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

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CYCLOSPORINE - OCULAR ABSORPTION,  PHARMACOKINETICS  & EFFECTS  ON U V E I T I S By GURSHARAN SINGH KALSI B.Sc,  The U n i v e r s i t y  of  B r i t i s h Columbia,  1983  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS  FOR THE DEGREE OF  MASTER OF SCIENCE  in THE FACULTY OF GRADUATE STUDIES (Department  of  Pathology,  We a c c e p t t h i s to  the  thesis  required  THE UNIVERSITY  Faculty of as  Medicine)  conforming  standard  OF BRITISH COLUMBIA  September,1986 (c)  Gursharan Singh K a l s i ,  1986  In presenting  this thesis in partial fulfilment  of the  requirements for an advanced  degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department  or  by  his  or  her  representatives.  It  is  understood  that  copying  or  publication of this thesis for financial gain shall not be allowed without my written permission.  Department of  PATHOLOGY  The University of British Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date  DE-6(3/81)  SEPTEMBER 2 5 ,  1986  ABSTRACT Inflammatory blindness  ocular  and u v e i t i s  disease  accounts  is  an i m p o r t a n t c a u s e  for  1.0%  of  blind  of  patients  i in  Canada.  treat, and  T h i s d i s e a s e c a n be p a r t i c u l a r l y t r o u b l e s o m e  because  mechanisms  the of  and  uveitis, locally use  the  causal  are u s u a l l y  they  with  may p r o d u c e  some  or  factors  unknown.  to  these  drugs  success  serious  and e l s e w h e r e i n t h e b o d y .  tolerance  factor  a n t i - i n f l a m m a t o r y agents have been  systemically  but  2 - 8  of  progression  Non-specific orally  nature  9 , 1 2  may  side  '  to  develop,  treat  effects  With  1 4  to  used  both  prolonged  making  them  ineffective. Recently  a  Cyclosporine  powerful  (Cy) ,  used  immunosuppressive  orally  treatment  of  uveitis  has  shown  However,  its  routine  use  is  therapeutic  index  Several injection ocular  studies  1 3 , 1 4  0  -  toxicity.  2  4  shown  in  Therefore,  subconjunctival ly  it  1 9  f  4 2  that  /  if  Cy  a  agents  p r o t o c o l  for  the ii  '  2 8  narrow  were  enhanced  pharmacological systemic  administered  to a v o i d the  side  and s y s t e m i c r o u t e s ,  a t t h e same t i m e p r o v i d e h i g h e r l e v e l s o f C y t o t h e A  1 6 - 1 9  subconjunctival  associated  m i g h t be p o s s i b l e  a s s o c i a t e d with the o r a l  of  the  4 3  a traditional the  in  results.  because  antineoplastic  and c i r c u m v e n t e d  effects.  effects  2  systemically  promising  limited  have  a number o f  absorption  sanctuary, side  of  and r e n a l  and  agent,  a d m i n i s t r a t i o n  and  eye. of  Cy  subconjunctivally  rabbits,  to  following  was  study  developed  toxicity,  in  New  ocular  Zealand  white  pharmacokinetics  equidose administration s u b c o n j u n c t i v a l l y  systemically  and t h e  effects  and  o f C y on a n a n i m a l m o d e l  of  uveitis. Subconjuntival (Sandimmune the  I.V.( ) R  50  mg/ml)  maximum t o l e r a t e d  s u p e r i o r to  minimizing  showed  that  response the  dose  intravenous  while  Cy was  and t h e  by  the  the  was  in  eye,  for ocular The  reducing  O.lcc to  be  and  was  penetration  uveitis  the  application  Cy i n  found  rabbits*  exposure.  effective  earlier  5mg o f  weekly  injection  systemic  of  of  model  inflammatory Cy t h e  milder  uveitis. The  local  results  from o u r s t u d y  administration  Cy a b s o r p t i o n  ocular  of  support  Cy w o u l d  and c i r c u m v e n t  administration. in  administration  the  disease.  contention  lead  to  side  effects  T h i s may f a c i l i t a t e  inflammatory  the  the  higher of  that  levels  of  systemic  r o u t i n e use  o f Cy  Contents Abstract List  of  ii Tables  vi  L i s t of  Figures  vii  List  Abbreviations  x  of  Acknowlegdements  xi  Introduction  1  C h a p t e r 1: O c u l a r t o x i c i t y study of possible vehicles for cyclosporine  5  Introduction Results Discussion Conclusions C h a p t e r 2:  Dose d e t e r m i n a t i o n  study  26  Introduction Results Discussion Conclusions C h a p t e r 3: P h a r m a c o k i n e t i c s o f c y c l o s p o r i n e following subconjunctival versus intravenous administration  48  Introduction Results Discussion Conclusions C h a p t e r 4: U v e i t i s  study  77  Introduction Results Discussion Conclusions Summary  100  Bibilography  102  iv  Appendix I : E f f e c t s o f m u l t i p l e dose s u b c o n j u n c t i v a l a n t i n e o p l a s t i c s on a c u t e immune u v e i t i s  Appendix I I : Combined l o c a l for spontaneously occurring anterior u v e i t i s i n cats.  chemotherapy  List  of  Tables  Table I. Grading system used t o assess o c u l a r i n f l a m m a t i o n due t o t h e v e h i c l e s I I . G r a d i n g system used t o assess o c u l a r i n f l a m m a t i o n due t o c y c l o s p o r i n e  III.Performance  data  I V . B i o a v a i l a b i l i t y of i n o c u l a r humors  on methods  1 &2  cyclosporine  V . G r a d i n g system used t o assess o c u l a r inflammatory response i n d u c e d b y b o v i n e serum a l b u m i n  List  of  Figures  Figure  Page  1.0 O c u l a r i n f l a m m a t o r y r e s p o n s e following subconjunctival i n j e c t i o n of v e h i c l e s .  12  1.1 R e p r e s e n t a t i v e p h o t o g r a p h s o c u l a r inflammatory response to a t o n e , f o u r and s e v e n d a y s .  of the each v e h i c l e 13  1.2 R e p r e s e n t a t i v e p h o t o g r a p h s o f the o c u l a r inflammatory response to each v e h i c l e a t one, f o u r and s e v e n d a y s C o n t ' d .  14  1.3 H i s t o l o g i c a l a t one week.  effects  15  1.4 H i s t o l o g i c a l a t one week.  effects  1.5 H i s t o l o g i c a l a t one week.  e f f e c t s of o l i v e  1.6 H i s t o l o g i c a l a t one week.  effects  of castor  oil  of c o t t o n seed  oil 16  oil 17  of peanut  oil 18  1.7 H i s t o l o g i c a l e f f e c t s g l y c o l a t one week.  of  1.8 H i s t o l o g i c a l a t one week.  o f sesame s e e d  effects  polyethylene 19 oil 20  2.0 O c u l a r i n f l a m m a t o r y response to d i f f e r e n t doses of c y c l o s p o r i n e subconjuctivally.  32  2 . 1 E f f e c t s o f 5 mg i n O . l c c a n d 10 mg i n 0 . 2 c c o f c y c l o s p o r i n e a t one, f o u r and s e v e n d a y s .  33  2 . 2 E f f e c t s o f 25 mg i n 0 . 5 c c o f cyclosporine a t one, f o u r and s e v e n d a y s and 5 mg i n O . l c c o n c e p e r week o f c y c l o s p o r i n e f o r two months.  34  2.3 H i s t o l o g i c a l a t one week.  effects  35  2.4 H i s t o l o g i c a l a t one week.  effects  2.5 H i s t o l o g i c a l a t one week.  effects  of  of  5 mg i n  10 mg i n  O.lcc  0.2cc 36  of  25 mg i n  0.2cc 37  vii  2 . 6 H i s t o l o g i c a l e f f e c t s o f 5 mg i n O . l c c o f c y c l o s p o r i n e o n c e p e r week i n a r a b b i t t h a t r e c i e v e d t h i s d o s e f o r two months.  38  2.7a H i s t o l o g i c a l e f f e c t s of a subconjunctival i n j e c t i o n o f 0 . 5 c c o f p e a n u t o i l , c o n t r o l , (A) and 5 mg of Cy i n 0.5cc of peanut o i l , experimental,(B) at 2 hours.  42  2.7b H i s t o l o g i c a l e f f e c t s of a s u b c o n j u n c t i v a l i n j e c t i o n o f 0 . 5 c c o f p e a n u t o i l , c o n t r o l , (A) and 5 mg o f Cy i n 0.5cc of peanut o i l , experimental,(B) at 4 hours.  43  2.7c H i s t o l o g i c a l e f f e c t s of a subconjunctival i n j e c t i o n o f 0 . 5 c c o f p e a n u t o i l , c o n t r o l , (A) and 5 mg o f Cy i n 0.5cc of peanut o i l , e x p e r i m e n t a l , ( B ) a t 12 h o u r s .  44  2.7d H i s t o l o g i c a l e f f e c t s of a subconjunctival i n j e c t i o n o f 0 . 5 c c o f p e a n u t o i l , c o n t r o l , (A) and 5 mg o f Cy i n 0.5cc of peanut o i l , e x p e r i m e n t a l , ( B ) a t 1 week.  45  3.0  A typical  chromatogram o f  a q u e o u s humor.  61  3.1  A typical  chromatogram o f  vitreous  62  3.2  A typical  chromatogram o f  urine.  63  3.3  A typical  chromatogram o f  blood.  64  3.4 S t a n d a r d c u r v e s o f u s i n g methods 1 & 2.  humor.  cyclosporine 65  3.5 L e v e l s o f cyclosporine i n t h e o c u l a r chambers o f t h e  eye.  66  3.6 B l o o d l e v e l s o f c y c l o s p o r i n e i n t h e r a b b i t a t a 5 mg i n O . l c c d o s e .  67  3.7 C u m m u l a t i v e u r i n e e l i m i n a t i o n c u r v e f o r c y c l o s p o r i n e i n the r a b b i t g i v e n a 5 mg i n O . l c c s y s t e m i c d o s e .  68  3.8  70  Structure  of  cyclosporine.  4.0 T h e e f f e c t o f s u b c o n j u n c t i v a l on u v e i t i s i n d u c e d b y b o v i n e serum a l b u m i n (BSA).  viii  cyclosporine 87  4.1 R e p r e s e n t a t i v e p h o t o g r a p h s o f the o c u l a r inflammatory response i n d u c e d by BSA and t h e e f f e c t s o f cyclosporine administered subconjunctival l y .  88  4.2 H i s t o l o g i c a l e f f e c t s o f i n d u c e d b y BSA i n G r o u p 1.  89  uveitis  4.3 H i s t o l o g i c a l e f f e c t s o f u v e i t i s i n d u c e d b y BSA i n G r o u p 1 C o n t ' d .  90  4.4 H i s t o l o g i c a l e f f e c t s o f c y c l o s p o r i n e on u v e i t i s i n d u c e d b y BSA i n G r o u p 2.  91  4.5 H i s t o l o g i c a l e f f e c t s of c y c l o s p o r i n e on u v e i t i s i n d u c e d b y BSA i n G r o u p 3.  92  ix  List BSA  : bovine  C :  cornea  Abbreviations  serum a l b u m i n  CE : c o n j u n c t i v a l CH :  choroid  Cy  cyclosporine  :  of  epithelium  CyC  : dihydrocyclosporin C  CyD  : cyclosporin D  EAU : e x p e r i m e n t a l EAU-BSA EAU-S-Ag FZ :  iris  L  :  lens : lamina  M -  muscle  zone o f  ON : o p t i c  area  nerve  polymorphonuclear  granulocyte  retina  RD SCL SF -  inflammation  propria  NA : n e c r o t i c  R -  uveitis  : e x p e r i m e n t a l autoimmune r e t i n a l S-Antigen  LP  PMN -  uveitis  : e x p e r i m e n t a l autoimmune b o v i n e serum a l b u m i n  : focal  I  autoimmune  retinal  detachment  sclera subretinal  fluid  x  induced  uveitis  induced  by by  Acknowlegdements  I would p a r t i c u l a r l y l i k e Gedy  Gudauskas  obstacles positive I  and  for  delays,  conclusion  would  guidance,  their  of  also  this  and t e a c h i n g  for  D r . J a c k Rootman and D r .  guidance, their  patience  faith  in  through  the  eventual  research.  to  of  thank  expert  and  like  to  thank  the  Dr.  skills  Nick  Bussanich  necessary  to  for  complete  the this  project. Thanks Campbell,  and  throughout To  also  the  Dr.  assistance  the  supervisory  Rollins  for  their  committee  Drs.  constructive  Autor,  suggestions  research.  Peter i n the  I would research  to  Dolman s p e c i a l histopathology  also  like  and Sandoz  to  thanks  for  and r e v i e w  thank M.R.C.  L t d . , for generously  for  the  many h o u r s  of  o f my t h e s i s . the  funding of  s u p p l y i n g the  this  Sandimmune  I.V. Thanks figures,  finally  to  my  my s i s t e r K a m a l j i t  Jasvinder  a n d my m o t h e r  father  Deep  Singh  for  drawing  the  f o r t y p i n g my t h e s i s a n d my b r o t h e r  Harbhajan  research.  xi  for their  support  d u r i n g my  INTRODUCTION Ocular  i n f l a m m a t o r y d i s e a s e c o n s t i t u t e s one o f t h e  important  causes  particular, Canada. and in  blindness  accounts  Uveitis  1  its  of  for  is  1.0%  exact e t i o l o g y  once t h e  disease  spite  treatment  specific  is  4 3  ,  may  directed  ultimately  to  the  of  '  all  for  traditional and  known • 'SO  end r e s u l t s  ocular  local  phenylbutazone,  and  in  and s y s t e m i c  the  eye  of  may  chronic  in uveitis and  are  cystoid  acuity.  application  administration such  such  In a  inflammatory disease  systemic  agents  ),  modalities  effects  l o s s of v i s u a l  agents  5 , 6  is  on an e m p i r i c  atrophic retina,  anti-inflammatory  immunosupprressive  Even  •  frequently  secondary  systemic  3 , 4  nature  Rheumatoid A r t h r i t i s ^  inflammation  media,  current therapy  corticosteroids,  non-specific  causation.  Often the  opacified  human c o n s i s t s  to  the  m a c u l a r edema w i t h e v e n t u a l The  in  anti-inflammatory agents.  the  inflammatory disease. cataracts,  patients  blind  i n f l a m m a t i o n may p e r s i s t  patients  control succumb  in  cause of u v e i t i s  disease is  number o f to  a  Uveitis,  •  established  with non-specific  fail  of  Juvenile  Treatment of t h i s  significant  the  •  Herpes S i m p l e x  basis,  people.  eludes c l i n i c a l investigators.  •  of  of  frequently  i n s t a n c e s where t h e  (eg.  in  most  as  as  of  in of  alternate  indomethacin,  administration azathioprine,  7  of and  cyclophosphamide. Although these in  the  treatment  of  agents have ocular 1  been used  with  some  inflammatory disease,  success  they  have  not  been  without  topical  a p p l i c a t i o n of  systemic  1 4  administration marrow  of  complications populations.  resulted is  secondary  other  another  tried  One o f t h e displays affect system. Cy  1 5  as  a possible  prevent  little  '  can  systemic  cause  and  bone  specific c e l l  the  Cyclosporine  candidate 1  6  ,  1  rejection  7  ,  1  9  ,  3  i n the  myelotoxicity  phagocytic  cells  (Cy),  treatment  Currently  0  of transplanted of  and i t  Cy  of  Cy  is  organs.  4 8  is  does not  that  it  adversely  of the mononuclear  phagocyte  5 6  causes immunosuppression d i r e c t e d  mediated  immunity.  mechanism achieved  with  proliferating  intriguing characteristics  the  while  Cushing's-like  tumors  immunosuppressive,  inflammatory d i s e a s e .  employed t o  in  which  normally  i n d u c e d by  1 0 , 1 1 , 1 4  associated  immunosuppressives  on  glaucoma  1 2 , 1 4 , 1 5  Recently  ocular  risk  suppression,  been  has  A greater  1 5  such as  corticosteroids,  administration  symptoms. '  has  complications  is by  not  5 6  Although  well  the  understood,  inhibition  of  r e l e a s e , and  inhibition  interleukin-2  by p r e c u s o r  of  towards  precise the  Cytotoxic  biochemical  effect  interleukin-2 receptor  T-cell-  may  be  synthesis  or  acquisition  T-cells,  as  diminished responsiveness of T - h e l p e r c e l l s to  well  for as  a  interleukin-  3^56,58,60 The response shown  effect is  retinal  of  Cy on s u p p r e s s i n g  T-cel1-mediated  particularly interesting, S-antigen  can  cause  because  experimental  immune  i t . has  been  autoimmune  uveitis  (EAU) i n a n i m a l s t h a t  ocular  inflammation  In view of for the  seen  this,  treatment  in  resembles the  T-cel1-mediated  humans.  Cy h a s b e e n g i v e n  systemically(I.M.) and i t  has  a l s o been employed o r a l l y i n t h e t r e a t m e n t  of patients  with  uveitis  however,  with  toxicity day.  encouraging  If  1 7 , 4 2 , 4 3  the  of  now,  side  of  while  effects  the  side  achieving  Local  absorption  subconjunctival  Using of  Cy,  we  the  be  applications;  use Cy  Cy  in  ocular  w o u l d be  within  of  one  the  eye  effects.  significantly  that  enhanced  t h e s e a g e n t s when compared  associated  subconjunctival vehicle  following  making i t  toxicities  route and  possible of  by to to  systemic  develop  f o r the drug  administration  toxicity,  ocular  equidose subconjunctival  administration,  subconjunctivally to  of  controlled  2 0 - 2 4  pharmacokinetics  uveitis  could  evaluated  intravenous  levels  administration,  the  administration.  a p p l i c a t i o n of  10 m g / k g p e r  be  its  renal  shown w i t h a n u m b e r o f a g e n t s  injection  systemic  circumvent  Cy c o u l d  limit  systemic side  Our l a b o r a t o r y has  equidose  of  effects  therapeutic  a v o i d i n g the  ocular  low dose of  i t may p r o v i d e w i d e c l i n i c a l  inflammatory disease. way  2 8 , 3 0 , 3 3  results;  occured at a r e l a t i v e l y  or circumvented, as  o f EAU w i t h s u c c e s s ,  administered a protocol  and  Cy on for the  the an  effects  animal  model  and of of  a d m i n i s t r a t i o n o f Cy  subconjunctivally. We  found  that  the  commercially  3  prepared  Cy i n  the  vehicle  Cremophor ( / E L p r o d u c e d l e s s  reaction the  and t h e  maximum  c o m m e r c i a l Cy  was  the pharmacokinetic that eye  significantly  equidose  study  in  subconjunctival l y The u v e i t i s  reduce  treatment  the  the  w i t h i n the  (experimental  time  greatly  study  systemic  side  effects  subconjunctivally the  treatment  the  of  a  possible  to  an  received  Cy  levels.  uveitis Cy  and t h e  induced was  able  earlier  the  uveitis.  making  candidate  Cy we were and a t  of  the  Cy for  the  able same  associated  administered routine  o c u l a r inflammatory disease.  4  the  the  showed t h a t  possibility Cy,  show  important,  recommended  concentrations  of  of  From  entered  that  administration of  ocular  reduce  eye  to  compared  Equally  inflammatory response  higher  Cy  autoimmune  w i t h Cy t h e m i l d e r t h e  achieve  able  of  rabbits,  With s u b c o n j u n c t i v a l to  by the  were  levels  injection.  were  dose  injection  by b o v i n e serum a l b u m i n ) to  we  higher  intravenous levels  tolerable  an i n f l a m m a t o r y  5 mg i n O . l c c o n c e p e r w e e k .  by s u b c o n j u n c t i v a l  blood  of  R  use  in  CHAPTER 1 O c u l a r t o x c i t y study of p o s s i b l e for  vehicles  eye1osporine  Introduction Cyclosporine been  used  treat also  disease,  '  resulted  Cy  significantly  order  to  when  treatment  resistant  toxicity. is  exact  a  vehicle  to  vehicle  are that  4 2  '  by  seed  oils  as  castor,  the  for  well  Cy  of  vehicle  Cy  '  produce l i t t l e  as  seed,  b o t h h y d r o p h o b i c and h y d r o p h i l i c  5  side  shown  route. be  to  were  In  2 0 - 2 4  injected  had t o  which  be  found  governed limited  requirements  for  by the the  o c u l a r r e a c t i o n and t h a t The v e h i c l e s  olive,  polyethylene  this  been  to  an a p p r o p r i a t e v e h i c l e for this  to  subconjunctival  systemic  formulation  cotton  conventional  locally,  local  be r e a d i l y a b s o r b e d by t h e e y e .  t e s t e d were  ocular  4 3  an o i l - b a s e d medium. O t h e r it  to  a b s o r p t i o n has  increased  chemistry  chronic  has  administration route  administered  Ocular  Cy. The r e q u i r e m e n t s lipophilic  of  agent,  c o n c e n t r a t i o n o f Cy n e e d e d  compared t o  subconjunctival ly,  the  the  w h i c h was  in renal  may be a v o i d e d .  injection,  it  in  immunosuppressive  the o c u l a r inflammation v i a t h i s  effect  for  an  However, the  Therefore,if  be  ,  systemically  inflammatory therapy.  (Cy)  peanut,  glycol  (PEG),  properties.  that  were  and sesame which  has  M a t e r i a l s & Methods P r i o r to study the  was  s t a r t i n g the  examined by  presence  five  of  rabbits  females  2.2  subconjunctivally,  attached  to  parallel  and  right  eye,  2.4  by  tuberculin posterior  f i l t r a t i o n filter  unit)  to  the  a  was  second  group peanut o i l , group  the  sesame seed o i l ,  and  inflammation presented  each  graded  grouping  injected  gauge  needle  shallow  angle  limbus  needle  of  injected.  0.22  micron  The  opposite  each r e c e i v e d  group c o t t o n  0.5  rabbit  the  third  f o u r t h group o l i v e  oil,  the  fifth  and t h e  sixth  group PEG. The eyes  b i o m i c r o s c o p y e a c h day f o r  photographed.  The  degree  examiners  of  different  6  average  evaluating  experiment.  2 other the  of  oil,  i n w h i c h an o v e r a l l  in this  cc  seed  i n e a c h e y e was g r a d e d a c c o r d i n g t o t h e I,  the  vehicle  5 rabbits,  serially  in Table  by  a  white  cc of a s t e r i l i z e d  slowly  i n f l a m m a t i o n was o b t a i n e d a f t e r for  at  25  Millex-FG(R)  were t h e n e x a m i n e d by s i i t - l a m p week,  a  of  inserted  group of the  out  was  The  oil,  rule  anaesthesia  superior  a non-treated control.  first  were  placing  syringe  throught  the  S i x groups  topical  s e r v e d as  castor  one  kg)  following  c o n j u n c t i v a a n d 0.5  disposable eye  -  (0.5%),  in  s u p p l i e d New Z e a l a n d  a n d w i t h a smooth m o t i o n t h e  under the (by  a  ocular disease.  ( locally  weighing  HC1  each r a b b i t  s i i t - l a m p biomicroscopy to  pre-existing  each  (proparacaine  experiments,  scheme  value  who h a d n o k n o w l e d g e No  of  each c r i t e r i o n  The r a b b i t s were  rabbits.  of  of  also the  significant  variability shown).  was  The groups  pentobarbital  in  sectioned  among  were  after  enucleated, fixed  found  the  examiners  sacrificed one  with  week.  The  (data  anoverdose globes  10%  buffered  vertically  Histologic  formalin,  and s t a i n e d  embedded by  in  were  paraffin,  Haematoxylin-Eosin  study.  assesment  of  density  and  area  inflammatory  i n f i l t r a t e was made a n d a n o v e r a l l  value  inflammation  of  assessments  obtained.  were made by m y s e l f  no k n o w l e d g e  of  significant  examiners  of  marked at the p o i n t of i n j e c t i o n w i t h a dye,  for histopathological  No  not  (data  the  not  shown).  7  average  histologic  and a p a t h o l o g i s t  grouping of  variability  The  the  was  rabbits noted  of  i n the between  who had study. the  Table I Conjunctiva Hyperemia Chemosis  Ocular  Erosion of  Discharge  Epithelium  none  none  none  none  trace  trace  trace  trace  mild  mild  mild  moderate  moderate  severe  severe  % Bleb present 0  Corneal Clarity  Iris Injection  Anterior Chamber  Overall Inflammation  clear  normal  normal  0  <10%  trace  trace  trace  1  mild  10%-40%  mild  mild  mild  2  moderate  moderate  40%-70%  moderate  moderate  moderate  3  severe  severe  70%-100%  severe  severe  severe  4  Results Clinical  Ocular  Toxicity  Subconjunctival resulted not  mucus  or  vitreous  did  not  seven  as  stain  almost  days  to  to  erosion  In g e n e r a l showed  the  the  moderate  to  the  of  conjunctival  conjunctiva,  groups  the  fourth  day,  and  cells  the  vehicles  In each  12 h o u r s ,  case  except  hyperemia  of  the  examination  in  all  the  and  c o r n e a was  anterior fibrin.  clear,  the  conjunctival depending seed,  chamber  The  and i t s  iris  surface  well  epithelium  inflammation  with hyperemia,  chemosis  peanut  present  and sesame s e e d  clinical to  moderate the  peaked  at  the  being  vehicle.  in  from which p o i n t  effects  epithelium  and m i l d as  approximately vehicles  olive,  hyperemia  took  of  on t h e  aggressive  as  bleb  The t o x i c  conjunctiva,  most  severe  of  first  within  vehicle  cotton  the  of  inflammation.  edema  be a b s o r b e d .  of  1.2)  0.5ml  biomicroscopic  flare,  every  to  of  and  fluorescein.  v a r y i n g degrees,  oils  mild  and t h e  with  were c o n f i n e d and  to  1.1  i n the  abnormalities  normal,  With  seen  Slit-lamp  showed no  appeared  was  secretion,  conjunctiva. cases  injection  i n v a r y i n g degrees of  much r e a c t i o n  for  (figures  reaction  moderate to  with  chemosis  severe  erosion  rabbits.  In  about  third  or  started  to  the  inflammation  these  decrease. Castor with  oil  mild  conjunctiva  to  showed a  less  moderate  with  aggressive  hyperemia  three 9  of  the  and  clinical  reaction  chemosis  rabbits  in  this  of  the  group  showing  mild  to  epithelium.  moderate  inflammation  1.0  one,  three  started  inflammation  at  of  The i n f l a m m a t i o n i n t h e  peaked between days  Figure  erosion  over  and  four  to the  one  representative  and s e v e n  period  are  shown  of  is  photographs  days  oil  group  also  from which p o i n t  The p l o t  week  conjunctival  castor  and f o u r ,  decrease.  the  the  the  overall  presented  of  each  in  vehicle  in Figures  1.1  and  1.2. T h e PEG g r o u p was been  absorbed  showing over  within  trace  the  the 24  hours  hyperemia  first  48  exception of  with  the  bleb  injection  and c h e m o s i s  hours,  the  from t h e r e  of  the  on t h e  having  and  only  conjunctiva eye  appeared  normal.  Histopathologic The  vehicles  inflammation, soft  Findings in  our  study  restricted  tissues.  The  to  rest  An i n t e r m e d i a t e  with  oil,  optic  nerve  and  infiltrate. seed,  typically  resulted with  caused  peanut  reached  the  the  eye  severe as  an  and  far  seed  of the  response.  reaction  occurred  posteriorly  o r no  inflammation.  10  as  the  inflammatory seen with  oil;  these  cotton agents  g l o b e and i n d u c e d a The  least  w i t h PEG w h i c h a p p e a r e d t o b e e n t i r e l y  little  posterior appeared  exuberant  sesame  and  of  itself  r e a c t i o n was  equator  inflammatory  conjunctiva  to  spread  An i n t e r m e d i a t e  olive,  moderate  which  the of  uninvolved. castor  produced v a r y i n g degrees  reaction absorbed  A l t h o u g h the degree of of  the  response  following and  was  injection,  lymphocytes  inflammation v a r i e d ,  similar a  few  could  be  seen  By t w e l v e h o u r s ,  oil  w h i c h were  Twenty  four  inflammatory  hours cells  after were  exhibited  occasional  foreign  lymphocytes  and p l a s m a  The  histological  shown i n f i g u r e s  cases.  streaming  hours  granulocytes dilated  by m a c r o p h a g e s ,  the  oil.  cells,  acute some  By o n e w e e k ,  foamy  the  posteriorly.  these  granulomatous  body g i a n t  Two  from  diffusing  injection,  joined  a  pattern  t h e s e had surrounded  gradually  w h i c h a p p e a r e d t o be i n g e s t i n g inflammation  a l l  polymorphonuclear  capillaries. droplets,  in  the  pattern,  of the  with  macrophages,  cells.  findings  1.3-1.8.  11  of  the  various  vehicles  are  Figure  1.0  OCULAR INFLAMMATORY SUBCONJUNCTIVAL  RESPONSE FOLLOWING  INJECTION  OF VEHICLES  4-,  0  1 ^ - 2  3  4  TIME (Days)  12  5  6  '  7  COLOUR PHOTOGRAPHS SHOULD NOT BE USED. THEY WILL APPEAR AS GREY OR BLACK. WE RECOMMEND THAT THE COPY OF THE THESIS SUBMITTED FOR MICROFILMING INCLUDE BLACK AND WHITE PHOTOGRAPHS REPRINTED FROM THE COLOUR PHOTOGRAPHS BY A PHOTOGRAPHER IF NECESSARY.  LORSQUE MICR0FILMEES, LES PHOTOGRAPHIES EN COULEUR PARAISSENT GRISES OU NOIRES. NOUS RECOMMANDONS QUE L E X E M P L A I R E DE LA THESE A MICROFILMER SOIT ACCOMPAGNE PLUTOT DE PHOTOGRAPHIES EN NOIR ET BLANC PRODUITES A PARTIR DES PHOTOGRAPHIES EN COULEURS PAR UN PHOTOGRAPHE , SI NECESSAIRE. 1  F i g u r e 1.1 C l i n i c a l Ocular  castor  o i l at  Inflammatory Response to  day 1  castor  o i l at  the  day 4  Vehicles  Tested  castor o i l at  day 7  c o t t o n seed o i l a t day 1  c o t t o n seed o i l a t day 4  c o t t o n seed o i l a t day 7  olive  olive  olive  o i l at  day 1  o i l at  13  day 4  at  day 7  Figure Clinical  peanut  1.2 Ocular  o i l at  Inflammatory Response to  day 1  sesame s e e d o i l a t day 1  peanut  o i l at  day 4  sesame s e e d o i l a t day 4  u  the  Vehicles Tested  peanut  Cont'd  o i l at  day 7  sesame s e e d o i l a t day 7  Figure  1.3*  Histological  effects  of Castor O i l at  15  one  week  Figure  1.4  Histological  effects  of Cotton  Seed  O i l at  one  week  F i g u r e 1.5 Histological  effects  o f O l i v e O i l at  one  week  Figure  1.6  Histological  effects  o f Peanut O i l at  one week  Figure  1.7  Histological  effects  of Polyethylene Glycol(PEG)  at  one  week  Figure  1.8  Histological  effects  o f Sesame  Seed O i l a t  one  week  DISCUSSION In had  previous  been  saline. be  studies  given '  in this  laboratory,  subconjunctivally  Most o f  the  were  drugs  soluble  the o c u l a r t o x i c i t y to the  c o n t r o l l e d by a d j u s t i n g t h e  amount o f  that in  drug c o u l d  drug  injected  subconjunctivally. Cyclosporine because  its  c h e m i s t r y demanded t h a t t h e v e h i c l e  lipophilic.  F o r Cy t o  ocular toxicity of  the  (Cy) p r e s e n t e d u s w i t h a u n i q u e p r o b l e m ,  vehicle  of  be  of  that  solvents  cotton seed,  These  had  that  were  candidates  selected  previously  been  and i n w h i c h Cy w o u l d be  selected  peanut,  polyethylene  of  f o r the  olive  glycol  ( t h i s being the  tolerate)  study  include:  and sesame s e e d o i l  (PEG).  These  amount t h e  were  from used  a as  readily  castor,  as w e l l  sterilized  as and  r a b b i t s ' c o n j u n c t i v a can  e a c h was i n j e c t e d s u b c o n j u n c t i v a l l y .  D a i l y o b s e r v a t i o n s were made b y s l i t to  the  4 6  The v e h i c l e s  0.5cc  were c h o s e n a s p o s s i b l e  injection.  vehicles  pharmacological soluble.  be  and o f C y .  subconjunctival  group  subconjunctival ly  two v a r i a b l e s h a d t o be d e t e r m i n e d ,  A number o f v e h i c l e s for  injected  had to  ascertain  the  effects  of  surrounding  the  eye.  confined  to  the  conjunctiva  chemosis  and  erosion  the  The major  of  O c c a s i o n a l l y the v e h i c l e s  the  lamp b i o m i c r o s c o p y  vehicles toxic  resulting  effects in  conjunctival  would cause a  within  and were  hypermia, epithelium.  c o r n e a l edema,  but  this  was  infrequent.  The with  vehicles  PEG  produced  causing  histologically  and  entirely  unexpected  toxicity  and t h e  was  made u p s o  found  in  within  not  it,  that  it  The o i l s  that  (data  Cy d i s s o l v e d  greater  when  that  to  sesame  reaction  seed  of oil  0.5  cc.  rate the  to  severe  of  eye.  findings  findings  low  to  a n d was  I attempted  that  absorbed  the to  use  of  dissolve  in  10  cc.  of  showed  f o r Cy (on a v e r a g e  oil),  but  also  tonicity  but the  clinical  a mild  to  well  with  moderate  be  peanut  inflammatory  findings  with castor  were  not  olive,  An i n t e r m e d i a t e  correlate  they  could  intermediate  5  produced a  p a r t l y because  an  reaction.  d i d not  which  of  equivalent  Cotton seed,  r e a c t i o n was s e e n h i s t o l o g i c a l l y these  not  subconjunctivally  NaCl)  affinity  and t h e  produced  histologically,  a moderate  is  compounds  Unfortunately,  inflammatory response,  adjusted  reaction  shown).  in  a slower  inflammation  result  milligrams dissolved  a greater  absorbed at  and  (0.9%  because 0.5  showed  are  had a t o n i c i t y  injection.  not  This  injected  chambers  possible  PEG s o l u t i o n  mg o f  PEG t h a t was  of  inflammatory  PEG'S  because  ocular  less  trace  degrees  clinically.  24 h o u r s a f t e r  PEG was Cy i n  the  a  varying  showed  to  oil,  severe  but  the  again  clinical  inflammatory  response. These spread it  of  discrepancies the  s p r e a d as  oil  may p a r t l y  posteriorly.  f a r back as  the 22  be  In the  explained  case  optic nerve,  of  by  castor  thereby  the oil,  causing  less  of  a reaction  limbus,  resulting  clinically. oil  with  was  the  superior  i n a m i l d to moderate response  visible  Because  exposed  to  the  the  conjunctiva  oil  the  had s p r e a d so  ocular  i n f l a m m a t o r y r e a c t i o n was s e e n With  cotton  the v e h i c l e s case the  of  seed,  olive,  tissue;  vehicles,  conjunctiva  environment.  the  that  peanut  over  most  the  of  we  Therefore, seen,  ocular  oil  the  since  reasoned  using  this  the castor  was  spread  to  of  In the  restricted exposing  severe  the  oil  to  this the  clinical  was  inflammatory  exposed reaction  less.  produced  oil  oil,  a  the  similar  furthest  response. posteriorly  c o u l d b e made a v a i l a b l e  to  the  eye  a vehicle  for  oil. using  the  delivery  oil  can be  of  castor  Cy,  the  oil  as  a base  advantageous  for  properties  of  castor  exploited.  Cremophor( ) EL  is  R  used  Cremophor( ) R  in  to  conjunctiva,  the  such I.V.  EL c o n s i s t  polyoxyethylene a cushion  oils  m o r e Cy  We f e l t  vehicle,  greater  p e r i o d of time to  less  ensuing  was  limbus,  a moderate  and b e c a u s e  tissues  average,  However,  a  and sesame seed  the  superior  was s e e n h i s t o l o g i c a l l y On  hence,  more  histologically.  p a r t of the c o n j u n c t i v a for a longer  to  f a r back,  s p r e a d up t o t h e e q u a t o r o f t h e g l o b e .  these  r e a c t i o n was  over  glycol  decrease and t h e  of  a form  of  Cy.  650mg.  effects  alcohol  available  The c o n t e n t s  33% a l c o h o l ,  (POEG) the  commercially  oil  and  The POEG may a c t  like  of  provides  castor  of  the two  vehicle  on  functions;  the it  allows  m o r e Cy t o b e d i s s o l v e d  i n the  castor  oil  so  less  v e h i c l e h a s t o b e u s e d , a n d a t t h e same t i m e i t r e d u c e s viscosity less  of  the  castor  uncomfortable to  properties  might  response  the  to  injections  be  oil  possibly  the  eye.  to  further  vehicle,  making the  The r e s u l t a n t  of  decrease  the  vehicle  all  these  inflammatory  and t o make p o s s i b l e  the  repeated  o f Cy s u b c o n j u n t i v a l l y .  Conclusions The  oils  on t h e a v e r a g e g a v e a s i m i l a r r e s p o n s e ,  from a c l i n i c a l less  severe.  perspective A  commercially  incorporated castor drug  in  less  castor o i l  oil  vehicle  a l o n e as a  On t h i s  only castor o i l  basis  a p p e a r e d t o be  available  vehicle  could  be  obtained  p r e p a r a t i o n s h o u l d produce l e s s of a l o c a l  the  Cremophor  only  R  speculate  experience  We h a v e I.V.( ) R  EL without the  the  the  obtained  was  indeed  received  inflammatory  be due t o  the  due t o  the  the  vehicle  found  obtain  used  most  of  in  our  much o f  the  and n o t Cy.  the  the  could  from  vehicle  vehicle. R  was  the  vehicle  Sandimmune I . V . ( )  response  of  One w o u l d e x p e c t  p r e p a r a t i o n and f o u n d t h a t  reaction  response.  C y . T h e r e f o r e , we  effects  oils.  reaction to  since  using  the commercial  a t t h e t i m e we w e r e n o t a b l e t o  about  with  inflammatory  that  ( )  by  the  vehicle.  we r e a s o n e d t h e o r e t i c a l l y  Unfortunately,  which  a l l o w e d f o r more d e l i v e r y o f  than  but  Sandimmune  inflammatory  In those  rabbits  subconjunctival ly, to  be  less  than  the with  the  vehicle  alone  The o r i g i n a l reaction I.V.( ) R  due  C y was  preparation allows vehicle  than  inflammatory  and e x p e r i m e n t s  assumption,  w o u l d be  and not  (data  could  to  the  that  most  vehicle  correct,  of  not the  used  and s i n c e  shown). inflammatory  i n Sandimmune the  commercial  f o r more d r u g t o be d e l i v e r e d be  obtained  using  r e a c t i o n c o u l d be g r e a t l y  25  the  oils  reduced.  in  alone,  less the  CHAPTER 2 Dose d e t e r m i n a t i o n  study  Introduction From t h e that  results  the  commercial  containing This  was  the  based  to  deliver  of  the  obtained  its  had  range  of  Cremophor ( )  more Cy (50 m g / m l ) .  to  be  for  determined.  was  as  well  as  its  the  was  I.V.( ) R  used. ability  formulation  C y , t h e maximum  This  decided  Sandimmune  To c o m p l e t e  dosage  it  E L w o u l d be  R  availability  subconjunctival  dose  preparation  vehicle on  i n C h a p t e r 1,  done  tolerated  by t e s t i n g  a  doses.  M a t e r i a l s and Methods Three groups of Zealand examined to  white  five  females  by s l i t - l a m p  rule  out  r a b b i t s each weighting  pre-existing  subconjunctivally  with  10  Cy  o r 25  mg o f  i n Cremphor)}  tuberculin  opposite unable  eye  to  one  o b t a i n the  week,  inflammation  and  posterior  a 25  topical  served  e y e s were e x a m i n e d  ocular  ( p r i o r to  anaesthetized with  -  2.4  as  kg.)  experiment  These  were  to the s u p e r i o r  limbus  injection  (50  mg/ml  the  right  eyes  R  by s l i t - l a m p  EL  control,  to  a  were  a s we w e r e  without  biomicroscopy  photographed.  Cy.  each  The  The  day  for  degree  of  was g r a d e d a c c o r d i n g t o t h e  26  of  p r o p a r a c a i n e HC1 (0.5%)}. T h e  a non-treated  serially  were  gauge n e e d l e a t t a c h e d  Cremophor ( )  i n e a c h eye  disease.  C y {SANDIMMUNE I . V .  using  syringe  2.2  s u p p l i e d New  biomicroscopy p r i o r to the  injected 5,  (locally  scheme  presented  in Table  II,  i n w h i c h an o v e r a l l  averaged  was o b t a i n e d by a s s e s s i n g e a c h c r i t e r i o n f o r t h e rabbits with  an  globes with  in this  overdose  of  a dye,  fixed  sectioned  in  The g r o u p s  pentobarbital  were e n u c l e a t e d ,  paraffin, Eosin  experiment.  after  were  10% b u f f e r e d  formalin,  individual sacrificed  one  marked at the p o i n t  value  of  week.  The  injection  embedded  in  v e r t i c a l l y and s t a i n e d b y H a e m a t o x y l i n -  for histopathological  27  study.'  Table II Conjunctiva Chemosis  Hyperemia  Ocular Discharge  Erosion of  % Bleb  Corneal  Iris  Anterior  Epithelium  present  Clarity  Injection  Chamber  Overall Inflammation  clear  normal  normal  0  <10%  trace  trace  trace  1  mild  10%-40%  mild  mild  mild  2  moderate  moderate  40%-70%  moderate  moderate  moderate  3  severe  severe  70%-100%  severe  severe  severe  4  none  none  none  none  trace  trace  trace  trace  mild  mild  mild  moderate  moderate  severe  severe  0  Results Clinical  Ocular Toxicity  The two h i g h e s t d o s e s o f toxic.  The  within  the  10 mg  of  secretion. erosion  of  the  six  days  to  found to  mild  trace within  be t h e  of  performed  with  noted  a single  that  upper  least  Observations  Most of  injection  in a d a i l y plot  of  the  5 mg i n O . l c c o f toxic  effects  It  of  mild  to  secondary  to  Over  the  resolved.  The  of  the  o n l y produced  trace  conjunctiva  with  the  these  the  more  changes  C y . From  frequency  than  effect  resolved  investigations injections  Cy  once  of  these  overall  we  o n c e p e r week noted  per  week  led  to  ( data  not  shown  ).  doses  is  summarized  inflammatory response  for  2.0.  study  of  the  subconjunctival  Cy o n c e a week that  of  had  f o r o u r s t u d i e s . We  inflammation  of the  each day i n f i g u r e The l o n g - t e r m  mucus  Cy p r o d u c e d  and  slowly  5 mg i n O . l c c d o s e o f  local on  of  trauma.  toxic.  and c h e m o s i s  regard to  injections  intolerable  too  produced  moderate  dose  local  w o u l d be c l i n i c a l l y t o l e r a b l e that  as  eyelid,  inflammation  secretion.  48 h o u r s  Cy  be  i n j e c t i o n o f a s i n g l e 5 mg i n O . l c c d o s e o f  hyperemia  mucus  the  of  epithelium  self-induced the  subconjunctival Cy was  of  to  s e v e r e h y p e r e m i a and  mg s u b c o n j u n c t i v a l  loss  and  to  well  conjunctival  hair  inflammation next  25  as  judged  dose  24 h o u r s m o d e r a t e  conjunctiva  The  moderate  subconjunctival  first  chemosis  Cy w e r e  were  29  f o r two months  not  present  injection showed with  of no the  single Cy,  subconjunctival of  conjunctiva  reacted  s l i g h t l y more t h a n  the  subconjunctival  single but  the  hours  reaction  after  This  eyelid  of  hours.  In  injection  largely  due  to  was of  resolved  the  O.lcc of  of  Cy  seen w i t h  the just  5 mg i n O . l c c  of  within  48  24 t o  the  causing  i n the the  various  doses  at  figures  2.1-2.2.  edematous  one,  needle  four,  of  the  resolved  within  reaction  injection  showing  of the  a l l  upper  the  was  upper  e y e l i d .  effect  and s e v e n days  48  which  p e n e t r a t i n g the  s w e l l i n g  photographs  seen with  edema  r a b b i t moving upon  resulted  Representative  severe  which u s u a l l y  instances,  to  inadvertently  moderate  rabbit,  some  e y e l i d ,  injections  a t r a n s i t o r y r e a c t i o n was  was  the  possibly  repeated  5 mg i n  injection.  Occasionally doses.  the  of  because  Cy,  except  injection  are  of  the  shown  in  Histopathological Findings From o u r o b s e r v a t i o n s some s u p p r e s s i o n  of  Sandimmune  I.V.( ).  At  hours,  two  experimental (PMN)  the  in Chapter  inflammatory response  eye  the  showed  b y 12 h o u r s ,  one  week  lymphocytes  suggests  when we  used  R  subconjunctival  the  tissues  of  the  few p o l y m o r p h o n u c l e a r g r a n u l o c y t e s  a n d b y 24 h o u r s ,  contained only a scattering By  1 our data  30  experimental  o f macrophages  experimental  and m a c r o p h a g e s .  the  eye  and  showed  eye  lymphocytes. only  a  few  I n c r e a s i n g t h e d o s a g e o f C y t o 10 a n d 25 mg r e s u l t e d additional caused  toxic  side  epithelial  collagen  and  localized  muscle.  with the These the  response  vehicle  2  and h e p a t i c  portal  long-term effects  a  denaturation  of  work  also  caused  tissues.  done  by  our  group indeed  was  less  the than  -  in  were 0.1  they in  less  cc  of  Cy  showed the  noticeable once  renal  cortex  in  weekly  lymphocyte  and  interstitium  triads.  of  findings  of  the  various  subconjunctival ly  d o s e s and  the  a d m i n i s t e r e d Cy a r e  2.3-2.6.  of  significant  variance  (nested)  difference  test  between  showed t h a t  the  various  t e s t e d o f t h e c o m m e r c i a l l y a v a i l a b l e p r e p a r a t i o n . The of  with  Analysis  An a n a l y s i s was  Cy  injections  effects  but  The h i s t o l o g i c a l  Statistical  O J  infiltrates  shown i n f i g u r e s  of  R  5 mg  months,  plasmocyte  injection  w i t h Sandimmune I . V . ( )  side  rabbits given  for  mg and  the p e r i o c u l a r  alone.  local  10  preparation, suggested that  R  inflammatory  25 mg  experimental  Sandimmune I . V . ( )  ,  irregularities  s c a r r i n g of  Subsequent  effects.  in  significance  between t h e  31  d o s e s was  found t o  be  there doses level  p=0.0001.  2.0  OCULAR INFLAMMATORY  RESPONSE TO DIFFERENT  CYCLOSPORINE (SANDIMMUNE I.V.)  TIME  (Days)  32  DOSES OF  SUBCONJUNCTIVALLY x  x  5mg/0.lcc OF CYCLOSPORINE  »  .  lOmg/O.Ecc OF CYCLOSPORINE  o  o 25mg/0.5cc OF CYCLOSPORINE  Figure  2.1  Effects  o f 5mg/0.1cc o f  a  t  d  a  ^  1  Cyclosporine  at  day 4  33  at  day 7  Figure  2.2  Effects  o f 25mg/0.5cc of  6&  y  5  Cyclosporine  at  d a y 30  at  day  60  Figure  2.3*  Histological  effects  o f 5mg/0.1cc o f C y c l o s p o r i n e at  one  week  36  Figure  2.5  Histological  effects  o f 25mg/0.5cc  o f C y c l o s p o r i n e at  one  week  lOx A mild inflammatory d i s r u p t e d and t h e r e  response i s present at i s a n e c r o t i c zone(NZ) 37  one w e e k . The c o l l a g e n i s p r e s e n t i n the lamina p r o p r i a .  Figure  2.6  H i s t o l o g i c a l e f f e c t s o f 5 m g / 0 . 1 c c o f C y c l o s p o r i n e o n c e p e r week t h a t r e c i e v e d t h i s d o s e f o r two months  Only a trace  inflammatory response  i s present  38  at  t h e end o f  two  DISCUSSION The of  the  dose  commercially  causing 24  to  determination  only  mild  48 h o u r s .  25 mg i n  prepared  hyperemia  C y was  the  side effects and  mucus d i s c h a r g e ,  loss  the  upper  the  doses  least  and  eyelid  (10  O.lcc toxic,  resolved  mg i n  0.2cc  of  the  at  the  due  to  with  conjunctiva h i g h e s t dose  local  by and  became more a p p a r e n t  chemosis  exuberant  5 mg i n  which had l a r g e l y  increasing  hyperemia  of  showed t h a t  With  0.5cc),  moderate  study  and hair  trauma  and  inflammation. Although almost  resolved  sensitive the  the  b y one week,  dose  conjunctiva  injection  (5  looked  investigations not  mg  the  with  almost  performed  shown),  this  once p e r  localized  t h e c o n j u n c t i v a were s t i l l  too  as  normal on  the  d o s e was  vehicle  (Sandimmune the  at  higher  at that  time.  With  administration  the  one  week,  frequency  found t o  of  and  is  denaturation  injection  be a c c e p t a b l e  some s u g g e s t i o n o f  for  response,  of  the  r e s p o n s e were  of  but  the  collagen  suppression particularly  conjunctival and m u s c l e  injected I.V.( ))  and  not  used  amount o f v e h i c l e 39  was  as  noted.  c a n l a r g e l y be a t t r i b u t e d t o t h e  R  from  week.  fibroblastic  50 m g / m l ,  the  O.lcc)  inflammatory  These e f f e c t s of  had  25 mg i n 0 . 5 c c i r r e g u l a r i t y  epithelium, well  doses  in  At h i g h e r doses there of  with  and c o u l d n o t be r e i n j e c t e d  low  (data  inflammation  to  Cy. only  Since  amount the  available  t h a t was g i v e n was  Cy as  governed  by  the  concentration  invariably causing  the  so,  tested, made  For  oil  cc  control right  in  peanut eye)  eye  supports The  of  the  cells,  the  increased, eye.  seen  as  the  control  cell  at  to the  indicates of  and  interleukin-II  oil  and  prepared  (which s e r v e d  as  the  of  to  the  peanut o i l eye).  T h e r e was  histological  (received oil  and  mechanism o f could  be  peanut Cy),  made,  research  oil  which  Cy a c t i o n .  between t h e  further  5 6 - 6 0  from  the  control  and  is  needed  to  f o l l o w i n g does o c c u r .  level  of  is  inhibition the able  interleukin-II blocking  peanut  between the  (peanut  cause  Cy  assumptions  s u b c o n j u n c t i v a l l y with  eye  populations  show t h a t t h e  the  vehicles  C y , w h i c h was  eye  eye  Though  of  experimental  differences  8 1  with  from the  Cy i n 0 . 5 c c  inferences  eye.  seemed  inhibition  left  current suggested  i n the  infiltration  increased  also  of  effect  were i n j e c t e d i n the  the  following  conclusively  was  commercial p r e p a r a t i o n .  mg/ml of  experimental  experimental  date  oil  from the  difference  Cy  10  (which s e r v e d  and  was  way  we t e s t e d t h e  a n d 5 mg o f  observations only)  indirect  containing  evidence,  to  an  Five rabbits  of  dose  response  Chapter 1 regarding the  locally. 0.5  vehicle  inflammatory  interest,  peanut  the  was much l e s s t h a n w i t h a n y o f t h e  shown  in  of  As  inflammatory response  the  higher doses  Cy.  amount  a greater  Even  of  the  T-cell. to  lymphocyte  Current  accomplish  production receptor  by p r e c u r s o r c y t o t o x i c 40  of  from  evidence this  T-helper  acquisition  T-cells,  as  by  well  for as  diminishing  the  responsiveness  of  T-helper  cells  to  interleukin-1.15,56-60 Normally  during  granulocytes  acute  (PMN's)  inflammation polymorphonuclear  predominate  in  h o u r s b e i n g r e p l a c e d by m o n o c y t e s This  i n f i l t r a t i o n was  experimental  seen  e y e s showed  became more e v i d e n t influence  the  injection,  and s i n c e  i n the  with time.  This  of  the  resulting  cascade  by Cy. T h i s  is  reflected  by t h e  experimental  could  eye.  interfere  interference  with  with  s u g g e s t e d as  The  6 1  '  through  from  the  cell  eye,  suppression presented The  with  of  in  Figures  dose  once  per  frequent  and  2.7a  of  in  administration.  the for  inhibited  by  in  w h i c h Cy  unclear,  but  has  been  the major a c t i o n s  of  the  5  8  -  6  0  the  tolerated  o f Cy  supported  a decrease  in  effect  These h i s t o l o g i c  of  is  conjunctiva  minor  the  of  the  on  the  findings  are  2.7h.  histological  was  of  factors  be  is  T-cells  through  (5 mg i n O . l c c ) week  which  Cy c o u l d  site  chemotaxis  possible  PMN a c t i v i t y .  clinical  single  seen a  the  p o p u l a t i o n seen  PMN's  observations  lymphocytes  the  4 7  6 2  suppression  histologic  number o f rabbits  the  to  24  but  suggests that  seems t o  The c u r r e n t l y h e l d p r e m i s e t h a t are  to  hours.  eyes,  chemotactic  C5a-induced  unlikely.  24 t o 48  mechanism  the  six  and macrophages  PMN's  PMN's r e l e a s e  first  control  PMN's  macrophages,  the  in  fewer  migration  the  findings  suggested  a  c o m m e r c i a l l y p r e p a r e d Cy better  To a s c e r t a i n  than  whether  more  repeated  F i g u r e 2.7a Histological effects of a subconjunctival injection of 0.5cc o f p e a n u t o i l , c o n t r o l , ( A ) a n d p e a n u t o i l a n d Cy 5 mg/ml, e x p e r i m e n t a l , ( B ) a t 2 h o u r s . At t h i s time t h e r e i s a d i f f e r e n c e i n t h e inflammatory i n f i l t r a t e , t h o u g h n o t d r a m a t i c , w i t h more PMN's p r e s e n t i n the control(A).  42  Figure  2.7b  H i s t o l o g i c a l effects of a subconjunctival i n j e c t i o n of 0.5cc o f p e a n u t o i l , c o n t r o l , ( A ) a n d p e a n u t o i l a n d Cy 5 mg/ml, e x p e r i m e n t a l , ( B ) a t 4 h o u r s . The d i f f e r e n c e i n t h e amount o f i n f l a m m a t o r y i n f i l t r a t e i s b e c o m i n g more o b v i o u s w i t h t h e c o n t r o l ( A ) s h o w i n g more PMN's.  43  Figure  2.7c  H i s t o l o g i c a l e f f e c t s of a s u b c o n j u n c t i v a l i n j e c t i o n of 0 . 5 c c o f p e a n u t o i l , c o n t r o l , ( A ) a n d p e a n u t o i l a n d Cy 5 mg/ml, e x p e r i m e n t a l , ( B ) a t 12 h o u r s . There i s a distinct difference i n t h e number o f i n f l a m m a t o r t y c e l l s p r e s e n t , m a i n l y PMN's a s w e l l as c o m p a r t m e n t a l i z a t i o n o f t h e o i l i n t h e c o n t r o l (A) a n d t h e experimental(B) appearing quiescent. A.  B.  44  Figure  2.7d  H i s t o l o g i c a l effects of a s u b c o n j u n c t i v a l i n j e c t i o n of 0 . 5 c c o f p e a n u t o i l , c o n t r o l , (A) a n d 5 mg o f C y i n 0.5cc o f p e a n u t o i l , e x p e r i m e n t a l , ( B ) a t 1 week. The d i f f e r e n c e i n t h e c e l l p o p u l a t i o n b e t w e e n t h e c o n t r o l and t h e e x p e r i m e n t a l eye i s q u i t e d r a m a t i c w i t h t h e e x p e r i m e n t a l one a p p e a r i n g a l m o s t a c e l l u l a r a n d t h e c o n t r o l showing q u i e t a heavy inflammatory i n f i l t r a t e . A.  45  administration of a group of once  five  p e r week  effects  this  d o s e w o u l d h a v e any a d v e r s e  rabbits received  f o r two m o n t h s .  5 mg o f Cy i n O . l c c o f Cy  T h e r a b b i t s s h o w e d no  c l i n i c a l l y t h a t were n o t p r e s e n t w i t h t h e  injection animals  of  5 mg i n  showed  histopathology epithelium, higher  did  show  but to  of  Cy.  changes  irregularities  can  Cy.  i r r e g u l a r i t y of  In  this  probably  injections  organs. the  in  the  portal  triads.  effects  when  it  is  to  Cy (see  Cy).  than to  renal  case  be the  the  more  the  Cy i s  alterations  have been  noted  treatment  of  chronic of  renal  intersitial  possible on,  but  injury,  3 8  in  mechanism f o r r e n a l  and  in a  for  which  and  the  ,  3  9  ,  4  0  ,  4  2  ,  4  side but  3  r a b b i t s are  effects,  These  moderate  were  injury  in  to  observed,  tubular  glomerular  decrease  7  Cy's  due of  they  effects  r e c e i v i n g Cy o r a l l y f o r  changes  fibrosis  changes  i n the  1  of  hepatic  i f Cy had b e e n g i v e n o r a l l y o r  in patients  histologic  infiltrates  been one  same p e r i o d o f t i m e .  uveitis,  the  i n Chapter 3 f o r l e v e l s  responsible  over the  the  to  i n t e r s t i t i u m and  given o r a l l y or sytemical l y ,  w o u l d h a v e b e e n much g r e a t e r systemically  However,  vehicle.  cortex  pharmacokinetic study if  the  epithelial  attributed  N e p h r o t o x i c i t y has  u n l i k e l y that  Even  of  conjunctival  In a d d i t i o n , l y m p h o c y t i c and p l a s m a c y t i c seen  single  A post-mortem  i n the  side  a l e s s e r d e g r e e t h a n was s e e n w i t h  of  repeated  O.lcc  no g r o s s  doses  were  effects,  c a n o n l y be  renal  severe  consisting  atrophy.  capillaries,  the  4 2 , 4 3  The  speculated 3 7  blood  tubular flow  4 1  have  been  renal  i m p l i c a t e d as b e i n g c a u s e d by C y , r e s u l t i n g  injury.  able  It  tissues. been  Cy,  i n high  that  Though t h e  are  degree,  or  These  of  aggregations  as  concern, to  of  brain  during  of  they  lymphocytes and  as  noted  Cy t h e r a p y .  hepatic  do n o t  structural  effects  discontinuation  presence  cyclophilins,  i n the  effects  interstitium  lead  mild  Cy may b e of  have  and k i d n e y ,  treatment  changes  with  observed  5 5  renal  rabbits  of the  e x p l a i n i n g the h i s t o l o g i c a l  kidney.  the  toxic  that  d i s c o v e r e d i n lymphoid  r e f e r r e d to  concentrations  display  possibly the  Cy b i n d i n g p r o t e i n s ,  These p r o t e i n s  found  organs  , in  been p o s t u l a t e d  to produce these e f f e c t s because  basic cytosolic  in  has a l s o  in  and  triads  seem t o  renal  or  have  been  5 mg  in  plasmacytes in  be o f  hepatic  the  a high damage.  reversible  on  4 4  Conclusions A  single  injection  commercially available the  maximum  injections Our r e s u l t s that to  were  eye  noted  0.1  to  tolerated cause  dose.  excessive  hypothesis, allow  for  correct.  47  that  is  repeated  of  the  found to  More local  using the commercially a v a i l a b l e  and  cc  C y o n c e p e r w e e k , was  clinically  our i n i t i a l  the  of  be  frequent response.  Cy s u g g e s t e d  i t w o u l d be l e s s administration,  toxic was  CHAPTER 3 Pharmacokinetics of cyclosporine f o l l o w i n g subconjunctival versus  intravenous administration.  Introduction With  systemic  central  nervous  sanctuaries.  ocular  of  of equidose  study)  that  able  chambers  Compared  to  injection  resulted  Cy  in  the  Cy  to  have  antineoplastic  Cy  were get  when  {the  shown  the  to  that  intravenous  agents.  To  2 0 - 2 4  in  O . l c c once  subconjunctival  compared i n r a b b i t s .  higher  amounts  given  0.1  the  times  of  o f Cy were u n d e t e c t a b l e  of  per dose  versus We f o u n d  Cy i n t o  the  subconjunctiva1 ly.  injection,  four times  and  5 mg i n  as d e t e r m i n e d from t h e  Cy was  intravenous  vitreous  Aqueous l e v e l s  and  pharmacologic  superior  following  injection  we w e r e  is  eye  t h i s was t r u e f o r C y , o c u l a r , b l o o d a n d  intravenous  ocular  represent  C y (SANDIMMUNE I . V . ) ,  determination  the  investigations  penetration  levels  week o f  system  administration  determine whether urine  administration  Previous  subconjunctival for  drug  subconjunctival  peak c o n c e n t r a t i o n that  in  i n the  the  of  blood.  rabbits  given  intravenously.  M a t e r i a l s and Methods Twelve New  groups  Zealand  tranquilized  of  white  three  rabbits  females  each  (locally  weighing  by i n t r a m u s c u l a r i n j e c t i o n s  48  supplied  2.0-2.2kg) of  0.2  were  m l / k g of  10:1 s o l u t i o n  (100 m g / m l )  acetylpromazine  (Rogar  Vancouver)  half  was  every  infused  into  the  30-40 m l / h o u r t o were  given  the  5 mg  with  right topical  the  left  is  S.T.B.  hour  f o r four  left  in  {posterior  eye  which  to  as  a bolus  the  ear vein).  i s by i n f u s i o n  Then a t 0.5,  1,  2,  It  superior  right  be n o t e d  a period of  ear.  8 a n d 12 h o u r s  The samples  later,  and f r o z e n .  were  placed  tubes  collected  d u r i n g e a c h i n t e r v a l was r e c o r d e d ,  u r i n e were p l a c e d At  with  the  appropriate  an o v e r d o s e  death  the  saline humour placed  The t o t a l  time  the animals  of pentobarbital  was  blotted aspirated  i n a microtest  and t h e a d h e r e n t  Later  into  1.5  of  and samples  t u b e s and f r o z e n . were  sacrificed  and i m m e d i a t e l y  after  dry.  About  0.10  of  through  the  inferior  blotted  the eyes 49  ml  were  allowed  to  and  enucleated,  T h e g l o b e s were  d r y and snap f r o z e n  with  aqueous  limbus  T h e e y e s were t h e n discarded.  ml  urine  rinsed  episclera  rinsed with saline,  artery of  and t h e c o r n e a s  tube.  this  1 ml b l o o d  volume  1.5 m l m i c r o t e s t  e y e s were p r o p t o s e d , and  nitrogen.  into  that  (in  time.  microtest  of  limbus  anaesthetized  s a m p l e s were o b t a i n e d by p u n c t u r e o f t h e m e d i a l the  either  f o r I . V . a d m i n i s t r a t i o n o f Cy,  over  4,  should  of  The r a b b i t s  p r o p a r a c a i n e HC1 (0.5%)} o r i n t r a v e n o u s l y  marginal  it  Cy  saline  at a rate  flow.  had been p r e v i o u s l y  n o t t h e s t a n d a r d method  rather  Normal  ear vein  urine of  Laboratories,  hours.  marginal  O.lcc  h y d r o c h l o r i d e and  and A y e r s t  maintain  subconjunctivally in  of ketamine  in  partially  then  liquid thaw,  a r e were t h e n i n c i s e d w i t h a s c a l p e l ; expressed  and p l a c e d  sonicator,  the  centrifuged  at  was  then  washed be  enucleation  thoroughly  with  them,  and  washed w i t h e t h a n o l  High-Pressure HPLC  analysis  Water A s s o c i a t e s  the  the  for  30  minutes.  tube  and  to  frozen. instruments  remove  sonicator  sonication  any  Cy t h a t  microtip  of  each  variable  the  samples  (Milford,  was  a  column heater  spectra  physics  Pure  Cy  Switzerland) courtesy of  of  Western  detector,  and  a and  may also  sample.  system  a  481  a  consisting  Intelligent Lambada-Max  M660 s o l v e n t control  with  programmer,  module,  and a  integrator.  donation pure  Ontario),  of  50  ug/ml  solution  of  the  ug/ml  100% m e t h a n o l .  Sandoz  Ltd,  Basle,  C  University  University  were of  from  dihydrocyc1osporin  Dr F r e e m a n ,  solution  in  a model  temperature  4290  (  was  c a r r i e d out  Massachusetts)  autosampler,  wavelength  were  C h r o m a t o g r a p h y (HPLC)  of  Processor)  and  supernatant  o f t w o M6000 p u m p s , a m o d e l 710B WISP ( W a t e r s Sample  vitreous  seconds,  The  surgical  ethanol  before  Liquid  used  five  in a microtest  frozen  tube and d i s r u p t e d w i t h a  being  2500 RPM f o r  each  coating  a test  microtip  placed  Before  in  the  used  Cy i n  internal  to  Hospital, prepare  100% m e t h a n o l  and  p r e p a r e d r a n g i n g from 2 5 n g / m l t o  10 u g / m l  -  stock stock  s t a n d a r d C y C was p r e p a r e d a s  Standard concentrations  50  the  (CyC  10  of  Cy were  from t h e  primary  stock  solution  standard  by  curves  serial  were  solution,  obtained  from which a p p r o p r i a t e  (figure  3.4).  E x t r a c t i o n Procedure  Two d i f f e r e n t  methods  levels  o f Cy i n t h e s a m p l e s  Method  #1.  For the detemining  the and  extraction  is  already  Cy l e v e l s Fields  as  Elut( ) R  the  100 mg o f  the  a  the  method used  for  on t h a t  few  developed  modifications.  with the m o d i f i c a t i o n s  use  s o l i d phase  in of  addition CN-Bond  columns  to  apparatus  The  apparatus  E l u t e s ( ) (B.E),  which  R  (of  1.0  ml  (both  by  identified:  the  c a p a c i t y and  c y a n o p r o p y l phase packing)  extraction  International,  was b a s e d  with  5  determine  collected.  required  mentioned  are disposable  6  follows  method  containing  r e q u i r e d to  u r i n e and o c u l a r s a m p l e s  Lensmeyer  This  were  from  and t h e V a c  Analytichem  Harbor C i t y , CA).  Reagents. HPLC  grade  acetonitrile  and m e t h a n o l  L a b o r a t o r i e s L t d . , Geogretown, O n t a r i o ) , prepared (Millipore  with  the  Corp.,  "Milli-Q" Bedford,  water  the  reagents  needed.  51  Caledon  deionized  purification  MA), and g l a c i a l  ( f r o m BDH C h e m i c a l s C a n a d a L t d . , preparing  (from  water system  acetic  acid  V a n c o u v e r ) were used  in  S o l u t i o n A: The d i l u e n t c o n s i s t i n g (70/30 by v o l )  Solution  B:  to  remove  Solution to  remove  Mobile vol),  C:  to  dilute  the  acid  (0.5  Acetic acid  (0.5  consisted  w h i c h was  filtered  of  Treatment  internal  ul  standard  5 minutes  internal  at  x  u r i n e or o c u l a r sample, ug/ml  of  100mm d i s p o s a b l e  2500 RPM.  {In  use.  50 u l  of  the  solution A  L e n s m e y e r ' s method  C y D a n d S o l u t i o n A was  Bond E l u t e C o n d i t i o n i n g -  CN-B.E.  (about  system  f o r 20 s e c o n d s a n d c e n t r i f u g e d  (70/30 by  volumes  by  centrifuge  CyC) and 2 m l o f  CyD i n w a t e r / a c e t o n i t r i l e  reservoir  used  (60/40  and d e g a s s e d p r i o r t o  of  s t a n d a r d was  (60/40)  /acetonitrile  To a  (10  16  used  CN - B . E .  -  were added. Then v o r t e x e d for  for  (80/20)  through a m i l l i p o r e f i l t e r  Sample  250  used  from CN - B . E .  water  a 22 urn d u r a p o r e f i l t e r )  tube  vol)  M) / a c e t o n i t r i l e  (using  test  from t h e  i n t e r f e r i n g substances  -  by  M) / a c e t o n i t r i l e  i n t e r f e r i n g substances  Phase  (80/20  CN - B . E .  Acetic  D:  water/acetonitrile  samples.  Water/acetonitrile  conditioning  Solution  used  of  1.0  52  ml)  350 u g / 1  of  vol).}  was of  the  primed  acetonitrile,  with  two  and  then  f o l l o w e d w i t h two r e s e r v o i r v o l u m e s diluted  sample  CN-B.E.,  making  between  was  passed  sure  that  passed  Elution  through the  last  packing,  of  residual  liquid  CN-B.E.  and  collected  -  left  into  Celsius,  the  did  not  dry  the  sample  CN-B.E. packing, the  CN-B.E.  then  from t h e  0.4  meniscus  of  the  of  solution top  washed  of  with  C.  16  x  from  two  minutes.  100  mm d i s p o s a b l e  under  and t h e  a  pressure  the  the  CN-B.E.  0.25  ml  centrifuged  dry extract  of  The e l u a t e  then  centrifuge  test  eluate  nitrogen  at  liquid was 40  f o r twenty The  reconstituted  e v a p o r a t e d u n d e r a s t r e a m o f a i r a t room t e m p e r a t u r e ,  and  was  reconstituted  phase.}  53  method  with  the  for was  dry extract  Lensmeyer's  with  elutant  the  {In  then  s e c o n d s and  was t h e n t r a n s f e r r e d t o a l i m i t e d - v o l u m e i n s e r t  chromatography.  was  degrees  was t h e n r e c o n s t i t u t e d  a t 2500 R P M .  the  was  The  vortexed  for 5 minutes  of  remove  was a d d e d t o  any r e s i d u a l  CN-B.E.  stream  of mobile phase,  After  CN-B.E.  ml o f a c e t o n i t r i l e  and u n d e r r e d u c e d  evaporated  extract  After  was  for  collected  ul  conditioned  D a n d r e d u c e d p r e s s u r e was m a i n t a i n e d t o  Elution  250  -  r e s e r v o i r volumes  CN-B.E.  Isolate  be  the  CN-B.E.  S o l u t i o n C had passed  the  Solution  tubes,  the  top of the  was washed w i t h two  any  through  the  applications.  Interference  the  o f S o l u t i o n B. T h e n  150  ul  of  mobile  Chromatography  50 into  microliters  the  HPLC  of  the  system  which  CCS/C8  (5 m i c r o n p a r t i c l e  mobile  phase  range  set  at  flow  heater  was  set  Lensmeyer's procedure the  x  x  was  4.6mm Whatman  was  set  integrator  and  degrees  Celsius.  {  with detector  214nm and r e c o r d e r c h a r t s p e e d  at  permaphase  range set 1  upon  extraction levels  thawing  that  impossible  i n the b l o o d  plugged  using  method#l  frozen,  the  blood  Freeman e t  al.  E T H (30 0.02  AUFS  to  substances  CN-B.E.,  making  determine  the  Cy  samples.  The method u s e d t o d e t e r m i n e t h e Cy l e v e l whole  at  and a  cm/min.}  B e c a u s e t h e w h o l e b l o o d s a m p l e s were formed  In  c o l u m n u s e d was a 25 x  4.6mm g u a r d c o l u m n p a c k e d w i t h size),  The  detector  speed  physics  analytical  column. with  chart  72  injected  Z o r b a x - c y a n o p r o p y 1 (5 m i c r o n p a r t i c l e s i z e )  micron p a r t i c l e at  at  25cm  ml/minute,  spectra  column  sample  analytical  c m / m i n u t e on t h e  the  5cm  1.5  a  210nm,  0.25  4.6 mm  size)  r a t e was AUFS  had  at  at  0.01  extracted  samples  was  that  The method i s  as  developed  i n the  by  frozen  Carruthers,  follows:  M e t h o d # 2: Reagents HPLC g r a d e Phillipsburg, (from  Diethyl N.J.),  ether  HPLC  (from J . T . B a k e r C h e m i c a l C o . ,  grade  acetonitrile  C a l e d o n L a b o r a t o r i e s L t d . , Geogretown, 54  and  methanol  Ontario),  and  deionized  water  purification used  to  prepared  system  prepare the  reagents  1:  0.2  M HCL  Solution  2:  0.2  M NaOH  3:  consisting  1.2 of  M (NH ) 4  20%  the  "Milli-Q"  ( M i l l i p o r e Corp.,  Solution  Solution  with  S  0  2  Bedford,  water  MA) w e r e  needed.  4  i  40.0  n  acetonitrile,  ml  20%  of  a  solution  methanol,  and  60%  water.  Reconstitution 2.0  Fluid  ml o f m e t h a n o l ,  -  consisted  5.0  of  3.0 m l o f  m l o f w a t e r a n d 1.0  acetonitrile,  ml of  Solution  3.  Mobile  Phase:  methanol, liters  33.05% w a t e r  of  mobile  m i l l i p o r e Durapore  consisted  (Millipore) ( ) R  Sample Treatment test  tube  microliters 2.0  ml  seconds.  of  250 of  To t h i s  system filter)  acetonitrile,  of  was  Solution filtered  (using  a  1 were  m i x t u r e 6.0  55  3 per  22  whole  standard added. ml o f  (10  to  Then Diethyl  a  use.  centrifuge  blood, ug/ml  2.0  micron  and d e g a s s e d p r i o r  of  20%  through  I n a 16 x 100 mm d i s p o s a b l e  internal  Solution  ml  which  microliters  the  46.95%  a n d 0.5  phase,  f i l t e r  of  50  o f CyC) and  vortexed ether  was  for  20  added  and 10  shaken  on  minutes.  a  Then  The aqueous  horizontal  centrifuged  a c i d phase  the  Diethyl  for  30  RPM.  The  ether  pipette was  to  then  degrees  2500  This  was  RPM 5  for  minutes.  was t h e n  for  ether-  vortexed  5 minutes  at  then t r a n s f e r r e d with a  a 16 x 100mm d i s p o s a b l e evaporated  RPM f o r  mixture  and c e n t r i f u g e d  phase  100  a n d 2.0 m l o f S o l u t i o n 2 was a d d e d  ether.  seconds,  at  at  was t h e n d i s c a r d e d w i t h a n  rinsed Pasteur pipette to  shaker  under  a  test  stream  tube.  of  2500  Pasteur  The  nitrogen  ether at  40  Celsius.  The fluid  dry extract and  was  vortexed  reconstituted  for  20  with  reconstitution  The  reconstituted  seconds.  e x t r a c t was t h e n t r a n s f e r r e d t o a l i m i t e d - v o l u m e i n s e r t  for  chromatography.  Chromatography The  analytical  containing  Spherisorb-C8  constructed University ml/min., column speed ul  of  each  column used  by  Dr.  Freeman  of Western  detector  the sample  elutors  on t h e  extracted there  from  the  of  Ontario.  settings  h e a t e r was s e t setting  packing  at  were  was (5  a  25cm x  micron  3.2mm  particle  University rate  0.01  at  72 d e g r e e s C e l s i u s  spectra physics  was  0.25  size),  Hospital,  The f l o w AUFS  I.D.  was  1.0  210nm,  the  and t h e  chart  cm/min.  50.0  s a m p l e was c h r o m a t o g r a p h e d and between  was  an a c e t o n i t r i l e  column.  c o n t r o l l e d by a M660 s o l v e n t  The  wash t o  remove  acetonitrile  programmer.  wash  late was  The M660 g r a d i e n t  c o n t r o l l e r was programmed a s 0-15  m i n 100% m o b i l e  15-17  m i n 100%  follows:  phase  acetonitrile  17 m i n 100% m o b i l e  phase.  T h e r u n n i n g t i m e was 17 m i n u t e s w i t h a n e q u i l i b r a t i o n delay of  eight  minutes  and a t o t a l  run time of  on  1 and 2  25  minutes.  Results Performance in  Table  twice The  the  III.  by  concentration  Method  The s e n s i t i v i t y  baseline  standard  developed  data  noise)  curves plotting  for  linearity.  sample  extracted  each  method  relative  area  squares  The chromatograms are  presented  57  (signals  ranged from  f o r C y , and l e a s t  assess  limit  are  15 t o (figure ratio  presented equal 25  ng/ml.  3.4)  (Cy/CyC)  r e g r e s s i o n was for  in figures  each  type  3.0-3.3.  to  were vs used of  Table III Performance Data on Methods 1 & 2 Method 1 2  n 20 20  Precision day to day mean concn, ng/ml 1 06 1 00 1 06 100  S.D. 6.8 7.5  C.V. % 9.9 113 .  Analytical recovery Mean Recovery % 90 70  Linearity (linear up to at least 1400 ng/ml) y=(5.109E-4)x + 4.165E-3 r=0.9980 y=(2.075E-4)x + 2.789E-5 r=0.9989 x= Cy concentration in ng/ml y= relative area ratio C y / C y C  After Subconjunctival The  levels  (that  injected  hour,  then  In  the  at by  Cy i n  decreased  1078 12  controls)  aqueous  subconjunctival ly)  vitreous  peaked ng/ml  of  administration  to  119  humor o f ng/ml  hours  the  at  two  (figure  levels  aqueous and v i t r e o u s  reaching hours  method,  of  is  4 5  levels  of  Cy i n  only  104  ng/ml,  (figure  (figure level  the  left  3.5).  to  eye  undetectable  one  o f Cy  then decreased  In  Cy were  the  eye  ng/ml at  hours  eye  hours,  Cy i n  subconjunctivally,  under-the-curve The  right  right  253  (  the  in  the  o c u l a r humors  when  humor.  The b i o a v a i l a b i l i t y administered  by 12  3.5).  of  the  p e a k e d a t 718  ng/ml  the  humor o f  3.6).  the  calculated  shown  whole  area-  at  hours  i n Table IV. blood  peaked  then decreased  Cy  by an  levels  in  to  4  54 n g / m l by  the  urine  12  were  undetectable.  After  Intravenous  The Cy l e v e l hours,  then  i n w h o l e b l o o d p e a k e d a t 1061 n g / m l a t  decreased  The c u m m u l a t i v e the  Administration  r a b b i t was  to  74  ng/ml  by 12  urine elimination less  than  1% o f  the  of  hours  (figure  Cy o v e r  injected  0.5 3.6).  12 h o u r s dose  in  (figure  3.7) . The d a t a were p o o l e d a paired t-test between them.  for  the  right  s h o w e d no s i g n i f i c a n t  The l e v e l s  of  Cy i n t h e  and  left  eyes,since  difference  (p=0.05)  a q u e o u s humor were  undetectable. The  levels  ng/ml  at  hours  (figure  The  of  Cy i n t h e  two h o u r s ,  then  humor p e a k e d a t  decreased  to  164  n g / m l b y 12  of  intravenously  is  Cy i n  the  ocular  humors  shown i n T a b l e I V .  Table IV Bioavailability of Cyclosporine in Ocular Humours Humour, Bioavailability (ng/ml.hr) Route of administration subconjunctival Intravenous  292  3.5).  bioavailability  administered  vitreous  Aqueous Humour Right 4.7  Vitreous Humour Right 1 0.4  not detectable  60  5.3  when  Figure  3.0  A typical  chromatogram of  Dihydrocyclosporin of  Cyclosporine  C (A)  (B)  DRUG-FREE EXTRACTED AQUEOUS HUMOR  aqueous eluting  eluting  B.  at  12  humor w i t h at  10  the  minutes  internal  and  a  standard  250ng/ml  standard  minutes.  EXTRACTED AQUEOUS HUMOR STANDARD  METHOD # I Column: 25cm x 0.46cm Whatman C C S / C ( 5 u m ) Injection Volume: 50ul Mobile Phase: 40% Acetonitrile, 60% Water Column Temperature: 72°C Flow Rate: 1.5 ml/min Detection: 210 nm 8  INJECT  61  Figure  3.1  A typical  chromatogram o f  Dihydrocyclosporin of  C.  Cyclosporine  (B)  DRUG-FREE EXTRACTED VITREOUS HUMOR  C (A)  vitreous eluting  eluting  D.  at  12  at  humor w i t h 10  the  minutes  internal  and  a  standard  25Qng/ml  standard  minutes.  EXTRACTED VITREOUS HUMOR STANDARD  METHOD # I Column: 25cm x 0.46cm Whatman CCS/C (5um) Injection Volume: 50ul Mobile Phase: 40% Acetonitrile, 60% Water Column Temperature: 72°C Flow Rate: 1.5 m l / min Detection: 210 nm 8  INJECT  62  Figure  3.2  A t y p i c a l chromatogram o f u r i n e w i t h the (A) e l u t i n g a t 10 m i n u t e s and a 2 5 0 n g / m l a t 12 m i n u t e s .  E.  DRUG - FREE EXRACTED URINE  F.  internal standard  standard Dihydrocyclosporin C o f C y c l o s p o r i n e (B) e l u t i n g  EXTRACTED URINE STANDARD  METHOD # I Column: 25cm x 0.46cm Whatman C C S / C (5 urn) Injection Volume: 50ul Mobile Phase: 40% Acetonitrile, 60% Water Column Temperature: 72°C Flow Rate: 1.5 ml / min Detection: 210 nm 8  INJECT  63  Figure  3.3  A t y p i c a l chromatogram o f whole blood w i t h the i n t e r n a l standard D i h y d r o c y c l o s p o r i n C ( A ) e l u t i n g a t 1 0 . 3 0 m i n u t e s and a 5 0 0 n g / m l s t a n d a r d o f C y c l o s p o r i n e (B) e l u t i n g a t 1 3 . 0 0 m i n u t e s .  G.  DRUG - FREE EXTRACTED WHOLE BLOOD  H.  EXTRACTED WHOLE BLOOD STANDARD  METHOD  #2  Column: 25cm x 0.32 I.D. (Spherisorb C , 5um) Injection Volume: 50ul Mobile Phase: 46.95% Acetonitrile, 20.00% Methanol, 33.05% Water Column Temperature: 72°C Flow Rate: 1.0 ml / min Detection: 210 nm 8  INJECT  64  Figure  3.4  STANDARD METHOD  CURVE  FOR  CYCLOSPORINE  USING  NO. I  Y = (5.IIE - 4) + 4.I7E-3  0  200  400  600  800  1000  CYCLOSPORINE CONCENTRATION (ng/ml)  STANDARD METHOD  CURVE  FOR  CYCLOSPORINE  USING  NO. 2  Y= (2.07E -4)X • 2.79E-5  CYCLOSPORINE CONCENTRATION  65  (ng/ml)  Figure  3.5  LEVELS  OF  CYCLOSPORINE  IN T H E  OCULAR  C H A M B E R S OF T H E  66  EYE  Figure  3.6  BLOOD L E V E L S OF C Y C L O S P O R I N E AT A 5 m g / O . I cc DOSE  IN T H E RABBIT  2000 1000-  100  TIME  (Hours)  Levels of cyclosporine in the blood following subconjunctival (x) or intravenous (•) administration  67  Figure  3.7  C U M M U L A T I V E URINE ELIMINATION C U R V E FOR CYCLOSPORINE IN T H E RABBIT GIVEN A 5 m g / 0 . l c c S Y S T E M I C DOSE 0.15 •  0.10-  /  /  /  /  /  s y  0.01 0  1  1  2  1  1  1  1  4  TIME  1  6  1  1  8  1  1  10  1  (Hours)  N O T E : - U r i n e levels were not detectable in rabbits given cyclosporine  68  subconjunctival^.  1—  12  DISCUSSION Cyclosporine undecapeptide inflatum. agent,  (Cy) obtained  it  proved  cause  early  stage  to  the  of  from  the  fungus  developed  a potent  when  1 5  '  given  transplants,  of  of  autoimmune  3.8);  in  of  it  of  1  it  anti-fungal  it  that at  has  been  intravenously  organ r e j e c t i o n promise  in  comprise  is  attributable.  Cy h a v e  in position  the  some  has  unique  a nine  amino a c i d s  to  l a c k of  Cy  also  features  carbon  exhibits  large  amino  which  found  leukcocytes  peak  In  serum  3.  1 5  the  rate.  first  for  w h i c h may  general  and  the  the  It  seven  contribute  orally.  displays  5 4  second  phase  are  the  achieved  the of in  in  a biphasic blood  40% i n p l a s m a ,  pharmacokinetics  concentrations  69  that  D-alanine  Finally,  representing  in erythrocytes,  ),  acids  its  inter-individual variations  and c l e a r a n c e  pattern,  tissues.  amino  except  in position  are N-methylated,  distribution  in  the  d e g r a d a t i o n when a d m i n i s t e r e d  bioavailability  50% i s  of  L configuration  8 and s a r c o s i n e  of the its  A l l  in the  a c i d , w h i c h was p r e v i o u s l y u n k n o w n a n d t o w h i c h much o f activity  an  1 5 , 5 1 - 5 3  reveals  position  an  or  shows  diseases. Cy  as  T-lymphocytes  orally  and  4 8  Tolypocladium  Currently,  5 6  cyclic  immunosuppressive  suppression  activation.  structure  (figure  hydrophobic  p r o p h y l a x i s and t r e a t m e n t  allogenic  The  be  selective  found b e n e f i c i a l  treatment  a  Although o r i g i n a l l y  could  for  is  (in  and 10%  peripheral Cy show 2 to  6  that hours  Figure  3.8  c  CH3 V  ^CHs CH  C H ^ ^CHs  MO ««4;  X  CH, ».H i »,H — N -N • C H , - N - C - CO - N — C — C CO  AAIO  m  s  AA11  CH3  CH2  H C- CO—N—C-—CI II A A1  AA2  0  CHs  -A— AAS  O i :  I  CO  CHs AA9  CH-CH2»«C  N-CH,  CHS CH,-N AAS OC-  ftr  AA7  I  N-CO-C —-N-CII I J *H H CH3 0  Structure  AAC  H  0  AAS  II  H  AA 4  C— N — C — C — N — C O — C 1*H I CHj C H s I /CH CH, CH3 CHS CHs CH3 CH3 CHs  i»  of Cyclosporine  70  ( M o l e c u l a r Weight o f  *  1202)  w i t h a T-jy of  34  °f  2  1  6  +/-11%.  5 6  p r i m a r i l y by t h e are  excreted  intact  into  In  The  4 00  5 7  into  ng/ml.  1 5  '  immunosuppression.  which has problem  of  Cy s u c h  of  liquid  this  immunosuppressed  limit  levels  of  excreted  safety  with and  nephrotoxicity  and  essential and  to  greater  (RIA)  6  3  (HPLC).  ,  6  to  avoid  maintain  have  of  25-50 n g / m l ) ,  o r had t o x i c  samples  method  was  metabolites. patients levels  that  it  easily,  of  that  o f Cy.  evolved and  4  high  Prior  6 5 - 6 7  to  to  Cartier,  6 7  it  the  major  could  The r e s u l t were The  not was  either advantage  with a t u r n - a r o u n d time  of  samples.  analysis  and was 71  but  c o u l d be p e r f o r m e d o n a  by HPLC w h i c h a l l o w s  i n w h o l e b l o o d t o b e d e t e r m i n e d was  Sawchuk a n d  1%  ng/ml,  procedures  that  for a batch of  The s e c o n d  Cy  procedure  R I A p r o c e d u r e was  6 hours  is  chromatography  impression  4 to  is  i n p l a s m a and b l o o d were measured by RIA (  with  number o f  than  and 400  as  radioimmunoassay  a  large  drug  concentrations  of  b e t w e e n Cy and i t s  the  at  Cy l e v e l s  distinguish  of  less  b e t w e e n 100  Two d i s t i n c t  a detection  false  this  narrow margin  effects  Cy l e v e l s :  Cy l e v e l s  of  5 6 , 5 7  side  performance  with  manifested  monitoring  toxic  1982  a  effects are  bioavailability  1 5 , 5 6  concentrations  Careful  measure  metabolism  bile,  Cy h a s  toxic  and an o r a l  y i e l d i n g numerous m e t a b o l i t e s w h i c h  the  urine.  hepatotoxicity  the  '  addition,  major  than  hours,  liver,  the  therapeutic the  +/-8  able  to  for  i n t r o d u c e d by  distinguish  Cy from  its metabolites depending  on  modified  the  sample.  others  in  6 5  analytical  of  of  (reverse  but  210nm. " 6 5  Important but  a l l  to  have  the  upon  Whole b l o o d  of  get  C  1  8  the  and  speed In  Cy for  a  is true  they  use  by  and t h e  that  the  of  of  high  temperature  is  not  plasma  reflect the  the  6 5  of  the  body,  at  '  (free  7 3  '  1 5 , 5 6  '  to  is,  since  levels 5 7  '  and 7 1  '  it  7 2  is  of  Cy,  the  free  amount o f is  drug  controversy  whether or whole  method o f measurement  g i v e s the  the  7 5  know  c l i n i c i a n a better  to  use  blood.  of  favoured based  immunosuppresion b e i n g o b t a i n e d .  the  because  7 4  the  levels)  the  variable  actual  that  only  separation.  there  currently being  this  widely  desirable  v a r i a b i l i t y associated  follow  type  cyanopropyl)  g  safety.  measurement;  setting is  of  picture  generally  as  is  preferable,  p l a s m a methods  Cy l e v e l  general,  or C ,  levels  plasma  is  rationale  and t o  in  it  clinical  a v o i d the  samples  Cy  narrow margin o f  traditional  level  was  h a s b e e n by a U . V . d e t e c t o r  temperature  r e g a r d i n g method o f  blood  the  its  of  available  Cy  analysis  to  Though  In the  procedure  the h a n d l i n g of the b l o o d samples,  dependent  levels  increase  required  monitoring  of  because  HPLC  7 0  concentration  important  to  phase  c h r o m a t o g r a p h y and d e t e c t i o n  method,  The  6 7  order  extraction  column  varied  about  '  l i m i t o f 10 t o 25 n g / m l  o f t h e d e t e r m i n a t i o n o f Cy l e v e l s .  methods  have  the  by  sensitivity  and had a d e t e c t i o n  whole on  the  picture  In order  with plasma l e v e l  of  to  methods,  s u g g e s t i o n s p r o v i d e d by MRC r e v i e w e r s  of  this  project,  we c h o s e t h e  Therefore, levels  the  was  whole b l o o d method.  method  HPLC,  chosen  because  this  d i s t i n g u i s h between Cy and i t s blood  samples.  Fields  6  samples, was  not  al.,  frozen  able  ° was  used  from t h e  samples,  Cy  aim  result  of  this  whole  These  method  b l o o d samples.  level  and Freeman  of  methods  most o f t h e  Cy i n  were  the  chosen  interfering  and h a d g o o d r e c o v e r y as w e l l  An  (70-90%)  as a C . V . o f  9-  the  local  intravenous  study  of  route  was  a d m i n i s t r a t i o n would  C y , and a v o i d t h e with systemic  levels  the to  enhance  i n the  blood  administration that  nephrotoxicity.  study  showed  that  peak c o n c e n t r a t i o n s  i n the v i t r e o u s  given  administered were  to  o c u l a r and u r i n e  pharmacokinetic  vs  whether  in  0.1 t i m e s was  the  25 n g / m l )  have been a s s o c i a t e d  and  able  and h a n d l e  by C a r r u t h e r s  determine  ( 10 t o  o c u l a r absorption of  The  is  ng/ml.  substantiate  can  frozen whole  samples.  subconjunctival  that  Cy i n t h e  t h e y were a b l e t o remove  100  The  method  o f Cy  d e v e l o p e d by L e n s m e y e r and  developed  to  blood  sensitivity  11% a t  measure  handle the  HPLC method  substances and  to  whole  because  to  analysis  but the e x t r a c t i o n procedure used i n t h i s  alternate et  used  the  metabolites  T h e HPLC method  was  5  for  undetectable  in  Cy were  and b l o o d r e s p e c t i v e l y  subconjunctivally  intravenously.  of  In  compared  addition,  rabbits  that  when  with  aqueous received  4  Cy  levels Cy  i n t r a v e n o u s l y , and t h e b i o a v a i l a b i l i t y o f s u b c o n j u n c t i v a l l y  administered that  Cy i n t h e  achieved  following  ocular  pharmacokinetics  showed  that  and the peak  and  vehicle which  in close the  results.  The  than  administration.  The  route  Cy p e a k e d a t  hours.  The d e l a y  findings  Since  the  bleb  hour  in  the  partly  from t h e  showed  also  1/2  vehicle  castor  oil  (containing  large  With vitreous  aqueous  expected  seen  to  from  the  of  Cy  amount  r o u t e may a l s o  the  chamber,  t h e Cy  peak b e f o r e  that  pharmacokinetic attained  by  the  h a v e been enhanced by  produced  by  in  the  subconjunctival  inflammatory response  would a l t e r  both blood  enhancing a b s o r p t i o n . regard  to  a n d aqueous  remained above  contrast,  the  a n d o c u l a r p e r m e a b i l i t y a n d may i n c r e a s e  thus  levels  the  trough  following the  dosage  were  below  obtained  subconjunctival  therapeutic  and r e m a i n e d j u s t but  levels  range  o b t a i n e d by s y s t e m i c  particular  12 h o u r s  of  study, which  are  was  The l o c a l  vascular  vitreous  greater  o c u l a r chambers can  histological  inflammation  injection.  this  two  proximity to  as  subconjunctival  they  times  subconjunctival  between t h e  aqueous  vitreous,  flow,  at  posteriorly.  in  l o c a l  the  levels  levels  by t h e  two  a n d Cy) f o r t h e f i r s t h o u r i s n e a r t h e l i m b i c a r e a ,  is  levels  of  determination  spreading  was  intravenous  aqueous  concentrations  dose  the  the  vitreous  be e x p l a i n e d  vitreous  were  only  above  the  for  in  the  injection,  12 h o u r s .  In  administration  at  detectable therapeutic  these obtained  in  in range  the  f o l l o w i n g a d m i n i s t r a t i o n o f Cy s u b c o n j u n c t i v a l l y .  the for  aqueous  Equally following below  important  this  route  (4 t i m e s )  toxic  side  the  control  the  quickly  sanctuary.  1 5  '  following systemic  affected or  levels  equidose  '  5 6  local is  therapy  based  with  the  i n the case  of  the  concept  population  general  local  that  disease  under  will  not  population  numerous d r u g s h a v e  been  following  systemic administration  in  shown  administration  administration,  lower  well  to a v o i d  on t h e  bring  the  systemic  remain  levels  5 7  following  equidose  blood  needed  lymphocyte  easily  better  ng/ml)  gradually  In a d d i t i o n ,  be a b s o r b e d  versus  (400  cy.  can  observed  a d m i n i s t r a t i o n w h i c h were  for pulsed  treatment  since  exchange  to  of  the  inflammatory disease  succesive  this  of  level  effects  The r a t i o n a l e ocular  are  and  than that  subconjunctival  i n many  drugs  2 0 - 2 4  .  Conclusions The  results  pharmacokinetic for  ocular  from  basis  that  of of  manner.  2 0 - 2 4  studied  but  ocular  absorption  oral  in or  the  i n the  other  ocular I.V.  use  of  Cy  disease.  drugs  obtained  case of is  experiment  Cy,  better  Local  provide  In  general,  vary with the  with  peaked  administered  75  in  this  drug  later  being  suggesting  h y d r o p h i l i c than  a d m i n i s t r a t i o n may h a v e  inflammatory  the  subconjunctival  administered  it  d i s e a s e which has Cy, but  a  subconjunctivally  Cy a d m i n i s t e r e d  The l e v e l s  hydrophobic drugs. play  for  inflammatory  pharmacokinetics resembles  this  which  with  a role  to  responded  to  had  to  be  discontinued toxic  because  systemic  of  the  l e v e l s o f Cy.  76  nephrotoxicity  associated  with  CHAPTER 4 Uveitis  study  Introduction Uveitis nature  is  of  an o c u l a r  the  difficult  to  causal  treat  succumb t o t h e during  types  basic  of  included  use 2  agents have been also  caused  infection, marrow  initially  mechanism  corticosteriods '  is of  autoimmune resembles  in  the  side  of oral  has  the  eye,  such  as  chronic  to  is  ultimately  inflammation  be  involved The  in  many  treatment  has  agents  like  such  2 - 8  as  These  5 - 8  to v a r y i n g degrees,  effects  but  have  susceptibility  ocular pressures  been  demonstrated  (EAU)  used  induced  successfully  because  T-cel 1 activation. doses  may  It  1 0 , 1 1  '  1 4  and  by  that  to  bone  experimental  retinal  S-antigen  immune-mediated o c u l a r i n f l a m m a t o r y  been  ^  eye  the  1 2 , 1 4 , 1 5  treatment  patients,  unknown.  immunosuppressives.  s e e n i n humans, w h i c h i s m a i n l y Cy h a s  of  thought  successful  uveitis  the  effects  and  1 4  suppression. it  and t h e  anti-inflammatory  increased  Recently  usually  immunological.  serious  9  is  i n which  recurrences.  uveitis the  factor  secondary  subsequent  The  inflammatory disease  needed t o resulted  nephrotoxicity,  of it  is  T-cel1-mediated. systemically  response 1 8 , 2 7 , 3 4  and  orally  T-cel1-mediated  uveitis  able  early  to b l o c k the  in stages  U n f o r t u n a t e l y the high systemic get  therapeutic  in  some  17  4?  'I^I^  levels  serious  o f Cy  and into  complications  From  the  pharmacokinetic  administration amounts  of  Cy,  penetrated  high blood levels To  assess  the  single  of  whether  subsided. might  in  intravitreal started  show t h a t while  effective  EAU was  bovine  higher  avoiding  the  serum  for  in  the  albumin  developed  at  least  one  group  injections.  one week a f t e r  rabbits  In another  intravitreal  of  into  within  a week,  a  the  six  and  to  then  inflammation  subconjunctival  of  of  possible  (BSA)  assess the degree to which the Cy  route  i n d u c e d by i n j e c t i o n  Acute u v e i t i s  suppressed,  initiated  to  subconjunctival  subconjunctival  be  and p e r s i s t e d To  be  of  able  chambers,  the  would  rabbits.  days,  ocular  uveitis,  dose  vitreous ten  of  were  of  of Cy.  administration treatment  we  study  therapy  immediately  was after  g r o u p Cy t h e r a p y  was  injections.  M a t e r i a l s and Methods Prior  to  examined  by  the  intravitreal  slit-lamp  presence of p r e - e x i s t i n g  injection  biomicroscopy  2.4kg) ml/kg  were t r a n q u i l i z e d by of  a  hydrochloride Ayerst  10:1  intramuscular  solution  (100  and a c e t y l p r o m a z i n e  previously  with  proparacaine  prepared s o l u t i o n  78  of  were  out  Fifteen  injection of  the  locally  (weighing  mg/ml)  maleate  eyes  rule  rabbits  Laboratories, Vancouver).  anaesthetized  to  ocular diseases.  s u p p l i e d New Z e a l a n d w h i t e f e m a l e  a l l  2.2 of  0.2  ketamine  (Roget S . T . B .  and  A f t e r b o t h e y e s were  HC1 (0.5%), 50 mg/ml o f  0.10 BSA i n  ml  ofa  Saline  (which  had been  sterilized  a Millex-Gs( )  through  injected  into  the  ciliary  body  injection 0.10  of  ml  of  used  of the  to  The  vitreous,  lens.  BSA s o l u t i o n  Prior  filter  unit)  taking care to  avoid  to  the  paracentesis was  intravitreal  was p e r f o r m e d and  removed  to  prevent  i n t r a v i t r e a l i n o c u l u m . The  received  perform  0.10  ml  of  saline  opposite  to  serve  as  a tuberculin syringe  was  aqueous  divided  2 received  paracentesis  and  into  three  after  groups  the  of  the  five  intravitreal  subconjunctival  injection  Cy i n O . l c c o n c e p e r week a n d G r o u p 3 r e c e i v e d i n j e c t i o n of  i n o c u l a t i o n w i t h BSA. received  saline  to  Immediately  Group  subconjunctival after  the  were  each.  injections, 5 mg o f  attached  injections.  animals  rabbits  no  5 mg o f  except  the  C y i n O . l c c o n e week  Group 1 s e r v e d a s t h e  treatment,  of  for  O.lcc  controls  of  normal  w h i c h was g i v e n s u b c o n j u n c t i v a l l y o n c e p e r week. The of  injected b y means syringe The  the  micron disposable  humour  gauge n e e d l e  intravitreal  5 mg  and  filtering  control. A 30  and  right  aqueous  vitreous  the  the  BSA an aqueous  extravasation left  0.22  R  was  by  Cy  in  O.lcc  posterior of once  rabbits  biomicroscopy, photography.  to  and  the  O.lcc  superior  a 25 g a u g e n e e d l e a  week were  upon  normal  limbus of  attached  to  intitiation  examined  indirect  of  daily  ophthalmoscopy  The r a b b i t s were  also  studied  saline  the  were  right  eye  a tuberculin of by  therapy. s i it-lamp  and b y two  serial other  examiners  who  h a d no  treatment  and  among  examiners  the  The  degree  according to Fox.  no  knowledge  of  the  An o v e r a l l  after  evaluating  of  significant  variability  (data not  presented  value  of  received  was  found  was  graded  shown).  inflammation  scheme  which r a b b i t s  in  each  eye  in  Table V modified  inflammation  was  from  obtained  each c r i t e r i o n f o r each each r a b b i t i n  this  experiment. The e x p e r i m e n t was t e r m i n a t e d a f t e r of  the  rabbits  pentobarbital. buffered  and  structural  of  cellular  with  in  an  overdose  of  i n 10%  sectioned  Haematoxy1in-Eosin  for  H i s t o l o g i c a s s e s m e n t was made on infiltrate,  (e.g. p o s t e r i o r  of  fixed  paraffin,  fibrin  the  specific  v a r i a b i l i t y was  80  content  synechiae,  T h i s was g r a d e d b y m y s e l f  who h a d no k n o w l e d g e No s i g n i f i c a n t  by  studies.  changes  detachment).  embedded  stained  histopathological amount  sacrificed  The g l o b e s were e n u c l e a t e d ,  formalin,  vertically  the  were  t h r e e weeks and a l l  and a  o r i g i n of  f o u n d among t h e  and  retinal  pathologist the  slides.  examiners.  Table V Conjunctiva Hyperemia  Iris  Ciliary  injection  Flush  NV of Cornea  Posterior Synechia  Anterior Chamber Cells Flares  Fibrin  Posterior Chamber Flares Cells  Overall Fibrin  Inflammation  none  none  none  none  none  none  none  none  none  none  none  0  trace  trace  trace  trace  trace  trace  trace  trace  trace  trace  trace  1  mild  mild  mild  mild  mild  mild  mild  mild  mild  mild  mild  2  mod  mod  mod  mod  mod  mod  mod  mod  mod  mod  mod  3  severe  severe  severe  severe  severe  severe  severe  severe  severe  severe  severe  4  NV = neovascuralization mod = moderate  Results Clinical  Ocular Findings  Immediately intravitreal the  after  and  anterior  became  pale  with a return to  mild  to  moderate,  saline.  aqueous  subconjunctival  chamber  reaction with  the  paracentesis,  injection  shallow  ( o f G r o u p 2)  and t h e  iris  normal w i t h i n 2 hours.  non-specific,  intravitreal  T h e r e was  transient  injections  appeared  inflammatory  w i t h b o t h BSA  and  T h e i n f l a m m a t o r y r e a c t i o n p e a k e d a t 24 h o u r s a n d  was c h a r a c t e r i z e d b y m i l d p e r i c o r n e a l h y p e r e m i a , m i l d moderate flare  vasodilation  and m i l d  of the  i r i s vessels,  i n the  anterior  fibrin  The i n f l a m m a t o r y r e a c t i o n s l o w l y two  days.  fifth  Thereafter,  day  degrees  at  which  of  been  the  point  reaction  noted  is  by  rabbits  subconjunctival  Cy  injection therapy  of  one  compared to  the  week a f t e r the  in  therapy  BSA) and  3  the 7 7  (that  controls  in  Except  for  the  2  injection  of the  for  on t h e  of  the  82  Cy  BSA) were onset of  d a i l y composites  inflammatory  the  received  course  f o r each day i n f i g u r e  transient  and  intravitreal  injection  Observations  from  (that after  the  varying  subconjunctival  ( G r o u p 1)  summarized i n a p l o t inflammation  next  transient  results  received  uveitis  overall  trauma o f  immediately  of  the  over the  This  Groups  development are  moderate  developed  The  intravitreal  uveitis.  resolved  uveitis. due t o  to  chamber.  animals  others.  experimental  m i l d to  eyes remained s t a b l e u n t i l  unilateral  inflammatory has  a  and the of  4.0.  response  to  the  trauma  of  received  the an  needle  aqueous  a l l  of  the  left  paracentesis  i n j e c t i o n o f 0.10 m l o f p h y s i o l o g i c a l  eyes  which had  and i n t r a v i t r e a l saline,  d e v e l o p e d no  uveitis. The which  right  were  treatment after and  of  injected  developed  conjunctival flare  uveitis become  the  visible  mild  posterior  flare,  iris  rabbits  mild  in  peaking  the  posterior  anterior  by t h e  The showed  and moderate  cells  quite and  fibrin;  after  were  received  mild  s t a r t e d t o s u b s i d e and a mainly  i n the  posterior  treatment,(Groups  the  uveitis  2,  intravitreal  2  and3)  inflammatory (Group 1).  was n o t e d  p e a k i n g by t h e  Group  83  lesser  i n the c o n t r o l s  groups t r a c e  thereafter. after  well,  b y d a y 21.  intravitreal injection,  immediately  moderate  observed.  response  t h a n was o b s e r v e d  subsiding  as  The  o f c o r n e a was n o t e d a n d i n t w o o f t h e  that  both the treated  and f i b r i n .  i n v o l v e d with  a s i m i l a r but s i g n i f i c a n t l y  response  The  point  inflammatory  groups  chamber.  The a n t e r i o r chamber a t t h i s  synechiae  chamber was p r e s e n t  injection,  i t had  A f t e r the 14th day the u v e i t i s moderate  perilimbal  1 4 t h d a y when  c e l l s  neovascularization  BSA a n d h a d no  T h i s was c h a r a c t e r i z e d b y m i l d  chamber h a d become  severe  (controls)  days  and f i b r i n  flare  1  trace to mild uveitis  hyperemia,  progressed  i n Group  six  quite severe.  showed  rabbits  i n t r a v i t r e a l l y with  the injections.  cells,  to  eyes  which injection,  seven  In days  14th day and was  treated  by t h e  14th  day  showed  a  mild  inflammatory treatment showed 14th  response  a slightly  the  response 14,  mainly  subsided  by day 21. both  involvement  of  to  the  a  trace  received  i n t r a v i t r e a l i n j e c t i o n o f BSA than  G r o u p 2. By t h e  m i l d to moderate  inflammatory  a trace  inflammmatory  to  2 and 3 showed  to  to  Group 3 which  A s w i t h G r o u p 1,  Groups  restricted  subsided  greater response  d a y Group 3 showed a which  which  by day 21.  one week a f t e r  response  and  response  mild  between days  that  posterior  the  seven  uveitis  chamber w i t h  slight  t h e a n t e r i o r chamber.  I n summary, s u b c o n j u n c t i v a l l y a d m i n i s t e r e d Cy was to  exhibit  the  treatment  case  with  an i m m u n o s u p r e s s i v e a c t i v i t y and t h e w i t h Cy t h e  Group  intraviteral showing seven,  the 14  was  2  that  m i l d e r the was  injection. progress  and 21 d a y s  of  uveitis.  treated  at  the  Representative uveitis  in  the  found  earlier  T h i s was  the  time  the  of  photographs  three  groups  at  inflammation depending  on  a r e shown i n f i g u r e  4.1.  H i s t o l o g i c a l Findings The the  3 g r o u p s were  amount  structural The  of  cellular  infiltrate,  fibrin  content  and  changes. common  polymorphonuclear and p l a s m o c y t e s , retinal  graded f o r  detachment  inflammatory granulocytes, while  the  macrophages,  structural  and p o s t e r i o r  84  c e l l s  i n c l u d e d lymphocytes  changes  synechiae.  included  Group chamber,  1  showed  and m o d e r a t e  region,  synechiae.  inflammation,  contained fibrin  or  the  group  angle,  2  to  perilenticular  rabbits also  detachment.  anterior  while  the  but  developed to  severe  inflamed  The  vitreous  moderate  trace  vitreous  contained  moderate  fibrin  to  severe  angle.  area,  and  One r a b b i t  choroid  retina  posterior  displayed  showed  trace  serous detachments.  inflammatory  was a s l i g h t l y  of  the  rabbits  synechia.  inflammatory inflammation  than  anterior  cells  and  with  deposits  The  mild  to  greater  inflammation  i n g r o u p 1. chamber,  T h e r e was  and m i l d  areas  showed  choroid  and of  to  iris  i n t h i s group a l s o developed  The  response  also  of  iris  inflammation of the p e r i l e n t i c u l a r r e g i o n , One o f t h e  vitreous  The  and t h e  b u t much l e s s  involvement  moderate  devoid  inflammation.  synechia.  focal  was  deposits.  g r o u p 2,  trace  mild  with  few  In group 3 t h e r e in  to  inflammation  inflammation  chamber  perilenticular  a posterior  moderate  mild  fibrin  anterior  was m o d e r a t e l y  inflammation  the  showed o n l y  had d e v e l o p e d  than  retina  complete  minimal  inflammation,  to  the  the  deposits.  In  mild  Two o f t h e  of  of  The c h o r o i d showed m o d e r a t e  while  partial  i n f lamination  inflammation  i r i s and a n g l e .  posterior  with  trace  the  focal  minimal  showed  a  moderate  retina  displayed  serous  detachments.  inflammation  with  a  mild The  moderate  present.  Representative  histological 85  photographs  comparing  the  v a r y i n g degrees of experimental  Statistical An showed the  rabbits  are  shown  in  figures  and  4.2-4.5.  Analysis  repeated that  measures  there  was  analysis  a  the  groups  was  groups  between  the  of  groups  rabbits, being  variance(nested)  be  with  the  of  level  test  between  significance  p=0.0001.  difference  p=0.047.  86  difference  The l e v e l  found to  f o u n d t o be a s i g n i f i c a n t  treated  of  significant  t r e a t e d and c o n t r o l r a b b i t s .  between also  i n f l a m m a t i o n between the c o n t r o l s  There  was  between the  two  of  significance  Figure  4.0  THE E F F E C T  OF SUBCONJUNCTIVAL  UVEITIS INDUCED BY BOVINE  87  SERUM  CYCLOSPORINE ALBUMIN  ON  Figure  4.1  E f f e c t s o f B o v i n e Serum A l b u m i n i n d u c e d u v e i t i s Cyclosporine administered subconjunctivally  lent 0) d a y 7  G r o u p 2 d a y 14  and 5mg/0 l e e  of  Group 2 day  21  Figure  4.2  Histological  effects  o f B o v i n e Serum A l b u m i n ( B S A )  i n Group 1  (received  2.5x  2.5x In some o f t h e n o n - t r e a t e d r a b b i t s p o s t e r i o r s y n e c h i a ( P S ) were p r e s e n t w i t h a m o d e r a t e t o s e v e r e i n f l a m m a t o r y r e s p o n s e and r e t i n a l d e t a c h m e n t ( R D ) * s e e l i s t of abbreviations for figures 4.2-4.5.  89  Figure  4:3  Histological  effects  o f BSA i n G r o u p 1 on t h e  The c h o r o i d ( C H ) i n t h e n o n - t r e a t e d non-granulomatous u v e i t i s .  rabbits  90  posterior  segment  showed a s e v e r e  of  chronic  the  Figure 4.4 H i s t o l o g i c a l e f f e c t s o f C y c l o s p o r i n e on u v e i t i s ( t r e a t m e n t s t r a t e d on d a y 0)  induced  by BSA i n G r o u p 2  A m i l d inflammatory response i s present i n the p e r i l e n t i c u l a r z o n e s ( F Z ) o f i n f l a m m a t i o n i n t h e c h o r i o d ( C H ) a s w e l l as f o c a l detachments(RD).  91  area w i t h focal serous r e t i n a l  Figure  4.5  H i s t o l o g i c a l e f f e c t s o f C y c l o s p o r i n e on u v e i t i s ( t r e a t m e n t s t a r t e d on day 7)  induced  by BSA i n G r o u p 3  2.5x  lOx A s l i g h t l y greater inflammatory response with focal zones(FZ) of inflammation i n t h e c h o r o i d ( C H ) and f o c a l s e r o u s r e t i n a l d e t a c h m e n t s ( R D ) are present i n t h e s e r a b b i t s c o m p a r e d t o G r o u p 2 ( t r e a t m e n t was s t a r t e d on d a y 0 ) . 92  DISCUSSION Uveitis diseases There  consists  of  have  which been  therapy  of  for  of  the  to  high  symptoms  administration application 1 0  '  1 1  to  these drugs,  8  ,  1  will  of  and  2  '  the  local 1  a  4  systemic  '  (autoimmune)  of  in  and  systemic  lternate  anti-  sanctuary.  side  due  systemic  to  of  levels  '  of  orally  an e f f e c t .  These  effects  such  as  due 1 2  to '  Cushing's-  and  1 4  The  compounded  because  have  found  current tolerance  therapy  and  resulted  prolonged  may d e v e l o p m a k i n g them  improve treatment, in uveitis  involved  with  been  has  is  been p o s t u l a t e d  origin.  Circulating 93  in some  to  be  use  of  ineffective.  an u n d e r s t a n d i n g required.  of  local  have  diseases  doses  systemic  1 5  also  In  and  administration  immunosuppressives  problem i s  1 4  within  drugs  myelotoxicity  1 5  1 3  corticosteroids  in  the  accepted  administration  these  observed  resulting  mechanism  potent  effective  Currently,  4  consisted  3 , 4  be  suitable  of  In order to processes  ,  find  uncertain.  5 - 8  inflammatory  resistant  7  remains  to  required to e l i c i t  in  corticosteroids,  ocular  ,  levels  are  resulted  glaucoma.  3  which  inflammatory-  obtain the necessary therapeutic  systemically  like  attempts  has  drugs,  ocular  represents a pharmacological  eye,  have  /  of  cort icoster iods,  immunosuppressives.  order  2  group  pathogenesis  drugs  uveitis  inflammatory  The eye  the  uveitis.  application  a  numerous  anti-inflammatory treatment  of  The  of  the  underlying  as  immunologic  and  intraocular  immune  complexes  uveitis  lends  lines  uveitis,  retinal  the  this  and h a v e  S-antigen  (S-ag).  humoral  and i s  in  contention.  resulted  in  '  i d e n t i f i e d as  In  experimental  c a n be i n d u c e d by S - a g  S-ag(EAU-S-ag)  particularly  isolation  i n the  (EAU-S-ag).  to  Experimental  has  d i f f i c u l t to  development  rats.  humoral the  This  of  not  Both  always  i n d u c t i o n of been  succesful  induce i n a r a b b i t model.  EAU.  This  resembles types  the  o f human u v e i t i s , segment  patients,  the  S-ag  in  This in  EAU.  the  specific  1  '  3  The  2  is  of  particularly  the  eye.  1 8  '  1 9  '  2 7  '  finding  1 9  '  T-cell  not necessary type  of  uveitis  of for  EAU-S-ag,  seen  in  some  i n v o l v i n g the  In  3 4  T - l y m p h o c y t e s were a l s o  vitro.  some  of  shown t o be  these  reactive  3 4  has  treatment  l e d to of  suppression  myelotoxicity  3  it  been  involvement  o c u l a r inflammatory response  posterior  to  of  that  major  p o s i t i o n has  occurred without the  immunity s u g g e s t i n g  development  the  S-ag causing a  s u p p o r t e d by t h e t r a n s f e r o f EAU f r o m u v e i t o g e n i c lines  of  systems,  S t u d i e s h a v e shown t h a t T - l y m p h o c y t e s p l a y t h e role  to  p r o t e i n f o u n d i n t h e o u t e r segments  hypersensitivity.  using  with  3 6  the  S-ag has been  2 5  3 5  patients  and c e l l u l a r immunity r e s p o n d t o  delayed-type uveitis  found  models have been d e v e l o p e d  photoreceptor.  autoimmune u v e i t i s  been  animal  48,000 m o l e c u l a r w e i g h t of  have  support to  Along these study  that  associated  the use  of Cyclosporine  uveitis,  of  T-cel 1s ,  with other  which 5  8  -  6  0  (Cy)  can  cause  without  causing  immunosuppressives  such  as  azathioprine  Cy h a s  '  been found e f f e c t i v e  inflammatory disease, suggest cells  that  for  the  The  cycle  affecting  the  increase  in  specific  the  cycle  for  discontinued.  cell  suppressor of  This  i n the  to  therapeutic  partly  and  3 1  be  because  because given  the  of  Tthe  its  Previously,  orally  use. the  1 7  '  4 2  local  '  The  may b e  without relative  due  to  the  that  the  after  disease  Cy  has  be may  been  subconjunctivally  uveitis  seen  the  due t o  and eye  doses  of  the  local  in  in renal  the  immune  Cy t h a t  systemically  result  disease  a cellular  immunosuppressive drugs  by b o v i n e  showed t h a t  produced  the  that  to  are reach  toxicity,  serum  albumin  toxicity,  of the EAU. which  of  a  injected  effectiveness  t h e s e d r u g s were a b l e  severity  value  were  were a s s e s s e d f o r t h e i r  EAU i n d u c e d  o n s e t and reduce  situation  4 3  antineoplastic  results  possible  population.  high  l e v e l s w i t h i n the  limiting  of  may be cells  clinical  may n o t be p r e d o m i n a t e l y  drugs  specific  1 8  clinic  The  findings  of T - h e l p e r  even  recurs,  against  S-ag  ocular  and c o n t i n u a t i o n  suggests  often  group  of  r e c r u i t m e n t w h i c h may a l s o  control  Unfortunately,  this  of  population  T-cell  S-ag.  under  required  D  EAU-S-ag,  by w h i c h t h i s  stimulation  T-suppressor  i n the  response,  1  treatment  case of  induction  mechanism  i n h i b i t i o n of the  remain  and i n t h e  ,  no  disease.  break  i n the  Cy c a n b r e a k t h e  needed OQ  is  and c y c l o p h o s p h a m i d e .  D  to  (  EAU-BSA). delay  However,  although  the  these  reversible  with  discontinuance  acceptable  in  the  the  subconjunctival the  of  drugs  would  c l i n i c a l  administration  side  effects  This  a p p r o a c h was  not  have  been  circumstance.  of  the  p r o d u c e d when g i v e n  drugs  Yet,  circumvented  systemically  (appendix  I) -  subconjunctival  used  injection  effects  associated  avoided,  making p o s s i b l e  inflammatory The  of  with  by t h e  Cy,  was  rabbits'  of  to  a  that  d i d not chose  causes  much  inflammatory  its  tried  side  may  be  ocular  J  Since  5 6 , 5 7  the  using 2 9  '  S - A g and 3 0  '  and  the  more  order  on w h i c h  rabbits,  likely  and c e l l u l a r  the  Unfortunately,  BSA,  is  in  3 3  by  uveitis  7 7  '  and  we  which  7 8  resulting due  to  immunity,  the 7 9  immune r e s p o n s e as  Cy c a u s e d  of  EAU-BSA.  human u v e i t i s  p r i m a r i l y by i n h i b i t i n g s t i m u l a t i o n minimal  in  uveitis  which  humoral  administered  the  acute  response  EAU-S-ag/°'"'  function,  the  Cy i n  i n any o f  a predominatly c e l l u l a r  a  of  subconjunctival ly.  induce  more  both  resembled  develop  to  by  and o t h e r s ,  2 5  contrast  has  use  effectiveness  of  in  that  the  O . l c c o n c e p e r week)  activation to  for  administration  routine  (5 mg i n  by W a c k e r  model  therefore a  hope  subconjunctivally  first  Cy a d m i n i s t e r e d  disease  Cy, i n the  the  test  EAU was  outlined  to t e s t  eye  to  procedure have  a protocol  disease.  used  Induction  develop  systemic  maximum t o l e r a b l e  dose  the  to  in seen  immunosuppression  of T - c e l l s ,  effect  on  T-cel1-independent  effect  o f  Cy  would  be  and  only  B-cell  reduced  in  EAU-BSA. at  Cy t h e r a p y was a d m i n i s t e r e d t o  the  time  of  inoculation  rabbits  received  and  control  a  received  no  The  treatment group  of  of  at  that  able  to  suggests  the  time less  that  Cy t h e r a p y s e v e n d a y s  the  effect  as  the  stages  the  of  effective  it  treatment  more  of  the  treated  control  the  treated  groups  and  the  that  of  is  side  received  h a d an i n f l a m m a t o r y of the group  activation of  of T -  and  had  primed  T-  the  likely  that  w i t h BSA.  suppression  earlier  is  a  application  to  there in by  was the  also  opportunity  subconjunctival ly  difference  significant  and e x p e r i m e n t a l  similarity explained  there  (treated)  a significant  inflammatory  the  fact  p r e d o m i n a t e l y m e d i a t e d by c e l l u l a r An  rabbits  inoculation  was o n l y a s l i g h t  groups,  between t h e  partly  apparent  the  be i n t h e  disease.  Although there two  any  suppression  Therefore,  the  subconjunctival  Cy c a u s e d t h e  early on  after  c e l l s . 15,56,58,59 Cy  of  inoculation,  o n s e t and r e d u c e  severe than that  be e x p e c t e d ,  during  the  inoculation  received  T h i s was t o  was  of  the  causing  that  l i t t l e  group  i n o c u l a t e d w i t h BSA  EAU-BSA  delay  response  cells  another  after  were  in  EAU w i t h o u t  The d a t a  Cy t h e r a p y  seven days which  showed  Cy was  severity effects.  BSA,  rabbits  treatment.  results  injection  with  a group of  arose  to  that  between  difference groups.  In  difference  response EAU-BSA  can is  be not  immunity. test  Cy  i n j e c t e d  i n a v e t e r i n a r y c l i n i c . Four cats  97  the  that  had  chronic  corticosteroid-resistant  (appendix  I I )  o  were  u  treated  subconjunctivalinjection developed cats to  i n the  received  l e s s e n the  uveitis after  0.1% t o p i c a l l y in  uveitis  was  uveitogenic resulting This of  Cy  the  the  the  showed  four  results  inhibiting  dexamethasone  the  months  twice  cats.  Possibly,  still  daily  uveitis  a  the  present  The  months on  flared  up  cause  of of  systemically,  recurrence. the  feasibility  subconjunctival ly.  support previous act  of routine  studies  synergistically.  the  activation  inhibits  of  T-helper  inflammation  In  that  Cy a c t s  3 3  application  by  later  activated.  98  after  the  Cy  cells,  though  stage  addition, and  predominatly  p r o d u c t i o n and r e s p o n s i v e n e s s o f  a  (BID)  population  prostaglandin at  the  maintained  the  were  protocol  b u t two  being  and  by  injection.  (BID)  origin  uveitis  being that  cats,  while  in  administered  dexamethasone by  of  T-celIs  when  in a l l  Cy t h e r a p y ,  systemic  in  only exception  subconjunctival  dexamethasone  three  two  dexamethasone  of the  had d i s a p p e a r e d  discontinuing  again  the  topical  effects  for  o f Cy a c c o r d i n g t o t h e  rabbits,  0.1%  anterior  T-cells  while  inhibiting T-cells, have  been  Conclusions Subconjuntival reducing results more  the  inflammatory  suggest  that  effective  injections treatment uveitis  of of  injections  Cy  it  the is  were  chronic  of  response  sooner  the  likely  to  also  Cy w e r e in  EAU-BSA  treatment be.  effective and  the  w i t h Cy  the  Subconjunctival  f o u n d t o be e f f e c t i v e  corticosteroid-resistant  i n cats without causing systemic  99  in  side  in  the  anterior  effects.  SUMMARY The  research  tried  administration while  avoiding  ocular and  route  was  the  toxic  study  able  limiting  provide  an a l t e r n a t i v e  to  factor  effects  The  of  results  demonstrated meet b o t h  being that  it  i n the  from t h e that  the  requirements,  with  Cy c o u l d o n l y be  less  in  Cy may n o t over  the  eye.  This  be  between  quiet  been  levels  shown  with  from  immunological  p o i n t of view the  it  to  f a r as  as  fast  as  treatment within the  eye  the  way  route  of  f o r use  of  o c u l a r inflammmatory d i s e a s e i n the or  as  an  adjunct.  case  of  agents  be  because  is  unique,  the uveitogenic  resulting  pulse  other  eye  therefore,  eye  in  T-cells  each  successive  T-cell  population  i n the  elimination  of  eye.  subconjunctival pave  i n the  may  the u v e i t o g e n i c  eventually  from the  This  replace  organ s i t e s ;  f u r t h e r reduces  disease  hopefully  not a b l e  other  the  The  is  be  administered  w i t h i n the  effective  subconjunctival ly.  so  would  injections,  injected an  only  vehicles  lipophilic  therapeutic  period  to  has  of  of subconjunctival l y  the  week  known  the  respect.  the l e v e l s  one is  are  this  remain at  the  therapy  that  of  administered  or  formulations  treatment  subconjunctival  New c o m b i n a t i o n s  Although  of  pharmacokinetic  o n c e p e r week b y t h i s r o u t e .  beneficial  route  f o r C y , t h a t w o u l d g i v e t h e b e n e f i t s o f Cy  inflammation.  uveitis  to  One,  however, 100  administration Cy i n t h e clinic must  be  of  Cy may  treatment  either aware  of  primarily that  the  d i s e a s e may n o t  be  in  was  origin  as  clinically  a  affected  disease  recurred  therapy  with  local  phenomenon,  possibly cats one  the we  month  subconjunctivally  101  but  case  treated after  rather  in in  systemic  three which  of  the the  discontinuing  a d m i n i s t e r e d Cy.  Bibliography 1.  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Kalsi  local  in  cats.  for  N,  Gudauskas  G,  et  al.  spontaneously occurring  Combined anterior  Submitted.  R o o t m a n J , B u s s a n i c h N , J o h n s t o n WH. S u b c o n j u n c t i v a l  Cyclosporine: in  Bussanich  chemotherapy  uveitis 81.  GS,  rabbits.  Suppresion  of  Submitted.  112  corneal  xenograft  rejection  APPENDIX  113  I  THE EFFECTS OF MULTIPLE DOSE SUBCONJUNCTIVAL ON ACUTE IMMUNE UVEITIS  J . Rootaan M.N. Bussanlch G. Gudauskas C. Kuml  114  ANTINEOPLASTICS  nmoDOCTZOM Hypersensitivity has b««n recognized as a f a c t o r In th« cause of some human ocular Inflammatory  diseases e s p e c i a l l y u v e i t i s , although the  amount and nature of antigen Involved are usually unknown.*  Corticosteroids  have been t r a d i t i o n a l l y used to treat ocular inflammations with success but 3  complications such as s u s c e p t i b i l i t y to i n f e c t i o n pressures**' say occur.  and Increased i n t r a o c u l a r  A l t e r n a t i v e non-toxic anti-inflammatory therapy,  e i t h e r as an adjunct or used Independently would be an advantage. Pharmacokinetic experiments suggest that penetration of subconjunctlvally administered drugs into the ocular compartments can be up to 100 times higher 6 7 8 9 than the same dose given systemically ' * ' .  Subconjunctival i n j e c t i o n s  may provide therapeutic drug levels t o the eye while avoiding the higher doses when given systemically. I t has been demonstrated  that an experimental a l l e r g i c u v e i t i s can  be induced by i n j e c t i o n of a single dose of bovine serum albumin i n t o the 2 v i t r e o u s of rabbits  and acute u v e i t i s develops within 6 to 10 days, and  p e r s i s t s f o r at least a week and then subsides.  The present experiments  were undertaken to determine whether subconjunctival a n t i n e o p l a s t i c therapy I n i t i a t e d immediately a f t e r the i n t r a v i t r e a l i n j e c t i o n s could suppress the degree of inflammation. MATERIALS AND METHODS P r i o r to the i n t r a v i t r e a l i n j e c t i o n a l l eyes were examined with a s l i t lamp to r u l e out the presence of pre-existing ocular diseases.  Twenty  l o c a l l y supplied Rev Zealand albino rabbits (2.2 - 2. A kg) were t r a n q u i l l z e d by i.m. i n j e c t i o n of Ketamine/Acepromazine maleate 115  (100 mg/ml) (10:1).  After  t o p i c a l anaesthesia with proparacain HC1 (O.SZ),  a s o l u t i o n of c r y s t a l i z e d  bovine serum albumin (SO ag/ml i n 0.92 NaCl solution) vaa f i l t e r e d a M i l lax  through  - GS 0.22um dlaposabla f l l t a r unit and 0.15 ml o f the f i l t r a t e  injected into the r i g h t v i t r e o u s .  The opposite l e f t v i t r e o u s  0.15 ml p h y s i o l o g i c a l s a l i n e to serve as controls. attached to a t u b e r c u l i n syringe was Inserted taking care to avoid  received  A 30 gauge needle  through the ocular coats  the p o s t e r i o r lena capsule.  Only a small amount of  injected f l u i d exuded from the injected s i t e s a f t e r removal of the needles. Animals were d i v i d e d i n t o 5 groups of 4 r a b b i t s each.  Immediately  a f t e r the I n t r a v i t r e a l i n j e c t i o n s , therapy with subconjunctival s a l i n e , methotrexate, 25 mg/ml; cystoslne arabinoside, 25 mg/ml and 6-Mercaptopurlne, 40 mg/ml was i n i t i a t e d . i n j e c t i o n s were made twice a week f o r 3 weeks.  i n j e c t i o n s of  150 mg/ml; 5 - F l u o r o u r a c i l , All  subconjunctival  A volume o f 0.5 ml of each  drug solution was i n j e c t e d p o s t e r i o r to the superior llmbus of the r i g h t eye by means of a 30 gauge needle attached to a tuberculin syringe. observations included c a r e f u l s l i t lamp observation  Daily  clinical  o f cornea f u r evidence o f  p e r l l l m b a l i n j e c t i o n and quantitation of anterior chamber c e l l s (expressed as 0 - 4+), f l a r e (expressed as 0 - 4+) and f i b r i n .  Conjunctival hyperemia,  edema, mucus secretion and I n j e c t i o n was noted.  The i r i s was monitored f o r  swelling, i n j e c t i o n and r e a c t i v i t y and the lens and v i t r e o u s were studied dally.  S e r i a l photography was taken to document the c l i n i c a l course.  Peripheral  blood counts yio n e on each animal once a week to monitor the extent of r  e  hematopoietic t o x i c i t y .  A d d i t i o n a l l y , a generalized  s e v e r i t y of Inflammatory  response was graded on a s c a l e of 0 - 4+ after a m o d i f i c a t i o n of the method of  116  Wlroesko and Halbert  as f e l l o w * :  0  •  normal.  1+  -  traca i r l a hyperemia, trace f l a r e and occasional  2+  -  mild I r i s hyperemia, c i l i a r y f l u s h , mild f l a r e , few In the a n t e r i o r chambers, peripheral  3+  -  cells. cells  injection.  moderate amount of f l a r e , c e l l s i n the gross, p r e c i p i t a t e over lens and cornea, f i b r i n i n the a n t e r i o r chamber, perlllmbal i n j e c t i o n , p o s t e r i o r synechia, v i t r e o u s opacity.  4+  -  extreme i r i s hyperemia, miotic pupils, Intense photophobia, f i b r i n i n the a n t e r i o r chambers, dense c e l l u l a r p r e c i p i t a t e , posterior synechia, p e r l l l m b a l i n j e c t i o n .  The  composite of the gradlngs of a) general c l i n i c a l observations, b)  flare  and c e l l s i n the anterior chamber f o r a l l animals i n each group were p l o t t e d against time.  ( F i g . 1).  The experiment was  terminated a f t e r 22 days.  r a b b i t s were s a c r i f i c e d with an overdose of pentobarbital sodium. eyes were enucleated,  All  Representative  f i x e d i n 10Z buffered formalin, sectioned and stained for  h i s t o l o g i c a l studies. RESULTS Immediately a f t e r the I n t r a v i t r e a l  injections the a n t e r i o r chambers  became shallow and the i r i s appeared pale with a return to normal w i t h i n 2 hours. There was  a mild, non-specific, transient inflammatory r e a c t i o n with I n t r a v i t r e a l  i n j e c t i o n s with both bovine serum albumin and s a l i n e . and was  This l a s t e d f o r a day  characterized by a mild p e r i c o r n e a l hyperemia, s l i g h t v a s o d i l a t a t i o n  of the i r i s vessels and f l a r e i n the a n t e r i o r chambers.  117  This r e a c t i o n  l a dua to tha trauma of i n j e c t i o n and has been noted by others.  Thereafter  a l l treated eyes remained quiet for at l e a s t 5 days at which point the animals developed varying degrees of u n i l a t e r a l u v e i t i s . The experimental r e s u l t s compared the onset and development  of u v e i t i s  i n r a b b i t s treated with subconjunctival s a l i n e versus those treated with a n t i n e o p l a s t i c agents.  Observations on the course of u v e i t i s are summarized  i n a p l o t of the d a i l y composites of the gradlngs 0 - 4+.  (Fig. 1 - l e f t ) .  Except f o r the transient inflammatory response to the trauma of the needle a l l l e f t eyes which received .15 ml normal s a l i n e developed no u v e i t i s . The r i g h t eyes of animals injected l n t r a v l t r e a l l y with bovine serum albunim and treated with subconjunctival s a l i n e developed grossly v i s i b l e u v e i t i s 6 days a f t e r the i n j e c t i o n s .  This was characterized by i r i s and  c o n j u n c t i v a l hyperemia, perlllmbal i n j e c t i o n , and c e l l s and f l a r e i n the a n t e r i o r chambers.  The inflammation peaked between the ninth and eleventh  days a f t e r the i n t r a v i t r e a l Injections.  A f t e r the t h i r t e e n t h day the  inflammation began to subside. With cytosine arabinoside, u v e i t i s was observed 8 days a f t e r the intravitreal injections. the  The u v e i t i s peaked between the thirteenth day and  sixteenth day and subsided thereafter.  Animals injected with methotrexate  developed an i n i t i a l l y mild u v e i t i s between the seventh and eleventh days with a moderate r i s e i n severity u n t i l the seventeenth day when i t began to subside.  With 5 - F l u o r o u r a c i l a mild u v e i t i s developed on the tenth day and  lasted f o r 6 days.  Animals injected with 6-Mercaptopurine had a milder  u v e i t i s throughout the period of the experiment.  118  Inflammation was minimal  u n t i l th« sixteenth day whan a c l i n i c a l l y s i g n i f i c a n t u v e i t i s davalopad l a a t l n g 4 days. In aummary, a l l 4 drugs vara found to e x h i b i t a n t i - i n f l a m a t o r y a c t i o n i n varying degreea with l a t e r onset and milder u v e i t i s than controls. 6-Mercaptopurlne appeared to be the most e f f e c t i v e In suppressing the degree and onset of the inflammation, followed by 5 - F l u o r o u r a c l l .  The eyes treated  with cytosine arabinoslde and methotrexate were more Inflamed than those treated with 6-Mercaptopurine and 5-Fluorouracil.  I t was noted that the  degree of inflammation i n a l l eyes, Including those treated with s a l i n e , waa about the same during the l a t t e r quarter of the experiment. TOXICITY STUDIES Systemic T o x i c i t y . There was no s i g n i f i c a n t sign of hemopoietic t o x i c i t y at the l e v e l s of drugs .injected as monitored once a week by p e r i p h e r a l blood counts f o r the duration of the experiment. Ocular T o x i c i t y . The multiple dose i n j e c t i o n of methotrexate r e s u l t e d i n no s i g n i f i c a n t signs of t o x i c i t y .  With cytosine arabinoslde there was a mild c o n j u n c t i v a l  hyperemia and djeoosis which resolved a f t e r the derapy was withdrawn.  Injections  with 6-Mercaptopurlne caused occasional hemorrhages i n the conjunctiva and In a number of animals there was f o c a l necrosis of the conjunctiva. 5 - F l u o r o u r a c i l was c l e a r l y t o x i c , leading to s i g n i f i c a n t corneal edema and subsequent v a s c u l a r i z a t i o n of peripheral cornea. chemosis and periocular h a i r loss was noted.  119  In a d d i t i o n , conjunctival  OCULAR triAMMATORY RESPONSE TOU.OWHQ SUBCONJUNCTIVAL ANTtCOFVASTICS  1  •HL 11 M  a  JL $*  1L  11  n  -jfTTt 11111 i iTTn J L J  D_aH,  UDJ  Tkn*(eayt>  120  to  C0MCL3SI0KS Thar* h«v« been numerous attempts to f i n d s u i t a b l e potent anti-inflammatory drugs which w i l l be e f f e c t i v e f o r the treatment of n o n - s p e c i f i c u v e i t i s  1 , 2  3 4 5 without e x h i b i t i n g the side e f f e c t s of c o r t i c o s t e r o i d s . ' ' ' Some of these side e f f e c t s  may be avoided by the use of non-steroidal anti-inflammatory  13 drugs.  Some cytotoxic agents, including cytosine arablnoside, methotrexate  and 6-Mercaptopurlne are known to have both anti-inflammatory and immunosuppressive p r o p e r t i e s * and may be u s e f u l i n the treatment/uveltls.  6-Mercaptopurine has  1  been used systemically to suppress immune u v e i t i s * 2  1 5  '  1 6  '  1 , 7  .  We have attempted to assess the l o c a l immunosuppressive value o f t h i s group of antineoplastic drugs when Injected s u b c o n j u n c t i v a l ^ .  Our experiments  have demonstrated that 6-Mercaptopurine end to a l e s s e r degree 5 - F l u o r o u r a c i l were able to delay the onset and reduce the severity of a l l e r g i c u v e i t i s induced by i n t r a v i t r e a l i n j e c t i o n of bovine serum albumin i n r a b b i t s . Cytosine arablnoside and methotrexate also appeared to reduce the s e v e r i t y and delay the onset of inflammatory response.  In the case of both 5-Fluorouracil  and 6-Mercaptopurlne we f e l t that there was s i g n i f i c a n t l o c a l t o x i c i t y at dose l e v e l s employed and although these t o x i c i t i e s were r e v e r s i b l e with discontinuance of the drugs they would not be acceptable i n the c l i n i c a l circumstance.  Further  i n v e s t i g a t i o n of subconjunctivally injected 6-Mercaptopurlne i n p a r t i c u l a r might be worthwhile, perhaps i n modified dose levels or as an adjuvant to other forms of therapy.  121  M u l t i p l e Dose Subconjunctival Antlnaoplaatlcs  REFERENCES 1.  Theodora FH, Schlosaman A:  Ocular a l l e r g y .  The William and V l l k l n s  Co., Baltimore, 1958. 2.  3.  Wiroatko E, Ralbert SP: captopurlne.  J Exp Med 116: 653, 1962.  Welnatein L:  C o r t i c o t r o p i n and corticosteroids i n human v i r a l i n -  fections. 4.  Suporesslon of a l l e r g i c u v e i t i s by 6-mer-  Med C l i n i c North Am 46: 1141 - 1661, 1962.  Becker B, M i l l s W:  C o r t i c o s t e r o i d s and i n t r a o c u l a r pressure.  Arch Ophthalmol 70: 500 - 507, 1963. 5.  Armaly MF:  E f f e c t s of c o r t i c o s t e r o i d s on i n t r a o c u l a r pressure and  f l u i d dynamics. 6.  Arch Ophthalmol 70 482 - 491, 1963.  Rootman J , Gudauskas G, Kumi C: cytosine arabinoslde: toxoclty.  7.  Subconjunctival versus  intravenous  e f f e c t of route of administration and ocular  Investigative Ophthalmol and Visual Science ( i n p r e s s ) .  Gudauskas G, Ostry A, Rootman J :  Concentrations of t r l t i a t e d metho-  trexate i n ocular compartments, serum and urine a f t e r subconjunctival and intravenous i n j e c t i o n . 8.  Can J Ophthalmol 15: 179 - 182, 1980.  Rootman J , Josephy PD, Adomat H, P a l c i c B:  Ocular absorption and  t o x i c i t y of a r a d l o s e n s i t i z e r and i t s effect on hypoxic  cells.  Arch Ophthalmol 100: 468 - 471, 1982. 9.  Rootman J , T i s d a l l J , Gudauskas G, Ostry A:  Intraocular penetration  14 of subconjunctivally administered  C-Fluorouracil i n r a b b i t s .  Arch Ophthalmol 97: 2375 - 2378, 1979.  122  M u l t i p l e Dose Subconjunctival Antineoplastics  10.  Barza M, Baum J : Am J Ophthalmol  11.  Intraocular penetration of c a r b e n l c l l l i n i n r a b b i t s . 75: 305 - 313,  Records RE, E l l i s PP: c i l l n and o x a c i l l i n .  12.  Hanna  1973.  Intraocular penetration of a a p l c l l i n , methiAm J Ophthalmol 64: 135,  C, Kaufman HE (ad.):  1967.  Nonsteroids anti-inflammatory agents.  Charles C Thomas publisher, S p r i n g f i e l d , 111: 136 - 149, 13.  Hanna C, Keats HC: Arch Ophthalmol  14.  77: 554,  e d i t i o n : 499 - 506,  In ocular inflammation i n r a b b i t s .  1967.  Meyer FH, Javetz E, Goldfein A: 4th  15.  Indomethacin  1970.  Review of medical pharmacology.  1974.  Schwartz R, Eisner A, Dameshek V:  The e f f e c t of 6-mercaptopurine  primary and secondary immune responses.  J C l i n Invest 38:  on  1393,  1959.  16.  Newell FW,  K r i l l AE, Thomson A:  mercaptopurine. 17.  Wong VG:  The treatment of u v e i t i s with 6-  Am J Ophthalmol 61: 1250,  1966.  Immunosuppressive therapy of ocular inflammatory diseases.  Arch Ophthalmol  81: 628,  1969.  123  APPENDIX  124  II  Combined l o c a l  chemotherapy f o r  occurring  anterior  G u r s h a r a n S.  uveitis  spontaneously in  cats  K a l s i , * BSc;  Nick Bussanich,  DVM;  A  Gedy G u d a u s k a s , -  PharmD;  J a c k R o o t m a n , * MD; A  From •pathology, General  the  departments  University  Hospital,  and  Cancer C o n t r o l Agency  Reprint Veterinary  of of  requests  British  the  A  to:  Dr.  ophthalmology  Columbia  -Advance  British  C l i n i c L t d , 7161  T h i s s t u d y was T r u s t Fund.  of  and  Vancouver  Therapeutics  Program,  Columbia,  Vancouver.  Nick  Bussanich,  Victoria.,  Vancouver,  s u p p o r t e d by the  125  and  Knight  Rd  BC.  Canadian V e t e r i n a r y  Research  Abstract Spontaneously occurring corticosteriod resistant anterior uveitis in four domestic cats was treated with subconjunctival injection of cyclosporin A and 0.1% dexamethasone topically for a period of eight weeks. Thereafter, the cats were maintained on 0.1% dexamethasone topically. The inflammation in all the cats had been stable for up to 2 months following treatment, but had relapsed while being maintained on topical dexamethasone twice daily.  Introduction We have previously demonstrated the superiority of ocular penetration following local injection of several commonly used antineoplastic agents24.25. Not only do these drugs penetrate in higher concentrations than are achieved with systemic administration of equal doses.20-23 ut the paraocular route D  avoids the metabolic breakdown of the active components of the drugs characteristically seen with systemic administration. Herein we report the treatment of chronic anterior uveitis by combined local therapy with Cyclosporin A and 0.1% dexamethasone.  126  Method The four cats chosen for the study had previously been treated topically and systemically with corticosteriods for anterior uveitis with an unsatisfactory response. They received Cyclosporin A subconjunctival^ once a week and 0.1% dexamethasone twice daily, for 8 weeks. The eye was anesthetized with proparacain HCL(0.5%), and injected with O.lcc of 5mg/0.1cc of Cyclosporin A  a  was given subconjunctival^ above the superior limbus using a 25 gauge needle attached to a tuberculin syringe. The cats were followed for a 8 weeks by slit-lamp biomicroscopy, indirect ophthalmoscopy and serial photography. The degree of inflammation was graded according to the following scheme modified from F o x : 1 2  Ciliary injection  corneal clarity  corneal erosion  iris injection  Ant. Chb. haze  Vitreous & Retina  HypODyon  none  clear  none  none  clear  chorioretinal detail share  none  0  tracs  trace  trace  trace  trace  chorioretinal trace detail blurred  1  mild  mild  mild  mild  mild  visible but no clear detail  mild  2  moderate  moderate moderate  moderate  moderate + cells  no detail + cells  moderate  3  severe  severe  severe  severe cells & tibrin  obscured  severe  4  severe  Overall intlammation  a SANDIMMUNE I.V. 50mg/ml; SANDOZ Ltd, BASLE, SWITZERLAND.  127  Cat 1 A domestic short haired 13 year old neutered male, was first seen in October 1984 with bilateral uveitis and hypopyon. It had a  normal C B C and  blood chemistry and the serum was negative for feline leukemia. The cat had been treated from October 1984 to September 13, 1985 with 0.1% topical dexamethasone alternating with oral prednisone at a dose of 5 mg/day for one month at a time, however, the disease was not subsiding and we had noted an increase in keratic percipitates (KP's) with inflammation of the iris (fig. 1a). In view of the increase in inflammation the cat was placed on Cyclosporin A subconjunctival^ once per week and 0.1% dexamethasone topical twice daily (BID), for 8 weeks. At the end of the treatment the C B C and blood chemistry were unchanged, the inflammation had decreased with only small KP's present and the cat was maintained on 0.1% topical dexamethasone (fig. 1b). Follow-up showed that the cat remained stable for 2 months but relapsed there after while on topical dexamethasone. The overall progress of the cat is documented in figure 1c.  128  QaLZ A domestic short haired 10 year old neutered male, was first seen in September, 1985 with bilateral uveitis, endothelial KP's, nodules on the iris and hypopyon in the right eye. Blood chemistry and C B C was normal and the serum was negative for Feline leukemia and toxoplasmosis. The initial treatment was with 0.1% topical dexamethasone BID for three weeks and at the end of the this period, examination showed only a slight decrease in the inflammation (fig. 2a). Since the disease was not responding to the  steriod treatment alone, the cat was placed on  Cyclosporin A  subconjunctivally once per week and 0.1% topical dexamethasone (BID), for 8 weeks; thereafter, maintained on 0.1% topical dexamethasone BID. At the end of the Cyclosporin A treatment, C B C and blood chemistry was normal except for the decrease in the white blood count from 7500/mm  3  to  5000/mm3. The progress of the cat on the Cyclosporin A and subsequent followups can be seen in figure 2c, demonstrating improvement of the uveitis while on the treatment (fig. 2b) and subsequent recurrence eight weeks after discontinuing therapy.  129  Cat 3 A domestic long haired 9 year old spayed female, was first seen on Februray 11, 1985 with bilateral uveitis, iris nodules and a fibrin clot attached to the right lens. The cat was placed on 0.1% topical dexamethasone and 5 mg of prednisone orally. The C B C and blood chemistry was normal and the serum negative for feline leukemia. On Februray 23, 1985 the eyes had become worse and by March 7 the anterior chamber had improved, but the cat had developed a superficial corneal erosion which slowly healed over one week. The eyes remained stable until early October 11,1985 when the cat had a relapse in both eyes with marked inflammation and a fibrin clot on the surface of the lens (fig. 3a). Because of the severe bilateral flare-up the cat was placed on Cyclosporin A subconjunctival^ once per week and 0.1% topical dexamethasone BID, for 8 weeks. By the end of the 8 week treatment there had been considerable improvement in both eyes with only a few small KP's (fig. 3b). The effects of Cyclosporin A can be seen in figure 3c. Blood chemistry and C B C done at end of the Cyclosporin A treatment was normal. The cat was maintained on 0.1 %topical dexamethasone BID and remains stable for one month.  130  Cat 4; A domestic short haired 8 year old neutered male was first seen on Nov 9, 1985 with right unilateral uveitis. The iris was congested, had nodules on it, and fibrin was noted on the lens capsule with a moderate amount of flare and cells in the anterior chamber vitreous (fig. 4a). This cat was placed on Cyclosporin A subconjunctival^ once per week and 0.1% topical dexamethasone BID, for 8 weeks. By December 7, 1985 it had developed a corneal erosion. The fifth injection of Cyclosporin A and topical dexamethasone was not given until the following week. The cat had improved considerably by the end of the treatment period (fig. 4b).  After discontinuing of the treatment the cat was placed on topical  dexamethasone BID. There was a minor relapse on the last week of therapy with Cyclosporin A and dexamethasone. The hypopyon had returned and the eye was becoming inflammed again, but on subsequent follow-ups the inflammation had regressed and one month following treatment with Cyclosporin A and dexamethasone there was only a trace of inflammation. The eye still remains stable 16 weeks afte treatment with Cyclosporin A and dexamethasone, the longest period of the four cats in this study. The progress of the cat while on the treatment and subsequent follow-ups can be seen in figure 4c.  131  Figure  la  Right  eye  ( arrow ) before  of  Cat 1  showing  i n i t i a t i o n of  132  Jceratic  treatment.  percipitates  Figure  Right completion and t o p i c a l  lb  eye  of  of  Cat  treatment  1  showing  with  lack  of  inflammation  subconjunctival  0.1% d e x a m e t h a s o n e  133  (eighth  week).  at  Cyclosporin A  Figure  lc  CAT 1 4.0  •  3.5  right eye  c 3.0 o  IS 2.5 |  2.0  J5 1.5 ^  1.0 0.5 J  0.0 0 1  I  2 3  l  I  I  I  1  4 5 6 7  Time (weeks) • Cyclosporin A treatment •  treatment ended  134  UJJ  1  8 16 24  Figure  Right percipitates  2a  eye  of  before  C a t 2 s h o w i n g Hypopyon a n d s m a l l i n i t i a t i o n of  135  treatment.  keratic  Figure  2b  Right of decrease completion and  in of  topical  Cat 2 showing the  number  treatment 0.1%  resorption of of  with  keratic  the  percipitates,  subconjunctival  dexamethsone 136  hypopyon  and at  Cyclosporin A  (eighth  week).  Figure  2c  CAT 2  c g  to E E  4.0 r 3.5 3.0 2.5 \ 2.0 1.5 N \ 1.0 Wo 0.5 i „i 0.0 0 1 2 3  \  •  A\  right eye  O left eye  \\ \  4 5 6 7  8 12 28  Time (weeks)  • • Cyclosporin A treatment •  treatment ended  137  Figure  3a  Right clot  eye  attached  nodules  on  initiation  to  the of  of  C a t 3 showing  the  lens,  surface  of  small the  treatment. 138  the  presence  of  a  fibrin  keratic  percipitates  iris  arrow  (  ),  and  before  Figure  Right at  the  end  and t o p i c a l  3b  eye of  of  Cat  treatment  3  showing  with  0.1% d e x a m e t h s o n e 139  decrease  of  subconjunctival (eighth  week).  inflammation Cyclosporin A  F i g u r e 3c  CAT 3 4.0 3.5  \ ^  J / \  • '9ht eye r  0 left eye  c 3.0  1  2.5  |  2.0  JS 1.5  -0—0-/M/^  -0-  •E 1.0 0.5 0.0  0  1  2  3  4  5  6  7  Time (weeks) • Cyclosporin A treatment •  treatment ended  140  8  •  12 16  Figure  Right clot  on  surface  the  4a  eye  of  surface  Cat of  ( arrow ) b e f o r e  4  showing  the  lens,  the  presence  of  a  with  nodules  on  the  i n i t i a t i o n of 141  treatment.  fibrin iris  Figure  Right at  the  end  and t o p i c a l  4b  eye of  of  Cat  treatment  0.1%  4  showing  with  decrease  of  subconjunctival  dexamethasone.  142  inflammation Cyclosporin A  CAT 4 •  right eye  O left eye  / O.Oo—o—o-o—o—o—o—o—o—o/y-o-o^— 0 1 •  2 3 •  •  4 5 6 7 Time (weeks) • •  • Cyclosporin A treatment •  treatment ended  143  8  9 13 21 2 5  Discussion  :  Because t h e e t i o l o g y it  c a n be d i f f i c u l t  not  suprising that  patients  case A  h a s been  treatment  o f treatment  7  from  inflammatory  t o be  retinal  1 0  '  cells  A  S-antigen  uveitis, ' ' 3  6  associated been  mode  with  be  of  of  of the release  of  suppressing  i s thought  treating  uveitis  which occurrs  to  mediated  effective  side-effects.  hepatotoxicity  i n the  action  thereby  in  e s p e c i a l l y i n the acute  1 5  i t ' s  mg/kg.  should  Cyclosporin  2  the  1 1  i n t h e development o f T - c e l l  Cyclosporin  i n some  and o r a l l y  inhibition  T-helper  reaction.  Since  human  effective i n treating  The  9  steroids,  agents.  (i.m.)  I t has been  6  inflammation. "  i s felt  interleukin-2  has been  systemically  of u v e i t i s . "  A  I t i s therefore  f o r 1.0% o f b l i n d  by immunosuppressive  3  mediated  defined,  1  employed  Cyclosporin  factor  u v e i t i s accounts  mainstay  supplemented  T-cell  t o t r e a t and r e c u r r e n t .  i n Canada.  The  of u v e i t i s i s not well  a  major  uveitis,  some  phase.  with  be  types  The  1 4  of  A  have  nephro  at a relatively  '  problems  Cyclosporin  Particularly  1 3  and  low d o s e o f 10  1 6 - 1 9  We  have  absorption injection  shown w i t h  a  number  c a n be s i g n i f i c a n t l y o f the drug  administration. circumvent  the  when  In  2 0 - 2 5  enhanced  compared  this  associated  o f agents  way  by  that  sunconjunctival  t o equidose  i t may  ocular  be  toxicities  systemic  possible of  to  systemic  administration. Subconjunctival developed toxicities of  by  studies  Cyclosporin  Cyclosporin  us  administration  using  a  rabbit  of Cyclosporin  model.  o f numerous v e h i c l e s  A that  As  a  t h e dose  result  subconjunctivally  144  was of  a n d makeup  we u s e d was 0.1 c c o f 5 mg/  A, a d m i n i s t e r e d  A  O.lcc  of  o n c e p e r week.  We w e r e a b l e resistant A  once  eight  week  weeks  and  dexamethasone  right 0.1%  auccesssfully  spontaneous  per  stable  to  for  and  0.1%  than  twice  about  topical  daily.  The  months  uveitis  with  eye 0.1%  t h a n 24  lid,  which  topical  would  acute phase of  the  on  except  last  control  2 months  it  on  steroid  Cyclosporin BID,  0.1%  cat  stable the  24  remained  4  to  which  date  had  ) .  The  Cyclosporin A, swelling  to  swelling  for  topical  average  for  about  way  would  48  of  the  hours,  lasted  for  but less  the be  the  more  responded  effective  combined t h e r a p y  dexamethasone a c t e d the  after  uveitis  in  to the  disease.  uveitis  discontinuing  of  Cyclosporin A  synergistically  successfully  F u r t h e r s t u d i e s on t h e of  eyes  given with  from t h e  study  a n d 0.1% t o p i c a l  to  cats  dexamethasone t h e  seem  From t h i s  to  the  would cause m i l d  would  Cyclosporin A that  able  by u s i n g  dexamethasone  remains  d e x a m e t h a s o n e was  chronic  hours. It  the  (  and  because C y c l o s p o r i n A alone upper  uveitis  maintaining  two  unilateral topical  anterior  control  for  and  a period of  was up  treatment.  subconjunctival  C y c l o s p o r i n A are under c u r r e n t  administration  investigation.  Acknowledgements:  The generously  authors  are  grateful  to  Sandoz,  s u p p l y i n g u s w i t h Sandimmune I . V .  145  Ltd.;  for  1.  Statistical  registered Institute  2.  with  Inc.  3.  -  WJ.  the in  1985;  1.  Uveitis, O'Connor  GR,  198-220.  Nussenblatt  Rb,  Palestine  systemic  Ophthalmol  Chan  induced  treatment  1985.  of  ed's.  AG,  of  Canadian  and  Publ.  Chan  Canada  National  Therapy.  In:  Thiene-Stratton  CC.  Cyclosporin  A  i n t r a o c u l a r inflammatory disease  corticosteroids  and c y t o x i c  a g e n t s . Am  1983;96:275-282.  CC, Palestine alteration  autoimmune  population  Pathophysiology  1983;  to  blind  CNIB  New Y o r k ,  resistant  4.  Blind  MacKiw,  therapy i n the  J  on  the  f o r the  Dinning  Kraus  studies  uveitis.  AG, Nussenblatt  of  humoral  Invest  RB.  response  Ophthalmol  Vis  Cyclosporine  -  in  experimental  Sci  1984;25:867-  870.  5.  Mochizuki  of  M, Nussenblatt  cyclosporine  experimental Vis  6.  Sci  T,  et  al.  immunosuppressive  uveitis  in  rats.  Effects  drugs  Invest  on  Ophthalmol  1985;26:226-232.  treatment  U.K.  other  autoimmune  Graham E M , S a n d e r s  the  7.  and  R B , Kuwabara  of  MD, James  posterior  DG, e t  uveitis.  al.  Cyclosporin A in  Trans  Ophthamol  Soc.  1985;104:146-151.  Lafferty  models  K J , Borel  for  J F , Hodgkin  mechanism  of  P.  Cyclosporine-A  action.  Transplant  (CsA): Proc  1983;15:2242-2247.  8.  T u t s c h k a P J , B e s c h o r n e r WE, H e s s A d , e t  to  prevent  patients  graft-versus-host recieing  allogenic  1983;61:318-325.  146  disease: marrow  a  al. pilot  Cyclosporin A study  transplants.  in  22  Blood  9.  Salisbury  allograft  JD,  Bebhardt  rejection  by  BM.  Suppression  cyclosporin  A.  of  Arch  corneal  Ophthalmol  1981;99:1640-1643.  10.  Hess  A d , Tutschxa P J , Santos  on  the  induction  interleukin  I  of  GW. E f f e c t  cytotoxic  and  of  cyclosporine  T-lymphocytes:  interleukin  II.  role  Transplant  of Proc  1983;15:2248-2258.  11.  Andrus  L , L a f f e r t y K J . I n h i b i t i o n of  T-cell  c u c l o s p o r i n A . S c a n d J Immunol  1982;14:449-458.  12.  et  Fox A ,  Hammer M E , L i l l  elicited  by  P,  al.  activity  Experimental  peptidoglycan-polysaccharide  lipopolysaccharide,  and  muramyl d i p e p t i d e .  by  uveitis:  complexes,  Arch  Ophthalmol  1984;102:1063-1067.  13.  Nussenblatt  Immune antigen.  14.  responsineness  of  of  M, Kuwabara  experimental  Invest Ophthalmol V i s S c i  15. of  Nussenblatt uveitis  patient  I,  Ballintine  uveitis  E J , et  patients  al.  to  cellular  retinal  s-  Am J O p h t h a l m o l 1 9 8 0 ; 8 9 : 1 7 3 - 1 7 9 .  Mochizuki  transfer  RB, Gery  and with  T,  McAllister  autoimmune  et  al.  uveoretinitis  Adoptive in  rats.  1985;26:1-9.  BR, P a l e s t i n e optic  C,  A , Chan C C , e t  nerve  multiple  disease  by  sclerosis.  al.  Improvement  cyclosporine  in  a  Am  J  Ophthalmol  JE,  et  al.  1984;97:790-791. 16.  Palestine  histopathologic cyclosporine  AG,  Austin  HA,  alterations  for uveitis.  in  N Engl  147  Balow  patients  treated  Renal with  J Med 1 9 8 6 ; 3 1 4 : 1 2 9 3 - 1 2 9 8 .  17.  Palestine  induced  A G , A u s t i n HA, N u s s e n b l a t t  nephrotoxicity  Transplant Proc  18.  in patients  bone  marrow  transplant  c y c l o s p o r i n A . B r J Haematol  Klintman  in  S,  66  20.  Ostry  A,  Nephrotoxicity treated  with  T E . Cyclosporin  subconjunctival  Can J Ophthalmol  A  recipients.  G. Pharmacokinetics  following  administration.  al.  allograft  Gudauskas  of 5-flurouracil  intravenous  Starzl  renal  1981;32:488-489.  J ,  et  recipients  Transplantation  metabolism  uveitis.  1983;54:69-78.  GBG, I w a t s u k i  hepatotoxicity  Rootman  autoimmune  1985;17(4):209-214.  Hows J M , C h i p p i n g PM, F a i r h e a d S ,  in  19.  with  RB. Cyclosporine-  and  versus  1984;19(4):187-  191.  21.  Gudauskas  tritiated urine  Ostry  methotrexate  after  in  Rootman ocular  J .  Concentrations  compartments,  and i n t r a v e n o u s  of  serum  injection.  and  Can J  1980;15:179-182.  Rootman  J ,  Josephy  absorption  and t o x i c i t y  on h y p o x i c  cells.  23.  A,  subconjunctival  Ophthalmol 22.  G,  Rootman  penetration  J ,  of  Tisdall  of  J ,  24.  J ,  Gudauskas  G, et  et  al.  Ocular  and i t s  effects  a  N,  Gudauskas  spontaneously  Can J O p h t h a l m o l  148  al.  Intraocular  administered  Arch Ophthalmol  Bussanich  for  tumour i n a c a t .  H,  a radiosensitzer  subconjunctivally  in rabbits.  chemotherapy  Adomat  Arch Ophthalmol 1982;100:468-471.  Fluorouracil Rootman  PO,  1  4  C -  1979;97:2375-2378. G.  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