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UBC Theses and Dissertations

Cyclosporine--ocular absorption, pharmacokinetics & effects on uveitis Kalsi, Gursharan Singh 1986

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CYCLOSPORINE - OCULAR ABSORPTION, PHARMACOKINETICS & EFFECTS ON UVEITIS By GURSHARAN SINGH KALSI B . S c , The U n i v e r s i t y o f B r i t i s h C o l u m b i a , 1983 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES (Department o f P a t h o l o g y , F a c u l t y o f M e d i c i n e ) We a c c e p t t h i s t h e s i s as conforming t o the r e q u i r e d s t a n d a r d THE UNIVERSITY OF BRITISH COLUMBIA September,1986 (c) G u r s h a r a n S i n g h K a l s i , 1986 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of PATHOLOGY The University of British Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date SEPTEMBER 25 , 1986 DE-6(3/81) ABSTRACT I n f l a m m a t o r y o c u l a r d i s e a s e i s an i m p o r t a n t c a u s e o f b l i n d n e s s and u v e i t i s accounts f o r 1.0% o f b l i n d p a t i e n t s i i n Canada. T h i s d i s e a s e can be p a r t i c u l a r l y t r o u b l e s o m e to t r e a t , because the n a t u r e o f the c a u s a l f a c t o r o r f a c t o r s and mechanisms o f p r o g r e s s i o n a r e u s u a l l y unknown. N o n - s p e c i f i c a n t i - i n f l a m m a t o r y a g e n t s h a v e been u s e d o r a l l y a n d s y s t e m i c a l l y w i t h some s u c c e s s t o t r e a t u v e i t i s , 2 - 8 but they may produce s e r i o u s s i d e e f f e c t s both l o c a l l y and e l s e w h e r e i n t h e b o d y . 9 , 1 2 ' 1 4 W i t h p r o l o n g e d u s e t o l e r a n c e t o t h e s e d r u g s may d e v e l o p , m a k i n g them i n e f f e c t i v e . R e c e n t l y a p o w e r f u l i m m u n o s u p p r e s s i v e a g e n t , C y c l o s p o r i n e (Cy) , u s e d o r a l l y and s y s t e m i c a l l y i n t h e t r e a t m e n t o f u v e i t i s has shown p r o m i s i n g r e s u l t s . 1 6 - 1 9 ' 2 8 H o w e v e r , i t s r o u t i n e u s e i s l i m i t e d b e c a u s e o f a n a r r o w t h e r a p e u t i c index and r e n a l t o x i c i t y . 1 9 f 4 2 / 4 3 S e v e r a l s t u d i e s h a v e shown t h a t s u b c o n j u n c t i v a l i n j e c t i o n o f a number o f a n t i n e o p l a s t i c a g e n t s e n h a n c e d o c u l a r a b s o r p t i o n 2 0 - 2 4 i n a t r a d i t i o n a l p h a r m a c o l o g i c a l s a n c t u a r y , 1 3 , 1 4 and c i r c u m v e n t e d t h e a s s o c i a t e d s y s t e m i c s i d e e f f e c t s . T h e r e f o r e , i f Cy w e r e a d m i n i s t e r e d s u b c o n j u n c t i v a l l y i t m i g h t be p o s s i b l e t o a v o i d t h e s i d e e f f e c t s a s s o c i a t e d w i t h t h e o r a l and s y s t e m i c r o u t e s , and a t t h e same t i m e p r o v i d e h i g h e r l e v e l s o f Cy t o t h e e y e . A p r o t o c o l f o r t h e a d m i n i s t r a t i o n o f C y i i s u b c o n j u n c t i v a l l y was d e v e l o p e d i n New Z e a l a n d w h i t e r a b b i t s , t o s t u d y t o x i c i t y , o c u l a r p h a r m a c o k i n e t i c s f o l l o w i n g e q u i d o s e a d m i n i s t r a t i o n s u b c o n j u n c t i v a l l y and s y s t e m i c a l l y and t h e e f f e c t s o f Cy on an a n i m a l m o d e l o f u v e i t i s . S u b c o n j u n t i v a l a d m i n i s t r a t i o n o f 5mg o f Cy i n O . l c c (Sandimmune I . V . ( R ) 50 mg/ml) week ly was found t o be t h e maximum t o l e r a t e d d o s e by t h e r a b b i t s * e y e , and was s u p e r i o r t o i n t r a v e n o u s i n j e c t i o n f o r o c u l a r p e n e t r a t i o n w h i l e m i n i m i z i n g s y s t e m i c e x p o s u r e . The u v e i t i s m o d e l showed t h a t Cy was e f f e c t i v e i n r e d u c i n g t h e i n f l a m m a t o r y re sponse and the e a r l i e r the a p p l i c a t i o n o f Cy the m i l d e r the u v e i t i s . The r e s u l t s from our s tudy s u p p o r t t h e c o n t e n t i o n t h a t l o c a l a d m i n i s t r a t i o n o f Cy would l e a d t o h i g h e r l e v e l s o f Cy a b s o r p t i o n and c i r c u m v e n t the s i d e e f f e c t s o f s y s t e m i c a d m i n i s t r a t i o n . T h i s may f a c i l i t a t e t h e r o u t i n e u s e o f Cy i n o c u l a r i n f l a m m a t o r y d i s e a s e . C o n t e n t s A b s t r a c t i i L i s t o f T a b l e s v i L i s t o f F i g u r e s v i i L i s t o f A b b r e v i a t i o n s x Acknowlegdements x i I n t r o d u c t i o n 1 C h a p t e r 1: O c u l a r t o x i c i t y s t u d y o f p o s s i b l e v e h i c l e s f o r c y c l o s p o r i n e 5 I n t r o d u c t i o n R e s u l t s D i s c u s s i o n C o n c l u s i o n s C h a p t e r 2: Dose d e t e r m i n a t i o n s t u d y 26 I n t r o d u c t i o n R e s u l t s D i s c u s s i o n C o n c l u s i o n s C h a p t e r 3: P h a r m a c o k i n e t i c s o f c y c l o s p o r i n e f o l l o w i n g s u b c o n j u n c t i v a l v e r s u s i n t r a v e n o u s a d m i n i s t r a t i o n 48 I n t r o d u c t i o n R e s u l t s D i s c u s s i o n C o n c l u s i o n s C h a p t e r 4: U v e i t i s s t u d y 77 I n t r o d u c t i o n R e s u l t s D i s c u s s i o n C o n c l u s i o n s Summary 100 B i b i l o g r a p h y 102 i v Appendix I : E f f e c t s o f m u l t i p l e dose s u b c o n j u n c t i v a l a n t i n e o p l a s t i c s on a c u t e immune u v e i t i s A p p e n d i x I I : C o m b i n e d l o c a l c h e m o t h e r a p y f o r s p o n t a n e o u s l y o c c u r r i n g a n t e r i o r u v e i t i s i n c a t s . L i s t o f T a b l e s T a b l e I . G r a d i n g system used t o a s s e s s o c u l a r i n f l a m m a t i o n due t o the v e h i c l e s I I . G r a d i n g system used t o a s s e s s o c u l a r i n f l a m m a t i o n due t o c y c l o s p o r i n e I I I . P e r f o r m a n c e d a t a on methods 1 & 2 I V . B i o a v a i l a b i l i t y o f c y c l o s p o r i n e i n o c u l a r humors V . G r a d i n g system used t o a s s e s s o c u l a r i n f l a m m a t o r y response i n d u c e d by b o v i n e serum a l b u m i n L i s t o f F i g u r e s F i g u r e Page 1.0 O c u l a r i n f l a m m a t o r y response f o l l o w i n g s u b c o n j u n c t i v a l i n j e c t i o n o f v e h i c l e s . 12 1.1 R e p r e s e n t a t i v e p h o t o g r a p h s o f t h e o c u l a r i n f l a m m a t o r y response t o each v e h i c l e a t one, f o u r and seven days . 13 1.2 R e p r e s e n t a t i v e photographs o f the o c u l a r i n f l a m m a t o r y response t o each v e h i c l e a t one, f o u r and seven days Cont 'd . 14 1.3 H i s t o l o g i c a l e f f e c t s o f c a s t o r o i l a t one week. 15 1.4 H i s t o l o g i c a l e f f e c t s o f c o t t o n seed o i l a t one week. 16 1.5 H i s t o l o g i c a l e f f e c t s o f o l i v e o i l a t one week. 17 1.6 H i s t o l o g i c a l e f f e c t s o f peanut o i l a t one week. 18 1.7 H i s t o l o g i c a l e f f e c t s o f p o l y e t h y l e n e g l y c o l a t one week. 19 1.8 H i s t o l o g i c a l e f f e c t s o f sesame seed o i l a t one week. 20 2.0 O c u l a r i n f l a m m a t o r y response t o d i f f e r e n t doses o f c y c l o s p o r i n e s u b c o n j u c t i v a l l y . 32 2 .1 E f f e c t s o f 5 mg i n O . l c c and 10 mg i n 0 .2cc o f c y c l o s p o r i n e a t one, f o u r and seven days . 33 2.2 E f f e c t s o f 25 mg i n 0.5cc o f c y c l o s p o r i n e a t one, f o u r and seven days and 5 mg i n O . l c c once p e r week o f c y c l o s p o r i n e f o r two months. 34 2.3 H i s t o l o g i c a l e f f e c t s o f 5 mg i n O . l c c a t one week. 35 2.4 H i s t o l o g i c a l e f f e c t s o f 10 mg i n 0.2cc a t one week. 3 6 2 .5 H i s t o l o g i c a l e f f e c t s o f 25 mg i n 0.2cc a t one week. 37 v i i 2.6 H i s t o l o g i c a l e f f e c t s o f 5 mg i n O . l c c o f c y c l o s p o r i n e once p e r week i n a r a b b i t t h a t r e c i e v e d t h i s dose f o r two months. 38 2 . 7 a H i s t o l o g i c a l e f f e c t s o f a s u b c o n j u n c t i v a l i n j e c t i o n o f 0 . 5 c c o f peanut o i l , c o n t r o l , (A) and 5 mg o f Cy i n 0.5cc o f peanut o i l , e x p e r i m e n t a l , ( B ) a t 2 h o u r s . 42 2 .7b H i s t o l o g i c a l e f f e c t s o f a s u b c o n j u n c t i v a l i n j e c t i o n o f 0 . 5 c c o f peanut o i l , c o n t r o l , (A) and 5 mg o f Cy i n 0 .5cc o f peanut o i l , e x p e r i m e n t a l , ( B ) a t 4 h o u r s . 43 2 . 7 c H i s t o l o g i c a l e f f e c t s o f a s u b c o n j u n c t i v a l i n j e c t i o n o f 0 . 5 c c o f peanut o i l , c o n t r o l , (A) and 5 mg o f Cy i n 0 . 5 c c o f peanut o i l , e x p e r i m e n t a l , ( B ) a t 12 h o u r s . 44 2 . 7 d H i s t o l o g i c a l e f f e c t s o f a s u b c o n j u n c t i v a l i n j e c t i o n o f 0 . 5cc o f peanut o i l , c o n t r o l , (A) and 5 mg o f Cy i n 0 . 5 c c o f peanut o i l , e x p e r i m e n t a l , ( B ) a t 1 week. 4 5 3.0 A t y p i c a l chromatogram o f aqueous humor. 61 3 .1 A t y p i c a l chromatogram o f v i t r e o u s humor. 62 3.2 A t y p i c a l chromatogram o f u r i n e . 63 3.3 A t y p i c a l chromatogram o f b l o o d . 64 3.4 S t a n d a r d c u r v e s o f c y c l o s p o r i n e u s i n g methods 1 & 2. 65 3.5 L e v e l s o f c y c l o s p o r i n e i n t h e o c u l a r chambers o f the eye. 66 3.6 B l o o d l e v e l s o f c y c l o s p o r i n e i n the r a b b i t a t a 5 mg i n O . l c c dose. 67 3.7 Cummulat ive u r i n e e l i m i n a t i o n c u r v e f o r c y c l o s p o r i n e i n the r a b b i t g i v e n a 5 mg i n O . l c c s y s t e m i c dose. 68 3.8 S t r u c t u r e o f c y c l o s p o r i n e . 70 4.0 The e f f e c t o f s u b c o n j u n c t i v a l c y c l o s p o r i n e on u v e i t i s i n d u c e d by b o v i n e serum a l b u m i n (BSA). 87 v i i i 4.1 R e p r e s e n t a t i v e photographs o f t h e o c u l a r i n f l a m m a t o r y r e s p o n s e i n d u c e d by BSA and the e f f e c t s o f c y c l o s p o r i n e a d m i n i s t e r e d s u b c o n j u n c t i v a l l y . 88 4.2 H i s t o l o g i c a l e f f e c t s o f u v e i t i s i n d u c e d by BSA i n Group 1. 89 4.3 H i s t o l o g i c a l e f f e c t s o f u v e i t i s i n d u c e d by BSA i n Group 1 C o n t ' d . 90 4.4 H i s t o l o g i c a l e f f e c t s o f c y c l o s p o r i n e on u v e i t i s i n d u c e d by BSA i n Group 2. 91 4 .5 H i s t o l o g i c a l e f f e c t s o f c y c l o s p o r i n e on u v e i t i s i n d u c e d by BSA i n Group 3. 92 i x L i s t o f A b b r e v i a t i o n s BSA : b o v i n e serum a lbumin C : c o r n e a CE : c o n j u n c t i v a l e p i t h e l i u m CH : c h o r o i d Cy : c y c l o s p o r i n e CyC : d i h y d r o c y c l o s p o r i n C CyD : c y c l o s p o r i n D EAU : e x p e r i m e n t a l autoimmune u v e i t i s EAU-BSA : e x p e r i m e n t a l autoimmune u v e i t i s i n d u c e d by b o v i n e serum a lbumin E A U - S - A g : e x p e r i m e n t a l autoimmune u v e i t i s i n d u c e d by r e t i n a l S - A n t i g e n FZ : f o c a l zone o f i n f l a m m a t i o n I : i r i s L : l e n s LP : l a m i n a p r o p r i a M - musc le NA : n e c r o t i c a r e a ON : o p t i c nerve PMN - p o l y m o r p h o n u c l e a r g r a n u l o c y t e R - r e t i n a RD - r e t i n a l detachment SCL - s c l e r a SF - s u b r e t i n a l f l u i d x Acknowlegdements I w o u l d p a r t i c u l a r l y l i k e t o thank Dr. J a c k Rootman and Dr. Gedy G u d a u s k a s f o r t h e i r e x p e r t g u i d a n c e , p a t i e n c e t h r o u g h o b s t a c l e s a n d d e l a y s , and f o r t h e i r f a i t h i n t h e e v e n t u a l p o s i t i v e c o n c l u s i o n o f t h i s r e s e a r c h . I w o u l d a l s o l i k e t o t h a n k D r . N i c k B u s s a n i c h f o r t h e g u i d a n c e , and t e a c h i n g o f the s k i l l s n e c e s s a r y t o comple te t h i s p r o j e c t . T h a n k s a l s o t o t h e s u p e r v i s o r y c o m m i t t e e D r s . A u t o r , C a m p b e l l , and R o l l i n s f o r t h e i r c o n s t r u c t i v e s u g g e s t i o n s t h r o u g h o u t t h e r e s e a r c h . To D r . P e t e r D o l m a n s p e c i a l t h a n k s f o r t h e many h o u r s o f a s s i s t a n c e i n the h i s t o p a t h o l o g y and r e v i e w o f my t h e s i s . I w o u l d a l s o l i k e t o t h a n k M . R . C . f o r t h e f u n d i n g o f t h i s r e s e a r c h and Sandoz L t d . , f o r g e n e r o u s l y s u p p l y i n g the Sandimmune I . V . T h a n k s f i n a l l y t o my f a t h e r Deep S i n g h f o r d r a w i n g t h e f i g u r e s , my s i s t e r K a m a l j i t f o r t y p i n g my t h e s i s and my b r o t h e r J a s v i n d e r a n d my m o t h e r H a r b h a j a n f o r t h e i r s u p p o r t d u r i n g my r e s e a r c h . x i INTRODUCTION O c u l a r i n f l a m m a t o r y d i s e a s e c o n s t i t u t e s one o f the most i m p o r t a n t c a u s e s o f b l i n d n e s s i n p e o p l e . U v e i t i s , i n p a r t i c u l a r , a c c o u n t s f o r 1.0% o f t h e b l i n d p a t i e n t s i n C a n a d a . 1 U v e i t i s i s f r e q u e n t l y o f a n o n - s p e c i f i c n a t u r e and i t s e x a c t e t i o l o g y e l u d e s c l i n i c a l i n v e s t i g a t o r s . Even i n i n s t a n c e s where t h e s p e c i f i c c a u s e o f u v e i t i s i s known • • • • • 'SO (eg . H e r p e s S i m p l e x 4 3 , J u v e n i l e R h e u m a t o i d A r t h r i t i s ^ ) , once t h e d i s e a s e i s e s t a b l i s h e d i n f l a m m a t i o n may p e r s i s t i n s p i t e o f t r e a t m e n t d i r e c t e d t o c a u s a t i o n . T r e a t m e n t o f t h i s d i s e a s e i s f r e q u e n t l y on an e m p i r i c b a s i s , w i t h n o n - s p e c i f i c a n t i - i n f l a m m a t o r y agents . In a s i g n i f i c a n t number o f p a t i e n t s a l l t r a d i t i o n a l m o d a l i t i e s may f a i l t o c o n t r o l t h e i n f l a m m a t i o n and t h e eye may u l t i m a t e l y succumb t o t h e s e c o n d a r y e f f e c t s o f c h r o n i c i n f l a m m a t o r y d i s e a s e . O f t e n the end r e s u l t s i n u v e i t i s are c a t a r a c t s , o p a c i f i e d m e d i a , a t r o p h i c r e t i n a , and c y s t o i d m a c u l a r edema w i t h e v e n t u a l l o s s o f v i s u a l a c u i t y . The c u r r e n t t h e r a p y f o r o c u l a r i n f l a m m a t o r y d i s e a s e i n t h e human c o n s i s t s o f l o c a l and s y s t e m i c a p p l i c a t i o n o f c o r t i c o s t e r o i d s , ' s y s t e m i c a d m i n i s t r a t i o n o f a l t e r n a t e a n t i - i n f l a m m a t o r y a g e n t s s u c h a s i n d o m e t h a c i n , p h e n y l b u t a z o n e , 3 , 4 a n d s y s t e m i c a d m i n i s t r a t i o n o f i m m u n o s u p p r r e s s i v e 5 , 6 a g e n t s s u c h as a z a t h i o p r i n e , 7 and c y c l o p h o s p h a m i d e . A l t h o u g h these agents have been used w i t h some success i n t h e t r e a t m e n t o f o c u l a r i n f l a m m a t o r y d i s e a s e , they have 1 not been w i t h o u t c o m p l i c a t i o n s such as g laucoma i n d u c e d by t o p i c a l a p p l i c a t i o n o f c o r t i c o s t e r o i d s , 1 0 , 1 1 , 1 4 w h i l e s y s t e m i c a d m i n i s t r a t i o n h a s r e s u l t e d i n C u s h i n g ' s - l i k e s y m p t o m s . 1 4 ' 1 5 A g r e a t e r r i s k i s a s s o c i a t e d w i t h s y s t e m i c a d m i n i s t r a t i o n o f immunosuppress ives which can cause bone m a r r o w s u p p r e s s i o n , s e c o n d a r y t u m o r s a n d s p e c i f i c c o m p l i c a t i o n s o n o t h e r n o r m a l l y p r o l i f e r a t i n g c e l l p o p u l a t i o n s . 1 2 , 1 4 , 1 5 R e c e n t l y another immunosuppress ive , C y c l o s p o r i n e (Cy), has been t r i e d as a p o s s i b l e c a n d i d a t e i n t h e t r e a t m e n t o f o c u l a r i n f l a m m a t o r y d i s e a s e . 1 6 , 1 7 , 1 9 , 3 0 C u r r e n t l y Cy i s employed t o p r e v e n t the r e j e c t i o n o f t r a n s p l a n t e d o r g a n s . 4 8 One o f t h e i n t r i g u i n g c h a r a c t e r i s t i c s o f Cy i s t h a t i t d i s p l a y s l i t t l e m y e l o t o x i c i t y and i t d o e s n o t a d v e r s e l y a f f e c t t h e p h a g o c y t i c c e l l s o f t h e m o n o n u c l e a r p h a g o c y t e s y s t e m . 1 5 ' 5 6 Cy causes immunosuppress ion d i r e c t e d towards T - c e l l -m e d i a t e d i m m u n i t y . 5 6 A l t h o u g h t h e p r e c i s e b i o c h e m i c a l m e c h a n i s m i s n o t w e l l u n d e r s t o o d , t h e e f f e c t may be a c h i e v e d by i n h i b i t i o n o f i n t e r l e u k i n - 2 s y n t h e s i s o r r e l e a s e , a n d i n h i b i t i o n o f r e c e p t o r a c q u i s i t i o n f o r i n t e r l e u k i n - 2 by p r e c u s o r C y t o t o x i c T - c e l l s , as w e l l as a d i m i n i s h e d r e s p o n s i v e n e s s o f T - h e l p e r c e l l s t o i n t e r l e u k i n -3^56,58,60 The e f f e c t o f Cy on s u p p r e s s i n g T - c e l 1 - m e d i a t e d immune response i s p a r t i c u l a r l y i n t e r e s t i n g , because i t . has been shown r e t i n a l S - a n t i g e n can cause e x p e r i m e n t a l autoimmune u v e i t i s (EAU) i n a n i m a l s t h a t r e s e m b l e s the T - c e l 1 - m e d i a t e d o c u l a r i n f l a m m a t i o n seen i n humans. I n v i e w o f t h i s , Cy h a s b e e n g i v e n s y s t e m i c a l l y ( I . M . ) f o r t h e t r e a t m e n t o f EAU w i t h s u c c e s s , 2 8 , 3 0 , 3 3 and i t has a l s o been employed o r a l l y i n the t r e a t m e n t o f p a t i e n t s w i t h u v e i t i s w i t h e n c o u r a g i n g r e s u l t s ; h o w e v e r , r e n a l t o x i c i t y o c c u r e d a t a r e l a t i v e l y l o w d o s e o f 10 m g / k g p e r d a y . 1 7 , 4 2 , 4 3 I f t h e s i d e e f f e c t s o f Cy c o u l d be c o n t r o l l e d o r c i r c u m v e n t e d , i t may p r o v i d e wide c l i n i c a l a p p l i c a t i o n s ; as o f now, t h e s i d e e f f e c t s l i m i t i t s u s e i n o c u l a r i n f l a m m a t o r y d i s e a s e . L o c a l a p p l i c a t i o n o f Cy would be one way o f a c h i e v i n g t h e r a p e u t i c l e v e l s o f Cy w i t h i n the eye w h i l e a v o i d i n g the s y s t e m i c s i d e e f f e c t s . Our l a b o r a t o r y h a s shown w i t h a number o f a g e n t s t h a t o c u l a r a b s o r p t i o n c o u l d be s i g n i f i c a n t l y e n h a n c e d by s u b c o n j u n c t i v a l i n j e c t i o n o f these agents when compared to e q u i d o s e s y s t e m i c a d m i n i s t r a t i o n , m a k i n g i t p o s s i b l e t o c i r c u m v e n t t h e a s s o c i a t e d t o x i c i t i e s o f s y s t e m i c a d m i n i s t r a t i o n . 2 0 - 2 4 U s i n g the s u b c o n j u n c t i v a l r o u t e f o r the a d m i n i s t r a t i o n o f C y , we e v a l u a t e d v e h i c l e and d r u g t o x i c i t y , o c u l a r p h a r m a c o k i n e t i c s f o l l o w i n g e q u i d o s e s u b c o n j u n c t i v a l and i n t r a v e n o u s a d m i n i s t r a t i o n , a n d t h e e f f e c t s o f s u b c o n j u n c t i v a l l y a d m i n i s t e r e d Cy on an a n i m a l model o f u v e i t i s t o d e v e l o p a p r o t o c o l f o r the a d m i n i s t r a t i o n o f Cy s u b c o n j u n c t i v a l l y . We found t h a t the c o m m e r c i a l l y p r e p a r e d Cy i n the 3 v e h i c l e C r e m o p h o r ( R / E L p r o d u c e d l e s s o f an i n f l a m m a t o r y r e a c t i o n and t h e maximum t o l e r a b l e d o s e by t h e eye o f t h e c o m m e r c i a l Cy was 5 mg i n O . l c c o n c e p e r week. From t h e p h a r m a c o k i n e t i c s t u d y we were a b l e t o show t h a t s i g n i f i c a n t l y h i g h e r l e v e l s o f Cy e n t e r e d t h e eye by s u b c o n j u n c t i v a l i n j e c t i o n c o m p a r e d t o an e q u i d o s e i n t r a v e n o u s i n j e c t i o n . E q u a l l y i m p o r t a n t , t h e b l o o d l e v e l s i n the r a b b i t s , t h a t r e c e i v e d Cy s u b c o n j u n c t i v a l l y were w i t h i n the recommended l e v e l s . The u v e i t i s ( e x p e r i m e n t a l autoimmune u v e i t i s induced by b o v i n e serum a l b u m i n ) s t u d y showed t h a t Cy was a b l e t o r e d u c e t h e i n f l a m m a t o r y r e s p o n s e and t h e e a r l i e r t h e t r e a t m e n t w i t h Cy the m i l d e r the u v e i t i s . W i t h s u b c o n j u n c t i v a l a d m i n i s t r a t i o n o f Cy we were a b l e t o a c h i e v e h i g h e r o c u l a r c o n c e n t r a t i o n s and a t t h e same t i m e g r e a t l y r e d u c e t h e p o s s i b i l i t y o f t h e a s s o c i a t e d s y s t e m i c s i d e e f f e c t s o f C y , m a k i n g Cy a d m i n i s t e r e d s u b c o n j u n c t i v a l l y a p o s s i b l e c a n d i d a t e f o r r o u t i n e use i n the t r e a t m e n t o f o c u l a r i n f l a m m a t o r y d i s e a s e . 4 CHAPTER 1 O c u l a r t o x c i t y s t u d y o f p o s s i b l e v e h i c l e s  f o r e y e 1 o s p o r i n e I n t r o d u c t i o n C y c l o s p o r i n e (Cy) , an i m m u n o s u p p r e s s i v e a g e n t , has been used s y s t e m i c a l l y i n the t r e a t m e n t o f c h r o n i c o c u l a r i n f l a m m a t o r y d i s e a s e , which was r e s i s t a n t t o c o n v e n t i o n a l t h e r a p y . ' H o w e v e r , t h e c o n c e n t r a t i o n o f Cy n e e d e d t o t r e a t the o c u l a r i n f l a m m a t i o n v i a t h i s a d m i n i s t r a t i o n r o u t e a l s o r e s u l t e d i n r e n a l t o x i c i t y . 4 2 ' 4 3 T h e r e f o r e , i f Cy i s a d m i n i s t e r e d l o c a l l y , t h i s s i d e e f f e c t may be a v o i d e d . O c u l a r a b s o r p t i o n has been shown to be s i g n i f i c a n t l y i n c r e a s e d b y l o c a l s u b c o n j u n c t i v a l i n j e c t i o n , when c o m p a r e d t o t h e s y s t e m i c r o u t e . 2 0 - 2 4 I n o r d e r t o e x a c t a f o r m u l a t i o n f o r Cy t o b e i n j e c t e d s u b c o n j u n c t i v a l l y , an a p p r o p r i a t e v e h i c l e had t o be found f o r Cy. The r e q u i r e m e n t s f o r t h i s v e h i c l e were governed by t h e l i p o p h i l i c c h e m i s t r y o f Cy ' w h i c h l i m i t e d t h e v e h i c l e t o an o i l - b a s e d medium. Other r e q u i r e m e n t s f o r the v e h i c l e a r e t h a t i t produce l i t t l e o c u l a r r e a c t i o n and t h a t i t be r e a d i l y a b s o r b e d by t h e e y e . The v e h i c l e s t h a t were t e s t e d were c a s t o r , c o t t o n seed, o l i v e , peanut , and sesame seed o i l s as w e l l as p o l y e t h y l e n e g l y c o l (PEG), which has b o t h h y d r o p h o b i c and h y d r o p h i l i c p r o p e r t i e s . 5 M a t e r i a l s & Methods P r i o r t o s t a r t i n g t h e e x p e r i m e n t s , e a c h r a b b i t i n t h e s t u d y was examined by s i i t - l a m p b i o m i c r o s c o p y t o r u l e out the p r e s e n c e o f p r e - e x i s t i n g o c u l a r d i s e a s e . S i x groups o f f i v e r a b b i t s e a c h ( l o c a l l y s u p p l i e d New Z e a l a n d w h i t e f e m a l e s w e i g h i n g 2 .2 - 2 .4 k g ) w e r e i n j e c t e d s u b c o n j u n c t i v a l l y , f o l l o w i n g t o p i c a l a n a e s t h e s i a ( p r o p a r a c a i n e HC1 (0.5%), by p l a c i n g a 25 gauge n e e d l e a t t a c h e d t o a t u b e r c u l i n s y r i n g e a t a s h a l l o w a n g l e p a r a l l e l and p o s t e r i o r t o t h e s u p e r i o r l i m b u s o f t h e r i g h t eye, and w i t h a smooth mot ion the n e e d l e was i n s e r t e d u n d e r t h e c o n j u n c t i v a and 0.5 c c o f a s t e r i l i z e d v e h i c l e (by f i l t r a t i o n t h r o u g h t a M i l l e x - F G ( R ) 0.22 m i c r o n d i s p o s a b l e f i l t e r u n i t ) was s l o w l y i n j e c t e d . The o p p o s i t e eye s e r v e d as a n o n - t r e a t e d c o n t r o l . The f i r s t g r o u p o f 5 r a b b i t s , e a c h r e c e i v e d 0.5 c c o f c a s t o r o i l , t h e s e c o n d g r o u p c o t t o n s e e d o i l , t h e t h i r d g r o u p p e a n u t o i l , t h e f o u r t h g r o u p o l i v e o i l , t h e f i f t h g r o u p sesame s e e d o i l , and t h e s i x t h g r o u p PEG. The e y e s were t h e n examined by s i i t - l a m p b i o m i c r o s c o p y each day f o r one week, and s e r i a l l y p h o t o g r a p h e d . The d e g r e e o f i n f l a m m a t i o n i n each eye was graded a c c o r d i n g t o the scheme p r e s e n t e d i n T a b l e I , i n which an o v e r a l l a v e r a g e v a l u e o f i n f l a m m a t i o n was o b t a i n e d a f t e r e v a l u a t i n g each c r i t e r i o n f o r each r a b b i t i n t h i s exper iment . The r a b b i t s were a l s o g r a d e d by 2 o t h e r e x a m i n e r s who h a d no k n o w l e d g e o f t h e g r o u p i n g o f t h e d i f f e r e n t r a b b i t s . No s i g n i f i c a n t 6 v a r i a b i l i t y was found among the examiners (data not shown) . The g r o u p s were s a c r i f i c e d w i t h a n o v e r d o s e o f p e n t o b a r b i t a l a f t e r one week. The g l o b e s were e n u c l e a t e d , m a r k e d a t t h e p o i n t o f i n j e c t i o n w i t h a d y e , f i x e d i n 10% b u f f e r e d f o r m a l i n , embedded i n p a r a f f i n , s e c t i o n e d v e r t i c a l l y and s t a i n e d by H a e m a t o x y l i n - E o s i n f o r h i s t o p a t h o l o g i c a l s tudy . H i s t o l o g i c assesment o f d e n s i t y and a r e a o f i n f l a m m a t o r y i n f i l t r a t e was made and an o v e r a l l a v e r a g e v a l u e o f i n f l a m m a t i o n o b t a i n e d . The h i s t o l o g i c assessments were made by m y s e l f and a p a t h o l o g i s t who had no knowledge o f the g r o u p i n g o f the r a b b i t s i n the s tudy . No s i g n i f i c a n t v a r i a b i l i t y was noted between the examiners ( d a t a n o t shown). 7 Table I Conjunctiva Ocular Discharge Erosion of Epithelium % Bleb present Corneal Clarity Iris Injection Anterior Chamber Overall Inflammation Hyperemia Chemosis none none none none 0 clear normal normal 0 trace trace trace trace <10% trace trace trace 1 mild mild mild mild 10%-40% mild mild mild 2 moderate moderate moderate moderate 40%-70% moderate moderate moderate 3 severe severe severe severe 70%-100% severe severe severe 4 R e s u l t s C l i n i c a l O c u l a r T o x i c i t y ( f i g u r e s 1.1 and 1.2) S u b c o n j u n c t i v a l i n j e c t i o n o f 0 .5ml o f t h e v e h i c l e s r e s u l t e d i n v a r y i n g degrees o f i n f l a m m a t i o n . In each case n o t much r e a c t i o n was s e e n i n t h e f i r s t 12 h o u r s , e x c e p t f o r mucus s e c r e t i o n , m i l d edema and h y p e r e m i a o f t h e c o n j u n c t i v a . S l i t - l a m p b i o m i c r o s c o p i c e x a m i n a t i o n i n a l l ca se s showed no a b n o r m a l i t i e s w i t h i n the a n t e r i o r chamber o r v i t r e o u s as t o f l a r e , c e l l s and f i b r i n . The i r i s appeared n o r m a l , and the cornea was c l e a r , and i t s s u r f a c e d i d no t s t a i n w i t h f l u o r e s c e i n . W i t h a l m o s t e v e r y v e h i c l e the b l e b took a p p r o x i m a t e l y s e v e n days t o be absorbed . The t o x i c e f f e c t s o f v e h i c l e s were c o n f i n e d t o the c o n j u n c t i v a , w i t h hyperemia , chemosis and e r o s i o n o f the c o n j u n c t i v a l e p i t h e l i u m b e i n g p r e s e n t t o v a r y i n g degrees , depending on the v e h i c l e . I n g e n e r a l c o t t o n s e e d , o l i v e , p e a n u t and sesame s e e d o i l s showed t h e most a g g r e s s i v e c l i n i c a l r e a c t i o n w i t h moderate t o s e v e r e hyperemia and m i l d t o moderate chemosis o f the c o n j u n c t i v a , as w e l l as moderate t o s e v e r e e r o s i o n o f c o n j u n c t i v a l e p i t h e l i u m i n t h e r a b b i t s . I n t h e s e g r o u p s t h e i n f l a m m a t i o n p e a k e d a t a b o u t t h e t h i r d o r f o u r t h d a y , f r o m w h i c h p o i n t t h e i n f l a m m a t i o n s t a r t e d t o d e c r e a s e . C a s t o r o i l showed a l e s s a g g r e s s i v e c l i n i c a l r e a c t i o n w i t h m i l d t o m o d e r a t e h y p e r e m i a and c h e m o s i s o f t h e c o n j u n c t i v a w i t h t h r e e o f t h e r a b b i t s i n t h i s g r o u p 9 s h o w i n g m i l d t o m o d e r a t e e r o s i o n o f t h e c o n j u n c t i v a l e p i t h e l i u m . The i n f l a m m a t i o n i n the c a s t o r o i l group a l s o p e a k e d b e t w e e n d a y s t h r e e and f o u r , f r o m w h i c h p o i n t t h e i n f l a m m a t i o n s t a r t e d t o d e c r e a s e . The p l o t o f the o v e r a l l i n f l a m m a t i o n o v e r t h e one week p e r i o d i s p r e s e n t e d i n F i g u r e 1.0 and r e p r e s e n t a t i v e photographs o f each v e h i c l e a t one , f o u r and s e v e n d a y s a r e shown i n F i g u r e s 1.1 and 1 .2 . The PEG g r o u p was t h e e x c e p t i o n w i t h t h e b l e b h a v i n g b e e n a b s o r b e d w i t h i n 24 h o u r s o f t h e i n j e c t i o n and o n l y s h o w i n g t r a c e h y p e r e m i a and c h e m o s i s o f t h e c o n j u n c t i v a o v e r t h e f i r s t 48 h o u r s , f r o m t h e r e on t h e eye a p p e a r e d n o r m a l . H i s t o p a t h o l o g i c F i n d i n g s The v e h i c l e s i n our s tudy produced v a r y i n g degrees o f i n f l a m m a t i o n , r e s t r i c t e d t o the c o n j u n c t i v a and p o s t e r i o r s o f t t i s s u e s . T h e r e s t o f t h e e y e i t s e l f a p p e a r e d u n i n v o l v e d . An i n t e r m e d i a t e t o s e v e r e r e a c t i o n o c c u r r e d w i t h c a s t o r o i l , w h i c h s p r e a d as f a r p o s t e r i o r l y as t h e o p t i c n e r v e a n d c a u s e d a n e x u b e r a n t i n f l a m m a t o r y i n f i l t r a t e . An i n t e r m e d i a t e r e a c t i o n was seen w i t h c o t t o n s e e d , o l i v e , p e a n u t and sesame s e e d o i l ; t h e s e a g e n t s t y p i c a l l y r e a c h e d t h e e q u a t o r o f t h e g l o b e and i n d u c e d a m o d e r a t e i n f l a m m a t o r y r e s p o n s e . T h e l e a s t r e a c t i o n r e s u l t e d w i t h PEG w h i c h a p p e a r e d t o be e n t i r e l y a b s o r b e d w i t h l i t t l e o r no i n f l a m m a t i o n . 10 A l t h o u g h the degree o f i n f l a m m a t i o n v a r i e d , t h e p a t t e r n o f t h e r e s p o n s e was s i m i l a r i n a l l c a s e s . Two h o u r s f o l l o w i n g i n j e c t i o n , a few p o l y m o r p h o n u c l e a r g r a n u l o c y t e s and l y m p h o c y t e s c o u l d be s e e n s t r e a m i n g f r o m d i l a t e d c a p i l l a r i e s . By t w e l v e h o u r s , t h e s e h a d s u r r o u n d e d t h e o i l d r o p l e t s , which were g r a d u a l l y d i f f u s i n g p o s t e r i o r l y . T w e n t y f o u r h o u r s a f t e r i n j e c t i o n , t h e s e a c u t e i n f l a m m a t o r y c e l l s were j o i n e d by m a c r o p h a g e s , some o f w h i c h a p p e a r e d t o be i n g e s t i n g t h e o i l . By one week, t h e i n f l a m m a t i o n e x h i b i t e d a granulomatous p a t t e r n , w i t h o c c a s i o n a l f o r e i g n body g i a n t c e l l s , foamy m a c r o p h a g e s , l ymphocytes and p lasma c e l l s . The h i s t o l o g i c a l f i n d i n g s o f the v a r i o u s v e h i c l e s are shown i n f i g u r e s 1 .3 -1 .8 . 11 F i g u r e 1.0 OCULAR INFLAMMATORY RESPONSE FOLLOWING SUBCONJUNCTIVAL INJECTION OF VEHICLES 4 - , 0 1 ^ - 2 3 4 5 6 ' 7 TIME (Days) 12 COLOUR PHOTOGRAPHS SHOULD NOT BE USED. THEY WILL APPEAR AS GREY OR BLACK. WE RECOMMEND THAT THE COPY OF THE THESIS SUBMITTED FOR MICROFILMING INCLUDE BLACK AND WHITE PHOTOGRAPHS REPRINTED FROM THE COLOUR PHOTOGRAPHS BY A PHOTOGRAPHER IF NECESSARY. L O R S Q U E M I C R 0 F I L M E E S , L E S PHOTOGRAPHIES EN COULEUR PARAISSENT GRISES OU NOIRES. NOUS RECOMMANDONS QUE L 1 E X E M P L A I R E DE LA THESE A MICROFILMER SOIT ACCOMPAGNE PLUTOT DE PHOTOGRAPHIES EN NOIR ET BLANC PRODUITES A PARTIR DES PHOTOGRAPHIES EN COULEURS PAR UN PHOTOGRAPHE , SI NECESSAIRE. F i g u r e 1.1 C l i n i c a l O c u l a r I n f l a m m a t o r y Response to the V e h i c l e s T e s t e d c a s t o r o i l a t day 1 c a s t o r o i l a t day 4 c a s t o r o i l a t day 7 c o t t o n seed o i l c o t t o n seed o i l c o t t o n seed o i l a t day 1 a t day 4 a t day 7 o l i v e o i l a t day 1 o l i v e o i l a t day 4 o l i v e a t day 7 13 F i g u r e 1.2 C l i n i c a l O c u l a r I n f l a m m a t o r y Response to the V e h i c l e s T e s t e d C o n t ' d peanut o i l a t day 1 peanu t o i l a t day 4 peanu t o i l a t day 7 sesame seed o i l sesame seed o i l sesame seed o i l a t day 1 a t day 4 a t day 7 u F i g u r e 1 .3* H i s t o l o g i c a l e f f e c t s o f C a s t o r O i l a t one week 15 F i g u r e 1.4 H i s t o l o g i c a l e f f e c t s o f C o t t o n Seed O i l a t one week F i g u r e 1.5 H i s t o l o g i c a l e f f e c t s o f O l i v e O i l a t one week F i g u r e 1.6 H i s t o l o g i c a l e f f e c t s o f Peanut O i l a t one week F i g u r e 1.7 H i s t o l o g i c a l e f f e c t s o f P o l y e t h y l e n e G l y c o l ( P E G ) a t one week F i g u r e 1.8 H i s t o l o g i c a l e f f e c t s o f Sesame Seed O i l a t one week DISCUSSION In p r e v i o u s s t u d i e s i n t h i s l a b o r a t o r y , the drugs t h a t h a d b e e n g i v e n s u b c o n j u n c t i v a l l y w e r e s o l u b l e i n s a l i n e . ' Most o f the o c u l a r t o x i c i t y t o the d r u g c o u l d be c o n t r o l l e d by a d j u s t i n g t h e amount o f d r u g i n j e c t e d s u b c o n j u n c t i v a l l y . C y c l o s p o r i n e (Cy) p r e s e n t e d us w i t h a u n i q u e p r o b l e m , b e c a u s e i t s c h e m i s t r y demanded t h a t t h e v e h i c l e had t o be l i p o p h i l i c . F o r Cy t o be i n j e c t e d s u b c o n j u n c t i v a l l y t h e o c u l a r t o x i c i t y o f two v a r i a b l e s had t o be d e t e r m i n e d , t h a t o f the v e h i c l e and o f Cy. A number o f v e h i c l e s were chosen as p o s s i b l e c a n d i d a t e s f o r s u b c o n j u n c t i v a l i n j e c t i o n . These were s e l e c t e d from a g r o u p o f v e h i c l e s t h a t h a d p r e v i o u s l y b e e n u s e d as p h a r m a c o l o g i c a l s o l v e n t s and i n which Cy would be r e a d i l y s o l u b l e . 4 6 The v e h i c l e s s e l e c t e d f o r t h e s t u d y i n c l u d e : c a s t o r , c o t t o n s e e d , p e a n u t , o l i v e and sesame s e e d o i l as w e l l as p o l y e t h y l e n e g l y c o l (PEG) . T h e s e were s t e r i l i z e d and 0 . 5 c c ( t h i s b e i n g t h e amount t h e r a b b i t s ' c o n j u n c t i v a c a n t o l e r a t e ) o f each was i n j e c t e d s u b c o n j u n c t i v a l l y . D a i l y o b s e r v a t i o n s were made by s l i t lamp b i o m i c r o s c o p y t o a s c e r t a i n t h e e f f e c t s o f t h e v e h i c l e s w i t h i n and s u r r o u n d i n g t h e e y e . T h e m a j o r t o x i c e f f e c t s w e r e c o n f i n e d t o t h e c o n j u n c t i v a r e s u l t i n g i n h y p e r m i a , c h e m o s i s and e r o s i o n o f t h e c o n j u n c t i v a l e p i t h e l i u m . O c c a s i o n a l l y the v e h i c l e s would cause a c o r n e a l edema, but t h i s was i n f r e q u e n t . The v e h i c l e s produced v a r y i n g degrees o f i n f l a m m a t i o n w i t h P E G c a u s i n g a t r a c e i n f l a m m a t o r y r e a c t i o n h i s t o l o g i c a l l y and c l i n i c a l l y . T h i s r e s u l t i s n o t e n t i r e l y u n e x p e c t e d b e c a u s e P E G ' S a r e compounds o f low t o x i c i t y and t h e PEG t h a t was i n j e c t e d s u b c o n j u n c t i v a l l y was made up so t h a t i t h a d a t o n i c i t y e q u i v a l e n t t o t h a t found i n the o c u l a r chambers (0.9% NaCl) and was absorbed w i t h i n 24 hours a f t e r i n j e c t i o n . U n f o r t u n a t e l y , the use o f PEG was not p o s s i b l e because when I a t tempted t o d i s s o l v e Cy i n i t , l e s s t h a t 0.5 m i l l i g r a m s d i s s o l v e d i n 10 c c . o f PEG s o l u t i o n (data not shown). The o i l s showed a g r e a t e r a f f i n i t y f o r Cy (on average 5 mg o f Cy d i s s o l v e d i n 0.5 c c . o f o i l ) , but a l s o produced a g r e a t e r i n f l a m m a t o r y r e s p o n s e , p a r t l y b e c a u s e t h e y were a b s o r b e d a t a s l o w e r r a t e and t h e t o n i c i t y c o u l d n o t be a d j u s t e d t o t h a t o f t h e eye . C o t t o n s e e d , o l i v e , p e a n u t and sesame seed o i l produced an i n t e r m e d i a t e i n f l a m m a t o r y r e a c t i o n h i s t o l o g i c a l l y , but the c l i n i c a l f i n d i n g s showed a moderate t o s e v e r e r e a c t i o n . An i n t e r m e d i a t e t o s e v e r e r e a c t i o n was seen h i s t o l o g i c a l l y w i t h c a s t o r o i l , but a g a i n t h e s e f i n d i n g s d i d n o t c o r r e l a t e w e l l w i t h t h e c l i n i c a l f i n d i n g s w h i c h showed a m i l d t o m o d e r a t e i n f l a m m a t o r y r e s p o n s e . T h e s e d i s c r e p a n c i e s may p a r t l y be e x p l a i n e d by t h e s p r e a d o f the o i l p o s t e r i o r l y . In the case o f c a s t o r o i l , i t s p r e a d as f a r b a c k as t h e o p t i c n e r v e , t h e r e b y c a u s i n g 22 l e s s o f a r e a c t i o n w i t h the c o n j u n c t i v a o v e r the s u p e r i o r l i m b u s , r e s u l t i n g i n a m i l d t o m o d e r a t e r e s p o n s e v i s i b l e c l i n i c a l l y . B e c a u s e t h e o i l h a d s p r e a d so f a r b a c k , more o i l was e x p o s e d t o t h e o c u l a r t i s s u e ; h e n c e , a g r e a t e r i n f l a m m a t o r y r e a c t i o n was seen h i s t o l o g i c a l l y . W i t h c o t t o n s e e d , o l i v e , p e a n u t and sesame s e e d o i l , t h e v e h i c l e s s p r e a d up t o t h e e q u a t o r o f t h e g l o b e . I n t h e case o f t h e s e v e h i c l e s , most o f the o i l was r e s t r i c t e d to t h e c o n j u n c t i v a o v e r t h e s u p e r i o r l i m b u s , e x p o s i n g t h i s p a r t o f t h e c o n j u n c t i v a f o r a l o n g e r p e r i o d o f t i m e t o t h e e n v i r o n m e n t . T h e r e f o r e , a m o d e r a t e t o s e v e r e c l i n i c a l r e a c t i o n was seen, and because l e s s o f the o i l was exposed t o t h e o c u l a r t i s s u e s t h e e n s u i n g i n f l a m m a t o r y r e a c t i o n t h a t was seen h i s t o l o g i c a l l y was l e s s . On a v e r a g e , t h e o i l s p r o d u c e d a s i m i l a r r e s p o n s e . However, s i n c e c a s t o r o i l spread the f u r t h e s t p o s t e r i o r l y we r e a s o n e d more Cy c o u l d be made a v a i l a b l e t o t h e eye u s i n g t h i s o i l . We f e l t u s i n g c a s t o r o i l as a b a s e f o r a v e h i c l e f o r t h e d e l i v e r y o f Cy, the advantageous p r o p e r t i e s o f c a s t o r o i l can be e x p l o i t e d . C r e m o p h o r ( R ) E L i s s u c h a c o m m e r c i a l l y a v a i l a b l e v e h i c l e , u s e d i n t h e I . V . f o r m o f C y . The c o n t e n t s o f C r e m o p h o r ( R ) E L c o n s i s t o f 33% a l c o h o l , c a s t o r o i l and p o l y o x y e t h y l e n e g l y c o l (POEG) 650mg. The POEG may a c t l i k e a c u s h i o n t o d e c r e a s e t h e e f f e c t s o f t h e v e h i c l e on t h e c o n j u n c t i v a , and t h e a l c o h o l p r o v i d e s two f u n c t i o n s ; i t a l l o w s more Cy t o be d i s s o l v e d i n t h e c a s t o r o i l so l e s s v e h i c l e h a s t o be u s e d , and a t t h e same t i m e i t r e d u c e s t h e v i s c o s i t y o f t h e c a s t o r o i l p o s s i b l y m a k i n g t h e v e h i c l e l e s s u n c o m f o r t a b l e t o the eye. The r e s u l t a n t o f a l l these p r o p e r t i e s m i g h t be t o f u r t h e r d e c r e a s e i n f l a m m a t o r y re sponse t o the v e h i c l e , and t o make p o s s i b l e the r e p e a t e d i n j e c t i o n s o f Cy s u b c o n j u n t i v a l l y . C o n c l u s i o n s The o i l s on t h e a v e r a g e g a v e a s i m i l a r r e s p o n s e , b u t from a c l i n i c a l p e r s p e c t i v e o n l y c a s t o r o i l appeared t o be l e s s s e v e r e . A c o m m e r c i a l l y a v a i l a b l e v e h i c l e which i n c o r p o r a t e d c a s t o r o i l a l l o w e d f o r more d e l i v e r y o f t h e d r u g i n l e s s v e h i c l e t h a n c o u l d be o b t a i n e d by u s i n g c a s t o r o i l a l o n e as a v e h i c l e . On t h i s b a s i s we reasoned t h e o r e t i c a l l y the commerc ia l p r e p a r a t i o n s h o u l d produce l e s s o f a l o c a l response . U n f o r t u n a t e l y , a t t h e t i m e we were n o t a b l e t o o b t a i n t h e C r e m o p h o r ( R) E L w i t h o u t t h e C y . T h e r e f o r e , we c o u l d o n l y s p e c u l a t e about the e f f e c t s o f the v e h i c l e from our e x p e r i e n c e w i t h t h e o i l s . One w o u l d e x p e c t much o f t h e i n f l a m m a t o r y r e a c t i o n t o be due t o the v e h i c l e and not Cy. We have s i n c e o b t a i n e d the v e h i c l e used i n the Sandimmune I . V . ( R ) p r e p a r a t i o n and found t h a t most o f the i n f l a m m a t o r y r e a c t i o n was indeed due t o the v e h i c l e . In those r a b b i t s t h a t r e c e i v e d Sandimmune I . V . ( R ) s u b c o n j u n c t i v a l l y , t h e i n f l a m m a t o r y response was found t o be l e s s than w i t h t h e v e h i c l e a l o n e (data and exper iments not shown). The o r i g i n a l as sumpt ion , t h a t most o f the i n f l a m m a t o r y r e a c t i o n w o u l d be due t o t h e v e h i c l e u s e d i n Sandimmune I . V . ( R ) and n o t Cy was c o r r e c t , and s i n c e t h e c o m m e r c i a l p r e p a r a t i o n a l l o w s f o r more d r u g t o be d e l i v e r e d i n l e s s v e h i c l e t h a n c o u l d be o b t a i n e d u s i n g the o i l s a l o n e , the i n f l a m m a t o r y r e a c t i o n c o u l d be g r e a t l y reduced . 25 CHAPTER 2 Dose d e t e r m i n a t i o n s t u d y I n t r o d u c t i o n From t h e r e s u l t s o b t a i n e d i n C h a p t e r 1, i t was d e c i d e d t h a t t h e c o m m e r c i a l p r e p a r a t i o n Sandimmune I . V . ( R ) c o n t a i n i n g the v e h i c l e Cremophor (R) EL w o u l d be used. T h i s was based on i t s a v a i l a b i l i t y as w e l l as i t s a b i l i t y t o d e l i v e r more Cy (50 mg/ml) . To comple te the f o r m u l a t i o n o f the s u b c o n j u n c t i v a l dosage f o r C y , t h e maximum t o l e r a t e d dose had t o be de termined . T h i s was done by t e s t i n g a range o f doses . M a t e r i a l s and Methods Three groups o f f i v e r a b b i t s each ( l o c a l l y s u p p l i e d New Z e a l a n d w h i t e f e m a l e s w e i g h t i n g 2.2 - 2.4 k g . ) w e r e examined by s l i t - l a m p b i o m i c r o s c o p y p r i o r t o the exper iment t o r u l e o u t p r e - e x i s t i n g o c u l a r d i s e a s e . T h e s e were i n j e c t e d s u b c o n j u n c t i v a l l y p o s t e r i o r t o the s u p e r i o r l i m b u s w i t h 5, 10 o r 25 mg o f Cy {SANDIMMUNE I . V . (50 m g / m l o f Cy i n Cremphor)} u s i n g a 25 gauge n e e d l e a t t a c h e d t o a t u b e r c u l i n s y r i n g e ( p r i o r t o i n j e c t i o n the r i g h t eyes were a n a e s t h e t i z e d w i t h t o p i c a l p r o p a r a c a i n e HC1 (0.5%)}. The o p p o s i t e eye s e r v e d as a n o n - t r e a t e d c o n t r o l , a s we were u n a b l e t o o b t a i n t h e C r e m o p h o r ( R) E L w i t h o u t Cy . The eyes were examined by s l i t - l a m p b i o m i c r o s c o p y each day f o r one week, and s e r i a l l y photographed . The degree o f i n f l a m m a t i o n i n each eye was graded a c c o r d i n g t o the scheme 26 p r e s e n t e d i n T a b l e I I , i n which an o v e r a l l a v e r a g e d v a l u e was o b t a i n e d by a s s e s s i n g each c r i t e r i o n f o r the i n d i v i d u a l r a b b i t s i n t h i s e x p e r i m e n t . The g r o u p s were s a c r i f i c e d w i t h an o v e r d o s e o f p e n t o b a r b i t a l a f t e r one week. The g l o b e s were e n u c l e a t e d , m a r k e d a t t h e p o i n t o f i n j e c t i o n w i t h a d y e , f i x e d i n 10% b u f f e r e d f o r m a l i n , embedded i n p a r a f f i n , s e c t i o n e d v e r t i c a l l y and s t a i n e d by H a e m a t o x y l i n -E o s i n f o r h i s t o p a t h o l o g i c a l s tudy . ' 27 Table II Conjunctiva Ocular Discharge Erosion of Epithelium % Bleb present Corneal Clarity Iris Injection Anterior Chamber Overall Inflammation Hyperemia Chemosis none none none none 0 clear normal normal 0 trace trace trace trace <10% trace trace trace 1 mild mild mild mild 10%-40% mild mild mild 2 moderate moderate moderate moderate 40%-70% moderate moderate moderate 3 severe severe severe severe 70%-100% severe severe severe 4 R e s u l t s C l i n i c a l O c u l a r T o x i c i t y T h e two h i g h e s t d o s e s o f Cy were j u d g e d t o be t o o t o x i c . The 10 mg s u b c o n j u n c t i v a l dose o f Cy produced w i t h i n t h e f i r s t 24 hours moderate t o s e v e r e hyperemia and c h e m o s i s o f c o n j u n c t i v a a s w e l l a s m o d e r a t e mucus s e c r e t i o n . The 25 mg s u b c o n j u n c t i v a l dose o f Cy produced e r o s i o n o f t h e c o n j u n c t i v a l e p i t h e l i u m and m i l d t o m o d e r a t e h a i r l o s s o f t h e u p p e r e y e l i d , s e c o n d a r y t o i n f l a m m a t i o n and s e l f - i n d u c e d l o c a l trauma. O v e r the nex t s i x days t h e i n f l a m m a t i o n s l o w l y r e s o l v e d . The s u b c o n j u n c t i v a l i n j e c t i o n o f a s i n g l e 5 mg i n O . l c c d o s e o f Cy was found t o be the l e a s t t o x i c . I t o n l y produced t r a c e t o m i l d h y p e r e m i a and c h e m o s i s o f t h e c o n j u n c t i v a w i t h t r a c e mucus s e c r e t i o n . Most o f these changes had r e s o l v e d w i t h i n 48 hours o f i n j e c t i o n o f the Cy. From i n v e s t i g a t i o n s p e r f o r m e d w i t h r e g a r d t o t h e f r e q u e n c y o f i n j e c t i o n s we no ted t h a t a s i n g l e 5 mg i n O . l c c dose o f Cy once p e r week w o u l d be c l i n i c a l l y t o l e r a b l e f o r o u r s t u d i e s . We n o t e d t h a t i n j e c t i o n s o f more than once p e r week l e d t o i n t o l e r a b l e l o c a l i n f l a m m a t i o n ( d a t a n o t shown ). O b s e r v a t i o n s on the e f f e c t o f t h e s e doses i s summarized i n a d a i l y p l o t o f t h e o v e r a l l i n f l a m m a t o r y r e s p o n s e f o r e a c h d a y i n f i g u r e 2.0. The l o n g - t e r m s tudy o f the s u b c o n j u n c t i v a l i n j e c t i o n o f 5 mg i n O . l c c o f Cy once a week f o r two months showed no t o x i c e f f e c t s t h a t were not p r e s e n t w i t h the 29 s i n g l e s u b c o n j u n c t i v a l i n j e c t i o n o f 5 mg i n O . l c c o f C y , e x c e p t b e c a u s e o f t h e r e p e a t e d i n j e c t i o n s o f Cy t h e c o n j u n c t i v a r e a c t e d s l i g h t l y more t h a n was seen w i t h j u s t t h e s i n g l e s u b c o n j u n c t i v a l i n j e c t i o n o f 5 mg i n O . l c c o f Cy, but the r e a c t i o n l a r g e l y r e s o l v e d w i t h i n 24 t o 48 hours a f t e r i n j e c t i o n . O c c a s i o n a l l y a t r a n s i t o r y r e a c t i o n was s e e n w i t h a l l d o s e s . T h i s was m o d e r a t e t o s e v e r e edema o f t h e u p p e r e y e l i d o f t h e r a b b i t , w h i c h u s u a l l y r e s o l v e d w i t h i n 48 h o u r s . I n some i n s t a n c e s , t h e edematous r e a c t i o n was p o s s i b l y due t o t h e r a b b i t m o v i n g upon i n j e c t i o n w h i c h i n a d v e r t e n t l y r e s u l t e d i n the n e e d l e p e n e t r a t i n g the upper e y e l i d , c a u s i n g t h e s w e l l i n g o f t h e e y e l i d . R e p r e s e n t a t i v e p h o t o g r a p h s s h o w i n g t h e e f f e c t o f t h e v a r i o u s d o s e s a t o n e , f o u r , and s e v e n d a y s a r e shown i n f i g u r e s 2 . 1 - 2 . 2 . H i s t o p a t h o l o g i c a l F i n d i n g s From o u r o b s e r v a t i o n s i n C h a p t e r 1 o u r d a t a s u g g e s t s some s u p p r e s s i o n o f the i n f l a m m a t o r y response when we used Sandimmune I . V . ( R ) . A t two h o u r s , t h e s u b c o n j u n c t i v a l t i s s u e s o f t h e e x p e r i m e n t a l eye showed few p o l y m o r p h o n u c l e a r g r a n u l o c y t e s (PMN) by 12 h o u r s , and by 24 h o u r s , t h e e x p e r i m e n t a l eye c o n t a i n e d o n l y a s c a t t e r i n g o f macrophages and lymphocytes . By one week t h e e x p e r i m e n t a l eye showed o n l y a few lymphocytes and macrophages. 30 I n c r e a s i n g t h e d o s a g e o f Cy t o 10 and 25 mg r e s u l t e d i n a d d i t i o n a l t o x i c s i d e e f f e c t s . , 10 mg i n j e c t i o n o f Cy caused e p i t h e l i a l i r r e g u l a r i t i e s and d e n a t u r a t i o n o f c o l l a g e n and m usc l e . 25 mg i n j e c t i o n s a l s o caused l o c a l i z e d s c a r r i n g o f the p e r i o c u l a r t i s s u e s . S u b s e q u e n t e x p e r i m e n t a l work done by o u r g r o u p w i t h Sandimmune I . V . ( R ) p r e p a r a t i o n , s u g g e s t e d t h a t i n d e e d t h e i n f l a m m a t o r y response w i t h Sandimmune I . V . ( R ) was l e s s than w i t h the v e h i c l e a l o n e . O J -These l o c a l s i d e e f f e c t s were l e s s n o t i c e a b l e i n t h e r a b b i t s g i v e n 5 mg i n 0.1 c c o f Cy o n c e w e e k l y f o r 2 m o n t h s , b u t t h e y s h o w e d l y m p h o c y t e a n d p l a s m o c y t e i n f i l t r a t e s i n the r e n a l c o r t e x i n t e r s t i t i u m and h e p a t i c p o r t a l t r i a d s . The h i s t o l o g i c a l f i n d i n g s o f the v a r i o u s doses and the l o n g - t e r m e f f e c t s o f s u b c o n j u n c t i v a l l y a d m i n i s t e r e d Cy are shown i n f i g u r e s 2 . 3 - 2 . 6 . S t a t i s t i c a l A n a l y s i s An a n a l y s i s o f v a r i a n c e (nested) t e s t showed t h a t t h e r e was a s i g n i f i c a n t d i f f e r e n c e be tween t h e v a r i o u s d o s e s t e s t e d o f the c o m m e r c i a l l y a v a i l a b l e p r e p a r a t i o n . The l e v e l o f s i g n i f i c a n c e between the doses was found t o be p=0.0001. 31 2.0 OCULAR INFLAMMATORY RESPONSE TO DIFFERENT DOSES OF CYCLOSPORINE (SANDIMMUNE I.V.) SUBCONJUNCTIVALLY x x 5mg/0.lcc OF CYCLOSPORINE » . lOmg/O.Ecc OF CYCLOSPORINE o o 25mg/0.5cc OF CYCLOSPORINE TIME (Days) 32 F i g u r e 2 . 1 E f f e c t s o f 5 m g / 0 . 1 c c o f C y c l o s p o r i n e a t d a ^ 1 a t day 4 a t day 7 33 F i g u r e 2 . 2 E f f e c t s o f 2 5 m g / 0 . 5 c c o f C y c l o s p o r i n e 6&y 5 a t day 30 a t day 60 F i g u r e 2 . 3 * H i s t o l o g i c a l e f f e c t s o f 5 m g / 0 . 1 c c o f C y c l o s p o r i n e a t one week 36 F i g u r e 2 . 5 H i s t o l o g i c a l e f f e c t s o f 2 5 m g / 0 . 5 c c o f C y c l o s p o r i n e a t one week lOx A m i l d i n f l a m m a t o r y r e s p o n s e i s p r e s e n t a t one week. The c o l l a g e n i s d i s r u p t e d and t h e r e i s a n e c r o t i c z o n e ( N Z ) p r e s e n t i n t he l a m i n a p r o p r i a . 37 F i g u r e 2 . 6 H i s t o l o g i c a l e f f e c t s o f 5 m g / 0 . 1 c c o f C y c l o s p o r i n e once p e r week t h a t r e c i e v e d t h i s dose f o r two months O n l y a t r a c e i n f l a m m a t o r y r e s p o n s e i s p r e s e n t a t t he end o f two 38 DISCUSSION The dose d e t e r m i n a t i o n s tudy showed t h a t 5 mg i n O . l c c o f t h e c o m m e r c i a l l y p r e p a r e d Cy was t h e l e a s t t o x i c , c a u s i n g o n l y m i l d hyperemia which had l a r g e l y r e s o l v e d by 24 t o 48 h o u r s . W i t h i n c r e a s i n g d o s e s (10 mg i n 0 .2cc and 25 mg i n 0 .5cc) , the s i d e e f f e c t s became more a p p a r e n t w i t h m o d e r a t e h y p e r e m i a and c h e m o s i s o f t h e c o n j u n c t i v a and exuberant mucus d i s c h a r g e , and a t the h i g h e s t dose h a i r l o s s o f the upper e y e l i d due t o l o c a l trauma and i n f l a m m a t i o n . A l t h o u g h t h e i n f l a m m a t i o n w i t h t h e h i g h e r d o s e s had a l m o s t r e s o l v e d by one week, the c o n j u n c t i v a were s t i l l too s e n s i t i v e a n d c o u l d n o t be r e i n j e c t e d a t t h a t t i m e . W i t h the low dose (5 mg i n O. lcc) a d m i n i s t r a t i o n the c o n j u n c t i v a l o o k e d a l m o s t n o r m a l a t one week, and f r o m i n v e s t i g a t i o n s p e r f o r m e d on t h e f r e q u e n c y o f i n j e c t i o n (data not shown), t h i s dose was found t o be a c c e p t a b l e f o r i n j e c t i o n once p e r week. A t h i g h e r doses t h e r e i s some s u g g e s t i o n o f s u p p r e s s i o n o f the l o c a l i z e d i n f l a m m a t o r y re sponse , but p a r t i c u l a r l y w i t h 25 mg i n 0 .5cc i r r e g u l a r i t y o f t h e c o n j u n c t i v a l e p i t h e l i u m , d e n a t u r a t i o n o f the c o l l a g e n and musc le as w e l l as f i b r o b l a s t i c response were noted . T h e s e e f f e c t s c a n l a r g e l y be a t t r i b u t e d t o t h e amount o f v e h i c l e i n j e c t e d a n d n o t t o C y . S i n c e t h e Cy (Sandimmune I . V . ( R ) ) used was o n l y a v a i l a b l e as 50 mg/ml , the amount o f v e h i c l e t h a t was g i v e n was governed 39 by t h e c o n c e n t r a t i o n o f Cy . As t h e d o s e was i n c r e a s e d i n v a r i a b l y the amount o f v e h i c l e was a l s o i n c r e a s e d , c a u s i n g a g r e a t e r i n f l a m m a t o r y response o f the e y e . 8 1 E v e n so , the i n f l a m m a t o r y response seen w i t h the h i g h e r d o s e s was much l e s s t h a n w i t h any o f t h e v e h i c l e s t e s t e d , shown i n an i n d i r e c t way from the assumpt ions made i n C h a p t e r 1 r e g a r d i n g the commerc ia l p r e p a r a t i o n . F o r i n t e r e s t , we t e s t e d t h e e f f e c t o f p e a n u t o i l and p e a n u t o i l c o n t a i n i n g 10 m g / m l o f C y , w h i c h was p r e p a r e d l o c a l l y . F i v e r a b b i t s were i n j e c t e d s u b c o n j u n c t i v a l l y w i t h 0.5 c c o f p e a n u t o i l i n t h e l e f t eye (wh ich s e r v e d as t h e c o n t r o l eye) and 5 mg o f Cy i n 0 .5cc o f p e a n u t o i l t o t h e r i g h t eye (which s e r v e d as the e x p e r i m e n t a l eye) . There was e v i d e n c e , f r o m t h e d i f f e r e n c e s be tween t h e h i s t o l o g i c a l o b s e r v a t i o n s o f the c o n t r o l eye ( r e c e i v e d peanut o i l o n l y ) and e x p e r i m e n t a l eye ( p e a n u t o i l and C y ) , w h i c h s u p p o r t s t h e c u r r e n t sugges ted mechanism o f Cy a c t i o n . 5 6 - 6 0 T h e f o l l o w i n g i n f e r e n c e s c o u l d be made, f r o m t h e d i f f e r e n c e i n the c e l l p o p u l a t i o n s between the c o n t r o l and e x p e r i m e n t a l eye . Though f u r t h e r r e s e a r c h i s n e e d e d t o c o n c l u s i v e l y show t h a t the f o l l o w i n g does o c c u r . Cy seemed t o cause i n h i b i t i o n o f lymphocyte i n f i l t r a t i o n a t the l e v e l o f the T - c e l l . C u r r e n t e v i d e n c e t o d a t e i n d i c a t e s Cy i s a b l e t o a c c o m p l i s h t h i s by i n h i b i t i o n o f i n t e r l e u k i n - I I p r o d u c t i o n f r o m T - h e l p e r c e l l s , and b l o c k i n g t h e r e c e p t o r a c q u i s i t i o n f o r i n t e r l e u k i n - I I by p r e c u r s o r c y t o t o x i c T - c e l l s , as w e l l as 40 d i m i n i s h i n g the r e s p o n s i v e n e s s o f T - h e l p e r c e l l s t o i n t e r l e u k i n - 1 . 1 5 , 5 6 - 6 0 N o r m a l l y d u r i n g a c u t e i n f l a m m a t i o n p o l y m o r p h o n u c l e a r g r a n u l o c y t e s (PMN's) p r e d o m i n a t e i n t h e f i r s t s i x t o 24 h o u r s b e i n g r e p l a c e d by m o n o c y t e s i n 24 t o 48 h o u r s . 4 7 T h i s i n f i l t r a t i o n was s e e n i n t h e c o n t r o l e y e s , b u t t h e e x p e r i m e n t a l eyes showed fewer PMN's and macrophages which became more e v i d e n t w i t h t i m e . T h i s sugges t s t h a t Cy c o u l d i n f l u e n c e t h e m i g r a t i o n o f PMN's t o t h e s i t e o f t h e i n j e c t i o n , and s i n c e PMN's r e l e a s e c h e m o t a c t i c f a c t o r s f o r macrophages, the r e s u l t i n g cascade seems t o be i n h i b i t e d by Cy. T h i s i s r e f l e c t e d by t h e c e l l p o p u l a t i o n seen i n t h e e x p e r i m e n t a l eye . The mechan i sm by w h i c h Cy c o u l d i n t e r f e r e w i t h the PMN's i s u n c l e a r , but i n t e r f e r e n c e w i t h C 5 a - i n d u c e d chemotaxis has been sugges ted as u n l i k e l y . 6 1 ' 6 2 The c u r r e n t l y h e l d premise t h a t the major a c t i o n s o f Cy a r e t h r o u g h the s u p p r e s s i o n o f T - c e l l s 5 8 - 6 0 i s s u p p o r t e d f r o m t h e h i s t o l o g i c o b s e r v a t i o n s o f a d e c r e a s e i n t h e number o f lymphocytes seen i n the c o n j u n c t i v a o f the r a b b i t s eye, w i t h a p o s s i b l e minor e f f e c t on the s u p p r e s s i o n o f PMN a c t i v i t y . These h i s t o l o g i c f i n d i n g s are p r e s e n t e d i n F i g u r e s 2.7a through 2.7h. The c l i n i c a l and h i s t o l o g i c a l f i n d i n g s s u g g e s t e d a s i n g l e dose (5 mg i n O. lcc) o f t h e c o m m e r c i a l l y p r e p a r e d Cy once p e r week was t o l e r a t e d b e t t e r t h a n more f r e q u e n t a d m i n i s t r a t i o n . To a s c e r t a i n whether repea ted F i g u r e 2.7a H i s t o l o g i c a l e f f e c t s o f a s u b c o n j u n c t i v a l i n j e c t i o n o f 0.5cc o f p e a n u t o i l , c o n t r o l , ( A ) and p e a n u t o i l and Cy 5 mg/ml, e x p e r i m e n t a l , ( B ) a t 2 h o u r s . A t t h i s t i m e t h e r e i s a d i f f e r e n c e i n t h e i n f l a m m a t o r y i n f i l t r a t e , though n ot d r a m a t i c , w i t h more PMN's p r e s e n t i n t h e c o n t r o l ( A ) . 42 F i g u r e 2.7b H i s t o l o g i c a l e f f e c t s o f a s u b c o n j u n c t i v a l i n j e c t i o n o f 0.5cc o f p e a n u t o i l , c o n t r o l , ( A ) and p e a n u t o i l and Cy 5 mg/ml, e x p e r i m e n t a l , ( B ) a t 4 h o u r s . The d i f f e r e n c e i n t h e amount o f i n f l a m m a t o r y i n f i l t r a t e i s b e c o m i n g more o b v i o u s w i t h t h e c o n t r o l ( A ) s h o w i n g more PMN's. 43 F i g u r e 2.7c H i s t o l o g i c a l e f f e c t s o f a s u b c o n j u n c t i v a l i n j e c t i o n o f 0.5cc o f p e a n u t o i l , c o n t r o l , ( A ) and p e a n u t o i l and Cy 5 mg/ml, e x p e r i m e n t a l , ( B ) a t 12 h o u r s . T h e r e i s a d i s t i n c t d i f f e r e n c e i n t h e n u m b e r o f i n f l a m m a t o r t y c e l l s p r e s e n t , m a i n l y PMN's as w e l l a s c o m p a r t m e n t a l i z a t i o n o f t h e o i l i n t h e c o n t r o l (A) and t h e e x p e r i m e n t a l ( B ) a p p e a r i n g q u i e s c e n t . A. B. 44 F i g u r e 2.7d H i s t o l o g i c a l e f f e c t s o f a s u b c o n j u n c t i v a l i n j e c t i o n o f 0.5cc o f p e a n u t o i l , c o n t r o l , (A) and 5 mg o f Cy i n 0.5cc o f peanut o i l , e x p e r i m e n t a l , ( B ) a t 1 week. The d i f f e r e n c e i n t h e c e l l p o p u l a t i o n between t h e c o n t r o l a n d t h e e x p e r i m e n t a l e y e i s q u i t e d r a m a t i c w i t h t h e e x p e r i m e n t a l one a p p e a r i n g a l m o s t a c e l l u l a r and t h e c o n t r o l showing q u i e t a heavy i n f l a m m a t o r y i n f i l t r a t e . A. 45 a d m i n i s t r a t i o n o f t h i s dose would have any a d v e r s e e f f e c t s , a g r o u p o f f i v e r a b b i t s r e c e i v e d 5 mg o f Cy i n O . l c c o f Cy o n c e p e r week f o r two months . The r a b b i t s showed no s i d e e f f e c t s c l i n i c a l l y t h a t were n o t p r e s e n t w i t h t h e s i n g l e i n j e c t i o n o f 5 mg i n O . l c c o f C y . A p o s t - m o r t e m o f t h e a n i m a l s showed no g r o s s c h a n g e s i n t h e o r g a n s . H o w e v e r , h i s t o p a t h o l o g y d i d show i r r e g u l a r i t y o f the c o n j u n c t i v a l e p i t h e l i u m , b u t t o a l e s s e r d e g r e e t h a n was s e e n w i t h t h e h i g h e r doses o f Cy. In t h i s case t h e e p i t h e l i a l i r r e g u l a r i t i e s can p r o b a b l y be a t t r i b u t e d more t o the r e p e a t e d i n j e c t i o n s t h a n t o t h e v e h i c l e . I n a d d i t i o n , l y m p h o c y t i c and p l a s m a c y t i c i n f i l t r a t e s were s e e n i n t h e r e n a l c o r t e x i n t e r s t i t i u m and h e p a t i c p o r t a l t r i a d s . N e p h r o t o x i c i t y has been one o f C y ' s s i d e e f f e c t s when g i v e n o r a l l y o r s y t e m i c a l l y , 1 7 , 3 9 , 4 0 , 4 2 , 4 3 but i t i s u n l i k e l y t h a t the a l t e r a t i o n s i n the r a b b i t s are due t o Cy (see p h a r m a c o k i n e t i c s tudy i n C h a p t e r 3 f o r l e v e l s o f C y ) . E v e n i f Cy i s r e s p o n s i b l e f o r t h e e f f e c t s , t h e y would have been much g r e a t e r i f Cy had been g i v e n o r a l l y o r s y s t e m i c a l l y o v e r t h e same p e r i o d o f t i m e . T h e s e e f f e c t s h a v e b e e n n o t e d i n p a t i e n t s r e c e i v i n g Cy o r a l l y f o r t h e t r e a t m e n t o f u v e i t i s , i n w h i c h m o d e r a t e t o s e v e r e c h r o n i c r e n a l h i s t o l o g i c changes were o b s e r v e d , c o n s i s t i n g o f i n t e r s i t i a l f i b r o s i s and t u b u l a r a t r o p h y . 4 2 , 4 3 The p o s s i b l e mechanism f o r r e n a l i n j u r y can o n l y be s p e c u l a t e d on, but changes i n g l o m e r u l a r c a p i l l a r i e s , 3 7 t u b u l a r i n j u r y , 3 8 and a decrease i n r e n a l b l o o d f l o w 4 1 h a v e b e e n i m p l i c a t e d as b e i n g c a u s e d by C y , r e s u l t i n g i n r e n a l i n j u r y . I t h a s a l s o b e e n p o s t u l a t e d t h a t Cy may be a b l e t o p r o d u c e t h e s e e f f e c t s b e c a u s e o f t h e p r e s e n c e o f b a s i c c y t o s o l i c Cy b i n d i n g p r o t e i n s , d i s c o v e r e d i n lymphoid t i s s u e s . These p r o t e i n s r e f e r r e d t o as c y c l o p h i l i n s , have been found i n h i g h c o n c e n t r a t i o n s i n the b r a i n and k i d n e y , organs t h a t d i s p l a y t o x i c e f f e c t s d u r i n g t r e a t m e n t w i t h Cy, p o s s i b l y e x p l a i n i n g the h i s t o l o g i c a l changes o b s e r v e d i n the k i d n e y . 5 5 Though t h e a g g r e g a t i o n s o f lymphocytes and p l a s m a c y t e s , i n t h e r e n a l i n t e r s t i t i u m and h e p a t i c t r i a d s i n t h e r a b b i t s a r e o f c o n c e r n , t h e y do n o t seem t o be o f a h i g h d e g r e e , o r l e a d t o s t r u c t u r a l r e n a l o r h e p a t i c damage. T h e s e m i l d e f f e c t s as n o t e d h a v e b e e n r e v e r s i b l e on d i s c o n t i n u a t i o n o f Cy t h e r a p y . 4 4 C o n c l u s i o n s A s i n g l e i n j e c t i o n o f 5 mg i n 0.1 c c o f t h e c o m m e r c i a l l y a v a i l a b l e Cy once p e r week, was f o u n d t o be the maximum c l i n i c a l l y t o l e r a t e d dose. More f r e q u e n t i n j e c t i o n s were noted t o cause e x c e s s i v e l o c a l response . Our r e s u l t s u s i n g the c o m m e r c i a l l y a v a i l a b l e Cy suggested t h a t our i n i t i a l h y p o t h e s i s , t h a t i s i t wou ld be l e s s t o x i c t o t h e eye and a l l o w f o r r e p e a t e d a d m i n i s t r a t i o n , was c o r r e c t . 47 CHAPTER 3 P h a r m a c o k i n e t i c s o f c y c l o s p o r i n e f o l l o w i n g s u b c o n j u n c t i v a l  v e r s u s i n t r a v e n o u s a d m i n i s t r a t i o n . I n t r o d u c t i o n W i t h s y s t e m i c d r u g a d m i n i s t r a t i o n t h e eye and t h e c e n t r a l n e r v o u s s y s t e m r e p r e s e n t p h a r m a c o l o g i c s a n c t u a r i e s . P r e v i o u s i n v e s t i g a t i o n s h a v e shown t h a t s u b c o n j u n c t i v a l a d m i n i s t r a t i o n i s s u p e r i o r t o i n t r a v e n o u s f o r o c u l a r p e n e t r a t i o n o f a n t i n e o p l a s t i c a g e n t s . 2 0 - 2 4 To d e t e r m i n e w h e t h e r t h i s was t r u e f o r C y , o c u l a r , b l o o d and u r i n e l e v e l s o f e q u i d o s e Cy {the 5 mg i n O . l c c o n c e p e r week o f Cy (SANDIMMUNE I . V . ) , as d e t e r m i n e d f r o m t h e d o s e d e t e r m i n a t i o n s t u d y ) f o l l o w i n g s u b c o n j u n c t i v a l v e r s u s i n t r a v e n o u s i n j e c t i o n were compared i n r a b b i t s . We found t h a t we were a b l e t o g e t h i g h e r amounts o f Cy i n t o t h e o c u l a r c h a m b e r s when Cy was g i v e n s u b c o n j u n c t i v a 1 l y . C o m p a r e d t o i n t r a v e n o u s i n j e c t i o n , s u b c o n j u n c t i v a l i n j e c t i o n r e s u l t e d i n f o u r t imes the peak c o n c e n t r a t i o n o f Cy i n t h e v i t r e o u s and 0.1 t i m e s o f t h a t i n t h e b l o o d . Aqueous l e v e l s o f Cy were u n d e t e c t a b l e i n the r a b b i t s g i v e n Cy i n t r a v e n o u s l y . M a t e r i a l s and Methods T w e l v e groups o f t h r e e r a b b i t s each ( l o c a l l y s u p p l i e d New Z e a l a n d w h i t e f e m a l e s w e i g h i n g 2 . 0 - 2 . 2 k g ) w e r e t r a n q u i l i z e d by i n t r a m u s c u l a r i n j e c t i o n s o f 0.2 m l / k g o f 48 10:1 s o l u t i o n (100 mg/ml) o f k e t a m i n e h y d r o c h l o r i d e and a c e t y l p r o m a z i n e (Rogar S . T . B . and A y e r s t L a b o r a t o r i e s , Vancouver ) e v e r y h a l f hour f o r f o u r h o u r s . Normal s a l i n e was i n f u s e d i n t o t h e l e f t m a r g i n a l e a r v e i n a t a r a t e o f 30 -40 m l / h o u r t o m a i n t a i n u r i n e f l o w . The r a b b i t s were g i v e n 5 mg i n O . l c c o f Cy as a b o l u s e i t h e r s u b c o n j u n c t i v a l l y { p o s t e r i o r t o the s u p e r i o r l imbus i n the r i g h t eye which had been p r e v i o u s l y a n a e s t h e t i z e d w i t h t o p i c a l p r o p a r a c a i n e HC1 (0.5%)} o r i n t r a v e n o u s l y ( i n the l e f t m a r g i n a l e a r v e i n ) . I t s h o u l d be noted t h a t t h i s i s not the s t a n d a r d method f o r I . V . a d m i n i s t r a t i o n o f Cy, r a t h e r i t i s by i n f u s i o n o v e r a p e r i o d o f t ime . T h e n a t 0 .5 , 1, 2, 4, 8 and 12 h o u r s l a t e r , 1 m l b l o o d s a m p l e s were o b t a i n e d by p u n c t u r e o f t h e m e d i a l a r t e r y o f t h e r i g h t e a r . T h e s a m p l e s w e r e p l a c e d i n t o 1.5 m l m i c r o t e s t t u b e s and f r o z e n . The t o t a l v o l u m e o f u r i n e c o l l e c t e d d u r i n g e a c h i n t e r v a l was r e c o r d e d , and s a m p l e s o f u r i n e were p l a c e d i n t o 1.5 ml m i c r o t e s t tubes and f r o z e n . A t t h e a p p r o p r i a t e t i m e t h e a n i m a l s were s a c r i f i c e d w i t h an o v e r d o s e o f p e n t o b a r b i t a l and i m m e d i a t e l y a f t e r d e a t h the eyes were p r o p t o s e d , and the c o r n e a s r i n s e d w i t h s a l i n e and b l o t t e d d r y . A b o u t 0.10 m l o f aqueous humour was a s p i r a t e d t h r o u g h t h e i n f e r i o r l i m b u s and p l a c e d i n a m i c r o t e s t tube . The eyes were t h e n e n u c l e a t e d , and the adherent e p i s c l e r a d i s c a r d e d . The g l o b e s were then r i n s e d w i t h s a l i n e , b l o t t e d d r y and snap f r o z e n i n l i q u i d n i t r o g e n . L a t e r the eyes were a l l o w e d t o p a r t i a l l y thaw, 49 a r e were t h e n i n c i s e d w i t h a s c a l p e l ; t h e f r o z e n v i t r e o u s e x p r e s s e d and p l a c e d i n a t e s t t u b e and d i s r u p t e d w i t h a s o n i c a t o r , t h e m i c r o t i p b e i n g u s e d f o r 30 s e c o n d s , and c e n t r i f u g e d a t 2500 RPM f o r f i v e minutes . The s u p e r n a t a n t was t h e n p l a c e d i n a m i c r o t e s t tube and f r o z e n . B e f o r e each e n u c l e a t i o n the s u r g i c a l i n s t r u m e n t s were washed t h o r o u g h l y w i t h e t h a n o l t o remove any Cy t h a t may be c o a t i n g them, and t h e s o n i c a t o r m i c r o t i p was a l s o washed w i t h e t h a n o l b e f o r e s o n i c a t i o n o f each sample . H i g h - P r e s s u r e L i q u i d Chromatography (HPLC) HPLC a n a l y s i s o f t h e s a m p l e s was c a r r i e d o u t w i t h a Water A s s o c i a t e s ( M i l f o r d , Massachuse t t s ) system c o n s i s t i n g o f two M6000 pumps, a m o d e l 710B WISP ( W a t e r s I n t e l l i g e n t S a m p l e P r o c e s s o r ) a u t o s a m p l e r , a m o d e l 481 Lambada-Max v a r i a b l e w a v e l e n g t h d e t e c t o r , a M660 s o l v e n t programmer, a c o l u m n h e a t e r and t e m p e r a t u r e c o n t r o l m o d u l e , and a s p e c t r a p h y s i c s 4290 i n t e g r a t o r . P u r e Cy ( a d o n a t i o n f r o m S a n d o z L t d , B a s l e , S w i t z e r l a n d ) a n d p u r e d i h y d r o c y c 1 o s p o r i n C (CyC -c o u r t e s y o f Dr Freeman, U n i v e r s i t y H o s p i t a l , U n i v e r s i t y o f W e s t e r n O n t a r i o ) , w e r e u s e d t o p r e p a r e t h e s t o c k s o l u t i o n o f 50 u g / m l o f Cy i n 100% m e t h a n o l and s t o c k s o l u t i o n o f t h e i n t e r n a l s t a n d a r d CyC was p r e p a r e d as 10 u g / m l i n 100% m e t h a n o l . S t a n d a r d c o n c e n t r a t i o n s o f Cy were p r e p a r e d r a n g i n g from 2 5 n g / m l t o 10 u g / m l from the p r i m a r y 50 s t o c k s o l u t i o n by s e r i a l s o l u t i o n , from which a p p r o p r i a t e s t a n d a r d c u r v e s were o b t a i n e d ( f i g u r e 3.4). E x t r a c t i o n P r o c e d u r e Two d i f f e r e n t methods were r e q u i r e d t o d e t e r m i n e t h e l e v e l s o f Cy i n the samples c o l l e c t e d . Method #1. F o r t h e u r i n e and o c u l a r s a m p l e s t h e method u s e d f o r d e t e m i n i n g t h e Cy l e v e l s was b a s e d on t h a t d e v e l o p e d by L e n s m e y e r and F i e l d s 6 5 w i t h a few m o d i f i c a t i o n s . The e x t r a c t i o n i s as f o l l o w s w i t h the m o d i f i c a t i o n s i d e n t i f i e d : T h i s method r e q u i r e d i n a d d i t i o n t o t h e a p p a r a t u s a l r e a d y ment ioned t h e use o f CN-Bond E l u t e s ( R ) (B.E), which a r e d i s p o s a b l e s o l i d phase columns (of 1.0 ml c a p a c i t y and c o n t a i n i n g 100 mg o f c y a n o p r o p y l phase packing) and the V a c E l u t ( R ) e x t r a c t i o n a p p a r a t u s ( b o t h f r o m A n a l y t i c h e m I n t e r n a t i o n a l , H a r b o r C i t y , CA). Reagent s . HPLC g r a d e a c e t o n i t r i l e and m e t h a n o l ( f rom C a l e d o n L a b o r a t o r i e s L t d . , G e o g r e t o w n , O n t a r i o ) , d e i o n i z e d w a t e r p r e p a r e d w i t h t h e " M i l l i - Q " w a t e r p u r i f i c a t i o n s y s t e m ( M i l l i p o r e C o r p . , B e d f o r d , MA), and g l a c i a l a c e t i c a c i d ( f r o m BDH C h e m i c a l s C a n a d a L t d . , V a n c o u v e r ) were u s e d i n p r e p a r i n g the r e a g e n t s needed. 51 S o l u t i o n A : The d i l u e n t c o n s i s t i n g o f w a t e r / a c e t o n i t r i l e (70/30 by v o l ) used t o d i l u t e samples . S o l u t i o n B: W a t e r / a c e t o n i t r i l e (80 /20 by v o l ) u s e d f o r c o n d i t i o n i n g the CN - B . E . S o l u t i o n C: A c e t i c a c i d (0.5 M) / a c e t o n i t r i l e (80/20) used t o remove i n t e r f e r i n g subs tances from the CN - B . E . S o l u t i o n D: A c e t i c a c i d (0.5 M) / a c e t o n i t r i l e (60/40) used t o remove i n t e r f e r i n g s u b s t a n c e s from CN - B . E . M o b i l e Phase - c o n s i s t e d o f water / a c e t o n i t r i l e (60/40 by v o l ) , which was f i l t e r e d t h r o u g h a m i l l i p o r e f i l t e r system ( u s i n g a 22 urn d u r a p o r e f i l t e r ) and degassed p r i o r t o use. Sample Treatment - To a 16 x 100mm d i s p o s a b l e c e n t r i f u g e t e s t t u b e 250 u l o f u r i n e o r o c u l a r s a m p l e , 50 u l o f t h e i n t e r n a l s t a n d a r d (10 u g / m l o f CyC) and 2 ml o f s o l u t i o n A were a d d e d . T h e n v o r t e x e d f o r 20 s e c o n d s and c e n t r i f u g e d f o r 5 m i n u t e s a t 2500 RPM. {In L e n s m e y e r ' s method t h e i n t e r n a l s t a n d a r d was CyD and S o l u t i o n A was 350 u g / 1 o f CyD i n w a t e r / a c e t o n i t r i l e (70/30 by v o l ) . } Bond E l u t e C o n d i t i o n i n g - C N - B . E . was pr imed w i t h two r e s e r v o i r vo lumes (about 1.0 ml) o f a c e t o n i t r i l e , and then 52 f o l l o w e d w i t h two r e s e r v o i r vo lumes o f S o l u t i o n B. Then the d i l u t e d s a m p l e was p a s s e d t h r o u g h t h e c o n d i t i o n e d C N - B . E . , making s u r e t h a t the C N - B . E . d i d not d r y between a p p l i c a t i o n s . I n t e r f e r e n c e E l u t i o n - A f t e r t h e m e n i s c u s o f t h e s a m p l e p a s s e d t h r o u g h t h e t o p o f t h e C N - B . E . p a c k i n g , t h e C N - B . E . was washed w i t h two r e s e r v o i r vo lumes o f s o l u t i o n C. A f t e r t h e l a s t o f S o l u t i o n C h a d p a s s e d t h e t o p o f t h e C N - B . E . p a c k i n g , the C N - B . E . was then washed w i t h 0.25 ml o f S o l u t i o n D and r e d u c e d p r e s s u r e was m a i n t a i n e d t o remove any r e s i d u a l l i q u i d from the C N - B . E . I s o l a t e E l u t i o n - 0.4 m l o f a c e t o n i t r i l e was addedto t h e C N - B . E . and l e f t f o r two m i n u t e s . The e l u a t e was t h e n c o l l e c t e d i n t o 16 x 100 mm d i s p o s a b l e c e n t r i f u g e t e s t t u b e s , and under reduced p r e s s u r e any r e s i d u a l l i q u i d was be c o l l e c t e d from the C N - B . E . The e l u a t e was then e v a p o r a t e d under a s tream o f n i t r o g e n a t 4 0 degrees C e l s i u s , and t h e d r y e x t r a c t was t h e n r e c o n s t i t u t e d w i t h 250 u l o f m o b i l e p h a s e , v o r t e x e d f o r t w e n t y s e c o n d s and c e n t r i f u g e d f o r 5 m i n u t e s a t 2500 RPM. The r e c o n s t i t u t e d e x t r a c t was then t r a n s f e r r e d t o a l i m i t e d - v o l u m e i n s e r t f o r c h r o m a t o g r a p h y . {In L e n s m e y e r ' s method t h e e l u t a n t was e v a p o r a t e d u n d e r a s t r e a m o f a i r a t room t e m p e r a t u r e , and t h e d r y e x t r a c t was r e c o n s t i t u t e d w i t h 150 u l o f m o b i l e phase .} 53 Chromatography 50 m i c r o l i t e r s o f t h e e x t r a c t e d s a m p l e was i n j e c t e d i n t o t h e HPLC s y s t e m w h i c h h a d a 25cm x 4.6mm Whatman C C S / C 8 (5 m i c r o n p a r t i c l e s i z e ) a n a l y t i c a l c o l u m n . The m o b i l e p h a s e f l o w r a t e was 1.5 m l / m i n u t e , w i t h d e t e c t o r r a n g e s e t a t 0.01 AUFS a t 210nm, c h a r t s p e e d was s e t a t 0.25 c m / m i n u t e on t h e s p e c t r a p h y s i c s i n t e g r a t o r and t h e c o l u m n h e a t e r was s e t a t 72 d e g r e e s C e l s i u s . { I n Lensmeyer's p r o c e d u r e the a n a l y t i c a l co lumn used was a 25 x 4.6 mm Z o r b a x - c y a n o p r o p y 1 (5 m i c r o n p a r t i c l e s i z e ) and a 5cm x 4.6mm g u a r d c o l u m n p a c k e d w i t h p e r m a p h a s e ETH (30 m i c r o n p a r t i c l e s i z e ) , w i t h d e t e c t o r range s e t a t 0.02 AUFS a t 214nm and r e c o r d e r c h a r t speed a t 1 cm/min.} Because the whole b l o o d samples were f r o z e n , subs tances f o r m e d u p o n t h a w i n g t h a t p l u g g e d t h e C N - B . E . , m a k i n g e x t r a c t i o n i m p o s s i b l e u s i n g method#l t o de termine the Cy l e v e l s i n the b l o o d samples . The method u s e d t o d e t e r m i n e t h e Cy l e v e l i n t h e f r o z e n w h o l e b l o o d s a m p l e s was t h a t d e v e l o p e d by C a r r u t h e r s , Freeman e t a l . The method i s as f o l l o w s : Method # 2: Reagents HPLC grade D i e t h y l e t h e r (from J . T . Baker C h e m i c a l Co . , P h i l l i p s b u r g , N . J . ) , HPLC grade a c e t o n i t r i l e and methanol (from C a l e d o n L a b o r a t o r i e s L t d . , Geogretown, O n t a r i o ) , and 54 d e i o n i z e d w a t e r p r e p a r e d w i t h t h e " M i l l i - Q " w a t e r p u r i f i c a t i o n s y s t e m ( M i l l i p o r e C o r p . , B e d f o r d , MA) were used t o p r e p a r e the r e a g e n t s needed. S o l u t i o n 1: 0.2 M HCL S o l u t i o n 2: 0.2 M NaOH S o l u t i o n 3: 1.2 M (NH 4) 2 S 0 4 i n 40.0 m l o f a s o l u t i o n c o n s i s t i n g o f 20% a c e t o n i t r i l e , 20% m e t h a n o l , and 60% w a t e r . R e c o n s t i t u t i o n F l u i d - c o n s i s t e d o f 3.0 ml o f a c e t o n i t r i l e , 2.0 m l o f m e t h a n o l , 5.0 m l o f w a t e r and 1.0 m l o f S o l u t i o n 3. M o b i l e P h a s e : c o n s i s t e d o f 46.95% a c e t o n i t r i l e , 20% m e t h a n o l , 33.05% w a t e r and 0.5 m l o f S o l u t i o n 3 p e r 2.0 l i t e r s o f m o b i l e p h a s e , w h i c h was f i l t e r e d t h r o u g h a m i l l i p o r e f i l t e r s y s t e m ( u s i n g a 22 m i c r o n Durapore ( M i l l i p o r e ) (R) f i l t e r ) and degassed p r i o r t o use. S a m p l e T r e a t m e n t - I n a 16 x 100 mm d i s p o s a b l e c e n t r i f u g e t e s t t u b e 250 m i c r o l i t e r s o f w h o l e b l o o d , 50 m i c r o l i t e r s o f the i n t e r n a l s t a n d a r d (10 u g / m l o f CyC) and 2.0 m l o f S o l u t i o n 1 were a d d e d . T h e n v o r t e x e d f o r 20 seconds . To t h i s m i x t u r e 6.0 ml o f D i e t h y l e t h e r was added 55 and shaken on a h o r i z o n t a l shaker a t 100 RPM f o r 10 m i n u t e s . T h e n c e n t r i f u g e d a t 2500 RPM f o r 5 m i n u t e s . The a q u e o u s a c i d p h a s e was t h e n d i s c a r d e d w i t h an e t h e r -r i n s e d P a s t e u r p i p e t t e and 2.0 m l o f S o l u t i o n 2 was a d d e d t o t h e D i e t h y l e t h e r . T h i s m i x t u r e was t h e n v o r t e x e d f o r 30 s e c o n d s , and c e n t r i f u g e d f o r 5 m i n u t e s a t 2500 RPM. The e t h e r phase was t h e n t r a n s f e r r e d w i t h a P a s t e u r p i p e t t e t o a 16 x 100mm d i s p o s a b l e t e s t t u b e . The e t h e r was t h e n e v a p o r a t e d u n d e r a s t r e a m o f n i t r o g e n a t 40 degrees C e l s i u s . The d r y e x t r a c t was r e c o n s t i t u t e d w i t h r e c o n s t i t u t i o n f l u i d and v o r t e x e d f o r 20 s e c o n d s . The r e c o n s t i t u t e d e x t r a c t was t h e n t r a n s f e r r e d t o a l i m i t e d - v o l u m e i n s e r t f o r chromatography . Chromatography The a n a l y t i c a l c o l u m n u s e d was a 25cm x 3.2mm I . D . c o n t a i n i n g S p h e r i s o r b - C 8 p a c k i n g (5 m i c r o n p a r t i c l e s i z e ) , c o n s t r u c t e d b y D r . F r e e m a n o f U n i v e r s i t y H o s p i t a l , U n i v e r s i t y o f W e s t e r n O n t a r i o . The f l o w r a t e was 1.0 m l / m i n . , d e t e c t o r s e t t i n g s were 0.01 AUFS a t 210nm, t h e co lumn h e a t e r was s e t a t 72 degrees C e l s i u s and the c h a r t speed s e t t i n g on the s p e c t r a p h y s i c s was 0.25 cm/min. 50.0 u l o f t h e e x t r a c t e d sample was chromatographed and between each sample t h e r e was an a c e t o n i t r i l e wash t o remove l a t e e l u t o r s f r o m t h e c o l u m n . T h e a c e t o n i t r i l e w a s h was c o n t r o l l e d by a M660 s o l v e n t programmer. The M660 g r a d i e n t c o n t r o l l e r was programmed as f o l l o w s : 0-15 min 100% m o b i l e phase 15-17 min 100% a c e t o n i t r i l e 17 min 100% m o b i l e phase. The r u n n i n g t i m e was 17 m i n u t e s w i t h an e q u i l i b r a t i o n d e l a y o f e i g h t minutes and a t o t a l r u n t ime o f 25 minutes . R e s u l t s Performance d a t a on Method 1 and 2 a r e p r e s e n t e d i n T a b l e I I I . The s e n s i t i v i t y l i m i t ( s i g n a l s e q u a l t o t w i c e t h e b a s e l i n e n o i s e ) r a n g e d f r o m 15 t o 25 n g / m l . The s t a n d a r d c u r v e s f o r e a c h method ( f i g u r e 3.4) were d e v e l o p e d by p l o t t i n g r e l a t i v e a r e a r a t i o ( C y / C y C ) v s c o n c e n t r a t i o n f o r Cy, and l e a s t squares r e g r e s s i o n was used a s s e s s l i n e a r i t y . The c h r o m a t o g r a m s f o r e a c h t y p e o f sample e x t r a c t e d a r e p r e s e n t e d i n f i g u r e s 3 .0-3.3. 57 Table III Performance Data on Methods 1 & 2 Method n Precision day to day S.D. C.V. % Analytical recovery Mean Recovery % Linearity (linear up to at least 1400 ng/ml) concn, ng/ml mean 1 20 1 00 1 06 6.8 9.9 90 y=(5.109E-4)x + 4.165E-3 r=0.9980 2 20 100 1 06 7.5 1 1 3 . 70 y=(2.075E-4)x + 2.789E-5 r=0.9989 x= Cy concentration in ng/ml y= relative area ratio Cy /CyC A f t e r S u b c o n j u n c t i v a l a d m i n i s t r a t i o n The l e v e l s o f Cy i n a q u e o u s humor o f t h e r i g h t eye ( tha t i n j e c t e d s u b c o n j u n c t i v a l l y ) peaked a t 718 n g / m l a t one h o u r , t h e n d e c r e a s e d t o 119 n g / m l by 12 hours ( f i g u r e 3.5). I n t h e v i t r e o u s humor o f t h e r i g h t eye t h e l e v e l o f Cy p e a k e d a t 1078 n g / m l a t two h o u r s , t h e n d e c r e a s e d t o 253 n g / m l by 12 h o u r s ( f i g u r e 3 .5 ) . I n t h e l e f t eye ( t h e c o n t r o l s ) t h e l e v e l s o f Cy were u n d e t e c t a b l e i n t h e aqueous and v i t r e o u s humor. The b i o a v a i l a b i l i t y o f Cy i n t h e o c u l a r humors when a d m i n i s t e r e d s u b c o n j u n c t i v a l l y , c a l c u l a t e d by an a r e a -u n d e r - t h e - c u r v e m e t h o d , 4 5 i s shown i n T a b l e I V . The l e v e l s o f Cy i n w h o l e b l o o d p e a k e d a t 4 h o u r s r e a c h i n g o n l y 104 n g / m l , t h e n d e c r e a s e d t o 54 n g / m l by 12 h o u r s ( f i g u r e 3 . 6 ) . Cy l e v e l s i n t h e u r i n e w e r e u n d e t e c t a b l e . A f t e r I n t r a v e n o u s A d m i n i s t r a t i o n The Cy l e v e l i n w h o l e b l o o d p e a k e d a t 1061 n g / m l a t 0.5 h o u r s , t h e n d e c r e a s e d t o 74 n g / m l by 12 hours ( f i g u r e 3.6). The c u m m u l a t i v e u r i n e e l i m i n a t i o n o f Cy o v e r 12 h o u r s i n t h e r a b b i t was l e s s t h a n 1% o f t h e i n j e c t e d d o s e ( f i g u r e 3.7) . The d a t a were p o o l e d f o r the r i g h t and l e f t e y e s , s i n c e a p a i r e d t - t e s t showed no s i g n i f i c a n t d i f f e r e n c e (p=0.05) b e t w e e n them. The l e v e l s o f Cy i n t h e a q u e o u s humor were u n d e t e c t a b l e . T h e l e v e l s o f Cy i n t h e v i t r e o u s humor p e a k e d a t 292 n g / m l a t two h o u r s , t h e n d e c r e a s e d t o 164 n g / m l by 12 h o u r s ( f i g u r e 3 .5 ) . The b i o a v a i l a b i l i t y o f Cy i n t h e o c u l a r humors when a d m i n i s t e r e d i n t r a v e n o u s l y i s shown i n T a b l e I V . Table IV Bioavailability of Cyclosporine in Ocular Humours Humour, Bioavailability (ng/ml.hr) Route of administration Aqueous Humour Right Vitreous Humour Right subconjunctival 4 .7 1 0.4 Intravenous not detectable 5.3 60 F i g u r e 3 . 0 A t y p i c a l ch romatogram o f aqueous humor w i t h the i n t e r n a l s t a n d a r d D i h y d r o c y c l o s p o r i n C (A) e l u t i n g a t 10 m i n u t e s and a 250ng/ml s t a n d a r d o f C y c l o s p o r i n e (B) e l u t i n g a t 12 m i n u t e s . DRUG-FREE EXTRACTED B. EXTRACTED AQUEOUS HUMOR AQUEOUS HUMOR STANDARD METHOD # I Column: 25cm x 0.46cm Whatman CCS/C 8 (5um) Injection Volume: 50ul Mobile Phase: 40% Acetonitrile, 60% Water Column Temperature: 72°C Flow Rate: 1.5 ml/min Detection: 210 nm INJECT 61 F i g u r e 3 .1 A t y p i c a l ch romatogram o f v i t r e o u s humor w i t h the i n t e r n a l s t a n d a r d D i h y d r o c y c l o s p o r i n C (A) e l u t i n g a t 10 m i n u t e s and a 25Qng/ml s t a n d a r d o f C y c l o s p o r i n e (B) e l u t i n g a t 12 m i n u t e s . C. DRUG-FREE EXTRACTED D. EXTRACTED VITREOUS HUMOR VITREOUS HUMOR STANDARD INJECT METHOD # I Column: 25cm x 0.46cm Whatman CCS/C 8 (5um) Injection Volume: 50ul Mobile Phase: 40% Acetonitrile, 60% Water Column Temperature: 72°C Flow Rate: 1.5 m l / min Detection: 210 nm 62 F i g u r e 3 . 2 A t y p i c a l chromatogram o f u r i n e w i t h t h e i n t e r n a l s t a n d a r d D i h y d r o c y c l o s p o r i n C (A) e l u t i n g a t 10 m i n u t e s and a 250ng/ml s t a n d a r d o f C y c l o s p o r i n e (B) e l u t i n g a t 12 m i n u t e s . E. DRUG - FREE F. EXTRACTED EXRACTED URINE URINE STANDARD METHOD # I Column: 25cm x 0.46cm Whatman CCS/C 8 (5 urn) Injection Volume: 50ul Mobile Phase: 40% Acetonitrile, 60% Water Column Temperature: 72°C Flow Rate: 1.5 ml / min Detection: 210 nm INJECT 63 F i g u r e 3 .3 A t y p i c a l ch romatogram o f w h o l e b l o o d w i t h t h e i n t e r n a l s t a n d a r d D i h y d r o c y c l o s p o r i n C (A) e l u t i n g a t 1 0 . 3 0 m i n u t e s and a 500ng/ml s t a n d a r d o f C y c l o s p o r i n e (B) e l u t i n g a t 1 3 . 0 0 m i n u t e s . G. DRUG - FREE EXTRACTED H. EXTRACTED WHOLE BLOOD WHOLE BLOOD STANDARD INJECT METHOD # 2 Column: 25cm x 0.32 I.D. (Spherisorb C8, 5um) Injection Volume: 50ul Mobile Phase: 46.95% Acetonitrile, 20.00% Methanol, 33.05% Water Column Temperature: 72°C Flow Rate: 1.0 ml / min Detection: 210 nm 64 F i g u r e 3 . 4 STANDARD CURVE FOR CYCLOSPORINE USING M E T H O D NO. I Y = (5.IIE - 4) + 4.I7E-3 0 200 400 600 800 1000 CYCLOSPORINE CONCENTRATION (ng/ml) STANDARD CURVE FOR CYCLOSPORINE USING METHOD NO. 2 Y= (2.07E -4)X • 2.79E-5 CYCLOSPORINE CONCENTRATION (ng/ml) 65 F i g u r e 3.5 L E V E L S OF C Y C L O S P O R I N E IN THE OCULAR C H A M B E R S OF THE E Y E 66 F i g u r e 3 . 6 BLOOD L E V E L S OF CYCLOSPORINE IN THE RABBIT AT A 5 mg/O. I cc DOSE 2000 1000-100 TIME (Hours) Levels of cyclosporine in the blood following subconjunctival (x) or intravenous (•) administration 67 F i g u r e 3.7 CUMMULATIVE URINE ELIMINATION CURVE FOR CYCLOSPORINE IN THE RABBIT GIVEN A 5 m g / 0 . l c c SYSTEMIC DOSE 0.15 • 0.10-0.01 / / / / / s y 1 1 1 1 1 1 1 1 1 1 1 1— 0 1 2 4 6 8 10 12 TIME (Hours) NOTE:-Urine levels were not detectable in rabbits given cyclosporine subconjunctival^. 68 DISCUSSION C y c l o s p o r i n e (Cy) i s a h y d r o p h o b i c c y c l i c u n d e c a p e p t i d e o b t a i n e d from the fungus T o l y p o c l a d i u m  i n f l a t u m . A l t h o u g h o r i g i n a l l y d e v e l o p e d as an a n t i - f u n g a l a g e n t , i t p r o v e d t o be a p o t e n t i m m u n o s u p p r e s s i v e t h a t c o u l d cause s e l e c t i v e s u p p r e s s i o n o f T - l y m p h o c y t e s a t an e a r l y s t a g e o f a c t i v a t i o n . 1 5 ' 5 6 C u r r e n t l y , i t h a s been f o u n d b e n e f i c i a l when g i v e n o r a l l y o r i n t r a v e n o u s l y f o r t h e p r o p h y l a x i s and t r e a t m e n t o f o r g a n r e j e c t i o n i n a l l o g e n i c t r a n s p l a n t s , 4 8 and i t shows promise i n the t r e a t m e n t o f autoimmune d i s e a s e s . 1 5 , 5 1 - 5 3 The s t r u c t u r e o f Cy r e v e a l s some u n i q u e f e a t u r e s ( f i g u r e 3 .8 ) ; i n p o s i t i o n 1 i t h a s a n i n e c a r b o n amino a c i d , w h i c h was p r e v i o u s l y unknown and t o w h i c h much o f i t s a c t i v i t y i s a t t r i b u t a b l e . A l l o f t h e amino a c i d s t h a t c o m p r i s e Cy have the L c o n f i g u r a t i o n except f o r D - a l a n i n e i n p o s i t i o n 8 and s a r c o s i n e i n p o s i t i o n 3. F i n a l l y , s even o f t h e amino a c i d s a r e N - m e t h y l a t e d , w h i c h may c o n t r i b u t e t o i t s l a c k o f d e g r a d a t i o n when a d m i n i s t e r e d o r a l l y . 5 4 Cy a l s o e x h i b i t s l a r g e i n t e r - i n d i v i d u a l v a r i a t i o n s i n b i o a v a i l a b i l i t y and c l e a r a n c e r a t e . I t d i s p l a y s a b i p h a s i c d i s t r i b u t i o n p a t t e r n , the f i r s t r e p r e s e n t i n g the b l o o d ( i n which 50% i s found i n e r y t h r o c y t e s , 40% i n p l a s m a , and 10% i n l e u k c o c y t e s 1 5 ) , and t h e s e c o n d p h a s e t h e p e r i p h e r a l t i s s u e s . In g e n e r a l the p h a r m a c o k i n e t i c s o f Cy show t h a t p e a k serum c o n c e n t r a t i o n s a r e a c h i e v e d i n 2 t o 6 h o u r s 69 F i g u r e 3 . 8 CH3 ^ C H s V C H C H ^ ^CHs » . H i » , H C H , - N - C - CO - N — C — C CO CHs CH-CH2»«C A A I O A A 9 AA11 CHS C H , - N A A S O C - ftr A A 7 N - C O - C — I J *H H CH3 c M O s « « 4 ; m C H , -N • H — N C -A A1 X C H 3 CH2 C O — N — C - — C -I II A A 2 0 H I - N - C -II 0 A A C C — N — 1*H I CHj CHs I CHs CH3 0 II A A S CHs - A — A A S H A A 4 O i : I CO N - C H , C — C — N — C O — C i» * / C H C H , CH3 CHS CH3 CHs S t r u c t u r e o f C y c l o s p o r i n e ( M o l e c u l a r W e i g h t o f 1202) 70 w i t h a T - j y 2 ° f 1 6 + / -8 h o u r s , and an o r a l b i o a v a i l a b i l i t y o f 34 + / - 1 1 % . 5 6 ' 5 7 T h e m e t a b o l i s m o f t h i s d r u g i s p r i m a r i l y by t h e l i v e r , y i e l d i n g numerous m e t a b o l i t e s which a r e e x c r e t e d i n t o t h e b i l e , w i t h l e s s t h a n 1% e x c r e t e d i n t a c t i n t o the u r i n e . 1 5 , 5 6 I n a d d i t i o n , Cy h a s a n a r r o w m a r g i n o f s a f e t y w i t h t h e r a p e u t i c c o n c e n t r a t i o n s between 100 and 400 n g / m l , and the major t o x i c e f f e c t s o f Cy such as n e p h r o t o x i c i t y and h e p a t o t o x i c i t y a r e m a n i f e s t e d a t c o n c e n t r a t i o n s g r e a t e r t h a n 4 00 n g / m l . 1 5 ' 5 6 , 5 7 C a r e f u l m o n i t o r i n g o f Cy l e v e l s i s e s s e n t i a l t o a v o i d t h e t o x i c s i d e e f f e c t s o f C y a n d t o m a i n t a i n immunosuppress ion. Two d i s t i n c t p r o c e d u r e s have e v o l v e d t o m e a s u r e Cy l e v e l s : r a d i o i m m u n o a s s a y (RIA) 6 3 , 6 4 and h i g h p e r f o r m a n c e l i q u i d c h r o m a t o g r a p h y ( H P L C ) . 6 5 - 6 7 P r i o r t o 1982 Cy l e v e l s i n p l a s m a and b l o o d were m e a s u r e d by RIA ( which has a d e t e c t i o n l i m i t o f 25-50 n g / m l ) , but the major p r o b l e m w i t h t h i s p r o c e d u r e was t h a t i t c o u l d n o t d i s t i n g u i s h between Cy and i t s m e t a b o l i t e s . The r e s u l t was a f a l s e i m p r e s s i o n t h a t p a t i e n t s w e r e e i t h e r immunosuppressed o r had t o x i c l e v e l s o f Cy. The advantage o f t h e RIA p r o c e d u r e was t h a t i t c o u l d be p e r f o r m e d on a l a r g e number o f samples e a s i l y , w i t h a t u r n - a r o u n d t ime o f 4 t o 6 hours f o r a b a t c h o f samples . The second method o f a n a l y s i s by HPLC which a l l o w s f o r l e v e l s i n w h o l e b l o o d t o be d e t e r m i n e d was i n t r o d u c e d by Sawchuk and C a r t i e r , 6 7 and was a b l e t o d i s t i n g u i s h Cy from 71 i t s m e t a b o l i t e s and had a d e t e c t i o n l i m i t o f 10 t o 25 n g / m l d e p e n d i n g on t h e s a m p l e . 6 5 ' 6 7 The HPLC p r o c e d u r e was m o d i f i e d by o t h e r s i n o r d e r t o i n c r e a s e t h e s p e e d and s e n s i t i v i t y o f the d e t e r m i n a t i o n o f Cy l e v e l s . In g e n e r a l , t h e methods o f e x t r a c t i o n o f t h e s a m p l e s and t h e t y p e o f a n a l y t i c a l co lumn ( r e v e r s e phase C 1 8 o r C g , c y a n o p r o p y l ) h a v e v a r i e d b u t a l l h a v e r e q u i r e d h i g h t e m p e r a t u r e chromatography and d e t e c t i o n has been by a U .V . d e t e c t o r a t about 2 1 0 n m . 6 5 " 7 0 I m p o r t a n t t o m o n i t o r i n g Cy l e v e l s i s n o t o n l y t h e method, but the h a n d l i n g o f the b l o o d samples , because the c o n c e n t r a t i o n o f Cy i n p l a s m a i s w i d e l y v a r i a b l e and dependent upon t h e t empera ture o f p lasma s e p a r a t i o n . 7 1 ' 7 2 W h o l e b l o o d a n a l y s i s i s p r e f e r a b l e , 6 5 ' 7 3 ' 7 4 s i n c e i t i s i m p o r t a n t t o g e t a t r u e p i c t u r e o f t h e l e v e l s o f C y , because o f i t s narrow marg in o f s a f e t y . 1 5 , 5 6 ' 5 7 ' 7 5 T h o u g h i t i s g e n e r a l l y d e s i r a b l e t o know t h e f r e e l e v e l s o f Cy as they r e f l e c t the a c t u a l amount o f drug a v a i l a b l e f o r use by the body, t h e r e i s c o n t r o v e r s y r e g a r d i n g method o f m e a s u r e m e n t ; t h a t i s , w h e t h e r t o u s e t r a d i t i o n a l p l a s m a methods ( f r e e l e v e l s ) o r w h o l e b l o o d . In t h e c l i n i c a l s e t t i n g the method o f measurement o f whole b l o o d Cy l e v e l i s c u r r e n t l y b e i n g f a v o u r e d b a s e d on t h e r a t i o n a l e t h a t t h i s g i v e s the c l i n i c i a n a b e t t e r p i c t u r e o f the l e v e l o f immunosuppresion b e i n g o b t a i n e d . In o r d e r t o a v o i d the v a r i a b i l i t y a s s o c i a t e d w i t h p lasma l e v e l methods, and t o f o l l o w the s u g g e s t i o n s p r o v i d e d by MRC r e v i e w e r s o f t h i s p r o j e c t , we chose the whole b l o o d method. T h e r e f o r e , t h e method c h o s e n f o r t h e a n a l y s i s o f Cy l e v e l s was H P L C , b e c a u s e t h i s m e t h o d i s a b l e t o d i s t i n g u i s h between Cy and i t s m e t a b o l i t e s and h a n d l e whole b l o o d samples . The HPLC method d e v e l o p e d by Lensmeyer and F i e l d s 6 5 was u s e d t o m e a s u r e Cy i n t h e o c u l a r and u r i n e s a m p l e s , b u t t h e e x t r a c t i o n p r o c e d u r e u s e d i n t h i s method was not a b l e t o h a n d l e the f r o z e n whole b l o o d samples . An a l t e r n a t e HPLC method d e v e l o p e d by C a r r u t h e r s and Freeman e t a l . , ° was u s e d t o d e t e r m i n e t h e l e v e l o f Cy i n t h e f r o z e n w h o l e b l o o d s a m p l e s . T h e s e methods were c h o s e n b e c a u s e t h e y were a b l e t o remove most o f t h e i n t e r f e r i n g s u b s t a n c e s from the samples , and had good r e c o v e r y (70-90%) and s e n s i t i v i t y ( 10 t o 25 n g / m l ) as w e l l as a C . V . o f 9 -11% a t 100 n g / m l . T h e a i m o f t h i s p h a r m a c o k i n e t i c s t u d y o f t h e s u b c o n j u n c t i v a l v s t h e i n t r a v e n o u s r o u t e was t o s u b s t a n t i a t e w h e t h e r l o c a l a d m i n i s t r a t i o n w o u l d e n h a n c e o c u l a r a b s o r p t i o n o f Cy, and a v o i d the l e v e l s i n the b l o o d t h a t have been a s s o c i a t e d w i t h s y s t e m i c a d m i n i s t r a t i o n t h a t can r e s u l t i n n e p h r o t o x i c i t y . The s t u d y showed t h a t peak c o n c e n t r a t i o n s o f Cy were 4 and 0.1 t i m e s i n t h e v i t r e o u s and b l o o d r e s p e c t i v e l y when Cy was g i v e n s u b c o n j u n c t i v a l l y c o m p a r e d w i t h Cy a d m i n i s t e r e d i n t r a v e n o u s l y . In a d d i t i o n , aqueous l e v e l s w e r e u n d e t e c t a b l e i n r a b b i t s t h a t r e c e i v e d Cy i n t r a v e n o u s l y , and the b i o a v a i l a b i l i t y o f s u b c o n j u n c t i v a l l y a d m i n i s t e r e d Cy i n the v i t r e o u s was two t imes g r e a t e r than t h a t a c h i e v e d f o l l o w i n g i n t r a v e n o u s a d m i n i s t r a t i o n . The o c u l a r p h a r m a c o k i n e t i c s o f the s u b c o n j u n c t i v a l r o u t e a l s o showed t h a t t h e aqueous l e v e l s o f Cy p e a k e d a t 1/2 h o u r a n d t h e v i t r e o u s l e v e l s a t two h o u r s . The d e l a y i n t h e peak c o n c e n t r a t i o n s between the o c u l a r chambers can p a r t l y be e x p l a i n e d by the h i s t o l o g i c a l f i n d i n g s from the v e h i c l e and d o s e d e t e r m i n a t i o n s t u d y , w h i c h showed c a s t o r o i l s p r e a d i n g p o s t e r i o r l y . S i n c e t h e b l e b ( c o n t a i n i n g t h e v e h i c l e and Cy) f o r t h e f i r s t h o u r i s n e a r t h e l i m b i c a r e a , which i s i n c l o s e p r o x i m i t y t o the aqueous chamber, the Cy l e v e l s i n the aqueous a r e expected t o peak b e f o r e t h a t i n t h e v i t r e o u s , a s was s e e n f r o m t h e p h a r m a c o k i n e t i c r e s u l t s . T h e l a r g e a m o u n t o f Cy a t t a i n e d b y t h e s u b c o n j u n c t i v a l r o u t e may a l s o h a v e b e e n e n h a n c e d by t h e l o c a l i n f l a m m a t i o n p r o d u c e d b y s u b c o n j u n c t i v a l i n j e c t i o n . The l o c a l i n f l a m m a t o r y response would a l t e r both v a s c u l a r and o c u l a r p e r m e a b i l i t y and may i n c r e a s e b l o o d f l ow , thus enhanc ing a b s o r p t i o n . W i t h r e g a r d t o t h e t r o u g h l e v e l s o b t a i n e d i n t h e v i t r e o u s and aqueous f o l l o w i n g s u b c o n j u n c t i v a l i n j e c t i o n , they remained above the t h e r a p e u t i c range f o r 12 h o u r s . In c o n t r a s t , l e v e l s o b t a i n e d by s y s t e m i c a d m i n i s t r a t i o n a t t h i s p a r t i c u l a r dosage were o n l y d e t e c t a b l e i n the v i t r e o u s and remained j u s t above the t h e r a p e u t i c range f o r 12 hours but were be low these o b t a i n e d i n the aqueous f o l l o w i n g a d m i n i s t r a t i o n o f Cy s u b c o n j u n c t i v a l l y . E q u a l l y i m p o r t a n t a r e t h e o b s e r v e d b l o o d l e v e l s f o l l o w i n g t h i s r o u t e o f a d m i n i s t r a t i o n w h i c h were w e l l b e l o w (4 t i m e s ) t h e l e v e l (400 n g / m l ) n e e d e d t o a v o i d t h e t o x i c s i d e e f f e c t s o f c y . 1 5 ' 5 6 ' 5 7 The r a t i o n a l e f o r p u l s e d l o c a l t h e r a p y i n t h e c a s e o f o c u l a r i n f l a m m a t o r y d i s e a s e i s b a s e d on t h e c o n c e p t t h a t s u c c e s i v e t r e a t m e n t can g r a d u a l l y b r i n g the d i s e a s e under c o n t r o l s i n c e the a f f e c t e d lymphocyte p o p u l a t i o n w i l l not exchange q u i c k l y o r e a s i l y w i t h the g e n e r a l p o p u l a t i o n i n t h i s s a n c t u a r y . In a d d i t i o n , numerous drugs have been shown t o be absorbed b e t t e r f o l l o w i n g l o c a l a d m i n i s t r a t i o n than f o l l o w i n g e q u i d o s e s y s t e m i c a d m i n i s t r a t i o n , and t h a t s y s t e m i c l e v e l s r e m a i n l o w e r f o l l o w i n g s u b c o n j u n c t i v a l v e r s u s equ idose s y s t e m i c a d m i n i s t r a t i o n i n many d r u g s 2 0 - 2 4 . C o n c l u s i o n s T h e r e s u l t s f r o m t h i s e x p e r i m e n t p r o v i d e a p h a r m a c o k i n e t i c b a s i s f o r t h e use o f Cy s u b c o n j u n c t i v a l l y f o r o c u l a r i n f l a m m a t o r y d i s e a s e . I n g e n e r a l , t h e p h a r m a c o k i n e t i c s o f Cy a d m i n i s t e r e d s u b c o n j u n c t i v a l r e s e m b l e s t h a t o f o t h e r d r u g s a d m i n i s t e r e d i n t h i s m a n n e r . 2 0 - 2 4 The l e v e l s o b t a i n e d v a r y w i t h the d r u g b e i n g s t u d i e d but i n the case o f Cy, i t peaked l a t e r s u g g e s t i n g o c u l a r a b s o r p t i o n i s b e t t e r w i t h h y d r o p h i l i c t h a n w i t h h y d r o p h o b i c drugs . L o c a l a d m i n i s t r a t i o n may have a r o l e t o p l a y i n o c u l a r i n f l a m m a t o r y d i s e a s e which has responded to o r a l o r I . V . a d m i n i s t e r e d Cy, but which had t o be 75 d i s c o n t i n u e d because o f the n e p h r o t o x i c i t y a s s o c i a t e d w i t h t o x i c s y s t e m i c l e v e l s o f Cy. 76 CHAPTER 4 U v e i t i s s t u d y I n t r o d u c t i o n U v e i t i s i s an o c u l a r i n f l a m m a t o r y d i s e a s e i n which the n a t u r e o f t h e c a u s a l f a c t o r i s u s u a l l y unknown. I t i s d i f f i c u l t t o t r e a t i n i t i a l l y and t h e eye may u l t i m a t e l y succumb t o t h e s e c o n d a r y e f f e c t s o f c h r o n i c i n f l a m m a t i o n d u r i n g subsequent r e c u r r e n c e s . The b a s i c mechan i sm t h o u g h t t o be i n v o l v e d i n many t y p e s o f u v e i t i s i s i m m u n o l o g i c a l . The t r e a t m e n t has i n c l u d e d the use o f a n t i - i n f l a m m a t o r y a g e n t s 2 - 8 such as c o r t i c o s t e r i o d s 2 ' 1 4 and i m m u n o s u p p r e s s i v e s . 5 - 8 T h e s e a g e n t s h a v e b e e n s u c c e s s f u l t o v a r y i n g d e g r e e s , b u t h a v e a l s o c a u s e d s e r i o u s s i d e e f f e c t s l i k e s u s c e p t i b i l i t y t o i n f e c t i o n , 9 i n c r e a s e d o c u l a r p r e s s u r e s 1 0 , 1 1 ' 1 4 and bone marrow s u p p r e s s i o n . 1 2 , 1 4 , 1 5 R e c e n t l y i t h a s b e e n d e m o n s t r a t e d t h a t e x p e r i m e n t a l auto immune u v e i t i s (EAU) i n d u c e d by r e t i n a l S - a n t i g e n r e s e m b l e s the immune-mediated o c u l a r i n f l a m m a t o r y response s e e n i n humans, w h i c h i s m a i n l y T - c e l 1 - m e d i a t e d . 1 8 , 2 7 , 3 4 Cy has been used s u c c e s s f u l l y s y s t e m i c a l l y and o r a l l y i n the t r e a t m e n t o f T - c e l 1 - m e d i a t e d u v e i t i s i n p a t i e n t s , ^ because i t i s a b l e t o b l o c k the e a r l y s tages o f T - c e l 1 a c t i v a t i o n . U n f o r t u n a t e l y the h i g h s y s t e m i c and o r a l doses needed t o ge t t h e r a p e u t i c l e v e l s o f Cy i n t o t h e eye, r e s u l t e d i n some s e r i o u s c o m p l i c a t i o n s 17 4? such as n e p h r o t o x i c i t y , 'I^I^ From t h e p h a r m a c o k i n e t i c s t u d y o f s u b c o n j u n c t i v a l a d m i n i s t r a t i o n o f C y , we were a b l e t o show t h a t h i g h e r amounts p e n e t r a t e d the o c u l a r chambers, w h i l e a v o i d i n g the h i g h b l o o d l e v e l s o f C y . T o a s s e s s w h e t h e r t h e s u b c o n j u n c t i v a l r o u t e o f a d m i n i s t r a t i o n w o u l d be e f f e c t i v e i n t h e p o s s i b l e t r e a t m e n t o f u v e i t i s , EAU was i n d u c e d by i n j e c t i o n o f a s i n g l e d o s e o f b o v i n e s e r u m a l b u m i n (BSA) i n t o t h e v i t r e o u s o f r a b b i t s . A c u t e u v e i t i s d e v e l o p e d w i t h i n s i x t o t e n d a y s , and p e r s i s t e d f o r a t l e a s t a week, and t h e n s u b s i d e d . To a s s e s s t h e d e g r e e t o w h i c h t h e i n f l a m m a t i o n m i g h t be s u p p r e s s e d , Cy s u b c o n j u n c t i v a l t h e r a p y was i n i t i a t e d i n one g r o u p o f r a b b i t s i m m e d i a t e l y a f t e r i n t r a v i t r e a l i n j e c t i o n s . I n a n o t h e r g r o u p Cy t h e r a p y was s t a r t e d one week a f t e r i n t r a v i t r e a l i n j e c t i o n s . M a t e r i a l s and Methods P r i o r t o t h e i n t r a v i t r e a l i n j e c t i o n a l l e y e s were e x a m i n e d by s l i t - l a m p b i o m i c r o s c o p y t o r u l e o u t t h e p r e s e n c e o f p r e - e x i s t i n g o c u l a r d i s e a s e s . F i f t e e n l o c a l l y s u p p l i e d New Z e a l a n d w h i t e f e m a l e r a b b i t s ( w e i g h i n g 2.2 -2.4kg) were t r a n q u i l i z e d by i n t r a m u s c u l a r i n j e c t i o n o f 0.2 m l / k g o f a 1 0 : 1 s o l u t i o n (100 m g / m l ) o f k e t a m i n e h y d r o c h l o r i d e and a c e t y l p r o m a z i n e m a l e a t e (Roget S .T .B . and A y e r s t L a b o r a t o r i e s , V a n c o u v e r ) . A f t e r b o t h e y e s were a n a e s t h e t i z e d w i t h p r o p a r a c a i n e HC1 (0.5%), 0.10 m l o f a p r e v i o u s l y p r e p a r e d s o l u t i o n o f 50 mg/ml o f BSA i n S a l i n e 78 (which had been s t e r i l i z e d by f i l t e r i n g the BSA s o l u t i o n t h r o u g h a M i l l e x - G s ( R ) 0.22 m i c r o n d i s p o s a b l e f i l t e r u n i t ) was i n j e c t e d i n t o the r i g h t v i t r e o u s , t a k i n g c a r e t o a v o i d t h e c i l i a r y b o d y and l e n s . P r i o r t o t h e i n t r a v i t r e a l i n j e c t i o n o f BSA an aqueous p a r a c e n t e s i s was per formed and 0 .10 m l o f a q u e o u s h u m o u r was r e m o v e d t o p r e v e n t e x t r a v a s a t i o n o f t h e i n t r a v i t r e a l i n o c u l u m . The o p p o s i t e l e f t v i t r e o u s r e c e i v e d 0.10 ml o f s a l i n e t o s e r v e as t h e c o n t r o l . A 30 gauge n e e d l e a t t a c h e d t o a t u b e r c u l i n s y r i n g e was u s e d t o p e r f o r m t h e a q u e o u s p a r a c e n t e s i s a n d t h e i n t r a v i t r e a l i n j e c t i o n s . The a n i m a l s were d i v i d e d i n t o t h r e e g r o u p s o f f i v e r a b b i t s e a c h . I m m e d i a t e l y a f t e r t h e i n t r a v i t r e a l i n j e c t i o n s , Group 2 r e c e i v e d s u b c o n j u n c t i v a l i n j e c t i o n o f 5 mg o f Cy i n O . l c c once p e r week and G r o u p 3 r e c e i v e d t h e s u b c o n j u n c t i v a l i n j e c t i o n o f 5 mg o f Cy i n O . l c c one week a f t e r i n o c u l a t i o n w i t h BSA. Group 1 s e r v e d as the c o n t r o l s and r e c e i v e d no t r e a t m e n t , e x c e p t f o r O . l c c o f n o r m a l s a l i n e which was g i v e n s u b c o n j u n c t i v a l l y once p e r week. The 5 mg o f Cy i n O . l c c and O . l c c o f normal s a l i n e were i n j e c t e d p o s t e r i o r t o the s u p e r i o r l i m b u s o f the r i g h t eye by means o f a 25 gauge n e e d l e a t t a c h e d t o a t u b e r c u l i n s y r i n g e once a week upon i n t i t i a t i o n o f t h e r a p y . The r a b b i t s were examined d a i l y by s i i t - l a m p b i o m i c r o s c o p y , i n d i r e c t ophthalmoscopy and s e r i a l p h o t o g r a p h y . The r a b b i t s were a l s o s t u d i e d by two o t h e r examiners who had no knowledge o f which r a b b i t s r e c e i v e d t r e a t m e n t and no s i g n i f i c a n t v a r i a b i l i t y was f o u n d among t h e e x a m i n e r s ( d a t a n o t shown). The d e g r e e o f i n f l a m m a t i o n i n e a c h eye was g r a d e d a c c o r d i n g t o the scheme p r e s e n t e d i n T a b l e V m o d i f i e d from Fox . An o v e r a l l v a l u e o f i n f l a m m a t i o n was o b t a i n e d a f t e r e v a l u a t i n g each c r i t e r i o n f o r each each r a b b i t i n t h i s e x p e r i m e n t . The exper iment was t e r m i n a t e d a f t e r t h r e e weeks and a l l o f t h e r a b b i t s w e r e s a c r i f i c e d w i t h a n o v e r d o s e o f p e n t o b a r b i t a l . The g l o b e s were e n u c l e a t e d , f i x e d i n 10% b u f f e r e d f o r m a l i n , e m b e d d e d i n p a r a f f i n , s e c t i o n e d v e r t i c a l l y a n d s t a i n e d by H a e m a t o x y 1 i n - E o s i n f o r h i s t o p a t h o l o g i c a l s t u d i e s . H i s t o l o g i c assesment was made on t h e amount o f c e l l u l a r i n f i l t r a t e , f i b r i n c o n t e n t and s t r u c t u r a l c h a n g e s ( e . g . p o s t e r i o r s y n e c h i a e , r e t i n a l d e t a c h m e n t ) . T h i s was g r a d e d by m y s e l f and a p a t h o l o g i s t who had no knowledge o f t h e s p e c i f i c o r i g i n o f the s l i d e s . No s i g n i f i c a n t v a r i a b i l i t y was found among t h e examiners . 80 Table V Conjunctiva Hyperemia Iris injection Ciliary Flush NV of Cornea Posterior Synechia Anterior Chamber Posterior Chamber Overall Inflammation Flares Cells Fibrin Flares Cells Fibrin none none none none none none none none none none none 0 trace trace trace trace trace trace trace trace trace trace trace 1 mild mild mild mild mild mild mild mild mild mild mild 2 mod mod mod mod mod mod mod mod mod mod mod 3 severe severe severe severe severe severe severe severe severe severe severe 4 NV = neovascuralization mod = moderate R e s u l t s C l i n i c a l O c u l a r F i n d i n g s I m m e d i a t e l y a f t e r t h e a q u e o u s p a r a c e n t e s i s , i n t r a v i t r e a l and s u b c o n j u n c t i v a l i n j e c t i o n (o f G r o u p 2) t h e a n t e r i o r chamber became s h a l l o w and the i r i s appeared p a l e w i t h a r e t u r n t o n o r m a l w i t h i n 2 h o u r s . T h e r e was a m i l d t o m o d e r a t e , n o n - s p e c i f i c , t r a n s i e n t i n f l a m m a t o r y r e a c t i o n w i t h i n t r a v i t r e a l i n j e c t i o n s w i t h b o t h BSA and s a l i n e . The i n f l a m m a t o r y r e a c t i o n p e a k e d a t 24 h o u r s and was c h a r a c t e r i z e d by m i l d p e r i c o r n e a l h y p e r e m i a , m i l d t o moderate v a s o d i l a t i o n o f the i r i s v e s s e l s , m i l d t o moderate f l a r e and m i l d f i b r i n i n the a n t e r i o r chamber. The i n f l a m m a t o r y r e a c t i o n s l o w l y r e s o l v e d o v e r the next two d a y s . T h e r e a f t e r , t h e e y e s r e m a i n e d s t a b l e u n t i l t h e f i f t h day a t w h i c h p o i n t t h e a n i m a l s d e v e l o p e d v a r y i n g d e g r e e s o f u n i l a t e r a l u v e i t i s . T h i s t r a n s i e n t i n f l a m m a t o r y r e a c t i o n i s due t o t h e trauma o f i n j e c t i o n and h a s b e e n n o t e d b y o t h e r s . 7 7 T h e r e s u l t s f r o m t h e e x p e r i m e n t a l r a b b i t s i n G r o u p s 2 ( t h a t r e c e i v e d s u b c o n j u n c t i v a l Cy t h e r a p y i m m e d i a t e l y a f t e r i n t r a v i t r e a l i n j e c t i o n o f BSA) and 3 ( that r e c e i v e d s u b c o n j u n c t i v a l Cy t h e r a p y one week a f t e r i n t r a v i t r e a l i n j e c t i o n o f BSA) were c o m p a r e d t o t h e c o n t r o l s i n (Group 1) f o r t h e o n s e t and deve lopment o f u v e i t i s . O b s e r v a t i o n s on the c o u r s e o f the u v e i t i s a r e summarized i n a p l o t o f the d a i l y compos i tes o f the o v e r a l l i n f l a m m a t i o n f o r each day i n f i g u r e 4.0. E x c e p t f o r the t r a n s i e n t i n f l a m m a t o r y response t o the 82 t r a u m a o f t h e n e e d l e a l l o f t h e l e f t e y e s w h i c h h a d r e c e i v e d a n a q u e o u s p a r a c e n t e s i s a n d i n t r a v i t r e a l i n j e c t i o n o f 0.10 ml o f p h y s i o l o g i c a l s a l i n e , d e v e l o p e d no u v e i t i s . The r i g h t e y e s o f t h e r a b b i t s i n G r o u p 1 ( c o n t r o l s ) w h i c h were i n j e c t e d i n t r a v i t r e a l l y w i t h BSA and h a d no t r e a t m e n t d e v e l o p e d v i s i b l e t r a c e t o m i l d u v e i t i s s i x days a f t e r t h e i n j e c t i o n s . T h i s was c h a r a c t e r i z e d by m i l d i r i s and c o n j u n c t i v a l h y p e r e m i a , m i l d p e r i l i m b a l i n j e c t i o n , c e l l s , f l a r e and f i b r i n i n t h e a n t e r i o r chamber . The u v e i t i s p r o g r e s s e d p e a k i n g by t h e 1 4 t h day when i t had become q u i t e s e v e r e . The a n t e r i o r chamber a t t h i s p o i n t showed m i l d f l a r e and m o d e r a t e c e l l s and f i b r i n . The p o s t e r i o r chamber had become q u i t e i n v o l v e d w i t h moderate t o s e v e r e f l a r e , c e l l s a n d f i b r i n ; a s w e l l , m i l d n e o v a s c u l a r i z a t i o n o f c o r n e a was n o t e d and i n two o f t h e r a b b i t s p o s t e r i o r s y n e c h i a e were o b s e r v e d . A f t e r t h e 1 4 t h day t h e u v e i t i s s t a r t e d t o s u b s i d e and a m o d e r a t e i n f l a m m a t o r y r e s p o n s e m a i n l y i n t h e p o s t e r i o r chamber was p r e s e n t by day 21. The g r o u p s t h a t r e c e i v e d t r e a t m e n t , ( G r o u p s 2 and3) showed a s i m i l a r b u t s i g n i f i c a n t l y l e s s e r i n f l a m m a t o r y r e s p o n s e t h a n was o b s e r v e d i n t h e c o n t r o l s (Group 1) . I n b o t h t h e t r e a t e d groups t r a c e u v e i t i s was noted s even days a f t e r i n t r a v i t r e a l i n j e c t i o n , p e a k i n g by the 14th day and s u b s i d i n g t h e r e a f t e r . G r o u p 2, w h i c h was t r e a t e d i m m e d i a t e l y a f t e r the i n t r a v i t r e a l i n j e c t i o n , by the 14th 83 day showed a m i l d r e s p o n s e w h i c h s u b s i d e d t o a t r a c e i n f l a m m a t o r y r e s p o n s e by day 21. G r o u p 3 w h i c h r e c e i v e d t r e a t m e n t one week a f t e r the i n t r a v i t r e a l i n j e c t i o n o f BSA showed a s l i g h t l y g r e a t e r r e s p o n s e t h a n G r o u p 2. By t h e 1 4 t h d a y G r o u p 3 showed a m i l d t o m o d e r a t e i n f l a m m a t o r y re sponse which s u b s i d e d t o a t r a c e t o m i l d inf lammmatory r e s p o n s e by day 21. As w i t h G r o u p 1, b e t w e e n d a y s s e v e n and 14, b o t h G r o u p s 2 and 3 showed t h a t t h e u v e i t i s was m a i n l y r e s t r i c t e d t o t h e p o s t e r i o r chamber w i t h s l i g h t i n v o l v e m e n t o f the a n t e r i o r chamber. In summary, s u b c o n j u n c t i v a l l y a d m i n i s t e r e d Cy was found t o e x h i b i t an i m m u n o s u p r e s s i v e a c t i v i t y and t h e e a r l i e r t h e t r e a t m e n t w i t h Cy the m i l d e r the u v e i t i s . T h i s was the c a s e w i t h G r o u p 2 t h a t was t r e a t e d a t t h e t i m e o f t h e i n t r a v i t e r a l i n j e c t i o n . R e p r e s e n t a t i v e p h o t o g r a p h s s h o w i n g t h e p r o g r e s s o f u v e i t i s i n t h e t h r e e g r o u p s a t s e v e n , 14 and 21 days a r e shown i n f i g u r e 4.1. H i s t o l o g i c a l F i n d i n g s The 3 groups were graded f o r i n f l a m m a t i o n depending on t h e amount o f c e l l u l a r i n f i l t r a t e , f i b r i n c o n t e n t and s t r u c t u r a l changes. T h e c o m m o n i n f l a m m a t o r y c e l l s i n c l u d e d p o l y m o r p h o n u c l e a r g r a n u l o c y t e s , macrophages, lymphocytes and p l a s m o c y t e s , w h i l e t h e s t r u c t u r a l c h a n g e s i n c l u d e d r e t i n a l detachment and p o s t e r i o r s y n e c h i a e . 84 G r o u p 1 showed t r a c e i n f l a m i n a t i o n o f t h e a n t e r i o r chamber, and moderate i n f l a m m a t i o n o f the p e r i l e n t i c u l a r r e g i o n , i r i s and a n g l e . Two o f t h e r a b b i t s a l s o d e v e l o p e d p o s t e r i o r s y n e c h i a e . The c h o r o i d showed moderate t o s e v e r e i n f l a m m a t i o n , w h i l e t h e r e t i n a was m o d e r a t e l y i n f l a m e d w i t h p a r t i a l o r c o m p l e t e d e t a c h m e n t . T h e v i t r e o u s c o n t a i n e d m i n i m a l i n f l a m m a t i o n b u t m o d e r a t e t o s e v e r e f i b r i n d e p o s i t s . I n g r o u p 2 t h e a n t e r i o r c h a m b e r was d e v o i d o f i n f l a m m a t i o n , w h i l e t h e p e r i l e n t i c u l a r a r e a , i r i s and a n g l e , showed o n l y t r a c e t o m i l d i n f l a m m a t i o n . One r a b b i t had d e v e l o p e d a p o s t e r i o r s y n e c h i a . The c h o r o i d d i s p l a y e d m i l d t o moderate i n f l a m m a t i o n and t h e r e t i n a showed t r a c e t o m i l d i n f l a m m a t i o n w i t h f o c a l s e r o u s d e t a c h m e n t s . The v i t r e o u s c o n t a i n e d few i n f l a m m a t o r y c e l l s and m i l d t o moderate f i b r i n d e p o s i t s . I n g r o u p 3 t h e r e was a s l i g h t l y g r e a t e r i n f l a m m a t i o n t h a n i n group 2, but much l e s s than i n group 1. There was t r a c e i n v o l v e m e n t o f t h e a n t e r i o r c h a m b e r , and m i l d t o m o d e r a t e i n f l a m m a t i o n o f t h e p e r i l e n t i c u l a r r e g i o n , i r i s a n g l e . One o f t h e r a b b i t s i n t h i s g r o u p a l s o d e v e l o p e d a p o s t e r i o r s y n e c h i a . The c h o r o i d showed a m o d e r a t e i n f l a m m a t o r y r e s p o n s e and t h e r e t i n a d i s p l a y e d m i l d i n f l a m m a t i o n w i t h areas o f f o c a l s erous detachments . The v i t r e o u s a l s o showed m i n i m a l i n f l a m m a t i o n w i t h moderate f i b r i n d e p o s i t s p r e s e n t . R e p r e s e n t a t i v e h i s t o l o g i c a l photographs comparing the 85 v a r y i n g d e g r e e s o f i n f l a m m a t i o n be tween t h e c o n t r o l s and e x p e r i m e n t a l r a b b i t s a r e shown i n f i g u r e s 4.2-4.5. S t a t i s t i c a l A n a l y s i s An r e p e a t e d measures a n a l y s i s o f v a r i a n c e ( n e s t e d ) t e s t showed t h a t t h e r e was a s i g n i f i c a n t d i f f e r e n c e between the t r e a t e d and c o n t r o l r a b b i t s . The l e v e l o f s i g n i f i c a n c e b e t w e e n t h e g r o u p s was f o u n d t o be p=0.0001. T h e r e was a l s o f o u n d t o be a s i g n i f i c a n t d i f f e r e n c e be tween t h e two t r e a t e d groups o f r a b b i t s , w i t h the l e v e l o f s i g n i f i c a n c e between the groups b e i n g p=0.047. 86 F i g u r e 4 . 0 THE EFFECT OF SUBCONJUNCTIVAL CYCLOSPORINE ON UVEITIS INDUCED BY BOVINE SERUM ALBUMIN 87 F i g u r e 4 . 1 E f f e c t s o f B o v i n e Serum A l b u m i n i n d u c e d u v e i t i s and 5mg/0 l e e o f C y c l o s p o r i n e a d m i n i s t e r e d s u b c o n j u n c t i v a l l y lent Group 2 day 14 Group 2 day 21 0) day 7 F i g u r e 4 . 2 H i s t o l o g i c a l e f f e c t s o f B o v i n e Serum A l b u m i n ( B S A ) i n Group 1 ( r e c e i v e d 2 . 5 x 2 . 5 x In some o f t h e n o n - t r e a t e d r a b b i t s p o s t e r i o r s y n e c h i a ( P S ) were p r e s e n t w i t h a modera te to s e v e r e i n f l a m m a t o r y r e s p o n s e and r e t i n a l de tachment (RD) * see l i s t o f a b b r e v i a t i o n s f o r f i g u r e s 4 . 2 - 4 . 5 . 89 F i g u r e 4 : 3 H i s t o l o g i c a l e f f e c t s o f BSA i n Group 1 on the p o s t e r i o r segment o f t h e The c h o r o i d ( C H ) i n t h e n o n - t r e a t e d r a b b i t s showed a s e v e r e c h r o n i c n o n - g r a n u l o m a t o u s u v e i t i s . 90 F i g u r e 4 . 4 H i s t o l o g i c a l e f f e c t s o f C y c l o s p o r i n e on u v e i t i s i n d u c e d by BSA i n Group 2 ( t r e a t m e n t s t r a t e d on day 0) A m i l d i n f l a m m a t o r y r e s p o n s e i s p r e s e n t i n t he p e r i l e n t i c u l a r a r e a w i t h f o c a l z o n e s ( F Z ) o f i n f l a m m a t i o n i n t h e c h o r i o d ( C H ) as w e l l as f o c a l s e r o u s r e t i n a l d e t a c h m e n t s ( R D ) . 91 F i g u r e 4 . 5 H i s t o l o g i c a l e f f e c t s o f C y c l o s p o r i n e on u v e i t i s i n d u c e d by BSA i n Group 3 ( t r e a t m e n t s t a r t e d on day 7) 2 . 5 x lOx A s l i g h t l y g r e a t e r i n f l a m m a t o r y r e s p o n s e w i t h f o c a l z o n e s ( F Z ) o f i n f l a m m a t i o n i n t h e c h o r o i d ( C H ) and f o c a l s e r o u s r e t i n a l d e t a c h m e n t s ( R D ) a r e p r e s e n t i n t h e s e r a b b i t s compared to Group 2 ( t r e a t m e n t was s t a r t e d on day 0 ) . 92 DISCUSSION U v e i t i s c o n s i s t s o f a g r o u p o f o c u l a r i n f l a m m a t o r y -d i s e a s e s o f w h i c h t h e p a t h o g e n e s i s r e m a i n s u n c e r t a i n . There have been numerous a t tempts t o f i n d s u i t a b l e p o t e n t a n t i - i n f l a m m a t o r y d r u g s w h i c h w i l l be e f f e c t i v e i n t h e t r e a t m e n t o f u v e i t i s . 2 / 3 , 7 , 8 , 1 4 C u r r e n t l y , t h e a c c e p t e d t h e r a p y f o r u v e i t i s has c o n s i s t e d o f l o c a l and s y s t e m i c a p p l i c a t i o n o f c o r t i c o s t e r i o d s , 2 ' 1 4 a l t e r n a t e a n t i -i n f l a m m a t o r y d r u g s , 3 , 4 and s y s t e m i c a d m i n i s t r a t i o n o f i m m u n o s u p p r e s s i v e s . 5 - 8 The eye r e p r e s e n t s a p h a r m a c o l o g i c a l s a n c t u a r y . 1 3 ' 1 4 In o r d e r t o o b t a i n t h e n e c e s s a r y t h e r a p e u t i c l e v e l s w i t h i n t h e e y e , h i g h l e v e l s o f t h e s e d r u g s o r a l l y a n d s y s t e m i c a l l y are r e q u i r e d t o e l i c i t an e f f e c t . These doses have r e s u l t e d i n o b s e r v e d s i d e e f f e c t s such as C u s h i n g ' s -l i k e s y m p t o m s d u e t o s y s t e m i c a d m i n i s t r a t i o n o f c o r t i c o s t e r o i d s , 1 5 m y e l o t o x i c i t y d u e t o s y s t e m i c a d m i n i s t r a t i o n o f i m m u n o s u p p r e s s i v e s 1 2 ' 1 5 and l o c a l a p p l i c a t i o n o f c o r t i c o s t e r o i d s h a v e a l s o r e s u l t e d i n g l a u c o m a . 1 0 ' 1 1 ' 1 4 The p r o b l e m i s compounded because some o c u l a r i n f l a m m a t o r y d i s e a s e s h a v e b e e n f o u n d t o be r e s i s t a n t t o c u r r e n t t h e r a p y and w i t h p r o l o n g e d u s e o f t h e s e d r u g s , t o l e r a n c e may d e v e l o p making them i n e f f e c t i v e . In o r d e r t o improve t r e a t m e n t , an u n d e r s t a n d i n g o f the p r o c e s s e s r e s u l t i n g i n u v e i t i s i s r e q u i r e d . The u n d e r l y i n g m e c h a n i s m i n v o l v e d has been p o s t u l a t e d as i m m u n o l o g i c (autoimmune) i n o r i g i n . C i r c u l a t i n g and i n t r a o c u l a r 93 immune c o m p l e x e s t h a t h a v e been f o u n d i n p a t i e n t s w i t h u v e i t i s l e n d s s u p p o r t t o t h i s c o n t e n t i o n . 3 5 ' 3 6 A l o n g t h e s e l i n e s a n i m a l models have been d e v e l o p e d to s t u d y u v e i t i s , and h a v e r e s u l t e d i n t h e i s o l a t i o n o f r e t i n a l S - a n t i g e n ( S - a g ) . 2 5 S-ag has been i d e n t i f i e d as the 48,000 m o l e c u l a r weight p r o t e i n found i n t h e o u t e r segments o f t h e p h o t o r e c e p t o r . I n e x p e r i m e n t a l s y s t e m s , autoimmune u v e i t i s can be i n d u c e d by S-ag (EAU-S-ag) . Both h u m o r a l a n d c e l l u l a r i m m u n i t y r e s p o n d t o S - a g c a u s i n g a d e l a y e d - t y p e h y p e r s e n s i t i v i t y . E x p e r i m e n t a l i n d u c t i o n o f u v e i t i s u s i n g S-ag(EAU-S-ag) has not a lways been s u c c e s f u l and i s p a r t i c u l a r l y d i f f i c u l t t o induce i n a r a b b i t mode l . S t u d i e s h a v e shown t h a t T - l y m p h o c y t e s p l a y t h e m a j o r r o l e i n t h e d e v e l o p m e n t o f E A U . T h i s p o s i t i o n h a s b e e n s u p p o r t e d by t h e t r a n s f e r o f EAU f r o m u v e i t o g e n i c T - c e l l l i n e s t o r a t s . T h i s o c c u r r e d w i t h o u t t h e i n v o l v e m e n t o f h u m o r a l i m m u n i t y s u g g e s t i n g t h a t i t i s n o t n e c e s s a r y f o r t h e d e v e l o p m e n t o f E A U . 3 1 ' 3 2 T h e t y p e o f E A U - S - a g , r e s e m b l e s t h e o c u l a r i n f l a m m a t o r y r e s p o n s e s e e n i n some t y p e s o f human u v e i t i s , p a r t i c u l a r l y u v e i t i s i n v o l v i n g the p o s t e r i o r segment o f the e y e . 1 8 ' 1 9 ' 2 7 ' 3 4 In some o f these p a t i e n t s , the T - l y m p h o c y t e s were a l s o shown t o be r e a c t i v e t o S -ag i n v i t r o . 1 9 ' 3 4 T h i s f i n d i n g has l e d t o the use o f C y c l o s p o r i n e (Cy) i n t h e t r e a t m e n t o f u v e i t i s , w h i c h c a n c a u s e s p e c i f i c s u p p r e s s i o n o f T - c e l 1 s , 5 8 - 6 0 w i t h o u t c a u s i n g m y e l o t o x i c i t y a s s o c i a t e d w i t h o t h e r i m m u n o s u p p r e s s i v e s such as a z a t h i o p r i n e ' D and cyc lophosphamide . , 1 D Cy has been found e f f e c t i v e i n the t rea tment o f o c u l a r i n f l a m m a t o r y d i s e a s e , and i n the case o f E A U - S - a g , f i n d i n g s s u g g e s t t h a t Cy c a n b r e a k t h e c y c l e o f S - a g s p e c i f i c T -c e l l s n e e d e d f o r t h e i n d u c t i o n and c o n t i n u a t i o n o f t h e OQ n o d i s e a s e . The mechanism by which t h i s may be p o s s i b l e i s i n h i b i t i o n o f the s t i m u l a t i o n o f T - h e l p e r c e l l s w i t h o u t a f f e c t i n g the T - s u p p r e s s o r c e l l p o p u l a t i o n . The r e l a t i v e i n c r e a s e i n t h e s u p p r e s s o r p o p u l a t i o n may be due t o t h e b r e a k i n the c y c l e o f T - c e l l r e c r u i t m e n t which may a l s o be s p e c i f i c f o r S - a g . T h i s s u g g e s t s t h a t t h e d i s e a s e may r e m a i n u n d e r c o n t r o l e v e n a f t e r C y h a s b e e n d i s c o n t i n u e d . 1 8 U n f o r t u n a t e l y , i n t h e c l i n i c a l s i t u a t i o n t h e d i s e a s e o f t e n r e c u r s , p a r t l y b e c a u s e t h e u v e i t i s s e e n i n t h e c l i n i c may n o t be p r e d o m i n a t e l y due t o a c e l l u l a r immune r e s p o n s e , 3 1 and b e c a u s e t h e h i g h d o s e s o f Cy t h a t a r e r e q u i r e d t o be g i v e n o r a l l y and s y s t e m i c a l l y t o r e a c h t h e r a p e u t i c l e v e l s w i t h i n the eye r e s u l t i n r e n a l t o x i c i t y , t h i s l i m i t i n g i t s u s e . 1 7 ' 4 2 ' 4 3 P r e v i o u s l y , t h e l o c a l i m m u n o s u p p r e s s i v e v a l u e o f a g r o u p o f a n t i n e o p l a s t i c d r u g s t h a t w e r e i n j e c t e d s u b c o n j u n c t i v a l l y were a s s e s s e d f o r t h e i r e f f e c t i v e n e s s a g a i n s t EAU i n d u c e d by b o v i n e serum a l b u m i n ( E A U - B S A ) . The r e s u l t s showed t h a t these drugs were a b l e t o d e l a y the o n s e t and r e d u c e t h e s e v e r i t y o f t h e E A U . H o w e v e r , t h e s e drugs produced l o c a l t o x i c i t y , which a l t h o u g h r e v e r s i b l e w i t h d i s c o n t i n u a n c e o f t h e d r u g s w o u l d n o t h a v e been a c c e p t a b l e i n t h e c l i n i c a l c i r c u m s t a n c e . Y e t , s u b c o n j u n c t i v a l a d m i n i s t r a t i o n o f t h e drugs c i r c u m v e n t e d t h e s i d e e f f e c t s produced when g i v e n s y s t e m i c a l l y (appendix I) -T h i s a p p r o a c h was u s e d t o d e v e l o p a p r o t o c o l f o r t h e s u b c o n j u n c t i v a l i n j e c t i o n o f Cy, i n the hope t h a t the s i d e e f f e c t s a s s o c i a t e d w i t h s y s t e m i c a d m i n i s t r a t i o n may be a v o i d e d , m a k i n g p o s s i b l e t h e r o u t i n e u s e o f Cy i n o c u l a r i n f l a m m a t o r y d i s e a s e . The maximum t o l e r a b l e s u b c o n j u n c t i v a l l y a d m i n i s t e r e d d o s e by t h e r a b b i t s ' eye (5 mg i n O . l c c o n c e p e r week) o f Cy, was used t o t e s t i t s e f f e c t i v e n e s s i n EAU-BSA. I n d u c t i o n o f EAU was f i r s t t r i e d by u s i n g S - A g and t h e p r o c e d u r e o u t l i n e d by W a c k e r 2 5 and o t h e r s , 2 9 ' 3 0 ' 3 3 i n o r d e r t o have a model t h a t r e s e m b l e d human u v e i t i s on which t o t e s t Cy a d m i n i s t e r e d s u b c o n j u n c t i v a l l y . U n f o r t u n a t e l y , the d i s e a s e d i d not d e v e l o p i n any o f the r a b b i t s , and we t h e r e f o r e c h o s e t o i n d u c e u v e i t i s by B S A , 7 7 ' 7 8 w h i c h causes a much more acute u v e i t i s and the r e s u l t i n g i n f l a m m a t o r y r e s p o n s e w h i c h i s more l i k e l y due t o t h e a c t i v a t i o n o f b o t h h u m o r a l and c e l l u l a r i m m u n i t y , 7 9 i n c o n t r a s t t o a p r e d o m i n a t l y c e l l u l a r immune response as seen i n E A U - S - a g / ° ' " ' J S i n c e Cy c a u s e d i m m u n o s u p p r e s s i o n p r i m a r i l y by i n h i b i t i n g s t i m u l a t i o n o f T - c e l l s , and o n l y has a m i n i m a l e f f e c t on T - c e l 1 - i n d e p e n d e n t B - c e l l f u n c t i o n , 5 6 , 5 7 the e f f e c t o f Cy would be reduced i n EAU-BSA. Cy t h e r a p y was a d m i n i s t e r e d t o a group o f r a b b i t s a t t h e t i m e o f i n o c u l a t i o n w i t h BSA, a n o t h e r g r o u p o f r a b b i t s r e c e i v e d t rea tment seven days a f t e r i n o c u l a t i o n , and a c o n t r o l group which were i n o c u l a t e d w i t h BSA r e c e i v e d no t r e a t m e n t . The r e s u l t s showed t h a t i n E A U - B S A s u b c o n j u n c t i v a l i n j e c t i o n o f Cy was a b l e t o d e l a y the onset and reduce the s e v e r i t y o f E A U w i t h o u t c a u s i n g a n y a p p a r e n t s i d e e f f e c t s . The d a t a sugges ts t h a t the r a b b i t s t h a t r e c e i v e d Cy t h e r a p y a t the t ime o f i n o c u l a t i o n had an i n f l a m m a t o r y r e s p o n s e t h a t was l e s s s e v e r e t h a n t h a t o f t h e g r o u p t h a t r e c e i v e d Cy t h e r a p y seven days a f t e r i n o c u l a t i o n w i t h BSA. T h i s was t o be expec ted , as Cy caused the s u p p r e s s i o n o f T -c e l l s d u r i n g t h e e a r l y s t a g e s o f a c t i v a t i o n and had l i t t l e e f f e c t o n t h e s u p p r e s s i o n o f p r i m e d T -c e l l s . 15,56,58,59 T h e r e f o r e , the e a r l i e r the a p p l i c a t i o n o f Cy t h e more e f f e c t i v e i t i s l i k e l y t o be i n t h e t r e a t m e n t o f the d i s e a s e . A l t h o u g h t h e r e was o n l y a s l i g h t d i f f e r e n c e between the two t r e a t e d g r o u p s , t h e r e i s a s i g n i f i c a n t d i f f e r e n c e between t h e c o n t r o l and e x p e r i m e n t a l ( t reated) groups . In t h e t r e a t e d groups t h e r e was a l s o a s i g n i f i c a n t d i f f e r e n c e a n d t h e s i m i l a r i t y i n t h e i n f l a m m a t o r y r e s p o n s e c a n be p a r t l y e x p l a i n e d by the f a c t t h a t EAU-BSA i s not p r e d o m i n a t e l y mediated by c e l l u l a r immunity. A n o p p o r t u n i t y a r o s e t o t e s t C y i n j e c t e d s u b c o n j u n c t i v a l l y i n a v e t e r i n a r y c l i n i c . F o u r c a t s t h a t 97 h a d c h r o n i c c o r t i c o s t e r o i d - r e s i s t a n t a n t e r i o r u v e i t i s ( a p p e n d i x I I ) o u w e r e t r e a t e d f o r t w o m o n t h s b y s u b c o n j u n c t i v a l i n j e c t i o n o f Cy a c c o r d i n g t o t h e p r o t o c o l d e v e l o p e d i n the r a b b i t s , the o n l y e x c e p t i o n b e i n g t h a t the c a t s r e c e i v e d 0.1% t o p i c a l dexamethasone t w i c e d a i l y (BID) t o l e s s e n the e f f e c t s o f the s u b c o n j u n c t i v a l i n j e c t i o n . The u v e i t i s h a d d i s a p p e a r e d i n a l l t h e c a t s , b u t two months a f t e r d i s c o n t i n u i n g Cy t h e r a p y , w h i l e b e i n g m a i n t a i n e d on 0.1% t o p i c a l l y dexamethasone (BID) the u v e i t i s f l a r e d up a g a i n i n t h r e e o f t h e f o u r c a t s . P o s s i b l y , t h e c a u s e o f u v e i t i s was s y s t e m i c i n o r i g i n and a p o p u l a t i o n o f u v e i t o g e n i c T - c e l I s were s t i l l p r e s e n t s y s t e m i c a l l y , r e s u l t i n g i n the r e c u r r e n c e . T h i s showed the f e a s i b i l i t y o f r o u t i n e a p p l i c a t i o n o f Cy when a d m i n i s t e r e d s u b c o n j u n c t i v a l l y . In a d d i t i o n , t h e r e s u l t s s u p p o r t p r e v i o u s s t u d i e s t h a t Cy and dexamethasone a c t s y n e r g i s t i c a l l y . 3 3 Cy a c t s p r e d o m i n a t l y by i n h i b i t i n g the a c t i v a t i o n o f T - h e l p e r c e l l s , w h i l e d e x a m e t h a s o n e i n h i b i t s i n f l a m m a t i o n by i n h i b i t i n g p r o s t a g l a n d i n p r o d u c t i o n and r e s p o n s i v e n e s s o f T - c e l l s , though a t a l a t e r s tage a f t e r the T - c e l l s have been a c t i v a t e d . 98 C o n c l u s i o n s S u b c o n j u n t i v a l i n j e c t i o n s o f Cy were e f f e c t i v e i n r e d u c i n g t h e i n f l a m m a t o r y r e s p o n s e i n E A U - B S A and t h e r e s u l t s suggest t h a t the sooner the t r e a t m e n t w i t h Cy the m o r e e f f e c t i v e i t i s l i k e l y t o b e . S u b c o n j u n c t i v a l i n j e c t i o n s o f Cy were a l s o f o u n d t o be e f f e c t i v e i n t h e t r e a t m e n t o f c h r o n i c c o r t i c o s t e r o i d - r e s i s t a n t a n t e r i o r u v e i t i s i n c a t s w i t h o u t c a u s i n g s y s t e m i c s i d e e f f e c t s . 99 SUMMARY The r e s e a r c h t r i e d t o p r o v i d e an a l t e r n a t i v e r o u t e o f a d m i n i s t r a t i o n f o r C y , t h a t w o u l d g i v e t h e b e n e f i t s o f Cy w h i l e a v o i d i n g the t o x i c e f f e c t s o f i t i n the t rea tment o f o c u l a r i n f l a m m a t i o n . The r e s u l t s from the p h a r m a c o k i n e t i c and u v e i t i s s tudy demonstrated t h a t the s u b c o n j u n c t i v a l r o u t e was a b l e t o meet b o t h r e q u i r e m e n t s , w i t h t h e o n l y l i m i t i n g f a c t o r b e i n g t h a t Cy c o u l d o n l y be a d m i n i s t e r e d o n c e p e r week by t h i s r o u t e . New c o m b i n a t i o n s o f v e h i c l e s o r f o r m u l a t i o n s t h a t a r e l e s s l i p o p h i l i c w o u l d be b e n e f i c i a l i n t h i s r e s p e c t . A l t h o u g h the l e v e l s o f s u b c o n j u n c t i v a l l y a d m i n i s t e r e d Cy may not remain a t the t h e r a p e u t i c l e v e l s w i t h i n the eye o v e r the one week p e r i o d between i n j e c t i o n s , p u l s e t h e r a p y i s known t o be q u i e t e f f e c t i v e i n t h e c a s e o f t h e e y e . T h i s h a s b e e n shown w i t h o t h e r a g e n t s i n j e c t e d s u b c o n j u n c t i v a l l y . T h i s may be because from an i m m u n o l o g i c a l p o i n t o f v i ew the eye i s u n i q u e , i n so f a r as i t i s no t a b l e t o r e p l a c e t h e u v e i t o g e n i c T - c e l l s as f a s t as o t h e r o r g a n s i t e s ; t h e r e f o r e , e a c h s u c c e s s i v e t r e a t m e n t f u r t h e r reduces the u v e i t o g e n i c T - c e l l p o p u l a t i o n w i t h i n the eye e v e n t u a l l y r e s u l t i n g i n the e l i m i n a t i o n o f the d i s e a s e from the eye. The s u b c o n j u n c t i v a l r o u t e o f a d m i n i s t r a t i o n o f Cy may h o p e f u l l y pave the way f o r use o f Cy i n the t rea tment o f o c u l a r inf lammmatory d i s e a s e i n the c l i n i c e i t h e r p r i m a r i l y o r as an a d j u n c t . One, h o w e v e r , must be aware t h a t t h e 100 d i s e a s e may not be a l o c a l phenomenon, but r a t h e r s y s t e m i c i n o r i g i n as was p o s s i b l y t h e c a s e i n t h r e e o f t h e c l i n i c a l l y a f f e c t e d c a t s we t r e a t e d i n which the d i s e a s e r e c u r r e d one month a f t e r d i s c o n t i n u i n g t h e r a p y w i t h s u b c o n j u n c t i v a l l y a d m i n i s t e r e d Cy. 101 B i b l i o g r a p h y 1. S t a t i s t i c a l S t u d i e s on t h e b l i n d p o p u l a t i o n o f C a n a d a r e g i s t e r e d w i t h t h e CNIB i n 1985. C a n a d i a n N a t i o n a l I n s t i t u t e f o r the B l i n d 1985; 1. 2. D i n n i n g WJ. U v e i t i s , P a t h o p h y s i o l o g y and T h e r a p y . T h i e n i n e - S t r a t t o n Inc . New York . 1983; 198-220. 3. Hanna C , Kaufman H E . N o n s t e r o i d s A n t i - I m f l a m m a t o r y Agents . C C . 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I m r a u n o p h a r m a c o l o g i c a l m o n i t o r i n g o f c y c l o s p o r i n A - t r e a t e d r e c i p i e n t s o f c a d a v e r i c k i d n e y a l l o g r a f t s . T r a n s p l a n t a t i o n 1982; 34: 36-45. 109 65 . L e n s m e y e r G L a n d F i e l d s B L . I m p r o v e d l i q u i d c h r o m a t o g r a p h i c d e t e r m i n a t i o n o f c y c l o s p o r i n e , w i t h c o n c o m i t a n t d e t e c t i o n o f a c e l l - b o u n d m e t a b o l i t e . C l i n Chem 1985; 31(2): 1967-301. 66 . C a r r u t h e r s S G , F r e e m a n D J , K o e g l e r J C , e t a l . S i m p l i f i e d 1 i q u i d - c h r o m a t o g r a p h i c a n a l y s i s f o r c y c l o s p o r i n A, and compar i son w i t h radioimmunoassay. C l i n Chem 1983; 29 (1): 180 -183 . 67. Sawchuk R J and C a r t i e r L L . L i q u i d C h r o m a t o g r a p h i c d e t e r m i n a t i o n o f c y c l o s p o r i n A i n b l o o d and p l a s m a . C l i n Chem 1981; 27: 1368-1371. 68. Yee GC, Gmur DJ , and Kennedy MS. L i q u i d - c h r o m a t o g r p h i c d e t e r m i n a t i o n o f c y c l o s p o r i n e i n serum w i t h use o f a r a p i d e x t r a c t i o n p r o c e d u r e . C l i n Chem 1982; 28: 2269-2271. 69. N i e d e r b e r g e r W, Schaub P and B e v e r i d g e T . H i g h -p e r f o r m a n c e l i q u i d c h r o m a t o g r a p h i c d e t e r m i n a t i o n o f c y c l o s p o r i n A i n human p l a s m a and u r i n e . J C h r o m a t o g r 1980; 182: 454-458. 7 0 . G a r r a f f o R a n d L a p a l u s P. S i m p l i f i e d l i q u i d c h r o m a t o g r a p h i c a n a l y s i s f o r c y c l o s p o r i n A i n b l o o d and p l a s m a w i t h u s e o f r a p i d e x t r a c t i o n . J C h r o m a t o g r 1985; 337: 416-422. 71. Wenk M, F o l l a t h F and A b i s c h E . Temperature dependency o f a p p a r e n t c y c l o s p o r i n A c o n c e n t r a t i o n s i n p l a s m a . C l i n Chem 1983; 29(10) : 1865. 110 72. D i e p e r i n k H . T e m p e r a t u r e d e p e n d e n c y o f c y c l o s p o r i n e p l a s m a l e v e l s . L a n c e t 1983; 1(8321): 416. 73. Bennet t M J , C a r p e n t e r KH, Worthy E , e t a l . C y c l o s p o r i n c o n c e n t r a t i o n s i n whole b l o o d and p l a s m a . C l i n Chem 1984; 30: 817. 74 . N i e d e r b e r g e r W, G r a t w o h l A , A b i s c h E , e t a l . D i s t r i b u t i o n and b i n d i n g s o f c y c l o s p o r i n e i n b l o o d and t i s s u e . T r a n s p l a n t P r o c 1983; 15(4): 2419-2421. 75. Bowers LD and C a n a f a x DM. C y c l o s p o r i n e : E x p e r i e n c e w i t h t h e r a p e u t i c m o n i t o r i n g . T h e r Drug M o n i t 1984; 6: 142-147. 76. Fox A, Hammer ME, L i l l P, e t a l . E x p e r i m e n t a l u v e i t i s e l i c i t e d b y p e p t i d o g 1 y c a n - p o 1 y s a c c h a r i d e c o m p l e x e s , l i p o p o l y s a c c h a r i d e , and muramyl d i p e p t i d e . A r c h O p h t h a l m o l 1984; 102: 1063-1067. 77. W i r o s t k o E and H a l b e r t SP . S u p p r e s s i o n o f a l l e r g i c u v e i t i s by 6 - m e r c a p t o p u r i n e . J Exp Med 1962; 116: 6 5 3 -654. 78. U u s i t a l o H. An e x p e r i m e n t a l u v e i t i s i n d u c e d by b o v i n e serum a l b u m i n a t r a n s m i s s i o n and s c a n n i n g e l e c t r o n m i c r o s c o p i c s tudy . A c t a O p h t h a l a m o l 1984; 62: 413-424. 79. U u s i t a l o H. An acute i n f l a m m a t o r y r e a c t i o n induced by i n t r a v i t r e a l b o v i n e serum a l b u m i n i n p r e s e n s i t i z e d r a b b i t s : t h e e f f e c t o f p h e n t o l a m i n e . A c t a O p h t h a l m o l 1984; 62: 636-642. I l l 80. K a l s i GS, B u s s a n i c h N , G u d a u s k a s G , e t a l . Combined l o c a l c h e m o t h e r a p y f o r s p o n t a n e o u s l y o c c u r r i n g a n t e r i o r u v e i t i s i n c a t s . Submi t t ed . 81 . Rootman J , B u s s a n i c h N, J o h n s t o n WH. S u b c o n j u n c t i v a l C y c l o s p o r i n e : S u p p r e s i o n o f c o r n e a l x e n o g r a f t r e j e c t i o n i n r a b b i t s . Submit ted . 112 APPENDIX I 113 THE EFFECTS OF MULTIPLE DOSE SUBCONJUNCTIVAL ANTINEOPLASTICS ON ACUTE IMMUNE UVEITIS J. Rootaan M.N. Bussanlch G. Gudauskas C. Kuml 114 nmoDOCTZOM Hypersensitivity has b««n recognized as a factor In th« cause of some human ocular Inflammatory diseases especially uveitis, although the amount and nature of antigen Involved are usually unknown.* Corticosteroids have been traditionally used to treat ocular inflammations with success but 3 complications such as susceptibility to infection and Increased intraocular pressures**' say occur. Alternative non-toxic anti-inflammatory therapy, either as an adjunct or used Independently would be an advantage. Pharmacokinetic experiments suggest that penetration of subconjunctlvally administered drugs into the ocular compartments can be up to 100 times higher 6 7 8 9 than the same dose given systemically ' * ' . Subconjunctival injections may provide therapeutic drug levels to the eye while avoiding the higher doses when given systemically. It has been demonstrated that an experimental a l l e r g i c uveitis can be induced by injection of a single dose of bovine serum albumin into the 2 vitreous of rabbits and acute uveitis develops within 6 to 10 days, and persists for at least a week and then subsides. The present experiments were undertaken to determine whether subconjunctival antineoplastic therapy Initiated immediately after the intravitreal injections could suppress the degree of inflammation. MATERIALS AND METHODS Prior to the intravitreal injection a l l eyes were examined with a s l i t lamp to rule out the presence of pre-existing ocular diseases. Twenty local l y supplied Rev Zealand albino rabbits (2.2 - 2. A kg) were tranquillzed by i.m. injection of Ketamine/Acepromazine maleate (100 mg/ml) (10:1). After 115 topical anaesthesia with proparacain HC1 (O.SZ), a solution of crystalized bovine serum albumin (SO ag/ml in 0.92 NaCl solution) vaa f i l t e r e d through a Mil lax - GS 0.22um dlaposabla f l l t a r unit and 0.15 ml of the f i l t r a t e injected into the right vitreous. The opposite l e f t vitreous received 0.15 ml physiological saline to serve as controls. A 30 gauge needle attached to a tuberculin syringe was Inserted through the ocular coats taking care to avoid the posterior lena capsule. Only a small amount of injected f l u i d exuded from the injected sites after removal of the needles. Animals were divided into 5 groups of 4 rabbits each. Immediately after the Intravitreal injections, therapy with subconjunctival injections of saline, methotrexate, 25 mg/ml; cystoslne arabinoside, 150 mg/ml; 5-Fluorouracil, 25 mg/ml and 6-Mercaptopurlne, 40 mg/ml was ini t i a t e d . A l l subconjunctival injections were made twice a week for 3 weeks. A volume of 0.5 ml of each drug solution was injected posterior to the superior llmbus of the right eye by means of a 30 gauge needle attached to a tuberculin syringe. Daily c l i n i c a l observations included careful s l i t lamp observation o f cornea fur evidence o f perlllmbal injection and quantitation of anterior chamber c e l l s (expressed as 0 - 4+), flare (expressed as 0 - 4+) and f i b r i n . Conjunctival hyperemia, edema, mucus secretion and Injection was noted. The i r i s was monitored for swelling, injection and reactivity and the lens and vitreous were studied dally. Serial photography was taken to document the c l i n i c a l course. Peripheral blood counts yiornee on each animal once a week to monitor the extent of hematopoietic toxicity. Additionally, a generalized severity of Inflammatory response was graded on a scale of 0 - 4+ after a modification of the method of 116 Wlroesko and Halbert as fellow*: 0 • normal. 1+ - traca i r l a hyperemia, trace flare and occasional c e l l s . 2+ - mild I r i s hyperemia, c i l i a r y flush, mild f l a r e , few c e l l s In the anterior chambers, peripheral injection. 3+ - moderate amount of f l a r e , cells in the gross, precipitate over lens and cornea, f i b r i n in the anterior chamber, perlllmbal injection, posterior synechia, vitreous opacity. 4+ - extreme i r i s hyperemia, miotic pupils, Intense photophobia, f i b r i n in the anterior chambers, dense ce l l u l a r precipitate, posterior synechia, perlllmbal injection. The composite of the gradlngs of a) general c l i n i c a l observations, b) flare and c e l l s i n the anterior chamber for a l l animals in each group were plotted against time. (Fig. 1). The experiment was terminated after 22 days. A l l rabbits were sacrificed with an overdose of pentobarbital sodium. Representative eyes were enucleated, fixed i n 10Z buffered formalin, sectioned and stained for h i s t o l o g i c a l studies. RESULTS Immediately after the Intravitreal injections the anterior chambers became shallow and the i r i s appeared pale with a return to normal within 2 hours. There was a mild, non-specific, transient inflammatory reaction with Intravitreal injections with both bovine serum albumin and saline. This lasted for a day and was characterized by a mild pericorneal hyperemia, slight vasodilatation of the i r i s vessels and flare i n the anterior chambers. This reaction 117 la dua to tha trauma of injection and has been noted by others. Thereafter a l l treated eyes remained quiet for at least 5 days at which point the animals developed varying degrees of unilateral uveitis. The experimental results compared the onset and development of uveitis in rabbits treated with subconjunctival saline versus those treated with antineoplastic agents. Observations on the course of uveitis are summarized in a plot of the daily composites of the gradlngs 0 - 4+. (Fig. 1 - l e f t ) . Except for the transient inflammatory response to the trauma of the needle a l l l e f t eyes which received .15 ml normal saline developed no u v e i t i s . The right eyes of animals injected l n t r a v l t r e a l l y with bovine serum albunim and treated with subconjunctival saline developed grossly v i s i b l e uveitis 6 days after the injections. This was characterized by i r i s and conjunctival hyperemia, perlllmbal injection, and c e l l s and flare in the anterior chambers. The inflammation peaked between the ninth and eleventh days after the i n t r a v i t r e a l Injections. After the thirteenth day the inflammation began to subside. With cytosine arabinoside, uveitis was observed 8 days after the i n t r a v i t r e a l injections. The uveitis peaked between the thirteenth day and the sixteenth day and subsided thereafter. Animals injected with methotrexate developed an i n i t i a l l y mild uveitis between the seventh and eleventh days with a moderate rise i n severity until the seventeenth day when i t began to subside. With 5-Fluorouracil a mild uveitis developed on the tenth day and lasted for 6 days. Animals injected with 6-Mercaptopurine had a milder uveitis throughout the period of the experiment. Inflammation was minimal 118 u n t i l th« sixteenth day whan a c l i n i c a l l y significant uveitis davalopad laatlng 4 days. In aummary, a l l 4 drugs vara found to exhibit anti-inflamatory action in varying degreea with later onset and milder uveitis than controls. 6-Mercaptopurlne appeared to be the most effective In suppressing the degree and onset of the inflammation, followed by 5-Fluorouracll. The eyes treated with cytosine arabinoslde and methotrexate were more Inflamed than those treated with 6-Mercaptopurine and 5-Fluorouracil. It was noted that the degree of inflammation i n a l l eyes, Including those treated with saline, waa about the same during the latter quarter of the experiment. TOXICITY STUDIES Systemic Toxicity. There was no significant sign of hemopoietic toxicity at the levels of drugs .injected as monitored once a week by peripheral blood counts for the duration of the experiment. Ocular Toxicity. The multiple dose injection of methotrexate resulted in no significant signs of toxicity. With cytosine arabinoslde there was a mild conjunctival hyperemia and djeoosis which resolved after the derapy was withdrawn. Injections with 6-Mercaptopurlne caused occasional hemorrhages i n the conjunctiva and In a number of animals there was focal necrosis of the conjunctiva. 5-Fluorouracil was clearly toxic, leading to significant corneal edema and subsequent vascularization of peripheral cornea. In addition, conjunctival chemosis and periocular hair loss was noted. 119 OCULAR triAMMATORY RESPONSE TOU.OWHQ SUBCONJUNCTIVAL ANTtCOFVASTICS • H L 1 11 M J L a 1L 11 $* n -jfTTt 11111 i iTTn J L J to D_aH, UDJ Tkn*(eayt> 120 C0MCL3SI0KS Thar* h«v« been numerous attempts to find suitable potent anti-inflammatory drugs which w i l l be effective for the treatment of non-specific u v e i t i s 1 , 2 3 4 5 without exhibiting the side effects of corticosteroids. ' ' ' Some of these side effects may be avoided by the use of non-steroidal anti-inflammatory 13 drugs. Some cytotoxic agents, including cytosine arablnoside, methotrexate and 6-Mercaptopurlne are known to have both anti-inflammatory and immunosuppressive properties 1* and may be useful i n the treatment/uveltls. 6-Mercaptopurine has been used systemically to suppress immune u v e i t i s 2 * 1 5 ' 1 6 ' 1 , 7 . We have attempted to assess the local immunosuppressive value of this group of antineoplastic drugs when Injected subconjunctival^. Our experiments have demonstrated that 6-Mercaptopurine end to a lesser degree 5-Fluorouracil were able to delay the onset and reduce the severity of a l l e r g i c uveitis induced by intravitreal injection of bovine serum albumin i n rabbits. Cytosine arablnoside and methotrexate also appeared to reduce the severity and delay the onset of inflammatory response. In the case of both 5-Fluorouracil and 6-Mercaptopurlne we f e l t that there was significant l o c a l toxicity at dose levels employed and although these toxicities were reversible with discontinuance of the drugs they would not be acceptable in the c l i n i c a l circumstance. Further investigation of subconjunctivally injected 6-Mercaptopurlne i n particular might be worthwhile, perhaps in modified dose levels or as an adjuvant to other forms of therapy. 121 Multiple Dose Subconjunctival Antlnaoplaatlcs REFERENCES 1. Theodora FH, Schlosaman A: Ocular allergy. The William and Vllklns Co., Baltimore, 1958. 2. Wiroatko E, Ralbert SP: Suporesslon of allergic uveitis by 6-mer-captopurlne. J Exp Med 116: 653, 1962. 3. Welnatein L: Corticotropin and corticosteroids in human v i r a l i n -fections. Med C l i n i c North Am 46: 1141 - 1661, 1962. 4. Becker B, Mills W: Corticosteroids and intraocular pressure. Arch Ophthalmol 70: 500 - 507, 1963. 5. Armaly MF: Effects of corticosteroids on intraocular pressure and f l u i d dynamics. Arch Ophthalmol 70 482 - 491, 1963. 6. Rootman J, Gudauskas G, Kumi C: Subconjunctival versus intravenous cytosine arabinoslde: effect of route of administration and ocular toxoclty. Investigative Ophthalmol and Visual Science (in press). 7. Gudauskas G, Ostry A, Rootman J : Concentrations of t r l t i a t e d metho-trexate i n ocular compartments, serum and urine after subconjunctival and intravenous injection. Can J Ophthalmol 15: 179 - 182, 1980. 8. Rootman J, Josephy PD, Adomat H, Palcic B: Ocular absorption and toxicity of a radlosensitizer and i t s effect on hypoxic c e l l s . Arch Ophthalmol 100: 468 - 471, 1982. 9. Rootman J, Tisdall J, Gudauskas G, Ostry A: Intraocular penetration 14 of subconjunctivally administered C-Fluorouracil in rabbits. Arch Ophthalmol 97: 2375 - 2378, 1979. 1 2 2 Multiple Dose Subconjunctival Antineoplastics 10. Barza M, Baum J: Intraocular penetration of c a r b e n l c l l l i n in rabbits. Am J Ophthalmol 75: 305 - 313, 1973. 11. Records RE, E l l i s PP: Intraocular penetration of a a p l c l l i n , methi-c i l l n and o x a c i l l i n . Am J Ophthalmol 64: 135, 1967. 12. Hanna C, Kaufman HE (ad.): Nonsteroids anti-inflammatory agents. Charles C Thomas publisher, Springfield, 111: 136 - 149, 1970. 13. Hanna C, Keats HC: Indomethacin In ocular inflammation i n rabbits. Arch Ophthalmol 77: 554, 1967. 14. Meyer FH, Javetz E, Goldfein A: Review of medical pharmacology. 4th edition: 499 - 506, 1974. 15. Schwartz R, Eisner A, Dameshek V: The effect of 6-mercaptopurine on primary and secondary immune responses. J C l i n Invest 38: 1393, 1959. 16. Newell FW, K r i l l AE, Thomson A: The treatment of uveitis with 6-mercaptopurine. Am J Ophthalmol 61: 1250, 1966. 17. Wong VG: Immunosuppressive therapy of ocular inflammatory diseases. Arch Ophthalmol 81: 628, 1969. 123 APPENDIX I I 124 Combined l o c a l chemotherapy f o r s p o n t a n e o u s l y o c c u r r i n g a n t e r i o r u v e i t i s i n c a t s G u r s h a r a n S. K a l s i , * BSc; N i c k B u s s a n i c h , A DVM; Gedy G u d a u s k a s , - PharmD; J a c k Rootman , A * MD; From the departments o f A o p h t h a l m o l o g y and • p a t h o l o g y , U n i v e r s i t y o f B r i t i s h Co lumbia and Vancouver G e n e r a l H o s p i t a l , and the -Advance T h e r a p e u t i c s Program, C a n c e r C o n t r o l Agency o f B r i t i s h C o l u m b i a , V a n c o u v e r . R e p r i n t r e q u e s t s t o : D r . N i c k B u s s a n i c h , K n i g h t Rd V e t e r i n a r y C l i n i c L t d , 7161 V i c t o r i a . , V a n c o u v e r , BC. T h i s s t u d y was s u p p o r t e d by the Canadian V e t e r i n a r y R e s e a r c h T r u s t F u n d . 125 Abstract Spontaneously occurring corticosteriod resistant anterior uveitis in four domestic cats was treated with subconjunctival injection of cyclosporin A and 0.1% dexamethasone topically for a period of eight weeks. Thereafter, the cats were maintained on 0.1% dexamethasone topically. The inflammation in all the cats had been stable for up to 2 months following treatment, but had relapsed while being maintained on topical dexamethasone twice daily. Introduction We have previously demonstrated the superiority of ocular penetration following local injection of several commonly used antineoplastic agents24.25. Not only do these drugs penetrate in higher concentrations than are achieved with systemic administration of equal doses.20-23 Dut the paraocular route avoids the metabolic breakdown of the active components of the drugs characteristically seen with systemic administration. Herein we report the treatment of chronic anterior uveitis by combined local therapy with Cyclosporin A and 0.1% dexamethasone. 126 Method The four cats chosen for the study had previously been treated topically and systemically with corticosteriods for anterior uveitis with an unsatisfactory response. They received Cyclosporin A subconjunctival^ once a week and 0.1% dexamethasone twice daily, for 8 weeks. The eye was anesthetized with proparacain HCL(0.5%), and injected with O.lcc of 5mg/0.1cc of Cyclosporin A a was given subconjunctival^ above the superior limbus using a 25 gauge needle attached to a tuberculin syringe. The cats were followed for a 8 weeks by slit-lamp biomicroscopy, indirect ophthalmoscopy and serial photography. The degree of inflammation was graded according to the following scheme modified from F o x 1 2 : Ciliary corneal corneal iris Ant. Chb. Vitreous HypODyon Overall injection clarity erosion injection haze & Retina intlammation none clear none none clear chorioretinal none 0 detail share tracs trace trace trace trace chorioretinal trace 1 detail blurred mild mild mild mild mild visible but no mild 2 clear detail moderate moderate moderate moderate moderate no detail moderate 3 + cells + cells severe severe severe severe severe obscured severe 4 cells & tibrin a SANDIMMUNE I.V. 50mg/ml; SANDOZ Ltd, BASLE, SWITZERLAND. 127 Cat 1 A domestic short haired 13 year old neutered male, was first seen in October 1984 with bilateral uveitis and hypopyon. It had a normal CBC and blood chemistry and the serum was negative for feline leukemia. The cat had been treated from October 1984 to September 13, 1985 with 0.1% topical dexamethasone alternating with oral prednisone at a dose of 5 mg/day for one month at a time, however, the disease was not subsiding and we had noted an increase in keratic percipitates (KP's) with inflammation of the iris (fig. 1a). In view of the increase in inflammation the cat was placed on Cyclosporin A subconjunctival^ once per week and 0.1% dexamethasone topical twice daily (BID), for 8 weeks. At the end of the treatment the CBC and blood chemistry were unchanged, the inflammation had decreased with only small KP's present and the cat was maintained on 0.1% topical dexamethasone (fig. 1b). Follow-up showed that the cat remained stable for 2 months but relapsed there after while on topical dexamethasone. The overall progress of the cat is documented in figure 1c. 128 QaLZ A domestic short haired 10 year old neutered male, was first seen in September, 1985 with bilateral uveitis, endothelial KP's, nodules on the iris and hypopyon in the right eye. Blood chemistry and CBC was normal and the serum was negative for Feline leukemia and toxoplasmosis. The initial treatment was with 0.1% topical dexamethasone BID for three weeks and at the end of the this period, examination showed only a slight decrease in the inflammation (fig. 2a). Since the disease was not responding to the steriod treatment alone, the cat was placed on Cyclosporin A subconjunctivally once per week and 0.1% topical dexamethasone (BID), for 8 weeks; thereafter, maintained on 0.1% topical dexamethasone BID. At the end of the Cyclosporin A treatment, CBC and blood chemistry was normal except for the decrease in the white blood count from 7500/mm 3 to 5000/mm3. The progress of the cat on the Cyclosporin A and subsequent follow-ups can be seen in figure 2c, demonstrating improvement of the uveitis while on the treatment (fig. 2b) and subsequent recurrence eight weeks after discontinuing therapy. 129 Cat 3 A domestic long haired 9 year old spayed female, was first seen on Februray 11, 1985 with bilateral uveitis, iris nodules and a fibrin clot attached to the right lens. The cat was placed on 0.1% topical dexamethasone and 5 mg of prednisone orally. The CBC and blood chemistry was normal and the serum negative for feline leukemia. On Februray 23, 1985 the eyes had become worse and by March 7 the anterior chamber had improved, but the cat had developed a superficial corneal erosion which slowly healed over one week. The eyes remained stable until early October 11,1985 when the cat had a relapse in both eyes with marked inflammation and a fibrin clot on the surface of the lens (fig. 3a). Because of the severe bilateral flare-up the cat was placed on Cyclosporin A subconjunctival^ once per week and 0.1% topical dexamethasone BID, for 8 weeks. By the end of the 8 week treatment there had been considerable improvement in both eyes with only a few small KP's (fig. 3b). The effects of Cyclosporin A can be seen in figure 3c. Blood chemistry and CBC done at end of the Cyclosporin A treatment was normal. The cat was maintained on 0.1 %topical dexamethasone BID and remains stable for one month. 130 Cat 4; A domestic short haired 8 year old neutered male was first seen on Nov 9, 1985 with right unilateral uveitis. The iris was congested, had nodules on it, and fibrin was noted on the lens capsule with a moderate amount of flare and cells in the anterior chamber vitreous (fig. 4a). This cat was placed on Cyclosporin A subconjunctival^ once per week and 0.1% topical dexamethasone BID, for 8 weeks. By December 7, 1985 it had developed a corneal erosion. The fifth injection of Cyclosporin A and topical dexamethasone was not given until the following week. The cat had improved considerably by the end of the treatment period (fig. 4b). After discontinuing of the treatment the cat was placed on topical dexamethasone BID. There was a minor relapse on the last week of therapy with Cyclosporin A and dexamethasone. The hypopyon had returned and the eye was becoming inflammed again, but on subsequent follow-ups the inflammation had regressed and one month following treatment with Cyclosporin A and dexamethasone there was only a trace of inflammation. The eye still remains stable 16 weeks afte treatment with Cyclosporin A and dexamethasone, the longest period of the four cats in this study. The progress of the cat while on the treatment and subsequent follow-ups can be seen in figure 4c. 131 F i g u r e l a R i g h t eye o f Cat 1 showing Jcera t i c p e r c i p i t a t e s ( arrow ) b e f o r e i n i t i a t i o n o f t r e a t m e n t . 132 F i g u r e l b R i g h t eye o f Cat 1 showing l a c k o f i n f l a m m a t i o n a t c o m p l e t i o n o f t r e a t m e n t w i t h s u b c o n j u n c t i v a l C y c l o s p o r i n A and t o p i c a l 0.1% dexamethasone ( e i g h t h week) . 133 F i g u r e l c CAT 1 4.0 3.5 c 3.0 o IS 2.5 | 2.0 J5 1.5 ^ 1.0 0.5 0.0 0 1 2 3 4 5 6 7 8 16 24 Time (weeks) • Cyclosporin A treatment • treatment ended • right eye J I l I I I 1 UJJ 1 134 F i g u r e 2a R i g h t eye o f C a t 2 showing Hypopyon and s m a l l k e r a t i c p e r c i p i t a t e s b e f o r e i n i t i a t i o n o f t r e a t m e n t . 135 F i g u r e 2b R i g h t o f Cat 2 showing r e s o r p t i o n o f t h e hypopyon and d e c r e a s e i n the number o f k e r a t i c p e r c i p i t a t e s , a t c o m p l e t i o n o f t r e a t m e n t w i t h s u b c o n j u n c t i v a l C y c l o s p o r i n A and t o p i c a l 0.1% dexamethsone ( e i g h t h week) . 136 F i g u r e 2c c g to E E CAT 2 4.0 r 3.5 \ 3.0 A 2.5 \ \ 2.0 \ \ 1.5 \ \ N -1.0 Wo 0.5 0.0 i „ i • right eye O left eye 0 1 2 3 4 5 6 7 8 12 28 Time (weeks) • • Cyclosporin A treatment • treatment ended 137 F i g u r e 3a R i g h t eye o f C a t 3 showing the p r e s e n c e o f a f i b r i n c l o t a t t a c h e d t o t h e l e n s , s m a l l k e r a t i c p e r c i p i t a t e s and n o d u l e s on t h e s u r f a c e o f the i r i s ( arrow ) , b e f o r e i n i t i a t i o n o f t r e a t m e n t . 138 F i g u r e 3b R i g h t eye o f C a t 3 showing d e c r e a s e o f i n f l a m m a t i o n a t t h e end o f t r e a t m e n t w i t h s u b c o n j u n c t i v a l C y c l o s p o r i n A and t o p i c a l 0.1% dexamethsone ( e i g h t h week) . 139 F i g u r e 3c 4.0 3.5 c 3.0 1 2.5 | 2.0 JS 1.5 •E 1.0 0.5 0.0 CAT 3 \ ^ J / \ • r '9ht eye 0 left eye -0- - 0 —0- /M/^ 0 1 2 3 4 5 6 7 8 12 16 Time (weeks) • • Cyclosporin A treatment • treatment ended 140 F i g u r e 4a R i g h t eye o f C a t 4 showing the p r e s e n c e o f a f i b r i n c l o t on the s u r f a c e o f t h e l e n s , w i t h n o d u l e s on t h e i r i s s u r f a c e ( arrow ) b e f o r e i n i t i a t i o n o f t r e a t m e n t . 141 F i g u r e 4b R i g h t eye o f C a t 4 showing decrease o f i n f l a m m a t i o n a t t h e end o f t r e a t m e n t w i t h s u b c o n j u n c t i v a l C y c l o s p o r i n A and t o p i c a l 0.1% dexamethasone. 142 CAT 4 • right eye O left eye / O . O o — o — o - o — o — o — o — o — o — o / y - o - o ^ — 0 1 2 3 4 5 6 7 8 9 13 21 25 Time (weeks) • • • • • • Cyclosporin A treatment • treatment ended 143 D i s c u s s i o n : Because the e t i o l o g y o f u v e i t i s i s not w e l l d e f i n e d , i t can be d i f f i c u l t t o t r e a t and r e c u r r e n t . I t i s t h e r e f o r e not s u p r i s i n g t h a t u v e i t i s accounts f o r 1.0% o f b l i n d human p a t i e n t s i n Canada. 1 The mainstay o f treatment has been s t e r o i d s , i n some case supplemented by immunosuppressive a g e n t s . 2 C y c l o s p o r i n A has been employed s y s t e m i c a l l y (i.m.) and o r a l l y i n the treat m e n t o f u v e i t i s . 3 " 6 I t has been e f f e c t i v e i n t r e a t i n g T - c e l l mediated i n f l a m m a t i o n . 7 " 9 The mode o f a c t i o n o f C y c l o s p o r i n A i s f e l t t o be i n h i b i t i o n o f the r e l e a s e o f i n t e r l e u k i n - 2 from T - h e l p e r c e l l s t h e r e b y s u p p r e s s i n g t h e inflammatory r e a c t i o n . 1 0 ' 1 1 S i n c e r e t i n a l S-antigen i s thought t o be a major f a c t o r i n t h e development o f T - c e l l mediated u v e i t i s , 1 3 ' 1 4 C y c l o s p o r i n A s h o u l d be e f f e c t i v e i n some t y p e s o f u v e i t i s , 3 ' 6 ' 1 5 e s p e c i a l l y i n the acute phase. The problems a s s o c i a t e d w i t h t r e a t i n g u v e i t i s w i t h C y c l o s p o r i n A have been i t ' s s i d e - e f f e c t s . P a r t i c u l a r l y nephro and h e p a t o t o x i c i t y which o c c u r r s a t a r e l a t i v e l y low dose o f 10 m g / k g . 1 6 - 1 9 We have shown w i t h a number o f agents t h a t o c u l a r a b s o r p t i o n can be s i g n i f i c a n t l y enhanced by s u n c o n j u n c t i v a l i n j e c t i o n o f t h e drug when compared t o equidose s y s t e m i c a d m i n i s t r a t i o n . 2 0 - 2 5 In t h i s way i t may be p o s s i b l e t o circumvent the a s s o c i a t e d t o x i c i t i e s o f s y s t e m i c a d m i n i s t r a t i o n . S u b c o n j u n c t i v a l a d m i n i s t r a t i o n o f C y c l o s p o r i n A was developed by us u s i n g a r a b b i t model. As a r e s u l t o f t o x i c i t i e s s t u d i e s o f numerous v e h i c l e s the dose and makeup o f C y c l o s p o r i n A t h a t we used was 0.1 cc o f 5 mg/ O. l c c o f C y c l o s p o r i n A, a d m i n i s t e r e d s u b c o n j u n c t i v a l l y once p e r week. 144 We were a b l e t o a u c c e s s s f u l l y c o n t r o l c h r o n i c s t e r o i d r e s i s t a n t spontaneous a n t e r i o r u v e i t i s by u s i n g C y c l o s p o r i n A once p e r week and 0.1% t o p i c a l dexamethasone BID, f o r e i g h t weeks and t h a n m a i n t a i n i n g t h e c a t s on 0.1% t o p i c a l dexamethasone t w i c e d a i l y . The eyes on a v e r a g e remained s t a b l e f o r about two months ( e x c e p t f o r c a t 4 w h i c h had r i g h t u n i l a t e r a l u v e i t i s and remains s t a b l e t o d a t e ) . The 0.1% t o p i c a l dexamethasone was g i v e n w i t h t h e C y c l o s p o r i n A , because C y c l o s p o r i n A a l o n e would cause m i l d s w e l l i n g o f t h e u p p e r eye l i d , w h i c h would l a s t about 24 t o 48 h o u r s , b u t w i t h 0.1% t o p i c a l dexamethasone t h e s w e l l i n g l a s t e d f o r l e s s t h a n 24 h o u r s . I t wou ld seem from t h e way t h e u v e i t i s r e s p o n d e d t o t h e C y c l o s p o r i n A t h a t i t would be more e f f e c t i v e i n t h e a c u t e phase o f t h e d i s e a s e . From t h i s s t u d y the combined t h e r a p y o f C y c l o s p o r i n A and 0.1% t o p i c a l dexamethasone a c t e d s y n e r g i s t i c a l l y and was a b l e t o c o n t r o l t h e u v e i t i s s u c c e s s f u l l y f o r a p e r i o d o f up t o 2 months a f t e r d i s c o n t i n u i n g t r e a t m e n t . F u r t h e r s t u d i e s on t h e s u b c o n j u n c t i v a l a d m i n i s t r a t i o n o f C y c l o s p o r i n A a r e under c u r r e n t i n v e s t i g a t i o n . Acknowledgements: The a u t h o r s a r e g r a t e f u l t o Sandoz , L t d . ; f o r g e n e r o u s l y s u p p l y i n g us w i t h Sandimmune I . V . 145 1. S t a t i s t i c a l s t u d i e s on t h e b l i n d p o p u l a t i o n o f Canada r e g i s t e r e d w i t h t h e CNIB i n 1985. C a n a d i a n N a t i o n a l I n s t i t u t e f o r t h e B l i n d 1985; 1. 2 . D i n n i n g WJ. U v e i t i s , P a t h o p h y s i o l o g y and T h e r a p y . I n : K r a u s - MacKiw, O ' C o n n o r GR, e d ' s . P u b l . T h i e n e - S t r a t t o n I n c . New Y o r k , 1983; 198-220. 3 . N u s s e n b l a t t R b , P a l e s t i n e A G , Chan C C . C y c l o s p o r i n A t h e r a p y i n t h e t r e a t m e n t o f i n t r a o c u l a r i n f l a m m a t o r y d i s e a s e r e s i s t a n t t o s y s t e m i c c o r t i c o s t e r o i d s and c y t o x i c a g e n t s . Am J Ophtha lmol 1983;96:275-282 . 4. Chan C C , P a l e s t i n e A G , N u s s e n b l a t t RB. C y c l o s p o r i n e -i n d u c e d a l t e r a t i o n o f humoral r e s p o n s e i n e x p e r i m e n t a l autoimmune u v e i t i s . I n v e s t Ophtha lmol V i s S c i 1984;25:867-870. 5. M o c h i z u k i M, N u s s e n b l a t t RB, Kuwabara T , e t a l . E f f e c t s o f c y c l o s p o r i n e and o t h e r immunosuppress ive drugs on e x p e r i m e n t a l autoimmune u v e i t i s i n r a t s . I n v e s t Ophtha lmol V i s S c i 1985;26:226-232 . 6. Graham EM, S a n d e r s MD, James DG, e t a l . C y c l o s p o r i n A i n t h e t r e a t m e n t o f p o s t e r i o r u v e i t i s . T r a n s Ophthamol S o c . U . K . 1985;104:146-151. 7 . L a f f e r t y K J , B o r e l J F , Hodgkin P . C y c l o s p o r i n e - A ( C s A ) : models f o r mechanism o f a c t i o n . T r a n s p l a n t P r o c 1983;15:2242-2247. 8. T u t s c h k a P J , B e s c h o r n e r WE, Hess A d , e t a l . C y c l o s p o r i n A t o p r e v e n t g r a f t - v e r s u s - h o s t d i s e a s e : a p i l o t s t u d y i n 22 p a t i e n t s r e c i e i n g a l l o g e n i c marrow t r a n s p l a n t s . B l o o d 1983;61:318-325. 146 9 . S a l i s b u r y J D , B e b h a r d t BM. S u p p r e s s i o n o f c o r n e a l a l l o g r a f t r e j e c t i o n by c y c l o s p o r i n A . A r c h Ophtha lmol 1981;99:1640-1643. 10. Hess A d , T u t s c h x a P J , Santos GW. E f f e c t o f c y c l o s p o r i n e on t h e i n d u c t i o n o f c y t o t o x i c T - l y m p h o c y t e s : r o l e o f i n t e r l e u k i n I and i n t e r l e u k i n I I . T r a n s p l a n t P r o c 1983;15:2248-2258. 11. A n d r u s L , L a f f e r t y K J . I n h i b i t i o n o f T - c e l l a c t i v i t y by c u c l o s p o r i n A . Scand J Immunol 1982;14:449-458. 12. Fox A , Hammer ME, L i l l P , e t a l . E x p e r i m e n t a l u v e i t i s : e l i c i t e d by p e p t i d o g l y c a n - p o l y s a c c h a r i d e complexes , l i p o p o l y s a c c h a r i d e , and muramyl d i p e p t i d e . A r c h O p h t h a l m o l 1984;102:1063-1067. 13 . N u s s e n b l a t t RB, G e r y I , B a l l i n t i n e E J , e t a l . c e l l u l a r Immune r e s p o n s i n e n e s s o f u v e i t i s p a t i e n t s t o r e t i n a l s -a n t i g e n . Am J O p h t h a l m o l 1980;89:173-179. 14. M o c h i z u k i M, Kuwabara T , M c A l l i s t e r C , e t a l . A d o p t i v e t r a n s f e r o f e x p e r i m e n t a l autoimmune u v e o r e t i n i t i s i n r a t s . I n v e s t Ophtha lmol V i s S c i 1985;26:1 -9 . 15. N u s s e n b l a t t BR, P a l e s t i n e A , Chan C C , e t a l . Improvement o f u v e i t i s and o p t i c n e r v e d i s e a s e by c y c l o s p o r i n e i n a p a t i e n t w i t h m u l t i p l e s c l e r o s i s . Am J O p h t h a l m o l 1984;97:790-791 . 16 . P a l e s t i n e A G , A u s t i n HA, Balow J E , e t a l . R e n a l h i s t o p a t h o l o g i c a l t e r a t i o n s i n p a t i e n t s t r e a t e d w i t h c y c l o s p o r i n e f o r u v e i t i s . N E n g l J Med 1986;314:1293-1298. 147 17. P a l e s t i n e A G , A u s t i n HA, N u s s e n b l a t t R B . C y c l o s p o r i n e -i n d u c e d n e p h r o t o x i c i t y i n p a t i e n t s w i t h autoimmune u v e i t i s . T r a n s p l a n t P r o c 1985;17(4 ) :209-214 . 18. Hows J M , C h i p p i n g PM, F a i r h e a d S, e t a l . N e p h r o t o x i c i t y i n bone marrow t r a n s p l a n t r e c i p i e n t s t r e a t e d w i t h c y c l o s p o r i n A . B r J Haematol 1983;54:69-78 . 19 . K l i n t m a n GBG, I w a t s u k i S , S t a r z l T E . C y c l o s p o r i n A h e p a t o t o x i c i t y i n 66 r e n a l a l l o g r a f t r e c i p i e n t s . T r a n s p l a n t a t i o n 1981;32:488-489. 20 . Rootman J , O s t r y A , Gudauskas G . P h a r m a c o k i n e t i c s and m e t a b o l i s m o f 5 - f l u r o u r a c i l f o l l o w i n g s u b c o n j u n c t i v a l v e r s u s i n t r a v e n o u s a d m i n i s t r a t i o n . Can J O p h t h a l m o l 1984 ;19 (4 ) :187 -191. 21 . Gudauskas G , O s t r y A , Rootman J . C o n c e n t r a t i o n s o f t r i t i a t e d m e t h o t r e x a t e i n o c u l a r compartments , serum and u r i n e a f t e r s u b c o n j u n c t i v a l and i n t r a v e n o u s i n j e c t i o n . Can J O p h t h a l m o l 1980;15:179-182. 22. Rootman J , J o s e p h y PO, Adomat H , e t a l . O c u l a r a b s o r p t i o n and t o x i c i t y o f a r a d i o s e n s i t z e r and i t s e f f e c t s on h y p o x i c c e l l s . A r c h Ophtha lmol 1982;100:468-471. 23 . Rootman J , T i s d a l l J , Gudauskas G , e t a l . I n t r a o c u l a r p e n e t r a t i o n o f s u b c o n j u n c t i v a l l y a d m i n i s t e r e d 1 4 C -F l u o r o u r a c i l i n r a b b i t s . A r c h Ophtha lmol 1979;97:2375-2378. 24. Rootman J , B u s s a n i c h N , Gudauskas G . Combined l o c a l chemotherapy f o r a s p o n t a n e o u s l y o c c u r r i n g i n t r a o c u l a r tumour i n a c a t . Can J Ophtha lmol 1983;18(4) :185-187 . 148 25. Rootman J , Gudauskas G . Treatment o f o c u l a r l eukemia w i t h l o c a l chemotherapy . Cancer T r e a t m e n t R e p o r t s 1985;69(1 ) :119-122 . 149 

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