bk-1983-0209.ch022

22 O s m i u m C a r b o h y d r a t e P o l y m e r s as P o t e n t i a l

Downloaded by UNIV OF PITTSBURGH on May 3, 2015 | http://pubs.acs.org Publication Date: January 26, 1983 | doi: 10.1021/bk-1983-0209.ch022

Antiarthritic D r u g s C. C. HINCKLEY and J. N. BEMILLER—Southern Illinois University at Carbondale, Department of Chemistry and Biochemistry, Carbondale, IL 62901 L. E. STRACK—Southern Illinois University at Carbondale, Department of Animal Industries, Carbondale, IL 62901 L. D. RUSSELL—Southern Illinois University at Carbondale, Department of Physiology, Carbondale, IL 62901 Osmium carbohydrate polymers, termed osmarins, have been investigated as potential antiarthritic agents. They are prepared in three steps from OsO , and have been characterized by elemental analysis, gel filtration, gel electrophoresis, solution density and viscosity measurements, and ultracentrifugation. The polymers are of variable composition dependent upon the details of preparation. They are polydisperse anionic polyelectrolytes, and preparations containing 30%-40% osmium have average molecular weights in the range, 50-100K daltons. The polymers react in strong base with the oxygen species O , O , and H O . They form complexes in solution with proteins. In antiarthritic application, dilute solutions of the compounds are injected directly into the synovial spaces of effected joints. Though some evidence of microscopic toxicity has been found, the materials have very low toxicity. In mice, IP injection at dose levels of 1g/Kg body weight produce no mortality. In pigs and rabbits, osmarins of high molecular weight stain the joint capsules and articular surfaces of injected joints irreversibly. In limited experimental treatments of arthritic dogs, improvements have been noted in five of six cases. It is proposed that, in vivo, osmarins may react with superoxide ion and remove this damaging species. 4

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New osmium compounds may prove to be antiinflammatory agents useful in the treatment of some forms of arthritis. The materials are osmium-carbohydrate polymers (1), termed osmarins. These compounds bind irreversibly with living tissue and exhibit other properties which suggest their use. 0097-6156/83/0209-0421$06.00/0 © 1983 American Chemical Society In Platinum, Gold, and Other Metal Chemotherapeutic Agents; Lippard, S.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.


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There i s a long standing connection between a r t h r i t i s treatment and osmium ( 2 ) . Osmium t e t r o x i d e has been used on a l i m i t e d b a s i s f o r the treatment of a r t h r i t i s i n humans, p r i n c i p a l l y i n Europe, f o r about 30 years. Osmium t e t r o x i d e treatment i s not used i n the United States and i s c o n t r o v e r s i a l everywhere. In theory, the treatment makes use of the t o x i c i t y of osmium t e t r o x i d e to achieve a "chemical synovectomy". D e t r a c t o r s b e l i e v e t h a t the damage t o j o i n t t i s s u e s inherent i n the procedure exacts a p r i c e too high f o r the method to be g e n e r a l l y acceptable. S t i l l , the s m a l l but p e r s i s t e n t l i t e r a t u r e which has developed over the years provides a growing l i s t of apparent successes. In 1976 a group of researchers i n S w i t z e r l a n d reported a study of over seventy people who had been helped by the t r e a t ment ( 3 ) . They found t h a t there were osmium c o n t a i n i n g d e p o s i t s i n the j o i n t s of these people long a f t e r the osmium t e t r o x i d e treatment. They i n c l u d e d i n t h e i r paper a suggestion, upon which our work i s p a r t l y based, t h a t the osmium c o n t a i n i n g deposits may c o n t r i b u t e to the long term e f f i c a c y of the procedure . Recent work provides s u p p o r t _ f o r t h e i r suggestion ( 4 ) . Involvement of superoxide i o n , 0^ , i n inflammation i s w e l l e s t a b l i s h e d (5,6). In a r t h r i t i s , superoxide may be present i n an e f f e c t e d j o i n t as p a r t of a response to the disease ( 4 ) . There i t may a t t a c k the s y n o v i a l f l u i d , d e s t r o y i n g i t s c a p a c i t y to l u b r i c a t e the j o i n t surfaces (7,8). Metal complexes have been shown to c a t a l y t i c a l l y remove superoxide ( 9 ) , and osmium c o n t a i n i n g d e p o s i t s could perform a s i m i l a r f u n c t i o n . Osmium e x h i b i t s many of the c h a r a c t e r i s t i c s b e l i e v e d necessary f o r these c a t a l y t i c p r o p e r t i e s . The f a c t t h a t the enzyme superoxide dismutase has been s u c c e s s f u l l y used to t r e a t a r t h r i t i s i n both animals (10) and humans (11,12) i s f u r t h e r support f o r the suggestion. In our proposed a p p l i c a t i o n , s o l u t i o n s of osmarins are t o be i n j e c t e d d i r e c t l y i n t o the s y n o v i a l spaces of a r t h r i t i c j o i n t s . The aim i s to provide osmium deposits s i m i l a r t o those which Bousinna, e t . a l . , (3) have suggested may be b e n e f i c i a l , w h i l e at the same time, a v o i d i n g the damage t h a t accompanies OsO^ i n j e c t i o n s . In t h i s paper, we f i r s t present the p r e p a r a t i o n and c h a r a c t e r i z a t i o n of osmium-carbohydrate polymers. This d i s c u s s i o n i s f o l l o w e d by a review of our s t u d i e s i n v o l v i n g animals. Mice have been used f o r t o x i c i t y s c r e e n i n g , and we have examined the e f f e c t of osmarins upon the t i s s u e s of the s y n o v i a l space i n both p i g s and r a b b i t s . F i n a l l y , we r e p o r t the r e s u l t s of a s m a l l number of experimental treatments of a r t h r i t i c dogs. Our f i n d i n g s are t h a t osmarins of high average molecular weight appear t o have the p r o p e r t i e s we seek, t h a t i s , they have minimal t o x i c i t y , no evident p e r s i s t e n t s i d e e f f e c t s , are r e t a i n e d f o r long periods w i t h i n the j o i n t , and are e f f e c t i v e

In Platinum, Gold, and Other Metal Chemotherapeutic Agents; Lippard, S.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

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i n some cases i n r e s t o r i n g freedom of movement of a r t h r i t i c j o i n t s and a l l o w i n g recovery of the j o i n t s u r f a c e s . Comments concerning apparent e f f i c a c y are not presented as a s s e r t i o n s o f proven f a c t , but r a t h e r as i n d i c a t i o n s which prompt our con t i n u i n g study.


Osmarins P r e p a r a t i o n and c h a r a c t e r i z a t i o n . Osmarins are prepared i n three s t e p s , the f i r s t of which begins w i t h osmium t e t r o x i d e . OsO^ (0.5 g ) , d i s s o l v e d i n a s m a l l amount (15 mL) of methanol c o n t a i n i n g 0.1M KOH, r e a c t s w i t h the a l c o h o l t o produce d i potassium tetramethylosmate(VI), K [ 0 s ( 0 C H ^ ) ^ 0 ] , which 2

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Elemental compositions f o r s e v e r a l osmarin pre parations. Preparation 1 2 3 4 5

% Os 9.7 13.6 15.0 23.0 35.0

% c 26.7 26.0 14.5 32.4 14.6

% H 4.5 4.3 3.6 5.6 5.8

% κ 11.7 10.0 0.7 6.9 4.9

p r e c i p i t a t e s as a green s o l i d (13). When t h i s product i s d i s s o l v e d i n a c e t i c a c i d (100 mL), blue potassium t r i a c e t a t o d i oxoosmate(VI) i s formed. Glucose (1.0 g ) , d i s s o l v e d f i r s t i n a small amount of water (20 ml) and then mixed w i t h a l a r g e r q u a n t i t y of a c e t i c a c i d (100 mL) i s added t o the blue s o l u t i o n and allowed t o r e a c t overnight a t room temperature t o form the osmarin. P u r i f i c a t i o n i s e f f e c t e d by g e l f i l t r a t i o n of an aqueous s o l u t i o n through Sephadex G-25 a f t e r the a c e t i c a c i d has been removed by evaporation under reduced pressure. Osmarins of w i d e l y d i f f e r i n g composition (Table I ) have been prepared by v a r y i n g the r e a c t i o n temperature, the concen t r a t i o n s of osmium and carbohydrate i n the p r e p a r a t i v e m i x t u r e , the nature of the carbohydrate, and the time of r e a c t i o n . F o r i n s t a n c e , when the q u a n t i t i e s of r e a c t a n t s i n d i c a t e d above are allowed t o r e a c t a t room temperature f o r twenty-four hours, the f i n a l p r e p a r a t i o n w i l l c o n t a i n 30 t o 40% osmium. Longer r e a c t i o n times (1 week) w i t h m i l d h e a t i n g on a steam bath y i e l d prepara t i o n s having 20 t o 30% osmiums. I n c r e a s i n g the q u a n t i t y of glucose (2-3 g) and h e a t i n g y i e l d s p r e p a r a t i o n s having l e s s than 20% osmium. The p r e p a r a t i o n s are brown or b l a c k depending upon t h e i r composition. P r e p a r a t i o n s c o n t a i n i n g l e s s than 20% osmium are brown, those c o n t a i n i n g more than 20% are b l a c k . The v e r y i n t e n s e c o l o r s are the consequence of broad a b s o r p t i o n



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throughout the v i s i b l e r e g i o n . E x t i n c t i o n c o e f f i c i e n t s are on the order of 2000 L/cm. mole o f Os. These values compare f a v o r a b l y t o those found f o r some charge t r a n s f e r complexes (14) . The r e l a t i v e l y h i g h percentages of carbon i n the polymers and t h e i r h i g h aqueous s o l u b i l i t y suggest the presence of bound carbohydrate. I n f r a r e d s p e c t r a o f - t h e polymers e x h i b i t broad bands i n the^regions 1610-1550 cm" , 1400-1300 cm" , and 1200-950 cm , c o n s i s t e n t w i t h the presence of gluconate and glucose. Permanganate t i t r a t i o n s of the polymers i n a c i d s o l u t i o n i n d i c a t e an o x i d a t i o n s t a t e f o r osmium of +4, but do not e l i m i n a t e the p o s s i b i l i t y of mixed o x i d a t i o n s t a t e s . These f a c t o r s are c o n s i s t e n t w i t h the e m p i r i c a l formula: K 0 s 0 ( g l u c o s e ) ( g l u c o n a t e ) O^O) . This formula embodies an i n t e r p r e t a t i o n of the materYals as glucose and/or gluconate s o l u b i l i z e d osmium d i o x i d e . In t h i s i n t e r p r e t a t i o n , d i r e c t Os-Os bonds are not assumed. B r i d g i n g between osmium ions through oxo and/or carbohydrate groups i s thought t o be most l i k e l y . R e c e n t l y , a new c l a s s of osmium(IV) complexes con t a i n i n g both oxide and c a r b o x y l a t e b r i d g e s has been reported (15) . The compounds, 0 β ( μ - 0 ) ( M - O ^ C H ^ X ^ P R ^ where X=Cl,Br, e x h i b i t b r i d g i n g between the osmium atoms of tne k i n d suggested f o r osmarins. Gel f i l t r a t i o n has i n d i c a t e d t h a t the components have r e l a t i v e l y l a r g e molecular volumes. They e l u t e as broad bands at or near the v o i d volume of a Sepharose £-B column. T h i s g e l excludes g l o b u l a r p r o t e i n s which exceed 10 d a l t o n s i n molecular weight. Since the g e l i s not c a l i b r a t e d f o r osmarins, g e l f i l t r a t i o n does not provide estimates of molecular weight. In g e l e l e c t r o p h o r e s i s , the p r e p a r a t i o n s migrate as s i n g l e broad bands toward the p o s i t i v e e l e c t r o d e . P l o t s of m i g r a t i o n d i s t a n c e versus g e l c o n c e n t r a t i o n (Ferguson p l o t s ) , measured f o r the same c o n d i t i o n s of temperature and e l e c t r i c f i e l d , show t h a t molecules i n the t r a i l i n g edge of the bands are l a r g e r than those i n the l e a d i n g edge. T h i s demonstrates t h a t the p r e p a r a t i o n s are p o l y d i s p e r s e , an i n d i c a t i o n which i s confirmed i n sedimentation v e l o c i t y experiments. Osmarins are extremely s o l u b l e ; aqueous s o l u t i o n s con t a i n i n g up to 10% osmarin were used f o r s o l u t i o n d e n s i t y , p a r t i a l s p e c i f i c volume, and v i s c o s i t y measurements. The e f f e c t of the d i s s o l v e d polymer upon the v i s c o s i t y o f the l i q u i d i s s l i g h t , even a t the extreme c o n c e n t r a t i o n s t u d i e d , i n d i c a t i n g t h a t , i n aqueous s o l u t i o n , the polymers are g e n e r a l l y s p h e r i c a l i n shape. Sedimentation v e l o c i t y experiments, supplemented by p a r t i a l s p e c i f i c volume and v i s c o s i t y measurements, p r o v i d e estimates of molecular weights ( F i g u r e 1). M o l e c u l a r weights may v a r y from 10,000 to 100,000 d a l t o n s or more i n a s i n g l e p r e p a r a t i o n . There i s a general r e l a t i o n s h i p between the composition of the osmarin p r e p a r a t i o n and the range of molecular weights of the components of the mixture. The molecular weights of osmarin u

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Sedimentation profiles for several osmarins plotted as absorbance vs. sedimentation coefficient.




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p r e p a r a t i o n s c o n t a i n i n g as l i t t l e as 15% osmium average 10K d a l t o n s , w h i l e the average molecular weight of those c o n t a i n i n g 35% i s 80K daltons or more. The combined r e s u l t s a l l o w us to draw the o u t l i n e s of s t r u c t u r e of b l a c k osmarins. The p r e p a r a t i o n s are p o l y d i p e r s e mixtures of osmium-carbohydrate polymers. The number of osmarin atoms v a r i e s from 10 to 50 per molecule most commonly, w i t h some having as many as 100 or more. W i t h i n the molecules, osmium atoms are l i n k e d together w i t h oxygen or carbohydrate bridges (gluconate or g l u c o s e ) . Osmium e x h i b i t s an average +4 o x i d a t i o n s t a t e , and the polymers are a n i o n i c . In s o l u t i o n , the molecules are d i s t o r t e d spheres. A suggested o r g a n i z a t i o n a l geometry i s t h a t of c o i l e d chains of v a r y i n g l e n g t h . The chains may be branched and/or c r o s s l i n k e d , but not l i n e a r l y extended. Chemical P r o p e r t i e s . Chemical p r o p e r t i e s of the polymers are c o n s i s t e n t w i t h those expected f o r the c o n s t i t u e n t s of the m a t e r i a l s , and w i t h those expected of a n i o n i c polymers. Osmarins are o x i d i z e d by a wide v a r i e t y of common o x i d i z i n g agents i n c l u d i n g potassium permanganate and e e r i e ammonium n i t r a t e . In these r e a c t i o n s , both the osmium and the bound carbohydrate are o x i d i z e d . Studies of osmarin-oxygen species chemistry are i n progress and w i l l be reported l a t e r . P r e l i m i n a r y experiments i n d i c a t e an extensive oxygen chemistry. The polymers r e a c t i n v i t r o w i t h the oxygen species H^O^, 0^ , and 0^, i n the presence of strong base. The r e a c t i o n w i t h hydrogen peroxide i s accompanied by vigorous gas e v o l u t i o n i n d i c a t i n g t h a t the peroxide has decomposed. Osmium i s o x i d i z e d to 0s0^ which may be removed by e x t r a c t i o n i n t o chloroform. In the r e a c t i o n of osmarins w i t h K 0 i n DMSO, no gas i s evolved i n s p i t e of the presence of water i n the s o l u t i o n . Gas i s evolved when osmarins are not present. F i g u r e 2 i s a p l o t of absorbance a t 480 αιμ as a f u n c t i o n of time, f o r the r e a c t i o n of an osmarin w i t h the oxygen i n a i r . The r e a c t i o n w i t h oxygen i s complicated by the f a c t t h a t both the osmium i n the compound and the attached carbohydrate r e a c t w i t h the oxygen. Product mixtures are complex, and c o n t a i n s e v e r a l osmium c o n t a i n i n g components as w e l l as a carbohydrate mix t h a t i n c l u d e s hexoses and evidence f o r pentoses. Osmarins r e a c t w i t h p r o t e i n s . Evidence f o r r e a c t i o n i s the formation of p r e c i p i t a t e s and the presence of p r o t e i n dependent bands i n e l e c t r o p h o r e s i s . P r o t e i n s s t u d i e d are albumin, cytochrome C, myglobin, and lysozyme. In these e x p e r i ments, s o l u t i o n s c o n t a i n i n g an osmarin p r e p a r a t i o n and the p r o t e i n are mixed and allowed t o stand f o r some time. Reaction i s not immediate, and r e a c t i o n time i s dependent upon the osmium content of the osmarin p r e p a r a t i o n . When the osmium percentage i s h i g h (25%), p r e c i p i t a t e s form i n two or three 2



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TIME (min.) Figure 2. Logarithms of absorbance (480 nm) are plotted vs. time for basic solution of an osmarin reacting with air. Key: I, no pretreatment; II, helium bubbled through prior to beginning reaction by adding strong base; and III, O gas pre treatment. t


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hours, i f the osmium percentage i s low (15%) p r e c i p i t a t e s may not form a t a l l . I n every case, however, e l e c t r o p h o r e s i s demonstrates the f o r m a t i o n of complexes which appear as d i s t i n c t and narrow bands, present o n l y when p r o t e i n i s p r e s e n t . P r o t e i n b i n d i n g i s a reasonable e x p e c t a t i o n f o r osmarins. I t i s c o n s i s t e n t w i t h the known s t r u c t u r e of the polymers and the p r o p e r t i e s of osmium ( I V ) . Osmarins are p o l y a n i o n i c , a s t r u c t u r a l f e a t u r e which p r o v i d e s the means t o form m u l t i p l e i o n i c l i n k a g e s w i t h p r o t e i n s , and a d d i t i o n a l l y , Os(IV) i s known to form complexes w i t h p r o t e i n sidegroups (16).


Animal s t u d i e s T o x i c i t y i n mice. Osmarin p r e p a r a t i o n s t o be used i n animal experiments are screened f o r acute t o x i c i t y . IP i n j e c t i o n s i n mice a t dose l e v e l s of l g osmarin/kg body weight produce no m o r t a l i t y . As y e t , no l e t h a l dose has been d e t e r mined. At the 1 g/kg.b.w. dose l e v e l the s k i n and eye c o l o r of the mice are darkened. No other e f f e c t s are observed. A f t e r about an hour, the u r i n e of i n j e c t e d mice i s darkened by osmarin, but s i g n i f i c a n t amounts are r e t a i n e d i n the animal. Gross examination of both l i v e r and kidney shows t h a t they are s l i g h t l y darkened. I n m i c r o s c o p i c examination of these t i s s u e s , dense deposits not seen i n u n i n j e c t e d mice are found i n the Kuppfer c e l l s of the l i v e r and i n the kidney p r o x i m a l convoluted t u b u l e c e l l s . These d e p o s i t s are presumed t o be osmarin w a l l e d o f f w i t h i n lysosomes. A f t e r about a month, mice which are not s a c r i f i c e d r e g a i n t h e i r normal c o l o r . S t a i n i n g and R e t e n t i o n i n p r o c i n e synovia. I f the Boussina, et a l . , (3) suggestion t h a t osmium c o n t a i n i n g d e p o s i t s have a long term b e n e f i c i a l e f f e c t i s c o r r e c t , then to be u s e f u l , osmarins should s t a i n t i s s u e w i t h i n s y n o v i a l spaces and be r e t a i n e d there f o r long p e r i o d s . I n i t i a l experiments w i t h p i g s and r a b b i t s have been d i r e c t e d toward examining t h i s and r e l a t e d i s s u e s . I n these experiments, osmarin s o l u t i o n s are i n j e c t e d i n t o the s y n o v i a l spaces of the l i v i n g animal, and some time l a t e r , the t i s s u e s of the j o i n t are examined a f t e r s a c r i f i c e of the animal. Our gross and m i c r o s c o p i c f i n d i n g s are t h a t osmarins w i l l s t a i n t i s s u e i n j o i n t s and be r e t a i n e d there. The e f f e c t i v e n e s s of both s t a i n i n g and r e t e n t i o n , however, i s dependent upon the composition of the osmarin. P i g s used i n t h i s study have been, f o r the most p a r t , a r t h r i t i c . Mycoplasmal a r t h r i t i s i s common i n l a r g e herds, and i s e n z o o t i c i n the SIU herd. I n i t i a l experiments w i t h p i g s were short term and i n v o l v e d four a r t h r i t i c p i g s which r e c e i v e d d i f f e r e n t osmarin preparat i o n s . J o i n t t i s s u e s were examined f o r r e t a i n e d osmarin and photographed one t o three days a f t e r i n j e c t i o n . A second group of p i g s i n c l u d i n g n o n a r t h r i t i c c o n t r o l s , were i n j e c t e d and



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examined one month a f t e r the i n j e c t i o n . Tissue samples f o r microscopic study were obtained from these animals. Osmarin s t a i n i n g was observed through the c o l o r imparted to the t i s s u e . In most cases, the s t a i n i n g was obvious as a grey or b l a c k c o l o r when the s t a i n i n g was due t o an osmarin p r e p a r a t i o n c o n t a i n i n g more than 30% osmium, or a brown c o l o r f o r l e s s osmium r i c h osmarins. The c o l o r of the s t a i n e d t i s s u e was c l e a r l y r e l a t e d to t h a t of the i n j e c t e d osmarin, which suggests t h a t the b i n d i n g i s a s s o c i a t i v e , and not accompanied by changes i n osmium o x i d a t i o n s t a t e or the c o n n e c t i v i t y of the osmium i o n s . F i g u r e 3 i s a photograph of a s t a i n e d j o i n t . The osmarin used i n t h i s case contained 35% osmium as a dry s o l i d . A l l surfaces w i t h i n the s y n o v i a l space were s t a i n e d , a r t i c u l a r c a r t i l a g e on the j o i n t s u r f a c e s and the j o i n t capsule which encloses the space. The most proximal lympth node was s t a i n e d (not shown). Except f o r the c o l o r , the s t a i n e d t i s s u e appears normal. There was no gross evidence of t o x i c i t y . In the month-long study from which F i g u r e 3 i s drawn, o n l y one t a r s a l and c a r p a l j o i n t of the p i g s were i n j e c t e d w i t h osmarin s o l u t i o n . The other j o i n t s were untreated and served as c o n t r o l s on the same animal. In the a r t h r i t i c animals, the untreated t a r s a l j o i n t s e x h i b i t e d l e s i o n s , c o n f i r m i n g the o r i g i n a l d i a g n o s i s . The t r e a t e d j o i n t s e i t h e r e x h i b i t e d no l e s i o n (one case) or s t r u c tures which are i n t e r p r e t e d as h e a l i n g l e s i o n s (two c a s e s ) . This l a s t assessment i s t e n t a t i v e and i s based upon m i c r o s c o p i c evidence of c a r t i l a g e growth i n t o the l e s i o n areas ( f o l l o w i n g section). In a f o l l o w i n g experiment, two a r t h r i t i c p i g s and one normal c o n t r o l were i n j e c t e d i n r i g h t t a r s a l and c a r p a l j o i n t s and s a c r i f i c e d one month l a t e r . The osmarin p r e p a r a t i o n used i n t h i s case contained o n l y 17% osmium as a dry s o l i d . On examination, the t r e a t e d j o i n t s were found t o be unstained. There was no d i f f e r e n c e between i n j e c t e d and u n i n j e c t e d j o i n t s . The e a r l i e r s h o r t term experiments (discussed above) i n d i c a t e d t h a t s t a i n i n g from low osmium percentage polymers was l i g h t . These f i n d i n g s i n d i c a t e a r a p i d c l e a r i n g time as w e l l , and i n these cases, no evidence of b e n e f i c i a l e f f e c t could be found. In subsequent experiments i n v o l v i n g r a b b i t s and dogs, o n l y osmarin p r e p a r a t i o n s c o n t a i n i n g more than 30% osmium i n the dry s o l i d were used. An i n t e r e s t i n g secondary f i n d i n g was made i n the experiment i n which one month separated i n j e c t i o n and s a c r i f i c e of the animal. In these cases, the s t a i n e d a r t i c u l a r s u r f a c e was marked w i t h a lacework p a t t e r n of unstained c a r t i l a g e (Figure 3). The p a t t e r n i s a t t r i b u t e d t o growth s i n c e p i g s used i n these s t u d i e s are young (3 mo) animals and double t h e i r weight i n the one month i n t e r v a l p r i o r to s a c r i f i c e .




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Figure 3.

The osmarin-stained surface of a pig tarsal joint. The pattern of white lines (unstained cartilage) is attributed to growth.


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The white l i n e d p a t t e r n observed appears t o be due t o the new c a r t i l a g e formed as the p i g s grew. Osmarin bound t o the c a r t i l a g e surface d i d not s t a i n the new c a r t i l a g e . The p a t t e r n i s an i n d i c a t i o n o f the s t r e n g t h and s t a b i l i t y o f the b i n d i n g . I t serves t o demonstrate t h a t once the osmium polymers have bound t o the c a r t i l a g e surface they do not move. The p a t t e r n i s not a r e f l e c t i o n o f normal growth and i s an e f f e c t o f the osmarin. H i s t o l o g y of s t a i n e d t i s s u e i n p i g s . H i s t o l o g i c a l s t u d i e s have had s e v e r a l o b j e c t i v e s . F i r s t i s the determination o f what the osmarin s t a i n s . Secondly, these s t u d i e s r e v e a l the extent o f microscopic t o x i c i t y . Stained t i s s u e appears h e a l t h y and e n t i r e l y undamaged. M i c r o s c o p i c a l l y , some dose r e l a t e d c e l l death i s observed. F i n a l l y , microscopic examination o f the p i g c a r t i l a g e confirmed t h a t the unusual l i n e d p a t t e r n i s due t o growth of c a r t i l a g e . Evidence o f c a r t i l a g e growth has been found, a s s o c i a t e d w i t h l e s i o n s i n osmarin t r e a t e d j o i n t s , which suggests regeneration. Tissue samples f o r microscopic study were obtained from p i g s s a c r i f i c e d one month a f t e r i n j e c t i o n . Samples were obtained from the j o i n t capsules and a r t i c u l a r s u r f a c e s . When a r t h r i t i c l e s i o n s were found, samples from these areas were obtained. The experiment i n c l u d e s two l e v e l s o f c o n t r o l . F i r s t , a l l o f the p i g s r e c e i v e d i n j e c t i o n s o f osmarin s o l u t i o n o n l y i n the r i g h t t a r s a l and c a r p a l j o i n t s . Thus, s t a i n e d and unstained t i s s u e was obtained from the same animal by examining both t a r s a l and c a r p a l j o i n t s . Secondly, three o f the p i g s were a r t h r i t i c and one was h e a l t h y . Assessments o f the a f f e c t s accompanying osmarin s t a i n i n g are made on the b a s i s o f comparisons o f s t a i n e d and unstained t i s s u e from both a r t h r i t i c and n o n - a r t h r i t i c animals. The d i s t r i b u t i o n o f osmarin i n a s t a i n e d s y n o v i a l space i s , f o r the most p a r t , independent o f a r t h r i t i c c o n d i t i o n s . J o i n t capsule and c a r t i l a g e o f a r t h r i t i c and h e a l t h y animals are s i m i l a r l y s t a i n e d . The growth r e l a t e d l i n e d p a t t e r n i s observed i n both a r t h r i t i c and h e a l t h y animals. Disease r e l a t e d d i f f e r e n c e s are found o n l y i n t i s s u e s r e l a t i n g t o a r t h r i t i c lesions. H i s t o l o g i c a l examination o f the s y n o v i a l membrane revealed osmarin t o be taken up by s u b s y n o v i a l macrophages (Figure 4 ) . Accumulations o f t h i s type are not seen i n c o n t r o l t i s s u e s . I n both osmarin t r e a t e d and u n i n j e c t e d j o i n t s the s y n o v i a l membrane appeared i n t a c t . Stained a r t i c u l a r c a r t i l a g e appears h e a l t h y and undamaged. M i c r o s c o p i c a l l y , s t a i n i n g o f the surface l a y e r o f the p i g c a r t i l a g e was confirmed by a strong a f f i n i t y o f t o l u i d i n e blue f o r t h i s r e g i o n which was not seen i n c o n t r o l t i s s u e s (Figure 5 ) . Most surface c e l l s o f the a r t i c u l a r c a r t i l a g e appeared healthy and were present as s i n g l e c e l l s . I n white l i n e r e g i o n s ,



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Figure 4. A tissue section taken through the synovial membrane and underlying connective tissue from a pig that received a single osmarin injection 30 d previously. Synovial cells (SC) appear healthy and line the joint cavity. Numerous sybsynovial macrophages (M) show dense osmarin deposits.

Figure 5. A section of the articular cartilage from a pig injected 30 d previously with osmarin. The surface zone (length of double-headed arrow) shows dense staining with toluidine blue to a depth of about 0.1 mm. Chrondrocyte nests (arrows) usually display one or two cells on the surface and one to ten cells in the deeper zones. At the right is a region that corresponds to a white line (arc) seen in Figure 3. Surface staining is not apparent at the white line region.



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a l l c e l l s appeared h e a l t h y and numerous m u l t i c e l l u l a r lacunae were present. C a r t i l a g e i n t h i s r e g i o n appears p i n k w i t h the metachromatic t o l u i d i n e blue s t a i n . The m a t r i x was l e s s dense w i t h the e l e c t r o n microscope. U l t r a s t r u c t u r a l l y the osmarin-treated c a r t i l a g e appeared normal except f o r evidence f o r an o c c a s i o n a l dead chondrocyte. These chondrocytes i n e v i t a b l y d i s p l a y e d e l e c t r o n dense i n c l u s i o n s which were most l i k e l y osmarin. For the most p a r t the c e l l u l a r r e a c t i o n t o osmarin appears to be phagocytosis. The compound i s absorbed and w a l l e d o f f i n s p e c i f i c i n c l u s i o n s . I n regions of a r t h r i t i c l e s i o n s the c a r t i l a g e was l e s s darkened by the osmarin. This i n d i c a t e d a c t i v e growth of c a r t i l a g e . This was confirmed by l i g h t microscope observations (Figure 6 ) . C a r t i l a g e nests contained numerous c e l l s , and the matrix s t a i n e d p i n k w i t h t o l u i d i n e b l u e . Rabbit s t u d i e s . An on going companion study i n v o l v i n g t h i r t y - f i v e r a b b i t s y i e l d e d p r e l i m a r y r e s u l t s somewhat s i m i l a r to those of the p i g s t u d i e s . Osmarin s o l u t i o n s i n j e c t e d i n t o the knee j o i n t s of the r a b b i t s s t a i n a r t i c u l a r c a r t i l a g e and the j o i n t capsule, as i n the p i g . In t h i s study, a measure o f osmarin r e t e n t i o n has been developed based upon the standard grey s c a l e . S e c t i o n s of s t a i n e d a r t i c u l a r c a r t i l a g e were removed and compared v i s u a l l y w i t h standard grey s c a l e s . I n t h i s study groups of ten r a b b i t s were i n j e c t e d a t the same time, and then animals were s a c r i f i c e d and the j o i n t s examined at i n t e r v a l s from one day to seven weeks post i n j e c t i o n . There appears to be v e r y l i t t l e change i n c a r t i l a g e d i s c o l o r a t i o n w i t h time, i n d i c a t i n g s t r o n g b i n d i n g of osmarin. G r o s s l y , the c a r t i l a g e and synovium of the j o i n t s , through s t a i n e d , appeared otherwise normal a t a l l time p e r i o d s . The i n t e r l a c i n g white l i n e s seen i n long term i n j e c t e d p i g s were not p r e s e n t , even when young growing r a b b i t s were examined. The darkening of the s y n o v i a l membrane appeared upon microscopic examinations t o be due to accumulation of osmarin i n s u b s y n o v i a l macrophages, s i m i l a r to those found i n p i g s . The a r t i c u l a r c a r t i l a g e was m i n i m a l l y d i s r u p t e d w i t h osmarin. L i t t l e evidence of c e l l death i n the c a r t i l a g e was noted. S y n o v i a l t i s s u e examined one day a f t e r osmarin i n j e c t i o n was d i s r u p t e d and contained numerous dead c e l l s . S y n o v i a l t i s s u e examined three weeks or l a t e r a f t e r i n j e c t i o n was normal. This i s a p o t e n t i a l l y important o b s e r v a t i o n . An e f f e c t upon the synovium suggests p o s s i b l e short term b e n e f i t s . In order t o provide comparisons w i t h osmarin t r e a t e d j o i n t t i s s u e s , a number of r a b b i t s were i n j e c t e d w i t h e q u i v a l e n t amounts (based upon osmium content) of osmium t e t r o x i d e s o l u t i o n s . The r a b b i t s were s a c r i f i c e d and the j o i n t s examined according to the same time i n t e r v a l s t h a t were employed i n the osmarin s t u d i e s . The r e s u l t s of these s t u d i e s were s i m i l a r t o those reported by others (17). H i s t o l o g i c a l l y , the osmium t e t r o x i d e



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Figure 6. Articular cartilage taken from a region less than 2 mm from an arthritic lesion. The surface is not differentially stained as shown in Figure 5. Chrondrocyte nests (arrows) contain from one to eight cells and suggest active growth in this region.


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treatments, were harsher on j o i n t t i s s u e s than were osmarin treatments and t h e i r d e l e t e r i o u s e f f e c t s were longer l a s t i n g . A f t e r one day the s y n o v i a l membranes of both groups appeared n e c r o t i c . However, the osmarin t r e a t e d j o i n t s regenerated a healthy appearing s y n o v i a l membrane by three weeks. The s y n o v i a l membrane of the osmium t e t r o x i d e group regenerated a f t e r seven weeks, although there remained much subsynovial f i b r o s i s and many f o r e i g n body g i a n t c e l l s . For the OsO^ t r e a t e d r a b b i t s , the e n t i r e c a r t i l a g e surface (about 2mm deep) showed n e c r o t i c c e l l s . In a d d i t i o n , i n the osmium t e t r o x i d e i n j e c t e d r a b b i t s , there was a surface exudate upon the c a r t i l a g e and a h i g h l y granulated c a r t i l a g e matrix. C l i n i c a l data i n dogs. Osmarins have been used i n a l i m i t e d number of c l i n i c a l s i t u a t i o n s i n dogs. S i x dogs have been t r e a t e d so f a r . With s p e c i a l p e r m i s s i o n from the owners of the dogs, i n t r a a r t i c u l a r i n j e c t i o n s of osmarin s o l u t i o n s were made i n t o j o i n t s a f f l i c t e d w i t h a r t h r i t i s . The e t i o l o g y ranged from trauma to probable genetic to o l d age. The t r e a t ments and c l i n i c a l e v a l u a t i o n s of the e f f e c t s of treatments were made by l i c e n s e d v e t e r i n a r i a n s i n p r i v a t e p r a c t i c e . I n some cases, pre- and post-treatment roentgenograms were used i n the c l i n i c a l e v a l u a t i o n s . There was no opportunity f o r necropsy and h i s t o l o g i c a l e v o l u t i o n of the t r e a t e d j o i n t s . No c l i n i c a l l y normal j o i n t s were i n j e c t e d . C r i t e r i a f o r improvement i n c l u d e d increased locomotor a c t i v i t y , increased freedom of movement o f the a f f e c t e d j o i n t , decreased l i m p i n g , and diminished evidence of p a i n upon p a l p a t i o n of the j o i n t . Where roentgenograms were made, a smoother, more c l e a r l y defined a r t i c u l a r surface and decreased r a d i o o p a c i t y i n the s o f t t i s s u e s immediately surroundi n g the j o i n t were i n t e r p r e t e d as post-treatment signs o f improvement. For a p e r i o d of 1 to 3 days f o l l o w i n g osmarin i n j e c t i o n the dogs e x h i b i t e d evidence of increased discomfort i n the t r e a t e d j o i n t . A s i m i l a r p e r i o d of increased discomfort was noted when normal r a b b i t j o i n t s were t r e a t e d w i t h osmarin, but was not seen i n e i t h e r normal or a r t h r i t i c osmarin t r e a t e d j o i n t s of p i g s . The authors do not a t t i r b u t e t h i s short p e r i o d of discomfort t o the p h y s i c a l trauma of the i n j e c t i o n . More l i k e l y explanations could be t h a t osmarins cause a temporary decrease i n s y n o v i a l f l u i d v i s c o s i t y , or t h a t the discomfort i s due to a type of chemical synovectomy which was p r e v i o u s l y noted i n the r a b b i t s . In f i v e of the s i x dogs, the temporary increase i n discomf o r t was followed by a p e r i o d of gradual improvement over the o r i g i n a l a r t h r i t i c c o n d i t i o n s . Two of the dogs have recovered normal f u n c t i o n i n the e f f e c t e d j o i n t s . Three of the dogs have improved, but s t i l l r e t a i n some l o s s of f u n c t i o n i n the a r t h r i t i c j o i n t s a t t h i s time. I n the dog which f a i l e d to show any improvement, the knee j o i n t was markedly deformed by e x o s t o s i s due to the long standing a r t h r i t i s .


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Conclusions The study of osmarins as potential arthritic agents follows directly, in this case, from the Boussina, et a l . , suggestion that osmium containing deposits may have long term beneficial effects in arthritis treatment. Osmarins exhibit properties, e.g. low toxicity and tissue staining and retention, which are thought necessary for such an application. In preliminary experiments involving dogs and pigs, favorable responses to treatment have been observed. We propose that osmarins in a joint, may react with superoxide ion and thereby protect the synovial fluid from damage by this species. A secondary finding in the study is that osmarins have a short term effect upon the synovial membrane. This effect may also contribute to the utility of these substances. It is important to emphasize the preliminary character of the research discussed in this paper. No claims of cures are implied. At the same time, i t is also important to recognize the potentialities that the research findings suggest. If the several uncertainties in the proposed application of osmarins can be successfully resolved, then new alternatives in arthritis treatment will be available. Acknowledgments This research has been supported by the departments listed and by the office of Research Development and Administration of Southern Illinois University. Drs. T. 0. Miller and D. M. Lane of Murphysboro, Illinois and L. F. Striegel of Carbondale, Illinois are veterinarians in private practice and have treated the dogs included in the study. Dr. G. H. Gass guided early studies of toxicity. Dr. W. J. Roth characterized osmarins. P. S. Ostenburg prepared many of the materials used in the study. A. M. Islam and P. A. Kibala are currently studying osmarin reactivities. Several undergraduate research assistants have contributed to the project. Literature Cited 1. 2. 3. 4. 5. 6.

Hinckley, C. C.; Ostenburg, P. S.; Roth, W. J. Polyhedron 1982, in press. Nissila, M. Scand. J. Rheumatology 1979, Suppl. 29, 1. Bonssina, I; Lagier, R.; Ott, H.; Fallet, G. H. Scand. J. Rheumatology 1976, 5, 53. Fridovitch, Irwin. Science 1978, 201, 875. Bannister, W. H.; Bannister, J. V., Eds,; "Biological and Clinical Aspects of Superoxide and Superoxide Dismutase", Elsevier/North Holland, New York, 1980, pp. 147-153. Bannister, W. H.; Bannister, J. V., Eds.; "Biological and Clinical Aspects of Superoxide and Superoxide Dismutase", Elsevier/North Holland, New York, 1980, pp. 154-159.



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7. McCord, J. M. Science 1974, 185, 530. 8. Greenwald, R. A.; Moy, W. W. Arthritis Rheum. 1980, 23, 455-463. 9. Spiro, T. G., Ed.; "Metals Ion Activation of Dioxygen", John Wiley and Sons, New York, 1980, pp. 209-237. 10. Michelson, A. M.; McCord, J. M.; Fridovitch, I., Eds.; "Superoxide and Superoxide Dismutases", Academic Press, New York, 1977, pp. 517-536. 11. Michelson, A. M.; McCord, J. M.; Fridovitch, I., Eds.; "Superoxide and Superoxide Dismutases", Academic Press, New York, 1977, pp. 537-550. 12. Bannister, W. H.; Bannister, J. V., Eds.; "Biological and Clinical Aspects of Superoxide and Superoxide Dismutase", Elsevier/North Holland, New York, 1980, pp. 424-430. 13. Criegee, R.; Marchand, B.; Wannowius, Η.; Ann. der Chem. 1942, 550, 99-133. 14. Cotton, F. A.; Wilkinson, G.; "Advanced Inorganic Chemistry", 4th Ed., "John Wiley and Sons, New York, 1980. 15. Armstrong, J. E . ; Robinson, W. R.; Walton, R.A. J. C. S. Chem. Comm. 1981, 1120-1121. 16. Nielson, A. J.; Griffith, W. P.; J. Chem. Soc. Dalton 1979, 1084. 17. Mitchell, N; Laurin, C.; Shepart, N.; J. Bone Joint Surgery (British) 1973, 55B, 814-821. RECEIVED October 13,

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