dichroism measurements 14 C i r c u l a r dichroism data of heparin (I), of methacrylated heparin polymer (II) and of the copolymer of methacrylated heparin with butyl methacrylate (III) in water solution are shown on Figure 4, measured with a "Dichrograph-II" JOUAN, P a r i s . In both polymers (II) and ( I I I ) , the (+) and (-) Cotton-effects are increased, when compared with heparin. However, for the polymer (II) the increase of the (-) Cotton-effect is greater (110 %) than the increase of the (+) Cotton-effect (69%). The reversed phenomenon is observed for the copolymer (III) . These changes in the Cotton-effects are due probably to a change in the geometry of the polymerized heparin molecule with methacrylate and/or with butyl methacrylate. This observation should also be taken in consideration to explain the dissociation of the antithrombic and antilipemic a c t i v i t i e s of the heparin polymers. Acknowledgement The authors are grateful to the Hoffmann-La Roche Co., Basle, Switzerland, for testing the heparin polymers . Abstract Heparin methacrylate is polymerized in 1,4-dioxane solution by heating with a z o d i i s o b u t y r o n i t r i l e as cata-
In Carbohydrate Sulfates; Schweiger, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
In Carbohydrate Sulfates; Schweiger, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
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Heparin
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COMPOUNDS
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Heparin
Derivatives
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+ 110%
ηm
Figure 4. Circular dichroism data in water (0.75 mg/mL) of heparin (I), methacryhted heparin polymer (II), and co-poly mer of methacryhted heparin with butyl methacrylate (III)
In Carbohydrate Sulfates; Schweiger, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
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120
carbohydrate sulfates
l y s t . Depending on the degree of polymerization, solu ble of gelatinous heparin preparations of high molecu lar weight are obtained, and separated on a column of Sephadex G-200 from unchanged heparin. A higher degree of polymerization of heparin methacrylate with alkyl methacrylates or v i n y l derivatives resulted in l i p o s o luble heparin preparations. Copolymerization with such r e t i c u l a n t s as divinylbenzene or Ν,N'-methylene-bisacrylamide changed completely the geometry of the molecule. C i r c u l a r dichroïsm measurements were used to follow the structural change of heparin methacrylate polymer and of i t s copolymer with butyl methacrylate. Some of the high molecular weight heparin preparations show a d i s s o c i a tion between the anticoagulant and antilipemic a c t i v i ties of heparin : the antithrombic a c t i v i t y decreased, while the antilipemic a c t i v i t y increased considerably or remained unchanged. Insoluble heparin preparations can be used for coating of surfaces. Literature cited 1. Foster A . B . and Huggard A.J., Adv.Carbohydr.Chem., 1955, 10, 335, Acad.Press, New-York. 2. Velluz L., Plotka C. and Nominé G . , Compt.Rend.Acad. Sci.Fr., 1958, 247, 2203. 3. Velluz L., Nominé G. and Mathieu J., Bull.Soc.Chim. Biol., 1959, 41, 415. 4. Nominé G . , Bucourt R. and Bertin D . , Bull.Soc.Chim. Fr., 1961, 561. 5. Adams S . S . , Heathcote B.V. and Macey P.E., J.Pharm. Pharmacol., 1961, 13, 240. 6. Inch T . D . , Ph.D.Thesis, Birmingham , 1963 . 7. Jacques L.B., Kavanagh L . W . , Mazurek M. and P e r l i n A . S . , Biochem.Biophys.Res.Comm., 1966, 24, 447. 8. P e r l i n A . S . , Ng Ying Kin N . M . K . , Bhattacharjee S.S. and Johnson L.F., Can.J.Chem., 1972, 50, 2437. 9. Jeanloz R.W., in "The Carbohydrates-Chemistry and Biochemistry" 2B, p.589, Ed. by Pigman W., Horton D. and Herp Α . , Acad.Press, New-York and London (1970). 10. Patat F. and E l i a s H., Naturwiss, 1959, 46, 322. 11. Laurent T.C., Arch.Biochem.Biophys., 1961, 92, 224. 12. Kiss J., in "Heparin-Chemistry and C l i n i c a l Usage" p . 3 , Ed.by Kakkar V . V . and Thomas D . P . , Acad.Press, New-York and London , 1976 . 13. Horner A . A . in "Heparin-Chemistry and C l i n i c a l Usa ge" p.37, Ed.by Kakkar V . V . and Thomas D . P . , Acad. Press, New-York and London, 1976 . 14. Stone A.L., in "Meth.Carbohydr.Chem.", 7, p.120, Ed.by Whistler R . L . and BeMiller J.N., Acad.Press, New-York and London , 1976 . Received February 6, 1978.
In Carbohydrate Sulfates; Schweiger, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
