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Chemical and biological studies of the radiosensitizer misonidazole Josephy, P. David 1981

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CHEMICAL  AND  BIOLOGICAL  STUDIES  OF  THE  RADIOSENSITIZER  MISONIDA'ZOLE  by P. B.  Sc.  David  (Hon.),  Josephy  U n i v e r s i t y of T o r o n t o , 1976  A T H E S I S S U B M I T T E D IN P A R T I A L REQUIREMENTS  FOR  THE  DEGREE  FULFILLMENT OF  DOCTOR  OF OF  THE PHILOSOPHY  in THE  F A C U L T Y OF G R A D U A T E THE  DEPARTMENT  OF  STUDIES  ZOOLOGY  We a c c e p t t h i s t h e s i s as c o n f o r m i n g to t h e r e q u i r e d s t a n d a r d  THE  U N I V E R S I T Y OF B R I T I S H June, ©  P.  COLUMBIA  1981  David Josephy,  1981  In p r e s e n t i n g t h i s  thesis  an advanced degree at the L i b r a r y I  in p a r t i a l  the U n i v e r s i t y  s h a l l make i t  freely  fulfilment of the requirements f o r of British  available  for  Columbia,  I agree  r e f e r e n c e and s t u d y .  f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e copying o f t h i s  for  that  thesis  s c h o l a r l y purposes may be granted by the Head o f my D e p a r t m e n t o r  by  his  of  this  written  representatives.  It  thesis for financial  i s understood that c o p y i n g o r p u b l i c a t i o n gain s h a l l  not be allowed without my  permission.  P.  Department o f The  University  Zoology of B r i t i s h  2075 Wesbrook P l a c e V a n c o u v e r , Canada V6T 1W5  Date  F e b . 2 6 , 1981  Columbia  David  Josephy  i i ABSTRACT  Misonidazole sitizes  hypoxic  radiation. limit  the  The  use  07-0582)  (Ro  is  a  (oxygen-deficient)  Tumours usefulness  contain of  cells  to the  radioresistant  radiotherapy  of m i s o n i d a z o l e a s  nitroheterocyclic  an  as  adjunct  lethal  of  Misonidazole absence  of  of  drug,  the  safely,  to  reductive  and,  such  as  activation  selective  the  in  sen-  ionising  which  cancer  in  hypoxic  may  may  treatment. improve  the  progress.  cells,  even  in  the  be related to t h e clinical t o x i c i t y misonidazole  of  limits  the  which  effectiveness  may  be the  of  t o x i c i t y of m i s o n i d a z o l e i s b e l i e v e d t o hypoxic  cells.  nitrobenzene,  of  to  dose  thus,  selective  metabolism  toxic  effect may  limits  The  its  compounds,  This  which  radiosensitizer.  We  selectively  radiation.  delivered  related  is  of  cells  radiotherapy  local c o n t r o l of s u c h t u m o u r s , a n d c l i n i c a l t r i a l s a r e  which  action  hypoxic  a modality to  drug  is  misonidazole to  Reduction  inhibited  by  a toxic species  of  nitroaromatic  oxygen; may  be  thus,  explain  the  a c t i o n of t h e d r u g a g a i n s t h y p o x i c c e l l s . have  metabolism,  studied the using  a  reductive  variety  of  chemistry  chemical  and  of m i s o n i d a z o l e , in v i t r o  and  biological  its  tech-  niques. Ascorbic  acid  hypoxic  Chinese  appears  to  be  (vitamin  hamster  caused  C)  ovary  by  e n h a n c e s t h e t o x i c i t y of m i s o n i d a z o l e to (CHO)  accelerated  cells.  This marked  d r u g metabolism in the  enhancement presence  of  ascorbate. Chemical  reduction  azo-misonidazole separated  by  and  of m i s o n i d a z o l e b y  z i n c d u s t y i e l d s a m i x t u r e of  azoxy-misonidazole.  preparative  reversed-phase  These  compounds  were  liquid chromatography,  char-  i ii  acterized  chemically,  Azo-misonidazole  is  and  almost  tested  for  non-toxic,  in v i t r o  but  biological  activity.  a z o x y - m i s o n i d a z o l e is  more  toxic than misonidazole itself. M i s o n i d a z o l e was system, major  under  product,  reduced  hypoxia. which  by  This  appears  the  xanthine/xanthine  enzymatic to  be  reduction  oxidase  yielded  a  (XO) single  hydroxylamino-misonidazole.  The  same e n z y m e s y s t e m also r e d u c e s azo- and a z o x y - m i s o n i d a z o l e .  14 The using  metabolic  dense  transformation  suspensions  converted  into  material  (presumably  organic-soluble chromatographic  several  of  polar  metabolite  CHO  of  C-misonidazole  cells  in  products,  and  macromolecules) fraction  hypoxia.  contains  b i n d i n g to was  studied,  Misonidazole  product,  is  acid-insoluble  observed.  a compound  p r o p e r t i e s to t h e x a n t h i n e / X O  was  with  The identical  b e l i e v e d to  be  hydroxylamino-misonidazole. The clinical The  s i g n i f i c a n c e of t h e s e r e s u l t s is d i s c u s s e d in t h e c o n t e x t of t h e  potential  of m i s o n i d a z o l e a n d  r e l a t e d d r u g s as r a d i o s e n s i t i z e r s .  p o s s i b i l i t y of e x p l o i t i n g h y p o x i c c y t o t o x i c i t y as a s e l e c t i v e  t h e r a p y f o r h y p o x i c t u m o u r c e l l s is c o n s i d e r e d .  chemo-  iv  T A B L E OF  CONTENTS  ABSTRACT  ii  T A B L E OF C O N T E N T S  iv  LIST OF F I G U R E S  vii  ABBREVIATIONS  ix  ABBREVIATIONS:  RADIOSENSITIZERS:  x  ACKNOWLEDGMENTS  , . . xi  INTRODUCTION: 1.1  Radiotherapy  and the o x y g e n effect  1  1.2  R a d i a t i o n a n d cell s u r v i v a l  2  1.3  T h e o x y g e n e f f e c t a n d cell s u r v i v a l  6  1.4  T h e o x y g e n e f f e c t - mechanisms  9  1.5  The oxygen effect - clinical significance  13  1.6  T h e r a p i e s w h i c h may o v e r c o m e t h e  radioresistance  of h y p o x i c c e l l s  16  1.7  Nitroimidazole radiosensitizers  20  1.8  T o x i c i t y of n i t r o a r o m a t i c c o m p o u n d s  23  1.9  H y p o x i c c y t o t o x i c i t y - mechanisms  26  MATERIALS &  METHODS:  2.1  C e l l c u l t u r e p r o c e d u r e s - C H O and C H 2 B  2.2  In v i t r o t o x i c i t y e x p e r i m e n t s  33  2.3  Z i n c r e d u c t i o n of m i s o n i d a z o l e  37  2.4  X a n t h i n e o x i d a s e - c a t a l y s e d r e d u c t i o n of m i s o n i d a z o l e . . . .  39  2.5  In v i t r o metabolism of m i s o n i d a z o l e  41  2  cell lines  32  V  MISONIDAZOLE  CYTOTOXICITY  IN  VITRO:  3.1  Introduction  46  3.2  A s c o r b a t e e n h a n c e m e n t of m i s o n i d a z o l e c y t o t o x i c i t y  47  3.3  A e r o b i c t o x i c i t y of a s c o r b a t e  49  3.4  Recent developments  53  CHEMICAL  REDUCTION  OF  MISONIDAZOLE:  4.1  Introduction  56  4.2  I d e n t i f i c a t i o n of z i n c r e d u c t i o n p r o d u c t s  60  4.3  Recent developments  69  4.4  In v i t r o t o x i c i t y of azo- a n d a z o x y - m i s o n i d a z o l e  70  4.5  M e t a b o l i c f o r m a t i o n of b i m o l e c u l a r p r o d u c t s  78  BIOCHEMICAL ITS  REDUCTION  AZO- AND AZOXY-  OF M I S O N I D A Z O L E  AND  DERIVATIVES:  5.1  Introduction  81  5.2  Xanthine oxidase  82  5.3  R e d u c t i o n of m i s o n i d a z o l e b y x a n t h i n e o x i d a s e  85  5.4  R e d u c t i o n of d e r i v a t i v e s b y x a n t h i n e o x i d a s e  88  5.5  Recent developments  94  vi  IN V I T R O M E T A B O L I S M O F  MISONIDAZOLE:  6.1  Introduction  97  6.2  In v i t r o metabolism of m i s o n i d a z o l e  99  6.3  Conclusions  104  DISCUSSION: 7.1  M i s o n i d a z o l e metabolism - in v i v o a n d in v i t r o  108  7.2  C l i n i c a l s i g n i f i c a n c e of h y p o x i c c y t o t o x i c i t y  112  7.3  New r a d i o s e n s i t i z e r s  114  7.4  Future studies  115  REFERENCES  117  vi i  LIST OF  FIGURES  INTRODUCTION: 1.  R a d i a t i o n s u r v i v a l c u r v e s in v i t r o  5  2.  Radiation s u r v i v a l c u r v e s f o r mixed populations  8  3.  R a d i o s e n s i t i v i t y as a f u n c t i o n of o x y g e n c o n c e n t r a t i o n  4.  In v i v o r a d i a t i o n s u r v i v a l  5.  curves: 15  S e l e c t i v e t o x i c i t y of m i s o n i d a z o l e t o h y p o x i c c e l l s  25  METHODS:  In v i t r o metabolism e x p e r i m e n t s : s c h e m a t i c  MISONIDAZOLE CYTOTOXICITY Ascorbate-enhancement  8.  E f f e c t of a s c o r b a t e c o n c e n t r a t i o n on e n h a n c e m e n t  of m i s o n i d a z o l e c y t o t o x i c i t y  48  of m i s o n i d a z o l e c y t o t o x i c i t y  50  T o x i c i t y of a s c o r b a t e t o a e r o b i c c e l l s in v i t r o  52  CHEMICAL 10.  43  IN V I T R O :  7.  9.  12  D e m o n s t r a t i o n of p r e s e n c e of r e s i s t a n t f r a c t i o n  MATERIALS & 6.  ....  R E D U C T I O N OF M I S O N I D A Z O L E :  U V - v i s i b l e s p e c t r a of m i s o n i d a z o l e , a z o - m i s o n i d a z o l e , and azoxy-misonidazole  64  11.  NMR s p e c t r u m of m i s o n i d a z o l e  65  12.  NMR s p e c t r u m of a z o - m i s o n i d a z o l e  66  13.  NMR s p e c t r u m of a z o x y - m i s o n i d a z o l e  67  vi i i  T o x i c i t y of b i m o l e c u l a r  derivatives:  14.  I.  C H O cells - aerobic incubation  72  15.  II.  C H O cells - h y p o x i c incubation  73  16.  III.  CH2B£ cells - aerobic incubation  74  17.  IV.  CH2B  75  18.  V.  DNA single - strand break production  19.  R a d i o s e n s i t i z i n g p r o p e r t i e s of d e r i v a t i v e s  BIOCHEMICAL  cells - h y p o x i c incubation  R E D U C T I O N OF M I S O N I D A Z O L E A N D  AZO- AND AZOXY20.  2  76 80  ITS  DERIVATIVES:  R e d u c t i o n of m i s o n i d a z o l e b y  hypoxanthine  a n d x a n t h i n e o x i d a s e in h y p o x i a  86  21.  H P L C a n a l y s i s of p r o d u c t s of m i s o n i d a z o l e r e d u c t i o n  89  22.  R e d u c t i o n of a z o - m i s o n i d a z o l e b y  hypoxanthine  a n d x a n t h i n e o x i d a s e in h y p o x i a 23.  R e d u c t i o n of a z o x y - m i s o n i d a z o l e b y  90 hypoxanthine  and x a n t h i n e oxidase in h y p o x i a : I. 24.  Kinetics  II.  Representative  92 chromatograms  IN V I T R O M E T A B O L I S M O F  93  MISONIDAZOLE:  25.  D i s t r i b u t i o n of r a d i o a c t i v i t y - C H O c e l l e x t r a c t s  101  26.  TLC  a u t o r a d i o g r a p h y of o r g a n i c - s o l u b l e e x t r a c t s  102  27.  H P L C r a d i o c h r o m a t o g r a m s of o r g a n i c - s o l u b l e e x t r a c t s  106  28.  H P L C dual-label radiochromatograms  107  DISCUSSION: 29.  M e t a b o l i s m of m i s o n i d a z o l e : S c h e m a t i c  111  ix  ABBREVIATIONS AF-2  2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide  CHO  Chinese hamster o v a r y (cell  CI  C h e m i c a l i o n i s a t i o n (mass s p e c t r o s c o p y )  Ci  C u r i e (radioactive decay  EDTA  E t h y l e n e diamine t e t r a a c e t i c a c i d  El  E l e c t r o n i o n i s a t i o n (mass s p e c t r o s c o p y )  ESR  Electron spin resonance  FAD  Flavin adenine dinucleotide  FCS  Foetal c a l f s e r u m  FMN  Flavin mononucleotide  GSH  Glutathione (reduced  GSSG  Glutathione (oxidized form)  Gy  G r a y ( u n i t of r a d i a t i o n d o s e )  HPLC  High pressure liquid chromatography  i.p.  intra-peritoneal  IR  Infra-red  LET  linear energy transfer  NAD  Nicotinamide adenine dinucleotide  NMR  Nuclear magnetic resonance  4-NQO  4-Nitroquinoline-N-oxide  ODS  Octadecyl sulfonate  OER  O x y g e n enhancement ratio  PAC  Polar amino-cyano  PNAP  Para-Nitroacetophenone  PBS  Phosphate-buffered saline  PE  Plating efficiency  SER  Sensitizer enhancement ratio  SOD  S u p e r o x i d e Dismutase  SSB  Single-strand break  TLC  Thin-layer chromatography  TNT  2,4,6-trinitrotoluene  XO  Xanthine oxidase  line)  rate)  (spectroscopy)  form)  (spectroscopy)  X  ABBREVIATIONS: RADIOSENSITIZERS: Ro 07-0582  Misonidazole: 3-Methoxy-1-(2-nitro-1-imidazolyI)-2-propanol  Ro 05-9963  Desmethylmisdnidazole: 1-(2,3-dihydroxypropyl)-2-n?troimidazole  Flagyl  (I)  (H)  Metronidazole: 1-2'-hydroxyethyl-2-methyl-5-nitroimidazole  (m)  •N  N0  N  2  c H C H(OH)CH OCH 2  2  ;  N  N  H  N O p  CH CH(OH)CH OH 2  2  N  O ? N ^ ^ I S T \ : H CH CH OH 2  2  m  xi  ACKNOWLEDGMENTS  The  research  oratories  of  reported  the  Medical  Biophysics  and  C.  scientific  support  of  Cancer  staff  Gedy  of  lab-  Research  the  Cancer  In p a r t i c u l a r , Gudauskas,  Stich.  T h e t e c h n i c a l a s s i s t a n c e of Isabel H a r r i s o n a n d Hans A d o m a t is  humour  of  thesis!).  gratitude,  Bev  Ersoy  (who  Thank  you,  Gedy,  as  is  did  the  not,  for  Kirsten Skov,  secretarial  however,  and D r .  Dr.  Korbelik, Dr.  with  Krystal, Dr.  Dr.  in t h e  Mladen  acknowledged  Gerry  the  B.  C e n t e r f o r t h e i r a s s i s t a n c e and e n c o u r a g e m e n t . interest  thank  Unit,  performed  Research  the  to  was  I  appreciate  like  thesis  Center.  I  would  in t h i s  skill  have  and  to  type 6  putting  the  IBM  system  Whillans,  and  Dr.  G.F.  Hans  good the word  p r o c e s s o r at my f i n g e r t i p s . Dr.  A.M.  Ontario  Cancer  findings  before  Rauth,  Dr.  D.W.  Institute,  Toronto,  publication;  their  gave  generous  hospitality  and  Whitmore,  access exchange  to  their  of  ideas  d u r i n g several v i s i t s were a p p r e c i a t e d .  and  Most of a l l ,  I extend  speed-chess  champion  research Unit.  supervisor,  They  semi-colon  of  and  the  Lloyd  participated  over the past four  Finally, modation  have  Dr.  my t h a n k s  support,  Dr.  Medical  Skarsgard, in  every  Branko  Biophysics Head,  project,  Palcic, Unit,  Medical paper,  scientist and  my  Biophysics figure,  and  years.  I w i s h to t h a n k t h e travel  to  B.  and  C. the  Canada for financial support (Research  Cancer National  Foundation for accomCancer  Studentship).  Institute  of  Tokusan the  hall,  b r o u g h t his notes on t h e " D i a m o n d S u t r a " to t h e f r o n t of  pointed  have exhausted vast  space.  world, burned  to them  the Even  with  abstruse though  a torch,  doctrines, you  have  and it  is  learned  said: like all  "Even  though  p u t t i n g a h a i r in a the  secrets  of  it is l i k e a d r o p of w a t e r d r i p p e d on t h e g r e a t o c e a n . " A n d his  notes.  Then,  making  bows,  teacher.  Mumonkan  he  you  took  his  leave  of  the he his  1  INTRODUCTION: 1.1  Radiotherapy and the o x y g e n effect: Roentgen  the  new  form  diagnosis. which  were  erythema  attention  in  performed  of  by  so f r e e l y  being  their  own  and  was  first  therapeutic  the  breast  was  Mme.  used  in  Curie.  by  the  a  tool  his in  in  earliest  subjects.  for  medical  investigators.  Roentgen  himself  h a n d s , a n d s o u g h t medical the  irradiation,  cancer  The  to  Later  (Morgan,  as  weeks,  c o u l d damage t h e t i s s u e s t h r o u g h  made  s k i n damage  1895, a n d w i t h i n  used  experimental  1896.  too,  isolated  in December  was  January,  the  carcinoma Radium,  radiation  passed  they  suffered  of  x-rays  The discovery that x-rays  they  Often,  discovered  same treating  Besancon,  therapy  month,  ed.,  almost  a  Grubbe  patient  1974, p.  as  soon as  for 404).  it  was  new s c i e n c e of r a d i a t i o n b i o l o g y and  the  medical d i s c i p l i n e of r a d i a t i o n o n c o l o g y d e v e l o p e d f r o m t h e w o r k of s u c h pioneers. Initially, bacteria, the  plant  study  of  1950's  (see  which  concern  radiation seeds,  biologists and  mammalian  so o n ,  cells  in  following section). radiation  "lower"  organisms:  curves,  influence  for of  were or  confined  entire  vitro  animals.  were  Nevertheless,  biologists  today  to  not  study  of  Techniques  for  developed  many  were  the  of t h e  first  until  phenomena  explored  e x a m p l e , t h e s h a p e and d e s c r i p t i o n of radiation  quality,  and  the  i d e n t i f i c a t i o n of  with  survival cellular  "targets". The  r a d i o s e n s i t i z i n g action  t h e 1920's,  of m o l e c u l a r o x y g e n  was  observed  b u t i t s s i g n i f i c a n c e was not a p p r e c i a t e d ( P e t r y , 1 9 2 3 ) .  in The  r e s i s t a n c e of p o o r l y o x y g e n a t e d c e l l s to r a d i a t i o n damage was t h o u g h t to  2  be a c o n s e q u e n c e of t h e metabolic state of s u c h c e l l s , f o r example inhibition general  of m i t o s i s .  than  the  to  Thus,  role  of  of  an  the  the  historical  "hypoxic  effect  hypoxic  To  in  I shall d i s c u s s the  radiation,  radiotherapy. problem,  continue  elements  perspective.  possible  that this  "oxygen effect"  cells  cell  problem"  I  shall  development  of  chronological my own  later.  a  now  modern  failures  on t h i s of  response,  local t u m o u r  and  the  control  by  e v a l u a t e t h e c l i n i c a l s i g n i f i c a n c e of t h e h y p o x i c cell  it will be n e c e s s a r y  describe  with  shall  from  a  r e s p o n s e of mammalian c e l l s a n d  of o x y g e n in  development,  to examine t h e b i o l o g y of t u m o u r g r o w t h ,  a n d to c o n s i d e r e v i d e n c e f r o m h i s t o l o g y a n d r a d i a t i o n t h e r a p y . I  was  s p e c i e s f o r m e d at t h e time of i r r a d i a t i o n , came much  Rather  tissues  realisation  p h e n o m e n o n , c a u s e d b y t h e i n t e r a c t i o n of m o l e c u l a r o x y g e n  t h e chemical  outline  The  the  attempts hypoxic  narrative;  to overcome cell  this  the  sensitizers. will  provide  problem, Here,  I  in  Finally,  particular,  shall  return  the to  a  the  immediate b a c k g r o u n d  to  of  clinical  importance,  is  b y cell s e n s i t i v i t y . T h e r a d i a t i o n s e n s i t i v i t y of an o r g a n  or  research.  1.2 R a d i a t i o n a n d cell s u r v i v a l : Tissue determined  sensitivity,  the  endpoint  t i s s u e d e p e n d s on two major f a c t o r s : t h e i n h e r e n t r a d i o s e n s i t i v i t y of t h e cells  of  capacity  which  it  is  composed  following exposure)  (as  measured  by  their  reproductive  a n d t h e i m p o r t a n c e of cell p r o l i f e r a t i o n in  m a i n t a i n i n g t i s s u e f u n c t i o n ( H a l l , 1978, c h a p t e r 1 0 ) .  The second factor  a c c o u n t s f o r most of t h e v a r i a t i o n in s e n s i t i v i t y among normal mammalian  3  tissues.  For example, the rapidly dividing stem cells of the hemopoietic  system and  the intestinal wall are particularly sensitive, whereas  non-  d i v i d i n g cells such as neurons are much more resistant to radiation. consequence, the  principal morbidity  radiation exposure are  syndromes following  hematological and  tumour is characterised  by  the  whole-body  gastrointestinal.  uncontrolled  In  A  (although not  malignant necessarily  rapid) growth of its cells. T h u s , although the tumour as a whole might be  expected to be  sensitive to radiation  (as measured by  decrease in  volume following radiation treatment, for example) its capacity to regrow from a small number of s u r v i v i n g  cells places a stringent  requirement  on  radiation therapy: the tumour must be almost completely sterilized.  At  the  same time, damage to surrounding normal tissue must be  kept  within acceptable limits. How  does cell survival depend on dose?  This question was  in a phenomenological manner at a time when little was mechanisms  by  which  molecules. It will be here. this  ionizing  radiation  studied  known about the  interacts  with  biological  useful to introduce such a phenomenological model  I shall assume cell s u r v i v a l to mean proliferative capacity, property  commonly.  (colony forming a b i l i t y ) is the  Also,  it may  be  the  since  endpoint measured most  endpoint of most direct relevance to  radiotherapy. Radiation dose, the independent variable in radiobiological models, is defined as  energy deposited per  dose is measured in Grays: 1 Gy 100  erg/gm = 0.01  that the  Gy,  unit mass.  = 1 Joule/kg. (The  is still widely used).  CGS  In SI  units,  unit, 1 rad =  It is important to note  Gray, a unit of absorbed dose, is not directly convertible  to  4  such  units  as  measure  of  ignores  the  Thus,  it  an  different not  differ  The  microscopic  those of  mammalian  cells  development  of  Marcus,  functions  of  not  for  S  is  Bacterial  a  plotted  constant.  as  for  survival  the  assumption  often  display  been  proposed of  multiple  hits,  A  that the  a  presence  "multitarget  function  curves.  kills  shouldered  a  a  of  single  cell.  or  a  survival theory"  as  this  targets  of  limited  curves. equation:  type  term of  (a  which  radiation.  of  1  Gy  of  cells  is  colony  on  free-living a  such  visible  experiments  mid  1950's, cells  are  using  following  in  culture  typically  yields of  as  survival  cell, repair  this  in  a  plot  type  such  behaviour  In  the  curves  illustrated  cellular  into  of  this  "hit",  per  Curie  the  (Puck  exponential  V  this  Actually,  explain  multiple  D:  grow  equation  response  "shoulder", to  an  of  Analogous  growth  (D/  Such  types  effects  ability  can  survival  e"  =  the  macroscopic  different  until  dose,  D Q is  molecule,  the  a  or  x-rays.  days.  performed  S  where  of  is  biological  forming  two  techniques  1956).  1 Gy  ionisation)  of  the  bacterium  about  were  effects  colony A  of  Dose  that  of  straightforward. in  measure  rate).  surprising  from  plate  (a  decay  measurement  agar  and  Roentgen  radioactive  is  neutrons  usually  the  is  fig.  for  1a.  a  single  I  of  a  Many  chap.5);  all  use  on  target  models  target  log used  mammalian  could  shall  if  anticipated  Alper,1979,  capacity  thesis,  be  line  always  ionisation  curves  or  almost  would  the  (e.g.  straight  cells have the  requiring account  the  for  so-called  5  Fig.1  Radiation s u r v i v a l c u r v e s in v i t r o  a) A typical s u r v i v a l c u r v e for mammalian c e l l s , modeled by the multi-target equation; the dashed line indicates the measurement of the extrapolation number, n. b) The effect of hypoxia on s u r v i v a l c u r v e s , of the oxygen enhancement ratio, OER.  i l l u s t r a t i n g the definition  6  Here,  a  = 1 - ( 1-  e'  a  constant  with  is  dimensionless Taylor's  S  constant  known  e x p a n s i o n of the  as  power  )  n  dimensions  the  target  1/Dose,  number.  and  For  n  large  is  a  D,  a  yields:  = n  S  a D  e  T h i s e q u a t i o n is a s t r a i g h t line w h i c h e x t r a p o l a t e s b a c k to t h e p o i n t S = n,  D  =  number. 1/e,  fig.  (see The  value is,  la). of  for  Thus,  D Q (the mammalian  n  is  dose  also  called  required  cells,  to  of o r d e r  the  extrapolation  reduce  survival  100 - 2 0 0 r a d s  by  (Hall,  p.37).  The  o x y g e n effect and cell  survival:  The  e f f e c t of o x y g e n on c e l l u l a r r a d i a t i o n r e s p o n s e is i l l u s t r a t e d in  1b.  In  cells  to  and  second  purging  this  x-rays for  with  similar  shape,  is v e r y  figure, is  the  plotted, medium  purified  radioprotective  kill  fig.  D Q = 1/a)  1978,  1.3  0  different  from  the  the  of  Chinese  which  oxic  dose  all  The  oxygen  and  hypoxic  required  OER  ratio ( O E R ) ,  =  has  ovary  to  survival  produce  This difference  defined  as:  2  equal  S  (CHO) medium,  removed  has a curves  a given  Dose in n i t r o g e n : : — Dose in a i r  TT  been  absence of o x y g e n  in the two cases.  the oxygen enhancement  hamster  f i r s t f o r i r r a d i a t i o n in a i r - s a t u r a t e d  nitrogen.  effect; but  response  level  by  dramatic are of  is m e a s u r e d  of cell by  7  If  oxygen  survival  'dose-modifying , can  is,  radiations,  S  =  such  as  be s u p e r i m p o s e d b y  0.1  is  commonly  x-rays,  the  OER  aerobic  a change  used. for  the  survival  levels  of each  p o p u l a t i o n e x p o s e d to a g i v e n d o s e . when  and  hypoxic  of t h e d o s e  scale,  For  sparsely  mammalian  cells  ionising  is a b o u t  3.  cell t y p e  in a  heterogeneous  T h i s is t h e s i t u a t i o n t h a t o b t a i n s  a t u m o u r is i r r a d i a t e d , s i n c e t h e s u r r o u n d i n g normal t i s s u e s  also e x p o s e d . aerobic  horizontal of t h e Thus,  Here, cell  cut  survival  at  a  given  curves  dose:  curves  implies t h a t  this  Kallman,  well-oxygenated curve.  This  1968).The  mathematical  r a t i o may  and  therapy,  it  in  family  studies  of  a  drastically  curves  shown  However, of  in  remarkably  tumours  m  rather  vivo.  The  than  a  steepness  1,000 o r  more.  of h y p o x i c c e l l s in an alters van  fig.  2  similar To  the  overall  Putten is  and  simply  a  curves  have  understand  these  to e v a l u a t e t h e i m p o r t a n c e of t h e o x y g e n e f f e c t in r a d i o is  necessary  to  review  the  r a d i o r e s i s t a n t h y p o x i c c e l l s in t u m o u r s . following  1%)  be  i l l u s t r a t e d in f i g . 2 ( a f t e r  construction.  obtained  results,  is  population  a vertical  of f i g . 1 b .  an a d m i x t u r e of a small f r a c t i o n ( s a y ,  survival  are  t h e r e l e v a n t p a r a m e t e r is t h e r a t i o between a n o x i c  t h r o u g h the s u r v i v a l  survival  otherwise  been  the  r a d i o t h e r a p i s t is c o n c e r n e d w i t h a d i f f e r e n t c o m p a r i s o n : he w i s h e s  to d e t e r m i n e  and  if  will be i n d e p e n d e n t of t h e v a l u e of S c h o s e n f o r c o m p a r i s o n ;  practice,  The  that  1  curves  then OER in  is  brief  oxygen effect.  d i s c u s s i o n of  the  evidence  for  the  presence  of  I s h a l l r e t u r n to t h i s q u e s t i o n  r a d i o b i o l o g i c a l mechanisms  of  the  8  I  o  10  20  DOSE Fig.  The  dashed  populations. resulting  2 Radiation  curves  show  The  overall  hypoxic fractions  survival  the  heavy survival of  1 % and  curves  response  curves curves 10%.  i  are  30  (Gy)  for  mixed  of f u l l y  anoxic,  theoretical  for  mixed  populations  and  fully  constructions populations  aerobic of  the  containing  9  1.4 T h e o x y g e n e f f e c t - m e c h a n i s m s : The  absorption  of  high  energy  radiation  in  matter  causes  ion-  i s a t i o n s a n d e x c i t a t i o n s of atoms a n d m o l e c u l e s .  R e g a r d l e s s of t h e t y p e  of  incident  principally  for  these  radiation effects.  or  particle,  Electron  electrons  impact  are  creates  free  responsible  radicals  (generally,  molecules w i t h an o d d n u m b e r of e l e c t r o n s )  by s t r i p p i n g electrons from  molecules.  d e p o s i t i o n is f o l l o w e d b y a  This  "chemical  "physical  stage"  radicals created  lasting  long-lived)  modified  by  reactions  alterations,  processes. agents  microseconds.  In  this  stage,  the  free  r a d i a t i o n i n t e r a c t w i t h u n d a m a g e d molecules a n d  These  chemical  recombination  a few  by the  w i t h one a n o t h e r .  s t a g e " of e n e r g y  that  can c a u s e " p e r m a n e n t "  or  The  they  can  biological  modify  this  terminate  effect  stage  of  of  ( o r at l e a s t , in  harmless  radiation  chemical  will  be  interactions.  E v e n if l o n g - l i v e d chemical c h a n g e s o c c u r , t h e s e may s t i l l be s u b j e c t to r e p a i r b y chemical o r e n z y m a t i c p r o c e s s e s .  Presumably,  t h e lethal a n d  mutagenic  effects  damage  macromolecules  (especially  D N A ) t h a t is not r e p a i r e d , o r is m i s r e p a i r e d .  Primary action  and  of  radiation  radiation indirect  events  action  are  may  (e.g.  due  be  to  divided  Bacq  in  into two c l a s s e s :  & Alexander,  1961, c h a p .  D i r e c t a c t i o n is t h e i o n i s a t i o n of a macromolecule b y a p r i m a r y event;  indirect  formed  by  as r ^ O ) .  the  action  is c a u s e d  absorption  by  the  reaction  of r a d i a t i o n e n e r g y  T h e s e s p e c i e s may t h e n a t t a c k  of  direct  chemical  2).  radiation species,  in small molecules  (such  macromolecules.  Water is t h e most common molecule in a l i v i n g c e l l , a n d t h e a c t i o n of r a d i a t i o n on w a t e r has been s t u d i e d in d e t a i l .  T h i s w o r k also dates  10  back  to  the  earliest  studies  o b s e r v e d t h e e v o l u t i o n of H (1901).  2  of  and 0  radioactivity:  Curie  and  Debierne  f r o m a q u e o u s s o l u t i o n s of Ra s a l t s  2  T h e c h e m i s t r y of w a t e r r a d i o l y s i s was s t u d i e d b y Weiss  who p r o p o s e d t h a t t h e i n i t i a l p r o c e s s was t h e p r o d u c t i o n of H ' radicals.  These  species then produce even-electron-number  H' OH  +  H"  -  +  OH'  OH'  +  H'  (1944)  and O H ' molecules:  H, H  -*  In t h e p r e s e n c e of o x y g e n ( 0 ) 2  2°2 H 0 2  o t h e r r e a c t i o n s can o c c u r , a l t e r i n g t h e  y i e l d s of t h e p r i m a r y r a d i o l y s i s p r o d u c t s , a n d c r e a t i n g r a d i c a l s s u c h as superoxide: H'  The  p r o d u c t s of  +  0  2  • H0  • H  2  +  +  0 " 2  r a d i o l y s i s of w a t e r can damage m a c r o m o l e c u l e s ,  OH'  +  RH  • H 0  +  2  e.g.:  R'  y i e l d i n g p r o d u c t s s i m i l a r to t h o s e p r o d u c e d b y d i r e c t d a m a g e :  RH  RH  +  R*  +  H  +  +  e~  O x y g e n may act as a r a d i o s e n s i t i z e r in a n u m b e r of w a y s : t h e importance  of  various  theoretically  p l a u s i b l e mechanisms  relative  remains  con-  11  troversial. 1979).  (For  a review,  see E w i n g a n d P o w e r s ,  in M e y n &  Withers,  G r a y p r o p o s e d t h a t o x y g e n c o n v e r t s H ' to s u p e r o x i d e :  0  (Gray,  +  2  1954).  e"  Alper  »  suggested  that  0 " 2  oxygen  interacts  with  direct  damage s i t e s to f o r m o r g a n i c p e r o x y r a d i c a l s : FT  (Alper,  1956).  +  0  2  » ROO*  :  0  Such radicals dissociate, yielding permanently  damaged  macromolecules. A l p e r and Howard-Flanders as  a  function  of  the  oxygen  studied the partial  t h r o u g h the suspensions ( A l p e r  r a d i o s e n s i t i v i t y of b a c t e r i a  pressure  in  & Howard-Flanders,  the  gas  1956).  bubbled  They  pro-  p o s e d an e m p i r i c a l r e l a t i o n s h i p to d e s c r i b e t h e d e p e n d e n c e :  S S  where pressures maximum Sensitivity P=K ( f i g .  S of  and  N  N  p  zero and  value rises 3).  S  of  m P + K  p  OER,  steeply  P + K are  P,  the  radiosensitivities  (a's)  m is a d i m e n s i o n l e s s c o n s t a n t and at  K  is  a  low o x y g e n  constant tension,  at  C>  partial  equal  to t h e  (dimension and  levels  2  pressure). off  above  12  air > LU >  3.0  CO  z  LU CC  LU CO  O  tt  2.0  hr /  y  1-01-  I  0  1  1  2 0  I  L.—I  4 0  i  '  »  6 0  O X Y G E N C O N C . (mm  Hg)  Fig. 3 R a d i o s e n s i t i v i t y as a function of oxygen concentration  The dashed c u r v e illustrates the form of the A l p e r equation ( t e x t , page 11), d e s c r i b i n g the radiosensitivity of cells at oxygen partial pressures ranging form complete anoxia to saturated oxygen.  13  K  Hg,  well below the oxygen tension of  venous blood (Hall, 1978, p. 85-87).  This suggests that normal tissues  are  is typically about 3 mm  almost  fully  oxygenated,  in terms  of their  However, it should be noted that the m out  in dilute  consumption  suspension;  iri vivo,  radiation  response.  vitro experiments are carried  it is likely  that cellular  oxygen  will reduce the effective oxygen tension in the cell.  1.5 The oxygen effect - clinical significance: The  presence  of a small fraction of r a d i o b i o l o g i c a l ^ hypoxic cells  (P S K, as defined above) greatly alters the overall radiation of a mixed population of cells. on  Thomlinson  response  and Gray (1955) suggested,  the basis of histological evidence, that hypoxic cells may  solid tumours. carcinoma.  The  tumour studied in this work was  Although  it may  exist in  human bronchial  appear paradoxical to search for hypoxic  cells in the lungs, Thomlinson  and  Gray pointed out that the tumour  tissue grew in solid cords or rods of epithelium. The capillary network of the  stroma  studied  the  found  that  did  histology cords  characterized by had were  tumour cells. was  of  penetrate the tumour. Thomlinson  radius  leaving  greater  a so-called  distinguishable from The  and  of tumour cords of various diameters. than  about  a core region of tissue in which  disintegrated, easily  not  the  "necrotic surrounding  0.2  mm  Gray They were  the cellular matrix  centre".  Such cells  "sheath" of viable  authors suggested that the sheath t h i c k n e s s , which  f a i r l y constant, represented the limit of oxygen diffusion  inward  from the surrounding stroma, and that the central necrosis was a con-  14  sequence and  of o x y g e n  viable  growth by  radiation)  was  from  tumours.  suspected.  studies  4).  mary  of t h e s e  Many  fraction  may  to  y  more t h a n  (fig.  the  indirect.  an  in  vivo  necrotic continued  4),  (1978,  reported  table  allowing,  if  curves.  Such  influence  on  model  radiation  12-1).  The  for  (1979)  measure  tumour  "tail"  the  range  results  back  effect  from  of  1% to  model  radiobiological  oxygenation  sum-  h y p o x i c cell  Thomlinson-Gray  modern  response technique  resistant  estimates  The  rodent  s i n c e 1963; a  necessary,  have produced ambiguous  has  research. somewhat  directly,  (Cater & Silver,  by 1960;  1963).  chronic with  to  killed  received  dilution assay  b y e x t r a p o l a t i o n of t h e  survival  Attempts  Evans & Naylor,  tumour  the  it must be a d m i t t e d t h a t i t s e x p e r i m e n t a l b a s i s is  polarography,  Brown  Hall  hypoxia"  measured the  20%, in d i f f e r e n t t u m o u r s .  Nonetheless,  as  between  r e s p o n s e of e x p e r i m e n t a l  (1963)  using  in  be estimated  tremendous  The  "chronic  radiation  Tolmach  is g i v e n  on  This  s i m i l a r s t u d i e s h a v e been  axis  shoulders  and,  of t h e  lymphosarcoma,  (fig.  had  the boundary  if t h e s u r r o u n d i n g a e r o b i c c e l l s w e r e to be  Powers and  a mouse  the  At  r e g i o n s , t h e p r e s e n c e of h y p o x i c c e l l s c a p a b l e of  (particularly  support  of  depletion.  hypoxia  most has  hypoxia,  model  dogmas, suggested  which  he  it  has  come to  has  attracted  that  there  refers  to  may  as  be  regarded  its be  "acute  share a  as of  heretics.  different  hypoxia".  dogma;  type  of  Acutely  h y p o x i c c e l l s may be located close to c a p i l l a r i e s ; t e m p o r a r y o c c l u s i o n of blood  flow  oxygenate and  in  the  capillary  if t h e c a p i l l a r y  c l o s i n g may  occur  on  induces  re-opened;  hypoxia.  Such  cells  would  re-  i t is s u g g e s t e d t h a t t h i s o p e n i n g  a time-scale  of  minutes.  The evidence  for  15  I000  I500  2000  2500  Dose (rod)  F i g . 4 In vivo radiation survival  curves:  Demonstration of presence of resistant fraction (from Powers and Tolmach, 1963, by permission)  X-ray survival  curve for mouse lymphosarcoma cells irradiated in vivo.  16  a c u t e h y p o x i a is s u m m a r i z e d in B r o w n ' s p a p e r . studies  (Yamaura  & Matsuzawa,  r a d i o s e n s i t i z e r s (see Thus, major  1979)  and  It i n c l u d e s h i s t o l o g i c a l  r e s u l t s of e x p e r i m e n t s  with  below).  a l t h o u g h t h e r e is g e n e r a l a g r e e m e n t t h a t h y p o x i c c e l l s a r e a  cause  of  tumour  radioresistance,  the  nature  of  hypoxia,  par-  t i c u l a r l y in human t u m o u r s , remains c o n t r o v e r s i a l .  1.6 T h e r a p i e s w h i c h may o v e r c o m e t h e r a d i o r e s i s t a n c e of h y p o x i c c e l l s : Various  approaches  proposed which may, cells.  These  to t h e  m o d i f i c a t i o n of r a d i o t h e r a p y  have  been  it is h o p e d , r e d u c e t h e r a d i o r e s i s t a n c e of h y p o x i c  include  the  use  of  new  forms  of  radiation,  different  f r a c t i o n a t i o n s c h e d u l e s , and chemical r a d i o s e n s i t i z a t i o n . The  b i o l o g i c a l e f f e c t i v e n e s s of a p a r t i c u l a r f o r m of r a d i a t i o n , s u c h  as X - r a y s o r n e u t r o n s , is r e l a t e d to i t s i o n i z a t i o n d e n s i t y . T h i s may be expressed  in  radiation. deposits been  terms  This energy  of  the  quantity as  or  describes  it t r a v e l s  linear the  through  as  low  as  modalities  in  hypoxia.  This  depth-dose  1.0. cancer is  matter.  seems  therapy  characteristics  particles  at  transfer,  which  The  v a l u e of  the  particle OER  has  ( B a r e n d s e n et a l . , 1 9 6 6 ) .  clear  that  should  the  reduce  use  of  the  effect  T h i s c o m b i n a t i o n of f e a t u r e s interact  such  radiation  of  tumour  in a d d i t i o n to lower O E R ,  of t h e r a d i a t i o n p e r m i t b e t t e r  which  the  of  is less t h a n 2, and f o r a - p a r t i c l e s it can  particularly likely if,  dose in t h e t u m o u r . (short-lived  It  energy  rate  f o u n d to d e c r e a s e w i t h i n c r e a s i n g L E T  For neutron i r r a d i a t i o n , OER be  LET,  strongly  the  l o c a l i s a t i o n of  makes t h e use of n  with  atomic  nuclei)  in  17  radiotherapy  appear  very  ever,  the  ning,  a n d many t e c h n i c a l  be  development  routinely  promising  of t h e s e  applied.  (Skarsgard  new  et a l . ,  1980).  How-  r a d i a t i o n modalities is j u s t  begin-  d i f f i c u l t i e s must be overcome b e f o r e t h e y  In  particular,  cost  is  certain  to  be  a  can  factor  obstructing their use. T h e most d i r e c t a p p r o a c h to o v e r c o m i n g t h e h y p o x i c cell p r o b l e m is hyperbaric radiated  oxygen  therapy.  With  this  technique,  in a p r e s s u r i z e d o x y g e n - r i c h e n v i r o n m e n t .  this approach  is s t r a i g h t f o r w a r d :  an i n c r e a s e  r e d u c e t h e s i z e of t h e h y p o x i c f r a c t i o n . pieces  of  therapy  evidence  for  the  is an a n a l y s i s  haemoglobin  levels  carcinoma. per  patients  100  on  Those ml  Bush  cure  patients  had  better  Nonetheless,  results  encouraging.  The  percentage  by  importance  increase  of  rates  with  blood  this  into the  depleted  by  problems,  hypoxic  than  are  metabolism  hyperbaric  in  oxygen  the is  effect  treated  levels those  oxygen  with  radio-  than  12 g levels.  been  two-fold.  capacity  cervical  lower  not  very  First,  is f a r  the  smaller  S e c o n d , o x y g e n is u n a b l e to  of t h e t u m o u r b e c a u s e periphery. also  on  for  greater  have  probably  oxygen-carrying  region  for  of t h e i n f l u e n c e of blood  patients  t h a n t h a t of t h e o x y g e n p a r t i a l p r e s s u r e . penetrate  oxygen  (1978)  hyperbaric for  rationale  in blood o x y g e n a t i o n may  of t h e  of  rates  ir-  I n d e e d , one of t h e most d i r e c t  haemoglobin  cure  with  reasons  et a l .  The  are  In  dangerously  i t is  addition toxic,  rapidly  to and  these the  p r o c e d u r e as a whole is d i f f i c u l t to p e r f o r m on a r o u t i n e b a s i s . The  newest  a n d most p r o m i s i n g a p p r o a c h to t h e c o n t r o l of h y p o x i c  c e l l s is t h e use of chemical r a d i o s e n s i t i z e r s , t h a t i s , d r u g s w h i c h mimic the  radiation-sensitizing properties  of o x y g e n .  One s u c h d r u g ,  miso-  18  nidazole,  is the focus of intense clinical and research interest.  discussing misonidazole, however,  I shall briefly  Before  outline the historical  development of this approach. Recent work  of  interest  Dr.  G.  Cancer Research  E.  in chemical radiosensitizers was stimulated by the Adams  and  colleagues  at  the  Campaign, Northwood, England.  Gray  Laboratory,  Adams observed that  the compounds known to be sensitizers (including, at the time, oxygen, NO,  and  certain  iodine-containing organics)  being efficient electron acceptors.  shared  Adams and Dewey  the  property  of  (1963) suggested  that: . . . an important contributory factor to radiosensitization is the capture and stabilisation of the (electron) . . . the negative radical ion so produced is longer lived than the free hydrated electron ... The sensitizer would be regarded, therefore, as an electron carrier and ideally would be a molecule combining a high electron affinity with a structure suitable for delocalisation of the attached electron . . . This criterion guided the search for effective variety  of in vitro test systems.  effective  in vitro,  1967, and Parker al.,  1971).  described Moroson  radiosensitizers, using a  Several compounds were found to be  including stable  nitroxyl free  radicals  (Emmerson,  et a l . , 1969) and aromatic nitro compounds (Adams et  Many of the early studies of chemical radiosensitization are in  and  the M.  Radiosensitization  volumes  Radiation  Quintilliani, (1974).  ed.,  However,  Protection 1970),  and  and  Sensitization  Advances  sensitization  irii  (H.  T_n Chemical was  not  achieved until 1971 (p-nitro-3-dimethylaminopropiophenone, NDPP),  and  the search for the ideal radiosensitizer continues.  vivo  19  What p r o p e r t i e s w o u l d s u c h a d r u g  possess?  First,  of c o u r s e ,  it  must be an e f f e c t i v e r a d i o s e n s i t i z e r , at a c l i n i c a l l y a c h i e v a b l e d r u g c o n centration. is,  if we  It is not n e c e s s a r y t h a t it be as e f f e c t i v e as o x y g e n . define  r a d i a t i o n doses  a s e n s i t i z e r enhancement r e q u i r e d to p r o d u c e  ratio,  SER,  a given effect  as t h e  1.3,  OER.  A  large  SER  is d e s i r a b l e ,  b u t an S E R  w o u l d s t i l l be of s i g n i f i c a n t c l i n i c a l b e n e f i t .  sensitizer  should  not  increase  the  r a t i o of  in t h e a b s e n c e  p r e s e n c e of t h e d r u g , t h e n we do not r e q u i r e t h a t t h e S E R as t h e  That  and  be as  large  as low a s ,  say,  A t t h e same t i m e ,  radiosensitivity  of  normal,  the  aerobic  c e l l s , o r t h e b e n e f i t s will be n u l l i f i e d . Second,  t h e d r u g m u s t be able to p e n e t r a t e i n t o t h e c e n t e r of t h e  t u m o u r ; t h i s implies t h a t it must be able to c r o s s t h e b a r r i e r by  the  cell  membranes  penetration  via  the  means  the  drug  that  of  the  extracellular must  have  surrounding space  is  tumour  possible).  presented  tissue  (unless  Generally,  lipophilic properties.  On  this  the  other  least,  much  h a n d , w a t e r - s o l u b i l i t y is r e q u i r e d f o r a d m i n i s t r a t i o n . Third,  the  drug  must  be  metabolically  stable,  or  at  more s t a b l e t h a n o x y g e n ; o t h e r w i s e , t h e d r u g will be u s e d u p b e f o r e it reaches the h y p o x i c target cells. Finally,  the  toxicity  within tolerable limits.  and  This  side-effects  of t h e  drug  must  be  kept  last c r i t e r i o n has p r o v e n to be one of t h e  most d i f f i c u l t to s a t i s f y . By known, scure  the  early  1970's, a number  of  on t h e b a s i s of in v i t r o t e s t s . all  but  the  first  criterion  effective  radiosensitizers  However,  in v i t r o s y s t e m s o b -  mentioned  above.  There  is o n l y  were  one  20  membrane to cross, bathing  the cells,  insignificant.  is present in excess in the medium  so metabolic depletion of the  Agnew  concentration including  and the drug  and Skarsgard  on the  (1974) studied the effect  sensitizing efficiency  para-Nitroacetophenone  drug concentration  (PNAP)  is  of cell  of a number of compounds, and nitrofurazone.  Although  SER's of 1.5 or more were achieved in dilute cell suspensions, most of this  enhancement  was  lost in concentrated  "cell  pellets".  This  effect  illustrated the importance of metabolic stability in the development of an effective  radiosensitizer.  1.7 Nitroimidazole radiosensitizers: Foster  and  screened for made  it  Willson  (1973)  suggested  that  available  structures with radiosensitizing potential.  possible to  circumvent  much  of  the  were already  electron-affinity 1954).  of  Chapman  time-consuming  process  Nitrofuran  in clinical use as antibacterial agents, and the  these  and  be  This approach  required for the development of an entirely new compound. derivatives  drugs  compounds  colleagues  had  showed  been that  demonstrated the  (Sasaki,  nitrofurans  nitro-  furazone and nitrofurantoin were good radiosensitizers in vitro (Reuvers et a l . ,  1972).  furans  and  studies  were  and  toxicity  Subsequently,  nitroimidazoles performed, were  mising  compound  studied a large  (Chapman  using mice;  considered,  The 5-nitroimidazole  they  as  well  etal.,  number of  1974).  Pharmacological  questions of metabolic as  in_ vitro  nitro-  stability  radiosensitization.  drug metronidazole appeared to be the most p r o -  studied.  Willson  has  published  an  interesting  his-  21  torical  a c c o u n t of t h e s e d e v e l o p m e n t s  (in  Finegold, e d . ,  1977, p. 147-  175). A t t h i s p o i n t , it is w o r t h w h i l e to d i g r e s s b r i e f l y , a n d c o n s i d e r t h e history drug  of  is  relevant  shortly. Poulenc find  metronidazole. to  the  In t h e e a r l y initiated  natural  parasites  The  an  rationale  problems  of  for  drug  the  development  t o x i c i t y to  be  of  this  considered  1950's, t h e F r e n c h p h a r m a c e u t i c a l f i r m of Rhoneextensive  products  with  Trichomonas.  screening  cytotoxic  Substantial  programme  activity activity  in an  against was  attempt  the  protozoal  discovered  e x t r a c t p r e p a r e d f r o m a s t r a i n of t h e f u n g u s S t r e p t o m y c e s .  to  in  The  an  active  p r i n c i p l e was i s o l a t e d a n d i d e n t i f i e d ; it was f o u n d to be i d e n t i c a l to t h e c o m p o u n d a z o m y c i n , o r 2 - n i t r o i m i d a z o l e ( d e s p i t e t h e t r i v i a l name, t h i s is not an azo c o m p o u n d ) .  In f a c t , t h e 'new' d r u g had a l r e a d y been  acterised  scientists  by  Japanese  Rhone-Poulenc tested further  them f o r  synthesised  a  (Maeda  variety  et a l . ,  of d e r i v a t i v e s  therapeutic effectiveness.  development,  1953).  char-  Chemists  of a z o m y c i n ,  The derivative  selected  1-2'-hydroxyethyl-2-methyl-5-nitroimidazole,  g i v e n t h e a p p r o v e d name m e t r o n i d a z o l e .  T h i s w o r k was r e v i e w e d b y  E.  p. 3-11).  Jolles  introduced vaginalis,  (in in  Finegold, Europe  ed.,  in  a n d u s e d in t h e  prescribed very  widely.  1960 U.S.A.  1977, for  the  Metronidazole  treatment  a f t e r 1963.  of  at and for was G. was  Trichomonas  S i n c e t h e n it has been  A n i n t e r e s t i n g r e v i e w of t h e use of t h i s d r u g  has been p u b l i s h e d r e c e n t l y ( G o l d m a n , 1980). Other therapeutic  nitroheterocyclic agents  even  compounds  earlier.  Dodd  had and  been  examined  colleagues  as  chemo-  described  the  22  antibiotic 1944).  properties However,  of  nitrofurans  the  introduction  a t t e n t i o n on n i t r o a r o m a t i c s .  in  the  of  1940's  (Dodd  metronidazole  &  Stillman,  focussed  new  A r e v i e w of t h e many n i t r o c o m p o u n d s now  i n v e s t i g a t e d has been p r e s e n t e d ( G r u n b e r g  & T i t s w o r t h , 1973).  T h e chemical s y n t h e s i s of a z o m y c i n was r e p o r t e d in 1965 ( L a n c i n i Lazzari,  1965; Beaman et a l . , 1 9 6 5 ) .  f i r m Hoffman therapeutic  workers  the  pharmaceutical  - La R o c h e i n i t i a t e d a s t u d y of t h e c h e m i s t r y and chemoproperties  of  2-nitroimidazole d e r i v a t i v e s .  compounds were s y n t h e s i s e d activity  Subsequently,  &  (e.g.  that:  11  such  Beaman et a l . , 1967) and t e s t e d f o r  a g a i n s t b a c t e r i a and p r o t o z o a ( G r u n b e r g concluded  D o z e n s of  etal.,  1967).  These  1 -(2-nitro-1 - i m i d a z o l y l ) - 3 - m e t h o x y - p r o p a n o l  is of s p e c i a l i n t e r e s t b e c a u s e of its low t o x i c i t y , m a r k e d a n t i t r i c h o m o n a l activity, As  a n d good a c t i v i t y a g a i n s t Entamoeba h i s t o l y t i c a soon as  d i s c o v e r e d , the  the  radiosensitizing properties  British  b r a n c h of Hoffman - La  of t h e a v a i l a b i l i t y of t h e s e  of  series  (Adams  correlated  et a l . ,  5-nitro  judged  compounds.  Again,  the  and  experimentation Ro-07-0582.  distributed  to  various  iin v i t r o a n d iri v i v o ,  It was f o u n d t h a t  In  general,  the  in  the  2-nitro-  r a d i o s e n s i t i z e r s t h a n t h e 4-  methoxy-propanol  to be t h e most p r o m i s i n g a g e n t .  quantity  advantage  with electron-affinity  1976).  imidazoles w e r e f o u n d to be more e f f e c t i v e or  took  were  nitroimidazole d e r i v a t i v e s and began a s t u d y  radiosensitizing ability  nitroimidazole  nitroaromatics  Roche  of t h e r a d i o s e n s i t i z i n g a b i l i t i e s of t h e s e c o m p o u n d s . i_Q v i t r o  ..."  The  d r u g was  laboratories  under the  derivative synthesised for  was in  further  Roche p r o d u c t number  23  At usable drug  the  same  time,  radiosensitizer, was  already  metronidazole  was  a process greatly  in c l i n i c a l u s e ,  developed  speeded  into a  by the fact that  The  were  col-  of  leagues  t h e same g r o u p , w o r k i n g at t h e C r o s s C a n c e r  in E d m o n t o n ,  the  albeit for a different p u r p o s e .  pharmacokinetics (1974);  clinically  metronidazole  studied  by  Urtasun  and  Institute  C a n a d a , b e g a n t h e f i r s t c l i n i c a l t r i a l of m e t r o n i d a z o l e as a  r a d i o s e n s i t i z e r ( U r t a s u n et a l . , 1 9 7 6 ) .  1.8 T o x i c i t y of N i t r o a r o m a t i c C o m p o u n d s : Anticancer rather than tissues  in many  d i r e c t l y to t h e t u m o u r .  are  imidazoles  drugs must,  frequently are  much  cases,  systemically,  T h u s , t o x i c s i d e e f f e c t s on normal  encountered. less  be d e l i v e r e d  effective  On  a  molar  sensitizers  basis,  than  is  the  nitro-  oxygen.  To 2  a c h i e v e s i g n i f i c a n t s e n s i t i z a t i o n , d r u g doses of t h e o r d e r of 10 g/m necessary.  This  is an  enormous  dose,  when  compared  to most  are other  pharmaceuticals.  It was c r i t i c a l , t h e n , t h a t d r u g s of o u t s t a n d i n g l y low  systemic  be  toxicity  promising:  it  is  trichomoniasis dry  mouth,  (Goldman, observed  selected.  " remarkable  On  this  since,  basis,  metronidazole  in t h e dose w h i c h  is c u r a t i v e  [ a b o u t 3 g ] t h e s i d e - e f f e c t s s u c h as n a u s e a , and  a  funny  1980)  high  neuropathy  a l . , e d . , 1973, p.  are  Nevertheless,  following  antoin-induced  taste  31-56).  neuropathic  doses was  relatively  also  of  mild a n d  known  (le  Quesne,  for  headache,  self-limited."  side-effects  metronidazole,  appeared  and in  had  been  nitrofurBaron,  et  24  T h e f i r s t c l i n i c a l t r i a l s of m e t r o n i d a z o l e r e v e a l e d n e u r o p a t h i c s i d e effects,  as well as n a u s e a a n d v o m i t i n g .  numbness, hearing Usually, avoided  and  loss,  r i n g i n g in t h e paralysis,  these by  and  side-effects  limiting the  ears. even  have  total  Patients experienced t i n g l i n g ,  In  severe cases,  psychosis been  drug  (Urtasun  temporary,  dose.  However,  there  may  etal.,  and  they  such  be  1977). can  be  limitations  r e d u c e t h e l i k e l i h o o d of a c h i e v i n g e f f e c t i v e r a d i o s e n s i t i z a t i o n . T o x i c i t y of n i t r o i m i d a z o l e s was o b s e r v e d d u r i n g in v i t r o s t u d i e s of radiosensitizers,  as well as in t h e c l i n i c a l t r i a l s . S u t h e r l a n d (1974) o b -  s e r v e d cell d e a t h a  workshop  Radiation these  in m u l t i c e l l " s p h e r o i d s " t r e a t e d w i t h m e t r o n i d a z o l e . A t  meeting  Research  effects  following  in S e a t t l e ,  were due  against hypoxic cells.  the  Washington,  Congress  1974, P a l c i c s u g g e s t e d  of that  T h i s p o s s i b i l i t y was q u i c k l y c o n f i r m e d (Moore et  et a l . i l l u s t r a t e t h e e f f e c t  misonidazole,  International  to a s p e c i f i c c y t o t o x i c a c t i o n of n i t r o i m i d a z o l e s  a l . , 1976; Hall & R o i z i n - T o w l e , 1975).  w e r e i n c u b a t e d at 37°  Fifth  : fig.5.  T h e f o l l o w i n g f i g u r e s f r o m Moore Chinese hamster o v a r y  (CHO)  cells  in medium c o n t a i n i n g t h e s t a t e d c o n c e n t r a t i o n s of  under either hypoxic or aerobic conditions.  produced by flowing purified  H y p o x i a was  gas o v e r t h e s t i r r e d cell s u s p e n s i o n s .  A t time i n t e r v a l s , a l i q u o t s of c e l l s w e r e r e m o v e d , w a s h e d f r e e of d r u g , a n d p l a t e d in p e t r i d i s h e s to d e t e r m i n e s u r v i v a l ( c o l o n y f o r m i n g a b i l i t y r e l a t i v e to c o n t r o l s ) .  T h e s e l e c t i v e k i l l i n g of h y p o x i c c e l l s is a p p a r e n t :  f o r e x a m p l e , 4 h r i n c u b a t i o n w i t h 15 mM m i s o n i d a z o l e , u n d e r h y p o x i a , reduces  survival  to  less  than  1%,  whereas  the  a e r o b i c c o n d i t i o n s has no m e a s u r a b l e t o x i c e f f e c t .  e x p o s u r e time  under  25  AEROBIC C H O (37°C)  100 OmM -£1rrM 50 15 mM  20 ' \ ^ 5 0 " 1  2  3  J  I  4  5  6  I  I  7  8  9  10  INCUBATION TIME AT 37°C (hours)  HYPOXIC C H O (37°C) U  Z 100  -,0mM  y  u. u. ui < Z  kl U  a.  5mM 0.1 1  2  3  INCUBATION  4  5 TIME  6  7  8  AT 3 7 ° C  9  10  (hours)  F i g . 5 Selective toxicity of misonidazole to hypoxic cells (from Moore et a l . , 1976, by permission)  Chinese hamster ovary ( C H O ) cells were exposed to the indicated concentrations of misonidazole, either in aerobic medium, or in medium made hypoxic by gassing with nitrogen. Colony forming ability was measured as a function of time of exposure to the d r u g .  26  The nitroimidazoles were originally developed for the treatment of anaerobic  infections.  T h u s , in hindsight, this selective action against  hypoxic mammalian cells phenomenon  by  pharmacology motivations: the  is not s u r p r i s i n g .  radiobiologists  of  has  nitroaromatic  of  stimulated  compounds.  on the one hand,  mechanism  The  're-discovery' much  This  research  hypoxic cytotoxicity  misonidazole's  neuropathic  interest  of the in  the  has  two  may be related to  side-effects,  and  an  understanding of this process could improve administration of the d r u g ; on the other hand, the selective toxicity raises the possibility of using misonidazole, hypoxic  or  tumour  a  similar d r u g ,  cells.  This  as a chemotherapeutic agent  possibility  has  been  reviewed  against recently  (Kennedy et a l . , 1980).  1.9 Hypoxic toxicity - mechanism: The radiosensitizing ability of misonidazole is a consequence of its electron-affinity nitro  anion  gested  that  toxicity. rather other  - that is, the ease with which it can be reduced to the  radical, a  R-NG^"  related  (Wardman,  radiolytic.  nitroaromatic  Early evidence  sug-  mechanism may be responsible for misonidazole  In this case, however,  than  1977).  the reduction chemistry is metabolic,  Much of this evidence came from studies with  compounds.  As discussed above, nitrofurans have  been studied as antibacterial agents since the 1950's - for a review, see McCalla, in Hahn, e d . , 1979. The metabolic and colleagues,  reduction of such compounds was studied by Asnis  using bacteria.  At  least two "nitroreductase" enzyme  27  systems  were  identified.  One  presence of oxygen (Asnis,  of  these  1957).  enzymes  is  inhibited by  the  Asnis and co-workers cultured E_.  coli in medium containing Furacin (5-nitro-2-furaldehyde semicarbazone) and  developed  a  drug-resistant  characterised  by  times  than  higher  However, strains clear  under  showed that  reductase  the  strain.  This  strain  was  a minimum inhibitory Furacin concentration about ten that  hypoxia  of  the  (E.  resistant  enzyme  parent  coli  no difference  (Peterson  oxygen-sensitive  bacterial  in  mutants et  al.,  have  is  strain  a facultative  susceptibility lack  the  to  et  al.,  anaerobe) Furacin.  1952). the  It  oxygen-independent  1979).  never  (Asnis  been  enzyme has some essential biological activity.)  (Mutants isolated:  two  is now nitro-  lacking presumably  the this  Asnis et al. studied the  rate of Furacin reduction by the two strains, and concluded that: The results of the Furacin reduction studies are somewhat paradoxical in their indication that Furacin is destroyed at a more rapid rate by the susceptible than by the resistant strain. It could be concluded that some reduction product of Furacin is the active inhibiting agent rather than Furacin itself. An analogous effect  was observed by D.  R.  McCalla and coworkers in  studies of bacterial mutagenesis by nitrofurans (McCalla and Voutsinos, 1974).  McCalla stated that:  . . . reduction of the nitro group is a prerequisite to the i n duction of mutations, since bacterial mutants which lack [ the oxygen-insensitive nitroreductase ] are much less extensively mutated than wild-type strains under aerobic conditions. (McCalla, in Hahn, e d . , 1979)  28  The metabolism of aromatic nitro compounds by mammalian systems was  studied by  began their rarely They  Fouts and  Brodie  paper with the  used as d r u g s , . . . " , made several  in the mid-1950's. (Ironically,  statement:  they  "Aromatic nitro compounds are  a situation which was to change shortly!)  important observations:  activity was found in both  the soluble fraction and the microsomal fraction of liver, there are large species rigid  differences  structural  1957).  in  activity,  requirements  Furthermore,  and for  the  its  enzyme  system showed  substrates."  (Fouts  &  "few  Brodie,  they observed that mammalian nitroreductase  was  strongly inhibited by air; there appeared to be no mammalian analogue of the oxygen-insensitive bacterial nitroreductase. The mechanism of oxygen inhibition of enzymatic nitroreduction was elucidated  by  the  Resonance (ESR) the  nitro  reduction nitro  of  Mason  and  Holtzman.  radical,  acid  process  compound,  during  (1975a). is and  This  the  hypoxic microsomal  shows  that  a one-electron transfer suggested  the  the  initial  (Biaglow et a l . , 1976) that,  oxygen depletion, was  suggested  by  to  the  Mason  one-electron however, parent  and  reduction step  in  of the  possibility  of  a  free  radical  The observation  under aerobic conditions, nitroheterocycles  stimulated  oxygen;  Spin  from the enzyme to the  mechanism in the oxygen inhibition of the reduction.  initial  Electron  spectroscopy was used to demonstrate the formation of  anion  nitrobenzoic  work  indirect evidence for the mechanism  Holtzman (1975b).  They  suggested that  the  step in nitroreduction continued in the presence of the nitro anion radical was re-oxidized very rapidly  compound.  Thus,  no  net  disappearance  of  the  nitro  29  compound under in  is o b s e r v e d ,  aerobic  oxygen  depletion  conditions.  depletion  superoxide  and  is  the  and  decreased.  Cofactor (reduced)  signal from the  Oxygen  and  dismutase  no ESR  is r e d u c e d  build-up  catalase  This  is  are  of  superoxide.  in  the the  Reductase ( o x i d ized)< +  e  Reductase (reduced)  R-N0„  If  rate  detected resulting exogenous  of  oxygen  following  scheme:  0  R-NO. Cofactor (oxid ized)  is  to s u p e r o x i d e ,  added,  illustrated  radical  (superoxide)  N  (hypoxia) R-N=X (reduced  products)  M e c h a n i s m of o x y g e n ' i n h i b i t i o n ' of n i t r o r e d u c t i o n : T h e i n i t i a l p r o d u c t of n i t r o r e d u c t i o n is t h e n i t r o a n i o n radical. U n d e r h y p o x i a , t h i s s p e c i e s is f u r t h e r r e d u c e d to u n i d e n t i f i e d p r o d u c t s , i n c l u d i n g , p e r h a p s , t o x i c a n d mutagenic species. Under aerobic conditions, the radical a n i o n is r e - o x i d i z e d q u i c k l y to t h e p a r e n t n i t r o c o m pound. T h e r e s u l t i n g s u p e r o x i d e is d e t o x i f i e d b y s u p e r o x i d e d i s m u t a s e a n d c a t a l a s e (see b e l o w ) .  30  H+  SOD  +  \ (0  +  CATALASE  NET:  H  +  +  e" + 0  2  —  H  +  *  \ 0  +  0 ~ 2  + H 0 )  2  2  2  + H 0  2  2  —»  3/4 0  2  + \ W£>  M e c h a n i s m of r e d u c e d o x y g e n c o n s u m p t i o n in p r e s e n c e s u p e r o x i d e d i s m u t a s e and c a t a l a s e :  of  S u p e r o x i d e production via the ' f u t i l e ' metabolic c y c l e o u t l i n e d a b o v e r e s u l t s in c o n s u m p t i o n of o x y g e n in c l o s e d microsomal incubation mixtures. In the presence of exogenous superoxide dismutase and catalase, the s u p e r o x i d e is c o n v e r t e d to o x y g e n a n d w a t e r , r e s u l t i n g in a 4-fold d e c r e a s e in o x y g e n c o n s u m p t i o n . ( A c t u a l l y , t h e s e r e a c t i o n s will o c c u r s p o n t a n e o u s l y at an a p p r e c i a b l e r a t e , so t h e o b s e r v e d d e c r e a s e in o x y g e n c o n s u m p t i o n may be less t h a n 4 - f o l d . ) Further reduction  s u p p o r t of t h i s mechanism f o r o x y g e n  was  provided  oxygen-independent  by  bacterial  a  study  enzyme  of t h e  i n h i b i t i o n of n i t r o -  anomalous  case,  ( P e t e r s o n et a l . , 1 9 7 9 ) .  viz.  the  ESR  and  o p t i c a l s p e c t r o s c o p y w e r e u s e d to d e m o n s t r a t e t h a t t h i s e n z y m e  reduces  n i t r o c o m p o u n d s v i a an i n i t i a l 2- ( o r more) e l e c t r o n s t e p , w i t h o u t f o r m ation of t h e n i t r o anion r a d i c a l as an In  three  different  intermediate.  biological systems  - the  "wild type"  bacterium  p o s s e s s i n g an o x y g e n - i n s e n s i t i v e n i t r o r e d u c t a s e , m u t a n t b a c t e r i a l a c k i n g this  enzyme,  the  formation  suggests  that,  a n d mammalian c e l l s , t h e r e  is a c l e a r c o r r e l a t i o n  of  and  as  nitroreduction  chemistry  of  b i o l o g i c a l damage.  A s n i s had p r o p o s e d , some p r o d u c t of  is t h e ultimate t o x i c s p e c i e s . the  products  reduction  However, of  between  nitroreduction  t h e n a t u r e of t h i s p r o d u c t ,  nitroheterocyclic  This  compounds,  has  and been  31  explored only sketchily.  The  e l u c i d a t i o n of t h e  reductive  metabolism of  misonidazole. is t h e c e n t r a l p r o b l e m c o n s i d e r e d in t h i s t h e s i s .  32  MATERIALS AND METHODS: 2.1 Cell culture procedures: The on  m vitro  Chinese  culture. grows  biological experiments reported here were performed  hamster  (Cricetulus  griseus)  The Chinese hamster• ovary  cell  (CHO)  lines  grown  in  tissue  cell line is used widely:  it  rapidly, can be cloned with high plating efficiency, and can be  grown in monolayer, suspension, and soft agar cultures (Thompson, in Jakoby  & Pastan,  ed.,  1979).  Also,  the  line has a relatively  stable  karyotype,  with 2n = 20-21 (22 in the animal; Worton & Duff, in Jakoby  & Pastan,  ed.,  1979).  We grew  CHO cells  in suspension culture in a  medium, supplemented with 10% foetal calf serum ( F C S ) , antibiotics, and bicarbonate  buffer.  (Incu-cover,  Associated Biomedic Systems) at 3 7 ° ;  7.4  by  diluted  continuous daily  to  The  cultures  gassing about  with  7 x 10^  were  5% CO^  grown  in air.  cells/ml,  in  an  incubator  pH is regulated to The cell culture  maintaining  is  asynchronous  exponential growth with a doubling time of about 12 hours. A  second  Chinese  hamster  CHEF-125 (Prescott & Bender, 25 cm  and carbonate buffer. and  transferred  CH2B2,  derived  from  line  1963) was grown in monolayer culture in  tissue culture flasks (Falcon).  essential medium (MEM,  week,  line,  The medium used was minimum  Gibco) supplemented with 10% F C S , antibiotics, Cells were harvested by trypsinization twice per into  new  flasks  (Agnew  & Skarsgard,  1972).  33  2.2 In vitro toxicity studies - cell survival experiments: The toxic effect of a d r u g , or drug combination, was measured by exposing cells to the drugs for various time intervals, then washing the cells free of the drug and plating aliquots of the cells into petri dishes to determine colony-forming ability solutions  were  prepared  bicarbonate buffer. filtered  (Nalgene).  fitted  syringe  central  port  for  such vessels,  experiment  The tube was  removal  of samples.  In  glass  through  a  was  kept  H^, or air)  "bubbler"  lacking  unit  short  eight  unit constructed locally.  was supplied from a tank,  tube  experiment,  drug combinations, were  in a 37° warm room.  filled  and a stoppered  The bath was maintained at 37° and set  up on top of a multi - magnetic stirrer  phase (C^,  a typical  including controls and various  apparatus  medium  sealed with a rubber stopper  needles to permit gas flow,  set up in a large water bath.  entire  in  From this stock, 29 ml was added to a wide-mouth  vessel.  two  each  Drug-medium  The solutions were adjusted to pH 7.4 and sterile  glass centrifuge with  before  (Moore et a l . , 1976).  with sterile  fitted  with  water. a  The appropriate  gas  and humidified in a  The gas was  plastic  The  syringe  admitted  tip.  The  drug-medium solutions were pre-gassed before addition of cells (for ^ 20 minutes) were  to achieve  harvested  from  temperature culture  by  and gas phase equilibrium. low-speed  centrifugation  Cells  (CHO)  or  trypsinization and centrifugation ( C H 2 B 2 ) and resuspended in drug-free medium at 6 x 10  cells/ml.  They were held at this concentration for  about 15 minutes, to ensure metabolic depletion of any remaining oxygen in the medium.  At zero time,  1 ml of cell suspension was added, to  34  give a volume of 30 ml, cell concentration of 2 x 10  cells/ml, and drug  concentration as desired. Aliquots (1 ml or less) were removed at zero time (immediately after adding cells) and at regular intervals  thereafter.  The aliquots were immediately diluted in 10 ml fresh medium at 0 ° . samples were spun to harvest  the cells;  supernatant  The  medium was de-  canted and replaced with fresh medium, and the cells were resuspended by vigorous pipetting and vortexing.  Samples of this suspension were  plated into petri dishes, using a micropipette. Each  petri  contained 5 ml medium (with bicarbonate  buffer)  and  5 10  'feeder' cells.  heavy  Feeder cells are cells of the same line, sterilized by  irradiation,  increase  the  overcome  overall  the  when widely  supply  plating  variation differing  pension is plated larger  which  in  nutrients  efficiency plating  inocula are  into a petri.  efficiency used.  If  inoculum volumes.  growth  factors  which  of the cloning procedure, which  Typically,  may  be  and  observed  10 \i\ of cell s u s -  significant toxicity was anticipated,  samples were plated (up to 2 ml);  several  and  replica plates were plated at  Following plating, the cell suspensions were  again vortexed  and a sample (2  phate-buffered  saline  (PBS),  ml)  and  was  removed, diluted with phos-  counted  with  an  electronic  cell  counter (Coulter Electronics) to determine cell concentration. Petri dishes were incubated at 37° in a tray incubator with 5% C C ^ flow  for  replaced off,  seven  days.  with stain  and the  petris  The  medium was  (methylene  blue).  then carefully  Finally,  the  decanted  stain was  washed  were counted to determine number of colonies.  clone of 50 or more cells is assumed to represent a survivor.  and  A  35  Typically,  each  sample  was  plated  in  triplicate.  The  average  number of colonies was calculated, and the number of cells plated was calculated from the Coulter count, dilution factor, and plating volume. Plating efficiency is then defined as: P.E.  -  Number of colonies Number of cells plated  Surviving  fraction is defined as plating efficiency of treated  relative to plating efficiency of controls.  In general, experiments were  repeated at least three times.  Values of log (surviving  averaged  and standard errors  for each time point,  calculated.  cells  fraction)  were  of the mean were  Results are presented as log S v s . time of drug exposure.  DNA single-strand breaks ( S S B ) : The alkaline sucrose gradient technique was deveoped by McGrath and Williams (1966)  as an assay for DNA damage in bacterial cells.  has since been adapted for  use with mammalian cells in a number of  laboratories (for example, Palcic and Skarsgard (1972,1981)). disrupted  by  intact  Cells are  lysis on top of the gradients on which separation of the  DNA molecules, according to size, is performed. of the  It  Thus,  fragmentation  DNA molecules, which occurs in the usual procedures of  chemical extraction  and handling, is minimized.  The alkaline gradient  (pH > 12) dissolves all the cell contents; DNA is freed from associated membranes and proteins, and becomes single-stranded DNA.  Molecules  of different molecular weight sediment at different speeds during cent-  36  rifugation.  Following  centrifugation,  t h e d i s t r i b u t i o n of t h e D N A had  been  detected (SSB)  labeled w i t h by  this  can  be  CH2B2  is m e a s u r e d .  activity.  The by  number a  periment, free  cells  H-thymidine,  of  specific  labeled  ization.  nucleotides.  Tritium-labeled  of  treated  (24  hr)  specific activity  by  DNA  DNA  breaks  and  control  41  in medium  were  Ci/mMole.  were  then  Prior  harvested  incubated  con-  52 C i / M o l e , o r 0.25 to an  1 h r i n c u b a t i o n in g r o w t h  Cells  cells  single-strand  incubation  activity  l a b e l i n g was t e r m i n a t e d  of  DNA  (M^).  w e r e labeled b y 14 t a i n i n g 0.05 p C i / m l C-thymidine, 3 uCi/ml  and  and t h e p r e s e n c e of  comparison  molecular weights  is f r a c t i o n a t e d ,  In o u r e x p e r i m e n t s , t h e  radioactive thymidine,  calculated  weight-averaged  the gradient  with  by  ex-  medium trypsin-  drug-containing  14 medium as  described  above,  and  d r u g - f r e e medium as c o n t r o l s . above  (toxicity  C-labeled  Incubations  experiments).  cells were incubated  with  w e r e p e r f o r m e d as d e s c r i b e d  Aliquots  of  experimental,  c o r r e s p o n d i n g c o n t r o l c e l l s w e r e pooled a f t e r w a s h i n g  and  and  resuspended  g at a c o n c e n t r a t i o n of 4 x 10 onto  0.5  ml  lysing  gradient,  in  a  17  cells/ml.  solution, ml  on  A 50 uj a l i q u o t was t h e n  top  nitrocellulose  of  a  5-20  ultracentrifuge  %  alkaline  tube.  layered sucrose  Cells  l y s e d at 20°C f o r 7 h r and c e n t r i f u g e d at 14,000 rpm f o r 11 h r , a B e c k m a n L-65B u l t r a c e n t r i f u g e a n d SW-27.1 r o t o r . fractionated  u s i n g an  Isco model " D " f r a c t i o n a t o r .  (Amersham  ACS,  5 ml)  using  Each g r a d i e n t  was  F r a c t i o n s (25 x 0.75  ml) w e r e c o l l e c t e d in s c i n t i l l a t i o n v i a l s a n d n e u t r a l i z e d w i t h cocktail  were  was  concentrated  HCI.  Scintillation  added  vial.  Samples w e r e c o u n t e d in a B e c k m a n s c i n t i l l a t i o n c o u n t e r .  to each  37  Weight-average molecular weights (M^)  of control and drug-treated DNA  were computed using the method of Palcic and Skarsgard  (1972).  2.3 Zinc reduction of misonidazole - chemical reduction procedure: Chemical in  the  work  presence  on the  nature nitro  reduction of misonidazole was performed using zinc dust of  CaC^.  reduction  of the  products obtained depends on a variety of factors:  the  temperature,  compounds.  solvent,  and  In  earlier the  used,  nitroaromatic  adapted from  general,  compound  of  This technique was  presence  of  salts.  Misonidazole used in all the studies reported in this thesis was a gift  of  U.K.,  Dr.  C.  Smithen,  and was  detected  by  Roche  used without  chromatography.  Products  Ltd.,  Welwyn Garden  further purification.  City,  No impurities were  Zn dust and C a C ^ were obtained from  Fisher Scientific. Misonidazole,  1 g,  double-distilled water. stirrer,  and  CaC^,  1 g,  temperature,  in colour.  and  then  dissolved  in  100 ml  The solution was rapidly stirred with a magnetic  and 2 g of Zn dust was added.  greenish-yellow  were  The solution quickly became  The mixture was  filtered  through  remove the Zn dust and precipitated ZnO.  stirred for 1-^ hr at room 1  Whatman #1 filter  paper  to  The solution was frozen in a  lyophilization flask, and concentrated by lyophilization.  Thin-layer chromatography ( T L C ) : The concentrated was  examined by  TLC.  reduction mixture, The  plates  prepared as outlined above,  used were type LK5DF,  Whatman,  38  Inc.,  Clifton,  formulated  N.J.  for  These  the  p l a t e s c o n s i s t of a 250 p l a y e r of s i l i c a gel  separation  substances.  The  area,  which  eliminates t h e  allows  volumes  TLC  product  moderately  to  strongly  polar  sample is a p p l i e d to a p r e a d s o r b e n t sample d i s p e n s i n g  as  large  guide,  acetone/methanol  of  need  for  careful  s p o t t i n g of  samples,  as 100 pi to be a p p l i e d to t h e plate  1978).  The  and  (Whatman  p l a t e s w e r e d e v e l o p e d in c h l o r o f o r m /  (45/45/10) and b a n d s w e r e d e t e c t e d  by colour, or  by  UV fluorescence quenching.  High-pressure liquid chromatography Analytical  H P L C was  (HPLC):  u s e d to m o n i t o r t h e p r o g r e s s of t h e  and t h e p u r i t y of t h e s e p a r a t e d  products.  SP-8000 c h r o m a t o g r a p h in i s o c r a t i c mode.  We u s e d a  c o l u m n : L i C h r o s o r b RP-8 r e v e r s e d - p h a s e ,  dimensions  4.6  mm x 250 mm; mobile p h a s e :  (80/20);  flow  rate:  Spectra-Physics  The following conditions were  employed:  4.5/methanol  reaction  p a r t i c l e s i z e 10 | j ,  10 mM acetate b u f f e r ,  pH  5 ml/min at a p r e s s u r e of a b o u t 2800  psi. Detection  of  variable-wavelength  the  eluted  components  was  performed  UV-visible detector, Schoeffel Spectra-Flow  with  a  770, set  to 370 n m .  Preparative  l i q u i d column c h r o m a t o g r a p h y :  Preparative reduction preparative  chromatography  products  on a  larger  reversed-phase  techniques  w e r e u s e d to s e p a r a t e  scale.  used  We  chromatography  colum:  a  the  recently-developed LiChroprep  LOBAR  39  RP-8, BDH  particle size 43-60 |j, 25 mm Chemicals,  Vancouver.  The  x 310 mm (size B ) , column  was  obtained from  adapted  with Swagelok  fittings to allow mobile phase to be supplied by the HPLC pump itself, rather than by a low-pressure peristaltic pump. H2O / methanol (70/30). at  the  allowing injected  maximum  through  Flow rates up to 13 ml/min could be achieved  operating  complete  Mobile phase used was  pressure  preparative  of  runs  in  a specially-prepared  the  glass  about  column hr.  V-*>  2 ml loop injector,  (90  psi),  Samples  were  made out of  Teflon tubing.  Spectroscopic techniques: UV-visible  spectroscopy  spectrophotometer.  was  performed  on  an  Aminco  DW-2  Nuclear magnetic resonance (H-NMR) was performed  on a Varian XL-100 Fourier transform spectrometer (NMR  lab, Chemistry  department,  or  analysis.  UBC). Mass  Samples  spectra  were  dissolved  were obtained  at  in  the  D^O  Chemistry  CD^OD  for  department,  U B C , using solid-probe injection and electron ionisation.  2.4 Xanthine oxidase - catalysed reduction of misonidazole: Enzymatic reduction procedures: Xanthine X-4500,  oxidase  was  from buttermilk,  purchased  from  chromatographically  Sigma,  St.  Louis  (type  purified, specific activity  1.25 units/mg protein), as were hypoxanthine, xanthine, and uric acid. Kinetics  of  spectrophotometer,  the  reduction  were  studied  using  an  Aminco  DW-2  equipped with an anaerobic cuvette and a controlled  temperature magnetic-stirrer cuvette holder.  4 0  M i s o n i d a z o l e r e d u c t i o n was s t u d i e d as f o l l o w s : All  solutions were dissolved  aerobic  cuvette  misonidazole enzyme 2 . 3  was f i l l e d w i t h  ( N H  substrate  and hypoxanthine.  solution,  M  in P B S .  )  4  2  S 0  The  cell  with  humidified  1 unit and  4  was then  mM E D T A  placed  solution,  T h e side-arm  of xanthine  1  T h e o p t i c a l cell o f t h e a n -  oxidase  1 . 9 m l , containing  was f i l l e d  in 0 . 5  with t h e  ml P B S c o n t a i n i n g  ( e t h y l e n e diamine t e t r a a c e t i c  in the spectrophotometer  N , with s t i r r i n g .  (37°)  acid).  and gassed  A f t e r 1 0 minutes e q u i l i b r a t i o n , t h e  2  contents of t h e side-arm were t i p p e d into t h e optical c e l l , a n d r e c o r d i n g started.  Initial  misonidazole, 210  concentrations  in  (jM; h y p o x a n t h i n e ,  1 0 0  uM; xanthine  oxidase,  by  activity  1 unit,  reaction  ( N H  4  )  2  S 0  i n a total  mixture  4  ,  mM;  4 8 0  volume  were: EDTA,  of 2 . 4  ml.  b y loss of a b s o r p t i o n a t 3 5 0 n m .  of t h e xanthine  oxidase  preparation  was s t u d i e d  measuring t h e conversion of xanthine to u r i c a c i d , in air-saturated  solution. cuvette  Xanthine maintained  (100  | J M i n 2 ml P B S ) , was a d d e d  at 3 7 ° .  Xanthine  a n d f o r m a t i o n o f u r a t e r e c o r d e d at  TLC  mM;  5  M i s o n i d a z o l e r e d u c t i o n was d e t e r m i n e d Specific  the  2 9 5  oxidase,  0.02  nm ( B r a y ,  to a  regular  u n i t s , was a d d e d ,  1 9 7 5 ) .  Autoradiography: 1 4  C-misonidazole, specific  activity  radioactive  labeled  9 . 2 uCi/mg,  impurities  chromatography  at t h e 2 p o s i t i o n o f t h e imidazole  systems  were  detected  used,  without f u r t h e r purification.  was a  gift above  of D r . C . a  level  and t h e radio-labeled  Smithen.  of 0 . 1 % drug  ring, No  in the  was u s e d  41  The nature of the reduced derivative of misonidazole formed in the 14 C-labeled d r u g .  reaction was studied using diluted  100  x  with  cold  misonidazole,  14  1  C-labeled misonidazole,  mg  total,  was  incubated  overnight with 2 mg hypoxanthine and 1 unit xanthine oxidase in 2 ml PBS,  N ,  37°.  2  residue was remove  The  solution  was  frozen and  resuspended in 1 ml methanol  insoluble  material.  Recovery  of  lyophilized. The  and centrifuged  the  radiolabel  dried  briefly  was  to  75-90 %.  The soluble fraction was studied by T L C and H P L C . T L C was performed as described above (Zn reduction), except that the  solvent  used was  acetone/methanol  (67/33).  The  plate  was  auto-  Whatman  PAC  phase:  ethyl  radiographed using Kodak X-OMAT-R x-ray film. HPLC  was  polar-bonded  performed  phase,  4.6  acetate/methanol  (50/50),  was  at  maintained  collected  in vials;  40°  as mm  flow with  follows: x  rate an  scintillation  250 2.0  air  cocktail  column, mm;  mobile  ml/min.  oven.  Column  Fractions  (Amersham  ACS  temperature  (0.4 II,  ml) 5 ml)  were was  added, and each vial was counted for 50 min. in a scintillation counter (Beckman LS-330).  2.5 In vitro metabolism of misonidazole CHO cells  were grown  in suspension culture as described above.  During the final 24 hr before an experiment, the cells were allowed to 5 reach  a concentration  vested volume.  by  of 5 x 10  centrif ugation,  and  8 cells/ml.  spun  to  Cells  form  a  (3 x 10 ) pellet  of  The pellet was resuspended with medium containing  were harabout 1 ml  42  C-misonidazole to give a total volume of 2 ml and a drug concentration of 0.38 mM. bath.  N  At zero time,  was  2  the tube was transferred  to a 37° water  flowed over the suspension throughout the experiment.  The suspension was vortexed occasionally to prevent sedimentation and adherence of the cells.  Samples of 0.3 ml were removed shortly  zero time (within two minutes) hours.  Each  after  and at each successive hour, up to 3  sample was treated  as shown in the schematic (fig.  6).  Distilled water (1.7 ml) was added, and the sample was sonicated for 10 seconds,  using  a  Branson  W-350  cell  disruptor  with  microtip.  An  aliquot of the sonicate was dissolved in 0.5 ml of 2M NaOH, neutralized with acetic acid, and counted to determine total activity. der of the  sonicate was frozen and lyophilized to dryness.  residue was  resuspended with ethyl acetate/methanol  minutes at 800 rpm), was  repeated  evaporated  The remain-  and the  supernatant  (63/37),  decanted.  The d r y spun  (5  This procedure  a total of 3 times - the supernatants were combined and  in a Buchler  vortex  evaporator  at 30°.  The dry  were stored at -15° until chromatography was performed.  samples  43  SAMPLE (0.3 ml - 4.5 x 10 CHO cells) 7  | DISRUPTED |  Sonicate CELLS Lyophilize  DRY RESIDUE Extract MeOH  ORGANIC-INSOLUBLE  Extract with MeOH/TCA. (10 times - combine)  with ethyl (3 times -  ORGANIC-SOLUBLE  Dry. Resuspend in 1 ml MeOH. Remove aliquot - T L C . Dry.  PELLET ubilize in NaOH Count.  acetate: combine)  Resuspend in acetate buffer for HPLC.  ORGANIC-INSOLUBLE Count aliquot.  Fig. 6 In vitro metabolism experiments: schematic  44  Extraction procedure: The was  pellet  further  material.  extracted  equal  to  extraction  separate  with ethyl  acid-soluble  acetate/methanol  and  acid-insoluble  The pellets were rinsed carefully into a cone of filter  (Whatman #1) of  remaining after  paper  and washed with 10 succesive 2 ml aliquots of a mixture  volumes  of  methanol  and  10% trichloroacetic  acid  (TCA).  Further washing did not extract additional radioactivity.  An aliquot of  the filtrate was counted to determine total acid-extractable  radioactivity.  Finally, with gentle counted.  the  remaining pellet was  heating. The  dissolved in 2 ml of 2M NaOH,  The sample was neutralized with acetic acid and  recovery  of  total  initial  radioactivity  in  the  three  fractions (organic-soluble, acid-soluble, and insoluble) was consistently 70-80%, due to losses in sample handling.  Chromatography (organic-soluble fraction): The  dried  supernatants  resulting from the  initial  extraction  into  ethyl acetate/ methanol were dissolved in 1 ml methanol, and an aliquot counted to determine total (50  ul)  dried,  was and  applied  to  developed  developed plate  was  organic-soluble activity.  a Whatman LK5DF immediately  in  TLC  A further  plate.  acetone/methanol  aliquot  The plate  was  (50/50).  The  d r i e d , and autoradiographed on Kodak X-OMAT-R  film. The remainder of the supernatant was evaporated, and the residue was  resuspended  carefully  to an  in  125  ul  acetate  buffer.  The  sample  was  added  Eppendorf micro centrifuge tube, and spun for 5 min-  45  utes  in  an  water-soluble lipid).  An  HPLC  Eppendorf supernatant  aliquot was  of  model from  the  5412 the  insoluble  supernatant  performed  as  centrifuge  was  follows:  to  separate  precipitate injected column,  into  the  (presumably the  Whatman  HPLC. ODS  reversed-phase, 4.6 mm x 250 mm; mobile phase, 10 mM acetate buffer, pH 4.5; flow rate, 2.5 ml/min. A second HPLC system was used for dual-label chromatography experiments  (see chap. 6).  For these experiments, samples were resus-  pended in ethyl acetate/methanol and centrifuged as above. The supernatants  were  amino-cyano  injected bonded  into  the  phase),  HPLC.  4.6  Column,  mm x  Whatman PAC  250 mm; mobile phase,  (polar ethyl  acetate/methanol (63/37); flow rate, 2.5 ml/min. In both systems, column temperature was maintained at 35°. A loop injector (50 ul) was used.  Fractions were collected in vials and counted  with 5 ml scintillation cocktail scintillation counter.  (Amersham  ACS)  in a Beckman  LS-330  46  MISONIDAZOLE 3.1  C Y T O T O X I C I T Y J_N V I T R O :  Introduction: Oxygen,  which  inhibits mammalian nitroreductase activity,  diminishes the toxicity of misonidazole in vitro. this thesis, by  greatly  In the first chapter of  I reviewed evidence that enzymatic nitroreduction proceeds  a one-electron, free-radical mechanism.  Oxygen reacts with nitro-  aromatic anion radicals at close to diffusion-limited rates (Greenstock & Dunlop,  1973; Wardman  regenerate  the  parent  &  Clarke,  nitro  1976) to produce superoxide, and  compound.  This  reaction  causes  oxygen  'inhibition' of nitroreduction. Superoxide is detoxified by the enzymes superoxide dismutase and catalase.  Ascorbic  acid  (vitamin  C)  is  oxidized by  demonstrated by Nishikimi and Yagi (1977).  superoxide,  as  It was suggested that this  reaction may be of comparable importance to the enzymatic de-activation of superoxide. It  appeared  reduction compound, the  in  reasonable to assume that the overall  the  and the  cell  depends  on the  electron-affinity  rate of of  the  nitronitro  net effect of agents that can transfer electrons to  nitro group and agents that can accept an electron from the nitro  anion radical (particularly,  oxygen).  We sought to test this hypothesis  by observing the effect of the former type of agent, and we selected ascorbate as a possible example.  47  3.2 Ascorbate enhancement of misonidazole cytotoxicity: The oxidation of ascorbate to dehydroascorbate can proceed via a free-radical intermediate, mono- (or  semi-) dehydroascorbate. Yamazaki  and colleagues demonstrated that this radical is formed enzymatically, in the  horseradish  techniques  peroxidase/  permitted  H  2®2  identification  s  V  of  s t e m  -  the  radical,  surements were made (Ohnishi et a l . , 1969). may  be  samples  the  main  (Borg,  in  component of Pryor,  the  ed.,  ESR  vol.  Continuous  ESR  kinetic  mea-  Indeed, ascorbate radical  signals  1,  and  flow  observed  1976).  Once  in  tissue  formed,  the  ascorbate radical is relatively nonreactive, and decays by disproportionate  (Bielski & Richter, 1975). We suspected that ascorbate could act as a one-electron donor to  misonidazole, perhaps via an enzyme intermediate; the resulting radical would be unlikely to reoxidize the nitro anion radical. was  supported  atically  with  by  the  the  observation  strongly  quinoline-N-oxide (4-NQO), (Biaglow et a l . , 1976). imidazoles,  that  ascorbate  electron-affinic  presumably via  This possibility  reacts non-enzym-  carcinogen  a free radical  4-nitro-  intermediate  4-NQO is more electron-affinic than the nitro-  and metabolic  reduction of 4-NQO can proceed even under  aerobic conditions (Matsushima & Sugimura, in Endo et a l . , e d . , 1971). These  properties  of  ascorbate  suggested that this  interact with misonidazole in a significant manner.  vitamin  might  We tested the effect  of ascorbate on the hypoxic toxicity of misonidazole to CHO cells, using the  techniques  misonidazole,  described  with  or  in  without  chapter ascorbate,  2.  Cells  were  for various  exposed  times,  and  to  then  48  0  1  2 T i m e (h)  F i g . 7 Ascorbate-enhancement of misonidazole cytotoxicity (from Josephy et a l . , 1978)  CHO cells were exposed to misonidazole in hypoxia, and colony forming ability was determined as described in Methods. A • O •  Misonidazole, Misonidazole, Misonidazole, Misonidazole,  5 mM 5 mM + Ascorbate, 5 mM 15 mM 15 mM + Ascorbate, 5 mM  49  plated  to  determine  periments.  survival.  Fig.  7 shows the  results of these  ex-  At 5 mlVI concentration, the 'shoulder , or lag period, in the 1  survival  curve of cells exposed to misonidazole in hypoxia, lasts about  7y hr.  The turning down of the curve can just be seen in the figure.  z  The addition of equimolar ascorbate hr,  thus  greatly  also  observed  The  effect  ascorbate:  is  shortens the  shoulder to about 1  enhancing the toxicity of the d r u g .  when 5 mM ascorbate probably  a  is added to 15 mM misonidazole.  consequence  gulonolactone,  a  Enhancement is  of  the  non-reducing  reducing  analogue  of  capacity  of  ascorbate,  produced no enhancement. In  later  experiments  (fig.  8,  unpublished data)  we studied  the  effect of varying the concentration of ascorbate, at a fixed misonidazole concentration (5 mM).  The main effect is a shortening of the shoulder,  dependent on ascorbate dose.  The effect appears to saturate above the  equimolar level of ascorbate. A  direct,  as  opposed to  eel I-mediated,  interaction  between  as-  corbate and misonidazole appears unlikely: pre-incubation of the mixture of the two agents in growth medium, H^, 3 7 ° , for up to 2 hours before addition of cells, did not affect the subsequent response. the enhanced toxicity  is strongly temperature  Furthermore,  dependent.  It  is more  pronounced above 37°, and almost absent at 0 ° . .  3.3 Aerobic toxicity of ascorbate: Nitroheterocyclic compounds are toxic in air, although much less so than  in  hypoxia.  This  superoxide in the  'futile'  toxicity  may  be mediated  reduction of the  by  production of  nitro group. As mentioned  50  CHO cells N  T  0  I  1  1  2  2  1  I  3  4  TIME (hours)  F i g . 8 Effect of ascorbate concentration on enhancement of misonidazole cytotoxicity  CHO cells were exposed to misonidazole, in hypoxia, with ascorbate added as indicated. Colony forming ability was determined as described in Methods. Ascorbate concentration: A 0 . 0 mM; • 0 . 2 mM; O 0 . 5 mM; • 2 . 0 mM; • 5 . 0 mM; • 5 0 . 0 mM. Misonidazole 5 mM.  51  earlier, that  ascorbate  ascorbate  can  reduce  inhibits  superoxide;  Mason et al.  superoxide-mediated  have shown  adrenochrome  formation  during aerobic microsomal incubations of nitrofurantoin (1977). the action of ascorbate  Thus,  may involve both reduction of the nitro group  and an interaction with the resulting superoxide. The cannot  effect  of  ascorbate  be studied directly,  on  the  in vitro,  aerobic  toxicity  of misonidazole  because of an additional  effect.  Ascorbate itself shows great toxicity in air, which is absent in hypoxia. This  surprising  result  is  illustrated  in f i g .  1978).  Ascorbate causes an immediate  cells.  This  effect  had  been  known. mation  et a l . ,  previously  & Prather,  in  other  systems  1977) but was not widely  The toxicity of ascorbate appears to be due entirely to f o r of  addition This  Josephy  rapid decline in survival of CHO  reported  (Benade et a l . , 1969; Peterkovsky  9 (from  peroxide  of  catalase  action  effect.  is  It  during  completely  specific  seems  ascorbate  to  eliminates  catalase  unlikely  oxidation.  that  -  The  ascorbate  bovine  catalase  toxicity  serum  crosses  simultaneous  the  (fig.  albumin cell  .9).  had  no  membrane,  suggesting that the peroxide is formed extracellularly and either enters the  cell,  or  damages  the  membrane.  were reported by Koch and Biaglow aerobic  toxicity  ubiquitous artifact. selective 1980).  of  enzyme;  ascorbate in  Nevertheless,  this the  is  Similar  (1978).  absent  sense,  of  and conclusions  Presumably, the observed  hn vivo,  the  possibility  results  [n  vitro  since  catalase  observation  achieving,  with  is a is  an  ascorbate,  killing of tumour cells in vivo has been raised (Park et a l . ,  52  I  0  1  I  1  i  I  2  i  L  3  T i m e (h)  F i g . 9 Toxicity of ascorbate to aerobic cells nn vitro (from Josephy et a l . , 1978)  CHO cells were incubated at 3 7 ° , under aerobic conditions. O • •  Ascorbate, 5 mM Ascorbate, 5 mM + Catalase, 0.3 mg/ml Misonidazole, 5 mM + Ascorbate, 5 mM + Catalase, 0.3 mg/ml - Hypoxic incubation  The dotted line represents the 5 mM misonidazole + 5 mM ascorbate response in hypoxia, from f i g . 7. Under hypoxia, the presence of catalase (or bovine serum albumin) increased slighth/ the toxic effects of the misonidazole - ascorbate combination.  53  3.4 Recent developments: In  this  papers  section,  which  I  shall  examine  the  present  effects  a  of  brief  review  ascorbate  of  and  some  other  recent  reducing  agents on misonidazole toxicity. Glutathione reducing  agent  (y-glutamylcysteinylglycine, distributed widely  cells exists mainly oxidized  form,  (Kosower  &  in the  Kosower,  dehydroascorbate  to  in  disulfide Pryor,  ascorbate,  glutathione dehydrogenase.  a  Generally,  tripeptide the  (GSSG)  may  vol.2,  reaction  be  present  1976).  catalysed  GSH by  the  activity  is also found in animal tissues (White et a l . , 1979, p. might  Colowick  expect  misonidazole toxicity  via  in  also  reduces enzyme  This enzyme has been isolated from plant  (Joslyn,  one  GSH  much smaller amounts of the  ed.,  a  is  sources  Thus,  in  in nature.  reduced form;  glutathione  GSH)  &  the  Kaplan,  addition  reduction  ed.,  of  1955,  p.  glutathione  847-9)  to  of endogenous ascorbate.  and  1182). enhance On  the  other hand, glutathione is believed to play an important role in detoxification of activated et a l . , could  ed., be  species in drug metabolism (Jakoby,  1976, p.  207-212) and on this basis, the opposite effect  anticipated.  misonidazole toxicity ethylamine).  in de Serres  Hall  et al.  (1977) observed  by the sulfhydryl  protection  agent cysteamine  against  (8-mercapto-  We have consistently observed enhanced toxicity  in  the  presence of glutathione,  although the effect is much less than that of  ascorbate.  of  toxicity other here.  was  Our  report  followed  reducing  by  agents,  the  effect  several  other  of  ascorbate  on  misonidazole  examinations of the effects of  and it is appropriate to review these  results  54  Koch and Biaglow (1979) observed similar ascorbate-enhancement of misonidazole toxicity, workers  used an elaborate  hypoxia;  they  of  the  These  deoxygenation procedure to ensure extreme  suggested that the ascorbate effect was greater  these conditions. slope  using the V79 Chinese hamster cell line.  under  Data were presented to suggest modification of the  exponential  shoulder modification, by  region  of  the  ascorbate.  survival  curve,  rather  than  Koch and Biaglow found that the  presence of ascorbate did not affect the radiosensitizing properties of misonidazole,  in agreeement  Finally,  reported that a variety  they  cysteamine,  cysteine,  with our own observations  and glutathione,  (unpublished).  of sulfhydryl species, including all  protect against misonidazole  toxicity (single time point only). Taylor  and  Rauth (1980a,b)  studied ascorbate and sulfhydryls as  modifiers of misonidazole toxicity and metabolism. (human) cells were compared;  CHO cells and HeLa  the two lines gave qualitatively  similar  results, although the effects were more pronounced in the hamster line. Again,  ascorbate  was  found  misonidazole  toxicity;  rather  slope-modification,  than  Glutathione  gave  shoulder  about 10%).  stantial cells.  by  a  the  to  very  produce  effect in  slight Taylor  misonidazole metabolism.  primarily  agreement  with  protective  effect  and  effect of ascorbate on the This important  was  substantial  enhancement  of  shoulder-reduction our  own  results.  (lengthening  of  Rauth also demonstrated a s u b -  metabolism of misonidazole by CHO  result will be considered in a later chapter, on  55  Stratford enhancement  (1979) by  also  observed  ascorbate.  Three  protection different  by  sulfhydryls  radiosensitizers  and were  examined, and Stratford concluded that: Vitamin C has the greatest effect on the toxicity of metronidazole, an intermediate effect on misonidazole, and no effect on nitrofurantoin. This is consistent with electron affinities in the order metronidazole < misonidazole < nitrofurantoin and hence, the involvement of Vitamin C and the nitrocompound in a redox reaction. Palcic  et  al.  (1980)  studied the effect of pH on the toxicity of  misonidazole and cysteamine + misonidazole; pH dependence was opposite in these two cases.  Greater misonidazole toxicity,  but less toxicity of  the combination, occurred at lower p H . The curves of misonidazole toxicity alone, and misonidazole plus cysteamine toxicity, cross at approximately pH 7. Consequently, cysteamine will be observed to increase or decrease the cytotoxicity of misonidazole depending on whether the pH is above or below this value. This effect may explain some of the discrepancies between different studies  of  sulfhydryl  effects,  since  several  groups  have  used  a  high-cell-density technique to produce hypoxia, and this results in low pH conditions.  56  CHEMICAL REDUCTION OF 4.1  MISONIDAZOLE:  Introduction: Nitroreduction of misonidazole appears to be a prerequisite, or at  least a co-requisite, to its toxicity.  Some of the evidence supporting  this  above.  hypothesis  several  been  investigators  least three the  has  in  Second,  have  of  an  misonidazole  effort  to  misonidazole has  properties  attempted  to  In  the  of  the  formed  in  demonstrate  been  resulting  it  hypoxic  that  few  years,  directly.  At  First, the nature of cells  nitroreduction  reduced chemically,  derivatives  last  demonstrate  distinct approaches have been tried.  metabolites  studied,  presented  measured.  has  been  occurs.  and the biological Finally,  since  the  active toxic species may be short-lived, these chemical procedures have been  carried  out  in the  presence of DNA, or even  whole cells,  and  biological damage measured (Rowley et a l . , 1980; Edwards, 1980). The  chemical  process,  since  Complete  reduction  resulting  in  variety  of  a  reduction  an  large of  amine  intermediates  (next page).  In  of  number a  nitro  functional may  general,  nitroaromatic  compounds is a complex  of  states  oxidation  be  reached.  six-electron  process,  compound  is  group.  In  between  as  shown  be formed,  a  may  these in  the  states,  a  schematic  the type of products formed during nitro-  reduction depends on many factors: the nature of the R group, temperature, concentration, solvent, and time.  57  0  R-N0  2  R-N=0  4  nitroso  R-NHOH  hydroxylamino  R-NH2  6  The  nitro  2  3  R-N=N(0)-R  azoxy  4  R-N=N-R  azo  5  R-N-N-R H H  amino  best-studied  system  hydrazo  is  the  reduction  of  nitrobenzene.  Certain  results are worth noting:  Although the evidence is overwhelming that nitrosobenzene is the initial reduction product of nitrobenzene . . . nitrosobenzene is reduced so much more rapidly than nitrobenzene that it cannot be isolated. The reduction of nitrosobenzene leads to phenylhydroxylamine, which, despite its reactivity, is the first isolable reduction product of nitrobenzene. (Brown, 1975, p. 740) Condensation of these intermediates can occur, particularly at high p H , resulting  in  the  formation  of  bimolecular  products.  reduction  of nitrobenzene with NaOH and CHgOH yields azoxybenzene.  Zn dust can further reduce this compound to azobenzene. NaOH  reduces  exhaustive occur.  list,  nitrobenzene  to  hydrazobenzene.  For  example,  Zn dust and  Without  giving  an  it is clear that a great variety of possible reactions can  A review of the chemistry of nitroreduction and  products is given by Wardman  (1977).  nitroreduction  58  In  addition  to  these  'classical'  chemical  syntheses,  two  other  techniques have been used in studies o f nitroreduction: pulse radiolysis and electrochemistry. Pulse species, iation  radiolysis  especially  energy.  (Adams  is a tool for the  free  A  radicals,  in  Pryor,  subject  ed.,  (usecond or less)  has been published  vol.3,  studied is contained in an irradiation spectrophotometer system.  molecular  produced by the absorption o f r a d -  review o f the  & Wardman  study o f short-lived  The cuvette  cell,  1977).  recently  The sample to be  and is also in line with a  is irradiated with a very  brief  pulse of electrons; simultaneously, a shutter opens to  allow the intense beam from the spectrophotometer to pass through the cuvette.  The beam is focussed on the entrance slit o f a monochromator,  and fast electronics produce a record of optical absorption as a function of  time  after  irradiation.  As  discussed earlier,  electron-affinity  has  been used as a criterion in the development o f hypoxic cell radiosensitizers.  Electron-affinity  Meisel  and  quinone  Neta  radical  (1975)  derivatives  R-NG^- + Q can  is  studied  of  known  R-NC>2 + Q . be  determined  the above equilibrium stant  can be measured by pulse radiolysis studies.  potentials  particular  virtue  electron-affinities The  of  to  the  the  equilibria and  concentration  spectrophotometrically,  measured.  proportional  reduction  electron-transfer  between  nitroaromatics:  o f the  and the  quinone  position of  The logarithm of the equilibrium condifference  nitro  between  compound  and  the  the  one-electron  quinone.  The  of pulse radiolysis is that such measurements can be  obtained on a very  short time-scale; thus, equilibrium can be achieved  59  before  secondary  irreversibly.  reactions  If  the  alter  nature  the  of  nature  these  of  the  secondary  chemical reactions  system can  be  controlled (for example, by the use of chemical "scavengers" which trap unwanted  intermediates)  technique.  radiolysis  can  be  used  as  a  synthetic  Whillans and Whitmore (1980) have studied the reduction of  misonidazole in this manner; their results will be discussed in chapter 5. The principles of electrochemical techniques, such as polarography and voltammetry, have  been  general,  reviewed  the  suitable  and their application to the study of radiosensitizers, by  electroactive  solvent,  in which  a potential  'working'  electrode  the  (in  Breccia  compound  under  et  al.,  study  ed., is  along with a 'supporting electrolyte',  solution conductivity.  allows  Roffia  1979).  dissolved which  In in a  increases  The solution is contained in a polarographic cell, difference (typically,  electroactive  is applied between a  surface  two electrodes  dropping mercury to  be  renewed  - a  electrode,  which  continuously),  and a  'counter' electrode.  The potential of the working electrode is measured  with  third  respect  reference placed  to  a  electrode  physically  is  electrode,  connected  to  the  'reference'  electrode.  the cell with a salt bridge,  adjacent to the working  electrode;  this  The and  circumvents  the effect of the resistive drop between the working and counter electrodes. between  To perform an electrochemical measurement, the potential drop the working  resulting current  flow  and reference electrodes may be varied, and the measured.  The  resulting trace is  known as a  'polarogram', and shows an increase in current flow at a potential cor-  60  responding to the  reduction or oxidation of the electroactive  From these  measurements,  measured.  However,  has  been  studied  irreversible on  under  than  factors  redox potential  of the  reaction can  be  such a determination is valid only if the reaction thermodynamically  reactions occur,  extraneous  rather  the  species.  such  representing  then as  reversible  conditions.  polarographic potentials  the  rate of  mercury  If  will depend  drop formation,  an intrinsic property of the substance being  studied. Polarographic  measurements  of nitroaromatic  radiosensitizers have  been performed; however, as Wardman notes: In aqueous conditions . . . for many of the compounds of interest to us, the intermediates in the overall reduction are unstable in water, and polarographic measurements often involve irreversible steps in water at pH 7. Whilst polarographic half-wave potentials for the reduction of some compounds in aprotic (non-aqueous) solvents may be a reliable quantitative guide to true reduction potentials, the complexity of the reduction of nitroaromatic compounds . . . detracts from [ their use ] . . . Electrochemical products.  methods  of  nitroreduction  For example, electrolytic  can  yield  a  variety  of  reduction of nitrobenzene leads to  the formation of bimolecular products, such as azoxybenzene (Lipsztajn et a l . , 1974). of partially  Presumably,  these products arise from the condensation  reduced intermediates, and, of course, the overall reaction  is irreversible.  4.2 Identification of zinc reduction products: Chemical reduction of misonidazole was described by Varghese and colleagues (1976).  They adapted a method used by Kuhn (1933) for the  61  reduction of 2-nitrofluorene to 2-aminofluorene: 1 hr reflux of the nitro compound with zinc dust,  in the  used  in  the  same  ethanol  as  isolated  and  conditions  solvent.  In  the  crystallized.  presence of CaClg.  their work  study, of  Varghese  including the  Kuhn, et  Varghese et al.  the  al.  amine  used  use  of 80%  product  was  a colorimetric  test  (formation of a red azo dye following treatment of the reaction mixture with  diazotized  sulfanilic  acid).  The  products  were  also  studied  by  14 paper chromatography of  C-labeled d r u g .  Using the conditions described by Varghese et a l . , we monitored the  reduction  by  greenish-yellow.  TLC  coloured product. gray-brown.  TLC.  Initially,  demonstrated  the the  reaction presence  mixture of  at  turned  least  one  After about 5 minutes, the reaction mixture became  Spectrophotometry  showed  loss  of  the  nitro  absorption above 300 nm, as stated by Varghese et al.  However,  group TLC  revealed the presence of a large number of products, including coloured and  fluorescent compounds.  conditions, Indeed,  the  compounds  misonidazole was  We concluded that,  under the  broken  variety  down  to  a  published  of products.  paper of Varghese et al. showed the presence of several on  paper  chromatograms,  despite the  limited resolution of  this technique; f u r t h e r , the quantity of zinc used was stoichiometrically insufficient  to  reduce  the  misonidazole to  complete conversion of Zn to ZnO.  the  amine,  even  assuming  We concluded that the reaction o b -  served was the breakdown of an intermediate in the reduction process, rather than quantitative reduction to the amine derivative.  62  We identify  attempted  to  the  products.  initial  modify  the conditions used, and to isolate  and  The amount of Zn used was increased;  water was used as solvent; the temperature was lowered to room temperature;  progress  temperature, colour).  the  TLC  compound,  of  the  had  was  proceeded  yellow  repeated  were unsuccessful. performed,  reaction  reaction  of  but  the  monitored  only  product  to  by  the  TLC.  first  appeared  to  At  stage give  room  (yellow a  single  attempts to obtain infra-red and NMR spectra  Finally, multiple development of the T L C plates was  in an attempt to increase  resolution.  The product,  which  appeared to be a single compound, was found to consist of  closely-spaced separation  bands,  had  been  yellow  and  achieved,  orange  isolation  in  colour.  and  Once  identification  of  two this the  products proceeded rapidly (Josephy et a l . , 1980). Since the capacity preparative  of T L C techniques  reversed-phase  liquid  chromatography  the two compounds, using a Merck " L O B A R " in  Methods.  Quantities  of  is limited, we developed a  order  system  to  separate  RP-8 column, as described  100 mg were  prepared  using  this  system. Products appropriate system.  A  and  elution  Product  both were quite  B  were  volumes A  was  obtained  On the  it appeared  by  from the  deep orange,  hygroscopic.  these two derivatives,  prepared  lyophilization liquid  product B basis of the  likely  that they  was  of  the  chromatography bright  yellow;  bright colours of were the azo and  azoxy derivatives of misonidazole.The analogous nitrobenzene derivatives are orange and yellow  respectively,  and both  azo- and azoxybenzene  63  are obtained by NaBH^ reduction of nitrobenzene (Nose & Kudo, 1977), for example.  The identification of products A and B was confirmed by  the following measurements: Solubility:  Both products are soluble in water, methanol, ethanol, and  (sparingly)  acetone and chloroform, but insoluble in less polar solvents  such as ether and carbon tetrachloride. Ultraviolet-visible spectroscopy: shown in f i g . 10.  Product A (azo-misonidazole) has a broad absorption  band with peak wavelength  400 nm.  390 nm; the peak wavelength nm in acetone.  The spectra of products A and B are  This  Product B has a single peak,  at  shifts to 386 nm in methanol, and to 379  shift towards  the  red  in more polar solvents is  known as bathochromic shift, and suggests that the absorption is due to a n-n  transition (Pasto & Johnson, 1969).  1 H-NMR Spectroscopy:  NMR  spectra  obtained as described in Methods. Misonidazole shows (-CH -0-CH ) 2  3  products  Spectra  are  peaks due to- the terminal at  (imid.-CH -CH(OH)-) 2  of  6 = 3.35-3.5;  shown  and  B  in f i g .  were 11-13.  side-chain carbon atoms  the  side-chain  carbons  give a doublet and multiplet at 6 = 4.5 and 4.15;  the imidazole H atoms give the doublet at 6 > 7. to solvent and water). to  A  (Other  peaks are due  The spectrum of product A is almost identical  that of misonidazole itself,  although the  and the shifts are slightly different.  peaks are  rather  broader  Product B is also similar, except  that the peak multiplicities are higher - for example, the methoxy peak is a doublet rather than a singlet, and the imidazole H peaks are split. We interpret these results as follows: azo-misonidazole is a symmetrical  64  Nitro:  misonidazole  Azo:  azo-misonidazole  Azoxy:  azoxy-misonidazole  All spectra were obtained function of  wavelength.  in H^O,  and show extinction  coefficient as a  65  Fig. 11 NMR spectrum of misonidazole Solvent:  CD 0D o  66  67  F i g . 13 NMR spectrum of azoxy-misonidazole Solvent:  D 0 o  68  dimer,  each  half  of  which  has  the  same arrangement of H atoms as  misonidazole; azoxy-misonidazole is similar, except that the presence of the asymmetrical azoxy O atom causes the chemical shifts from the two halves of the molecule to be very slightly different. Mass spectroscopy: were  Attempts  unsuccessful,  to obtain mass spectra of the  presumably  because  of  products  insufficient  volatility.  Therefore, the products were derivatized to more volatile compounds by acetylation. acetic by  Each  anhydride  TLC,  using  product was at  dissolved in pyridine and treated with  room temperature.  chloroform/methanol  The acetylation  (95/5)  as  was monitored  solvent.  Shortly  after  addition of acetic anhydride, both mono- and diacetate derivatives were obtained gave  (higher  on T L C than the  a single monoacetate,  compound.  In  materials).  Product  A  as expected from the symmetry of the azo  the case of product B,  bands were resolved.  starting  two closely-spaced monoacetate  Again, this is due to the asymmetry  introduced  by the azoxy O atom: the monoacetates with the acetyl group on the same side, and the opposite side of the molecule to the O atom, form a pair  of  geometrical  characteristics. complete.  The  isomers,  After  with  30 minutes,  diacetates  slightly  conversion  to  the  chromatographic diacetates  were recrystallized from methylene  and submitted for mass spectroscopy. diacetate:  different  422; product B diacetate:  Parent peaks were:  438.  was  chloride  product A  These molecular weights  are  in agreement with the molecular formulae Cjgh^gOgNg and C^gHggOyNg for  the  azo- and  azoxy-diacetate  respectively.  spectroscopy of the azo-diacetate gave M  +  High-resolution  mass  = 422.1908 (calc. 422.1908).  69  Elemental analysis:  Elemental  analysis  was  performed  by  Dr.  C.  Smithen and colleagues, Roche Products L t d . , and the results are given here with permission. Azo derivative of misonidazole: found C 42.86 C  14 22°4 6 H  "  N  2°  3 H  H 6.22  requires C 42.85  N 21.3%  H 7.19  N 21.4%  Azoxy derivative of misonidazole: found C 46.17 C  1 4 2 2 ° 5 6 ' " 2° H  N  1  z H  r  e  q  u  i  r  e  s  c  4  6-28  H 6.54 H 6.26  N 23.35% N 23.72%.  On the basis of the above evidence, we conclude that product A is azo-misonidazole, and product B is azoxy-misonidazole.  4.3 Recent developments: In a recent publication (1980), Varghese and Whitmore present the results of a study of the  products of zinc reduction of misonidazole,  using conditions similar to those described in our work.  The reduction  was carried out in aqueous solution, at 3 7 ° , but with NH^CI in place of CaC^.  Spectrophotometric  absorbing  in  the  region  measurements  of  demonstrated that a product  400 nm was formed initially.  This  yellow  product was not isolated, but, presumably, it is a mixture of azo- and azoxy-misonidazole.  At  colour  in  disappeared,  eratures.  HPLC  a  later  agreement  analysis  of  the  time with  (about our  colourless  10 minutes) results  the  yellow  at elevated  temp-  product  mixture demon-  70  strated  the  suggested  presence of at least three components. that  derivatives analysis  of  these  included the amine,  misonidazole.  The  have been circulated  mass  Mass spectroscopy  hydroxylamine and spectral  in pre-print form.  data  hydrazo  and  related  This work supports  our evidence that bimolecular products are formed initially, with further reduction leading to the formation of a number of other species. Koch  and  Goldman  (1979)  studied  the- zinc  reduction  of  metro-  nidazole, under conditions very similar to those used (independently) our  studies:  the  aqueous conditions, room temperature.  reduction  products  reversed-phase ethyl)-oxamic others  studied  chromatography. acid  were  were  was  The  identified  formed).  This  hydrolysis of a reactive  by  as  product  appears  2 hours,  ion-exchange  compound  one  product  After  to  in  and  N-(2-hydroxy(although  be formed  reduced intermediate of metronidazole.  several by  the  Similar  results were obtained by enzymatic reduction of metronidazole - this will be  discussed  in  the  misonidazole behave  following  chapter.  quite differently  Clearly,  metronidazole  and  in these reduction procedures, a  phenomenon which will also be discussed later in this thesis.  4.4 In vitro toxicity of azo- and azoxy-misonidazole: The use of preparative separate to  the  produce  each.  bimolecular reduction enough  Preliminary  Modality  reversed-phase column chromatography to  pure  products of misonidazole allowed us  compound to  test  the  biological  activity  of  results were presented at the conference "Combined  Cancer Treatment:  Radiation Sensitizers and Protectors",  Key  71  Biscayne,  Florida, October 1979.  A detailed report has been submitted  for publication (Josephy et a l . , 1981c). The toxicity the  of each derivative  to CHO cells was measured using  system described in Methods, and used in the studies reported in  chapter  3.  Results  of a typical  experiment are  shown  in f i g . 14-15.  Azo-misonidazole shows little toxicity in aerobic or hypoxic incubations certainly,  much  less  azoxy-misonidazole conditions; for  short  is  that  of  misonidazole itself.  more toxic than  the  parent drug  In  contrast,  under  aerobic  in hypoxia, it is also more toxic than misonidazole, at least times.  responses  than  seen  misonidazole  There with  shows  a marked difference  misonidazole and a  Azoxy-misonidazole  is  biphasic  toxicity  the  azoxy-misonidazole.  response,  curves  between  are  with  purely  an  toxicity  Typically,  initial  shoulder.  exponential,  and  show  significant cell kill after one hour or less. The results of experiments with the C H 2 B fig.  16-17.  periments. hypoxic general, to  These CH2B  toxicity  show  the  averaged  cell line are shown in results  of  three  ex-  cells are considerably more sensitive to misonidazole  than  however,  those  2  figures  2  are  CHO cells  (Palcic & Skarsgard,  1978).  In  the results obtained with these cells are comparable  observed  with  CHO's.  The  dramatic  toxicity  of  azoxy-misonidazole is again demonstrated: the slopes of the exponential portions  of  the  misonidazole and  azoxy-misonidazole hypoxic  survival  curves are about equal, and the curves do not cross. The  alkaline  sucrose  gradient  technique,  described  in  Methods,  was used to measure production of DNA damage (single-strand breaks,  72  CHO CELLS: AEROBIC  TIME (hours) F i g . 14 Toxicity of bimolecular derivatives CHO cells - aerobic  incubation  O - control; V - azo-misonidazole; A - misonidazole; • - azoxy-misonidazole. All drugs 5 mM.  73  CHO C E L L S : HYPOXIC  '  I  2  4  TIME  i  I  i  6  (hours)  Fig. 15 Toxicity of bimolecular derivatives C H O cells - hypoxic  incubation  O - control; V - azo-misonidazole; A - misonidazole; • - azoxy-misonidazole. All drugs 5 mM. %  L  8  74  CH2B2 CELLS: AEROBIC control  i  0  i  i  \  2  4  ,  i  6  \  TIME (hours) F i g . 16 Toxicity of bimolecular derivatives CH2B  0  cells - aerobic  incubation  • - control; f - azo-misonidazole; • - misonidazole; • - azoxy-misonidazole. All drugs 5 mM.  75  CH2B2 C E L L S : HYPOXIC  '  0  •  i  I  2  I  I  4 TIME  I  I  6  (hours)  Fig. 17 Toxicity of bimolecular derivatives CH2B,, cells - hypoxic  incubation  • - control; • - azo-misonidazole; • - misonidazole; • - azoxy-misonidazole. All drugs .5 mM.  L  8  76  CH2B2  CELLS  CO  c  TIME  (hours)  Fig. 18 Toxicity of bimolecular derivatives DNA single-strand break production  All drugs 5 mM - see text for details of procedure. Misonidazole  (aerobic)  and  azo-misonidazole 9  induced fewer than 0.2 breaks per 10  (aerobic  daltons in 4 hours.  and  hypoxic)  77  SSB)  by  parallel  these the  remarkably  compounds, in C H 2 B 2 cells  survival  curve  (fig.  measurements.  18).  These  results  Azoxy-misonidazole  shows  high production of DNA damage, whereas the azo derivative  is innocuous.  The production of SSB  by azoxy-misonidazole is  linear,  with no initial lag. Dr. these  C . Smithen and colleagues,  compounds in another  Roche Products  system.  L t d . , have tested  Chinese hamster V-79-379A cells  are grown in the presence of the drug (chronic aerobic toxicity), the  inhibition  of protein  synthesis  is measured.  and  Azo-misonidazole was  less toxic and azoxy-misonidazole more toxic than misonidazole itself, on the basis of concentration  required to produce 50% reduction in protein  content relative to controls. The  three  compounds  studied  in  these  experiments  form  a  homologous series of chemicals, differing only in the level of reduction of  the  nitro  functional  increasing toxicity  as:  group.  We  azo < nitro  may  < azoxy.  correlation of toxicity with electron-affinity least These  as  values  Smithen, (0.1M  measured have  using  polarographic  been  samples  compounds  Saturated  by  measured supplied  dissolved  Calomel Electrode  in  arrange  by  phosphate  reduction  C.J.  the  385 mV 150 180  Little  values  buffer,  (SCE)):  misonidazole azo-misonidazole azoxy-misonidazole  order  of  among these compounds, at  Dr.  us;  in  There appears to be no  half-wave by  them  pH  potentials.  and  were 7.4  Dr.  as  C.  follows  - mV  vs  78  As  discussed  earlier,  thermodynamically  such  values  reversible  are  only  electron-affinities.  a  rough  guide  Nonetheless,  to  the  there  is  no correlation between these values and the observed toxicity. The  bimolecular  derivatives  radiation-sensitizing ability,  of  misonidazole  showed  very  little  when tested using CHO cells irradiated in  hypoxia (drug concentration = 5 mM); (fig.  19).  4.5 Metabolic formation of bimolecular products: The formation of azo- and azoxy- compounds during the reduction of  misonidazole  with  zinc  may  be  due  to  condensation  of  partially  reduced, monomeric intermediates such as the nitroso and hydroxylamine derivatives,  although  these  were  not  isolated.  Indeed,  the  synthesis  may be regarded as evidence for the instability of these intermediates. Are  similar  products  formed  in  biological  systems?  There  is  some  suggestion of this in the literature. Some patients  receiving  metronidazole excrete red-brown pigments  in their urine. The chemical nature of this material is unclear.  Manthei  and Feo (1964) described an attempt at characterisation:  The dark material was concentrated to a brown oil . . . . and its IR spectrum e x a m i n e d . . . . It had many peaks in common with the IR spectrum of Flagyl (metronidazole) and in addition it had a peak at 6.25 u characteristic of a N=N linkage. Therefore, authentic azometronidazole was prepared by cautious reduction of Flagyl. This was found to be reasonably similar chromatographically to the relatively impure dark material isolated from urine . . . . In a later publication from the same group (Stambaugh was stated that:  et a l . , 1968) it  79  Further studies showed that the chemical nature of this brown pigment was not the same in the various urines studied. Comparison of synthetic reduction products with the urinary isolates indicated that these dark pigments may represent azoxy h y d r o c h l o r i d e s . . . The  details of the  however, very  as  mentioned  different  initial  "cautious"  yellow  results  does  above, from  procedure are  reduction of  a brown  of  metronidazole There  gives is  no  solution is produced, with a  We were unable to characterize this material, but  products  analogous  reduction of misonidazole. obtained  not described;  misonidazole.  not appear to be a pure compound. It  breakdown  are  zinc  reduction  colour - instead,  characteristic odour. it  reduction  even  at  to those obtained by  Since,  low  may be a mixture of high-temperature  with metronidazole, the same results  temperatures,  there  may  be  significant  differences between the reactivities of partially reduced intermediates of the two nitroimidazoles. As mentioned earlier,  Koch and Goldman obtain-  ed results which support this hypothesis. Dr. series  R.C.  of  Urtasun, Cross Cancer Institute,  urine  and  serum  samples  obtained  Edmonton, provided a  from  patients  receiving  misonidazole,  and  metabolites.  These specimens also showed red-brown coloration, which  was  most  we examined these for the formation of bimolecular  pronounced  administration.  in  However,  samples these  taken  using  conditions  azoxy-misonidazole,  gave  hours  after  pigments are water-soluble  not be extracted into organic solvents. samples,  8-12  suitable uniformly  for  drug  and could  Direct HPLC examination of the the  negative  detection results.  of Thus,  metronidazole, the nature of these products remains unknown.  azo-  and  as  with  80  100 \  \  '••  **• \ *  * *  X  *  A *• " \ \  v.  10  < > >  DC D CO  *  \ \  C H O Cells  \r>  \  • • •  *  * -  a  \  • Azoxy  \ \  \\  10mM  • Azo  5mM  X  \  *^^^\  \  • \  \  l  *  1  \  \  " 1  1  \  \  \  •  t  \  \  • '• \ N  2  \MISO\lmM  AIRl 01  \  •  10  •. L- •  20  '  30  D O S E (Gy)  Fig. 19: Radiosensitizing. properties of derivatives  CHO cells were irradiated to various doses ( Co y) and colony forming ability determined. Misonidazole or derivatives added during irradiation, as indicated. All irradiations performed under hypoxia, except curve marked " A I R " .  81  BIOCHEMICAL  REDUCTION OF MISONIDAZOLE  AND  ITS  AZO- AND AZOXY- D E R I V A T I V E S : 5.1  Introduction: The  reduction of nitroaromatic  observed  early  in  this  century  compounds by  (Neuberg  &  microorganisms was  Welde,  1914);  I  have  discussed the mechanism of bacterial nitroreduction in the introduction. There  were few  studies  of  mammalian nitroreduction  increase in the use of nitro d r u g s . in  rabbits  was  studied  (Channon  et  until the  recent  Reduction of trinitrotoluene  (TNT)  al.,  1944),  and in vitro  homogenate techniques were also used (Bueding & Jolliffe, and Brodie (1956) were the first workers to study systematically,  using  subcellular fractions.  a variety  of  compounds,  tissue  1946).  Fouts  this enzyme activity species,  Reduction of nitrobenzoic acid was  organs,  and  measured by  formation of aminobenzoic acid, using a colorimetric assay for the amine; no attempt was  made to isolate intermediates.  Activity  was  found in  both the supernatant soluble fraction and in the microsomal fraction of rabbit liver.  Both fractions were NADPH dependent; some activity  also observed with N A D H . proportional  to  the  The amount of reduced product formed was  concentration  of  drug,  even  gesting that the enzyme systems have a very compounds. reduced  reviews  Also, a wide variety  at comparable rates.  and even of  the  work  on  Brodie & Gillette, e d . , 1971;  up to 10 mM,  of nitrobenzene derivatives  The  identity  nitroreduction  of the enzymes  are  Mitchard, 1971.  sug-  low affinity for the nitro  number of distinct enzymes, was  early  was  could be involved,  not determined.  available  - Gillette,  Two in  82  Gillette and colleagues attempted to identify the enzyme responsible for the reduction of nitrobenzoate by liver microsomes. They concluded that cytochrome P-450 was involved, since the activity was CO inhibited (Gillette,  ibid.)  A CO - insensitive activity was also found, and this is  believed to be NADPH - cytochrome c reductase (Feller et a l . , 1971); despite  its  reduction 1978).  name, of  the  probable  cytochrome  Both cytochrome  proteins,  and  the  P-450  biological function  of  this  (Bock  in  Aldridge,  P-450 and the  purification  of  the  &  Remmer,  enzyme is ed.,  reductase are membrane-bound components of  the  cytochrome  P-450 system remains problematical.  5.2 Xanthine oxidase: In  contrast to the  rather  intractable  cytochrome  P-450,  xanthine  oxidase ( X O , xanthine: oxygen oxidoreductase, EC is a soluble enzyme  which  including The  is  milk.  enzyme  It  was  contains  review  of  (Bray,  in Boyer,  XO,  purified  the  very  from  a  variety  structure  and  vol.  iron-sulfur  properties  78, 1975).  of  groups,  XO  has  and been  discussed recently (Krenitsky, catalyses  the  FAD.  A  presented was  1978).  reduction  of  oxygen  and  the  simultaneous  oxidation of hypoxanthine to xanthine, and in a second step, to uric acid.  sources,  The biological significance of  and the closely-related enzyme aldehyde oxidase (EC  XO  of  one of the first enzymes to be crystallized.  molybdenum,  ed.,  easily  xanthine  83  The  reducing  mixed  one-  generates  equivalents and  (e  + H )  two-electron  are transferred  process.  The  to oxygen,  xanthine/XO  in a  system  both H^C^ and O^: indeed, it was the study of this  system  that led to the discovery of superoxide dismutase. In  addition  to  hypoxanthine  and  xanthine,  a variety  reducing agents can serve as electron donors to X O , acetaldehyde, from the  and  system,  benzaldehyde it  (Bray,  op_. c i t . ) .  is possible to demonstrate  including If  the  of  other NADH,  is excluded  reduction of many  other compounds by the xanthine/XO system. These include the azo dye methylene Brown,  blue,  cytochrome  1969) and the  (Pan & Bachur, Taylor  equivalents  were transferred the  and  xanthine/XO  per  hydroxylamine  co-workers reduction  (1976 of  suggested  mole of level. &  nitrofurazone was  nitrofuran,  This  1978).  studied.  that  work  In  the  and  adriamycin  found to be reduced with about 6-electron 4- electron as reported by Taylor et al.) as the amine derivative. tentatively,  reduces  TLC,  4  reducing  consistent  was 1976  AF-2  and IR spectroscopy were used to identify products.  identified,  drug  &  Although the products were not  measurements  3- (5-nitro-2-furyl)acrylamide)  was  (Stohrer  showed that the xanthine/XO system  stoichiometric  Tatsumi  chemotherapeutic  under anoxic conditions.  to  purine-N-oxide  1980).  identified,  reduction  (ibid.),  anthracycline  et al. (1951)  nitrofurans,  c  mass  extended  with by  publication, (2-(2-furyl)spectroscopy,  Nitrofurazone was  stoichiometry  (rather than  and the product was identified  In the case of AF-2, the reduced as a re-arrangement  derivative  product of the amine.  84  In the second paper, Tatsumi et al. studied the reduction of a series of nitrobenzene  derivatives;  corresponding  the  products  hydroxylamines,  were  prepared  by  compared  reduction  with  of  the  the nitro  group with Zn and NH^CI.  The authors concluded that: "aromatic nitro  compounds were ultimately  reduced to the  hydroxylamines, but not to  the a m i n e s . . . " by the enzymatic system (Tatsumi et a l . , 1978). Thus,  the  differences AF-2  and  must  be  in  nitrobenzenes  and  reduction by this  nitrofurazone studied  gave  nitrofurans  system; even  different  individually  before  show  within the nitrofurans,  products. a  characteristic  Clearly,  general  each  conclusion  can  case be  reached. Dr.  P.  Goldman  metronidazole  by  been  colleagues  bacteria  perimental animals. not  and  studied  isolated  extensively;  obligate anaerobes, gut  from  studied  the  which  bacteria  may  a  review  of  number  of  administration  small of  acetamide  (Koch  (Koch  al.,  et  al.,  1979b).  excreta of germ-free rats, formation.  metabolism of tracts  of  ex-  literature  has  been  Most of the bacteria found in the gut are implies that the play  molecules  labeled et  intestinal  the  an  intestinal  important  particularly following oral drug administration. a  the  The role of intestinal flora in drug metabolism has  presented (Goldman, 1978).  Thus,  have  excreta  metronidazole.  These  These  and  in  nitroreduction,  Goldman et al. isolated  the  1979a)  from  role  tract is hypoxic.  of  rats,  following  products  included  N-(2-hydroxyethyl)-oxamic  metabolites  were  not  present  in  acid the  which implicates bacterial metabolism in their  It was suggested that the metabolites arose from the hydro-  85  lysis and ring cleavage of a reactive reduced intermediate, as discussed earlier  in the consideration of Zn  reduction of metronidazole (chapter  4). In a subsequent paper (Chrystal  et a l . , 1980), XO was used as a  model system for the study of metronidazole reduction. these  results  after  a  presentation  of  our  studies  I shall consider of  XO-catalysed  reduction of misonidazole and its derivatives.  5.3 Reduction of misonidazole by xanthine oxidase: The  results of our investigations on the reduction of misonidazole  and its derivatives al.,  by xanthine oxidase (XO)  1981a) and will be summarized in this  are in press (Josephy et and the following section.  The methods used have been presented in chapter 2. A typical  measurement of misonidazole reduction by xanthine/XO,  under hypoxia, is shown in f i g . 20.  Reduction is measured by loss of  nitro group UV absorption at 350 nm.  Even at the highest misonidazole  concentration linear,  used  the  curves  were  exponential  rather  than  suggesting that the Michaelis constant for misonidazole is higher  than this value. in  (1mM),  the  enzyme xanthine  absence had as  No reduction was observed under aerobic conditions, of  been  the  reducing  inactivated  reducing  substrate  by  substrate,  brief  under  (hypoxanthine), boiling.  aerobic  In  or  studies  conditons,  the  if  the  using initial  rate of xanthine oxidation was 1.75 uMoles/min/unit; using misonidazole as  oxidizing  substrate  under  hypoxia, the  rate of xanthine oxidation  86  .4 .3  \  \  \  M I S O N I D A Z O L E  \  V  O . Q  350  2 1A  20  40 M l  60  80  N U T E S  F i g . 20 Reduction of misonidazole by hypoxanthine and xanthine oxidase, in hypoxia  Reduction was monitored by loss of absorption at 350 nm. Initial misonidazole concentration was 100 uM. See text for details of enzymatic reduction procedure.  87  was only 6 nMoles/min/unit. much  less  oxygen.  effective The  in  T h u s , the nitro group  accepting  rate is of the  electrons  from  of misonidazole is  the  enzyme  than  is  same order as that reported by Goldman  for metronidazole (see below). Stoichiometry  was  determined  by  comparing  the  rates  of  miso-  nidazole reduction and uric acid formation, using xanthine as substrate. Reversed-phase incubation  HPLC  mixture.  misonidazole,  was  used  to  separate  the  components  of  the  We found a ratio of 2.06 moles xanthine per mole  consistent  with  reduction  to  the  hydroxylamine  level  (4-electron). 14 We  used  C-labeled  misonidazole reduction, soluble  in  band at slightly 90%  of  the  R.p = 0.73; The  higher  the  of  HPLC  not a  R^. In  thus,  results  but  showed  activity.  to  as described  methanol,  radiochromatograms  drug  characterize in Methods.  in  single  acetone band  at  or  the  products  of  The derivative  was  chloroform.  TLC  R^ = 0.15,  and a minor  The main band accounted for more than this  product  TLC is  system,  much  misonidazole  itself  had  more polar than misonidazole.  radiochromatography,  using  phase system described in Methods (section 2.4),  the  polar  bonded  are shown in f i g . 21.  A single major product peak contained over 80% of the total activity. An additional sample was r u n , and this peak was collected and studied by mass spectroscopy. ionisation (El)  Successful  results  were obtained in both electron  and chemical ionisation (CI)  modes. El mass spectra gave  parent mass m/e = 187, consistent with hydroxylamino-misonidazole;  the  complete spectrum was identical to that of a compound isolated by Var-  88  ghese  and  Whitmore,  using  described in chapter 4. as  the  modified  zinc  reduction  procedure  This compound has been tentatively  hydroxylamino-misonidazole.  However,  attempts  to  identified  obtain  NMR  spectra were unsuccessful, both in our lab and in the work of Dr. D. Whillans  at  the  Ontario  Cancer  Institute  (see  below).  The  product  appears to be unstable in concentrated solutions, perhaps due to disproportionation,  which  amines (Wardman,  is  reported  to  occur  with  aromatic  hydroxyl-  1977).  5.4 Reduction of derivatives of misonidazole by xanthine oxidase: We  have  also  examined  the  reduction  of  azoxy-misonidazole by xanthine/xanthine oxidase ( X O ) ,  azo-  and  (Josephy et a l . ,  1981a). The  disappearance  of azo-misonidazole from a hypoxic  mixture similar to that used for fig.  20, misonidazole).  incubation  misonidazole is shown in f i g . 22 (cf.  The reduction is faster than for misonidazole,  and the linear kinetics suggest a much lower Michaelis constant for the azo  compound.  The  product  was  colourless,  but  was  not  studied  further. Preliminary as  an  intermediate  intriguing the  experiments  reduction  was  was  the  reduction  of  azoxy-misonidazole.  This  result meant that it was difficult to measure the kinetics of spectrophotometrically,  azoxy-misonidazole HPLC  in  showed that azo-misonidazole was formed  chromophores  used to separate  performed  as  for  overlap.  since  Therefore,  the derivatives.  misonidazole,  the  except  azo-  and  reversed-phase  The enzymatic reduction that azoxy-misonidazole,  89  1  10  20  30  40 F  50 R  A  C  T  60 I  O  70  80  90  100  N  F i g . 21 HPLC analysis of products of misonidazole reduction  C-misonidazole was reduced with xanthine and xanthine oxidase, and the product mixture extracted and analysed by H P L C , using PAC column. Fractions eluted from the column were collected and counted to determine radioactivity. Misonidazole itself is eluted near solvent front (fraction 12). For further details, see text.  90  1.6. 1.2J  O.D. .8J 4 0 0  .4. 0  Fig.  \  AZO\MISONIDAZOLE \ \ \ Y \ V 10  20  MINUTES  30  22 Reduction of azo-misonidazole by hypoxanthine and xanthine oxidase in hypoxia  Reduction was monitored by loss azo-misonidazole concentration was enzymatic reduction procedure.  of absorption at 400 nm. Initial 100 uM. See text for details of  91  1 mM, was the oxidizing substrate. regular The  intervals  samples  400  were 4.6  studied  mm  x  Retention  times  of  3.2,  9.0,  rate  of  separable.  were 2.0  constructed;  HPLC.  mm;  Whatman  phase:  10  mM  ODS  acetate  azoxy-misonidazole,  and 13.0 minutes, All  curves  Column:  Optical absorption was measured at  for  three  respectively,  compounds  azo- and  response (peak height vs  the range studied.  mobile  misonidazole,  ml/minute.  Calibration  by  250  (75/25).  azo-misonidazole flow  then  pH 4.5/methanol  nm.  at  following addition of the enzyme, and placed on ice.  reversed-phase, buffer,  Samples (100 ul) were withdrawn  were  and at a  baseline  azoxy-misonidazole were  concentration)  was  linear over  At equimolar concentrations, the ratio of azoxy- to  azo-misonidazole peak height was 100 to 88. The  disappearance  subsequent reduction,  of  disappearance is  shown  shown in f i g . 24.  in  azoxy-misonidazole, and the formation and of  fig.  azo-misonidazole 23.  formed  Representative  during  the  chromatograms  are  The level of azo-misonidazole was never higher than  about 50 uM; it is barely detectable in the right-hand chromatogram of fig.  24.  Presumably,  the  kinetics  of  azo-misonidazole formation  and  disappearance during the experiment result from competition between it and  the  starting  material  Identification azoxy-misonidazole  for  reducing equivalents  of  the  intermediate  in  as  azo-misonidazole was  the  from the enzyme.  enzymatic  made on the  reduction  of  basis of  the  retention time and spectrum of the product, which are identical to those of  authentic  compound.  azo-misonidazole prepared The  nature  of  the  final  by  Zn  product  reduction was  not  of the  nitro  studied,  but,  presumably, it is the same as the product of azo-misonidazole reduction.  92  k  AZOXY-MISONIDAZOLE •  REL. PEAK HT.  \•  AZOXY  30  60  MINUTES  Fig.  90  23 Reduction of azoxy-misonidazole by hypoxanthine and xanthine oxidase, in hypoxia I.  Kinetics  Reduction of azoxy-misonidazole, and formation and reduction of azo-misonidazole (formed as an intermediate in the reduction) were measured by H P L C . Initial azoxy-misonidazole concentration was 1.0 mM. See text for further details.  93  1  A.U. 400 nm  1K J 8  j.  16  0  8  16  MINUTES  F i g . 24 Reduction of azoxy-misonidazole by hypoxanthine and xanthine oxidase, in hypoxia II.  Representative  chromatograms  Reduction of azoxy-misonidazole was monitored by HPLC - see text for details. Left: sample obtained immmediately after adding enzyme to start reduction. Right: sample obtained after 30 minutes incubation. Retention times: azoxy-misonidazole, 9 m i n . ; azo-misonidazole, 13 min. Note reduction of azoxy-misonidazole peak, and appearance of trace of azo-misonidazole, in the right hand panel.  94  5.5 Recent developments: th We presented the preliminary results of these studies at the 28 annual  meeting of the  1980.  In a paper presented during the same session, Dr.  described  the  technique  Radiation  reduction  (Whillans  &  of  Research  these  Society,  New Orleans, June  compounds by  Whitmore,  1980;  a  D. Whillans  radiation  1981).  In  chemical  these  studies,  misonidazole was prepared in dilute aqueous solution, at neutral p H , in the presence of 100 mM Na formate. and y-irradiated.  The solutions were de-oxygenated  In this system, the H' and O H ' radicals produced by  radiolysis of water are scavenged by formate to produce COr, ; this species, and e , reduce the d r u g . Stoichiometry of the reduction can aq be calculated from the appropriate G-values.  Misonidazole was reduced  with 4-electron stoichiometry, and over 80% of a  single  HPLC  peak.  This  product  appears  activity was found in to  be  identical  to  our  enzymatic product (HPLC and mass spectrum). In  addition, Whillans and Whitmore  studied the  reduction of azo-  and azoxy-misonidazole, which we had provided for these experiments. Again, the radiolytic results were analogous to those obtained with the enzymatic nidazole,  system.  Azoxy-misonidazole  to a colourless product.  was  reduced  via  azo-miso-  This product was suggested to be  hydrazo-misonidazole, on the basis of mass spectroscopy. Dr. catalysed This  Goldman and colleagues studied the reduction of metronidazole by  research  appears  that  XO, was the  concurrently  with  reported  recently  two  nitroimidazoles  our  own  work  (Chrystal behave  very  et  on misonidazole. al.,  1980).  differently  in  It this  95  system,  as well as in the Zn reduction.  Chrystal  et  incubation products  al.  isolated  mixture, formed,  a wide  variety  reminiscent  Instead of a single product, of  small  of the results  and a schematic  pathway  molecules  from the  of Zn reduction.  for their  formation,  The are  shown in the following figure (from Chrystal et a l . , 1980):  OH NH 0. -OH NHj  OH  OH  xNH„  \  NH  OH  / N .  NH  «\— N  >  2  2  NH  2  r' 1  HjN HO  _ o  0  0  '  N  Fragmentation patterns of metronidazole. The pairs of complementary fragments formed in the cleavage schemes are: cleavage a, A/-(2-hydroxyethyl)-oxamic acid and acetamide; cleavage b, N-acetylethanolamine and glycine; cleavage c, ethanolamine and N-acetylglycine; and cleavage d, N-glycoylethanolamine and acetic acid.  In a second paper bimolecular  products  (Goldman  may  reduction of metronidazole.  be  et a l . , 1980) it was suggested that  formed  during  the  The authors stated that:  XO  -  catalysed  96  . . . we can only account for approximately one-third of the products of metronidazole reduction ... [S]ome products of the reduction of metronidazole by xanthine oxidase may be the result of a bimolecular reaction. Perhaps these are azo or azoxy compounds comparable to those isolated in the chemical reduction of misonidazole by Josephy et al. . . . Indeed, Tatsumi et al. (1978) had suggested that azoxy formed during the XO - catalysed reduction of nitrobenzene  derivatives derivatives:  In the case of p-nitrobenzenesulfonamide, a small amount of azoxy-compound was detected by T L C as one of the reduction products It is most probable that the azoxy-compound was formed during the isolation procedure and was not originally formed by the action of xanthine oxidase. In  a  reduction  recent of  a  paper,  variety  of  Clarke  et  al.  nitroimidazoles  (1980) by  have  reduced  examined FMN  and  the by  xanthine oxidase. They concluded that: the stoichiometry of reduction of nitroimidazoles by both free and a protein-bound flavin has provided evidence for the production of hydroxylamines during biochemical nitroreduction . . . Clearly, more work is needed to clarify the differences observed in the reduction of these nitrobenzene and nitroimidazole compounds.  97  J_N VITRO METABOLISM OF 6.1  MISONIDAZOLE:  Introduction: In the preceding two chapters,  nidazole by  I discussed the reduction of miso-  chemical and biochemical techniques.  These studies  light on the nature of the reduced derivatives of the d r u g , tified  a  possible  enzymatic  hypoxic mammalian cells.  mechanism  for  In this chapter,  misonidazole  shed  and iden-  reduction  in  I shall describe our studies  14 of  the  metabolism of  1981b).  First,  C-misonidazole in  however,  along these lines.  CHO cells  (Josephy  et a l . ,  I shall describe briefly, some previous studies  Much of this work overlaps studies of the chemical  reduction and toxicity of misonidazole, described earlier. Varghese et al.  (1976)  was discussed, in part,  reduced misonidazole with Z n ; this study  in chapter 4.  These workers also studied the  metabolism of labeled drug in CHO cells and KHT (mouse fibrosarcoma) tumour cells.  The  KHT  cells were studied both in vitro and in vivo,  following injection of the d r u g , tumour.  The  water-soluble graphy  lower both  with which in vitro  R^ than the tumour  were  supernatants  system  Hypoxic cells  cells  and  intraperitoneally, isolated  were the  studied, Zn  converted  parent d r u g . normal  tissues  suggested that these metabolites (desmethylmisonidazole,  and  into mice bearing the  homogenized,  using  the  and  paper  the  chromato-  reduction products were analysed.  misonidazole into several  products of  Similar product peaks were found in of  the  i.p.-injected  mice.  It  was  included the O-demethylation product  Ro 05-9963) as well as a glucuronide conjugate.  Another product ("peak 2")  was thought to be the amine derivative, on  98  the  basis  However, of the this  of  co-chromatography  with  the  Zn  reduction  product.  as discussed in chapter 4, there is doubt about the  Zn  reduction  paper.  The  identity  products obtained using the method described in  resolution of the  paper chromatograms is inferior to  that of more modern methods, such as  HPLC,  and definitive chemical  identification of none of the metabolites was achieved. Taylor  and  misonidazole  in  Rauth CHO  (1978)  cells  studied the toxicity  and human (HeLa)  and metabolism of  cells.  Toxicity  studies  were performed using a similar method to that described in this thesis; metabolism was examined using the chromatography system described by Varghese et al. (1976). activity of  14  is accumulated in hypoxic, but not aerobic cells, as a function  incubation  time.  metabolite peaks. was  Taylor and Rauth showed that C-misonidazole  extended.  The  hypoxic  cells  showed production of  In a later paper (Taylor Ascorbate,  cysteamine,  & Rauth,  several  1980a) this  work  and glutathione were shown to  modify the metabolism of misonidazole in hypoxic CHO and HeLa cells; all of the agents increased the amounts of activity in some of the polar product  peaks,  although  the  resulting  patterns  were quite  different.  Ascorbate showed the greatest effect, in accord with its profound effect on  misonidazole  toxicity.  production of certain controversy however,  over  The  metabolites,  the  the metabolite  effects  of  sulfhydryl  agents  also  enhanced  an interesting result in view of the these  agents  on  peaks were not identified.  toxicity.  Again,  The entire water-  soluble cell extract was run together, without fractionation according to organic-solubility.  99  In vitro metabolites. and  Rauth  metabolism  studies  For example, (e.g.  metabolite activity  chart  yield  very  small  quantities  in the chromatograms published by 7,  1978)  the  largest  peak  of  of  Taylor  intracellular  is little higher than the free misonidazole peak, even  following 5 hour exposure to 5 mM d r u g .  Some metabolites were found  in extracellular medium (chart 7, panels G & H), but they represented only about 1% of the radioactivity in the medium. It is not clear whether this material diffused out of intact cells, or was released by the lysis of dead cells.  The total amount of intact misonidazole contained i n , say, 5  x 1o7 CHO cells, at 5 mM, is only about 100 ugm.  T h u s , the amount of  any particular metabolite that could be recovered in this sytem is very small. The  studies  characterize  a  reported  reduced  matography systems;  in  the  derivative  previous of  chapter  misonidazole  allowed  in  various  us  to  chro-  with this knowledge, it was possible to search for  the presence of this compound in cell extracts.  6.2 In vitro metabolism of misonidazole: The techniques (Methods).  used in these  Several  principles  studies are described in chapter 2  were  considered  in  designing  these  14 experiments.  We used  C-misonidazole of much higher specific activity  than that used in the studies of Varghese et al. and Taylor et al. labeled drug was activity. very  The  used without dilution, to obtain the highest possible  Second, recovery of metabolites was maximized by the use of  dense cell  suspensions, so that all of the added drug would be  100  converted partially  to metabolites. purify  solubility  the  Third,  an extraction procedure was used to  anticipated  properties.  metabolite  Finally,  we  on the  used  basis of its  HPLC  known  to  separate  the  under  hypoxia,  and  metabolites. Cells  were  incubated  with  labeled  drug  aliquots were removed at various times. fractionated into three extracts, chapter 2.  radioactivity  fractions.  activity  organic-insoluble  acid-extractable, after  Fig.  25  shows  among these fractions.  organic-soluble  Even  according to the methods described in  These extracts were the organic-soluble, acid-soluble, and  acid-insoluble  and  The cells were disrupted and  all  distribution  of  total  As a function of incubation time,  (misonidazole) products.  the  is  converted to organic-soluble  Most  of  the  latter  material  is  but some is associated with the acid-insoluble pellet. free  misonidazole  has  disappeared  incubation) the percentage of radioactivity less than 10% of the total.  (about  3  hours  associated with the pellet is  Further incubation beyond 3 hours produced  little change in the distribution. The detail. may  nature  of the acid-soluble material  has not been studied  in  These metabolites are highly polar (R.^ almost zero on T L C ) and  include  ionic  (probably covalently)  conjugates.  The  pellet  contains  to nucleic acid and protein.  been observed both in vitro and in vivo (Varghese The  organic-soluble fractions  obtained  at  activity  Similar results have & Whitmore,  successive  were studied by T L C and HPLC as described in chapter 2. of T L C autoradiography are shown in f i g . 26.  bound  time  1980). points  The results  Misonidazole ( R  f  = 0.78)  101  DISTRIBUTION O F  RADIOACTIVITY  CHO CELL EXTRACTS 100 ORGANIC soluble 80  ORGANIC insoluble  PELLET  ACID soluble  % TOTAL CPM 40  20-1  0 1 2 3  0 1 2 3 I N C U B A T I O N TIME  F i g . 25 Distribution of radioactivity  0 1 2 3 (HOURS)  - CHO cell extracts  CHO cells were incubated under hypoxia with C-misonidazole for the indicated lenghts of time, ' as described in text. Samples were dried and extracted with ethyl acetate/methanol (3 x 2 ml) and then with methanol/TCA (10 x 2 ml). The total activity in the organic-soluble, acid-soluble, and acid-insoluble fractions is shown as a function of time of incubation.  102  Fig. 26 T L C autoradiography of organic-soluble extracts  CHO cells were incubated under hypoxia with C-misonidazole for up to 3 hours, and the organic-soluble metabolites separated by T L C , as described in text. Samples were run on a single pre-channeled T L C plate, which was then autoradiographed on Kodak X - r a y film for ten days. From left: 2 m i n . , 1 h r . , 2 h r . , 3 h r . incubation. Misonidazole is indicated by letter M. Dashed lines mark R = 0 and 1. f  103  is depleted by about 50% in one hour and completely metabolised in 3 5  hours. cell  This corresponds to the conversion of about 10  per  second.  molecules per  Several metabolites of misonidazole can be resolved.  All have much lower  than misonidazole itself.  This is in agreement  with the general pattern of metabolism to more polar derivatives. The results of reversed-phase HPLC analysis are shown in f i g . 27. The data shown  in this figure parallel the  results obtained by  TLC.  Misonidazole (which is eluted between fractions 60 and 80) is depleted during  incubation, and converted to a variety  (shorter  retention times).  minutes  (Fig.  incubation, results,  27a)  further  products  incubation  pattern.  component was  xanthine  After an incubation time of as little as two are  detectable.  After  one  hour  a complex pattern of metabolites is seen; as with the T L C  effect on the This  two  of more polar products  oxidase  The  shown  -  affects largest to  catalysed  the  peak  heights,  but  has  little  peak is at or near fraction  co-chromatograph reduction  of  with the  #21.  product of  misonidazole,  which  is  believed to be hydroxylamino-misonidazole.  This was demonstrated by  the  described  dual-label  Results  are  chromatography  presented  in  fig.  technique 28.  in  chapter  2.  The enzymatic reduction was  not  carried to completion, so that the extracted material contains both 3 3 H-misonidazole and H-hydroxylamino-misonidazole as markers. These markers gave peaks in fractions 52 and 18 respectively (system A ) and 15 and 60 respectively  (system B ) .  In both systems, a major peak of  14 C  activity  coincided  was particularly  with the enzymatically  clear in system B;  apparently,  reduced product.  This  some of the metabolites  104  isolated along with the  hydroxylamino  insoluble in ethyl acetate/methanol  product in system A are almost  (used in system B).  The material in  the product peak in system B was collected, concentrated, TLC.  Fractions  14  Again,  were  3  C and  scraped,  H activity  eluted  with  methanol,  and run on  and  counted.  ran together (data not shown).  6.3 Conclusions: In  chapters  misonidazole by using  zinc  however,  4 and 5 of this thesis, a variety  metal,  these  led  of systems.  to  the  I discussed the reduction of  Chemical  formation  of  bimolecular  derivatives;  do not appear to be formed in biological systems.  enzymatic reduction of misonidazole (chapter reduction (Whillans & Whitmore, 4-electron  reduction of the drug  stoichiometry.  This  5),  and radiation  The  chemical  1980) yield a single major product with product  is  probably  hydroxylamino-  misonidazole, but may be of limited stability. In  this  chapter,  I have shown that a metabolite of misonidazole,  produced in hypoxic cells, is identical to the enzymatic product. metabolite (fig.  can  27a)  be detected after a very  and,  following  the  This  brief exposure to misonidazole  disappearance  of  the  parent  drug,  it  remains the major organic-soluble metabolite. 14 The  metabolism  (Varghese al.,  1978).  100  times  et  al., In  C-misonidazole  1976; Taylor  this  higher  of  work,  & Rauth,  has  been  studied  previously  1978, 1980a,b; Whitmore  et  we have used labeled drug with more than  specific  activity.  Also,  we  have  separated  organic-soluble material from the cell extracts, prior to chromatography.  105  The organic-insoluble, acid-soluble material the metabolite activity;  represents at least half of  since this material is very polar, it would elute  early  on a reversed-phase  column, and might obscure the metabolites  seen  in  We  fig.  27 and  28.  consider  it  unlikely  that  the  reduced  product identified in this chapter corresponds to one of the peaks ( e . g . P1,  P2)  observed  in paper chromatography of crude extracts of CHO  cells (Taylor & Rauth, 1978).  106  REVERSED-PHASE CHROMATOGRAPHY  2 Q.  O  O o o  50  FRACTION  F i g . 27 HPLC Radiochromatograms of organic-soluble extracts  CHO cells were incubated under hypoxia with C-misonidazole and extracted as described in text. Samples were analysed by reversed-phase HPLC as described in Methods. Chromatograms show counts per minute per fraction (0.5 ml). Incubation times were: a: 2 minutes; b: 1 h r . ; c: 2 h r . ; d: 3 hr.  107  DUAL-LABEL  Fig.  CHO  cells  described reduced catalysed  28 HPLC dual-label radiochromatograms  were  incubated  hypoxia  with  C-misonidazole,  as  Extracted samples were combined with aliquots of 3 misonidazole ( H label) prepared by xanthine oxidase reduction  collected,  HPLC  (see (a)  counted  text).  Combined  samples  or polar bonded phase HPLC for  background and spillover. shown  under  in text.  reversed-phase were  CHROMATOGRAPHY  as dashed lines, a:  3  3  H  and  14  C  activity,  were (b).  run  on  Fractions  and corrected  H data are offset vertically for clarity,  for and  2 hour incubation (same experiment as f i g .  27). b: 3 hour incubation (different  experiment).  108  DISCUSSION: 7.1 Misonidazole metabolism - in vivo and in vitro: In  the  previous  chapters,  I  have  described  the  reduction  of  misonidazole by chemical and biochemical techniques, and the metabolism of  the  drug  in vitro.  Here,  I wish to discuss these  results  in  the  context of the clinical use of radiosensitizers, including such matters as the  pharmacokinetics  radiosensitizers  with  of misonidazole in man, the development of improved  properties,  and  the  new  possibility  of  exploiting cytotoxicity as a form of chemotherapy. The  use of in vitro  metabolism under  biological techniques allow us to study  controlled  conditions; but such model systems  drug ignore  many aspects of in vivo pharmacology: drug absorption, distribution in the body, excretion, and the effects of the drug and its metabolites on particular  body  tissues,  for  example.  Flockhart  et  al.  (1978a,b)  various  mammals,  in  has  14 studied  the  metabolism  including  man.  The  reviewed  recently  of  C-misonidazole  pharmacology  (Workman,  of  1980).  in  misonidazole Some  results  vivo  will  be  been  presented  here. Misonidazole is absorbed rapidly, following oral administration. The half-life  of the  drug in plasma is about 12 hours in man; in rats and  mice it is much shorter (1-2 hours),  a fact which makes it difficult to  extrapolate  from these  animals to man.  The drug is excreted free in  the  as a glucuronide conjugate,  and as desmethylmisonidazole.  urine,  Together, istered  these  metabolites  misonidazole  Presumably,  the  dose  remainder  account for (Flockhart of  the  drug  less than  half of the admin-  et  1978a,  is  al.,  converted  to  Table  I).  unidentified  109  products,  or  is  bound.  remainder  of  the  Flockhart  excreted  et  al.  metabolites  (1978a)  has  state  eluded  that  "The  characterization,  mainly due to their extreme water solubility, By  analogy  with  other  nitro  compounds, one  product is the amine derivative. catalytic shows  This derivative  hydrogenation (Flockhart  et a l . , 1978a).  no significant toxicity |n vitro,  conditions  (B.  Palcic,  unpublished  possible was  excretion  synthesized by  The amine compound  under either aerobic or hypoxic observations),  but  its  presence  serves as an indicator of reductive metabolism of the d r u g .  Flockhart  et  amine  al.  (1978b)  tumour  observed  extracts  from  evidence  drug-treated  Flockhart  et  al.  concluded  patients,  but  ...  the excretion  administered  dose."  for  Also,  that:  the mice,  "amine  levels  presence as  of  did  the  Varghese  formation  is  in  (1980).  occurring  in  do not exceed about 2% of the  some evidence for  reductive  metabolism of  the drug by gut flora was obtained. Our  in  vitro  studies  are  consistent  with  the  view  that  nitro-reduced metabolites of misonidazole are formed in cells, but are of limited  stability,  bound  to  and are further  macromolecules  metabolised to polar compounds, or  (chapter  6).  This  would  account  for  the  observation of highly water-soluble metabolites in the urines of patients receiving the d r u g ,  despite the relatively low levels of amine metabolite  in the urine. We  have  misonidazole presented  by  presented  metabolism Taylor  a (fig.  and  schematic 29).  Rauth  illustrating  This  (1980b).  scheme  the is  pathways similar  Misonidazole may  to  of one  be con-  110  verted  to  polar  demethylation,  for  importance. relatively  derivatives example.  Reductive  stable  nitroreduction the  detected.  analogous  benzene reduction be further  This  activation  hydroxylamine, An  without  reductive  pathway proceeds  in  of  by  pharmacokinetic  hypoxia.  The  first  product to be formed is probably  nitroso compound being mechanism has  (Wardman,  is  activation,  1977, p.  the  reduced too rapidly to be  been demonstrated 371).  The  for  nitro-  hydroxylamine may  reduced to the amine, which appears to be a urinary meta-  bolite in man. One may  or  reactive  be responsible for  toxic  and  what  stage  identitiy the  more  mutagenic in  has  the  reduction  proved  (chapter  elusive, Indeed,  4)  formed during the  binding to nucleic acid and  properties  intermediates.  reduction  intermediates  of the  drug.  pathway  this  It  protein,  is still  product  is  reduction and  the  not clear  at  formed.  Its  due in part to the difficulty of isolating the  is probably  formation  of  dimers  a consequence of the  during  zinc  reactivity  of  29 are  not  be followed  by  intermediates such as the nitroso compound. It mutually  should be noted that the exclusive;  reduction, products  or may  for  reduction be  example, by  pathways  demethylation  conjugation.  quantitatively  shown  organic-insoluble, acid-soluble fraction.  could  Such  predominant,  in f i g .  "twice-metabolised" particularly  in  the  111  R-NO,  conjugation ^  polar metabolites (intact NC^ group)  demethylation  reduction (O^ inhibited or  reversed)  R-N=0 (unstable?)  R-NHOH  Binding  to macromolecules?  polar reduced metabolites  conjugation R-NH,  Fig. 29 Metabolism of misonidazole: Schematic  112  7.2 Clinical significance of hypoxic cytotoxicity: The  relationship between the hypoxic cytotoxicity of misonidazole,  observed in vitro,  and the clinical effects of the d r u g , remains unclear  (Denekamp & McNally,  1978).  an  shoulder  initial  zero-slope  recovered 1978). cycle  Hypoxic cytotoxicity is characterised (chapter  3);  this  shoulder  if hypoxic cells are subsequently exposed to air  Brown has between  by  may  be  (Stratford,  suggested (1979) that acutely hypoxic cells,  oxygenated and hypoxic states (see section 1.5)  which will be  unaffected by misonidazole cytotoxicity, for these reasons. Animal  studies  have  not resolved this  radiosensitization and cytotoxicity which  the  animals given  drug  bearing the  without  is  tumours.  drug  following  drug)  is  radiosensitization. experiments. obtained  In  with  cytotoxicity  administered  due, Fowler  a  versus  before,  of  killing in the  significant  to  Denekamp SER  cell  irradiation  (compared to controls  presumably,  post-irradiation  plays  after,  irradiation  some cases,  The effects of  may be separated by experiments in  Enhancement  and  question.  values  drug role.  cytotoxicity (1979)  have  In  other  animals  irradiated  rather  than  reviewed  such  as high as 1.4  administration,  of  have been  suggesting  experiments,  that  no such  enhancement was seen. Korbelik et al. hypoxic decreases  cytotoxicity the  (1980,  1981) have demonstrated that misonidazole  interacts  with  damage.  Radiation  duration of the zero-slope shoulder of the  cytotoxicity  response, in a dose-dependent manner.  radiation  Cells which have been exposed  to doses greater than about 14 Gy show no shoulder at all when they  113  are subsequently exposed to misonidazole, in hypoxia. be  due  to  an  misonidazole  and  result  significant  is  a  interaction by  between  radiation.  the  DNA  Whatever the  damage  caused  by  mechanism may be,  the  enhancement of the toxicity  cells that have been irradiated.  This effect may  of misonidazole to  If such an interaction occurs in vivo,  it would increase the importance of cytotoxicity as a mechanism of cell death in tumours treated by radiation and misonidazole. Chapman  et  al.  (1981)  have  explored  misonidazole, labeled at high specific activity, cells  in  tumours.  This  approach  takes  the  possible  use  of  as a marker for hypoxic  advantage  of the  preferential  binding of misonidazole metabolites in hypoxic cells, discussed earlier. Experimental  tumours  was given an i.p. mouse  was  examined  were grown  in a mouse (subcutaneously)  dose of labeled misonidazole.  sacrificed,  which  Three hours later,  and the tumours were excised,  by microscopy and autoradiography.  In  sectioned,  the and  small tumours  (1.5  mm x 0.5 mm) a clearly defined rim of hypoxic cells was observed, as a region of heavy 10 cells  in  from  exposure in the autoradiograph. The rim began about the edge of the tumour,  linson-Gray model of chronic hypoxia. more complex pattern. be  very  Chapman  useful et  radionuclides,  suggest  could a  be  Administered  to  metabolically  reduced  Thom-  Larger tumours showed a much  It is likely that this phenomenon will prove to  in clarifying  al.  in accord with the  that  used  patient and  the  in  nature  of hypoxic cells  sensitizers, in  a clinical  small  bound  in  doses,  labeled assay the  with  for  y-emitting  hypoxic  sensitizer  hypoxic cells;  then be detected by y-scanning techniques.  in tumours.  these  cells.  would cells  be  could  114  7.3 New radiosensitizers: The  dose  misonidazole sensitizing  limitations  have  imposed  inhibited  property.  the  Less  by  full  the  neurotoxic  side  effects  of  exploitation of the d r u g ' s radio-  lipophilic compounds penetrate  the  blood/  brain barrier less effectively than does misonidazole, and yet they  are  taken up by tumour tissue almost as well (Brown & Workman, 1980).  It  is hoped that such compounds will be less neurotoxic than misonidazole, without sacrificing radiosensitizing ability. urinary  excretion  of polar drugs  In addition, the more rapid  lowers the total  body exposure  re-  quired ("area under the curve" of a plot of drug concentration versus time).  A  variety  of  low-lipophilicity  thesized (Brown & Workman, 1980).  Also,  there  is  1980; Astor et a l . ,  renewed  interest  misonidazole,  desmethylmisonidazole.  water-soluble  than  (White  &  radiosensitizers  in  This  the  has  been  1980; Adams et a l . , polar  derivative  metabolite is  much  misonidazole, and offers pharmacokinetic  Workman,  1980).  The  high  syn-  of  more  advantages  water-solubility  permits  subconjunctival administration of concentrated solutions into the eye, a route of administration which may be useful in retinoblastoma (Rootman et a l . , 1981). local  hemorrhaging  lowing studied itself.  Unfortunately, desmethylmisonidazole was found to cause  oral  (ibid.);  administration  drugs  may  not  similar results were observed in mice, f o l (Chao  et  necessarily  al., be  1980).  more  Thus,  these  newly  useful than misonidazole  115  7.4 Future studies: Finally,  I wish to suggest some areas of application of the results  and techniques that I have described in this thesis.  These studies may  clarify further the mechanism and significance of the toxicity of nitroaromatic radiosensitizers. The  effect  of  ascorbate  has  not  been  studied  in  vivo;  if  the  combination of ascorbate with misonidazole produces much more effective killing  of  hypoxic  cells,  as  indicated  by  the  m vitro  studies,  then  clinical trial of the combination might be warranted. The synthesis of reduced monomeric derivatives of misonidazole by reduction earlier.  of  the  nitro  compound has  proven  difficult,  as  described  It is possible that a better route to, for example, the nitroso  derivative,  would be via oxidation of the bimolecular compounds.  synthesis  of  this  compound would  chemistry  of  the  reduced  improve  derivatives  of  our  the  The  understanding of  drug,  and facilitate  the its  detection as a metabolite. The techniques developed for the study of misonidazole metabolism in vitro may be extended to studies with animals.  For example, binding  14 of  C-misonidazole  and  formation  of  reduced  metabolites  could  be  studied in different tissues of animals, following administration of the drug. ever, and  Previous  work has involved study of excreted metabolites; how-  it may well be the the  excretion.  reduced  bound products that result in toxic effects,  derivatives  may  be  further  metabolised  before  116  The techniques adapted and  developed for the  study  of misonidazole could b  to the study of related d r u g s , such as desmethylmisonidazole  comparisons . of  the  metabolism and chemistry  of such compound  may help in the choice of drugs for further development.  117  REFERENCES: Adams, G . E . , and Dewey, D.L. (1963) Hydrated electrons and radiobiological sensitization. Biochem. Biophys. Res. Comm. 1_2/ 473. Adams, G . E . , A s q u i t h , J . C . , Dewey, D . L . , Foster, J . L . , Michael, B . D . , and Willson, R.L. 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