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UBC Theses and Dissertations

Percutaneous penetration and anti-inflammatory activity of desfluorotriamcinolone acetonide Verma, Subhash Chander 1972

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PERCUTANEOUS PENETRATION AND ANTI-INFLAMMATORY A C T I V I T Y OF DESFLUOROTRIAMCINOLONE ACETONIDE by SUBHASH CHANDER VERMA B. Ph a r m . , M. Pharm. ( P h a r m . T e c h . ) G u j a r a t U n i v e r s i t y , I n d i a A THESIS SUBMITTED IN PAR T I A L FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE I N PHARMACY i n t h e D i v i s i o n o f P h a r m a c e u t i c s o f t h e F a c u l t y o f P h a r m a c e u t i c a l S c i e n c e s We a c c e p t t h i s t h e s i s a s c o n f o r m i n g t o t h e r e q u i r e d s t a n d a r d THE UNIVERSITY OF B R I T I S H COLUMBIA M a r c h 1972 In presenting this thesis In partial fulfilment o f the requirements f o r 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 f o r extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by his representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Faculty of Pharmaceutical Sciences, The University of British Columbia, Vancouver, 8, Canada. i ABSTRACT D e s o n i d e , a new t o p i c a l a n t i - i n f l a m m a t o r y a n d a n t i -p r u r i t i c s t e r o i d , h a s b e e n i n v e s t i g a t e d f o r i t s c l i n i c a l , v a s o c o n s t r i c t o r a n d i n v i t r o p e r c u t a n e o u s p e n e t r a t i o n p r o p -e r t i e s , a n d c o m p a r e d t o b e t a m e t h a s o n e 1 7 - v a l e r a t e , t r i a m -c i n o l o n e a c e t o n i d e a n d h y d r o c o r t i s o n e . The c l i n i c a l a n d v a s o c o n s t r i c t o r b i o a s s a y t e s t s p l a c e d e s o n i d e q u a n t i t a t i v e l y among t h e most e f f e c t i v e t o p i c a l a n t i - i n f l a m m a t o r y a g e n t s , p o s s i b l y b e c a u s e o f i t s r e l a t i v e l y r a p i d s k i n p e n e t r a t i o n r a t e , The s i g n i f i c a n c e o f t h e s t u d y i s i ( a ) i t p r o v i d e s d e f i n i t i v e d a t a o n t o p i c a l a n t i - i n f l a m m a t o r y e f f e c t i v e n e s s o f d e s o n i d e a n d ( b ) i t r e v e a l s t h a t , c o n t r a r y t o c u r r e n t o p i n i o n , f l u o r i n a t i o n o f t h e s t e r o i d m o l e c u l e may be u n n e c e s s a r y f o r t o p i c a l a n t i - i n f l a m m a t o r y a c t i v i t y , a n d t h a t 9 o C - f l u o r i n a t i o n i n p r e d n i s o l o n e a c e t o n i d e s i m p e d e s r a t h e r t h a n f a v o u r s t h e i r s k i n p e n e t r a t i o n r a t e s . New d a t a o n o c t a n o l / w a t e r p a r t i t i o n c o e f f i c i e n t s a n d a n u n s u c c e s s f u l e f f o r t o f a d o p t i n g t h e M a r t i n ( 1 9 6 8 ) o x i m e d e r i v a t i v e s p e c t r o p h o t o f l u o r o m e t r i c t e c h n i q u e f o r d e s o n i d e a s s a y s a r e a l s o i n c l u d e d . S u p e r v i s o r . 11 TABLE OF CONTENTS P a g e I . INTRODUCTION 1 I I . LITERATURE SURVEY 5 A. I n f l a m m a t i o n a n d A n t i - i n f l a m m a t o r y S t e r o i d s 5 1 . I n f l a m m a t i o n 7 2 . A n t i - i n f l a m m a t o r y m e c h a n i s m o f c o r t i c o s t e r o i d s 1 0 3 . S t r u c t u r e - a c t i v i t y r e l a t i o n s h i p s 1 4 4 . T o p i c a l a n t i - i n f l a m m a t o r y c o r t i c o -s t e r o i d s 1 9 B. S k i n P e n e t r a t i o n 2 4 1 . B i o p h y s i c a l f a c t o r s i n s k i n p e n e t r a t i o n 2 4 a . A v e n u e s o f p e n e t r a t i o n 2 4 b. B a r r i e r c h a r a c t e r i s t i c s 2 8 c. F a c t o r s i n f l u e n c i n g p e n e t r a t i o n 30 d. P h y s i c a l c h e m i s t r y o f d i f f u s i o n 3 3 2 ; M e t h o d s f o r m e a s u r i n g p e r c u t a n e o u s a b s o r p t i o n 4 1 3 . I s o l a t i o n o f e p i d e r m a l s h e e t s f r o m human a u t o p s y s k i n 4 3 C. • A n a l y t i c a l M e t h o d s 4 7 1 . S p e c t r o p h o t o m e t r i c 4 7 2 . F l u o r o m e t r i c 4 7 3 . O t h e r methods (GLC a n d r a d i o i s o t o p i c ) 4 8 4 . V a s o c o n s t r i c t i o n a s s a y 4 9 i i i Page I I I . STATEMENT OF PROBLEM 51 IV. EXPERIMENTAL METHODS 52 A. Apparatus 52 1. Poulsen s k i n d i f f u s i o n c e l l 52 2. Spectrophotofluorometer (SPF) 5 2 3 . P i c k e r nuclear l i q u i m a t 55 4. Spectrophotometer ( H i t a c h i & Beckman) 55 5 . Dermatome 55 B. Procedures 55 1. A n a l y s i s of s t e r o i d s 55 a) Spectrophotofluorometric a n a l y s i s of desonide 55 b) Spectrophotometric 58 2. S o l u b i l i t y and p a r t i t i o n c o e f f i c i e n t determination 58 3 . I n ^ v i t r o p e n e t r a t i o n s t u d i e s 60 a) P r e p a r a t i o n of membranes f o r d i f f u s i o n c e l l 60 i ) F u l l t h ickness of mouse s k i n 60 i i ) Epidermal sheets from human autopsy 60 b) To check i n t a c t n e s s of epidermal sheets by t r i t i a t e d water 62 c) I n ^ v i t r o s t e r o i d p e n e t r a t i o n 63 4. V a s o c o n s t r i c t i o n t e s t 64 i v P a g e V. RESULTS AND DISCUSSION 66 A. S p e c t r o p h o t o f l u o r o m e t r i c M e t h o d o f t h e E s t i m a t i o n o f D e s o n i d e 66 B. S p e c t r o p h o t o m e t r y 67 C. S o l u b i l i t y a n d P a r t i t i o n C o e f f i c i e n t 68 D. R e l i a b i l i t y o f H e a t S e p a r a t i o n M e t h o d f o r t h e R e m o v a l o f E p i d e r m i s f r o m A u t o p s y S k i n 74 E. T e s t f o r I n t a c t n e s s o f S k i n w i t h T r i t i a t e d W a t e r 74-P. P e r c u t a n e o u s P e n e t r a t i o n o f S t e r o i d s 79 G. V a s o c o n s t r i c t i o n B i o a s s a y 83 H. C l i n i c a l S t u d i e s o f E f f e c t i v e n e s s o f D e s o n i d e 91 V I . SUMMARY AND CONCLUSION 99 V I I . BIBLIOGRAPHY .102 APPENDIX 111 V LIST OF TABLES Page 1. Summary of the inflammatory response 9 2. Enhancement factors for various functional groups of corticosteroids 20 3. Anti-inflammatory congeners of hydrocortisone 22 4. Solubility of steroids in two different solvents 71 5. Octanol/water and ether/water partition coefficient of several steroids 72 6. Rate of diffusion of tr i t i a t e d water through human abdominal skin 77 7. Rate of diffusion of tr i t i a t e d water through human epidermis and hairless mice skin 78 8. Cumulative penetration of desonide and triam-cinolone acetonide through human abdominal skin at room temperature. The steroids were applied in drug deposit form 84 9.. Cumulative penetration of betamethasone 17-valerate, desonide, triamcinolone acetonide and hydrocortisone, through human abdominal skin. The steroids were in 40# ethanol 87 10. Cumulative penetration of betamethasone 17-valerate, desonide, triamcinolone acetonide and hydrocortisone, through human skin. The steroids were in hydrophllic ointment base, 89 11. Vasoconstriction produced by triamcinolone acetonide and desonide 92 12. Effectiveness of desonide vs betamethasone 17-valerate, in contact dermatitis 95 13. Effectiveness of desonide vs betamethasone valer-ate in atopic dermatitis 96 14. Effectiveness of desonide vs betamethasone valer-ate in psoriasis 97 v i L I S T OF FIGURES P a g e 1. S t r u c t u r e o f a n a n t i - i n f l a m m a t o r y c o r t i c o s t e r o i d 11 2. P o s s i b l e mode o f i n t e r a c t i o n o f s e r o t o n i n a n d h i s t a m i n e w i t h C o r t i s o l r e c e p t o r ( s ) 13 3 . N u m b e r i n g a n d s i t e s m o d i f i e d i n h y d r o c o r t i s o n e m o l e c u l e 17 4 . S t r u c t u r e o f f o u r a n t i - i n f l a m m a t o r y s t e r o i d s s t u d i e d 1 7(a) 5. A n a t o m i c a l z o n e s e n c o u n t e r e d i n p e r c u t a n e o u s a b s o r p t i o n 25 6. P o s s i b l e a v e n u e s o f p e n e t r a t i o n t h r o u g h t h e u n b r o k e n s k i n 26 7. D i a g r a m m a t i c r e p r e s e n t a t i o n o f e p i d e r m i s u l t r a s t r u c t u r e 29 8. R e p r e s e n t a t i v e p e n e t r a t i o n p r o f i l e f o r d r u g d i f f u s i n g t h r o u g h human s k i n 40 9. A s s e m b l e d s i d e v i e w o f P o u l s e n ' s s k i n d i f f u s i o n c e l l 53 10. D i f f e r e n t p a r t s o f t h e d i f f u s i o n c e l l 54 11. Top v i e w o f t h e d i f f u s i o n c e l l , s h o w i n g t h e e x p o s e d e p i d e r m a l a r e a 54 12. R e m o v a l o f e p i d e r m i s f r o m a u t o p s y s k i n 6 l 13. S t a n d a r d c u r v e f o r c o r t i c o s t e r o i d s i n n o r m a l s a l i n e 69 14. R e l a t i o n s h i p o f p a r t i t i o n c o e f f i c i e n t t o c u m u l a t i v e p e n e t r a t i o n 73 15. Dermatome a n d h e a t s e p a r a t e d ; s e c t i o n s o f human e p i d e r m i s 75 16. S t e r o i d p e n e t r a t i o n t h r o u g h human a b d o m i n a l s k i n . The s t e r o i d s w e r e i n d r u g d e p o s i t f o r m 85 17. Amount o f d e s o n i d e and t r i a m c i n o l o n e p e n e t r a t e d t h r o u g h human s k i n 18. S t e r o i d p e n e t r a t i o n t h r o u g h human e p i d e r m i s a t room t e m p e r a t u r e . The s t e r o i d s were i n 40$ e t h a n o l 19. S t e r o i d p e n e t r a t i o n t h r o u g h human e p i d e r m i s a t room t e m p e r a t u r e . The s t e r o i d s were i n h y d r o -p h i l i c o intment base 20. V a s o c o n s t r i c t i o n b i o a s s a y , l o g c o n c e n t r a t i o n vs % dose re s p o n s e ACKNOWLEDGEMENTS To D r . J.O. R u n i k i s , t h e s i s s u p e r v i s o r , f o r h i s e n c o u r a g e -ment t h r o u g h o u t t h e c o u r s e o f t h i s s t u d y . To D r . W i l l i a m D. S t e w a r t , f o r h i s a d v i c e i n t h e c l i n i c a l a s p e c t o f t h i s t h e s i s . T o K i s s S y l v i a W a l l a c e , f o r h e r v e r y v a l u a b l e comments a n d c o n s t r u c t i v e c r i t i c i s m . T o D r . T.H. B r o w n , D r . A.M. G o o d e v e a n d M r s . P a t H a u g e n f o r t h e i r v a l u a b l e d i s c u s s i o n s . T o my w i f e , A s h a , f o r h e r u n c e a s i n g e n c o u r a g e m e n t , w h i c h h e l p e d me i n c o m p l e t i n g t h i s p r o j e c t . To D ean 3 . E . R i e d e l e s p e c i a l l y , who e x t e n d e d h i s a d v i c e a n d s u p p o r t t o me, a n d p r o v i d e d new i d e a s i n t h e o r g a n i z a t i o n o f t h i s t h e s i s . To my b e l o v e d p a r e n t s whose i n s p i r a t i o n h a s made t h i s w o r k p o s s i b l e . I INTRODUCTION The I n t r o d u c t i o n o f t o p i c a l h y d r o c o r t i s o n e i n d e r m a -t o l o g y b y S u l z b u r g e r a n d W i t t e n (195 2), s h o r t l y a f t e r -w a r d s f o l l o w e d b y more e f f e c t i v e h y d r o c o r t i s o n e d e r i v a t i v e s , p a r t i c u l a r l y f l u o r i n a t e d d e r i v a t i v e s , w e r e m a j o r t h e r a p e u t i c e v e n t s i n t h e t r e a t m e n t o f n o n - i n f e c t i v e i n f l a m m a t o r y a n d p r u r i t i c s k i n c o n d i t i o n s . T h e i r e f f e c t i v e n e s s s u r p a s s e d b y f a r o t h e r t r e a t m e n t s . S i n c e i n f l a m m a t i o n i s t h e commonest p a t h o l o g i c a l p r o c e s s e n c o u n t e r e d i n d e r m a t o l o g y ( C a l n a n , 1970) a n d s i n c e t h e more p o t e n t t o p i c a l c o r t i c o s t e r o i d s a r e e f f e c t i v e a g a i n s t o t h e r d i s e a s e s l i k e p s o r i a s i s , t h e i r e c o -n o m i c i m p o r t a n c e i s a s g r e a t a s t h e i r t h e r a p e u t i c i m p o r t a n c e . T o d a y , a s a c o n s e q u e n c e , t h e r e i s a d r i v e o n t h e p a r t o f m a n u f a c t u r e r s o f t o p i c a l m e d i c a t i o n s , a n d a l s o d e r m a -t o l o g i c a l i n v e s t i g a t o r s t o d e v e l o p more p o t e n t t o p i c a l a n t i -i n f l a m m a t o r y c o r t i c o s t e r o i d s i n more d i v e r s e d o s a g e f o r m s . T h i s d r i v e f o r 'more* c o u p l e d t o a l a c k o f c l e a r u n d e r s t a n d -i n g o f t h e e t i o l o g y o f t h e i n f l a m m a t o r y p r o c e s s o r t h e p h y s -i c a l - c h e m i c a l a n d b i o - c h e m i c a l f a c t o r s d e t e r m i n i n g t h e t o p i c a l e f f e c t i v e n e s s o f t h e c o r t i c o s t e r o i d s , h a s p r o d u c e d a s i t u a t i o n w h e r e t h e n e w e r , more p o t e n t , a n d more t o x i c f l u o r i n a t e d c o r t i c o s t e r o i d s , a r e u s e d t o t h e e x c l u s i o n o f t h e l e s s p o t e n t c o r t i c o s t e r o i d s , a n d p h a r m a c i e s h a v e t o s t o c k more t h a n 30 c o r t i c o s t e r o i d s i n s e v e r a l h u n d r e d d o s e -v e h i c l e c o m b i n a t i o n s . G e n e r a l i z a t i o n s r a n g i n g f r o m p r o b a b l y e r r o n e o u s t o d e m o n s t r a b l y e r r o n e o u s h a v e become w i d e s p r e a d among t h e med-i c a l a n d p h a r m a c e u t i c a l p r a c t i t i o n e r s . A l i t e r a t u r e s u r v e y was u n d e r t a k e n t o i d e n t i f y what n e e d t h e r e i s f o r u s e o f more p o t e n t a n d more t o x i c f l u o r i n a t e d c o r t i c o s t e r o i d s , what I n f o r m a t i o n t h e r e e x i s t s o n t h e b i o l o g i c a l e q u i v a l e n c e o f t h e m u l t i t u d e o f t o p i c a l c o r t i c o s t e r o i d p r e p a r a t i o n s a n d t o i n q u i r e i n t o e x p e r i m e n t a l p o s s i b i l i t i e s t o p r o v i d e o b j e c t i v e i n f o r m a t i o n on t h e r e l a t i v e e f f e c t i v e n e s s o f t h e s e p r e p a r a -t i o n s . The s u r v e y r e v e a l e d : l o n g - t e r m u s e o f h i g h l y p o t e n t t o p i c a l compounds c a n c a u s e s i g n i f i c a n t s y s t e m i c a b s o r p t i o n a n d a d v e r s e e f f e c t s l i k e i n c r e a s e d e p i d e r m a l f r a g i l i t y , t e l a n g i e c t a s i s , s t r i a e , e c c h y m o s e s a n d f i b r a b l a s t i c r e p a i r . W a d d i n g t o n e t a l (I968) r e p o r t e d t h a t t h e u s e o f f l u o r i n a t e d s t e r o i d s s h o u l d be a v o i d e d i n t h e t r e a t m e n t o f i n f a n t i l e e c z ema. E p s t e i n ^ t _ a l (1963), G r l c e (I966), I v e a n d M a r k s (1968) r e p o r t e d t h a t l o n g - t e r m u s e o f p o t e n t f l o u r i n a t e d c o r t i c o s t e r o i d s c a u s e a t r o p h y o f t h e s k i n . S n e d d o n (1969) h a s r e p o r t e d a d v e r s e e f f e c t s a f t e r p r o l o n g e d t o p i c a l a p p l i c a -t i o n o f b e t a m e t h a s o n e i n p a t i e n t s s u f f e r i n g f r o m r o s a c e a . New n o n - f l u o r l n a t e d s t e r o i d ' D e s o n i d e ' (1'6'oC- h y d r o x y p r e d n i s o l o n e - 16,17 a e e & o n i d e o r d e s f l u o r o t r i a m c i n o l o n e a c e t o n i d e ) h a s b e e n d e v e l o p e d b y M i l e s L a b o r a t o r i e s , I n c . a n d p r o m o t e d a s a p o t e n t a n t i - i n f l a m m a t o r y s t e r o i d . I t d i f -f e r s f r o m i t s f l u o r i n a t e d a n a l o g , t r i a m c i n o l o n e a c e t o n i d e , o n l y 3 b y t h e a b s e n c e o f 9<X. - f l u o r i n e i n r i n g B. ( P i g . 4 ) . M a n t i c a (1970) h a s s t u d i e d t h e c h e m i c a l a n d p h y s i c a l c h a r -a c t e r i s t i c s o f d e s o n i d e . M a s c i t e l l i - C o r i a n d o l i (1970) h a v e s t u d i e d t h e b i o c h e m i c a l a n d p h a r m a c o l o g i c a l a c t i v i t y o f d e s o n i d e . P h i l l i p s e t a l ( 1 9 7 1 ) f r o m M i l e s L a b o r a t o r i e s h a v e r e p o r t e d on t h e p h y s i c a l , p h a r m a c o l o g i c a l a n d t o x l c o -l o g i c a l p r o p e r t i e s . T h e i r s t u d i e s showed d e s o n i d e t o be a s e f f e c t i v e a s f l u o r i n a t e d compounds a n d h a v i n g l o w e r t o x i c i t y s y s t e m i c a l l y , b u t t h e y h a d f e w d a t a o n t o p i c a l a c t i v i t y . P o l a n o (1970) h a s shown t h a t a n o t h e r n o n - f l u o r i n a t e d s t e r o i d h y d r o c o r t i s o n e b u t y r a t e , i s a s e f f e c t i v e t o p i c a l l y a s t r i a m -c i n o l o n e a c e t o n i d e . A d o u b l e b l i n d c o m p a r i s o n c a r r i e d o u t b y R e i d a n d B r o o k s (1968) o f 3 c o r t i c o s t e r o i d s , r e v e a l e d t h a t h y d r o c o r t i s o n e a n o n - f l u o r i n a t e d s t e r o i d i n a v e r y l a r g e number o f eczema p a t i e n t s , was a s e f f e c t i v e a s t r i a m c i n o l o n e a c e t o n i d e o r f l u o c i n o l o n e a c e t o n i d e , b o t h f l u o r i n a t e d s t e r -o i d s . T h e s e r e s u l t s c o n f l i c t w i t h t h e w i d e l y a c c e p t e d g e n e r a l -i z a t i o n t h a t f l u o r l n a t i o n i n c r e a s e s antIT i n f l a m m a t o r y a c t i v i t y t o a d e g r e e w h e r e f l u o r i n a t e d c o r t i c o s t e r o i d s , a s a g r o u p , a r e more e f f e c t i v e t o p i c a l a n t i - i n f l a m m a t o r y a g e n t s t h a n n o n -f l u o r i n a t e d s t e r o i d s . The g e n e r a l i z a t i o n may be i n c o r r e c t a n d d e s e r v e s i n v e s t i g a t i o n . No s t u d y h a s s o f a r b e e n r e p o r t e d i n v o l v i n g t h e p e n e t r a -4 tion of desonide through human skin. The rate of penetra-tion of desonide through human skin shall be compared to triamcinolone acetonide, betamethasone «r> 1? - valerate and hydrocortisone. Vasoconstriction test shall be carried out to compare desonide and triamcinolone acetonide in their a b i l i t i e s to blanch the skin. The combination of in-vltro studies and human biological testing provides a valuable correlation between theory and therapy. This work should be regarded as a preparatory study for a comprehensive investigation into the biological and thera-peutic equivalence of topical anti-inflammatory corticoster-oids. 2 I I LITERATURE SURVEY A. Inflammation and Ant1-Inflammatory S t e r o i d s C o r t i c o s t e r o i d s are used p r i m a r i l y f o r the n o n - s p e c i f i c , p a l i a t i v e treatment of a l a r g e a r r a y of dermatoses w i t h an inflammatory component i n which the primary e t l o l o g i c f a c t o r i s unknown or u n a s s a i l a b l e . C o r t i c o s t e r o i d s are i n d i c a t e d f o r use i n diseases as v a r i e d as hand eczemas, contact der-m a t i t i s , pemphigus, p s o r i a s i s , e x f o l i a t i v e d e r m a t i t i s (Brown, 1970). The mechanism and causes of inflammation i n these diseases are complex and vary w i t h the disease. I t i s ex-pected that the e f f e c t i v e n e s s of d i f f e r e n t , drugs, i n c l u d i n g d i f f e r e n t d e r i v a t i v e s of c o r t i c o s t e r o i d s , v a r i e s a c c o r d i n g l y . The observation, t h a t the t o p i c a l a d m i n i s t r a t i o n of potent f l u o r i n a t e d c o r t i c o s t e r o i d i s e f f e c t i v e i n p s o r i a s i s , whereas t o p i c a l hydrocortisone i s not, i s an example. To evaluate the r e l a t i v e e f f e c t i v e n e s s of t o p i c a l c o r t i c o -s t e r o i d s and the r o l e of f l u o r i n a t i o n i n enhancing t h a t e f f e c -t i v e n e s s , i t i s necessary t o f i n d model inflammation systems which i m i t a t e t o p i c a l c o r t i c o s t e r o i d a c t i o n i n the c l i n i c a l s i t u a t i o n . I n absence of a known e t i o l o g i c f a c t o r , the search must be f o r models which in c l u d e some steps i n the inflamma-t o r y process of the disease which i s blocked by the drugs. As pointed out by Weiner and P i l l e r o (1970), there i s too much of a rush t o evaluate drugs i n f a v o u r i t e anti-inflammatory 6 models. This understandable p a t t e r n tends to d i r e c t the de-velopment of anti-inflammatory drug design along a l i n e which w i l l s e l e c t agents working by the same mechanism as p r e v i o u s l y discovered drugs which perhaps c o r r e l a t e too w e l l w i t h each other and not w e l l enough with c l i n i c a l a p p l i c a t i o n s . That such has been the case i n the development of c o r t i c o s t e r o i d s i s h a r d l y i n doubt. Since the d i s c o v e r y by P r i e d and Sabo ( 1 9 5 3 ) of g r e a t e r systemic anti-inflammatory e f f e c t i v e n e s s of 9<?C - f l u o r o h y d r o -c o r t i s o n e by the c r o t t o n p e l l e t and l i v e r glycogen deposit assays, these model assays have been used as the b a s i s f o r the e v a l u a t i o n of the potency of new c o r t i c o s t e r o i d s . I n -c l u s i o n of f l u o r i n e i n p r a c t i c a l l y a l l anti-inflammatory cor-t i c o s t e r o i d s produced i n the l a t e f i f t i e s and s i x t i e s , f o l l o w e d , as d i d the u n c r i t i c a l acceptance t h a t 9<*C - f l u o r i n a t i o n i n -creases a l l b i o l o g i c a l a c t i v i t i e s of the c o r t i c o s t e r o i d s . ! h. The f a c t that 9oC - f l u o r i n e i n t r i a m c i n o l o n e produces a sys-t e m i c a l l y a c t i v e but t o p i c a l l y i n a c t i v e anti-inflammatory c o r -t i c o s t e r o i d , i n d i c a t i n g t h a t d i f f e r e n t anti-inflammatory models are r e q u i r e d f o r the e v a l u a t i o n of e f f e c t i v e n e s s of t o p i c a l and systemic anti-inflammatory agents has been overlooked. The f o l l o w i n g s e c t i o n emphasizes the events i n inflamma-t i o n which have been thought t o be blocked by t o p i c a l c o r t i c o -s t e r o i d s and are of value i n the i n t e r p r e t a t i o n of t h e i r e f f e c t i v e n e s s . Less a t t e n t i o n has been devoted t o events which may be of importance i n the development of the theory of inflammation but whose r e l a t i o n s h i p s t o the a c t i v i t y of c o r t i c o s t e r o i d s have not been c l e a r l y demonstrated. The ex-c e l l e n t reviews of^ffiibMson^' and A n g e l l (1971) and Weiner and P i l l e r o (1970) should be here consulted. These reviews and that of Wlnkelman ( 1 9 7 D t together w i t h the ol d e r p u b l i c a -t i o n s of Dougherty and F r y i n g ( 1 9 5 5 ) t form the b a s i s of the f o l l o w i n g survey of the nature of inflammation. 1. Inflammation Inflammation i s a dynamic process which f o l l o w s a v a r i -ety of pathways. There i s agreement only on a d e s c r i p t i v e d e f i n i t i o n of inflammation. Attempts t o define inflammation i n mechanistic p h y s i c a l - c h e m i c a l or b i o l o g i c a l terms have f a i l e d ( F l o r e y , 195*+). The current d e f i n i t i o n of inflamma-t i o n i s as f o l l o w s : "Inflammation i s a r e a c t i v e t r a i n of morphologic and biochemical events a f f e c t i n g both blood ves-s e l s and c e l l s which occurs i n the v i t a l t i s s u e s surrounding a s i t e of i n j u r y " ( BObinson, 1971). This d e f i n i t i o n i n c l u d e s the c l a s s i c a l c l i n i c a l d e s c r i p t i o n of the successive steps In inflammation as heat, redness, p a i n and s w e l l i n g , due t o the progressive development of changes i n v a s c u l a r perme-a b i l i t y , c e l l u l a r m i g r a t i o n , edema formation and u l t i m a t e l y l e a d i n g t o e i t h e r the removal of the inflammatory s t i m u l i or c e l l u l a r death. The t i m i n g of these events and the b i o -chemical mediators thought to be r e s p o n s i b l e are summarized i n Table 1. The t i m i n g of the changes described i n Table 1 v a r i e s w i t h the s e v e r i t y and nature of the i n j u r y . The i n -j u r i o u s s t i m u l i may be p h y s i c a l e.g. a f o r e i g n body such as a c o t t o n p e l l e t or croton o i l , chemical, e.g. such as an a l l e r g i c response to a s e n s i t i z i n g drug, or m i c r o b i o l o g i c a l . The f o l l o w i n g d e t a i l e d events have at one time or another acquired prominence i n attempts t o e x p l a i n the mode of a c t i o n of c o r t i c o s t e r o i d s . Histamine appears to be a c t i v e p r i n c i p a l l y d u r i n g the e a r l y phase of inflammatory response, when i t induces a r t e r i o -l a r d i l a t i o n and the f i r s t wave of increased p e r m e a b i l i t y f o l l o w i n g i n j u r y , a f f e c t i n g c h i e f l y the venules. Serotonin (5 - hydroxy - tryptamine) d i s t r i b u t e d widely i n most c e l l s and p l a t e l e t s can produce v a s o d i l a t i o n . Proteoses and p o l y -peptides (the k i n i n s ) are important mediators. They are among the most potent v a s o d i l a t o r s known. They are capable of causing s t r i k i n g increase i n venular and c a p i l l a r y perme-a b i l i t y . Moreover k i n i n s have shown to a t t r a c t leukocytes and alone can evoke many of the c a r d i n a l m a n i f e s t a t i o n s of inflammation (Menkln, 1 9 5 6 ) . T a b l e i . Summary o f t h e I n f l a m m a t o r y R e s p o n s e T e m p o r a l S e q u e n c e M e d i a t o r S i t e Hemodynamic Changes P e r m e a b i l i t y C hanges W h i t e C e l l V i s i b l e C h a n g e s Change I m m e d i a t e ( t r a n s i e n t ) : 0 t o 5 m i n . N e u r o g e n i c A r t e r i o l e s Va s oc ons t r i c t i ve i s c h e m i a None None B l a n c h i n g E a r l y P h a s e : 5 t o 30 m i n . H i s t a m i n e S e r o t o n i n A r t e r i o l e s V a s o d i l a t i o n I n c r e a s e d None K i n i n s P r o t e a s e s M i l e s f a c t o r G l o b u l i n p e r -m e a b i l i t y f a c t o r Complement e s t e r a s e s C a p i l l a r i e s V e n u l e s New c h a n n e l s opened E n g o r g e m e n t -o v e r a l l i n c r e a s e i n b l o o d f l o w I n c r e a s e d I n c r e a s e s -e n d o t h e l i a l j o i n t s o pened None P a v e m e n t i n g A d h e s i o n B e g i n n i n g e m i g r a t i o n R u b o r ( r e d n e s s ) C a l o r ( h e a t ) D o l o r ( p a i n ) Tumor ( s w e l l i n g ) O t h e r m e d i a t o r s D e l a y e d P h a s e : I t o 2 h r . ? L y s o l e c i t h i n ? P r o t e i n p r o d -u c t s V e n u l e s L e u k o c y t e em- C a p i l l a r i e s i g r a t i n g f a c t o r s E n g o r g e m e n t -o v e r a l l i n c r e a s e i n b l o o d f l o w E m i g r a t i o n A s a b o v e I n c r e a s e d P e r i v a s c u l a r f o r m a t i o n l e u k o c y t e o f f l u i d a g g r e g a t i o n a n d c e l l u -l a r e x u d a t e \o ( Prom : Robinson, and Angell, 1971 ) 10 Among hemodynamic changes there i s an increase i n the permeability of vessels and leukocytic change. Another point i n inflammation i s the destruction of connective t i s -sue. Frying and Dougherty (1955) proposed that the inten-s i t y of inflammation i s enhanced by the destruction of f i b r o b l a s t s . In t h i s respect the destruction of one c e l l adds to the amount of the phlogogenic substance produced which leads to the destruction of another c e l l . The stages of inflammation (Winkelmann, 1971) have been studied by combined s t r u c t u r a l and pharmacologic-physiologic methods. Many investigators prefer to c l a s s i f y inflammation i n two stages, an early stage related to venous permeability and a l a t e r stage to c e l l u l a r damage. 2. Anti-inflammatory mechanism of c o r t i c o s t e r o i d s Anti-inflammatory mechanisms can be considered from the viewpoints of b i o l o g i c a l and s t r u c t u r e - a c t i v i t y mechanisms. The proposed s t r u c t u r e - a c t i v i t y r e l a t i o n s h i p and the chem-i s t r y of c o r t i c o s t e r o i d s are discussed i n the next chapter. Corticosteroids exert t h e i r anti-inflammatory effect^by i n -terrupting the chain of reactions of c e l l u l a r destruction, that i s triggered by the i n i t i a t i n g stimulus. I t has been suggested that the anti-inflammatory e f f e c t of the c o r t i c o -steroids i s related to t h e i r e f f e c t on the metabolism of selected c e l l s p a r t i c i p a t i n g i n the process of inflammation. Dougherty and Schneebeli (1955) propose that an a n t i - i n -flammatory hormone probably acts by s t a b i l i z i n g the imperme-able state of the c e l l membrane, thus preventing c e l l u l a r destruction. The concept that the f i b r o b l a s t i s the primary c e l l involved i n inflammation and that i t i s the primary target of hydrocortisone a c t i v i t y i s further substantiated by the observed s t r u c t u r e - a c t i v i t y r e l a t i o n s h i p of an t i - I n -flammatory steroids and t h e i r f i b r o b l a s t i n h i b i t i n g a b i l i t y . Steroids that have shown to have an anti-inflammatory a c t i v -i t y i n human skin can be represented by the following struc-ture: - c -0 Fig; 1. /± ~ Pregnene - l l | ^ 1 7 c C - d i o l - 3,20 dione B e r l i n e r and Ruhmann (I967) indicated a d i r e c t r e l a t i o n s h i p between the structure of steroids, t h e i r f i b r o b l a s t i n h i b i t -ing potency, and t h e i r a b i l i t y to suppress Inflammation. Schayer (1964) indicated that the p r i n c i p a l physiolog-i c a l function of c o r t i c o s t e r o i d s i n Inflammation would In-volve a vasoconstrictive e f f e c t as well as a moderation of the histamine action. Corticosteroids s t a b i l i z e endothelial c e l l s against changes induced by histamine. Cline and Melman (1966) explained that anti-inflammatory steroids control i n -flammation by preventing the release of active k i n i n by pre-venting the i n t e r a c t i o n between k a l l l k r e i n and i t s substrate. Greaves and Shuster (1970) explained that the anti-inflamma-tory a c t i v i t y of c o r t i c o s t e r o i d s may be due to reduced forma-t i o n of kinins. Corticosteroids i n h i b i t the delayed inflam-matory response to streptokinase, a powerful a c t i v a t o r of k i n i n forming enzymes. The mechanism of anti-Inflammatory a c t i v i t y of these compounds, l i e , not i n the a c t i o n of k i n i n on blood vessels, but on enzymic formation of k i n i n . Keir (I967) proposed a model F i g . 2 explaining how the binding of C o r t i s o l with hypothetical receptors may i n t e r -fere with serotonin and histamine, functioning as an antago-^ n i s t to these amine inflammation mediators. The manner i n which steroids interact with t h e i r recep-tors was suggested by Bush (I962). He proposed that i n t r i n s i c action r e s u l t s not from a chemical reaction but from r e l a -t i v e l y firm "physical" association with t h e i r receptors that involve l i t t l e or no movement of the parts of the molecules -13 Pig. 2 . I l l u s t r a t i o n of possible mode of in t e r a c t i o n of serotonin and histamine with C o r t i s o l re-c e p t o r s ) . From Kier, L.B.: J . Med. Chem., 11, 915 (1968). — 14 t h a t are i n c l o s e a p p o s i t i o n . Wolf and c o l l a b o r a t o r s ( 1964 ) suggested that i n the s t e r o i d - r e c e p t o r complex the s t e r o i d i s i n contact w i t h the rece p t o r surface i n two d i s c r e t e areas: the0 -face of r i n g A, B and C and °C -face of r i n g D. They claimed t h a t the e f f e c t of the s t e r o i d i s t o induce a conformational change i n the r e c e p t o r , since no chemical r e a c t i o n as such takes place. The -face" theory has been widely accepted as the b a s i s f o r the design of new molecular m o d i f i c a t i o n s and t o e x p l a i n d i f f e r e n c e s i n pharmacological a c t i v i t i e s of hydrocortisone congeners ( G o l d s t e i n , 1968; Wagner, 1971). 3. S t r u c t u r e a c t i v i t y r e l a t i o n s h i p s The search f o r mediators of inflammation, s p e c i f i c t a r -get c e l l s and receptors f o r c o r t i c o s t e r o i d s continues. L i t t l e i s known on the nature and l o c a t i o n of rec e p t o r s . Recently Lev-I 'KSonrr (1972) announced a rec e p t o r f o r g l u c o c o r t l c o c o i d ac-t i v i t y of hydrocortisone i n the a c i d p r o t e i n of c e l l s . I t has been proposed that anti-inflammatory e f f e c t s of c o r t i c o -s t e r o i d s depend on l o c a l a c t i o n of s t e r o i d s . The understand-i n g of molecular mechanisms by which c o r t i c o s t e r o i d s exert t h e i r c h a r a c t e r i s t i c e f f e c t s on the i n h i b i t i o n of inflammatory processes i n e i t h e r the e a r l y inflammatory stage (edema, f i b r i n 15 d e p o s i t i o n , c a p i l l a r y d i l a t i o n , m i g r a t i o n of phagocytes i n t o the inflamed area) and the l a t e r m a n i f e s t a t i o n s ( c a p i l l a r y p r o l i f e r a t i o n , f i b r o b l a s t p r o l i f e r a t i o n , d e p o s i t i o n of c o l -lagen and c i c a t r i z a t i o n ) i s incomplete. In view of l a c k of s p e c i f i c knowledge of receptor s i t e , s t r u c t u r e a c t i v i t y r e -l a t i o n s h i p (abbreviated SAR) s t u d i e s have been g e n e r a l l y d i r e c t e d towards the e v a l u a t i o n of b i o l o g i c a l e f f e c t s e.g. thymus i n v o l u t i o n and l i v e r glycogen neogenesis assays i n animals. S t r u c t u r e - a c t i v i t y r e l a t i o n s h i p s t u d i e s however cannot deepen the understanding of molecular mechanisms by which c o r t i c o s t e r o i d s act i n man. For t h a t the i s o l a t i o n and i d e n t i f i c a t i o n of receptors are needed., Therefore con-c l u s i o n from SAR s t u d i e s must be regarded as s p e c u l a t i v e . The SAR s t u d i e s however have provided u s e f u l i n f o r m a t i o n i n research f o r new and more e f f e c t i v e s t e r o i d s . B a s i c a l l y , SAR s t u d i e s are done as f o l l o w s : A s u i t a b l e b i o l o g i c a l e f f e c t of a drug i s chosen. A proto-type drug, which e l i c i t s the c h a r a c t e r i s t i c e f f e c t , i s then modified s y s t e m a t i c a l l y i n i t s molecular s t r u c t u r e . Sub-s t i t u e n t s are added or subtracted at v a r i o u s p o s i t i o n s and i n d i f f e r e n t s t e r i c c o n f i g u r a t i o n . A s e r i e s of such chemi-c a l l y r e l a t e d drugs i s known as a congeneric s e r i e s . By t e s t i n g the congeners and observing how the s u b s t i t u e n t groups of each member a f f e c t the b i o l o g i c a l potency, i t i s p o s s i b l e 16 t o p o s t u l a t e a n i m a g i n a r y r e c e p t o r s u r f a c e a n d d r a w c o n -c l u s i o n s a b o u t t h e p r e c i s e mode o f c o m b i n a t i o n o f a d r u g w i t h i t s r e c e p t o r s u r f a c e ( G o l d s t e i n J l S ? ) . The m a i n l i m i t a -t i o n s o f SAR i n t h e e v a l u a t i o n o f c o r t i c o s t e r o i d s a r e i n t h e a r b i t r a r y c h o i c e o f t h e b i o l o g i c a l e f f e c t e m p l o y e d t o e v a l u a t e s u b s t i t u e n t g r o u p - e f f e c t a n d t h e s p e c u l a t i v e n a t u r e o f r e c e p t o r s . The SAR s t u d i e s c a n n o t d i s t i n g u i s h r e a d i l y be-t w e e n s u b s t i t u e n t g r o u p e f f e c t s w h i c h a r e due t o a l t e r e d a f f i n i t y o f a d r u g f o r t h e r e c e p t o r o r a l t e r e d t r a n s p o r t p r o p e r t i e s . I n no c a s e SAR d a t a p r e c l u d e t h a t t h e r e c o u l d n o t be a n a l t e r n a t i v e g r o u p w h i c h w o u l d i n c r e a s e t h e e f f e c t i v e n e s s s t i l l more, a n d t h e r e i s a l w a y s t h e u n c e r t a i n t y t h a t c o n c l u -s i o n s d r a w n f o r one b i o l o g i c a l e f f e c t may mot h o l d f o r a n -o t h e r . The s t r u c t u r e o f t h e p r o t o t y p e , o r p a r e n t , m o l e c u l e o f a n t i - i n f l a m m a t o r y s t e r o i d i s h y d r o c o r t i s o n e . The n u m b e r i n g a n d t h e s i t e s most f r e q u e n t l y m o d i f i e d f o r i n c r e a s e d a n t i -i n f l a m m a t o r y a c t i v i t y a r e g i v e n i n F i g . 3 The r o l e o f v a r i o u s s u b s t i t u e n t g r o u p s i n f l u e n c i n g t h e a n t i - i n f l a m m a t o r y i s t h o u g h t t o be a s f o l l o w s : T h e y a l l c o n t a i n t h e 4-ere3-one s y s t e m i n r i n g A, a n o x y g e n f u n c t i o n ( e i t h e r k e t o o r h y d r o x y l ) a t G'^lll i n r i n g C, a n g u l a r m e t h y l g r o u p s a t C-10 a n d C-13, a n d a d i h y d r o x y a c e t o n e f u n c -17 F i g . 3 . S u b s t i t u e n t g r o u p s i n c r e a s i n g t h e a n t i - i n f l a m m a t o r y -a c t i v i t y o f h y d r o c o r t i s o n e . CH OH I 2 D e s o n i d e B e t a m e t h a s o n e 1 7 - v a l e r a t e P i g . 4 '. The s t r u c t u r e o f f o u r s t e r o i d s i n v e s t i g a t e d i n t h i s s t u d y . 18 t i o n a t C-17 i n r i n g D. The r i n g s t r u c t u r e and the f u n c -t i o n a l groups mentioned must remain e s s e n t i a l l y i n t a c t i f the compound i s t o have a n t i - i n f l a m m a t o r y a c t i v i t y . Molec-u l a r m o d i f i c a t i o n s to enhance the a n t i - i n f l a m m a t o r y e f f e c t have taken p l a c e i n the shadowed a r e a , shown on the hydro-c o r t i s o n e s t r u c t u r e . The f o l l o w i n g changes, e i t h e r s i n g l y or i n combination, appear to be the most e f f i c a c i o u s i n In-c r e a s i n g the a n t i - i n f l a m m a t o r y a c t i v i t y (Brown, 1 9 7 0 ; B e r l i n e r , 1 9 6 8 ) : 1. I n t r o d u c t i o n of a double bond between C - l and C - 2 . 2 . I n t r o d u c t i o n of f l u o r o groups a t C-9 and/or C-6 i n the a l p h a c o n f i g u r a t i o n . 3 . I n t r o d u c t i o n of an a l p h a h y d r o x y l group a t C -16 . 4 . I n t r o d u c t i o n of methyl group e i t h e r a l p h a or beta a t C - 1 6 . 5 . I n t r o d u c t i o n of the a c e t o n i d e group on the h y d r o x y l s a t C-16 and C -17 . 6 . E s t e r i f i c a t i o n of e i t h e r or both hydroxyls, i n the C-17 s i d e c h a i n r e.g. S t r u c t u r e of s i d e c h a i n : H H I ( I I — C — C — CH? -C— C - CHo -C — C — CHw I II I I I I I I 3 OH 0 OH OH OH OH OH OH ; ' : — / 17 Ketogenic S t e r o i d s 19 The enhancement factors measure the potency increment that a p a r t i c u l a r function contributes to the compounds a c t i v i t y . The vast majority of s t r u c t u r a l modifications that r e s u l t i n increased b i o l o g i c a l a c t i v i t y of steroids probably produce t h e i r e f f e c t by reducing the rate of elim-ination of steroids from the body, by a l t e r i n g t h e i r d i s t r i -bution, e f f e c t i v e s o l u b i l i t y and p a r t i t i o n c o e f f i c i e n t . Table (2 shows an example of the r e l a t i v e influence of sub-stituent groups i n enhancing the anti-inflammatory a c t i v i t y r e l a t i v e to hydrocortisone. Note that the tests are f o r systemic responses i n rats and may lead to wrong conclusions that the enhancement increments are applicable to t o p i c a l anti-inflammatory a c t i v i t y as well. 4. Topical anti-inflammatory c o r t i c o s t e r o i d s Manufacturers of t o p i c a l s t e r o i d medications and derma-t o l o g i c a l investigators are constantly attempting to develop more potent t o p i c a l anti-inflammatory drugs. This has been achieved by f l u o r l n a t i o n at the 9 °z s i t e on the st e r o i d mole-cule, and more recently by further f l u o r l n a t i o n at the 6 c<-s i t e as well. Added effectiveness i s produced by a l t e r i n g side chains at 6 , 1 6 , 1 7 , and 21 positions. Topical c o r t i c o -steroids a v a i l a b l e f o r dermatologic therapy are considered Table 2 Enhancement F a c t o r s f o r V a r i o u s F u n c t i o n a l Groups of C o r t i c o s t e r o i d s F u n c t i o n a l Glycogen Ant 1 -inflammatory E f f e c t s on group d e p o s i t i o n a c t i v i t y u r i n a r y sodium a 9 - F l u o r o 10 7-10 +++ 9 OL - Chi or o 3-5 ^ 3 ++ 9 OL - Bromo 0 . 4 b + 12 oc - F l u o r o 6-8° 12 oC - C h l o r o 4 C 1 - Dehydro 3-4 3-4 6 - Dehydro 0.5-0.7 + 2 oL - Methyl 3-6 1-4 ++ 6 «c - Methyl 2-3 1-2 l6o6 - Hydroxy 0 . 4 - 0 . 5 0.1-0.2 l ? o c - Hydroxy 1-2 4 -21 - Hydroxy 4-7 25 ++ 21 - F l u o r o 2 2 a+ = r e t e n t i o n ; - = e x c r e t i o n . b I n 1-dehydrosteroids t h i s v alue i s 4. c I n the presence of a 17 oC - h y d r o x y l group t h i s v alue i s <£ 0.01. . ( From; Burger,A., 1970 ) by medical investigators as belonging to one of the four classes according to halogenation (Bluefarb, 1 9 7 0 ) . 1 . Nonhalogenated c o r t i c o s t e r o i d s : - hydrocortisone, pred-nisolone, desonide. 2. Monofluorinated c o r t i c o s t e r o i d s : - fluorohydrocortisone, fluoromethalone, fluorandrenolone, triamcinolone, dexa-methasone and betamethasone. 3. Difluorinated c o r t i c o s t e r o i d s : - fluocinolone, flumethasone. 4. Chlorinated c o r t i c o s t e r o i d s : - beclomethasone. Other substituent groups than halogens however are also present. Table 3 gives the therapeutically most frequently used congeners of hydrocortisone. Most of them have t o p i c a l as well as systemic a c t i v i t y . Pew have equal t o p i c a l and systemic a c t i v i t y and some have equal a c t i v i t y . It i s seen that most steroids contain f l u o r i n e group i n addition to other modifications. This has made i t easy to ascribe the main potency enhancing properties to f l u o r l n a -t i o n since, whether needed or not,fluorine had been added to a l l potent steroids. In fact many fluorinated steroids have l i t t l e t o p i c a l a c t i v i t y as i n the cases for Instance, SfV; triamclnolone and fluocinolone. Desonide, containing no f l u o r i n e , i f proven to be as potent as the most potent t o p i c a l fluorinated steroids would demonstrate the lack of need f o r f l u o r l n a t i o n and t h i s would aid Table 3 Anti-Inflammatory Congeners of Hydrocortisone No. Corticosteroid and abbreviations used Substituent —^r-4,5-double bond In A-Rlng 1. Hydrocortisone (HC) 2 . Hydrocortisone acetate (HCOAc) 3 . Hydrocortisone butyrate (HCOBu) 4. Fludrocortisone (9FHC) 5 . Fludrocortisone acetate (9FHC0Ac) 6 . Flurandrenolone 7. Flurandrenolone acetonide 1,2-double bond & 4 ,5-double  bond i n A-Rlng 8. Prednisolone 9 . Prednisolone acetate 1 0 . Methyl prednisolone 1 1 . Methyl prednisolone acetate 1 2 . Fluorometholone 1 3 . Paramethasone acetate 14. Paramethasone 1 5 . Dexamethasone 16 . Betamethasone 17. Betamethasone 1 7-valerate (B-17-V) 18. Beclomethasone diproplonate 19* Flumethasone 2 0 . Flumethasone plvalate 2 1 . Desonide (DA) 2 2 . Triamcinolone acetonide (TA) 2 3 . Triamcinolone 24. Fluocinolone 2 5 . Fluocinolone acetonide (FA) 26. Fluocinolone acetonide acetate (FAOAc) 2 7 . Deprodone of Hydrocortisone In Position 9oC- 16oC- 160 - 16*-, 17*6-or 17c^-21 fluoro fluoro methyl methyl methyl fluoro fluoro fluoro fluoro fluoro fluoro fluoro fluoro fluoro hydroxy fluoro fluoro fluoro fluoro chloro fluoro fluoro fluoro fluoro fluoro fluoro fluoro fluoro methyl methyl methyl methyl methyl hydroxy hydroxy acetate butyrate acetate acetonide acetate acetate deoxy acetate methyl methyl valerate methyl propionate acetonide acetonide propionate trimethyl acetate acetonide acetonide propionate acetate deoxy *Fluorometholone has a 17 fl -hydroxy instead of a 17«?C-hydroxy group as the other steroids have. 23 i n establishing the t r u l y relevant properties of steroids which determine the t o p i c a l anti-Inflammatory a c t i v i t y . B. Skin Penetration 1. Biophysical factors i n skin penetration The skin i s under constant assault by a huge variety of noxious chemicals as well as from substances applied to the skin as cosmetics or medlcinals. The degree of penetra-t i o n i s dependent primarily on physiologic factors of the skin and physical chemical factors due to the penetrant and somewhat secondarily on the vehicle and formulation. (a) Avenues of penetration: Molecules moving from the environment on to the intact skin of l i v i n g man have three potential portals of entry; v i a the f o l l i c u l a r region, through the sweat ducts, or through the unbroken stratum corneum i n between these appendages (Idson, 1968). Grasso (1971) has explained the r o l e of skin appendages i n per-cutaneous absorption. According to him, skin appendages form the major pathway fo r absorption^ f o r a b r i e f but v a r i -able period, Immediately a f t e r percutaneous a p p l i c a t i o n of the test substance. A f t e r t h i s period percutaneous absorp-t i o n occurs mainly through the stratum corneum, but a sub-s t a n t i a l proportion of the test material passes through the appendages. Skin appendages influence percutaneous absorption v i a the stratum corneum through t h e i r secretions, i n p a r t i c u l a r 2-5 P i g . 5» A n a t o m i c a l z o n e s e n c o u n t e r e d by a p e n e t r a t i n g s u b s t a n c e i n t r a n s i t f r o m t h e s k i n s u r f a c e t o t h e b l o o d v e s s e l s ( f r o m G r i e s e m e r , R.D., J . S o c . C o s m e t i c C h e m i s t s , 11, 80 (i960)). CO Betweon the Cells ot the Stratum Corneum. Through the Wall* ol the • H a i r Follicle Through the Sweat Gland Through the Sebaceoum Gland B • Through the Celli o f the Sirolum Corneum, FN--'-' F i g . 6 . P o s s i b l e a v e n u e s o f p e n e t r a t i o n i n t o a n d t h r o u g h t h e u n b r o k e n s k i n ( f r o m G r i e s e m e r , R.D., J . S o c . C o s m e t i c C h e m i s t s , 11, 81 (I960)). 27 by a l t e r i n g the state of hydration of stratum corneum and by a l t e r i n g skin pH. The epidermis presents a surface area 100 or 1000 times greater than other routes of absorption. The appendages, sweat glands and hair f o l l i c l e s probably contribute only 0.1 to 1.0 per cent of the area of the skin. The two p r i n c i p a l secretions from the skin appendages (sebum and sweat) a f f e c t the l i p i d and water content of the stratum corneum, i n d i r e c t l y a f f e c t i n g percutaneous absorption. With the exception of sodium ions and water which may be a c t i v e l y diffused into the skin,most substances are thought to penetrate the skin by passive d i f f u s i o n , and the degree of absorption Is primarily a function of the combined properties of the drug preparation and of the skin. Scheuplein (1965)])maintains that i t i s wrong to assume that a p r i n c i p a l route of penetration exists without a re-quirement to further specify other conditions. He proposes two stages i n the d i f f u s i o n of drugs. An i n i t i a l stage when d i f f u s i o n occurs primarily v i a f o l l i c l e s and ducts and l a t e r stage or steady state d i f f u s i o n primarily through intact stratum corneum. Once a substance passes through stratum corneum, there i s apparently no s i g n i f i c a n t hindrance to penetration through the remaining epidermal layers and the dermis. 28 (b) Barrier characteristics: The stratum corneum is the rate limiting barrier which restricts inward and out-ward movement of chemical substances. Structurally, the stratum corneum is a heterogeneous tissue composed of f l a t -tened keratinised c e l l s , the outer layers of which are less densely packed than those adjacent to the underlying granu-lar layer. This greater impermeability in the lower horny layer has led to the suggestion that a separate barrier ex-ists at this level, the so-called "stratum conjunctivum" (Idson, 1971). No real evidence exists for the localization of the functional barrier. Analysis of penetration data, evidence from controlled stripping experiments and the de-tailed picture of the stratum corneum gained from electron microscopy, a l l support the idea that the barrier to penetra-tion consists of a keratln-phospholipld complex In the dead and relatively dry cells of the entire stratum corneum. The proposed mechanisms by which the stratum corneum serves as a barrier also vary. Rein (1924) originated the concept of the barrier being electronegative thereby attracting cations and repelling anions. According to Rothman (1956) the bar-r i e r layer has the characteristics of an electric double layer, the outer horny layer having a strongly acid, and the inner epidermal layer, a slightly alkaline reaction while the proteins of the Interposed membrane layer are at the F i g . 7' D i a g r a m m a t i c r e p r e s e n t a t i o n o f e p i d e r m a l u l t r a s t r u c t u r e ( f r o m S e l b y , C.C.J J . S o c . C o s m e t i c C h e m i s t , 2*594, 1956). SMx S t r a t u m m a l p i g h i i S L : S t r a t u m l u c l d u m SG: S t r a t u m g r a n u l o s u m SC: S t r a t u m c o r n e u m 30 i s o e l e c t r i c p o i n t . T r e g e a r (1962) c o n c e i v e d t h e r e s i s t a n c e o f t h e b a r r i e r t o be a p h y s i c a l p r o p e r t y . T h e r e i s a l i m i t e d k n o w l e d g e o f t h e c o m p o s i t i o n o f t h e b a r r i e r . The m a i n c e l l u l a r c o m p o n e n t s a r e t h e p r o t e i n s , l i p i d s a n d w a t e r c o m b i n e d i n t o a n o r d e r e d s t r u c t u r e . S k i n w i t h a d i s r u p t e d e p i d e r m a l b a r r i e r w i l l a l -l o w up t o 80 p e r c e n t o f h y d r o c o r t i s o n e t o p a s s i n t o t h e d e r m i s . W i t h a f u n c t i o n a l l y i n t a c t e p i d e r m i s t h e a b s o r p t i o n o f t h e s t e r o i d i s a b o u t 1 p e r c e n t ( K e i p e r t , 1971). ( c ) F a c t o r s i n f l u e n c i n g p e n e t r a t i o n : F a c t o r s i n f l u e n c -i n g p e n e t r a t i o n h a v e b e e n r e v i e w e d b y S h e l m i r e (i960), H i g u c h i (I960), Wagner (1961), B a r r (1962), T r e g e a r (1966), B l a n k a n d S c h e u p l e i n (1969), S a r k a n y a n d H a d g r a f t (I969), S c h e u p l e i n (1971). T h e s e f a c t o r s c a n be d i v i d e d i n t o d i f f e r e n t c a t e -g o r i e s : a ) n a t u r e o f t h e s k i n , b ) n a t u r e o f t h e d r u g , c ) v e -h i c l e , a s f o l l o w s : a ) N a t u r e o f s k i n : i S p e c i e s d i f f e r e n c e i i A ge o f t h e s k i n i i i S k i n t e m p e r a t u r e i v The s t a t e o f t h e s k i n ( n o r m a l , a b r a d e d , d i s e a s e d ) v P r e t r e a t m e n t o f s k i n 31 v i The degree of hydration of skin v i i Skin appendages b) Nature of drug: 1 Molecular size and shape i i D i s s o l u t i o n c h a r a c t e r i s t i c s of drug i i i Polymorphic forms iv P a r t i c l e s i z e , i n case of drugs i n suspension or emulsion ^ v S o l u b i l i t y and p a r t i t i o n c o e f f i c i e n t v i Thermodynamic properties of drugs v i i Concentration of the medicament c) Nature of v e h i c l e : i P a r t i t i o n c o e f f i c i e n t vehicle/stratum corneum i i Moisture i n vehicle and skin b a r r i e r i i i Nature of the base of the vehicle i v Presence of surfactants i n vehicle v V i s c o s i t y of the vehicle d) Miscellaneous factors of importance are: i Area of a p p l i c a t i o n , contact time and frequency of r e a p p l i c a t i o n i i Route of absorption i i i Thickness of skin b a r r i e r phase i v Manner of a p p l i c a t i o n as under occlusion McKenzie and Stoughton have shown that the penetration 32 of corticosteroids may be increased one hundredfold by oc-cluding the site of application. Occluding the skin prevents evaporation of water that passes across the epidermis, and the watery secretions of sweat ducts leading to an increase in the water content of the stratum corneum, thus increasing percutaneous absorption. Wet epidermis may show up to lOOOx greater diffusion constant (Scheuplein, 1965) . Vickers (1963) has demonstrated that occlusion not only enhances penetration of corticosteroids, but creates a depot of corticosteroids in the stratum corneum. A large reservoir f»or the gluco-cortlcosteroids oan be established in the stratum corneum very rapidly by using dimethysulfoxide (DMSO) as a vehicle. "State of hydration" of skin i s very important. According to Blank ( 1965)t dry stratum corneum presents a very high resis-tance to penetrations. It has been claimed that skin pH can be made less acidic i f the water content is increased (Blank, 1965). The shift in pH can affect the absorption of some chemical agents, but not steroids which are non-electrolytes. Fritsch and Stoughton ( i 9 6 0 ) found a tenfold Increase in penetration when the environmental temperature was raised from 10° to 37° and about a tenfold Increase in the completely hydrated skin as compared to a relative humidity of 50 per cent. 33 (d) Physical chemistry of diffusion: The transport of drugs across the skin barrier may be considered as a process of passive diffusion. The flux, J, for transport across a membrane is proportional to the product of force and concentration. J = gg f =$&~J (1) d In a RT L d x J " -DC ~ ™ a ( u = uo + RT In a) (2) (3) (4) -D dC = — 5 J ~ (for a constant y ) (5) In the above equations: (Ostrenga, 1971) D is the diffusion coefficient for the drug in the barrier, R Is the gas constant, T is the absolute temperature, du Is ~ dx the chemical potential gradient across the barrier, a is the thermodynamic activity and J£_ is the activity coefficient. The rate of penetration, dg>, is then given by: dt h ( ( 6 ) J 1* h Is the effective thickness of the barrier, and is the amount penetrated per unit area. Since the concentration of drug In the membrane surface on the vehicle side, Cg, is related to the concentration in the vehicle, C v, by K = C 2/C vt and i f the concentration at the membrane surface on the oppo-site side, C^, is maintained at zero concentration such that the concentration gradient of diffusion, dC/dx, is equal to the ratio of C£» over the thickness of the membrane, Cg/h, then: d£ = D(K)C * t = h" ( 7) dQ can be obtained from the penetration studies by calcu-dt lating the slope in the steady-state region from plots of amount of drug penetrated versus time. Higuchi (i960) i n -itiated the basic equations, describing the variables af-fecting the rate of release of solid drugs suspended in vehicles. He pointed out that the driving force behind drug movement through the skin is the difference in the thermodynamic potential between the vehicle and deeper t i s -sues. The direction of flow for systems Is always from higher thermodynamic potential to lower thermodynamic po-t e n t i a l . i ) S o l u b i l i t y and p a r t i t i o n c o e f f i c i e n t c h a r a c t e r i s t i c s of the penetrant: The aqueous s o l u b i l i t y of a drug determines the con-centration presented to the absorption s i t e , and the par-t i t i o n c o e f f i c i e n t strongly Influences the rate of transport across the absorption s i t e . Katz and Shaikh (1965) indicate that the e f f i c i e n c y of percutaneous absorption may be a function of the product of the p a r t i t i o n c o e f f i c i e n t and the square root of the aqueous s o l u b i l i t y , i n agreement with t h e o r e t i c a l considerations developed by Higuchl (196,0). The lipid/water p a r t i t i o n c o e f f i c i e n t per se i s not as s i g n i f i -cant as the stratum corneum/vehicle p a r t i t i o n c o e f f i c i e n t . Treherne (1956) related permeability constant to ether/water p a r t i t i o n c o e f f i c i e n t . A s i m i l a r r e l a t i o n s h i p between vaso-d i l a t o r a c t i v i t y and lipid/water p a r t i t i o n c o e f f i c i e n t was demonstrated for.a series of esters of n i c o t i n i c acid by Stoughton et a l ( i 9 6 0 ) . They also found a s i m i l a r c o r r e l a -t i o n between benzene/water p a r t i t i o n c o e f f i c i e n t s and the penetration of epidermis by a series of c l o s e l y related boronic a c i d derivatives. The effects of s o l u b i l i t y and p a r t i t i o n c o e f f i c i e n t on skin penetration are perhaps best i l l u s t r a t e d with c o r t i c o -36 steroids. Triamcinolone possesses f i v e times the systemic a c t i v i t y of hydrocortisone, but only one-tenth i t s t o p i c a l a c t i v i t y . Conversion of triamcinolone to i t s acetonide y i e l d s a more favourable llpid/water p a r t i t i o n c o e f f i c i e n t and enhances the t o p i c a l a c t i v i t y one thousandfold (McKenzie, 1 9 6 2 ) . Similarly, betamethasone has 30 times the systemic a c t i v i t y of hydrocortisone, but only 10 times i t s t o p i c a l a c t i v i t y . Conversion to betamethasone-17-valerate increases t o p i c a l a c t i v i t y over tenfold (Idson, 1971). i i ) Molecular c h a r a c t e r i s t i c s of penetrant: An inverse r e l a t i o n s h i p appears to exist between ab-sorption rate and molecular weight (Tregear, 1 9 6 4 ) . Small molecules penetrate more rapidl y than large molecules, but within a narrow range of molecular size there i s l i t t l e cor-r e l a t i o n between siz e and penetration rate. The ef f e c t on penetration rate of the size and shape of the penetrating molecules can be determined only i f the ef f e c t of size and shape can be separated from the ef f e c t of s o l u b i l i t y char-a c t e r i s t i c s . According to Blank and Scheuplein (1964) water s o l u b i l i t y decreases and l i p i d s o l u b i l i t y Increases with an increase i n molecular weight i n the series of analogs such as a l i p h a t i c alcohols. The rate of penetration increases as the molecular weight increases. But higher molecular weight materials also show variable penetration. L i t t l e i s known o f t h e e f f e c t o f t h e m o l e c u l a r s h a p e , i i i ) V e h i c l e e f f e c t s : The l i t e r a t u r e o n t h e I n f l u e n c e o f v e h i c l e s on s k i n p e n e t r a t i o n i s c o n f u s i n g a n d o f t e n c o n t r a d i c t o r y . A l a c k o f a w a r e n e s s o f p o s s i b l e d r u g - v e h i c l e i n t e r a c t i o n a n d t h e f u n c t i o n o f t h e t h e r m o d y n a m i c s i n v o l v e d i n t h e i n t e r p r e t a t i o n o f r e s u l t s ( , h a s a d d e d t o t h e d i f f i c u l t i e s . R o thman (1954) r e v i e w e d t h e l i t e r a t u r e o n v e h i c l e s up t o 195^« a n d - B a r r t o 1962. More r e c e n t r e v i e w s a r e t h o s e o f B a r r e t e t a l . ( 1 9 6 9 ) , Munro (1969) a n d S a r k a n y a n d H a d g r a f t (I969). P h y s -i o l o g i c a l a v a i l a b i l i t y o f t h e t o p i c a l l y a p p l i e d d r u g d e p e n d s on b o t h t h e r a t e o f r e l e a s e f r o m t h e v e h i c l e a n d t h e p e r m e a -b i l i t y o f t h e s k i n . R e l e a s e o f a s u b s t a n c e w i l l be f a v o u r e d b y t h e s e l e c t i o n o f v e h i c l e s h a v i n g a l o w a f f i n i t y f o r t h e p e n e t r a n t , o r i n w h i c h t h e d r u g i s l e a s t s o l u b l e . T h i s i s c o n s i s t e n t w i t h t h e v i e w t h a t t h e r a t e o f r e l e a s e i s g o v e r n e d b y t h e v e h i c l e t o r e c e p t o r p h a s e ( s t r a t u m corneum) p a r t i t i o n c o e f f i c i e n t ( S c h u t z , 1951). A n o t h e r i m p o r t a n t f a c t o r i s how f i r m l y o r l o o s e l y t h e s o l u t e i s h e l d b y t h e v e h i c l e . S o l u t e s h e l d f i r m l y b y t h e v e h i c l e , s u c h a s when t h e d r u g f o r m s a s o l u b l e c o m p l e x w i t h t h e v e h i c l e , e x h i b i t l o w a c t i v i t y c o e f f i c i e n t s ; h e n c e t h e r a t e o f r e l e a s e f r o m s u c h d r u g - v e h i c l e c o m b i n a t i o n s w i l l be s l o w . S o l u t e s h e l d " l o o s e l y " b y t h e v e h i c l e , e x h i b i t h i g h 38 a c t i v i t y c o e f f i c i e n t ; t h e r e f o r e the r a t e of r e l e a s e from such drug-vehicle combination w i l l be f a s t e r (Poulsen, 1968). Dempskl (19695) et al„demonstrated that i n v i t r o r e l e a s e of a medicinal agent i s a f u n c t i o n of the degree of s o l u b i l -i t y of that agent i n both the base and i t s surrounding media. P e n e t r a t i o n of an ointment f i l m such as i s a p p l i e d t o s k i n occurs i n two phases. I n i t i a l l y , the p e n e t r a t i n g agent par-t i t i o n s between i t s c a r r y i n g medium. This i s fol l o w e d by the second phase of p e n e t r a t i o n , the d i f f u s i o n of the penetrat-i n g agent through the ointment f i l m . The second phase of the permeation may be f u r t h e r subdivided i n t o two stages. The f i r s t stage i s the establishment of a uniform c o n c e n t r a t i o n gradient of penetrant across the b a r r i e r , and second i s the constant, uniform d i f f u s i o n of penetrant through the b a r r i e r a f t e r the uniform c o n c e n t r a t i o n gradient has been e s t a b l i s h e d . The second phase i s known as steady s t a t e d i f f u s i o n . The steady s t a t e d i f f u s i o n l a s t s as long as the chemical remains i n adequate supply on top surface and Is removed from the lower surface (Lueck, 1957)• Iv) "Accelerant" s o l v e n t : An a c c e l e r a n t increases cutaneous p e r m e a b i l i t y by caus-ing the k e r a t i n to s w e l l and by l e a c h i n g out e s s e n t i a l s t r u c -t u r a l m a t e r i a l from the stratum corneum, thus reducing the d i f f u s i o n a l r e s i s t a n c e . V a r i e d agents have been reported t o 38 activity coefficient; therefore the rate of release from such drug-vehicle combination w i l l be faster (Poulsen, 1968). Dempski (1969.) et al,. demonstrated that in vitro release of a medicinal agent is a function of the degree of solubil-i t y of that agent in both the base and i t s surrounding media. Penetration of an ointment film such as is applied to skin occurs in two phases. I n i t i a l l y , the penetrating agent par-titions between i t s carrying medium. This is followed by the second phase of penetration, the diffusion of the penetrat-ing agent through the ointment film. The second phase of the permeation may be further subdivided into two stages. The f i r s t stage is the establishment of a uniform concentration gradient of penetrant across the barrier, and second is the constant, uniform diffusion of penetrant through the barrier after the uniform concentration gradient has been established. The second phase is known as steady state diffusion. The steady state diffusion lasts as long as the chemical remains in adequate supply on top surface and is removed from the lower surface (Lueck, 1957)• iv) "Accelerant" solvent: An accelerant increases cutaneous permeability by caus-ing the keratin to swell and by leaching out essential struc-tural material from the stratum corneum, thus reducing the diffusional resistance. Varied agents have been reported to 39 have accelerant action, p a r t i c u l a r l y propylene g l y c o l , sur-face active agents, and aprotic materials such as urea, DMSO, DMP, and DMA. Work with these solvents has contributed to a greater understanding of the chemical nature of the skin bar-r i e r i n r e l a t i o n to a sp e c i f i e d penetrant and the transport mechanism of various compounds across skin. DMSO, DMP, and DMA are a l l strongly hygroscopic and hence increase the hydra-t i o n and therefore i t s permeability. There are possible re-v e r s i b l e configuration changes i n skin protein structure brought about by substitution of int e g r a l water molecules by DMSO. DMSO can extract soluble components from the stratum corneum, suggesting u l t r a s t r u c t u r a l modifications consistent with an increase i n permeability (Plfbaum, 1968). Surface active agents appear to play a major role i n promoting transappendageal absorption. Penetration of cer-t a i n antimicrobial substances appears to be enhanced by the addition of surface active agents. The penetration of f a t t y acid soaps varies inversely with pH (Sprott, 1970). v) The pattern of skin penetration: The t h e o r e t i c a l l y expected pattern of skin penetration i s as follows (Tregear, 1966): When a substance i s applied to skin, alone or i n solution, i t only reaches the c i r c u l a t i o n , or i n the case of d i f f u s i o n c e l l , the washing saline, gradually; the rate of transfer DMSO '(Demethylsulfoxidei)), DMF, (Dimethylformamide), DMA (Dime thy lac etamide) 4 U R e p r e s e n t a t i v e P e n e t r a t i o n P r o f i l e f o r Drug D i f f u s i n g Through Human S k i n Time P i g . : 8 .. ; A g e n e r a l i z e d p e n e t r a t i o n c u r v e showing d e l a y time, t d and s t e a d y s t a t e c o n d i t i o n . through the skin r i s e s to reach a steady rate. This steady penetration rate i s maintained thereafter i n d e f i n i t e l y , pro-vided that (a) a constant concentration of the penetrant i s maintained on the skin, and (b) the penetrant, or i t s vehicle, does not attack the skin surface. The time taken to reach t h i s steady state i s given by the delay period, t d (Pig. 8 )• The steady state penetration, Q_, i s obtained from the slope of the straight l i n e portion i n a plot of penetration rate vs time. Its units are mass per unit time per area. 2. Methods f o r measuring percutaneous absorption Blank ( I 9 6 0 ) , Gemmel (1957) t Ainsworth ( i 9 6 0 ) and Stoughton (1964) have reviewed methods f o r measuring percu-taneous absorption. Most authors f i n d i t convenient to c l a s s i f y these methods as in-vivo, i n ^ v i t r o and autoradio-graphy methods. Blank ( i 9 6 0 ) has observed more s p e c i f i c a l l y that most percutaneous absorption investigations have i n -volved the measurement of: i) penetration i n model systems i i ) h i s t o l o g i c a l studies i i i ) the use of tracers 42 The measuring methods using models a l l are of In vitro type. Membranes composed of cellulose film e.g. dimethyl-polysiloxane (Flynn, 1971) sheep bladder etc., have been used as models, alternatively human and animal excised skin is used. The assumption is made that the process of penetra-tion in the skin is similar to the diffusion through a mem-brane. Various designs of skin diffusion cells have been used for drug penetration studies (Poulsen, 1969). The use of the diffusion c e l l is very convenient and gives a satisfac-tory representation of the relative a b i l i t y of drugs to pene-trate the skin. They have a limited value, however, because the models represent either a r t i f i c i a l or metabolically i n -active systems which lack circulating blood. Histological studies have also been ut i l i z e d to measure. absorption of medicaments (Duemling, 1941). Goldzieher (1952) used histologic sections in their study of the penetration of estrogens. Kosch (1944) studied the penetration of s a l i -c ylic acid, sulfur, resoroinol and other substances by ex-amining histological sections for evidence of keratolysis. The most common methods of studying percutaneous absorp-tion In vivo have been done by analysis of circulating blood, urine or feces. In certain oases organs or tissues have been excised and analyzed for drug content. Hlynka (I969) studied the intracutaneous absorption of drugs, u t i l i z i n g the latter 4 3 approach. Extraneous dyes, fluorescent materials, radioactive tracers, have been ut i l i z e d for a long time to trace drug penetration inppercutaneous absorption studies. The use of dyes and fluorescent materials in penetration studies is open to criticism. One cannot be certain that the dye or fluorescent material remains with the substance whose pene-tration is being studied. Radioactive tracers have become Increasingly popular for studying percutaneous absorption. The use of radioactive tracers is carried out by one of the following techniques: (1) direct counting at the surface (11) autoradiographic technique ( i l l ) measurement of radioisotope within the body. 3. Isolation of epidermal sheets The closest approximation to In vivo diffusion conditions In model systems is through the use of human autopsy skin for a diffusion membrane either in the form of Isolated epidermal sheets or through the use of whole thickness skin. The iso-1 lated epidermal sheets give more reproducible results. The human epidermal sheets can be removed mechanically, chemically or by heat separation: 44 i ) Mechanical method: Wolf (1939) f i r s t i n t r o d u c e d a ce l l o p h a n e tape s t r i p -p i n g technique f o r the removal of epidermal sheets. Van S c o t t (1952) showed t h a t i f a s t r i p of human s k i n i s s t r e t c h e d to approximately double i t s o r i g i n a l l e n g t h , the epidermis can be s t r i p p e d away q u i t e e a s i l y . U n f o r t u n a t e l y , the t e n s i o n r e q u i r e d t o ac h i e v e t h i s purpose i s very l i t t l e l e s s than the br e a k i n g p o i n t of the s k i n . G i l b e r t et a l . ? (1963) • proposed an improved technique f o r the i s o l a t i o n of epidermis from human s k i n . They found i t necessary t o s t r e t c h the s k i n t o 1 . 7 5 times i t s r e l a x e d l e n g t h . The epidermis c o u l d then be separated by b l u n t d i s s e c t i o n w i t h a s u i t a b l e instrument. i i ) Chemical method: I n the l e a t h e r i n d u s t r y , the epidermis i s removed from dermis by e f f i c i e n t chemical methods. Baumberger (1939) used chemical methods f o r s e p a r a t i o n of epidermis from der-mis. They used d i l u t e a c e t i c a c i d , or one normal ammonium hydroxide f o r 20 minutes a t room temperature. They a l s o found sodium carbonate s o l u t i o n to be e q u a l l y e f f e c t i v e a l -though slower i n a c t i o n . The e f f e c t s of the compounds s t u d i e d was^ gthat an a c i d or a base which can pe n e t r a t e the t i s s u e with s u f f i c i e n t speed and w i l l not r e a c t with the t i s s u e proteins,., can form a compound having an a c i d d i s -s o c i a t i o n constant near the i s o e l e c t r i c p o i n t of c o l l a g e n . *5 This may cause the collagen to swell, permitting the de-tachment of the epidermis. Wood and Bettley (197D removed the stratum corneum by suspending the skin on the glass rods of a chromatogram tank above 0.88 ammonia solution for 60 minutes, after which the stratum corneum was l i f t e d off. According to Medawar (1941), epidermal sheets can be isolated by incubation of the skin with enzymes. He ob-served that epidermal sheets could be prepared from human skin Incubated in Tyrode's solution containing commercial trypsin. Epidermis is thought to be secured to the dermis by elastic fibres and the enzyme elastase breaks down elas-t i c fibres, i l l ) Heat separation: Scheuplein (I965) removed the whole epidermal sheets from autopsy skin by Immersing It in a water bath at 60° for 30 seconds. These membranes carry the intact stratum corneum, and can be obtained in unruptured pieces, Allenby (1969) et a l removed epidermal sheets by dipping human autopsy skin (after removing the subcutaneous adipose tissue) in a water bath at 60° for one minute, blotting dry, and peeling the epidermis off with forceps. Marrs (1971) removed the epidermis by immersing the skin in a water bath at 55° for 30 seconds and immediately cooling with ice water. The 46 skin was spread on an Iced petrl-dlsh cover and epidermis was easily peeled away. Stratum corneum Is shown to undergo Irreversible structural changes when heated above 65° (Bernstein, 1970). Depending upon the purpose for which the epidermal sheet Is required, a choice from these various techniques can be made. For the purposes of penetration studies, Scheupleints (I965) method was found to be most useful because It ren-dered large Intact epidermal sheets which could be stored easily. The complex chemical, physical and biological prop-erties of l i v i n g skin preclude adequate simulation by In vitro models. Therefore, In vivo methods should also be carried out for complete Information. 47 G. A n a l y t i c a l M e t h o d s A n a l y s i s o f c o r t i c o s t e r o i d s i n b i o l o g i c a l s a m p l e s c a n be p e r f o r m e d b y s p e c t r o p h o t o m e t r y , f l u o r o m e t r i c , g a s c h r o m a -t o g r a p h i c a n d r a d i o i s o t o p i c t e c h n i q u e s . 1. S p e c t r o p h o t o m e t r i c a s s a y s 4 S o l u t i o n s o f s t e r o i d s c o n t a i n i n g t h e A - 3 k e t o g r o u p 240-242 nm. a n d 310 nm. S u b s t i t u e n t s g r o u p s may c h a n g e t h e p e a k i n t e n s i t y w a v e l e n g t h s t o w a r d s 250 nm. The 240-250 nmii r e g i o n c a n be u s e d f o r q u a n t i t a t i o n . The g r e a t a d v a n t a g e o f t h i s t y p e o f a s s a y i s i t s r a p i d i t y . The d i s a d v a n t a g e s a r e l o w s e n s i t i v i t y a n d l o w s p e c i f i c i t y . 2. F l u o r o m e t r i c a s s a y N o u j a i m a n d J e f f e r y (1970) h a v e r e v i e w e d t h e s p e c t r o -p h o t o f l u o r o m e t r i c m ethods f o r t h e e s t i m a t i o n o f c o r t i c o s t e r o i d s . The s p e c t r o p h o t o f l u o r o m e t r i c ( S P F ) method i d e n t i f i e s a n d q u a n t i t a t e s compounds t h r o u g h e i t h e r t h e i r f l u o r e s c e n t o r p h o s p h o r e s c e n t p r o p e r t i e s . The S PF method i s a c c u r a t e , 48 precise and s p e c i f i c . Corticosteroids do not possess native fluorescence but can be made to fluoresce by treatment with strong ac i d . The functional groups responsible f o r promot-ing the acid-induced fluorescence are the 11 - hydroxyl and the A - 3 - keto groups ( sweat, 1954). C o r t i s o l , c o r t i c o -sterone, 20 -|J - hydroxycortisol and t h e i r 11 - hydroxy epimers possess these groups. Fluorescence of steroids i s further Increased by the presence of a 17 - or 21 - hydroxyl group. The functional groups responsible f o r i n h i b i t i n g f l u o r e s -cence are the 11: - keto or 16<<- hydroxyl group. Reduction of the double bond at the 4th p o s i t i o n ( Shimo, 1967 ) also de-creases- fluorescence. 3. Other methods (a) Gas l i q u i d chromatography assay: %X£f$ ft ) Gas l i q u i d chromatography (GLC) i s a useful technique f o r the analysis of complex mixtures because i t affords a means of separation and quantitation i n one procedure. The use of GLC with steroids requires columns with low concentrations of stationary phase. Determination of steroids i n b i o l o g i c a l samples necessitates the use of detectors with high s e n s i t i v -i t i e s because of very low concentrations of s t e r o i d present. 49 (b) Radioisotopic assay: Noujaim and Jeffery (1971) have recently reviewed the analysis of corticosteroids in biological samples by radioisotopic methods. Radioisotopic methods are reported to be far superior in sensitivity, pre-cision and specificity when compared to nonradioactive tech-niques. Their main disadvantages are expense of the Isotopes, precautionary measures necessary especially in human experi-mentation and their Inapplicability for routine testing of commercially available steroids. 4 . Vasoconstriction assay Topical or systemic anti-inflammatory substances, have been evaluated in a variety of procedures, which apparently represent different modes of action and/or different stages of inflammatory process. The most commonly used method is the vasoconstriction test (McKenzle, 1962) . The mechanism of steroid-induced dermal vasoconstric-tion remains unexplained. Some authors have proposed that steroids increase the sensitivity of vascular smooth muscles to normal levels of nor-epinephrine (Solomon, 1965). More recent theories include: 1. a direct effect of steroids, similar to sympathomimetics, on sodium transport across smooth muscle, 2 . a release of locally bound nor-epinephrine from cutaneous 50 s t o r e s , o r 3. e f f e c t s on v a r i o u s enzyme s y s t e m s i n v o l v e d i n t h e r e -l e a s e o f a number o f s u b s t a n c e s , i n c l u d i n g n o r - e p i n e p h r i n e , s e r o t o n i n , b r a d y k l n i n , t h u s i n t e r r u p t i n g n o r m a l v a s c u l a r t o n e ( C l i n e , 1 9 6 6 ) . S t i l l a n o t h e r p r o p o s a l i s t h a t s t e r o i d s a c t b y d e c o n -g e s t i n g t h e c a p i l l a r i e s o f t h e n o r m a l p a p i l l a e . I n v e s t i g a t i o n s I n t o ,the m e c h a n i s m o f a n t 1 - i n f l a m m a t o r y a c t i o n o f C o r t i s o l h a v e r e v e a l e d t h a t s t e r o i d s may e x e r t t h e i r e f f e c t b y i n h i b i t i n g t h e r e l e a s e o f p l a s m a k i n i n s a n d a l s o b y p r e v e n t i n g i n t e r a c t i o n b e t w e e n t h e a c t i v a t e d p l a s m a enzymes a n d t h e i r p l a s m a p r o t e i n s u b s t r a t e ( C l i n e , 1966) w h i c h a r e v a s o d i l a t i n g s u b s t a n c e s . I t seems p o s s i b l e t h a t t h i s m e c h a n i s m e x p l a i n s t h e e x c e l l e n t a g r e e m e n t b e t w e e n v a s o c o n s t r i c t i o n a c t i v i t y a n d t h e a n t i - i n f l a m m a t o r y a c t i v i t y o f t h e s t e r o i d s . C e r t a i n d i f f i c u l t i e s a r e e n c o u n t e r e d w i t h v a s o c o n s t r i c -t i o n t e s t s , a n d e r r o r s a r e i n h e r e n t . C a r e must be t a k e n i n v a s o c o n s t r i c t i o n a s s a y s t o a l l o w f o r s i t e t o s i t e a n d i n d i -v i d u a l t o i n d i v i d u a l v a r i a t i o n s ( M c K e n z i e , I 9 6 6 ) . D e s p i t e t h e l o w p r e c i s i o n o f t h e a s s a y , most a u t h o r s a g r e e t h a t i t i s a v e r y u s e f u l a n d a q u i t e r e l i a b l e m e t hod f o r s c r e e n i n g new compounds b e f o r e c l i n i c a l t r i a l s . 51 III STATEMENT OF PROBLEM The objectives of this Investigation ares (1) to determine whether the recently introduced non-fluorinated steroid desonide (desfluorotriamcinolone aceto-nide) is c l i n i c a l l y as acceptable for the topical treatment of steroid-responsive dermatoses as the fluorinated beta-methasone 17-valerate which is regarded as the therapeutically most potent anti-inflammatory steroid. (2) To explore the possibilities for the development of spec-trophptofluorometrlc technique for the assay of desonide. ( 3 ) To develop a convenient and reproducible In vitro method for the measurement of epidermal penetration rates of anti-inflammatory corticosteroids. (4) To interpret the results with the aid of auxiliary parti-tion coefficient and solubility determinations and by compar-ing results with those obtained for two other steroids, hydrocortisone which is known to be less active than triam-cinolone acetonide and betamethasone 17-valerate, which is known to be more active than triamcinolone acetonide. (5) To evaluate the effects of 9-alpha fluorinatlon on the biological activity by determining the ED^Q of the vasocon-strictor test for desonide and i t s fluorinated analog, triam-cinolone acetonide. IV EXPERIMENTAL METHODS A. Apparatus 1. Poulsen skin d i f f u s i o n c e l l The Poulsen skin d i f f u s i o n c e l l shown i n F i g . 9 , consists of an upper donor chamber i n which the penetrating agent i s placed and a lower glass chamber with a side arm to allow sampling of the receptor phase. A t e f l o n coated bar attached to a polyethylene s a i l provides e f f i c i e n t mix.-ing within the lower receptor chamber. Two t e f l o n discs holding the skin are clamped onto the f l a t ground glass surface at the top of the receptor chamber,/ leaving an ex-posed central c i r c u l a r area of 1.0 cm t. 0.05 cm/through which the penetration i s measured. The volume of the re-ceptor chamber i s 10 ml, and that of donor chamber 0.4 ml. F i g . 9 ., shows the assembled d i f f u s i o n c e l l . F i g . 10? shows a l l the d i f f e r e n t parts of the d i f f u s i o n c e l l , and F i g . II shows the top view of the d i f f u s i o n c e l l . 2. Spectrophotofluorometer Aminco-Bowman spectrophotofluorometer Model 4-802^ was used. This Instrument consists of a s o l i d state xenon lamp, D.C. power supply unit, I.?> 21 photomultiplier tube of 1 American Instrument Company, Inc., S i l v e r Spring, Maryland. 53 P o u l s e n S k i n D i f f u s i o n C e l l F i g . 9 A, S k i n s p e c i m e n . B, T e f l o n p i e c e s h o l d i n g s k i n . C, R e c e p t o r chamber. D, S i d e arm. E, T e f l o n c o a t e d B a r . F, P o l y e t h y l e n e s a i l . G, Clamp. F i g . 11 S h o w i n g t o p v i e w o f t h e d i f f u s i o n c e l l , h a v i n g e p i d e r m i s e x p o s e d , t h r o u g h w h i c h p e n e t r a t i o n o c c u r s . 55 a wave length range of 300 - 700 millimicrons. S l i t ar-rangement #3 (33331) was used. The c e l l s were fused quartz of 1.0 cm path length. 3. Picker Nuclear Llqulmat, Model-650-503 was used. D i v i s i o n of Picker X-ray Corporation. Manufactured by: Intertech. Inc., North Haven, Conn., U.S.A. 4. A. Hitachi-Cabman 124 - Spectrophotometer B. Beckman - DGBT Spectrophotometer 5. Dermatome,ccastroviejo electro-keratotome was used i n modified form (Stewart and Runlkis, 1967). B. Procedures 1. Analysis of steroids (a) Spectrophotofluormetric analysis of desonide: The f o l -lowing procedures were t r i e d : i ) Modified Mattingly's method (1962) i i ) MacKenzie's method (1967) i l l ) Modified Martin's method (1968) Mattingly's and MacKenzie's method: The fluorometric method o r i g i n a l l y established f o r 56 hydrocortisone was t r i e d for analysis of desonide. The following were the steps involved: (A) 15 ml of methylene chloride was added to 2 ml of deso-nide standard sol u t i o n (5 ^ug/ml). The extract ion\&was a l -lowed to proceed f o r 20 minutes. (B) The tubes were removed from the shaker and the two layers allowed to separate. The upper layer was discarded and the methylene chloride layer f i l t e r e d . (C) 10 .0 ml of the f i l t r a t e was transferred to clean test tubes. 5»° ml of fluorescence reagent was added and mixed thoroughly. The fluorescence of the acid layer was measured at an e x c i t a t i o n wavelength of 395 JHftt an emission wavelength of 450 nm with s l i t arrangement #3 ( 3 3 3 D . Step C was also modified s l i g h t l y by evaporating the methylene chloride i n a warm current of nitrogen at 48°C before the addition of fluorescar»^seagent. To the dry tubes 5.0 ml of fluorescence reagent was added, mixed f o r 30 sec-onds and the fluorescence read immediately. Modified Martin's method: Martin proposed an oxime formation method to d i f f e r e n -t i a t e C o r t i s o l and corticosterone i n human plasma. The oxime formation was very rapid and f i n a l fluorescence could be read even a f t e r 60 minutes. 57 C + NH9OH II -C— NHOH 0 OH Their method was t r i e d to analyze desonide, as follows: (a) To the standard solution of desonide, 15 ml of methylene chloride was added and ste r o i d extracted by gentle shaking. The tubes were centrifuged and the upper layer discarded. (b) Duplicate 5 ml aliquots of methylene chloride were transferred into clear centrifuge tubes, a c i d i f i e d with a drop of 25$ acetic acid, and evaporated to dryness i n a water bath at a temperature not exceeding 40°c. (c) Duplicate tubes were prepared, - the front row serving as a sample, the back row of tubes as "-k.blank. To the front row of tubes, 0.3 ml of blank reagent was added and to the back row of tubes 0.3 ml of hydroxylamine reagent was added. The solutions were mixed and then air-lowed to stand f o r one hour at room temperature. At the end of one hour 1.7 ml of d i s t i l l e d water was added to a l l tubes. (d) 10.0 ml of methylene chloride was added to a l l tubes. The tubes were shaken, centrifuged and the aqueous layer discarded, care being taken to remove a l l water from the tubes. (e) 1.2 ml of the fluorescence reagent was added. 5« The tubes were shaken v i g o r o u s l y f o r 30 seconds and t h e e x t r a c t a l l o w e d t o s t a n d f o r one hour. The f l u o r e s c e n c e o f t h e e t h a n o l i c s u l f u r i c a c i d phase was r e a d . (b) S p e c t r o p h o t o m e t r i c methods The s t e r o i d s namely, h y d r o c o r t i s o n e , d e s o n i d e , t r i a m -c i n o l o n e a c e t o n i d e , betamethasone 1 7 - v a l e r a t e , w e r e d i s s o l v e d i n a minimum q u a n t i t y o f e t h a n o l and d i l u t e d w i t h normal s a l i n e . The d i l u t i o n s ranged from 0 . 5 t o 10.0 ug/ml. Spec-t r a f o r a l l s t e r o i d s were o b t a i n e d i n t h e r e g i o n o f 200-340 nm. F o r a n a l y t i c a l purposes the absorbance was measured a t t h e peak f o r each s t e r o i d . 2. S o l u b i l i t y and p a r t i t i o n c o e f f i c i e n t d e t e r m i n a t i o n s 1) S o l u b i l i t y d e t e r m i n a t i o n s The s o l u b i l i t i e s of h y d r o c o r t i s o n e , t r i a m c i n o l o n e a c e t o n i d e , d e s o n i d e and b e t a -methasone 1 7 - v a l e r a t e were d e t e r m i n e d i n d i s t i l l e d w a t e r , normal s a l i n e and 40$ e t h a n o l a t 25°C, a c c o r d i n g t o Dempskl (1970). An e x c e s s o f s t e r o i d was added t o a p p r o x i m a t e l y 100 ml. o f s o l v e n t i n g l a s s b o t t l e s . The b o t t l e s were t i g h t l y capped and p l a c e d on a r o t a t i n g - b o t t l e a p p a r a t u s i n a 25°C i 0.1 w a t e r b a t h f o r p e r i o d s o f not l e s s t h a n 24 hours o r more t h a n 72 h o u r s . E q u i l i b r i u m was d e t e r m i n e d by r e p e t l -59 t i v e sampling. Before the bottles were sampled f o r assay, the r o t a t i n g apparatus was turned off to allow the excess of s t e r o i d to s e t t l e i n the solvent. The l i q u i d was f i l -tered through Millipore f i l t e r s (0.22 AH pore size) to ensure the absence of any s o l i d p a r t i c l e s . The f i l t r a t e was d i -luted with the appropriate solvent f o r assay. The UPV, absorbance of each solu t i o n was determined and the steroid concentration calculated from previously determined absorp-t i v i t y , ifa 5) values. i i ) P a r t i t i o n c o e f f i c i e n t : The p a r t i t i o n c o e f f i c i e n t of hydrocortisone, triamcinolone acetonide, desonide and betamethasone 17-valerate were determined i n octanol/water system. 20 mg. of each stero i d was weighed and dissolved i n 25 ml of octanol. 25 ml water was added to the octanol solutions, the bottles capped t i g h t l y and placed on a ro-t a t i n g b o t t l e apparatus i n a 25 - 0.1°C water bath f o r 3 days. Equilibrium was determined by r e p e t i t i v e sampling. A f t e r the t h i r d day, contents of the bottles were poured into separatory funnels and l e f t undisturbed f o r 2k hours. The aqueous layer was separated and f i l t e r e d through Whatman #1 f i l t e r paper. The stero i d concentration i n the aqueous layer was calculated from the absorptivity, a, value. The concentration of s t e r o i d i n octanol could be calculated by 60 s u b t r a c t i n g the c o n c e n t r a t i o n of s t e r o i d i n aqueous l a y e r from the o r i g i n a l c o n c e n t r a t i o n used. .- c o n c e n t r a t i o n i n organic phase c o n c e n t r a t i o n i n aqueous phase 3. In v i t r o p e n e t r a t i o n s t u d i e s (a) P r e p a r a t i o n of membranes f o r d i f f u s i o n c e l l : i ) P u l l t h i c k n e s s h a i r l e s s mouse s k i n : H a i r l e s s mice (HRS/J s t r a i n ) from Jackson L a b o r a t o r i e s , Bar Harbour, U.S.A., were used f o r t r i t i a t e d water d i f f u s i o n s t u d i e s . The f u l l t h i c k n e s s of the mice s k i n was used,, age 8 t o 32 weeks. i i ) Epidermal sheet of human autopsy s k i n : Human autopsy s k i n samples were obtained from Vancouver General H o s p i t a l . The autopsy s k i n was from the abdomen of the p a t i e n t s , who were not on s t e r o i d a l therapy f o r one week p r i o r t o death. The samples were from autopsies done w i t h i n 24-48 hours a f t e r the death of the p a t i e n t s . Epidermal sheets from the autopsy s k i n were obtained, f i r s t by removing the subcutaneous a d i -pose t i s s u e . The s k i n was then immersed i n a water bath a t 60° f o r 30 seconds. I t was pressed between the two g l a s s sheets. The epidermis was s l o w l y l i f t e d o f f as shown i n F i g . |12 . The epidermal sheets were stored i n the r e f r i g e r a t o r a t F i g . 1 2 . S h o w i n g how t h e e p i d e r m i s was r e m o v e d f r o m t h e a u t o p s y s k i n . 62. 4 ° c , and rehydrated when required f o r experiment by dipping them i n d i s t i l l e d water at 37° f o r 30 minutes (Comaish,1971 ). (b) To check intactness of epidermal sheets by t r i t i a t e d water: Hydrated epidermal sheets were sandwiched between the two t e f l o n pieces of the d i f f u s i o n c e l l and 8.0 ml of physiological s o l u t i o n was added to the receptor chamber. 0.4 ml (25/# c i ) of t r i t i a t e d water was put on top of the epidermis fixed i n the d i f f u s i o n c e l l . 0.5 ml samples were withdrawn from the lower chamber at varying time i n t e r v a l s . Each sample was replaced by 0.5 ml of saline i n order to keep the volume i n the lower chamber constant. The 0.5 ml samples were added to 12.5 ml of the s c i n t i l l a t i o n f l u i d . The solu t i o n i n the v i a l s was allowed to stand u n t i l the sodium chloride had se t t l e d at the bottom of the v i a l s . Appropriate standard and background samples were pre-pared and assayed with each set of unknowns. A known amount of t r i t i a t e d water was added to a series of previously as-sayed samples, the increase i n counts were within experimen-t a l error, showing there was no quenching. Rate of d i f f u s i o n of t r i t i a t e d water was calculated by using Downes (1967 ) equation. Rate of d i f f u s i o n = C A x B where i s the area of the skin i n contact with the solu-t i o n i n the lower chamber, B_ i s the s p e c i f i c a c t i v i t y 63 (counts/min/mg.) of water on top of the s k i n , C_ i s the t o t a l amount of r a d i o a c t i v i t y (counts/min.) which has accumulated on the sampled s i d e d u r i n g one hour. (c) Inevitro s t e r o i d p e n e t r a t i o n : The^penetration of s t e r o i d s ; namely.hydrocortisone, t r i a m c i n o l o n e acetonide, desonide.and betamethasone 1 7-valerate,through human epidermis was s t u d i e d u s i n g the f o l l o w i n g v e h i c l e s : i ) S t e r o i d deposit on epidermal sheets: 0.4 ml of the s t e r o i d s o l u t i o n s c o n t a i n i n g 2.0 mg. of hydrocortisone, t r i a m -cinolone acetonide and desonide and betamethasone 1 7-valerate was s l o w l y deposited on top of the epidermal sheet f i x e d i n the d i f f u s i o n c e l l . Ethanol was evaporated by a current of c o l d a i r from the blower. i i ) S t e r o i d s i n 40$ ethanol: 0.4 ml of s t e r o i d s o l u t i o n s , i n 40$ e t h a n o l , r e p r e s e n t i n g O.56 mg of each s t e r o i d , was p i p e t t e d on top of the epidermis. i i i ) S t e r o i d s i n h y d r o p h i l i c ointment base: 0.15 gm/ of the s t e r o i d ointments, r e p r e s e n t i n g 1.5 mg of each s t e r o i d was spread on the epidermal sheet. P e n e t r a t i o n s t u d i e s : The s t e r o i d s , hydrocortisone, t r i a m c i n o l o n e acetonide, desonide, betamethasone 1 7-valerate i n drug d e p o s i t , 40$ ethanol and h y d r o p h i l i c ointment base, were a p p l i e d on top of the epidermis which was sandwiched between the two t e f l o n 64 p i e c e s , l e a v i n g a n e x p o s e d c i r c u l a r a r e a o f 1 . 0 cm , t h r o u g h w h i c h t h e p e n e t r a t i o n was m e a s u r e d . 8 . 0 m l . o f t h e s o l u t i o n o f 0.9% N a C l was p i p e t t e d i n t o t h e r e c e p t o r c h a m b e r . The s i d e armswas c l o s e d w i t h p a r a f i l m , a n d a g l a s s c o v e r s l i p was p l a c e d on t o p o f t h e e x p o s e d s u r f a c e t o p r e v e n t e v a p o r a t i o n . 2 . 0 m l . o f s a m p l e s o l u t i o n was w i t h d r a w n f r o m t h e l o w e r c hamber a t r e g u l a r i n t e r v a l s f o r a n a l y s i s . The s a m p l e s o l u t i o n was r e p l a c e d b y f r e s h 2 . 0 m l . o f s o l u t i o n o f n o r m a l s a l i n e i n t h e d i f f u s i o n c e l l . 4. V a s o c o n s t r i c t i o n b i o a s s a y The M c K e n z i e - S t o u g h t o n v a s o c o n s t r i c t i o n a s s a y a s i m -p r o v e d b y P l a c e ( '1970) a n d h i s S y n t e x g r o u p o f c o - w o r k e r s was u s e d . The t e s t was b a s e d on t h e o b s e r v a t i o n t h a t u p o n p e n e t r a t i o n i n t o t h e s k i n a n a n t i - i n f l a m m a t o r y c o r t i c o s t e r o i d , a b o v e i t s t h r e s h o l d d o s e , c a u s e d s k i n b l a n c h i n g due t o v a s o -c o n s t r i c t i o n . We d e t e r m i n e d t h e E l | g o f d e s o n i d e a n d t r i a m -c i n o l o n e a c e t o n i d e a s f o l l o w s ; 50 a d u l t s w i t h no s k i n d i s -o r d e r s w e r e s e l e c t e d , t h e s t e r o i d s w e r e a p p l i e d i n e t h a n o l s o l u t i o n s a n d i n c r e a m s i n t e n f o l d s e r i a l d i l u t i o n s f r o m 0 . 0 0 0 1 t o 0.1%. The s k i n was p r e p a r e d b y w a s h i n g w i t h s o a p a n d w a t e r . 2 S i x t e e n s q u a r e s o f one cm a r e a w e r e m a r k e d o f f o n b o t h f o r e -65 arms by means of a s i l i c o n e g r e a s e d stamp. The s o l u t i o n s and t h e creams were coded by a n o n p a r t i c i p a n t i n t h e a s s a y . T a b l e s o f random numbers were used t o a s s i g n the® t e s t a r e a s . S o l u t i o n s and creams were randomized d i f f e r e n t l y . Each arm had a row o f f o u r s o l u t i o n s and a row o f f o u r creams. S i n c e t h e s t e r o i d s at.«higher c o n c e n t r a t i o n s show c o n s i d e r a b l e l a t -e r a l s p r e a d i n g , t h e s p a c i n g between t e s t a r e a s had t o be about 1 . 5 cm. Thus, th e number o f t e s t p r e p a r a t i o n s were e q u a l t o t h e number of t e s t a r e a s so t h a t e v e r y t e s t a r e a had an e q u a l chance t o r e c e i v e e v e r y t e s t p r e p a r a t i o n . S o l u t i o n s were a p p l i e d i n 20 /til. volumes, The s o l v e n t was a l l o w e d t o e v a p o r a t e and t h e s t e r o i d d e p o s i t s c o v e r e d 2 w i t h m a t c h i n g p l a s t i c cups. U n i f o r m l a y e r s o f 20 ng/cm -20,^/g/cm of s o l i d drugs were t h u s o b t a i n e d . The creams were f i l l e d f l u s h i n p l a s t i c cups and p r e s s e d f i r m l y on t h e i r t e s t a r e a s . Blenderm t a p e and S a r a n wrap were used t o s e c u r e t h e cups and t o o c c l u d e t h e a r e a s f o r 16 t o 24 h o u r s . Two i n v e s t i g a t o r s r e a d t h e r e s p o n s e s one hour a f t e r t h e removal o f d r e s s i n g s . 66 TfV RESULTS AND DISCUSSION A. S p e c t r o p h o t o f l u o r o m e t r i c Methods f o r t h e E s t i m a t i o n o f Desonide A d a p t a t i o n o f M a t t i n g l y ' s (1962),MacKenzie's (1967), and M a r t i n ' s methods (1968) f o r t h e e s t i m a t i o n o f h y d r o -c o r t i s o n e were t r i e d f o r d e s o n i d e . W i t h t h e f i r s t two methods t h e a d d i t i o n o f f l u o r e s c e n c e r e a g e n t t o methylene c h l o r i d e e x t r a c t r e s u l t e d i n f o r m a t i o n o f an e m u l s i o n , which was d i f f i c u l t t o break. The method had t o be m o d i f i e d & s l i g h t l y i n o r d e r t o a v o i d t h e f o r m a t i o n of t h i s e m u l s i o n . M ethylene c h l o r i d e was f i r s t e v a p o r a t e d from t h e f i n a l e x t r a c t o f d e s o n i d e , and the f l u o r e s c e n c e r e a g e n t was t h e n added t o t h e d r y t u b e s . The c o n t e n t s were mixed, and t h e f l u o r e s c e n c e was measured. W i t h t h i s m o d i f i c a t i o n no emul-s i o n was formed. However, f l u o r e s c e n c e d e c r e a s e d r a p i d l y . I n f a c t , no f l u o r e s c e n c e c o u l d be r e c o r d e d a f t e r one minute. The m o d i f i e d method was thus found u n s a t i s f a c t o r y . E f f o r t s were c o n t i n u e d t o f i n d an a l t e r n a t i v e SPP method, and t h i s t i m e , M a r t i n ' s method was t r i e d . Most o f t h e g l u c o c o r t i c o s t e r o i d s h a v i n g a k e t o group a t t h e 20th p o s i t i o n r e a c t v e r y r a p i d l y w i t h h y d r o x y l a m i n e h y d r o c h l o r i d e t o form t h e r e s p e c t i v e oxime d e r i v a t i v e . M a r t i n has used t h i s t e c h n i q u e t o a n a l y z e C o r t i s o l and c o r t i c o s t e r o n e . 67 E v e n t h i s m e t h o d was u n s a t i s f a c t o r y f o r d e s o n i d e . A s M a r t i n o b s e r v e d i n h i s w o r k w i t h C o r t i s o l , i m m e d i a t e l y a f t e r a d d i -t i o n o f f l u o r e s c e n c e r e a g e n t , t h e f l u o r e s c e n c e i n t e n s i t y o f t h e h y d r o c o r t i s o n e o x i m e d e r i v a t i v e d e c r e a s e d a n d t h e n l e v e l e d o f f a n d r e m a i n e d c o n s t a n t f o r 60 m i n u t e s . H o w e v e r , a d d i t i o n o f f l u o r e s c e n c e r e a g e n t t o d e s o n i d e a n d b l a n k t u b e s r e s u l t e d i n a n i n c r e a s e i n f l u o r e s c e n c e i n t e n s i t y i n b o t h s e t s . The p o s s i b i l i t y o f u s i n g M a r t i n ' s m e t h o d f o r e s t i m a t i o n o f d e s o n i d e was : t h e r e f o r e r u l e d o u t . F l u o r o m e t r i c m e t h o d s f o r e s t i m a t i o n o f d e s o n i d e w e r e u n s u c c e s s f u l b e c a u s e s f ( a ) t h e p r e s e n c e o f -OH g r o u p a t t h e C-16 p o s i t i o n , d o e s n o t f a v o u r f l u o r e s c e n c e ( S h i m o , 1967 )? ( b ) t h e a d d i t i o n o f f l u o r e s c e n c e r e a g e n t c a u s e s r a p i d h y d r o l y s i s o f t h e compound a t C-16 a n d C-17 p o s i t i o n s ( c ) t h e a d d i t i o n o f s t r o n g a c i d c a u s e s t h e f o r m a t i o n o f d i f f e r e n t s p e c i e s o r d e c o m p o s i t i o n o f t h e d r u g . S p e c t r o p h o t o f l u o r o m e t r i c m e t h o d s w e r e a l s o u n s a t i s f a c -t o r y f o r t r i a m c i n o l o n e a c e t o n i d e . B. S p e c t r o p h o t o m e t r i c M e t h o d S p e c t r o p h o t o m e t r i c m e t h o d s a r e most f r e q u e n t l y u s e d . The two m a i n d i s a d v a n t a g e s o f s p e c t r o p h o t o m e t r i c a s s a y s a r e 6 8 l o w s e n s i t i v i t y a n d l a c k o f s p e c i f i c i t y . F o r t u n a t e l y t h e s t e r o i d s s t u d i e d h a d a h i g h a b s o r p t i o n i n t e n s i t y a n d s t a b l e a p s o r p t i o n p e a k s . T h e r e was no i n t e r f e r e n c e b y o t h e r ma-t e r i a l s i n o u r s p e c t r o p h o t o m e t r i c a s s a y . The minimum amount o f s t e r o i d d e t e c t a b l e was i n t h e r a n g e o f 0 . 5 ug t o 2 . 0 u g , d e p e n d i n g u p o n t h e s t e r o i d a n d s o l v e n t u s e d . T h i s e n a b l e d us t o m e a s u r e t h e s t e r o i d d i f f u s e d t h r o u g h t h e s k i n t o t h e l o w e r c hamber o f t h e d i f f u s i o n c e l l . C. S o l u b i l i t y a n d P a r t i t i o n C o e f f i c i e n t s o f S t e r o i d s The s o l u b i l i t y o f a d r u g i n t h e v e h i c l e d e t e r m i n e s t h e c o n c e n t r a t i o n p r e s e n t e d t o t h e a b s o r p t i o n s i t e , a n d t h e p a r -t i t i o n c o e f f i c i e n t s t r o n g l y i n f l u e n c e s t h e r a t e o f t r a n s p o r t a c r o s s t h e a b s o r p t i o n s i t e . T h e o r e t i c a l c o n s i d e r a t i o n s d e -v e l o p e d b y H i g u c h i (I965), K a t z a n d S h a i k h (1965) i n d i c a t e d t h a t e f f i c i e n c y o f p e r c u t a n e o u s a b s o r p t i o n may b e a f u n c t i o n o f t h e p r o d u c t o f t h e p a r t i t i o n c o e f f i c i e n t a n d t h e s q u a r e r o o t o f t h e a q u e o u s s o l u b i l i t y . The w a t e r s o l u b i l i t y o f s t e r o i d s i s g i v e n i n T a b l e 4. H y d r o c o r t i s o n e h a s maximum w a t e r s o l u b i l i t y w h e r e a s B e t a -m e t h a s o n e 1 7 - v a l e r a t e h a s t h e minimum. I t i n d i c a t e s t h e p r o g r e s s i v e d e c r e a s e i n p e r m e a b i l i t y a s t h e s t e r o i d becomes i n c r e a s i n g l y more p o l a r . o 70 The octanol/water partition coefficients of the steroids were determined. The partition coefficient in this system are reported by us for the f i r s t time. The ether/water partition data are taken from Plynn ( 1 9 7 1 ) . Ether/water partition coefficient for desonide was calculated from Plynn*s equation. These partition coefficients in both the systems are given in Tabl& 5. Fig. 14 shows the linear relationship of partition coefficient to cumulative penetration. Betamethasone 17-valerate has the highest partition coefficient and hydrocortisone the lowest. It Indicates that the greater the partition coefficient of a drug, the more rapidly i t diffuses from aqueous fluids to membranes. Theoretical calculation of ether/water partition co-efficient for desonide according to Plynn ( 1 9 7 1 ) . F Partition coefficient of prednisolone (Ky) = 1 .13 Value of group constant factor ( £ ) of 16 ©c hydroxy «s 0 . 4 5 Partition coefficient of 16 ochydroxy prednisolone 1 .13 x 0 . 4 5 8 0 . $ 5 9 Value of group constant factor ( £ ) of 16 and 17 - aceto-nide = 1 9 . 3 Partition coefficient of 16 oc - hydroxy, prednisolone, 1 6 - 1 7 , acetonide O .559 x 1 9 . 3 = 1 0 . 7 0 8 . 71 T a b l e 4 S o l u b i l i t y o f s t e r o i d s I n two d i f f e r e n t s o l v e n t s Name o f t h e s t e r o i d H y d r o c o r t i s o n e D e s o n i d e T r i a m c i n o l o n e A c e t o n i d e B e t a m e t h a s o n e 1 7 - v a l e r a t e S o l u b i l i t y (mg./ml.) a t 2 5 °  D i s t i l l e d w a t e r 40% E t h a n o l 0.280 0 . 0 7 5 0 . 0 1 2 0 . 0 0 5 5.48 5 . 2 0 1.44 1.47 0 S o l u b i l i t y o f s t e r o i d s a s r e p o r t e d i n t h e l i t e r a t u r e H y d r o c o r t i s o n e D e s o n i d e T r i a m c i n a l o n e A c e t o n i d e B e t a m e t h a s o n e 1 7 - v a l e r a t e 0.280s 0 . 0 1 0 ' a T h e M e r c k I n d e x , e i g h t h e d . , 1968, p. 542. ^ M a l k i n s o n a n d Klrschenbaum,(1905)• 72 T a b l e 5 O c t a n o l / w a t e r and e t h e r / w a t e r p a r t i t i o n c o e f f i c i e n t s o f s e v e r a l s t e r o i d s Name o f S t e r o i d P C ( K | ) P C ( K ^ ) H y d r o c o r t i s o n e 2 8 . 8 1 . 6 Desonide 61.0 1 0 . 8 T r l a m c i n a l o n e a c e t o n i d e 8 8.4 14 . 6 Betamethasone 1 7 - v a l e r a t e 1 4 9 . 8 509.0 * E t h e r / w a t e r p a r t i t i o n c o e f f i c i e n t s r e p o r t e d by G . L . B l y n n ( 1 9 7 D . * * E t h e r / w a t e r p a r t i t i o n c o e f f i c i e n t t h e o r e t i c a l l y c a l c u l a t e d from t h e e q u a t i o n o f E l y n n ( 1 9 7 1 ) . D. R e l i a b i l i t y of Heat S e p a r a t i o n Method f o r t h e Removal of E p i d e r m i s from Autopsy S k i n The e p i d e r m i s was removed by a heat s e p a r a t i o n t e c h n i q u e . I n o r d e r t o check i f t h e e p i d e r m i s s e p a r a t e d by heat was dam-aged, t h e whole s k i n was exposed t o h e a t f o r e x a c t l y 30 s e c -onds a t 6 0 °c . Prom one a u t o p s y s k i n , dermatome s e c t i o n s o f about 0.1 mm. t h i c k n e s s were c u t and from t h e r e s t o f t h e a u t o p s y s k i n , t h e e p i d e r m i s was removed by h e a t . Sample s e c t i o n s were o b t a i n e d u s i n g a c r y o s t a t microtome. M i c r o -s c o p i c e x a m i n a t i o n of b o t h samples r e v e a l e d t h a t the e p i d e r -mal s h e e t s were not damaged by t h e heat s e p a r a t i o n t e c h n i q u e ( P i g . 1 5 ) • The iheat s e p a r a t i o n t e c h n i q u e i s v e r y s a f e and s a t i s f a c t o r y f o r removing t h e e p i d e r m i s from t h e a u t o p s y s k i n . L a r g e , u n r u p t u r e d , i n t a c t p i e c e s c o u l d be o b t a i n e d . E. T e s t s f o r I n t a c t n e s s o f S k i n w i t h T r i t i a t e d Water The i n t a c t n e s s o f t h e e p i d e r m a l s h e e t s was checked w i t h t r i t i a t e d w a t e r . Downes (1967) e t a l . d e t e r m i n e d t h e r a t e o f d i f f u s i o n o f t r i t i a t e d w ater t h r o u g h h a i r l e s s mice s k i n and a u t o p s y s k i n . H i s r e s u l t s showed marked v a r i a t i o n , r a n g i n g from 0.18 t o 1.08 mg/cm / h r . f o r h a i r l e s s mice s k i n Dermatome s e c t i o n o f human s k i n I 1 — " H e a t s e p a r a t e d e p i d e r m a l s h e e t f r o m human a u t o p s y s k i n F i g . 1 5 . Dermatome a n d h e a t s e p a r a t e d s e c t i o n s o f human e p i d e r m i s . 76 and 0.01 to O.69 mg/cm /hr. for human autopsy skin. Table 7 shows the rate of diffusion of t r i t i a t e d water through hair-less mice skin and human epidermis. The results obtained were very close to the reported values. Downes et a l com-pared the diffusion rates of tr i t i a t e d water from the dermal side with those of the epidermal side of the skin. Their results suggested that diffusion in either direction is ap-proximately equal. The var i a b i l i t y in the rate of diffusion of water existed not only among specimens of skin from d i f f e r -ent body regions of the same animal. Blank (1952), and Mali (1956) have also noted variation in water diffusion rates through different samples of human skin In vitro. Accurate measurements of the rate of diffusion of t r i t i -ated water through human epidermis can be made within a period of two hours. Moreover, the data obtained are highly repro-ducible. Our main object was to check the intactness of the epidermis. If our results of rate of diffusion of tr i t i a t e d water f e l l within the reported range, i t was assumed that the epidermal sheet under test was intact and could be used for steroid penetration studies. Table 6 Rate of d i f f u s i o n of t r i t i a t e d water through human abdominal autopsy skin  Specimen number Number of hours Rate of d i f f u s i o n and age (Years) a f t e r death (mg/cm /hour) # age 4 39 8 0 . 4 5 0 t 0 . 0 3 2 12 41 47 0 . 4 5 9 - 0 . 0 4 5 10 56 7 0 . 3 8 7 - 0 . 0 5 1 9 58 24 0.332 - 0 . 0 3 8 6 58 7 0 . 4 3 0 i 0 . 0 3 1 20 70 11 0 . 4 5 1 - 0.047 24 66 44 0 . 5 2 0 - 0 . 0 6 0 29 32 23 0 . 3 8 2 t 0 . 0 3 8 43 68 12 0 . 4 6 3 - 0 . 0 3 7 38 7 24 0 . 5 8 2 - 0.040 Mean (n = 4) - standard deviation. T a b l e 7 D i f f u s i o n o f t r i t i a t e d w a t e r 2 Rate of d i f f u s i o n (mg./cm / h r . ) Specimen # H a i r l e s s mice s k i n Human e p i d e r m i s 1 0 . 3 8 - 0 . 0 3 2 0 . 6 1 - 0 . 0 2 1 2 0.40 i 0.03.8 0 . 6 0 t 0.028 3 0 . 3 7 - 0.040 0 . 4 5 t 0 . 0 3 1 4 0.41 - 0 . 0 3 2 0 . 4 3 - 0 . 0 3 7 5 0 . 5 0 t 0.028 0 . 3 8 ± 0.040 6 O.36 - 0 . 0 2 5 0 . 4 4 ± 0 . 0 6 0 L i t e r a t u r e v a l u e s ; Rate o f d i f f u s i o n o f t r i t i a t e d w a t e r : (a) h a i r l e s s mice > w, 0.18 - 1.8, (b) a u t o p s y 0.01 - 0 . 7 0 . mg./cm2/hr. P e r m e a b i l i t y c o n s t a n t of t r i t i a t e d w a t e r t h r o u g h t h e - 3 human e p i d e r m i s as r e p o r t e d i n t h e l i t e r a t u r e : 0.1 - 0.5x10 C/w P e r m e a b i l i t y c o n s t a n t o f t r i t i a t e d w a t e r t h r o u g h human e p i -- 3 dermis as o b t a i n e d i n our e x p e r i m e n t s : 0.2 t o 0.45 x 10 o ~ The r a t e o f d i f f u s i o n o f t r i t i a t e d w a ter i s t h e mean o f f o u r d i f f u s i o n r e s u l t s from each specimen of s k i n , h a i r l e s s mice/human e p i d e r m i s . 79 S a m p l e c a l c u l a t i o n f o r m e a s u r i n g r a t e o f d i f f u s i o n o f t r i t i a t e d w a t e r t h r o u g h human, s k i n : 1 m i c r o c u r i e ( u c i ) = 2 .220 x 10 dpm E f f i c i e n c y o f t h e m a c h i n e f o r t r i t i u m , E = 4 9 . 1 7 $ F i g . o f m e r i t = ( E ) 2 = ( 4 9 . 1 7 ) 2 = 161.00 B 15 R a t e o f d i f f u s i o n o f w a t e r = C A x B A = 1 .0 cm 2 B = 69375 c o u n t s / m i n . / m g . C = 16085 R a t e o f d i f f u s i o n = 16085 = 0.23 mg/cm 2/hr. 1.0 x 69375 F. P e r c u t a n e o u s P e n e t r a t i o n o f S t e r o i d s The most i m p o r t a n t g o a l i n p e r c u t a n e o u s r e s e a r c h i s t o e x p r e s s a l l r e s u l t s i n t e r m s o f c o m p a r a b l e m e a s u r e m e n t s . T y p i c a l c u m u l a t i v e p e n e t r a t i o n c u r v e s f o r b e t a m e t h a s o n e 17-v a l e r a t e , d e s o n i d e a n d t r i a m c i n o l o n e a c e t o n i d e , h y d r o c o r t i -s o n e a r e shown i n F i g . 16 . 1 7 , 1 8 . 19 . T h e ^ p e n e t r a -t i o n c u r v e s show a n i n i t i a l r a p i d p e n e t r a t i o n o f s t e r o i d s . T h i s c o u l d be due t o t h e f a c t t h a t s h u n t d i f f u s i o n m i g h t be d o m i n a t i n g i n t h e i n i t i a l t r a n s i e n t s t a g e o f d i f f u s i o n . Penetration slows progressively with time, not quite reach-ing steady state penetration within 25 to 50 hours, the duration of our observations. It has been suggested that for drugs having the same intrinsic pharmacological activity, drug transport rates to active sites determine their rela-tive potency. By this hypothesis, one would.expect beta-methasone 17-valerate to have the fastest penetration rate, hydrocortisone the lowest, with desonide and triamcinolone acetonide giving intermediate penetration. In 25 hours, rather substantial amounts —1.5 to 3»5%—of the steroid penetrated the skin barrier. Tables 8, 9t 10 and graphs 16, 18, 19 show that desonide penetrated the skin,barrier faster than triamcinolone acetonide. The only structural difference between desonide and triamcinolone acetonide is the absence of a fluorine mole-cule in desonide. The strongly electronegative fluorine atom decreases the redox potential of the ketone-hydroxyl pair stabilizing the 11-betahydroxyl group and making triamcinolone acetonide more polar than desonide. In other words, the presence of fluorine at the 9o^ position in triamcinolone acetonide decreases i t s l i p i d solubility. Water solubility of triamcinolone acetonide is less than that of b l desonide (Table 4). This contradicts the theoretical as-sumption that triamcinolone acetonide Is more polar than desonide. One possible explanation of i t s lower water solu-b i l i t y is that an interaction between the C - l l hydroxyl group and an electron on the C-l double bond takes place, forming a reasonably stable six-membered ring structure. Another pos-s i b i l i t y is that an interaction occurs between the C - l l hydroxyl group and the C-20 carboxyl group, yielding a seven-membered ring. However, this is less l i k e l y to occur (Brown, personal communication). The penetration curves (Pig. 16, 18, 19) indicate a pro-gressive decrease in the permeability of steroids as they be-come increasingly polar. More polar and less mobile compounds have lower potencies when applied topically. Our results are in agreement with this. Our penetration curves show that topical activity could be Increased by reducing the polarity of the molecules. This effect has been attained most fre-quently by esterlflcation of hydroxyl groups at C-21 and C-17 or by linking the hydroxyl group at C-16 and C-17 with an acetonide bond (Schlagel, 1965). Desonide shares the a b i l i t y of triamcinolone to pene-trate epidermis because of the presence of the acetonide moiety. Our results show that of the four ant1-Inflammatory agents studied, betamethasone 17-valerate Is the most effective. f o l l o w e d by d e s o n i d e , t r i a m c i n o l o n e a c e t o n i d e and h y d r o -c o r t i s o n e r e s p e c t i v e l y . The main o b j e c t i v e o f t h e work was a c o m p a r a t i v e e v a l -u a t i o n of s t e r o i d s w i t h r e s p e c t t o t h e i r a b i l i t y t o p e n e t r a t e $h$ s k i n b a r r i e r . No emphasis was g i v e n t o t h e use of d i f f e r e n t v e h i c l e s . Sample c a l c u l a t i o n s : To c a l c u l a t e t h e c u m u l a t i v e d i f f u s i o n o f d r u g i n time i n t e r v a l , t : Say, i n t h e 1 s t hour, t h e sample gave an absorbance o f 0 . 0 2 , C = A/a = 0 . 0 2 = . 0 0 0 5 9 mg/ml (a i s c a l c u l a t e d from s t . c u r v e ) 3378" The sample drawn was 2 ml. c o n c e n t r a t i o n i n 2 m l , .00059 x 2 = 0.00118 The t o t a l volume o f l i q u i d i n t h e l o w e r chamber o f t h e c e l l was 8 ml. c o n c e n t r a t i o n i n 8 m l , 0 . 0 0 0 5 9 x 8 = .00472 mg/8 ml. But 2 ml. o f t h e sample was r e p l a c e d by f r e s h 2 ml. o f s a l i n e s o l u t i o n . T h e r e f o r e t h e d r u g l e f t i n the l o w e r chamber o f th e d i f f u s i o n c e l l , was 0.00472 - 0.0018 = 0 . 0 0 3 5 ^ mg. The second sample was drawn a t a d e f i n i t e i n t e r v a l and r e a d a b s o r b a n c e , Cone. A/a = 0 . 0 2 5 = . 0 0 0 7 3 mg/ml " 3 3 T 8 Cone, i n 2 m l , 0 . 0 0 7 3 x 2 = 0.00146 mg. 83 Cone, i n 8 m l , 0.0073 x 8 = 0.00584 mg. D r u g l e f t i n t h e l o w e r chamber o f t h e d i f f u s i o n c e l l 0.00584 -0.00146 =o.00438 mg. D r u g d i f f u s e d i n t h e 2nd h o u r w o u l d be 0.00584 - 0.00354 mg = 0.0023 mg. C u m u l a t i v e d i f f u s i o n i n t h e 2nd h o u r w o u l d b e : 0.00472 + 0.0023 = 0.00702 mg. o r 7.02 u g . G. V a s o c o n s t r i c t i o n B i o a s s a y C o r t i c o s t e r o i d s , a b o v e t h e i r t h r e s h o l d d o s e , c a u s e s k i n b l a n c h i n g due t o v a s o c o n s t r i c t i o n . A p p l i e d a t s e v e r a l c o n -c e n t r a t i o n s t h e t e s t g i v e s t h e t y p i c a l s i g m o i d d o s e - r e s p o n s e c u r v e o f a s t a n d a r d b i o a s s a y . U s i n g s t a t i s t i c a l t e c h n i q u e s w h i c h t r a n s f o r m t h e d o s e - r e s p o n s e c u r v e t o a s t r a i g h t l i n e , t h e ED^Q c a n be c a l c u l a t e d . When two a n t i - i n f l a m m a t o r y s t e r o i d s g i v e p a r a l l e l d o s e - r e s p o n s e c u r v e s , i t i s e v i d e n t t h a t t h e same m e c h a n i s m o f a c t i o n i s o p e r a t i v e ( G o l d s t e i n , 1968). The r a t i o o f t h e i r ED^Q 'S e x p r e s s e s t h e i r r e l a t i v e p o t e n c y . E x t r a p o l a t i o n t o ED<^ g i v e s a n e s t i m a t e o f t h e maximum d o s e b e y o n d w h i c h no g r e a t e r p h y s i o l o g i c a l r e s p o n s e c a n be a c h i e v e d . B o t h d e s o n i d e ( O— P ~ C 7 ) a n d t r i a m c i n o l o n e a c e t o n i d e ( A — A — A ) g&ve p r a c t i c a l l y c o i n c i d e n t d o s e - r e s p o n s e - 4 c u r v e s a n d t h e same E D ^ ( a p p r o x i m a t e l y , 2 x 10 %). The max-Table 8 Cumulative penetration of desonide and triamcinolone acetonide through human abdominal skin (epidermis) at room temperature.. The steroids were applied i n drug deposit form. Time i n t e r v a l Amount of drug penetrated (mg./cm x 1(H) Hours Desonide Triamcinolone acetonide 1 10.0 ± 0 .50 9.0 + 0 . 6 3 2 15.5 ± 0 .50 13.3 + 0 .50 3 21.7 - 0.63 16.0 + 0 . 5 0 5 2 5 . 0 t 0 .60 1928 + 0 . 5 0 10 3 2 . 2 - ± 0.60 26.5 + 0.60 15 40.0 t 0 .50 38.5 0 . 6 0 20 47 .2 t 0.48 42.5 + 0.48 25 52.7 - 0 . 5 0 47 .2 ± 0.48 30 63.5 -0.37 58.0 + 0.60 Mean (n = 10) - standard deviation P i g . 17. P e n e t r a t i o n o f d e s o n i d e a n d t r i a m c i n o l o n e a c e t o n i d e t h r o u g h human e p i d e r m i s ( a g e 58 y e a r s , 2k h o u r s a f t e r d e a t h ) . v v D e s o n i d e • T r i a m c i n o l o n e a c e t o n i d e T a b l e 9 C u m u l a t i v e p e n e t r a t i o n o f b e t a m e t h a s o n e 1 7 - v a l e r a t e . d e s o n i d e , t r i a m c i n o l o n e a c e t o n i d e , h y d r o c o r t i s o n e t h r o u g h human a b d o m i n a l s k i n ( e p i d e r m i s ) a t room t e m p e r a t u r e . The s t e r o i d s w e r e a p p l i e d i n 40$ e t h a n o l . 2 3 * Time i n t e r v a l Amount p e n e t r a t e d (mg./cm x 10 J) H o u r s B e t a m e t h a s o n e D e s o n i d e T r i a m c i n o l o n e H y d r o c o r t i s o n e 1 7 - v a l e r a t e a c e t o n i d e 1 7.4 + 0.44 6.9 t 0 . 5 0 6.6 + 0 . 6 0 4.8 ± 0 . 5 0 2 1U8 + 0 . 5 0 1 0 . 2 ± 0 . 6 1 8 .0 + 0.5 0 7.2 + 0 . 5 2 h 1 17.5 + 0.62 11.4 t 0.60 1 1 . 0 + 0.47 9.6 + 0.47 5 23.2 + 0.69 2 2 . 0 t 0 . 5 0 19.1 + 0.34 14.1 + 0.47 10 40.4 + 0.5.0 31.6 t 0.63 27.0 + 0.44 22.3 + 0.12 15 48.6 + 0 . 5 0 45.6 t 0 . 5 0 40.0 + 0 . 5 0 2 5 . 0 + 0 . 3 1 20 86.6 + 0.48 53.0 - 0 . 5 0 48.0 + 0.53 28.3 + 0 .62 25 65.O + 0.38 60.8 * 0.50 55.2 + 0 .60 3 0 . 0 + 0.40 30 80.0 + 0.40 74.3 - 0.48 68.2 + 0.49 38.0 + 0 . 3 9 * + Mean ( n = 10) - s t a n d a r d d e v i a t i o n . Table 10 Cumulative penetration of betamethasone 17-valerate, desonide, triamcinolone acetonide  and hydrocortisone through human abdominal skin (epidermis) at room temperature. The steroids were applied In hydrophlllc ointment base. Amount of drug penetrated (mg./cm x 10^) Time i n t e r v a l Hours Betamethasone Desonide Triamcinolone Hydrocortisone 17 - valerate acetonide 1 4 . 7 * 0 . 5 0 4 . 4 ± 0 . 3 3 3 . 8 t 0.40 2 . 0 * 0 . 6 3 2 7 . 4 1 0 . 3 1 6 . 7 ± 0 . 3 9 5 . 8 t 0 . 4 3 4 . 7 - 0.42 3 1 0 . 6 t 0 . 3 1 9 . 0 t 0 . 6 6 8.5 ± 0 . 4 4 6 . 2 ± 0 . 3 8 5 14.6 ± 0.42 + 1 3 . 1 - 0 . 5 9 1 2 . 8 t 0 . 5 0 9 . 3 4 0 . 4 3 10 2 5 . 5 1 0 . 6 0 2 3 . 0 t 0 . 5 0 2 1 . 0 - 0 . 5 0 1 3 . 4 ± 0 . 6 0 15 28.2 t 0 . 5 0 2 5 . 2 ± 0 . 5 0 24.0 * 0 . 5 0 18.0 t O.56 20 3 3 . 0 ± 0 . 7 3 3 1 . 0 - 0 . 5 6 2 5 . 5 - 0 . 6 0 2 2 . 0 ± 0 . 4 9 25 3 8 . 0 i O.56 3 5 . 0 ± 0.48 3 2 . 4 ± 0 . 4 3 2 5 . 4 t 0 . 5 0 30 5 0 . 0 ± 0 . 5 8 48.2 ± 0 . 6 1 ; 4 3 . 1 i 0 . 4 3 3 1 . 8 ± 0 . 5 0 Mean (n = 10) - standard deviation. 91 imum e f f e c t i v e n e s s o f t h e drugwa:s r e a c h e d a t d o s e s o f 0.01$ when a p p r o x i m a t e l y 90$ o f t h e s i t e s o f a p p l i c a t i o n v a s o c o n -s t r i c t e d . We c o n c l u d e t h a t 9 a l p h a - f l u o r o g r o u p h a s no e f f e c t o n t h e p o t e n c y o f t r i a m c i n o l o n e a c e t o n i d e . The r e m o v a l o f t h e g r o u p l e a v e s t h e p h y s i o l o g i c a l a c t i v i t y o f t h e m o l e c u l e u n -i m p a i r e d . P r o n o u n c e d v e h i c l e e f f e c t i s n o t i c e d o n l y a t t h e l o w e s t c o n c e n t r a t i o n s . T h i s may be due t o l a c k o f u n i f o r m d i s p e r s i o n u p o n d i l u t i o n . A t h i g h e r c o n c e n t r a t i o n s , 0.001$ a n d u p, v e h i c l e a n d d i l u t i o n e f f e c t s d i s a p p e a r . O i n t m e n t f o r m u l a t i o n s show n e a r l y t h e same e f f e c t i v e n e s s a s t h e s o l u -t i o n s . I t seems c l e a r , h o w e v e r , t h a t a t a c o n c e n t r a t i o n o f a p p r o x i m a t e l y 0.01$, d e s o n i d e a n d t r i a m c i n o l o n e a c e t o n i d e r e a c h t h e maximum i n t h e d o s e - r e s p o n s e c u r v e . The u s e o f h i g h e r c o n c e n t r a t i o n s w i l l n o t c a u s e a p p r e c i a b l y g r e a t e r numbers o f p e r s o n s t o r e s p o n d . T h u s , c o n c e n t r a t i o n s u s e d i n p r a c t i c e , 0.05$ f o r d e s o n i d e a n d 0.1$ f o r t r i a m c i n o l o n e a c e t o n i d e , r e p r e s e n t a 50 t o one h u n d r e d f o l d e x c e s s o v e r t h e maximum r e s p o n s e o b t a i n a b l e . H. C l i n i c a l S t u d i e s o f E f f e c t i v e n e s s o f D e s o n i d e The c l i n i c a l s t u d i e s o f d e s o n i d e w e re done b y one o f t h e T a b l e '11 V a s o c o n s t r i c t i o n p r o d u c e d b y T r i a m c i n o l o n e a c e t o n i d e a n d D e s o n i d e  C o n c e n t r a t i o n Number o f p a t i e n t s w i t h p o s i t i v e r e s p o n s e 0.0001$ 0.001$ D e s o n i d e 0.01% 0.1% S o l u t i o n 17A3 27/43 44/47 40/46 39.5$ 62.8$ 93.6$ 87.0$ Cream 6/43 28/43 40/46 35/46 14.0$ 65.0$ 87.0$ 76.0$ 0.0001$ 16/44 36.0$ 11/44 25.0$ 0.001$ 27/44 62.8$ 28/43 65.1$ T r i a m c i n o l o n e a c e t o n i d e 0.01$ 44/47 93.6$ 42/47 89.4$ 0.1$ 42/48 87.5$ 38/48 79.2$ 93 V A S O C O N S T R I C T I O N B I O A S S A Y O O m - 5 - 3 - 2 L O G C O N C (%) -1 n—-DESONIDE (SOLUTION) — " (CREAM) A —;TRIAMCINOLONE ACETONIDE (SOLUTION) - 4 E D = 2 X 1 0 % 5 0 . Pig. 20. Vasoconstriction bioassay, log concentration vs $ response 94 c o - i n v e s t I g a t o r s , D r . W.D. S t e w a r t , H e a d , D i v i s i o n o f Derma-t o l o g y , F a c u l t y o f M e d i c i n e , U.B.C., V a n c o u v e r 8, B.C. The c l i n i c a l e f f e c t i v e n e s s o f d e s o n i d e i n t h r e e s k i n d i s o r d e r s , c o n t a c t d e r m a t i t i s , a t o p i c d e r m a t i t i s a n d p s o r i a s i s , , was c o m p a r e d t o t h a t o f a n o t h e r p o t e n t f l u o r i n a t e d t o p i c a l c o r t i c o s t e r o i d , b e t a m e t h a s o n e 1 7 - v a l e r a t e , u s i n g S a l z b u r g e r ' s t e c h n i q u e (1946). T h i s t e c h n i q u e i n v o l v e s t h e c o m p a r i s o n o f two d r u g s b y use o f b i l a t e r a l l y s y m m e t r i c a l l e s i o n s , r a n d o m i z e d a n d d o u b l e b l i n d a p p l i c a t i o n a n d e v a l u a t i o n . C o m m e r c i a l l y s u p p l i e d c r e a m s o f d e s o n i d e 0.05$ a n d b e t a m e t h a s o n e 1 7 - v a l e r a t e 0.1$ were u s e d -t h e two c o n c e n t r a t i o n s most commonly p r e s c r i b e d i n p r a c t i c e . E a c h d r u g was a p p l i e d t w i c e d a i l y w i t h o u t o c c l u s i o n . P a t i e n t s w e r e e x a m i n e d w e e k l y f o r f o u r weeks f o r a n t i - i n f l a m m a t o r y a n d a n t i - p r u r i t l c a c t i v i t i e s o f e a c h d r u g . T a b l e 12, r e p r e s e n t s t h e r e s u l t s o b t a i n e d w i t h p a t i e n t s h a v i n g c o n t a c t d e r m a t i t i s a n d T a b l e 13,' shows t h e r e s u l t s °n o f a t o p i c d e r m a t i t i s . B o t h t h e t a b l e s show p e r c e n t a g e ? o f p a -t i e n t s w i t h a b e t t e r r e s p o n s e t o d e s o n i d e ^ a n d b e t t e r r e s p o n s e t o b e t a m e t h a s o n e 1 7 - v a l e r a t e anH e q u a l r e s p o n s e t o b o t h a n d t h o s e i n w h i c h n e i t h e r was e f f e c t i v e . T a b l e 14, shows t h e c o m p a r i s o n o f d e s o n i d e a n d b e t a -m e t h a s o n e 1 7 - v a l e r a t e i n p a t i e n t s w i t h p s o r i a s i s . The a b o v e Table 12 Week E f f e c t i v e n e s s of desonide 0.05% vs betamethasone 1 7-valerate 0.1? Contact D e r m a t i t i s T o t a l # P a t i e n t s Desonide Superi o r Betamethasone Superior Equal E f f e c t N e ither E f f e c t i v e F i r s t 48 32$ 33$ 27$ 8 Second 44 25 3 6 37 2 T h i r d 37 32 32 35 0 Fourth 29 28 38 34 0 T a b l e 13 E f f e c t i v e n e s s o f d e s o n i d e 0 . 0 5 $ v s B e t a m e t h a s o n e 1 7 - v a l e r a t e 0 . 1 $ A t o p i c D e r m a t i t i s Week T o t a l # P a t i e n t s D e s o n i d e S u p e r i o r B e t a m e t h a s o n e S u p e r i o r E q u a l E f f e c t N e i t h e r E f f e c t i v e F i r s t S e c o n d T h i r d F o u r t h 34 31 27 23 12$ 16 22 13 26$ 19 19 17 59$ 65 59 70 3$ 0 0 0 T a b l e 14 E f f e c t i v e n e s s o f d e s o n i d e 0 . 0 5 $ vs betamethasone 1 7 - v a l e r a t e 0 . 1 $ P s o r i a s i s Week T o t a l # P a t i e n t s Desonide S u p e r i o r Betamethasone S u p e r i o r E q u a l E f f e c t N e i t h e r E f f e c t i v e F i r s t Second T h i r d F o u r t h 35 31 30 30 14$ 10 10 10 26^ 35 33 37 43$ 39 34 33 17$ 16 23 20 98 T a b l e s 12 , 13 , Ik, , show t h a t d e s o n i d e i s i n t h e same r a n g e o f e f f e c t i v e n e s s a s b e t a m e t h a s o n e , a l t h o u g h p e r h a p s v e r y s l i g h t l y l e s s e f f e c t i v e i n s e l e c t e d s u b j e c t s . 9 9 VI SUMMARY AND CONCLUSION The vasoconstriction potency, penetration across human epidermis and the c l i n i c a l effectiveness of desonide, a new non-fluorinated c o r t i c o s t e r o i d have been compared to those of two fluorinated steroids, triamcinolone acetonide and betamethasone 17-valerate. A. An attempt to establish spectrophotofluorometric method for analysis of desonide was unsuccessful. This was due to the presence ofa'l6«C hydroxy1 group i n desonide, which provided unstable fluorescence. Therefore,all subsequent steroids were analyzed by a spectrophotometric method. B. S o l u b i l i t y of four steroidssnamely, hydrocortisone, triamcinolone acetonide, and betamethasone 17-valerate, was determined i n d i s t i l l e d water, normal saline and k0% ethanol. The r e l a t i v e s o l u b i l i t y of steroids i n water and i n k0% ethan-o l were of the following order: Hydrocortisone > desonide triamcinolone acetonide betamethasone 17-valerate. Octanol/water p a r t i t i o n c o e f f i c i e n t s of the above sa-M steroids were determined at 25°± 0.1. The values obtained experimentally were compared to ether/water p a r t i t i o n coef-f i c i e n t s reported i n the l i t e r a t u r e . A l i n e a r r e l a t i o n s h i p was obtained when octanol/water and ether/water p a r t i t i o n xuu c o e f f i c i e n t s were p l o t t e d vs cumulative p e n e t r a t i o n . Par-t i t i o n c o e f f i c i e n t of the s t e r o i d s followed the order i n -d i c a t e d by t h e i r r e l a t i v e s o l u b i l i t y . C. The p e n e t r a t i o n of hydrocortisone, t r i a m c i n o l o n e aceto-n i d e , desonide, and betamethasone 17-valerate were st u d i e d using human epidermal sheets i n Poulsen s k i n d i f f u s i o n c e l l 3 -Epidermal sheets were removed by heat se p a r a t i o n method. The i n t a c t n e s s of epidermal sheets was checked by comparing the p e r m e a b i l i t y constant of t r i t i a t e d water w i t h the r e -ported l i t e r a t u r e values. The order of p e n e t r a t i o n of s t e r o i d s were: Betamethasone 17-valerate > desonide > t r i a m c i n o l o n e aceto-n i c e > hydrocortisone. There seems to be a l i n e a r r e l a t i o n s h i p between pene-t r a t i o n and p a r t i t i o n c o e f f i c i e n t of s t e r o i d s . D. V a s o c o n s t r i c t i o n a c t i v i t y of desonide was compared to t r i a m c i n o l o n e acetonide. Both desonide and t r i a m c i n o l o n e acetonide gave c o i n c i d e n t dose-response curves and the same ED5o*' of 2 x 10" 6g/ml. E. C l i n i c a l e f f e c t i v e n e s s of desonide was compared to betamethasone 17-valerate In diseases l i k e contact derma-t i t i s , a t o p i c d e r m a t i t i s and p s o r i a s i s . Desonide was found i n the same range of e f f e c t i v e n e s s 101 as betamethasone 17-valerate although perhaps very slightly less effective in selected subjects. In conclusion, desonide has been demonstrated to have an effective degree of anti-inflammatory activity, skin pene-tration and substantial vasoconstrictive action. 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Forsch., 46:170, 1939. Wood, D.C.F. and Bettley, F.R.: The effect of various deter-gents on human epidermis, Brit. J. Derm., 84:321, 1971. Wolff, M.E. and Winston, H.O.s The steroid-receptor complex. Some considerations based on Sp - hybridized systems, J. Med. Chem., 2 s577. 1964. APPENDIX M a t e r i a l s P h a r m a c e u t i c a l s The p h a r m a c e u t i c a l s w e re u s e d a s r e c e i v e d f r o m t h e m a n u f a c t u r e r w i t h o u t f u r t h e r p u r i f i c a t i o n . H y d r o c o r t i s o n e . U.S.P. X V I I I , 11,1?,21-trihydroxy - 4 -p r e g n e n e - 3.20 - d i o n e . L o t #17.17k, M e r c k S h a r p a n d Dohme o f Ca n a d a L t d . , M o n t r e a l , C a n a d a . M o l . Wt. 362.5 M e l t i n g P o i n t ~215-220°C ( w i t h d e c o m p o s i t i o n ) S o l u b i l i t y i n w a t e r 280 ug/ml. A b s o r p t i o n max. 249 nm T r i a m c i n o l o n e A c e t o n i d e , 9 - f l u o r o - 16 oC h y d r o x y p r e d -n i s o l o n e 16,17 - a c e t o n i d e . Cod #20-282, L o t #L-902. E.R. S q u i b b a n d Sons L t d . , M o n t r e a l , C a n a d a . •CHoOH C=0 112 Mol. Wt. 434.4 Melting Point 274-278°c (with decomposition) Solubility in water 10-12 ug/ml. Absorption max. —242 nm Desonide (Tridesllon ) (Desfluorotriamcinolone acetonide), l6^?C?-hydroxyprednisolonet 16, 17 - acetonide. Code #CS-l-37. Dome Laboratories. Division of Miles Laboratory Inc., West Haven, Conn. 06516, U.S.A. CHgOH C = 0 0 Mol. Wt. >--394.5 Melting Point 274-275°C (with decomposition) Solubility in water 75.8 ug/ml. Absorption max.———242 nm Betamethasone 17-Valerate. N.F. XVII, 9 oL~ fluoro - 16 o{_-methylprednlsolone - 17 - valerate. Code #12301, Lot #RL-7/ll6. Schering Corporation Ltd., Pointe Claire, Quebec. P 113 M o l . Wt. 476.6 M e l t i n g P o i n t d e c o m p o s i t i o n S o l u b i l i t y i n w a t e r 5»5 u g / m l . A b s o r p t i o n max. 242 nm T r i t i a t e d W a t e r (5 C i / m l . ) TRS - 1, c a t a l o g 70/71. A m e r s h a m / S e a r l e , 2636 C l e a r b r o o k D r i v e , A r l i n g t o n , I l l i n o i s . H y d r o p h i l i c O i n t m e n t , U.S.P. X V I I I was p r e p a r e d i n t h i s l a b o r a t o r y . P r e c a u t i o n s w e r e t a k e n t o p r e v e n t w a t e r l o s s d u r i n g t h e p r e p a r a t i o n o f t h e o i n t m e n t , b e c a u s e l o s s o f e v e n s m a l l a m o u n t s o f w a t e r a f f e c t s t h e c o n s i s t e n c y o f t h e o i n t -ment. The c o m p o s i t i o n o f t h e o i n t m e n t i s a s f o l l o w s : M e t h y l p a r a b e n 0.25 gnu P r o p y l p a r a b e n 0.15 gm. S o d i u m L a u r y l S u l f a t e 10.0 gm. P r o p y l e n e g l y c o l 120.0 gm. S t e a r y l a l c o h o l 250.0 gm. W h i t e s o f t p a r a f f i n 250.0 gm. P u r i f i e d w a t e r 370.0 gm. To make a b o u t 1000 gm. The f i n i s h e d o i n t m e n t was p a s s e d t h r o u g h t h e P a s c a l l o i n t -ment m i l l t o a s s u r e b a t c h u n i f o r m i t y i n t e x t u r e a n d c o n -s i s t e n c y . S t e r o i d s I n H y d r o p h i l i c O i n t m e n t 1$, h y d r o c o r t i s o n e , d i e s o n i d e , t r i a m c i n o l o n e a c e t o n i d e a n d b e t a m e t h a s o n e 1 7 - v a l e r a t e w e r e made i n H y d r o p h i l i c o i n t -114 merit base. These were used f o r p e n e t r a t i o n s t u d i e s a c r o s s t h e human e p i d e r m i s . 0.1$ s t o c k o f t h e d e s o n i d e and t r i a m c i n o l o n e a c e t o n i d e were a l s o made i n t h e same way d e s c r i b e d above f o r v a s o c o n -s t r i c t i o n s t u d i e s . S t e r o i d s i n 40$ Eithanol From t h e s o l u b i l i t y s t u d i e s o f s t e r o i d s i n 40$ e t h a n o l , h y d r o c o r t i s o n e , t r i a m c i n o l o n e a c e t o n i d e , d e s o n i d e and b e t a -methasone 1 7 - v a l e r a t e , each were weighed 14.0 mg, added 4.0 ml. of 100$ e t h a n o l and t h o r o u g h l y a g i t a t e d , added d i s t i l l e d w a t e r s l o w l y and made up t h e volume t o 10 ml. The s o l u t i o n s were f i l t e r e d t h r o u g h (m i l l i p o r e f i l t e r s . S o l v e n t s a n d R e a g e n t s S o l v e n t s a n d r e a g e n t s w e r e s e l e c t e d f o r minimum i m -p u r i t i e s . T h e y w e r e f o u n d t o be g e n e r a l l y o f a c c e p t a b l e q u a l i t y . R o u t i n e p u r i f i c a t i o n was n e c e s s a r y o n l y f o r t h e e t h y l a l c o h o l . E t h y l a l c o h o l , 100$, was p u r i f i e d b y r e d i s t i l l a t i o n o v e r 2 , 4 - d i n i t r o p h e n y l h y d r a z i n e ( S w e a t , 195*0 t o remove a l d e -h y d e s a n d k e t o n e s . The e t h y l a l c o h o l c o n t a i n i n g 5 grams o f 2, 4 - d i n i t r o p h e n y l h y d r a z i n e a n d 10 m l o f h y d r o c h l o r i c a c i d p e r 1000 m l was r e f l u x e d f o r f o u r t o e i g h t h o u r s i n a vacuum j a c k e t e d a l l - g l a s s u n l u b r i c a t e d p a c k e d d i s t i l l a t i o n c o l u m n w i t h CORAD h e a d . I t was t h e n d i s t i l l e d , t h e f i r s t a n d l a s t 20$ b e i n g d i s c a r d e d , t h e n r e d i s t i l l e d , a g a i n d i s c a r d i n g t h e f i r s t a n d l a s t 2 0 $ . The a l c o h o l was s t o r e d i n 100 m l w e l l f i l l e d a l u m i n u m f o i l c a p p e d b o t t l e s a t 2°C t o m i n i m i z e r e -f o r m a t i o n o f a l d e h y d e s . S u l f u r i c A c i d ( A l l i e d C h e m i c a l o f C a n a d a , L t d . , M o n t r e a l , Q u ebec) was f o u n d - t o v a r y i n f l u o r e s c e n c e i m p u r i t i e s f r o m l o t t o l o t . S c r e e n i n g was d one t o e l i m i n a t e u n s a t i s f a c t o r y l o t s , b y c h e c k i n g t h e i r f l u o r e s c e n c e . F l u o r e s c e n c e r e a g e n t , c o n s i s t i n g o f 85 p a r t s o f s u l f u r i c a c i d a n d 15 p a r t s o f e t h y l a l c o h o l by w e i g h t was p r e p a r e d a s f o l l o w s : 30 grams o f c o l d d i s t i l l e d e t h y l a l c o h o l was 116 weighed into a tared glass stoppered flask packed in ice. About 90 ml. of cold sulfuric acid was added slowly with constant agitation so that the solution remained cool. The solution was made up to 200 grams with cold acid. This reagent was prepared freshly just before use. Its fluorescence characteristics were reproducible from day to day so long as the same batch of acid was used. Preparation by weight was adopted because i t was found that the method used by Mattingly (1962) calling for slow addition of 7 volumes of concentrated acid to 3 volumes of ethyl alcohol while cooling under the cold water tap, gave less reproduc-ible results. This was attributed to the inherent lack of accuracy in the volumetric measurement of a viscous liquid such as sulfuric acid, and to insufficient cooling. This reagent for practical purposes Is 70% acid by volume. Methylene Chloride (Matheson, Colemaniand Bell, Norwood, Ohio) was used as received from the manufacturer. This solvent is reported by the manufacturer to have a f l u -orescence of 0.3 parts per b i l l i o n as quinine base. 2 . 5 - diphenyloxazole (PPO) 1,4 -bis Q2-(5-phenyloxazole)^ benzene (P0P0P) Naphthalene, recrystallized 117 Dioxane, analytical grade Gctanol 1 (primary) Hydroxylamine hydrochloride Formula of S c i n t i l l a t i o n f l u i d (Downes, 1967) PPO 4.0 gm. POPOP 25.0 mg. Naphthalene 50«0 gm» Dioxane to make 1000.0 ml. 

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