C H E M I C A L A N D B I O L O G I C A L S T U D I E S OF T H E RAD IOSENS IT I ZER MISONIDA'ZOLE by P. Dav id Josephy B. S c . ( H o n . ) , U n i v e r s i t y of T o r o n t o , 1976 A THES I S S U B M I T T E D IN P A R T I A L F U L F I L L M E N T OF T H E REQU IREMENTS FOR T H E DEGREE OF D O C T O R OF PH I LOSOPHY in T H E F A C U L T Y OF G R A D U A T E STUD I ES T H E D E P A R T M E N T OF Z O O L O G Y We accept th i s thes is as conforming to the r equ i r ed s tandard T H E U N I V E R S I T Y OF BR IT I SH C O L U M B I A J une , 1981 © P. Dav id J o sephy , 1981 In presenting th is thes is in pa r t i a l fu l f i lment of the r e q u i r e m e n t s f o r an advanced degree at the Univers i ty of B r i t i s h Columbia, I agree t h a t the L ibrary sha l l make it f ree ly ava i lab le for r e f e r e n c e and study . I fur ther agree that permission for extensive copying o f t h i s t h e s i s for scho lar ly purposes may be granted by the Head o f my Department or by his representat ives . It i s understood that c o p y i n g o r p u b l i c a t i o n o f th is thes is fo r f inanc ia l gain sha l l not be allowed without my written permission. P. David Josephy Department of Z o o l o g y The Univers i ty of B r i t i s h Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 Date Feb. 26, 1981 i i A B S T R A C T M i s o n i d a z o l e (Ro 07-0582 ) i s a n i t r o h e t e r o c y c l i c d r u g w h i c h s e n -s i t i z e s h y p o x i c ( o x y g e n - d e f i c i e n t ) c e l l s t o t h e l e t h a l a c t i o n o f i o n i s i n g r a d i a t i o n . T u m o u r s c o n t a i n 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 w h i c h may l im i t t h e u s e f u l n e s s o f r a d i o t h e r a p y as a m o d a l i t y o f c a n c e r t r e a t m e n t . T h e u s e of m i s o n i d a z o l e as an a d j u n c t t o r a d i o t h e r a p y may i m p r o v e t h e loca l c o n t r o l o f 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 i n p r o g r e s s . M i s o n i d a z o l e i s s e l e c t i v e l y t o x i c t o h y p o x i c c e l l s , e v e n in t h e a b s e n c e of r a d i a t i o n . T h i s e f f e c t may be r e l a t e d to t h e c l i n i c a l t o x i c i t y o f t h e d r u g , w h i c h l i m i t s t h e d o s e of m i s o n i d a z o l e w h i c h may be d e l i v e r e d s a f e l y , a n d , t h u s , l i m i t s t h e e f f e c t i v e n e s s of t h e r a d i o s e n s i t i z e r . T h e 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 i s b e l i e v e d to be r e l a t e d t o i t s m e t a b o l i s m i n h y p o x i c c e l l s . R e d u c t i o n of n i t r o a r o m a t i c c o m p o u n d s , s u c h as n i t r o b e n z e n e , i s i n h i b i t e d b y o x y g e n ; t h u s , r e d u c t i v e a c t i v a t i o n of m i s o n i d a z o l e to a t o x i c s p e c i e s may e x p l a i n t h e s e l e c t i v e 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 . We h a v e s t u d i e d t h e r e d u c t i v e c h e m i s t r y of m i s o n i d a z o l e , a n d i t s m e t a b o l i s m , u s i n g a v a r i e t y of c h e m i c a l a n d i n v i t r o b i o l o g i c a l t e c h -n i q u e s . A s c o r b i c a c i d ( v i t a m i n C ) e n h a n c e s t h e t o x i c i t y o f m i s o n i d a z o l e t o h y p o x i c C h i n e s e h a m s t e r o v a r y ( C H O ) c e l l s . T h i s m a r k e d e n h a n c e m e n t a p p e a r s to be c a u s e d b y a c c e l e r a t e d d r u g m e t a b o l i s m i n t h e p r e s e n c e of a s c o r b a t e . C h e m i c a l r e d u c t i o n 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 a z o - m i s o n i d a z o l e a n d a z o x y - m i s o n i d a z o l e . T h e s e c o m p o u n d s w e r e s e p a r a t e d b y p r e p a r a t i v e r e v e r s e d - p h a s e l i q u i d c h r o m a t o g r a p h y , c h a r -i i i a c te r i zed chemica l l y , and tested fo r in v i t r o b io logica l a c t i v i t y . Azo-misonidazole is almost non- tox i c , but azoxy-misonidazo le is more t ox i c than misonidazole i t se l f . Misonidazole was reduced by the xan th ine/xan th ine ox idase ( X O ) s y s t em, unde r h y p o x i a . T h i s enzymat ic reduc t ion y ie lded a s ing le major p r o d u c t , wh ich appears to be hydroxy lamino-mison idazo le . The same enzyme system also reduces azo- and azoxy-mison idazo le . 14 The metabolic t rans format ion of C-misonidazole was s t u d i e d , u s ing dense suspens ions of CHO cel ls in h y p o x i a . Misonidazole is conve r ted into severa l polar p r o d u c t s , and b i n d i n g to ac id- inso lub le material ( p resumab ly macromolecules) was o b s e r v e d . The o rgan i c-so lub le metabolite f r a c t i on conta ins a compound wi th ident ica l ch romatograph ic p rope r t i e s to the x a n t h i n e / X O p r o d u c t , be l ieved to be hydroxy lamino-mison idazo le . The s ign i f i cance of these resu l t s is d i s cussed in the context of the c l in ica l potent ia l of misonidazole and re lated d r u g s as r a d i o s e n s i t i z e r s . The poss ib i l i t y of exp lo i t i ng h y p o x i c c y t o t o x i c i t y as a se lec t ive chemo-t h e r a p y fo r h y p o x i c tumour cel ls is c o n s i d e r e d . i v T A B L E OF C O N T E N T S A B S T R A C T ii T A B L E OF C O N T E N T S iv L IST OF F IGURES v i i A B B R E V I A T I O N S ix A B B R E V I A T I O N S : R A D I O S E N S I T I Z E R S : x A C K N O W L E D G M E N T S , . . x i I N T R O D U C T I O N : 1.1 Rad io the rapy and the oxygen ef fect 1 1.2 Radiat ion and cell s u r v i v a l 2 1.3 The o x y g e n ef fect and cell s u r v i v a l 6 1.4 The o x y g e n ef fect - mechanisms 9 1.5 The oxygen ef fect - c l in ica l s ign i f i cance 13 1.6 The rap i e s which may overcome the rad io res i s tance of h y p o x i c cel ls 16 1.7 Ni t ro imidazole r ad iosens i t i ze r s 20 1.8 T o x i c i t y of n i t roaromat ic compounds 23 1.9 Hypox i c c y t o tox i c i t y - mechanisms 26 M A T E R I A L S & M E T H O D S : 2.1 Cel l cu l t u r e p rocedures - CHO and C H 2 B 2 cell l ines 32 2.2 In v i t r o t o x i c i t y exper iments 33 2.3 Z inc reduc t ion of misonidazole 37 2.4 Xan th i ne ox idase - ca ta lysed reduc t ion of misonidazole . . . . 39 2.5 In v i t r o metabolism of misonidazole 41 V MISON IDAZOLE C Y T O T O X I C I T Y IN V I T R O : 3.1 In t roduct ion 46 3.2 Asco rba te enhancement of misonidazole c y to tox i c i t y 47 3.3 Ae rob i c t o x i c i t y of ascorbate 49 3.4 Recent developments 53 C H E M I C A L R E D U C T I O N OF M I S O N I D A Z O L E : 4.1 In t roduct ion 56 4.2 Ident i f i cat ion of z inc reduc t ion p roduc 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- and azoxy-misonidazole 70 4.5 Metabol ic format ion of b imolecular p roduc t s 78 B I O C H E M I C A L R E D U C T I O N OF M I SON IDAZOLE A N D ITS AZO- A N D A Z O X Y - D E R I V A T I V E S : 5.1 In t roduct ion 81 5.2 X a n t h i n e ox idase 82 5.3 Reduct ion of misonidazole by xan th ine ox idase 85 5.4 Reduct ion of de r i v a t i v e s by xan th ine ox idase 88 5.5 Recent developments 94 v i IN V I T R O M E T A B O L I S M OF M I S O N I D A Z O L E : 6.1 In t roduc t ion 97 6.2 In v i t r o metabolism of misonidazole 99 6.3 Conc lus ions 104 D I S C U S S I O N : 7.1 Misonidazole metabolism - in v i vo and in v i t r o 108 7.2 C l in i ca l s ign i f i cance of h y p o x i c c y t o tox i c i t y 112 7.3 New rad iosens i t i ze r s 114 7.4 Fu tu re s tud ies 115 R E F E R E N C E S 117 v i i L IST OF F IGURES I N T R O D U C T I O N : 1. Radiat ion s u r v i v a l c u r v e s in v i t r o 5 2. Radiat ion s u r v i v a l c u r v e s fo r mixed populat ions 8 3. Rad iosens i t i v i t y as a func t i on of oxygen concent ra t ion . . . . 12 4. In v i vo rad ia t ion s u r v i v a l c u r v e s : Demonstrat ion of presence of r es i s t an t f r ac t i on 15 5. Se lec t ive t o x i c i t y of misonidazole to h y p o x i c cel ls 25 M A T E R I A L S & M E T H O D S : 6. In v i t r o metabolism expe r imen t s : schematic 43 M I SON IDAZOLE C Y T O T O X I C I T Y IN V I T R O : 7. Ascorbate-enhancement of misonidazole c y t o tox i c i t y 48 8. Ef fect of ascorbate concent ra t ion on enhancement of misonidazole c y t o tox i c i t y 50 9. T o x i c i t y of ascorbate to aerobic cel ls in v i t r o 52 C H E M I C A L R E D U C T I O N OF M I S O N I D A Z O L E : 10. UV-v i s i b l e spec t ra of mison idazo le , azo-misonidazole , and azoxy-misonidazo le 64 11. NMR spec t rum of misonidazole 65 12. NMR spec t rum of azo-misonidazole 66 13. NMR spec t rum of azoxy-misonidazo le 67 v i i i T o x i c i t y of b imolecu lar d e r i v a t i v e s : 14. I. C H O cel ls - aerobic incuba t ion 72 15. II. C H O cel ls - h y p o x i c incuba t ion 73 16. III. CH2B£ ce l ls - ae rob ic i ncuba t ion 74 17. IV. C H 2 B 2 ce l ls - h y p o x i c incuba t ion 75 18. V . DNA s ing le - s t r a n d b reak p roduc t i on 76 19. R a d i o s e n s i t i z i n g p rope r t i e s of d e r i v a t i v e s 80 B I O C H E M I C A L 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 ITS A Z O - A N D A Z O X Y - D E R I V A T I V E S : 20 . Reduc t ion of misonidazole by h y p o x a n t h i n e and x a n t h i n e ox idase in h y p o x i a 86 21 . H P L C ana l y s i s of p r o d u c t s of misonidazole r educ t i on 89 22. Reduc t ion of azo-misonidazole by h y p o x a n t h i n e and x a n t h i n e ox idase in h y p o x i a 90 23 . Reduc t ion of azoxy-mison idazo le by h y p o x a n t h i n e and x a n t h i n e ox idase in h y p o x i a : I. K i ne t i c s 92 24. II. Rep resen ta t i v e chromatograms 93 IN V I T R O M E T A B O L I S M OF M I S O N I D A Z O L E : 25 . D i s t r i b u t i o n of r ad i oac t i v i t y - C H O cel l e x t r a c t s 101 26. T L C a u t o r a d i o g r a p h y of o rgan i c- so lub l e e x t r a c t s 102 27. H P L C rad iochromatograms of o rgan i c-so lub l e ex t r a c t s 106 28. H P L C dua l- labe l rad iochromatograms 107 D I S C U S S I O N : 29. Metabol ism of mison idazo le : Schemat ic 111 i x A B B R E V I A T I O N S AF-2 2- (2- fury l ) -3- (5-n i t ro-2-fury l ) ac ry l amide CHO Ch inese hamster o v a r y (cel l l ine) CI Chemical ionisat ion (mass spec t roscopy ) C i C u r i e ( rad ioac t i ve decay rate) E D T A Ethy lene diamine te t raacet i c ac id El E lect ron ionisat ion (mass spec t roscopy ) ESR E lec t ron sp in resonance ( spec t ro s copy ) FAD F lav in adenine d inuc leot ide FCS Foetal calf serum FMN F lav in mononucleot ide GSH Gluta th ione ( r educed form) GSSG Gluta th ione ( ox id i zed form) G y G r a y ( un i t of rad ia t ion dose) H P L C H igh p r e s s u r e l i qu id ch romatography i . p . in t ra-per i tonea l IR In f ra-red ( spec t roscopy ) LET l inear ene rgy t r a n s f e r NAD Nicot inamide adenine d inuc leot ide NMR Nuc lear magnetic resonance 4-NQO 4-Ni t roqu ino l ine-N-ox ide ODS Octadecy l su l fonate OER O x y g e n enhancement rat io P A C Polar amino-cyano P N A P Para-Ni t roacetophenone PBS Phospha te-buf fe red sa l ine PE P la t ing e f f i c i ency SER Sens i t i ze r enhancement rat io SOD Supe rox ide Dismutase SSB S i ng l e-s t r and break T L C T h i n - l a y e r ch romatography T N T 2 ,4 ,6- t r i n i t ro to luene X O Xan th i ne ox idase X ABBREVIATIONS: RADIOSENSITIZERS: Ro 07-0582 Misonidazole: 3-Methoxy-1-(2-nitro-1-imidazolyI)-2-propanol (I) Ro 05-9963 Desmethylmisdnidazole: 1-(2,3-dihydroxypropyl)-2-n?troimidazole (H) Flagyl Metronidazole: 1-2'-hydroxyethyl-2-methyl-5-nitroimidazole (m) •N N N 0 2 c H 2 C H ( O H ) C H 2 O C H ; N N N O p C H 2 C H ( O H ) C H 2 O H N O ? N ^ ^ I S T \ : H C H 2 C H 2 O H H m x i A C K N O W L E D G M E N T S The research repor ted in th i s thes is was per formed in the l a b -ora tor ies of the Medical B i ophys i c s U n i t , B. C . Cance r Research C e n t e r . I would l ike to t hank the sc i en t i f i c s ta f f of the Cance r Research Cen te r f o r t he i r ass is tance and encouragement . In p a r t i c u l a r , I apprec ia te the in te res t and s u p p o r t of D r . Gedy G u d a u s k a s , D r . Mladen K o r b e l i k , D r . G e r r y K r y s t a l , D r . K i r s t en S k o v , and D r . Hans S t i c h . The techn ica l ass is tance of Isabel Ha r r i son and Hans Adomat is acknowledged wi th g r a t i t u d e , as is the secre ta r ia l sk i l l and good humour of Bev E rsoy (who d id no t , however , have to t ype the t h e s i s ! ) . T h a n k y o u , G e d y , fo r pu t t i ng the IBM system 6 word p rocessor at my f i n g e r t i p s . D r . A . M . R a u t h , D r . D.W. Whi l l ans , and D r . G . F . Whitmore, Ontar io Cance r Ins t i tu te , T o r o n t o , gave generous access to t he i r f i n d i n g s before p u b l i c a t i o n ; t h e i r hosp i t a l i t y and exchange of ideas d u r i n g severa l v i s i t s were app re c i a t ed . Most of a l l , I ex tend my t h a n k s to D r . B r anko Pa l c i c , s c i en t i s t and speed-chess champion of the Medical B i ophys i c s U n i t , and my research s u p e r v i s o r , D r . L loyd S k a r s g a r d , Head , Medical B i ophys i c s U n i t . T h e y have pa r t i c ipa ted in e ve r y pro jec t , p a p e r , f i g u r e , and semi-colon ove r the past f ou r y e a r s . F i na l l y , I w ish to t hank the B. C . Cance r Foundat ion fo r accom-modation and t r ave l s u p p o r t , and the National Cance r Inst i tute of Canada fo r f inanc ia l s u p p o r t (Research S t u d e n t s h i p ) . T o k u s a n b r o u g h t his notes on the "Diamond S u t r a " to the f r o n t of the h a l l , po inted to them with a t o r c h , and s a i d : " E ven though you have exhaus ted the abs t ruse d o c t r i n e s , it is l ike pu t t i ng a ha i r in a vas t space . Even though you have learned all the secre ts of the w o r l d , it is l ike a d r o p of water d r i p p e d on the grea t o c e a n . " A n d he b u r n e d his notes . T h e n , making bows , he took his leave of h is t eache r . Mumonkan 1 I N T R O D U C T I O N : 1.1 Rad io the rapy and the oxygen e f fec t : Roentgen d i s cove red x- rays in December 1895, and wi th in weeks , the new form of rad iat ion was be ing used as a tool f o r medical d i agnos i s . The d i s c o v e r y that x- rays could damage the t i s sues t h r o u g h which they passed so f r ee l y was made by the ear l ies t i n v e s t i g a t o r s . O f t e n , they were the i r own exper imenta l sub jec t s . Roentgen himself su f f e r ed ery thema and sk in damage to his h a n d s , and sought medical a t tent ion in J a n u a r y , 1896. Later in the same month , G r u b b e per formed the f i r s t the rapeu t i c i r r a d i a t i o n , t r ea t i ng a pat ient fo r carc inoma of the breas t (Mo rgan , in Besancon , e d . , 1974, p. 404) . Rad ium, too , was used in cancer t h e r a p y almost as soon as it was isolated by Mme. C u r i e . The new sc ience of rad iat ion b io logy and the medical d i s c i p l i ne of rad iat ion onco logy deve loped from the work of such p ionee rs . I n i t i a l l y , rad iat ion b io log is ts were conf ined to the s t u d y of bac t e r i a , p lant seeds , and so o n , or ent i re animals . Techn iques fo r the s t u d y of mammalian ce l ls in v i t r o were not deve loped unt i l the 1950's (see fo l lowing sec t i on ) . Neve r the l e s s , many of the phenomena which conce rn rad ia t ion b io log is ts today were f i r s t exp lo red wi th " l ower " o rgan i sms : fo r example , the shape and desc r i p t i on of s u r v i v a l c u r v e s , in f luence of rad ia t ion q u a l i t y , and ident i f i ca t ion of ce l lu la r " t a r g e t s " . The r ad iosens i t i z i ng act ion of molecular oxygen was obse r ved in the 1920's, but i ts s ign i f i cance was not apprec ia ted ( P e t r y , 1923). The res is tance of poor l y oxygena ted cel ls to rad iat ion damage was t h o u g h t to 2 be a consequence of the metabolic state of such c e l l s , f o r example the i nh ib i t i on of mi tos i s . The rea l isat ion that th i s " o x y g e n e f fec t " was a genera l phenomenon, caused by the in terac t ion of molecular oxygen wi th the chemical species formed at the time of i r r a d i a t i o n , came much l a te r . Ra ther than cont inue in an h i s to r i ca l deve lopment , I shal l now out l ine the elements of the " h y p o x i c cel l p rob lem" from a modern p e r s p e c t i v e . T h u s , I shal l d i s cuss the response of mammalian cel ls and t i s sues to r ad i a t i on , the ef fect of oxygen on th i s r e sponse , and the poss ib le role of h y p o x i c ce l ls in f a i l u res of local tumour contro l by r ad io the r apy . To evaluate the c l in ica l s ign i f i cance of the h y p o x i c cell p rob l em, it wi l l be necessa ry to examine the b io logy of tumour g r o w t h , and to cons ide r ev idence from h is to logy and rad iat ion t h e r a p y . F i na l l y , I shal l desc r i be attempts to overcome the p rob lem, in p a r t i c u l a r , the development of h y p o x i c cell s e n s i t i z e r s . He re , I shal l r e t u r n to a chrono log ica l n a r r a t i v e ; th i s wi l l p rov ide the immediate b a c k g r o u n d to my own r e s e a r c h . 1.2 Radiat ion and cel l s u r v i v a l : T i s s u e s e n s i t i v i t y , the endpo in t of c l in ica l impor tance , is determined by cel l s e n s i t i v i t y . The rad ia t ion s e n s i t i v i t y of an o rgan or t i s sue depends on two major f a c t o r s : the inhe ren t r ad io sens i t i v i t y of the cel ls of which i t is composed (as measured by the i r r ep roduc t i v e capac i t y fo l lowing exposu re ) and the importance of cell p ro l i f e ra t ion in mainta in ing t i s sue func t i on ( H a l l , 1978, chapte r 10 ) . The second fac to r accounts fo r most of the va r i a t ion 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-dividing cells such as neurons are much more resistant to radiation. In consequence, the principal morbidity syndromes following whole-body radiation exposure are hematological and gastrointestinal. A malignant tumour is characterised by the uncontrolled (although not necessarily rapid) growth of its cells. Thus, 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 surviving 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 studied in a phenomenological manner at a time when little was known about the mechanisms by which ionizing radiation interacts with biological molecules. It will be useful to introduce such a phenomenological model here. I shall assume cell survival to mean proliferative capacity, since this property (colony forming ability) is the endpoint measured most commonly. Also, it may be the endpoint of most direct relevance to radiotherapy. Radiation dose, the independent variable in radiobiological models, is defined as energy deposited per unit mass. In SI units, dose is measured in Grays: 1 Gy = 1 Joule/kg. (The CGS unit, 1 rad = 100 erg/gm = 0.01 Gy, is still widely used). It is important to note that the Gray, a unit of absorbed dose, is not directly convertible to 4 s u c h u n i t s a s t h e R o e n t g e n ( a m e a s u r e o f i o n i s a t i o n ) o r t h e C u r i e ( a m e a s u r e o f r a d i o a c t i v e d e c a y r a t e ) . D o s e i s a m a c r o s c o p i c t e r m w h i c h i g n o r e s t h e d i f f e r e n t m i c r o s c o p i c e f f e c t s o f d i f f e r e n t t y p e s o f r a d i a t i o n . T h u s , i t i s n o t s u r p r i s i n g t h a t t h e b i o l o g i c a l e f f e c t s o f 1 G y o f n e u t r o n s d i f f e r f r o m t h o s e o f 1 G y o f x - r a y s . T h e m e a s u r e m e n t o f c o l o n y f o r m i n g a b i l i t y o f f r e e - l i v i n g c e l l s i s u s u a l l y s t r a i g h t f o r w a r d . A b a c t e r i u m c a n g r o w i n t o a v i s i b l e c o l o n y o n a n a g a r p l a t e i n a b o u t t w o d a y s . A n a l o g o u s e x p e r i m e n t s u s i n g m a m m a l i a n c e l l s w e r e n o t p e r f o r m e d u n t i l t h e m i d 1 9 5 0 ' s , f o l l o w i n g t h e d e v e l o p m e n t o f t e c h n i q u e s f o r t h e g r o w t h o f s u c h c e l l s i n c u l t u r e ( P u c k a n d M a r c u s , 1 9 5 6 ) . B a c t e r i a l s u r v i v a l c u r v e s a r e t y p i c a l l y e x p o n e n t i a l f u n c t i o n s o f d o s e , S = e" ( D / V w h e r e D Q i s a c o n s t a n t . S u c h a n e q u a t i o n y i e l d s a s t r a i g h t l i n e i f l o g S i s p l o t t e d a s a f u n c t i o n o f D : t h i s t y p e o f p l o t i s a l m o s t a l w a y s u s e d f o r s u r v i v a l c u r v e s . A r e s p o n s e o f t h i s t y p e w o u l d b e a n t i c i p a t e d o n t h e a s s u m p t i o n t h a t a s i n g l e " h i t " , s u c h a s t h e i o n i s a t i o n o f a t a r g e t m o l e c u l e , k i l l s t h e c e l l . A c t u a l l y , s u r v i v a l c u r v e s f o r m a m m a l i a n c e l l s o f t e n d i s p l a y a " s h o u l d e r " , a s i l l u s t r a t e d i n f i g . 1 a . M a n y m o d e l s h a v e b e e n p r o p o s e d t o e x p l a i n t h i s b e h a v i o u r ( e . g . A l p e r , 1 9 7 9 , c h a p . 5 ) ; t h e p r e s e n c e o f m u l t i p l e t a r g e t s p e r c e l l , o r a s i n g l e t a r g e t r e q u i r i n g m u l t i p l e h i t s , o r a l i m i t e d c e l l u l a r r e p a i r c a p a c i t y c o u l d a l l a c c o u n t f o r s h o u l d e r e d s u r v i v a l c u r v e s . In t h i s t h e s i s , I s h a l l u s e t h e s o - c a l l e d " m u l t i t a r g e t t h e o r y " e q u a t i o n : 5 Fig .1 Radiation survival curves in v i t ro a) A typical surv iva l curve for mammalian cel ls , modeled by the multi-target equation; the dashed line indicates the measurement of the extrapolation number, n. b) The effect of hypoxia on survival curves , i l lustrat ing the definition of the oxygen enhancement rat io, OER. 6 S = 1 - ( 1- e ' a D ) n H e r e , a i s a c o n s t a n t w i t h d i m e n s i o n s 1 / D o s e , a n d n i s a d i m e n s i o n l e s s c o n s t a n t k n o w n as t h e t a r g e t n u m b e r . F o r l a r g e D , a T a y l o r ' s e x p a n s i o n o f t h e p o w e r y i e l d s : S = n e T h i s e q u a t i o n i s a s t r a i g h t l i n e w h i c h e x t r a p o l a t e s b a c k t o t h e p o i n t S = n , D = 0 ( s e e f i g . l a ) . T h u s , n i s a l s o c a l l e d t h e e x t r a p o l a t i o n n u m b e r . T h e v a l u e o f D Q ( t h e d o s e r e q u i r e d t o r e d u c e s u r v i v a l b y 1/e, D Q = 1/a) i s , f o r mammal i an c e l l s , o f o r d e r 100 - 200 r a d s ( H a l l , 1 9 7 8 , p . 3 7 ) . 1.3 T h e o x y g e n e f f e c t a n d c e l l s u r v i v a l : T h e e f f e c t o f o x y g e n o n 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 i s i l l u s t r a t e d i n f i g . 1 b . In t h i s f i g u r e , t h e r e s p o n s e o f C h i n e s e h a m s t e r o v a r y ( C H O ) c e l l s t o x - r a y s i s p l o t t e d , f i r s t f o r i r r a d i a t i o n i n a i r - s a t u r a t e d m e d i u m , a n d s e c o n d f o r m e d i u m f r o m w h i c h a l l o x y g e n has b e e n r e m o v e d b y p u r g i n g w i t h p u r i f i e d n i t r o g e n . T h e a b s e n c e o f o x y g e n h a s a d r a m a t i c r a d i o p r o t e c t i v e e f f e c t ; t h e o x i c a n d h y p o x i c s u r v i v a l c u r v e s a r e o f s i m i l a r s h a p e , b u t t h e d o s e r e q u i r e d t o p r o d u c e a g i v e n l e v e l o f c e l l k i l l i s v e r y d i f f e r e n t i n t h e t w o c a s e s . T h i s d i f f e r e n c e is m e a s u r e d b y t h e o x y g e n e n h a n c e m e n t r a t i o ( O E R ) , d e f i n e d a s : D o s e i n n i t r o g e n O E R = TT : : 2 — D o s e in a i r e q u a l S 7 If oxygen is ' dose-mod i f y ing 1 , tha t i s , if the aerobic and h y p o x i c s u r v i v a l c u r v e s can be super imposed by a change of the dose sca le , then OER wil l be independent of the va lue of S chosen fo r compar i son ; in p r a c t i c e , S = 0.1 is commonly u s e d . For spa r se l y ion i s ing r ad i a t i ons , such as x - r a y s , the OER fo r mammalian cel ls is about 3. The r ad io the rap i s t is concerned wi th a d i f f e r en t compar i son : he wishes to determine the s u r v i v a l levels of each cel l t ype in a heterogeneous popula t ion exposed to a g i ven dose . T h i s is the s i tuat ion that obta ins when a tumour is i r r a d i a t e d , s ince the s u r r o u n d i n g normal t i s sues are also e x p o s e d . He re , the re levant parameter is the rat io between anox ic and aerobic cel l s u r v i v a l at a g i ven dose : a ve r t i ca l r a ther than a hor izonta l cu t t h r o u g h the s u r v i v a l cu r ve s of f i g . 1b . The steepness of the s u r v i v a l c u r v e s implies tha t th i s rat io may be 1,000 or more. T h u s , an admix tu re of a small f r a c t i on ( s a y , 1%) of h y p o x i c cel ls in an o therwise we l l-oxygenated populat ion d r a s t i c a l l y a l ters the overa l l s u r v i v a l c u r v e . T h i s is i l l u s t r a t ed in f i g . 2 (a f ter van Put ten and Ka l lman, 1968 ) .The fami ly of c u r v e s shown in f i g . 2 is s imply a mathematical c o n s t r u c t i o n . However , r emarkab ly s imi lar c u r v e s have been obta ined in s tud ies of tumours m v i v o . To unde r s t and these r e s u l t s , and to evaluate the importance of the oxygen ef fect in r a d i o -t h e r a p y , it is necessa ry to rev iew the ev idence fo r the presence of r ad io res i s t an t h y p o x i c cel ls in t umour s . I shal l r e t u r n to th i s quest ion fo l lowing a b r i e f d i s cuss ion of the rad iobio logica l mechanisms of the oxygen e f fec t . 8 I i o 1 0 2 0 3 0 DOSE (Gy) F i g . 2 R a d i a t i o n s u r v i v a l c u r v e s f o r m i x e d p o p u l a t i o n s T h e d a s h e d c u r v e s s h o w t h e r e s p o n s e o f f u l l y a n o x i c , a n d f u l l y a e r o b i c p o p u l a t i o n s . T h e h e a v y c u r v e s a r e t h e o r e t i c a l c o n s t r u c t i o n s o f t h e r e s u l t i n g o v e r a l l s u r v i v a l c u r v e s f o r m i x e d p o p u l a t i o n s c o n t a i n i n g h y p o x i c f r a c t i o n s o f 1 % a n d 1 0 % . 9 1.4 The oxygen ef fect - mechanisms: The absorp t ion of h igh ene rgy rad iat ion in matter causes ion-isat ions and exc i ta t ions of atoms and molecules. Regard less of the t y p e of i nc iden t rad ia t ion or pa r t i c l e , e lec t rons are p r i n c i p a l l y respons ib le f o r these e f f e c t s . E lect ron impact creates f ree rad ica ls ( g en e r a l l y , molecules wi th an odd number of e l ec t rons ) by s t r i p p i n g e lec t rons from molecules . T h i s " p h y s i c a l s tage " of ene rgy depos i t ion is fo l lowed b y a "chemical s tage " l as t ing a few mic roseconds . In th i s s tage , the f ree rad ica ls c reated by the rad iat ion in te rac t wi th undamaged molecules and wi th one ano the r . These react ions can cause "pe rmanent " (or at least , l ong- l i ved ) chemical a l t e ra t i ons , o r t he y can terminate in harmless recombinat ion p rocesses . The biological e f fect of rad iat ion wil l be modif ied by agents that modify th i s stage of chemical i n t e rac t i ons . Even if long- l i ved chemical changes o c c u r , these may s t i l l be subject to r epa i r by chemical o r enzymat ic p rocesses . P r esumab l y , the lethal and mutagenic e f fects of rad iat ion are due to damage in macromolecules (espec ia l l y D N A ) tha t is not r e p a i r e d , or is m i s r epa i r ed . P r imary rad ia t ion events may be d i v i ded into two c l a sses : d i r e c t act ion and i n d i r e c t act ion ( e . g . Bacq & A l e x a n d e r , 1961, c h a p . 2 ) . D i rec t act ion is the ionisat ion of a macromolecule by a p r ima ry rad iat ion even t ; i nd i r e c t act ion is caused by the react ion of chemical spec i es , formed by the absorp t ion of rad iat ion ene rgy in small molecules ( such as r ^ O ) . These spec ies may then a t tack macromolecules. Water is the most common molecule in a l i v i ng c e l l , and the act ion of rad ia t ion on water has been s tud ied in de ta i l . T h i s work also dates 10 back to the ear l i es t s tud ies of r a d i o a c t i v i t y : C u r i e and Debierne obse r ved the evo lut ion of H 2 and 0 2 f rom aqueous so lut ions of Ra sal ts (1901) . The chemis t r y of water r ad io l y s i s was s tud ied by Weiss (1944) who proposed that the in i t ia l p rocess was the p roduc t ion of H ' and O H ' r ad i ca l s . These species then p roduce even-e lec t ron-number molecules : H ' + H" O H - + O H ' O H ' + H ' In the presence of oxygen ( 0 2 ) o ther react ions can o c c u r , a l t e r ing the y i e lds of the p r ima ry r ad io l y s i s p r o d u c t s , and c rea t ing rad ica ls such as s u p e r o x i d e : H ' + 0 2 • H 0 2 • H + + 0 2 " The p roduc t s of r ad io l y s i s of water can damage macromolecules, e . g . : O H ' + RH • H 2 0 + R' y i e l d i ng p roduc t s s imi lar to those p roduced by d i r e c t damage: R* + H + + e~ O x y g e n may act as a r ad iosens i t i ze r in a number of ways : the re la t i ve importance of v a r i ous theore t i ca l l y p laus ib le mechanisms remains con-H, H2°2 -* H 2 0 RH RH + 11 t r o v e r s i a l . ( Fo r a r ev i ew , see Ewing and Powers , in Meyn & W i thers , 1979). G r a y proposed that oxygen conve r t s H ' to s u p e r o x i d e : 0 2 + e" » 0 2 " ( G r a y , 1954). A l p e r sugges ted that oxygen in te rac ts wi th d i r e c t damage s i tes to form o rgan i c pe roxy r ad i ca l s : FT + 0 2 : » ROO* 0 ( A l p e r , 1956) . Su ch rad ica ls d i s soc i a t e , y i e l d i n g permanent ly damaged macromolecules. A l p e r and Howard-F landers s tud ied the r ad io sens i t i v i t y of bacter ia as a f unc t i on of the oxygen par t ia l p r e s s u r e in the gas bubb led t h r o u g h the suspens ions ( A l p e r & Howard-F l ande r s , 1956). T h e y p r o -posed an empir ica l re l a t ionsh ip to desc r ibe the dependence : S p m P + K S N P + K where S N and S p are the r ad iosens i t i v i t i e s (a 's ) at C>2 par t ia l p r e s s u r e s of zero and P, m is a d imensionless constant equal to the maximum va lue of O E R , and K is a constant (d imension p r e s s u r e ) . S e n s i t i v i t y r i ses s teep ly at low oxygen t e n s i o n , and levels of f above P=K ( f i g . 3 ) . 12 a i r > 3 . 0 LU > LU CC CO z LU CO O 2 . 0 hr / y tt 1-01-I 1 1 L .— I I i ' » 0 2 0 4 0 6 0 OXYGEN CONC. (mm Hg) Fig. 3 Radiosensitivity as a function of oxygen concentration The dashed curve illustrates the form of the Alper equation (text, page 11), describing the radiosensitivity of cells at oxygen partial pressures ranging form complete anoxia to saturated oxygen. 13 K is typically about 3 mm Hg, well below the oxygen tension of venous blood (Hall, 1978, p. 85-87). This suggests that normal tissues are almost fully oxygenated, in terms of their radiation response. However, it should be noted that the m vitro experiments are carried out in dilute suspension; iri vivo, it is likely that cellular oxygen consumption will reduce the effective oxygen tension in the cell. 1.5 The oxygen effect - clinical significance: The presence of a small fraction of radiobiological^ hypoxic cells (P S K, as defined above) greatly alters the overall radiation response of a mixed population of cells. Thomlinson and Gray (1955) suggested, on the basis of histological evidence, that hypoxic cells may exist in solid tumours. The tumour studied in this work was human bronchial carcinoma. Although it may 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 did not penetrate the tumour. Thomlinson and Gray studied the histology of tumour cords of various diameters. They found that cords of radius greater than about 0.2 mm were characterized by a core region of tissue in which the cellular matrix had disintegrated, leaving a so-called "necrotic centre". Such cells were easily distinguishable from the surrounding "sheath" of viable tumour cells. The authors suggested that the sheath thickness, which was fairly constant, represented the limit of oxygen diffusion inward from the surrounding stroma, and that the central necrosis was a con-14 sequence of oxygen dep le t i on . A t the bounda r y between the necrot i c and v iab le r e g i o n s , the presence of h ypox i c cel ls capable of cont inued g rowth ( p a r t i c u l a r l y if the s u r r o u n d i n g aerobic cel ls were to be k i l l ed by rad ia t ion ) was s u s p e c t e d . T h i s " c h r o n i c h y p o x i a " model rece ived s u p p o r t f rom s tud ies of the rad ia t ion response of exper imenta l rodent t umour s . Powers and Tolmach (1963) measured the rad ia t ion response of a mouse lymphosarcoma, us ing an in v i vo d i lu t ion assay t echn ique ( f i g . 4 ) . Many s imi lar s tud ies have been repor ted s ince 1963; a s u m -mary of these is g i ven in Hall (1978, table 12-1) . The h y p o x i c cell f r a c t i on may be est imated by ex t rapo la t ion of the res i s tan t " t a i l " back to the y ax is ( f i g . 4 ) , a l l ow ing , if necessa r y , f o r the ef fect of shou lde rs on the s u r v i v a l c u r v e s . Such estimates range from 1% to more than 20%, in d i f f e r en t t umour s . The Thoml inson-Gray model has had t remendous in f luence on modern rad iob io log ica l r e s e a r c h . Nonethe less , it must be admitted tha t i ts exper imenta l basis is somewhat i n d i r e c t . A t tempts to measure tumour oxygena t ion d i r e c t l y , by p o l a r o g r a p h y , have p roduced ambiguous resu l t s (Ca te r & S i l v e r , 1960; Evans & N a y l o r , 1963). The ch ron i c h ypox i a model has come to be rega rded as dogma; a n d , as wi th most dogmas, it has a t t rac ted its share of he r e t i c s . B rown (1979) has sugges ted that there may be a d i f f e r en t t ype of tumour h y p o x i a , which he re fe rs to as "acute h y p o x i a " . A c u t e l y h y p o x i c ce l ls may be located close to c ap i l l a r i e s ; t empora ry occ lus ion of blood f low in the c ap i l l a r y induces h y p o x i a . Such cel ls would re-oxygena te if the cap i l l a r y r e-opened ; i t is sugges ted that th i s open ing and c los ing may occu r on a t ime-scale of minutes . The ev idence fo r 15 I000 I500 2 0 0 0 2 5 0 0 Dose (rod) Fig. 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 acute h ypox i a is summarized in B rown ' s pape r . It inc ludes h is to logica l s tud ies (Yamaura & Matsuzawa, 1979) and resu l t s of exper iments with r ad iosens i t i ze r s (see be low) . T h u s , a l though the re is genera l agreement tha t h y p o x i c ce l ls are a major cause of tumour r ad io res i s t ance , the na ture of h y p o x i a , p a r -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 The rap i e s wh ich may overcome the rad iores i s tance of h y p o x i c c e l l s : Va r ious approaches to the modif icat ion of r ad io the rapy have been proposed which may, it is h o p e d , reduce the rad io res i s tance of h y p o x i c ce l l s . These inc lude the use of new forms of r ad i a t i on , d i f f e r en t f rac t iona t ion s chedu l e s , and chemical r ad iosens i t i za t i on . The bio logica l e f fec t i veness of a pa r t i cu l a r form of r ad i a t i on , such as X - r a y s or n e u t r o n s , is re lated to i ts ionizat ion d e n s i t y . T h i s may be exp res sed in terms of the L E T , or l inear ene rgy t r a n s f e r , of the r ad i a t i on . T h i s quan t i t y desc r ibes the rate at which the par t i c le depos i ts ene rgy as it t r a ve l s t h r o u g h matter . The va lue of OER has been found to decrease wi th i nc reas ing LET ( Ba rendsen et a l . , 1966). For neut ron i r r a d i a t i o n , OER is less than 2, and f o r a-part ic les it can be as low as 1.0. It seems c lear that the use of such rad iat ion modalit ies in cancer t h e r a p y shou ld reduce the ef fect of tumour h y p o x i a . T h i s is p a r t i c u l a r l y l i ke l y i f , in add i t ion to lower O E R , the depth-dose cha rac t e r i s t i c s of the rad iat ion permit bet ter local isat ion of dose in the tumour . T h i s combinat ion of fea tures makes the use of n ( sho r t- l i v ed par t i c les wh ich in te rac t s t r o n g l y wi th atomic nuc le i ) in 17 r ad io the rapy appear v e r y p romis ing ( S k a r s g a r d et a l . , 1980). How-e v e r , the development of these new radiat ion modalit ies is j us t b e g i n -n i n g , and many techn ica l d i f f i cu l t i e s must be overcome before they can be rou t i ne l y a p p l i e d . In p a r t i c u l a r , cost is ce r ta in to be a fac tor o b s t r u c t i n g the i r use . The most d i r e c t approach to overcoming the h y p o x i c cell problem is h y p e r b a r i c oxygen t h e r a p y . With th i s t e c h n i q u e , pat ients are i r -rad ia ted in a p r e s s u r i z e d o x y g e n - r i c h env i ronment . The rat ionale fo r th i s approach is s t r a i g h t f o r w a r d : an increase in blood oxygena t ion may reduce the s ize of the h y p o x i c f r a c t i o n . Indeed, one of the most d i r e c t pieces of ev idence fo r the importance of the oxygen ef fect on r a d i o -t h e r a p y is an ana lys i s by B u s h et a l . (1978) of the in f luence of blood haemoglobin levels on cu re rates of pat ients t rea ted f o r ce rv i ca l ca rc inoma. Those pat ients wi th haemoglobin levels g rea te r than 12 g per 100 ml had bet ter cu re rates than those wi th lower l eve l s . Nonethe less , resu l t s wi th h y p e r b a r i c o x y g e n have not been v e r y e n c o u r a g i n g . The reasons f o r th i s are p robab l y two- fo ld . F i r s t , the percentage increase of blood o x y g e n - c a r r y i n g capac i t y is f a r smaller than that of the oxygen par t ia l p r e s s u r e . S e cond , o x y g e n is unable to penetra te into the h y p o x i c reg ion of the tumour because i t is r a p i d l y depleted by metabolism in the p e r i p h e r y . In add i t ion to these p rob lems , h y p e r b a r i c oxygen is also dange rous l y t o x i c , and the p rocedu re as a whole is d i f f i c u l t to per form on a rout ine bas i s . The newest and most p romis ing approach to the cont ro l of h y p o x i c cel ls is the use of chemical r a d i o s e n s i t i z e r s , that i s , d r u g s which mimic the r ad i a t i on-sens i t i z ing p rope r t i e s of o x y g e n . One such d r u g , miso-18 nidazole, is the focus of intense clinical and research interest. Before discussing misonidazole, however, I shall briefly outline the historical development of this approach. Recent interest in chemical radiosensitizers was stimulated by the work of Dr. G. E. Adams and colleagues at the Gray Laboratory, Cancer Research Campaign, Northwood, England. Adams observed that the compounds known to be sensitizers (including, at the time, oxygen, NO, and certain iodine-containing organics) shared the property of being efficient electron acceptors. Adams and Dewey (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 radiosensitizers, using a variety of in vitro test systems. Several compounds were found to be effective in vitro, including stable nitroxyl free radicals (Emmerson, 1967, and Parker et a l . , 1969) and aromatic nitro compounds (Adams et a l . , 1971). Many of the early studies of chemical radiosensitization are described in the volumes Radiation Protection and Sensitization (H. Moroson and M. Quintilliani, ed . , 1970), and Advances T_n Chemical Radiosensitization (1974). However, sensitization irii vivo was not achieved until 1971 (p-nitro-3-dimethylaminopropiophenone, NDPP), and the search for the ideal radiosensitizer continues. 19 What p rope r t i e s would such a d r u g possess? F i r s t , of c o u r s e , it must be an e f fec t i ve r ad i o sens i t i z e r , at a c l i n i ca l l y ach ievab le d r u g c o n -c e n t r a t i o n . It is not necessa ry tha t it be as e f fec t i ve as o x y g e n . T h a t i s , if we def ine a sens i t i ze r enhancement r a t i o , S E R , as the rat io of rad iat ion doses r equ i r ed to p roduce a g i ven ef fect in the absence and presence of the d r u g , then we do not r equ i r e that the SER be as large as the OER . A large SER is d e s i r a b l e , but an SER as low as , s ay , 1 .3 , would s t i l l be of s i gn i f i c an t c l in ica l bene f i t . A t the same t ime, the sens i t i ze r shou ld not increase the r ad io sens i t i v i t y of norma l , aerobic c e l l s , o r the benef i ts wil l be n u l l i f i e d . S e cond , the d r u g must be able to penetrate into the center of the t umour ; t h i s implies tha t it must be able to c ross the b a r r i e r p resented by the cel l membranes of the s u r r o u n d i n g tumour t i s sue (un less penet ra t ion v ia the ex t r a ce l l u l a r space is p o s s i b l e ) . Gene ra l l y , th i s means tha t the d r u g must have l ipoph i l i c p r o p e r t i e s . On the o ther h a n d , wa te r-so lub i l i t y is r equ i r ed f o r admin i s t r a t i on . T h i r d , the d r u g must be metabol ica l ly s tab le , o r at least , much more stable than o x y g e n ; o the rw i se , the d r u g wil l be used up before it reaches the h y p o x i c t a rge t c e l l s . F i na l l y , the t o x i c i t y and s ide-ef fects of the d r u g must be kept w i th in to lerab le l imi ts . T h i s last c r i t e r i on has p roven to be one of the most d i f f i c u l t to s a t i s f y . B y the ea r l y 1970's, a number of e f fec t i ve r ad iosens i t i ze r s were k n o w n , on the bas is of in v i t r o t e s t s . However , in v i t r o systems o b -scu re all bu t the f i r s t c r i t e r i on mentioned above . T h e r e is on l y one 20 membrane to cross, and the drug is present in excess in the medium bathing the cells, so metabolic depletion of the drug concentration is insignificant. Agnew and Skarsgard (1974) studied the effect of cell concentration on the sensitizing efficiency of a number of compounds, including para-Nitroacetophenone (PNAP) 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 Willson (1973) suggested that available drugs be screened for structures with radiosensitizing potential. This approach made it possible to circumvent much of the time-consuming process required for the development of an entirely new compound. Nitrofuran derivatives were already in clinical use as antibacterial agents, and the electron-affinity of these compounds had been demonstrated (Sasaki, 1954). Chapman and colleagues showed that the nitrofurans nitro-furazone and nitrofurantoin were good radiosensitizers in vitro (Reuvers et a l . , 1972). Subsequently, they studied a large number of nitro-furans and nitroimidazoles (Chapman e t a l . , 1974). Pharmacological studies were performed, using mice; questions of metabolic stability and toxicity were considered, as well as in_ vitro radiosensitization. The 5-nitroimidazole drug metronidazole appeared to be the most pro-mising compound studied. Willson has published an interesting his-21 to r i ca l account of these developments ( in F i nego ld , e d . , 1977, p. 147-175). A t th i s po in t , it is wor thwhi le to d i g r e s s b r i e f l y , and cons ide r the h i s t o r y of metron idazo le . The rat ionale fo r the development of th i s d r u g is re levant to the problems of d r u g t o x i c i t y to be cons ide red s h o r t l y . In the ea r l y 1950's, the F rench pharmaceut ica l f i rm of Rhone-Poulenc in i t ia ted an ex tens i ve s c r een ing programme in an attempt to f i nd natura l p roduc t s wi th c y to tox i c a c t i v i t y aga ins t the protozoal paras i tes T r i chomonas . Subs tan t i a l a c t i v i t y was d i s cove red in an ex t r a c t p repa red f rom a s t r a in of the f u n g u s S t rep tomyces . The ac t i ve p r i n c i p l e was isolated and i d e n t i f i e d ; it was found to be ident ica l to the compound azomyc in , or 2-nitroimidazole (desp i te the t r i v i a l name, th i s is not an azo compound ) . In f a c t , the 'new' d r u g had a l ready been c h a r -ac te r i sed by Japanese sc ien t i s t s (Maeda et a l . , 1953) . Chemists at Rhone-Poulenc s yn thes i sed a v a r i e t y of de r i v a t i v e s of azomyc in , and tested them fo r the rapeu t i c e f f e c t i veness . The de r i v a t i v e selected fo r f u r t h e r deve lopment , 1-2 '-hydroxyethy l-2-methy l-5-n i t ro imidazo le , was g i v en the app roved name metron idazo le . T h i s work was rev iewed by G . E. Jol les ( in F i nego ld , e d . , 1977, p. 3-11). Metronidazole was i n t roduced in Europe in 1960 f o r the t reatment of Tr i chomonas v a g i n a l i s , and used in the U . S . A . a f ter 1963. S ince then it has been p r e s c r i b e d v e r y w ide l y . A n i n t e res t i ng rev iew of the use of th i s d r u g has been pub l i shed recen t l y (Go ldman, 1980). O the r n i t r ohe te rocyc l i c compounds had been examined as chemo-the rapeu t i c agents even ea r l i e r . Dodd and col leagues desc r i bed the 22 ant ib io t i c p rope r t i e s of n i t r o f u r a n s in the 1940's (Dodd & S t i l lman , 1944). However , the i n t roduc t ion of metronidazole focussed new at tent ion on n i t roa romat i cs . A rev iew of the many n i t ro compounds now inves t iga ted has been presented ( G r u n b e r g & T i t s w o r t h , 1973). The chemical s yn thes i s of azomycin was repor ted in 1965 ( Lanc in i & L a z z a r i , 1965; Beaman et a l . , 1965). S u b s e q u e n t l y , the pharmaceut ica l f i rm Hoffman - La Roche in i t ia ted a s t u d y of the chemis t ry and chemo-the rapeu t i c p rope r t i e s of 2-nitroimidazole d e r i v a t i v e s . Dozens of such compounds were s yn thes i s ed ( e . g . Beaman et a l . , 1967) and tested fo r a c t i v i t y aga inst bac te r ia and protozoa ( G r u n b e r g e t a l . , 1967). These wo rke r s conc luded tha t : 11 1 -(2-nitro-1 - imidazoly l )-3-methoxy-propanol is of specia l i n te res t because of its low t o x i c i t y , marked ant i t r i chomonal a c t i v i t y , and good a c t i v i t y aga inst Entamoeba h i s to l y t i ca . . . " A s soon as the r ad iosens i t i z i ng p rope r t i e s of n i t roaromat ics were d i s c o v e r e d , the B r i t i s h b r anch of Hoffman - La Roche took advantage of the ava i l ab i l i t y of these n i t ro imidazole de r i v a t i v e s and began a s t udy of the r ad iosens i t i z i ng ab i l i t ies of these compounds . It was found that i_Q v i t r o r ad iosens i t i z i ng ab i l i t y co r re l a ted with e l ec t ron-a f f i n i t y in the n i t ro imidazole ser ies (Adams et a l . , 1976). In g e n e r a l , the 2-nitro-imidazoles were found to be more e f fec t i ve r ad iosens i t i ze r s than the 4-or 5-nitro compounds . A g a i n , the methoxy-propano l de r i v a t i v e was j udged to be the most promis ing agent . The d r u g was syn thes i sed in q u a n t i t y and d i s t r i b u t e d to va r ious laborator ies f o r f u r t h e r exper imentat ion iin v i t r o and iri v i v o , under the Roche p r o d u c t number Ro-07-0582. 23 A t the same t ime, metronidazole was deve loped into a c l i n i ca l l y usable r ad i o sens i t i z e r , a process g rea t l y speeded by the fact tha t the d r u g was a l ready in c l in ica l use , a lbei t f o r a d i f f e r en t p u r p o s e . The pharmacok inet i cs of metronidazole were s tud ied by U r t a sun and c o l -leagues (1974) ; the same g r o u p , w o r k i n g at the C ross Cance r Inst i tute in Edmonton, C a n a d a , began the f i r s t c l in i ca l t r i a l of metronidazole as a r ad iosens i t i ze r ( U r t a s u n et a l . , 1976) . 1.8 T o x i c i t y of N i t roaromat ic Compounds : A n t i c a n c e r d r u g s mus t , in many cases , be de l i ve red sys temica l l y , r a ther than d i r e c t l y to the tumour . T h u s , tox i c s ide ef fects on normal t i s sues are f r e q u e n t l y encoun te r ed . On a molar ba s i s , the n i t ro-imidazoles are much less e f fec t i ve sens i t i ze r s than is o x y g e n . To 2 achieve s i gn i f i c an t s ens i t i z a t i on , d r u g doses of the o r d e r of 10 g/m are neces sa r y . T h i s is an enormous dose , when compared to most o ther pharmaceut i ca l s . It was c r i t i c a l , t h e n , that d r u g s of ou t s t and ing l y low systemic t o x i c i t y be se lec ted . On th i s b a s i s , metronidazole appeared p r o m i s i n g : it is " remarkable s i n ce , in the dose which is cu r a t i v e fo r t r i chomonias is [ about 3 g ] the s ide-ef fects such as nausea , headache, d r y mouth , and a f u n n y taste are re l a t i ve l y mild and s e l f - l i m i t e d . " (Go ldman , 1980) Neve r the l e s s , neuropa th i c s ide-ef fects had been obse r ved fo l lowing h igh doses of metron idazo le , and n i t r o f u r -anto in- induced neuropa thy was also known (le Quesne , in B a r o n , et a l . , e d . , 1973, p. 31-56). 24 The f i r s t c l in ica l t r i a l s of metronidazole revealed neuropa th i c s ide-e f f ec t s , as well as nausea and vom i t i ng . Pat ients expe r i enced t i n g l i n g , numbness , and r i n g i n g in the ea r s . In severe cases , there may be hea r ing l oss , p a r a l y s i s , and even psychos i s ( U r t a s u n e t a l . , 1977). U s u a l l y , these s ide-ef fects have been t empora r y , and they can be avo ided by l imi t ing the total d r u g dose . However , such l imitat ions reduce the l ike l ihood of ach iev ing e f fec t i ve r ad iosens i t i za t i on . T o x i c i t y of n i t ro imidazoles was obse r ved d u r i n g in v i t r o s tud ies of r a d i o s e n s i t i z e r s , as well as in the c l in ica l t r i a l s . Su the r l and (1974) o b -se r ved cel l death in mult icel l " s p h e r o i d s " t reated with metron idazole . A t a wo rkshop meeting fo l lowing the F i f th Internat ional Cong res s of Radiat ion Research in Seat t le , Wash ing ton , 1974, Palc ic sugges ted that these ef fects were due to a spec i f i c c y to tox i c act ion of n i t ro imidazoles aga inst h y p o x i c c e l l s . T h i s poss ib i l i t y was q u i c k l y conf i rmed (Moore et a l . , 1976; Hall & Ro iz in-Towle , 1975). The fo l lowing f i g u r e s from Moore et a l . i l l u s t ra te the ef fect : f i g . 5 . Ch inese hamster o v a r y ( C H O ) cel ls were incubated at 37° in medium conta in ing the stated concent ra t ions of mison idazo le , unde r e i ther h y p o x i c or aerobic cond i t i ons . Hypox i a was p roduced by f low ing p u r i f i e d gas ove r the s t i r r e d cel l 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 iquots of cel ls were removed , washed f ree of d r u g , and plated in pe t r i d i shes to determine s u r v i v a l ( co lony fo rming ab i l i t y re la t i ve to c o n t r o l s ) . The se lect ive k i l l i ng of h y p o x i c cel ls is appa ren t : f o r example , 4 h r incubat ion wi th 15 mM misonidazole , unde r h y p o x i a , reduces s u r v i v a l to less than 1%, whereas the exposu re time unde r aerobic cond i t ions has no measurable tox i c e f fec t . 25 100 50 20 AEROBIC CHO (37°C) OmM -£1rrM 15 mM ' \ ^ 5 0 " J I I I 1 2 3 4 5 6 7 8 9 10 INCUBATION TIME AT 37°C (hours) U Z 100 y u. u. ui < Z kl U a. 0.1 HYPOXIC CHO (37°C) -,0mM 5mM 1 2 3 4 5 6 7 8 9 10 I N C U B A T I O N TIME AT 3 7 ° C (hours) Fig. 5 Selective toxicity of misonidazole to hypoxic cells (from Moore et a l . , 1976, by permission) Chinese hamster ovary (CHO) 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 drug. 26 The nitroimidazoles were originally developed for the treatment of anaerobic infections. Thus, in hindsight, this selective action against hypoxic mammalian cells is not surprising. The 're-discovery' of the phenomenon by radiobiologists has stimulated much interest in the pharmacology of nitroaromatic compounds. This research has two motivations: on the one hand, hypoxic cytotoxicity may be related to the mechanism of misonidazole's neuropathic side-effects, and an understanding of this process could improve administration of the drug; on the other hand, the selective toxicity raises the possibility of using misonidazole, or a similar drug, as a chemotherapeutic agent against hypoxic tumour cells. This possibility has been reviewed recently (Kennedy et a l . , 1980). 1.9 Hypoxic toxicity - mechanism: The radiosensitizing ability of misonidazole is a consequence of its electron-affinity - that is, the ease with which it can be reduced to the nitro anion radical, R-NG^" (Wardman, 1977). Early evidence sug-gested that a related mechanism may be responsible for misonidazole toxicity. In this case, however, the reduction chemistry is metabolic, rather than radiolytic. Much of this evidence came from studies with other nitroaromatic compounds. As discussed above, nitrofurans have been studied as antibacterial agents since the 1950's - for a review, see McCalla, in Hahn, ed . , 1979. The metabolic reduction of such compounds was studied by Asnis and colleagues, using bacteria. At least two "nitroreductase" enzyme 27 systems were identified. One of these enzymes is inhibited by the presence of oxygen (Asnis, 1957). Asnis and co-workers cultured E_. coli in medium containing Furacin (5-nitro-2-furaldehyde semicarbazone) and developed a drug-resistant bacterial strain. This strain was characterised by a minimum inhibitory Furacin concentration about ten times higher than that of the parent strain (Asnis et a l . , 1952). However, under hypoxia (E. coli is a facultative anaerobe) the two strains showed no difference in susceptibility to Furacin. It is now clear that the resistant mutants lack the oxygen-independent nitro-reductase (Peterson et a l . , 1979). (Mutants lacking the oxygen-sensitive enzyme have never been isolated: presumably this enzyme has some essential biological activity.) 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 in-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, ed . , 1979) 28 The metabolism of aromatic nitro compounds by mammalian systems was studied by Fouts and Brodie in the mid-1950's. (Ironically, they began their paper with the statement: "Aromatic nitro compounds are rarely used as d r u g s , . . . " , a situation which was to change shortly!) They made several important observations: activity was found in both the soluble fraction and the microsomal fraction of liver, there are large species differences in activity, and the enzyme system showed "few rigid structural requirements for its substrates." (Fouts & Brodie, 1957). Furthermore, 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 work of Mason and Holtzman. Electron Spin Resonance (ESR) spectroscopy was used to demonstrate the formation of the nitro anion radical, during the hypoxic microsomal reduction of nitrobenzoic acid (1975a). This shows that the initial step in the reduction process is a one-electron transfer from the enzyme to the nitro compound, and suggested the possibility of a free radical mechanism in the oxygen inhibition of the reduction. The observation (Biaglow et a l . , 1976) that, under aerobic conditions, nitroheterocycles stimulated oxygen depletion, was indirect evidence for the mechanism suggested by Mason and Holtzman (1975b). They suggested that the initial one-electron step in nitroreduction continued in the presence of oxygen; however, the nitro anion radical was re-oxidized very rapidly to the parent compound. Thus, no net disappearance of the nitro 29 compound is o b s e r v e d , and no ESR s igna l f rom the rad ica l is de tec ted u n d e r ae rob ic c o n d i t i o n s . O x y g e n is r educed to s u p e r o x i d e , r e s u l t i n g in o x y g e n dep le t ion and the b u i l d - u p of s u p e r o x i d e . If exogenous s u p e r o x i d e d ismutase and cata lase are a d d e d , the rate of o x y g e n dep le t ion is d e c r e a s e d . T h i s is i l l u s t r a t e d in the fo l low ing scheme: C o f a c t o r ( r e d u c e d ) C o f a c t o r ( o x i d i z e d ) R e d u c t a s e ( o x i d ized)< + e R e d u c t a s e ( r e d u c e d ) ( h y p o x i a ) R-NO. R-N0„ N 0 ( s u p e r o x i d e ) R-N=X (re d u c e d p r o d u c t s ) Mechanism 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 in 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 the n i t r o anion r a d i c a l . U n d e r h y p o x i a , t h i s spec ies is f u r t h e r r educed 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 and mutagen ic s p e c i e s . U n d e r ae rob ic c o n d i t i o n s , the rad ica l an ion is r e - o x i d i z e d q u i c k l y to the pa ren t n i t r o com-p o u n d . T h e r e s u l t i n g s u p e r o x i d e is de tox i f i ed by s u p e r -o x i d e d ismutase and cata lase (see be low ) . 30 H + + SOD + \ ( 0 2 + H 2 0 2 ) C A T A L A S E * \ 0 2 + H 2 0 N E T : H + + e" + 0 2 — H + + 0 2 ~ — » 3/4 0 2 + \ W£> Mechanism of reduced o x y g e n consumpt ion in presence of supe rox ide d ismutase and cata lase : Supe rox ide p roduc t i on v ia the ' fu t i l e ' metabolic cyc le out l ined above resu l t s in consumpt ion of oxygen in c losed microsomal incubat ion m i x t u r e s . In the presence of exogenous supe rox ide dismutase and cata lase , the s u p e r -ox ide is conve r ted to oxygen and wate r , r e su l t i ng in a 4-fold decrease in oxygen consumpt ion . ( A c t u a l l y , these react ions wi l l o ccu r spontaneous ly at an apprec iab le ra te , so the obse r ved decrease in oxygen consumpt ion may be less than 4- fo ld . ) F u r t h e r s u p p o r t of th i s mechanism fo r oxygen inh ib i t i on of n i t r o -reduc t ion was p rov i ded by a s t u d y of the anomalous case , v i z . the oxygen- independen t bacter ia l enzyme (Pe terson et a l . , 1979). ESR and opt ica l spec t roscopy were used to demonstrate that th i s enzyme reduces n i t ro compounds v ia an in i t ia l 2- (or more) e lect ron s t ep , w i thout f o r m -ation of the n i t ro anion radica l as an in termediate . In th ree d i f f e r en t biological systems - the "w i ld t y p e " bacter ium possess ing an oxygen- insens i t i v e n i t r o r e d u c t a s e , mutant bac ter ia l a ck ing th i s enzyme, and mammalian ce l l s , there is a c lear cor re la t ion between the format ion of n i t r o r educ t i on p roduc t s and bio logica l damage. T h i s sugges t s t ha t , as A s n i s had p r o p o s e d , some p roduc t of n i t r o r educ t i on is the ult imate t ox i c spec ies . However , the nature of t h i s p r o d u c t , and the chemis t r y of reduc t ion of n i t r ohe te rocyc l i c compounds , has been 31 exp lo red on ly s k e t c h i l y . The e luc idat ion of the r educ t i ve metabolism of misonidazole. is the cent ra l problem cons ide red in th i s t h e s i s . 32 MATERIALS AND METHODS: 2.1 Cell culture procedures: The m vitro biological experiments reported here were performed on Chinese hamster (Cricetulus griseus) cell lines grown in tissue culture. The Chinese hamster• ovary (CHO) cell line is used widely: it grows 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 (FCS), antibiotics, and bicarbonate buffer. The cultures were grown in an incubator (Incu-cover, Associated Biomedic Systems) at 37°; pH is regulated to 7.4 by continuous gassing with 5% CO^ in air. The cell culture is diluted daily to about 7 x 10^ cells/ml, maintaining asynchronous exponential growth with a doubling time of about 12 hours. A second Chinese hamster line, C H 2 B 2 , derived from line CHEF-125 (Prescott & Bender, 1963) was grown in monolayer culture in 25 cm tissue culture flasks (Falcon). The medium used was minimum essential medium (MEM, Gibco) supplemented with 10% FCS, antibiotics, and carbonate buffer. Cells were harvested by trypsinization twice per week, and transferred into new flasks (Agnew & Skarsgard, 1972). 33 2.2 In vitro toxicity studies - cell survival experiments: The toxic effect of a drug, 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 (Moore et a l . , 1976). Drug-medium solutions were prepared before each experiment in medium lacking bicarbonate buffer. The solutions were adjusted to pH 7.4 and sterile filtered (Nalgene). From this stock, 29 ml was added to a wide-mouth glass centrifuge vessel. The tube was sealed with a rubber stopper fitted with two syringe needles to permit gas flow, and a stoppered central port for removal of samples. In a typical experiment, eight such vessels, including controls and various drug combinations, were set up in a large water bath. The bath was maintained at 37° and set up on top of a multi - magnetic stirrer unit constructed locally. The entire apparatus was kept in a 37° warm room. The appropriate gas phase (C^, H^, or air) was supplied from a tank, and humidified in a glass "bubbler" unit filled with sterile water. The gas was admitted through a short tube fitted with a plastic syringe tip. The drug-medium solutions were pre-gassed before addition of cells (for ^ 20 minutes) to achieve temperature and gas phase equilibrium. Cells were harvested from culture by low-speed centrifugation (CHO) or trypsinization and centrifugation ( CH2B2 ) 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 ° . The samples were spun to harvest the cells; supernatant 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. Feeder cells are cells of the same line, sterilized by heavy irradiation, which supply nutrients and growth factors which increase the overall plating efficiency of the cloning procedure, and overcome the variation in plating efficiency which may be observed when widely differing inocula are used. Typically, 10 \i\ of cell sus-pension is plated into a petri. If significant toxicity was anticipated, larger samples were plated (up to 2 ml); replica plates were plated at several inoculum volumes. Following plating, the cell suspensions were again vortexed and a sample (2 ml) was removed, diluted with phos-phate-buffered saline (PBS), and counted with an electronic cell counter (Coulter Electronics) to determine cell concentration. Petri dishes were incubated at 37° in a tray incubator with 5% CC^ flow for seven days. The medium was then carefully decanted and replaced with stain (methylene blue). Finally, the stain was washed off, and the petris were counted to determine number of colonies. A clone of 50 or more cells is assumed to represent a survivor. 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 cells relative to plating efficiency of controls. In general, experiments were repeated at least three times. Values of log (surviving fraction) were averaged for each time point, and standard errors of the mean were calculated. Results are presented as log S vs. time of drug exposure. DNA single-strand breaks (SSB): The alkaline sucrose gradient technique was deveoped by McGrath and Williams (1966) as an assay for DNA damage in bacterial cells. It has since been adapted for use with mammalian cells in a number of laboratories (for example, Palcic and Skarsgard (1972,1981)). Cells are disrupted by lysis on top of the gradients on which separation of the DNA molecules, according to size, is performed. Thus, fragmentation of the intact 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 r i f u g a t i o n . Fol lowing c e n t r i f u g a t i o n , the g rad ien t is f r a c t i o n a t e d , and the d i s t r i b u t i o n of the DNA is measured . In our expe r imen t s , the DNA had been labeled wi th rad ioac t i ve t h y m i d i n e , and the presence of DNA detected by th i s a c t i v i t y . The number of s i ng l e-s t r and DNA b reaks ( S SB ) can be ca lcu lated by a compar ison of t rea ted and cont ro l we ight-averaged molecular we ights ( M ^ ) . CH2B2 cel ls were labeled by incubat ion (24 h r ) in medium con-14 t a in ing 0.05 pCi/ml C- thymid ine , spec i f i c a c t i v i t y 52 C i/Mo le , or 0.25 3 uCi/ml H-thymid ine , spec i f i c a c t i v i t y 41 Ci/mMole. P r i o r to an e x -per iment , labe l ing was terminated by 1 h r incubat ion in g rowth medium f ree of labeled nuc leo t ides . Ce l l s were then ha rves ted by t r y p s i n -i za t i on . T r i t ium- labe led cel ls were incubated wi th d rug-con ta i n i ng 14 medium as desc r i bed above , and C-labeled cel ls were incubated wi th d rug- f r ee medium as con t ro l s . Incubat ions were per formed as desc r i bed above ( t ox i c i t y e x p e r i m e n t s ) . A l i quo t s of expe r imen ta l , and c o r r e s p o n d i n g cont ro l cel ls were pooled a f ter wash ing and resuspended g at a concent ra t ion of 4 x 10 ce l l s /ml . A 50 uj a l iquot was then layered onto 0.5 ml l y s i n g so l u t i on , on top of a 5-20 % a lka l ine sucrose g r a d i e n t , in a 17 ml n i t roce l lu lose u l t r a c e n t r i f u g e t u b e . Ce l l s were l ysed at 20°C fo r 7 h r and c en t r i f uged at 14,000 rpm fo r 11 h r , us ing a Beckman L-65B u l t r a c e n t r i f u g e and SW-27.1 ro to r . Each g rad i en t was f rac t iona ted us ing an Isco model " D " f r a c t i ona to r . F rac t ions (25 x 0.75 ml) were col lected in sc in t i l l a t ion v ia l s and neu t ra l i zed wi th concent ra ted HCI . Sc in t i l l a t ion cockta i l (Amersham A C S , 5 ml) was added to each v i a l . Samples were counted in a Beckman sc in t i l l a t ion coun te r . 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 reduction of misonidazole was performed using zinc dust in the presence of CaC^ . This technique was adapted from earlier work on the reduction of nitroaromatic compounds. In general, the nature of the products obtained depends on a variety of factors: the nitro compound used, temperature, solvent, and presence of salts. Misonidazole used in all the studies reported in this thesis was a gift of Dr. C. Smithen, Roche Products L td . , Welwyn Garden City, U.K., and was used without further purification. No impurities were detected by chromatography. Zn dust and CaC^ were obtained from Fisher Scientific. Misonidazole, 1 g, and C a C ^ , 1 g, were dissolved in 100 ml double-distilled water. The solution was rapidly stirred with a magnetic stirrer, and 2 g of Zn dust was added. The solution quickly became greenish-yellow in colour. The mixture was stirred for 11-^ hr at room temperature, and then filtered through Whatman #1 filter paper to remove the Zn dust and precipitated ZnO. The solution was frozen in a lyophilization flask, and concentrated by lyophilization. Thin-layer chromatography (TLC ) : The concentrated reduction mixture, prepared as outlined above, was examined by T L C . The plates used were type LK5DF, Whatman, 38 I n c . , C l i f t o n , N . J . These plates cons i s t of a 250 p layer of s i l i ca gel formulated f o r the separat ion of moderate ly to s t r o n g l y polar subs t ances . The sample is app l ied to a p readso rben t sample d i spens i ng a r ea , wh ich el iminates the need f o r ca re fu l spo t t ing of samples , and allows volumes as large as 100 pi to be appl ied to the plate (Whatman T L C p r o d u c t g u i d e , 1978). The plates were deve loped in ch lo ro fo rm/ acetone/methanol (45/45/10) and bands were detected by co l ou r , or by UV f luorescence q u e n c h i n g . H i g h - p r e s s u r e l i qu id ch romatography ( H P L C ) : Ana l y t i c a l H P L C was used to monitor the p rog ress of the react ion and the p u r i t y of the separated p r o d u c t s . We used a Spec t r a-Phys i c s SP-8000 chromatograph in i soc ra t i c mode. The fo l lowing cond i t ions were employed : co lumn: L i C h r o s o r b RP-8 r eve r sed-phase , par t i c l e s ize 10 | j , d imensions 4.6 mm x 250 mm; mobile phase : 10 mM acetate b u f f e r , pH 4.5/methanol (80/20) ; f low ra te : 5 ml/min at a p r e s s u r e of about 2800 p s i . Detect ion of the e luted components was per formed wi th a va r i ab le-wave length UV-v i s i b l e de tec to r , Schoeffe l Spect ra-F low 770, set to 370 nm. P repa ra t i ve l i qu id column ch romatog raphy : P repa ra t i ve ch romatography techn iques were used to separate the reduc t ion p roduc t s on a l a rge r sca le . We used a recent l y-deve loped p repa ra t i v e reve rsed-phase ch romatography co lum: L i C h r o p r e p L O B A R 39 RP-8, particle size 43-60 |j, 25 mm x 310 mm (size B), obtained from BDH Chemicals, Vancouver. The column was adapted with Swagelok fittings to allow mobile phase to be supplied by the HPLC pump itself, rather than by a low-pressure peristaltic pump. Mobile phase used was H2O / methanol (70/30). Flow rates up to 13 ml/min could be achieved at the maximum operating pressure of the glass column (90 psi), allowing complete preparative runs in about V-*> hr. Samples were injected through a specially-prepared 2 ml loop injector, made out of Teflon tubing. Spectroscopic techniques: UV-visible spectroscopy was performed on an Aminco DW-2 spectrophotometer. Nuclear magnetic resonance (H-NMR) was performed on a Varian XL-100 Fourier transform spectrometer (NMR lab, Chemistry department, UBC). Samples were dissolved in D^O or CD^OD for analysis. Mass spectra were obtained at the Chemistry department, UBC, using solid-probe injection and electron ionisation. 2.4 Xanthine oxidase - catalysed reduction of misonidazole: Enzymatic reduction procedures: Xanthine oxidase was purchased from Sigma, St. Louis (type X-4500, from buttermilk, chromatographically purified, specific activity 1.25 units/mg protein), as were hypoxanthine, xanthine, and uric acid. Kinetics of the reduction were studied using an Aminco DW-2 spectrophotometer, equipped with an anaerobic cuvette and a controlled temperature magnetic-stirrer cuvette holder. 4 0 Misonidazole reduc t ion was s tud ied as fo l lows : A l l so lut ions were d i s so l ved in P B S . The opt ica l cel l of the a n -aerobic cuve t te was f i l l ed wi th subs t r a t e so lu t i on , 1 . 9 m l , con ta in ing misonidazole and h y p o x a n t h i n e . The side-arm was f i l l ed wi th the enzyme so lu t i on , 1 un i t of xan th ine ox idase in 0 . 5 ml PBS conta in ing 2 . 3 M ( N H 4 ) 2 S 0 4 and 1 mM EDTA (e thy lene diamine te t raacet i c a c i d ) . The cel l was then placed in the spect rophotometer ( 3 7 ° ) and gassed wi th humid i f i ed N 2 , wi th s t i r r i n g . A f t e r 1 0 minutes e q u i l i b r a t i o n , the contents of the side-arm were t i pped into the opt ica l c e l l , and reco rd ing s t a r t e d . Init ial concent ra t ions in the react ion mix tu re were : mison idazo le , 1 0 0 (jM; h y p o x a n t h i n e , 5 mM; ( N H 4 ) 2 S 0 4 , 4 8 0 mM; E D T A , 2 1 0 uM; xan th ine o x i d a s e , 1 u n i t , in a total volume of 2 . 4 ml . Misonidazole reduc t ion was determined by loss of absorp t ion at 3 5 0 nm. Spec i f i c a c t i v i t y of the xan th ine ox idase p repara t ion was s tud ied by measur ing the conve rs ion of xan th ine to u r i c a c i d , in a i r-sa tu ra ted so lu t i on . Xan th i ne ( 1 0 0 | J M in 2 ml P B S ) , was added to a regu la r cuve t te maintained at 3 7 ° . X an th i ne ox idase , 0 . 0 2 u n i t s , was a d d e d , and format ion of urate recorded at 2 9 5 nm ( B r a y , 1 9 7 5 ) . T L C A u t o r a d i o g r a p h y : 1 4 C-mison idazo le , labeled at the 2 pos i t ion of the imidazole r i n g , spec i f i c a c t i v i t y 9 . 2 u C i / m g , was a g i f t of D r . C . Smi then . No rad ioact i ve impur i t i es were detected above a level of 0 . 1 % in the chromatography systems u s e d , and the radio-labeled d r u g was used wi thout f u r t h e r p u r i f i c a t i o n . 41 The nature of the reduced derivative of misonidazole formed in the 14 14 reaction was studied using C-labeled drug. C-labeled misonidazole, diluted 100 x with cold misonidazole, 1 mg total, was incubated overnight with 2 mg hypoxanthine and 1 unit xanthine oxidase in 2 ml PBS, N 2 , 37°. The solution was frozen and lyophilized. The dried residue was resuspended in 1 ml methanol and centrifuged briefly to remove insoluble material. Recovery of the radiolabel was 75-90 %. The soluble fraction was studied by TLC and HPLC. TLC was performed as described above (Zn reduction), except that the solvent used was acetone/methanol (67/33). The plate was auto-radiographed using Kodak X-OMAT-R x-ray film. HPLC was performed as follows: column, Whatman PAC polar-bonded phase, 4.6 mm x 250 mm; mobile phase: ethyl acetate/methanol (50/50), flow rate 2.0 ml/min. Column temperature was maintained at 40° with an air oven. Fractions (0.4 ml) were collected in vials; scintillation cocktail (Amersham ACS II, 5 ml) 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 8 reach a concentration of 5 x 10 cells/ml. Cells (3 x 10 ) were har-vested by centrif ugation, and spun to form a pellet of about 1 ml volume. The pellet was resuspended with medium containing 42 C-misonidazole to give a total volume of 2 ml and a drug concentration of 0.38 mM. At zero time, the tube was transferred to a 37° water bath. N 2 was 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 after zero time (within two minutes) and at each successive hour, up to 3 hours. Each 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. The remain-der of the sonicate was frozen and lyophilized to dryness. The dry residue was resuspended with ethyl acetate/methanol (63/37), spun (5 minutes at 800 rpm), and the supernatant decanted. This procedure was repeated a total of 3 times - the supernatants were combined and evaporated in a Buchler vortex evaporator at 30°. The dry samples were stored at -15° until chromatography was performed. 43 SAMPLE (0.3 ml - 4.5 x 10 7 CHO cells) | Sonicate DISRUPTED CELLS | Lyophilize DRY RESIDUE Extract with ethyl acetate: MeOH (3 times - combine) ORGANIC-INSOLUBLE Extract with MeOH/TCA. (10 times - combine) ORGANIC-SOLUBLE Dry. Resuspend in 1 ml MeOH. Remove aliquot - TLC . Dry. Resuspend in acetate buffer for HPLC. PELLET ubilize in NaOH Count. ORGANIC-INSOLUBLE Count aliquot. Fig. 6 In vitro metabolism experiments: schematic 44 Extraction procedure: The pellet remaining after extraction with ethyl acetate/methanol was further extracted to separate acid-soluble and acid-insoluble material. The pellets were rinsed carefully into a cone of filter paper (Whatman #1) and washed with 10 succesive 2 ml aliquots of a mixture of equal 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, the remaining pellet was dissolved in 2 ml of 2M NaOH, with gentle heating. The sample was neutralized with acetic acid and counted. The 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 organic-soluble activity. A further aliquot (50 ul) was applied to a Whatman LK5DF TLC plate. The plate was dried, and developed immediately in acetone/methanol (50/50). The developed plate was dried, and autoradiographed on Kodak X-OMAT-R film. The remainder of the supernatant was evaporated, and the residue was resuspended in 125 ul acetate buffer. The sample was added carefully to an Eppendorf micro centrifuge tube, and spun for 5 min-45 utes in an Eppendorf model 5412 centrifuge to separate the water-soluble supernatant from the insoluble precipitate (presumably lipid). An aliquot of the supernatant was injected into the HPLC. HPLC was performed as follows: column, Whatman 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 ex-periments (see chap. 6). For these experiments, samples were resus-pended in ethyl acetate/methanol and centrifuged as above. The super-natants were injected into the HPLC. Column, Whatman PAC (polar amino-cyano bonded phase), 4.6 mm x 250 mm; mobile phase, 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 (Amersham ACS) in a Beckman LS-330 scintillation counter. 46 MISONIDAZOLE CYTOTOXICITY J_N VITRO: 3.1 Introduction: Oxygen, which inhibits mammalian nitroreductase activity, greatly diminishes the toxicity of misonidazole in vitro. In the first chapter of this thesis, I reviewed evidence that enzymatic nitroreduction proceeds by a one-electron, free-radical mechanism. Oxygen reacts with nitro-aromatic anion radicals at close to diffusion-limited rates (Greenstock & Dunlop, 1973; Wardman & Clarke, 1976) to produce superoxide, and regenerate the parent nitro 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 superoxide, as demonstrated by Nishikimi and Yagi (1977). It was suggested that this reaction may be of comparable importance to the enzymatic de-activation of superoxide. It appeared reasonable to assume that the overall rate of nitro-reduction in the cell depends on the electron-affinity of the nitro compound, and the net effect of agents that can transfer electrons to the 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 peroxidase/ H2®2 s V s t e m - Continuous flow ESR techniques permitted identification of the radical, and kinetic mea-surements were made (Ohnishi et a l . , 1969). Indeed, ascorbate radical may be the main component of the ESR signals observed in tissue samples (Borg, in Pryor, ed . , vol. 1, 1976). Once formed, the ascorbate radical is relatively nonreactive, and decays by disproportion-a t e (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. This possibility was supported by the observation that ascorbate reacts non-enzym-atically with the strongly electron-affinic carcinogen 4-nitro-quinoline-N-oxide (4-NQO), presumably via a free radical intermediate (Biaglow et a l . , 1976). 4-NQO is more electron-affinic than the nitro-imidazoles, and metabolic reduction of 4-NQO can proceed even under aerobic conditions (Matsushima & Sugimura, in Endo et a l . , ed . , 1971). These properties of ascorbate suggested that this vitamin might interact with misonidazole in a significant manner. We tested the effect of ascorbate on the hypoxic toxicity of misonidazole to CHO cells, using the techniques described in chapter 2. Cells were exposed to misonidazole, with or without ascorbate, for various times, and then 48 0 1 2 T i m e (h) Fig. 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 Misonidazole, 5 mM • Misonidazole, 5 mM + Ascorbate, 5 mM O Misonidazole, 15 mM • Misonidazole, 15 mM + Ascorbate, 5 mM 49 plated to determine survival. Fig. 7 shows the results of these ex-periments. At 5 mlVI concentration, the 'shoulder 1, or lag period, in the survival curve of cells exposed to misonidazole in hypoxia, lasts about 7yz hr. The turning down of the curve can just be seen in the figure. The addition of equimolar ascorbate shortens the shoulder to about 1 hr, thus greatly enhancing the toxicity of the drug. Enhancement is also observed when 5 mM ascorbate is added to 15 mM misonidazole. The effect is probably a consequence of the reducing capacity of ascorbate: gulonolactone, a non-reducing analogue 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^, 37°, for up to 2 hours before addition of cells, did not affect the subsequent response. Furthermore, the enhanced toxicity is strongly temperature 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 toxicity may be mediated by production of superoxide in the 'futile' reduction of the nitro group. As mentioned 50 CHO cells N 2 T I 1 1 I 0 1 2 3 4 TIME (hours) Fig. 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, ascorbate can reduce superoxide; Mason et al. have shown that ascorbate inhibits superoxide-mediated adrenochrome formation during aerobic microsomal incubations of nitrofurantoin (1977). Thus, the action of ascorbate may involve both reduction of the nitro group and an interaction with the resulting superoxide. The effect of ascorbate on the aerobic toxicity of misonidazole cannot be studied directly, in vitro, because of an additional effect. Ascorbate itself shows great toxicity in air, which is absent in hypoxia. This surprising result is illustrated in f ig. 9 (from Josephy et a l . , 1978). Ascorbate causes an immediate rapid decline in survival of CHO cells. This effect had been reported previously in other systems (Benade et a l . , 1969; Peterkovsky & Prather, 1977) but was not widely known. The toxicity of ascorbate appears to be due entirely to for-mation of peroxide during ascorbate oxidation. The simultaneous addition of catalase completely eliminates ascorbate toxicity (fig. .9). This action is specific to catalase - bovine serum albumin had no effect. It seems unlikely that catalase crosses the cell membrane, suggesting that the peroxide is formed extracellularly and either enters the cell, or damages the membrane. Similar results and conclusions were reported by Koch and Biaglow (1978). Presumably, the observed aerobic toxicity of ascorbate is absent hn vivo, since catalase is a ubiquitous enzyme; in this sense, the [n vitro observation is an artifact. Nevertheless, the possibility of achieving, with ascorbate, selective killing of tumour cells in vivo has been raised (Park et a l . , 1980). 52 I 1 I i I i L 0 1 2 3 Time (h) Fig. 9 Toxicity of ascorbate to aerobic cells nn vitro (from Josephy et a l . , 1978) CHO cells were incubated at 37°, 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 ig. 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 section, I shall present a brief review of some recent papers which examine the effects of ascorbate and other reducing agents on misonidazole toxicity. Glutathione (y-glutamylcysteinylglycine, GSH) is a tripeptide reducing agent distributed widely in nature. Generally, the GSH in cells exists mainly in the reduced form; much smaller amounts of the oxidized form, glutathione disulfide (GSSG) may be present also (Kosower & Kosower, in Pryor, ed . , vol.2, 1976). GSH reduces dehydroascorbate to ascorbate, a reaction catalysed by the enzyme glutathione dehydrogenase. This enzyme has been isolated from plant sources (Joslyn, in Colowick & Kaplan, ed . , 1955, p. 847-9) and activity is also found in animal tissues (White et a l . , 1979, p. 1182). Thus, one might expect the addition of glutathione to enhance misonidazole toxicity via reduction of endogenous ascorbate. On the other hand, glutathione is believed to play an important role in detox-ification of activated species in drug metabolism (Jakoby, in de Serres et a l . , ed . , 1976, p. 207-212) and on this basis, the opposite effect could be anticipated. Hall et al. (1977) observed protection against misonidazole toxicity by the sulfhydryl agent cysteamine (8-mercapto-ethylamine). We have consistently observed enhanced toxicity in the presence of glutathione, although the effect is much less than that of ascorbate. Our report of the effect of ascorbate on misonidazole toxicity was followed by several other examinations of the effects of other reducing agents, and it is appropriate to review these results here. 54 Koch and Biaglow (1979) observed similar ascorbate-enhancement of misonidazole toxicity, using the V79 Chinese hamster cell line. These workers used an elaborate deoxygenation procedure to ensure extreme hypoxia; they suggested that the ascorbate effect was greater under these conditions. Data were presented to suggest modification of the slope of the exponential region of the survival curve, rather than shoulder modification, by ascorbate. Koch and Biaglow found that the presence of ascorbate did not affect the radiosensitizing properties of misonidazole, in agreeement with our own observations (unpublished). Finally, they reported that a variety of sulfhydryl species, including cysteamine, cysteine, and glutathione, 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. CHO cells and HeLa (human) cells were compared; the two lines gave qualitatively similar results, although the effects were more pronounced in the hamster line. Again, ascorbate was found to produce substantial enhancement of misonidazole toxicity; the effect was primarily shoulder-reduction rather than slope-modification, in agreement with our own results. Glutathione gave a very slight protective effect (lengthening of shoulder by about 10%). Taylor and Rauth also demonstrated a sub-stantial effect of ascorbate on the metabolism of misonidazole by CHO cells. This important result will be considered in a later chapter, on misonidazole metabolism. 55 Stratford (1979) also observed protection by sulfhydryls and enhancement by ascorbate. Three different radiosensitizers 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 < miso-nidazole < 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 pH. The curves of misonidazole toxicity alone, and miso-nidazole 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 MISONIDAZOLE: 4.1 Introduction: Nitroreduction of misonidazole appears to be a prerequisite, or at least a co-requisite, to its toxicity. Some of the evidence supporting this hypothesis has been presented above. In the last few years, several investigators have attempted to demonstrate it directly. At least three distinct approaches have been tried. First, the nature of the metabolites of misonidazole formed in hypoxic cells has been studied, in an effort to demonstrate that nitroreduction occurs. Second, misonidazole has been reduced chemically, and the biological properties of the resulting derivatives measured. 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 reduction of nitroaromatic compounds is a complex process, since a large number of oxidation states may be reached. Complete reduction of a nitro compound is a six-electron process, resulting in an amine functional group. In between these states, a variety of intermediates may be formed, as shown in the schematic (next page). In general, the type of products formed during nitro-reduction depends on many factors: the nature of the R group, temp-erature, concentration, solvent, and time. 57 0 R-N0 2 nitro 2 R-N=0 nitroso 3 R-N=N(0)-R azoxy 4 R-NHOH hydroxylamino 4 R-N=N-R azo 6 R-NH2 amino 5 R-N-N-R hydrazo H H The best-studied system is the reduction of nitrobenzene. Certain results are worth noting: Although the evidence is overwhelming that nitroso-benzene 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 pH, resulting in the formation of bimolecular products. For example, reduction of nitrobenzene with NaOH and CHgOH yields azoxybenzene. Zn dust can further reduce this compound to azobenzene. Zn dust and NaOH reduces nitrobenzene to hydrazobenzene. Without giving an exhaustive list, it is clear that a great variety of possible reactions can occur. A review of the chemistry of nitroreduction and nitroreduction products is given by Wardman (1977). 58 In addition to these 'classical' chemical syntheses, two other techniques have been used in studies of nitroreduction: pulse radiolysis and electrochemistry. Pulse radiolysis is a tool for the study of short-lived molecular species, especially free radicals, produced by the absorption of rad-iation energy. A review of the subject has been published recently (Adams & Wardman in Pryor, ed . , vol.3, 1977). The sample to be studied is contained in an irradiation cell, and is also in line with a spectrophotometer system. The cuvette is irradiated with a very brief (usecond or less) 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 of 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 of hypoxic cell radiosens-itizers. Electron-affinity can be measured by pulse radiolysis studies. Meisel and Neta (1975) studied electron-transfer equilibria between quinone derivatives of known electron-affinities and nitroaromatics: R-NG^- + Q R-NC>2 + Q . The concentration of the quinone radical can be determined spectrophotometrically, and the position of the above equilibrium measured. The logarithm of the equilibrium con-stant is proportional to the difference between the one-electron reduction potentials of the nitro compound and the quinone. The particular virtue of pulse radiolysis is that such measurements can be obtained on a very short time-scale; thus, equilibrium can be achieved 59 before secondary reactions alter the nature of the chemical system irreversibly. If the nature of these secondary reactions can be controlled (for example, by the use of chemical "scavengers" which trap unwanted intermediates) radiolysis can be used as a synthetic technique. 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, and their application to the study of radiosensitizers, have been reviewed by Roffia (in Breccia et a l . , ed . , 1979). In general, the electroactive compound under study is dissolved in a suitable solvent, along with a 'supporting electrolyte', which increases solution conductivity. The solution is contained in a polarographic cell, in which a potential difference is applied between two electrodes - a 'working' electrode (typically, a dropping mercury electrode, which allows the electroactive surface to be renewed continuously), and a 'counter' electrode. The potential of the working electrode is measured with respect to a third electrode, the 'reference' electrode. The reference electrode is connected to the cell with a salt bridge, and placed physically adjacent to the working electrode; this circumvents the effect of the resistive drop between the working and counter elec-trodes. To perform an electrochemical measurement, the potential drop between the working and reference electrodes may be varied, and the resulting current flow 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 species. From these measurements, the redox potential of the reaction can be measured. However, such a determination is valid only if the reaction has been studied under thermodynamically reversible conditions. If irreversible reactions occur, then polarographic potentials will depend on extraneous factors such as the rate of mercury drop formation, rather than representing 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 com-pounds . . . detracts from [ their use ] . . . Electrochemical methods of nitroreduction can yield a variety of products. For example, electrolytic reduction of nitrobenzene leads to the formation of bimolecular products, such as azoxybenzene (Lipsztajn et a l . , 1974). Presumably, these products arise from the condensation of partially 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 presence of CaClg. Varghese et al. used the same conditions in their study, including the use of 80% ethanol as solvent. In the work of Kuhn, the amine product was isolated and crystallized. Varghese et al. used 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 drug. Using the conditions described by Varghese et a l . , we monitored the reduction by T L C . Initially, the reaction mixture turned greenish-yellow. TLC demonstrated the presence of at least one coloured product. After about 5 minutes, the reaction mixture became gray-brown. Spectrophotometry showed loss of the nitro group absorption above 300 nm, as stated by Varghese et al. However, TLC revealed the presence of a large number of products, including coloured and fluorescent compounds. We concluded that, under the published conditions, misonidazole was broken down to a variety of products. Indeed, the paper of Varghese et al. showed the presence of several compounds on paper chromatograms, despite the limited resolution of this technique; further, the quantity of zinc used was stoichiometrically insufficient to reduce the misonidazole to the amine, even assuming complete conversion of Zn to ZnO. We concluded that the reaction ob-served was the breakdown of an intermediate in the reduction process, rather than quantitative reduction to the amine derivative. 62 We attempted to modify the conditions used, and to isolate and identify the initial products. The amount of Zn used was increased; water was used as solvent; the temperature was lowered to room temp-erature; progress of the reaction was monitored by T L C . At room temperature, the reaction proceeded only to the first stage (yellow colour). TLC of the yellow product appeared to give a single compound, but repeated attempts to obtain infra-red and NMR spectra were unsuccessful. Finally, multiple development of the TLC plates was performed, in an attempt to increase resolution. The product, which had appeared to be a single compound, was found to consist of two closely-spaced bands, yellow and orange in colour. Once this separation had been achieved, isolation and identification of the products proceeded rapidly (Josephy et a l . , 1980). Since the capacity of TLC techniques is limited, we developed a preparative reversed-phase liquid chromatography system to separate the two compounds, using a Merck "LOBAR" RP-8 column, as described in Methods. Quantities of order 100 mg were prepared using this system. Products A and B were prepared by lyophilization of the appropriate elution volumes obtained from the liquid chromatography system. Product A was deep orange, product B was bright yellow; both were quite hygroscopic. On the basis of the bright colours of these two derivatives, it appeared likely that they 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: The spectra of products A and B are shown in f ig. 10. Product A (azo-misonidazole) has a broad absorption band with peak wavelength 400 nm. Product B has a single peak, at 390 nm; the peak wavelength shifts to 386 nm in methanol, and to 379 nm in acetone. This 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 of products A and B were obtained as described in Methods. Spectra are shown in f ig. 11-13. Misonidazole shows peaks due to- the terminal side-chain carbon atoms (-CH 2 -0-CH 3 ) at 6 = 3.35-3.5; the side-chain carbons (imid.-CH 2-CH(OH)-) give a doublet and multiplet at 6 = 4.5 and 4.15; the imidazole H atoms give the doublet at 6 > 7. (Other peaks are due to solvent and water). The spectrum of product A is almost identical to that of misonidazole itself, although the peaks are rather broader and the shifts are slightly different. 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 in H^O, and show extinction coefficient as a function of wavelength. 65 Fig. 11 NMR spectrum of misonidazole Solvent: C D o 0 D 66 67 Fig. 13 NMR spectrum of azoxy-misonidazole Solvent: D o 0 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: Attempts to obtain mass spectra of the products were unsuccessful, presumably because of insufficient volatility. Therefore, the products were derivatized to more volatile compounds by acetylation. Each product was dissolved in pyridine and treated with acetic anhydride at room temperature. The acetylation was monitored by T L C , using chloroform/methanol (95/5) as solvent. Shortly after addition of acetic anhydride, both mono- and diacetate derivatives were obtained (higher on TLC than the starting materials). Product A gave a single monoacetate, as expected from the symmetry of the azo compound. In the case of product B, two closely-spaced monoacetate bands were resolved. 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 isomers, with slightly different chromatographic characteristics. After 30 minutes, conversion to the diacetates was complete. The diacetates were recrystallized from methylene chloride and submitted for mass spectroscopy. Parent peaks were: product A diacetate: 422; product B diacetate: 438. These molecular weights are in agreement with the molecular formulae Cjgh^gOgNg and C^gHggOyNg for the azo- and azoxy-diacetate respectively. High-resolution mass spectroscopy of the azo-diacetate gave M + = 422.1908 (calc. 422.1908). 69 Elemental analysis: Elemental analysis was performed by Dr. C. Smithen and colleagues, Roche Products L td . , and the results are given here with permission. Azo derivative of misonidazole: found C 42.86 H 6.22 N 21.3% C 1 4 H 2 2 ° 4 N 6 " 3 H 2 ° requires C 42.85 H 7.19 N 21.4% Azoxy derivative of misonidazole: found C 46.17 H 6.54 N 23.35% C 1 4 H 2 2 ° 5 N 6 ' 1 " z H 2 ° r e q u i r e s c 46-28 H 6.26 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 37°, but with NH^CI in place of C aC ^ . Spectrophotometric measurements demonstrated that a product absorbing in the region of 400 nm was formed initially. This yellow product was not isolated, but, presumably, it is a mixture of azo- and azoxy-misonidazole. At a later time (about 10 minutes) the yellow colour disappeared, in agreement with our results at elevated temp-eratures. HPLC analysis of the colourless product mixture demon-70 strated the presence of at least three components. Mass spectroscopy suggested that these included the amine, hydroxylamine and hydrazo derivatives of misonidazole. The mass spectral data and related analysis have been circulated in pre-print form. 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) in our studies: aqueous conditions, room temperature. After 2 hours, the reduction products were studied by ion-exchange and reversed-phase chromatography. The compound N-(2-hydroxy-ethyl)-oxamic acid was identified as one product (although several others were formed). This product appears to be formed by the hydrolysis of a reactive reduced intermediate of metronidazole. Similar results were obtained by enzymatic reduction of metronidazole - this will be discussed in the following chapter. Clearly, metronidazole and misonidazole behave quite differently 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 reversed-phase column chromatography to separate the bimolecular reduction products of misonidazole allowed us to produce enough pure compound to test the biological activity of each. Preliminary results were presented at the conference "Combined Modality 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 of each derivative to CHO cells was measured using the system described in Methods, and used in the studies reported in chapter 3. Results of a typical experiment are shown in f ig. 14-15. Azo-misonidazole shows little toxicity in aerobic or hypoxic incubations -certainly, much less than that of misonidazole itself. In contrast, azoxy-misonidazole is more toxic than the parent drug under aerobic conditions; in hypoxia, it is also more toxic than misonidazole, at least for short times. There is a marked difference between the toxicity responses seen with misonidazole and azoxy-misonidazole. Typically, misonidazole shows a biphasic response, with an initial shoulder. Azoxy-misonidazole toxicity curves are purely exponential, and show significant cell kill after one hour or less. The results of experiments with the CH2B 2 cell line are shown in f ig . 16-17. These figures show the averaged results of three ex-periments. CH2B 2 cells are considerably more sensitive to misonidazole hypoxic toxicity than are CHO cells (Palcic & Skarsgard, 1978). In general, however, the results obtained with these cells are comparable to those 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) Fig. 14 Toxicity of bimolecular derivatives CHO cells - aerobic incubation O - control; V - azo-misonidazole; A - misonidazole; • - azoxy-misonidazole. All drugs 5 mM. 73 CHO CELLS: HYPOXIC ' I i I i L 2 4 6 8 TIME (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. % 74 CH2B2 CELLS: AEROBIC control i i i \ , i \ 0 2 4 6 TIME (hours) Fig. 16 Toxicity of bimolecular derivatives CH2B 0 cells - aerobic incubation • - control; f - azo-misonidazole; • - misonidazole; • - azoxy-misonidazole. All drugs 5 mM. 75 CH2B2 CELLS : HYPOXIC ' • i I I I I I L 0 2 4 6 8 TIME (hours) Fig. 17 Toxicity of bimolecular derivatives CH2B,, cells - hypoxic incubation • - control; • - azo-misonidazole; • - misonidazole; • - azoxy-misonidazole. All drugs .5 mM. 76 CH2B2 C E L L S 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 (aerobic and hypoxic) 9 induced fewer than 0.2 breaks per 10 daltons in 4 hours. 77 SSB) by these compounds, in CH2B2 cells (fig. 18). These results parallel the survival curve measurements. Azoxy-misonidazole shows remarkably 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. C. Smithen and colleagues, Roche Products L td . , have tested these compounds in another system. Chinese hamster V-79-379A cells are grown in the presence of the drug (chronic aerobic toxicity), and the inhibition of protein synthesis is measured. 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 group. We may arrange them in order of increasing toxicity as: azo < nitro < azoxy. There appears to be no correlation of toxicity with electron-affinity among these compounds, at least as measured by polarographic half-wave reduction potentials. These values have been measured by Dr. C. J . Little and Dr. C. Smithen, using samples supplied by us; the values were as follows (0.1M compounds dissolved in phosphate buffer, pH 7.4 - mV vs Saturated Calomel Electrode (SCE)): misonidazole azo-misonidazole azoxy-misonidazole 385 mV 150 180 78 As discussed earlier, such values are only a rough guide to the thermodynamically reversible electron-affinities. Nonetheless, there is no correlation between these values and the observed toxicity. The bimolecular derivatives of misonidazole showed very little radiation-sensitizing ability, 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 examined... . 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 et a l . , 1968) it was stated that: 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 hydrochlorides... The details of the "cautious" reduction procedure are not described; however, as mentioned above, zinc reduction of metronidazole gives very different results from reduction of misonidazole. There is no initial yellow colour - instead, a brown solution is produced, with a characteristic odour. We were unable to characterize this material, but it does not appear to be a pure compound. It may be a mixture of breakdown products analogous to those obtained by high-temperature reduction of misonidazole. Since, with metronidazole, the same results are obtained even at low 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. R.C. Urtasun, Cross Cancer Institute, Edmonton, provided a series of urine and serum samples obtained from patients receiving misonidazole, and we examined these for the formation of bimolecular metabolites. These specimens also showed red-brown coloration, which was most pronounced in samples taken 8-12 hours after drug administration. However, these pigments are water-soluble and could not be extracted into organic solvents. Direct HPLC examination of the samples, using conditions suitable for the detection of azo- and azoxy-misonidazole, gave uniformly negative results. Thus, as with metronidazole, the nature of these products remains unknown. 80 100 v. 10 < > > DC D CO * 1 0 1 \ '•• * * X \ **• * \r> \ * A *• " \ CHO Cells \ • • \ \ • * \ • Azoxy 10mM \ * \ \ a- \ • Azo 5mM \ \ X \ * ^ ^ ^ \ \ \ • \ l * \ 1 \ \ " 1 • t \ \ \ '•• \ N 2 \MISO\lmM AIRl \ • •. L- • ' 10 20 DOSE (Gy) 30 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 "AIR". 81 BIOCHEMICAL REDUCTION OF MISONIDAZOLE AND ITS AZO- AND AZOXY- DERIVATIVES: 5.1 Introduction: The reduction of nitroaromatic compounds by microorganisms was observed early in this century (Neuberg & Welde, 1914); I have discussed the mechanism of bacterial nitroreduction in the introduction. There were few studies of mammalian nitroreduction until the recent increase in the use of nitro drugs. Reduction of trinitrotoluene (TNT) in rabbits was studied (Channon et a l . , 1944), and in vitro tissue homogenate techniques were also used (Bueding & Jolliffe, 1946). Fouts and Brodie (1956) were the first workers to study this enzyme activity systematically, using a variety of compounds, species, organs, and subcellular fractions. Reduction of nitrobenzoic acid was 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 was also observed with NADH. The amount of reduced product formed was proportional to the concentration of drug, even up to 10 mM, sug-gesting that the enzyme systems have a very low affinity for the nitro compounds. Also, a wide variety of nitrobenzene derivatives could be reduced at comparable rates. The identity of the enzymes involved, and even the number of distinct enzymes, was not determined. Two reviews of early work on nitroreduction are available - Gillette, in Brodie & Gillette, ed . , 1971; Mitchard, 1971. 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 name, the probable biological function of this enzyme is reduction of cytochrome P-450 (Bock & Remmer, in Aldridge, ed . , 1978). Both cytochrome P-450 and the reductase are membrane-bound proteins, and the purification of the components of the cytochrome P-450 system remains problematical. 5.2 Xanthine oxidase: In contrast to the rather intractable cytochrome P-450, xanthine oxidase (XO, xanthine: oxygen oxidoreductase, EC 220.127.116.11) is a soluble enzyme which is purified very easily from a variety of sources, including milk. It was one of the first enzymes to be crystallized. The enzyme contains molybdenum, iron-sulfur groups, and FAD. A review of the structure and properties of XO has been presented (Bray, in Boyer, ed . , vol. 78, 1975). The biological significance of XO, and the closely-related enzyme aldehyde oxidase (EC 18.104.22.168) was discussed recently (Krenitsky, 1978). XO catalyses the reduction of oxygen and the simultaneous oxidation of hypoxanthine to xanthine, and in a second step, xanthine to uric acid. 83 The reducing equivalents (e + H ) are transferred to oxygen, in a mixed one- and two-electron process. The xanthine/XO system generates 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 of other reducing agents can serve as electron donors to XO, including NADH, acetaldehyde, and benzaldehyde (Bray, op_. c i t . ) . If is excluded from the system, it is possible to demonstrate the reduction of many other compounds by the xanthine/XO system. These include the azo dye methylene blue, cytochrome c ( ibid.) , purine-N-oxide (Stohrer & Brown, 1969) and the anthracycline chemotherapeutic drug adriamycin (Pan & Bachur, 1980). Taylor et al. (1951) showed that the xanthine/XO system reduces nitrofurans, under anoxic conditions. Although the products were not identified, stoichiometric measurements suggested that 4 reducing equivalents were transferred per mole of nitrofuran, consistent with reduction to the hydroxylamine level. This work was extended by Tatsumi and co-workers (1976 & 1978). In the 1976 publication, xanthine/XO reduction of nitrofurazone and AF-2 (2-(2-furyl)-3- (5-nitro-2-furyl)acrylamide) was studied. T L C , mass spectroscopy, and IR spectroscopy were used to identify products. Nitrofurazone was found to be reduced with about 6-electron stoichiometry (rather than 4- electron as reported by Taylor et al.) and the product was identified as the amine derivative. In the case of AF-2, the reduced derivative was identified, tentatively, as a re-arrangement product of the amine. 84 In the second paper, Tatsumi et al. studied the reduction of a series of nitrobenzene derivatives; the products were compared with the corresponding hydroxylamines, prepared by reduction of 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 amines..." by the enzymatic system (Tatsumi et a l . , 1978). Thus, the nitrobenzenes and nitrofurans show characteristic differences in reduction by this system; even within the nitrofurans, AF-2 and nitrofurazone gave different products. Clearly, each case must be studied individually before a general conclusion can be reached. Dr. P. Goldman and colleagues have studied the metabolism of metronidazole by bacteria isolated from the intestinal tracts of ex-perimental animals. The role of intestinal flora in drug metabolism has not been studied extensively; a review of the literature has been presented (Goldman, 1978). Most of the bacteria found in the gut are obligate anaerobes, which implies that the intestinal tract is hypoxic. Thus, gut bacteria may play an important role in nitroreduction, particularly following oral drug administration. Goldman et al. isolated a number of small molecules from the excreta of rats, following administration of labeled metronidazole. These products included acetamide (Koch et a l . , 1979a) and N-(2-hydroxyethyl)-oxamic acid (Koch et a l . , 1979b). These metabolites were not present in the excreta of germ-free rats, which implicates bacterial metabolism in their formation. 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. I shall consider these results after a presentation of our studies 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 by xanthine oxidase (XO) are in press (Josephy et a l . , 1981a) and will be summarized in this 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 ig. 20. Reduction is measured by loss of nitro group UV absorption at 350 nm. Even at the highest misonidazole concentration used (1mM), the curves were exponential rather than linear, suggesting that the Michaelis constant for misonidazole is higher than this value. No reduction was observed under aerobic conditions, in the absence of the reducing substrate (hypoxanthine), or if the enzyme had been inactivated by brief boiling. In studies using xanthine as reducing substrate, under aerobic conditons, the 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 O . Q 3 5 0 2 1A \ \ \ M I S O N I D A Z O L E \ V 20 40 M l N U T E S 60 80 Fig. 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. Thus, the nitro group of misonidazole is much less effective in accepting electrons from the enzyme than is oxygen. The rate is of the 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 HPLC was used to separate the components of the incubation mixture. We found a ratio of 2.06 moles xanthine per mole misonidazole, consistent with reduction to the hydroxylamine level (4-electron). 14 We used C-labeled drug to characterize the products of misonidazole reduction, as described in Methods. The derivative was soluble in methanol, but not in acetone or chloroform. TLC radiochromatograms showed a single band at R^ = 0.15, and a minor band at slightly higher R^ . The main band accounted for more than 90% of the activity. In this TLC system, misonidazole itself had R.p = 0.73; thus, the product is much more polar than misonidazole. The results of HPLC radiochromatography, using the polar bonded phase system described in Methods (section 2.4), are shown in f ig. 21. A single major product peak contained over 80% of the total activity. An additional sample was run, and this peak was collected and studied by mass spectroscopy. Successful results were obtained in both electron ionisation (El) 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 the modified zinc reduction procedure described in chapter 4. This compound has been tentatively identified as hydroxylamino-misonidazole. However, attempts to 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 dis-proportionation, which is reported to occur with aromatic hydroxyl-amines (Wardman, 1977). 5.4 Reduction of derivatives of misonidazole by xanthine oxidase: We have also examined the reduction of azo- and azoxy-misonidazole by xanthine/xanthine oxidase (XO), (Josephy et a l . , 1981a). The disappearance of azo-misonidazole from a hypoxic incubation mixture similar to that used for misonidazole is shown in f ig. 22 (cf. f ig. 20, misonidazole). 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 experiments showed that azo-misonidazole was formed as an intermediate in the reduction of azoxy-misonidazole. This intriguing result meant that it was difficult to measure the kinetics of the reduction spectrophotometrically, since the azo- and azoxy-misonidazole chromophores overlap. Therefore, reversed-phase HPLC was used to separate the derivatives. The enzymatic reduction was performed as for misonidazole, except that azoxy-misonidazole, 89 1 10 20 30 40 50 60 70 80 90 100 F R A C T I O N Fig. 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 HPLC, 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. 4 0 0 . 8 J .4. \ A Z O -\ M I S O N I D A Z O L E \ \ \ Y \ V 0 10 20 30 MINUTES Fig. 22 Reduction of azo-misonidazole by hypoxanthine and xanthine oxidase in hypoxia Reduction was monitored by loss of absorption at 400 nm. Initial azo-misonidazole concentration was 100 uM. See text for details of enzymatic reduction procedure. 91 1 mM, was the oxidizing substrate. Samples (100 ul) were withdrawn at regular intervals following addition of the enzyme, and placed on ice. The samples were then studied by HPLC. Column: Whatman ODS reversed-phase, 4.6 mm x 250 mm; mobile phase: 10 mM acetate buffer, pH 4.5/methanol (75/25). Optical absorption was measured at 400 nm. Retention times of misonidazole, azoxy-misonidazole, and azo-misonidazole were 3.2, 9.0, and 13.0 minutes, respectively, at a flow rate of 2.0 ml/minute. All three compounds were baseline separable. Calibration curves for azo- and azoxy-misonidazole were constructed; response (peak height vs concentration) was linear over the range studied. At equimolar concentrations, the ratio of azoxy- to azo-misonidazole peak height was 100 to 88. The disappearance of azoxy-misonidazole, and the formation and subsequent disappearance of azo-misonidazole formed during the reduction, is shown in f ig. 23. Representative chromatograms are shown in f ig. 24. The level of azo-misonidazole was never higher than about 50 uM; it is barely detectable in the right-hand chromatogram of f ig. 24. Presumably, the kinetics of azo-misonidazole formation and disappearance during the experiment result from competition between it and the starting material for reducing equivalents from the enzyme. Identification of the intermediate in the enzymatic reduction of azoxy-misonidazole as azo-misonidazole was made on the basis of the retention time and spectrum of the product, which are identical to those of authentic azo-misonidazole prepared by Zn reduction of the nitro compound. The nature of the final product was not studied, but, presumably, it is the same as the product of azo-misonidazole reduction. 92 k A Z O X Y - M I S O N I D A Z O L E REL. PEAK HT. • \ • AZOXY 30 60 90 MINUTES Fig. 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 HPLC. Initial azoxy-misonidazole concentration was 1.0 mM. See text for further details. 93 A.U. 1 400 nm 1 K J j . 8 16 0 8 16 M I N U T E S Fig. 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 min.; 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 Radiation Research Society, New Orleans, June 1980. In a paper presented during the same session, Dr. D. Whillans described the reduction of these compounds by a radiation chemical technique (Whillans & Whitmore, 1980; 1981). In these studies, misonidazole was prepared in dilute aqueous solution, at neutral pH, in the presence of 100 mM Na formate. The solutions were de-oxygenated and y-irradiated. In this system, the H' and OH' radicals produced by radiolysis of water are scavenged by formate to produce COr, ; this species, and e , reduce the drug. Stoichiometry of the reduction can aq be calculated from the appropriate G-values. Misonidazole was reduced with 4-electron stoichiometry, and over 80% of activity was found in a single HPLC peak. This product appears 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 system. Azoxy-misonidazole was reduced via azo-miso-nidazole, to a colourless product. This product was suggested to be hydrazo-misonidazole, on the basis of mass spectroscopy. Dr. Goldman and colleagues studied the reduction of metronidazole catalysed by XO, concurrently with our own work on misonidazole. This research was reported recently (Chrystal et a l . , 1980). It appears that the two nitroimidazoles behave very differently in this 95 system, as well as in the Zn reduction. Instead of a single product, Chrystal et al. isolated a wide variety of small molecules from the incubation mixture, reminiscent of the results of Zn reduction. The products formed, and a schematic pathway for their formation, are shown in the following figure (from Chrystal et a l . , 1980): OH NH 0. -OH NHj OH OH OH x N H „ / N . NH \ NH 2 « \— 2 N > NH 2 r 1 ' _ o ' N HjN 0 0 HO 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 (Goldman et a l . , 1980) it was suggested that bimolecular products may be formed during the XO - catalysed reduction of metronidazole. The authors stated that: 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 derivatives formed during the XO - catalysed reduction of nitrobenzene derivatives: In the case of p-nitrobenzenesulfonamide, a small amount of azoxy-compound was detected by TLC 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 recent paper, Clarke et al. (1980) have examined the reduction of a variety of nitroimidazoles by reduced FMN and 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 MISONIDAZOLE: 6.1 Introduction: In the preceding two chapters, I discussed the reduction of miso-nidazole by chemical and biochemical techniques. These studies shed light on the nature of the reduced derivatives of the drug, and iden-tified a possible enzymatic mechanism for misonidazole reduction in hypoxic mammalian cells. In this chapter, I shall describe our studies 14 of the metabolism of C-misonidazole in CHO cells (Josephy et a l . , 1981b). First, however, I shall describe briefly, some previous studies along these lines. Much of this work overlaps studies of the chemical reduction and toxicity of misonidazole, described earlier. Varghese et al. (1976) reduced misonidazole with Zn; this study was discussed, in part, 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 drug, intraperitoneally, into mice bearing the tumour. The cells were isolated and homogenized, and the water-soluble supernatants were studied, using the paper chromato-graphy system with which the Zn reduction products were analysed. Hypoxic cells in vitro converted misonidazole into several products of lower R^ than the parent drug. Similar product peaks were found in both tumour and normal tissues of the i.p.-injected mice. It was suggested that these metabolites included the O-demethylation product (desmethylmisonidazole, Ro 05-9963) as well as a glucuronide conjugate. Another product ("peak 2") was thought to be the amine derivative, on 98 the basis of co-chromatography with the Zn reduction product. However, as discussed in chapter 4, there is doubt about the identity of the Zn reduction products obtained using the method described in this paper. The 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 Rauth (1978) studied the toxicity and metabolism of misonidazole in CHO cells and human (HeLa) 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). Taylor and Rauth showed that 1 4C-misonidazole activity is accumulated in hypoxic, but not aerobic cells, as a function of incubation time. The hypoxic cells showed production of several metabolite peaks. In a later paper (Taylor & Rauth, 1980a) this work was extended. Ascorbate, cysteamine, 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. The sulfhydryl agents also enhanced production of certain metabolites, an interesting result in view of the controversy over the effects of these agents on toxicity. Again, however, the metabolite peaks were not identified. The entire water-soluble cell extract was run together, without fractionation according to organic-solubility. 99 In vitro metabolism studies yield very small quantities of metabolites. For example, in the chromatograms published by Taylor and Rauth (e.g. chart 7, 1978) the largest peak of intracellular metabolite activity is little higher than the free misonidazole peak, even following 5 hour exposure to 5 mM drug. 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 in, say, 5 x 1o7 CHO cells, at 5 mM, is only about 100 ugm. Thus, the amount of any particular metabolite that could be recovered in this sytem is very small. The studies reported in the previous chapter allowed us to characterize a reduced derivative of misonidazole in various chro-matography systems; 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 used in these studies are described in chapter 2 (Methods). Several principles 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. The labeled drug was used without dilution, to obtain the highest possible activity. Second, recovery of metabolites was maximized by the use of very dense cell suspensions, so that all of the added drug would be 100 converted to metabolites. Th i rd , an extraction procedure was used to partially purify the anticipated metabolite on the basis of its known solubility properties. Finally, we used HPLC to separate the metabolites. Cells were incubated with labeled drug under hypoxia, and aliquots were removed at various times. The cells were disrupted and fractionated into three extracts, according to the methods described in chapter 2. These extracts were the organic-soluble, acid-soluble, and acid-insoluble fractions. Fig. 25 shows the distribution of total radioactivity among these fractions. As a function of incubation time, organic-soluble activity (misonidazole) is converted to organic-soluble and organic-insoluble products. Most of the latter material is acid-extractable, but some is associated with the acid-insoluble pellet. Even after all free misonidazole has disappeared (about 3 hours incubation) the percentage of radioactivity associated with the pellet is less than 10% of the total. Further incubation beyond 3 hours produced little change in the distribution. The nature of the acid-soluble material has not been studied in detail. These metabolites are highly polar (R.^ almost zero on TLC ) and may include ionic conjugates. The pellet contains activity bound (probably covalently) to nucleic acid and protein. Similar results have been observed both in vitro and in vivo (Varghese & Whitmore, 1980). The organic-soluble fractions obtained at successive time points were studied by TLC and HPLC as described in chapter 2. The results of TLC autoradiography are shown in f ig. 26. Misonidazole (R f = 0.78) 101 100 80 D I S T R I B U T I O N O F 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 % T O T A L C P M 40 20-1 ORGANIC soluble ORGANIC insoluble ACID soluble PELLET 0 1 2 3 0 1 2 3 0 1 2 3 I N C U B A T I O N TIME ( H O U R S ) Fig. 25 Distribution of radioactivity - 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 TLC plate, which was then autoradiographed on Kodak X-ray film for ten days. From left: 2 min., 1 h r . , 2 h r . , 3 hr. incubation. Misonidazole is indicated by letter M. Dashed lines mark R f = 0 and 1. 103 is depleted by about 50% in one hour and completely metabolised in 3 5 hours. This corresponds to the conversion of about 10 molecules per cell per second. 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 ig. 27. The data shown in this figure parallel the results obtained by T L C . Misonidazole (which is eluted between fractions 60 and 80) is depleted during incubation, and converted to a variety of more polar products (shorter retention times). After an incubation time of as little as two minutes (Fig. 27a) two products are detectable. After one hour incubation, a complex pattern of metabolites is seen; as with the TLC results, further incubation affects the peak heights, but has little effect on the pattern. The largest peak is at or near fraction #21. This component was shown to co-chromatograph with the product of xanthine oxidase - catalysed reduction of misonidazole, which is believed to be hydroxylamino-misonidazole. This was demonstrated by the dual-label chromatography technique described in chapter 2. Results are presented in f ig. 28. 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 with the enzymatically reduced product. This was particularly clear in system B; apparently, some of the metabolites 104 isolated along with the hydroxylamino product in system A are almost insoluble in ethyl acetate/methanol (used in system B). The material in the product peak in system B was collected, concentrated, and run on T L C . Fractions were scraped, eluted with methanol, and counted. 14 3 Again, C and H activity ran together (data not shown). 6.3 Conclusions: In chapters 4 and 5 of this thesis, I discussed the reduction of misonidazole by a variety of systems. Chemical reduction of the drug using zinc metal, led to the formation of bimolecular derivatives; however, these do not appear to be formed in biological systems. The enzymatic reduction of misonidazole (chapter 5), and radiation chemical reduction (Whillans & Whitmore, 1980) yield a single major product with 4-electron stoichiometry. This 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. This metabolite can be detected after a very brief exposure to misonidazole (fig. 27a) and, following the disappearance of the parent drug, it remains the major organic-soluble metabolite. 14 The metabolism of C-misonidazole has been studied previously (Varghese et a l . , 1976; Taylor & Rauth, 1978, 1980a,b; Whitmore et a l . , 1978). In this work, we have used labeled drug with more than 100 times higher specific activity. Also, we have separated organic-soluble material from the cell extracts, prior to chromatography. 105 The organic-insoluble, acid-soluble material represents at least half of the metabolite activity; since this material is very polar, it would elute early on a reversed-phase column, and might obscure the metabolites seen in f ig. 27 and 28. We 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 Fig. 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 D U A L - L A B E L C H R O M A T O G R A P H Y Fig. 28 HPLC dual-label radiochromatograms CHO cells were incubated under hypoxia with C-misonidazole, as described in text. Extracted samples were combined with aliquots of 3 reduced misonidazole ( H label) prepared by xanthine oxidase -catalysed reduction (see text). Combined samples were run on reversed-phase HPLC (a) or polar bonded phase HPLC (b). Fractions 3 14 were collected, counted for H and C activity, and corrected for 3 background and spillover. H data are offset vertically for clarity, and shown as dashed lines, a: 2 hour incubation (same experiment as f ig . 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 of misonidazole in man, the development of new radiosensitizers with improved properties, and the possibility of exploiting cytotoxicity as a form of chemotherapy. The use of in vitro biological techniques allow us to study drug metabolism under controlled conditions; but such model systems 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) 14 studied the metabolism of C-misonidazole in various mammals, including man. The pharmacology of misonidazole in vivo has been reviewed recently (Workman, 1980). Some results will be 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 urine, as a glucuronide conjugate, and as desmethylmisonidazole. Together, these metabolites account for less than half of the admin-istered misonidazole dose (Flockhart et a l . , 1978a, Table I). Presumably, the remainder of the drug is converted to unidentified 109 products, or is bound. Flockhart et al. (1978a) state that "The remainder of the excreted metabolites has eluded characterization, mainly due to their extreme water solubility, By analogy with other nitro compounds, one possible excretion product is the amine derivative. This derivative was synthesized by catalytic hydrogenation (Flockhart et a l . , 1978a). The amine compound shows no significant toxicity |n vitro, under either aerobic or hypoxic conditions (B. Palcic, unpublished observations), but its presence serves as an indicator of reductive metabolism of the drug. Flockhart et al. (1978b) observed evidence for the presence of the amine in tumour extracts from drug-treated mice, as did Varghese (1980). Flockhart et al. concluded that: "amine formation is occurring in patients, but . . . the excretion levels do not exceed about 2% of the administered dose." Also, 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, and are further metabolised to polar compounds, or bound to macromolecules (chapter 6). This would account for the observation of highly water-soluble metabolites in the urines of patients receiving the drug, despite the relatively low levels of amine metabolite in the urine. We have presented a schematic illustrating the pathways of misonidazole metabolism (fig. 29). This scheme is similar to one presented by Taylor and Rauth (1980b). Misonidazole may be con-110 verted to polar derivatives without reductive activation, by demethylation, for example. This pathway is of pharmacokinetic importance. Reductive activation proceeds in hypoxia. The first relatively stable nitroreduction product to be formed is probably the hydroxylamine, the nitroso compound being reduced too rapidly to be detected. An analogous mechanism has been demonstrated for nitro-benzene reduction (Wardman, 1977, p. 371). The hydroxylamine may be further reduced to the amine, which appears to be a urinary meta-bolite in man. One or more reactive intermediates formed during the reduction may be responsible for binding to nucleic acid and protein, and the toxic and mutagenic properties of the drug. It is still not clear at what stage in the reduction pathway this product is formed. Its identitiy has proved elusive, due in part to the difficulty of isolating the intermediates. Indeed, the formation of dimers during zinc reduction (chapter 4) is probably a consequence of the reactivity of intermediates such as the nitroso compound. It should be noted that the pathways shown in f ig. 29 are not mutually exclusive; for example, demethylation could be followed by reduction, or reduction by conjugation. Such "twice-metabolised" products may be quantitatively predominant, particularly in the organic-insoluble, acid-soluble fraction. 111 R-NO, conjugation ^ polar metabolites (intact NC^ group) demethylation reduction (O^ inhibited or reversed) polar reduced conjugation metabolites R-N=0 (unstable?) R-NHOH Binding to macromolecules? 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 drug, remains unclear (Denekamp & McNally, 1978). Hypoxic cytotoxicity is characterised by an initial zero-slope shoulder (chapter 3); this shoulder may be recovered if hypoxic cells are subsequently exposed to air (Stratford, 1978). Brown has suggested (1979) that acutely hypoxic cells, which cycle between oxygenated and hypoxic states (see section 1.5) will be unaffected by misonidazole cytotoxicity, for these reasons. Animal studies have not resolved this question. The effects of radiosensitization and cytotoxicity may be separated by experiments in which the drug is administered after, versus before, irradiation of animals bearing tumours. Enhancement of cell killing in the animals given the drug following irradiation (compared to controls irradiated without drug) is due, presumably, to cytotoxicity rather than radiosensitization. Fowler and Denekamp (1979) have reviewed such experiments. In some cases, SER values as high as 1.4 have been obtained with post-irradiation drug administration, suggesting that cytotoxicity plays a significant role. In other experiments, no such enhancement was seen. Korbelik et al. (1980, 1981) have demonstrated that misonidazole hypoxic cytotoxicity interacts with radiation damage. Radiation decreases the duration of the zero-slope shoulder of the cytotoxicity response, in a dose-dependent manner. 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. This effect may be due to an interaction between the DNA damage caused by misonidazole and by radiation. Whatever the mechanism may be, the result is a significant enhancement of the toxicity of misonidazole to cells that have been irradiated. 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 the possible use of misonidazole, labeled at high specific activity, as a marker for hypoxic cells in tumours. This approach takes advantage of the preferential binding of misonidazole metabolites in hypoxic cells, discussed earlier. Experimental tumours were grown in a mouse (subcutaneously) which was given an i.p. dose of labeled misonidazole. Three hours later, the mouse was sacrificed, and the tumours were excised, sectioned, and examined by microscopy and autoradiography. In small tumours (1.5 mm x 0.5 mm) a clearly defined rim of hypoxic cells was observed, as a region of heavy exposure in the autoradiograph. The rim began about 10 cells in from the edge of the tumour, in accord with the Thom-linson-Gray model of chronic hypoxia. Larger tumours showed a much more complex pattern. It is likely that this phenomenon will prove to be very useful in clarifying the nature of hypoxic cells in tumours. Chapman et al. suggest that sensitizers, labeled with y-emitting radionuclides, could be used in a clinical assay for hypoxic cells. Administered to a patient in small doses, the sensitizer would be metabolically reduced and bound in hypoxic cells; these cells could then be detected by y-scanning techniques. 114 7.3 New radiosensitizers: The dose limitations imposed by the neurotoxic side effects of misonidazole have inhibited the full exploitation of the drug's radio-sensitizing property. Less 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. In addition, the more rapid urinary excretion of polar drugs lowers the total body exposure re-quired ("area under the curve" of a plot of drug concentration versus time). A variety of low-lipophilicity radiosensitizers has been syn-thesized (Brown & Workman, 1980; Astor et a l . , 1980; Adams et a l . , 1980). Also, there is renewed interest in the polar metabolite of misonidazole, desmethylmisonidazole. This derivative is much more water-soluble than misonidazole, and offers pharmacokinetic advantages (White & Workman, 1980). The high 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). Unfortunately, desmethylmisonidazole was found to cause local hemorrhaging ( ibid.) ; similar results were observed in mice, fol-lowing oral administration (Chao et a l . , 1980). Thus, these newly studied drugs may not necessarily be more useful than misonidazole itself. 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 nitro-aromatic 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 of the nitro compound has proven difficult, as described earlier. It is possible that a better route to, for example, the nitroso derivative, would be via oxidation of the bimolecular compounds. The synthesis of this compound would improve our understanding of the chemistry of the reduced derivatives of the drug, and facilitate 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. 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UBC Theses and Dissertations
Chemical and biological studies of the radiosensitizer misonidazole Josephy, P. David 1981
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