Fritz, Hans

Jochum, Marianne

Siebeck, Matthias

Tschesche, H.

Welter, H. F.

English

Universität München: Elektronischen Publikationen

Proteinase inhibitor therapy of severe inflammation in pigs: First results with eglin, a potent inhibitor of granulocyte elastase and cathepsin G.

Open Access LMU

Proteinases in Inflammation and Tumor Invasion Review Articles including those from an International Conference Bielefeld, Federal Republic of Germany March 14-16,1985 Editor Harald Tschesche W DE G Walter de Gruyter • Berlin • New York 1986 Editor Harald Tschesche, Dr. rer. nat. Professor für Biochemie Lehrstuhl für Biochemie Fakultät für Chemie Universität Bielefeld D-4800 Bielefeld Federal Republic of Germany Universitäts-Bibliothek München Library of Congress Cataloging in Publication Data Proteinases in inflammation and tumor invasion Includes bibliographies and index. 1. Inflammation—Congresses. 2. Proteinase—Physiological effect—Congresses. Cancer invasiveness— Congresses. 4. Collagen diseases—Congresses. I. Tschesche, Harald. [DNLM: 1. Inflammation—congresses. 2. Neoplasm Invasiveness—congresses. 3. Peptide Peptidohydrolases—congresses. QZ 202 P967 1985] RB131.P77 1986 616'.0473 86-16708 ISBN 0-89925-216-8 (U.S.) CIP-Kurztitelaufnahme der Deutschen Bibliothek Proteinases in inflammation and tumor invasion : review articles including those from an international conference, Bielefeld, Fed. Republic of Germany, March 14 -16,1985 / ed. Harald Tschesche. - Berlin ; New York : de Gruyter, 1986. ISBN 3-11-010530-6 NE: Tschesche, Harald [Hrsg.] Copyright © 1986 by Walter de Gruyter & Co., Berlin 30. All rights reserved, including those of translation into foreign languages. No part of this book may be reproduced in any form - by photoprint, microfilm or any other means nor transmitted nor translated into a machine language without written permission from the publisher. Printing: Gerike GmbH, Berlin. - Binding: D. Mikolai, Berlin. - Printed in Germany. CONTENTS G r a n u l o c y t e P r o t e i n a s e s as M e d i a t o r s o f U n s p e c i f i c P r o t e o l y s i s i n I n f l a m m a t i o n : A R e v i e w H. F r i t z , M. J o c h u m , K.H. D u s w a l d , H. D i t t m e r , H. K o r t m a n n , S. Neumann, H. Lang 1 E f f e c t o f G r a n u l o c y t e E l a s t a s e on t h e M e t a b o l i s m o f P r o t e o g l y c a n s i n I n f l a m m a t o r y J o i n t D i s e a s e s K. K l e e s i e k , R. R e i n a r d s , H. G r e i l i n g 25 P r o t e i n a s e I n h i b i t o r T h e r a p y o f S e v e r e I n f l a m m a t i o n i n P i g s : F i r s t R e s u l t s w i t h E g ! i n , a P o t e n t I n h i b i t o r o f G r a n u l o c y t e E l a s t a s e and C a t h e p s i n G M. J o c h u m , H.F. W e l t e r , M. S i e b e c k , H. F r i t z 53 Mast C e l l s and M a t r i x D e g r a d a t i o n i n t h e R h e u m a t o i d J o i n t D.E. W o o l l e y , J.R. Y o f f e , J.M. E v a n s o n 6 1 Human C a t h e p s i n B, H and L and t h e i r E n d o g e n o u s P r o t e i n I n h i b i t o r s : S t r u c t u r e , F u n c t i o n and B i o l o g i c a l R o l e V. T u r k , J . B r z i n , M. K o p i t a r , M. K o t n i k , B. L e n a r c i c , T. P o p o v i c , A. R i t o n j a , M. T r s t e n j a k , B. Rozman, W. M a c h l e i d t 77 C a t h e p s i n B: A P r o t e i n a s e L i n k e d t o M e t a s t a s i s ? B. F. S l o a n e , R.E. R y a n , J . R o z h i n , T.T. L a h , J.D. C r i s s m a n , K. V. Honn 93 The C o l l a g e n s - M o l e c u l a r and M a c r o m o l e c u l a r S t r u c t u r e s K. Kühn 107 P r o t e o l y t i c M o d i f i c a t i o n s o f T y p e V I C o l l a g e n J . R a u t e r b e r g , R. J a n d e r , D. T r o y e r 145 D e g r a d a t i o n o f S p e c i f i c C o l l a g e n s by M a c r o p h a g e s and N e u t r o p h i 1 s C. L. M a i n a r d i , M.S. H i b b s , K.A. H a s t y 161 C o n n e c t i v e T i s s u e M e t a l 1 o p r o t e i n a s e s and M a t r i x D e g r a d a t i o n G. M u r p h y , J . G a v r i l o v i c , Ch. M c A l p i n e 173 VIII T i s s u e I n h i b i t o r o f M e t a 1 1 o p r o t e i n a s e s T.E. C a w s t o n , E. M e r c e r , C. Mooney, J.K. W r i a h t 189 C o l l a g e n D e g r a d a t i o n i n P e r i o d o n t a l D i s e a s e s V . - J . U i t t o 211 I n t e r s t i t i a l C o l l a g e n a s e , G e l a t i n a s e and a S p e c i f i c T y p e I V / V ( B a s e m e n t Membrane) C o l l a g e n D e g r a d i n q P r o t e i n a s e f r o m Human L e u k o c y t e s H. T s c h e s c h e , J . F e d r o w i t z , U. K o h n e r t , H.W. M a c a r t n e y , J . M i c h a e l i s , K. K ü h n , H. Wiedemann 2 2 5 The R o l e o f C e l l M o t i l i t y i n Tumor I n v a s i o n G. H a e m m e r l i 2 4 5 P e r i t u m o r a l E x t r a c e l l u l a r M a t r i x D e g r a d a t i o n i n t h e L o o s e C o n n e c t i v e T i s s u e o f t h e M e s e n t e r y W. M ü l 1 e r - G l a u s e r 2 6 1 P l a s m i n o g e n A c t i v a t o r s and Tumor I n v a s i o n L. S k r i v e r , L.R. L u n d , P.A. A n d r e a s e n , P. K r i s t e n s e n , K. Dan0 2 8 1 The R o l e o f P r o t e i n a s e I n h i b i t o r s i n C e l l u l a r I n v a s i v e n e s s : P u r i f i c a t i o n and C h a r a c t e r i z a t i o n o f a S e r i n e P r o t e i n a s e I n h i b i t o r f r o m R a t C h o n d r o s a r c o m a H. L e v y , G. F e i n s t e i n 3 0 5 C h a r a c t e r i z a t i o n o f a C o l l a g e n a s e I n h i b i t o r f r o m Human A m n i o t i c F l u i d w h i c h I n h i b i t s Tumor C e l l I n v a s i o n H. L e v y , P. M i g n a t t i , D.B. R i f k i n 3 2 1 D e v e l o p m e n t o f a D i r e c t M e a s u r e m e n t A s s a y f o r C o l l a g e n a s e a g a i n s t Type I and Type IV C o l l a g e n s i n T i s s u e H o m o g e n a t e and i t s A p p l i c a t i o n i n Stomach and Lung C a n c e r s K. K u b o c h i , K. Maruyama, I . O k a z a k i 3 3 7 M e t a s t a t i c A b i l i t y o f L e w i s Lung C a r c i n o m a C e l l s and E x t r a c e l l u l a r P r o t e o l y s i s . I n t e r a c t i o n s b e t w e e n Tumour C e l l s and H o s t M a c r o p h a g e s i n t h e D e g r a d a t i o n o f Type I and T y p e IV C o l l a g e n G. V a e s , N. H e n r y , A.L. van L a m s w e e r d e , Y. E e c k h o u t 3 5 7 IX A T-Lymphoma D e r i v e d P r o t e i n a s e , S y n e r g i z i n g w i t h an E n d o g l y c o s i d a s e i n t h e D e g r a d a t i o n o f S u l p h a t e d P r o -t e o g l y c a n s i n S u b e n d o t h e l i a l E x t r a c e l l u l a r M a t r i x M.D. K r a m e r , V. S c h i r r m a c h e r , M. B a r - N e r , I . V l o d a v s k y 373 P r o t e o l y t i c A c t i v i t y i n C u l t u r e d Human Lung C a n c e r C e l l s O.L. S c h o e n b e r q e r , H. S c h w ö b e l , W. E b e r t , P. A u l e n b a c h e r , H.O. W e r l i n g , N. P a w e l e t z 395 L i s t o f A u t h o r s 409 S u b j e c t I ndex 411 PROTEINASE INHIBITOR THERAPY OF SEVERE INFLAMMATION IN PIGS: FIRST RESULTS WITH EGLIN, A POTENT INHIBITOR OF GRANULOCYTE ELASTASE AND CATHEPSIN G M. Jochum, H. F. We l t e r * , M. Siebeck* and H. F r i t z Dept. o f C l i n i c a l C h e m i s t r y and C l i n i c a l B i o c h e m i s t r y i n t h e *Surgery C l i n i c C i t y , U n i v e r s i t y of Munich, Munich, FRG I n t r o d u c t i o n Lysosomal enzymes r e l e a s e d d u r i n g i n f l a m m a t i o n may enhance the i n f l a m m a t o r y response by s p e c i f i c a c t i v a t i o n of humoral systems ( c l o t t i n g , f i b r i n o l y s i s , complement, k a l l i k r e i n / k i n i n system) o r by u n s p e c i f i c d e g r a d a t i o n and o x i d a t i o n o f plasma f a c t o r s and t i s s u e p r o t e i n s . R e c e n t l y , the pa t h o b i o c h e m i c a l e f f e c t s of enzymes of the a z u r o p h i l i c and s p e c i f i c g r a n u l e s o f polymorpho-n u c l e a r g r a n u l o c y t e s ( e l a s t a s e , c a t h e p s i n G, collagenase, ca-t h e p s i n B, lysozyme, myeloperoxidase etc.) were i n v e s t i g a t e d i n more d e t a i l ( f o r r e v i e w o f l i t e r a t u r e see ( 1 ) ) . N o r m a l l y , t h e p r o t e o l y t i c a c t i o n o f the lysosomal p r o t e i n a s e s i s balanced by p r o t e i n a s e i n h i b i t o r s p r e s e n t i n plasma or o t h e r body f l u i d s . D uring severe i n f l a m m a t i o n , however, these p r o t e i n a s e s may be l i b e r a t e d i n amounts exceeding the n a t u r a l i n h i b i t o r l e v e l s so t h a t d e s t r u c t i v e processes can occur e i t h e r l o c a l l y or system-i c a l l y . Evidence c o u l d be presented t h a t l e u k o c y t i c e l a s t a s e i s indeed r e l e a s e d d u r i n g s e p t i c e m i a and o t h e r i n f l a m m a t o r y pro-cesses ( 1 ) . The c o r r e l a t i o n o b s e r v e d between t h e r e l e a s e o f t h i s p r o t e i n a s e and the consumption of plasma f a c t o r s i n d i c a t e s t h a t l y s o s o m a l enzymes may s i g n i f i c a n t l y c o n t r i b u t e t o t h e consumption of plasma f a c t o r s by u n s p e c i f i c p r o t e o l y s i s . Fur-t h e r o n , a d m i n i s t r a t i o n of an exogenous i n h i b i t o r of lysosomal p r o t e i n a s e s i n an e x p e r i m e n t a l a n i m a l model ( e n d o t o x e m i a ) caused a s i g n i f i c a n t r e d u c t i o n o f t h e c o n s u m p t i o n o f plasma f a c t o r s ( 2 ) . Proteinases in Inflammation and Tumor Invasion © 1986 Walter de Gruyter & Co., Berlin • New York - Printed in Germany 54 E g l i n — a p r o t e i n a s e i n h i b i t o r which o r i g i n a l l y has been i s o -l a t e d from t h e l e e c h Hirudo m e d i c i n a l i s — i s a p o t e n t i n h i b i t o r of the g r a n u l o c y t e p r o t e i n a s e s e l a s t a s e and c a t h e p s i n G ( 3 ) . With regard t o t h e r o l e o f these p r o t e i n a s e s i n i n f l a m m a t o r y s t a t e s l i k e s e p t i c shock and trauma e v a l u a t i o n o f a t h e r a p e u t i c approach by p r o t e i n a s e i n h i b i t i o n seemed t o be d e s i r a b l e . Since e g l i n i s now a v a i l a b l e i n s u f f i c i e n t q u a n t i t i e s (Fa. Ciba Geigy/Basel) by g e n e t i c e n g i n e e r i n g ( 4 ) e x p e r i m e n t a l animal s t u d i e s were performed i n o r d e r t o e l u c i d a t e the i n h i b i t o r y potency of t h i s i n h i b i t o r i n sepsis and s e p t i c shock. Methods Septicemia was induced i n p i g s by i . v . i n f u s i o n of E . c o l i f o r 2 hours ( 5 ) . Three d i f f e r e n t p r e p a r a t i o n s of e g l i n were i n -t r a v e n o u s l y a d m i n i s t e r e d f o r 4 hours: cloned e g l i n c ( b a t c h I or batch I I ) o r e g l i n b ( i s o l a t e d from the l e e c h ) . Animals (16.5 - 23 kg) were d i v i d e d i n t o 7 groups (Tab. 1 ) . Hematologic, hemodynamic, r e s p i r a t o r y , h i s t o l o g i c a l and bioche-m i c a l parameters were measured by r o u t i n e methods; e x t r a v a s c u -l a r lung water (EVLW) was e v a l u a t e d u s i n g the thermo-dye-technique ( 6 ) . E g l i n l e v e l s i n plasma and u r i n e were e s t i m a t e d by r a d i a l i m m u n o d i f f u s i o n a p p l y i n g s p e c i f i c a n t i b o d i e s ( 7 ) . Results Pharmacokinetic d a t a : E g l i n was e x c r e t e d i n u r i n e t o 75-95 % w i t h i n 12 h i n animals w i t h normal kidney f u n c t i o n . Animals w i t h r e n a l i n s u f f i c i e n c y due t o s e p t i c e m i a showed a s i g n i f i c a n t accumulation of e g l i n i n the c i r c u l a t i o n i n d i c a t i n g the p r e -v a i l i n g r e n a l e l i m i n a t i o n of t h i s i n h i b i t o r . 55 S u r v i v a l time (Tab. 1 ) : Animals of group I I and I I I d i e d w i t h i n 7.2 _+ 3.0 h and 5.3 _+ 1.5 h, r e s p e c t i v e l y . One p i g o f each of these two groups s u r v i v e d 20 hours. Four animals o f group IV s u r v i v e d an e x p e r i m e n t a l p e r i o d o f 30 h d e s p i t e f e v e r due t o t r a c e s o f e n d o t o x i n p r e s e n t i n batch I o f t h e c l o n e d e g l i n c. The mean s u r v i v a l time i n group V was 19.1 + 6 h. I n group VI and V I I e n d o t o x i n - f r e e e g l i n was used and a remarkable p r o l o n -g a t i o n o f s u r v i v a l time was observed i n s p i t e o f t y p i c a l hema-t o l o g i c a l , hemodynamic and r e s p i r a t o r y d i s o r d e r s due t o s e p t i -cemia . Group B a c t e r i a dosage ( E . c o l i c e l l s ) E g l i n dosage (mg/kg x h) S u r v i v a l time No n i n 12 ml phys. NaCl/h f o r 2 h i n 50 ml phys.NaCl/h f o r 4 h h I 10 - - 30 I I 8 3 x 10 9 - 7.2 + 3.0 I I I 9 3 x 1 0 1 0 - 5.3 + 1 .5 IV 5 - 3.85 ( E g l i n c; charge I ) 3 0 ( 4 x ) ; 6. 5(1x) V 5 3 x 1 0 9 3.85 ( E g l i n c; charge I ) 19.1 + 9.6 VI 2 3 x 10 9 4.55 ( E g l i n b) 3 0 ( 1 x ) ; 4. 5(1x) V I I 7 3 x 1 0 1 0 3.85 ( E g l i n c; charge I I ) 30(5x);15. 2(2x) Table 1: S u r v i v a l time o f p i g s i n e x p e r i m e n t a l bacteremia as a f u n c t i o n of b a c t e r i a ( E . c o l i ) and i n h i b i t o r ( E g l i n ) dosages. 56 Lung d i s o r d e r s : A s i g n i f i c a n t i n c r e a s e i n e x t r a v a s c u l a r lung water was measured i n u n t r e a t e d s e p t i c a n i m a l s , whereas d u r i n g and a f t e r e g l i n - a d m i n i s t r a t i o n o n l y a minor r i s e i n EVLW oc-cured ( F i g , 1 ) . Moreover, i n c o n t r a s t t o u n t r e a t e d s e p t i c p i g s e g l i n - t r e a t e d animals showed o n l y modest m o r p h o l o g i c a l lung a l t e r a t i o n s ( i n t e r s t i t i a l edema). •= i t 7 • 7 4 3 2 I I I I I » 1 * * ? ? ? 1 ? ? ? ? l ? ? ? 7 7 l • -—I , — c • t C C t t t C t t t * • L t t t t C t t t 1 J » • - « F i g . 1: Mean e x t r a v a s c u l a r lung water l e v e l s (EVLW) i n s e p t i c p i g s w i t h o u t (a) and w i t h i n h i b i t o r (b) t r e a t m e n t . C l o t t i n g f a c t o r s : I n the e g l i n - t r e a t e d groups I V - V I I f a c t o r X I I I and a n t i t h r o m b i n I I I l e v e l s decreased t r a n s i e n t l y ( F i g . 2b, 3b), whereas i n the u n t r e a t e d bacteremic animals (group I I and I I I ) a s i g n i f i c a n t consumption of f a c t o r X I I I ( F i g . 2a) and a n t i t h r o m b i n I I I ( F i g . 3a) was measured u n t i l death. 57 a Bacteremia E.coii 3 • i m 1 s L * S 1 7 4 4 2 4 • 1 • I # » » Bacteremia * Eglin E.cli 3 i i m - Efll. 3.95 t ^ t U T-H, I TT" : S 5 $ 5 S 5 5 5 • 5 • 5 • $ 4 J * * " * *Ttat (Urn) F i g . 2: Mean plasma l e v e l s of f a c t o r X I I I i n s e p t i c p i g s w i t h -out (a) and w i t h i n h i b i t o r (b) t r e a t m e n t . a Bacteremia E.e.n 31 i m Bactereiia - Eglin E.coli 3 i i m - Uli» 3J5 wA, U F i g . 3: Mean plasma l e v e l s o f a n t i t h r o m b i n I I I i n s e p t i c p i g s w i t h o u t (a) and w i t h i n h i b i t o r (b) t r e a t m e n t . 58 Conclusions Treatment of e x p e r i m e n t a l s e p t i c e m i a i n pi g s e x c l u s i v e l y by e g l i n r e v e a l e d a c o n v i n c i n g p r o t e c t i v e e f f e c t of t h i s p r o t e i n -ase i n h i b i t o r on lun g t i s s u e and consumption o f plasma p r o t e i n s probably due t o i n h i b i t i o n o f u n s p e c i f i c p r o t e o l y s i s . Since, however, some severe organ f a i l u r e s (e.g. r e n a l i n s u f f i c i e n c y ) due t o septicemia c o u l d not be overcome, a c o m b i n a t i o n o f the i n h i b i t o r a d m i n i s t r a t i o n w i t h an approved i n t e n s i v e care t h e r a -py (e.g. catecholamines, r e s p i r a t o r y t h e r a p y ) i s needed f o r an o p t i m a l outcome. Acknowledgement We are very g r a t e f u l t o Drs. H.-P. S c h n e b l i , W. Märkl and M. L i e r s c h (Fa. Ciba Geigy, Basel, S w i t z e r l a n d ) f o r s u p p l y i n g us w i t h cloned E g l i n c (^-N 0^-Acetyl-Eglin c, CGP 32968) as w e l l as t o Dr. R. Maschler and Prof. Dr. Dr. E. Fink (Fa. P l a n t o r g a n , Bad Zwischenahn, FRG) f o r E g l i n b from Hirudo m e d i c i n a i i s . We a l s o thank Drs. U. Seemüller and W. Müller-Esterl f o r making a v a i l a b l e anti-serum t o E g l i n c. 59 References 1 F r i t z , H. , M. Jochum, K.-H. Duswald, H. D i t t m e r , H. K o r t -mann, S. 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A large fragment approach to DNA synthesis: total synthesis of a gene for the protease inhibitor eglin c from the leech Hirudo medicinalis and its expression in E.coli.

Bedside measurement of lung water.

Clotting and other plasma factors in experimental endotoxemia: inhibition of degradation by exogenous proteinase inhibitors.

Granulocyte proteinases as mediators of unspecific proteolysis in inflammation. A review. In:

Immunochemical quantitation of antigens by single radial immunodiffusion.

Isolation and characterization of a low molecular weight inhibitor (of chymotrypsin and human granuloytic elastase and cathepsin G) from leeches.

Study on the treatment of shock lung with superoxide dismutase (SOD) and C1-inactivator (C1-INA).

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Proteinase inhibitor therapy of severe inflammation in pigs: First results with eglin, a potent inhibitor of granulocyte elastase and cathepsin G.

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