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UBC Theses and Dissertations

Radiosensitizing and toxic effects of Ro-07-0582 in hypoxic mammalian cells Moore, Brian A. 1976

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RADIOSENSITIZING AND TOXIC EFFECTS OF Ro-07-05 82 IN HYPOXIC MAMMALIAN CELLS by BRIAN A. MOORE B . S c , U n i v e r s i t y o f B r i t i s h Columbia, 1973 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES (Department of M e d i c a l Genetics) • We accept t h i s t h e s i s as conforming to the r e q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA January, 1976 In p resent ing t h i s t he s i s in p a r t i a l f u l f i l m e n t o f the requirements f o r an advanced degree at the U n i v e r s i t y of. B r i t i s h Columbia, I agree that the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e fo r reference and study. I f u r t h e r agree that permiss ion for ex tens i ve copying of t h i s t he s i s f o r s c h o l a r l y purposes may be granted by the Head of my Department or by h i s r ep re sen ta t i ve s . It i s understood that copying or p u b l i c a t i o n of t h i s t he s i s f o r f i n a n c i a l gain s h a l l not be a l lowed without my w r i t t e n permi s s ion . Department of The U n i v e r s i t y of B r i t i s h Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 ^y Date ,a , /?7C MASTER OF SCIENCE (197 6) UNIVERSITY OF B R I T I S H COLUMBIA ( D e p a r t m e n t o f M e d i c a l G e n e t i c s ) V a n c o u v e r , B.C. T I T L E : R a d i o s e n s i t i z i n g a n d T o x i c E f f e c t s o f Ro-07-0582 i n H y p o x i c Mammalian C e l l s AUTHOR: B r i a n A. M o o r e , B . S c . ( U n i v e r s i t y o f B r i t i s h C o l u m b i a ) SUPERVISOR: L.D. S k a r s g a r d , Ph.D. NUMBER OF PAGES: x i , 81 ABSTRACT: C e l l s e x p e r i e n c i n g a l o w o x y g e n t e n s i o n show r e l a t i v e r e s i s t a n c e t o t h e l e t h a l e f f e c t s o f r a d i a t i o n . I t i s b e l i e v e d t h a t t h e e f f e c t i v e n e s s o f t h e t r e a t m e n t o f c e r t a i n human t u m o r s i s h i n d e r e d by t h e e x i s t e n c e o f s u c h r a d i o r e s i s t a n t c e l l s w i t h i n t h e t u m o r . The p u r p o s e o f t h i s w o r k was t o s t u d y t h e d r u g Ro-07-0582 b o t h f o r i t s t o x i c e f f e c t s a n d i t s a b i l i t y t o p r e f e r e n t i a l l y s e n s i t i z e h y p o x i c c e l l s t o t h e l e t h a l e f f e c t s o f r a d i a t i o n ( r a d i o s e n s i t i z e ) . T h e s e p r o p e r t i e s w e r e e x a m i n e d i n v i t r o i n two C h i n e s e h a m s t e r c e l l l i n e s , CHO and C H 2 B 2 , a n d a l s o i n t h e mouse t u m o u r c e l l l i n e EMT6. Ro-07-0582 i s shown t o ha v e a c h e m o t h e r a p e u t i c p o t e n t i a l i n t h a t i t d e m o n s t r a t e s a v e r y s e l e c t i v e t o x i c i t y f o r h y p o x i c c e l l s a f t e r a few h o u r s e x p o s u r e . I t i s much l e s s t o x i c t o a e r o b i c c e l l s . T h e s e t o x i c p r o p e r t i e s w e r e s t u d i e d e x t e n s i v e l y , b o t h i n h y p o x i c a n d a e r o b i c c e l l s u s p e n s i o n s . The m e a s u r e d e n d p o i n t was t h e a b i l i t y o f a c e l l t o m u l t i p l y a n d f o r m a c o l o n y o f 50 o r more c e l l s w i t h i n an a l l o t t e d i n c u b a t i o n t i m e . H y p o x i c t o x i c i t y was g r e a t e r a t 37°C t h a n a t 22°C a n d was a f f e c t e d b y s m a l l c h a n g e s (~30ppm) i n 0^ c o n c e n t r a t i o n i n t h e c e l l s u s p e n s i o n . The t o x i c e f f e c t s w e r e s i m i l a r i n t h e t h r e e c e l l l i n e s . The r a d i o s e n s i t i z i n g c a p a b i l i t y o f Ro-07-0582 was d e t e r m i n e d by m e a s u r i n g t h e Dose M o d i f y i n g F a c t o r s (DMF's) f o r v a r i o u s d r u g c o n c e n t r a t i o n s w i t h e a c h c e l l l i n e . DMF's w e r e c a l c u l a t e d by c o m p a r i s o n o f s u r v i v a l c u r v e s f o r c e l l s u s p e n s i o n s i r r a d i a t e d u n d e r h y p o x i a i n t h e p r e s e n c e o f d r u g w i t h t h e s u r v i v a l c u r v e f o r c e l l s u s p e n s i o n s i r r a d i a t e d u n d e r h y p o x i a i n t h e a b s e n c e o f d r u g . The DMF f o r t h e i r r a d i a t i o n o f a e r o b i c c e l l s i n t h e a b s e n c e o f d r u g i s c a l l e d t h e Oxygen E n h a n c e m e n t R a t i o (OER) a n d was a p p r o x i m a t e l y 3.0 i n a l l t h r e e c e l l l i n e s . Ro-07-0582 was f o u n d t o s e l e c t i v e l y r a d i o s e n s i t i z e h y p o x i c c e l l s i n s u s p e n s i o n w i t h h i g h e f f i c i e n c y . F o r e a c h c e l l l i n e , s e n s i t i z a t i o n was o b s e r v e d w i t h d r u g c o n c e n t r a t i o n s a s l o w a s O.lmM, w h i l e c o n c e n t r a t i o n s o f lOmM o r g r e a t e r y i e l d e d DMF's w i t h i n t h e m e a s u r e d r a n g e o f OER v a l u e s . The p r e s e n c e o f ImM Ro-07-0582 duri n g i r r a d i a t i o n o f hypoxic c e l l s y i e l d s a DMF of 1.8. I n t r o d u c t i o n of the drug b e f o r e o r a f t e r i r r a d i a t i o n , i n s t e a d o f d u r i n g i r r a d i a t i o n , had l i t t l e i f any e f f e c t . R a d i o s e n s i t i z a t i o n measurements were a l s o c a r r i e d out a t hig h c e l l c o n c e n t r a t i o n s ( c e l l p e l l e t s ) , where many s e n s i t i z e r s are i n e f f e c t i v e . R e s u l t s showed t h a t the 0582 r a d i o s e n s i t i z a t i o n a t t a i n e d i n c e l l p e l l e t s i s q u i t e comparable with t h a t a t t a i n e d i n d i l u t e suspension. The a t t r i b u t e s o f Ro-07-0582 as a p o t e n t i a l r a d i o s e n s i -t i z e r were c o n s i d e r e d . The s e n s i t i z a t i o n achieved by Ro-07-0582 i s very good, and surpasses t h a t of metronidazole, a chemical under study f o r c l i n i c a l use. For drug doses necessary to achieve high l e v e l s o f s e n s i t i z a t i o n the t o x i c i t y o f Ro-07-0582 to a e r o b i c c e l l s i s q u i t e a c c e p t a b l e . The t o x i c i t y to hypoxic c e l l s , however, i s much i n c r e a s e d over the t o x i c i t y to a e r o b i c c e l l s , and t h i s may prove t o be a u s e f u l adjunct to the drug's s e n s i t i z i n g p r o p e r t i e s i n d e s t r o y i n g hypoxic tumour c e l l s . i v TABLE OF CONTENTS Page 1. INTRODUCTION 1 1.1 Cancer and R a d i a t i o n Therapy 1 1.2 R a d i a t i o n Damage 4 1.3 Mechanisms of R a d i o s e n s i t i z a t i o n 7 1.4 R a d i o s e n s i t i z e r s 12 2. MATERIALS AND METHODS 14 2.1 T i s s u e C u l t u r e Techniques 14 2.1.1 C e l l L i n e s 14 2.1.2 CH2B 2 C e l l C u l t u r e 14 2.1.3 CHO C e l l C u l t u r e 15 2.1.4 EMT6 C e l l C u l t u r e . 16 2.2 R a d i a t i o n Source 17 2.3 C e l l Suspension Experiments 18 2.3.1 P r e p a r a t i o n of the C e l l Suspension . . . . 18 2.3.2 Hypoxic and A e r o b i c C o n d i t i o n s 20 2.3.3 P l a t i n g and Colony Formation 21 2.3.4 P l a t i n g E f f i c i e n c y and S u r v i v i n g F r a c t i o n 2 2 2.3.5 S u r v i v a l Curves and Dose M o d i f y i n g F a c t o r s 22 2.4 C e l l P e l l e t S u r v i v a l Experiments 24 2.4.1 Formation of the C e l l P e l l e t . . . . . . . 24 v TABLE OF CONTENTS (continued) Page 2.4.2 Ir r a d i a t i o n and C e l l Survival of P e l l e t Samples 24 2.5 Ro-07-0582 27 RESULTS 29 3.1 Tox i c i t y Tests With Ro-07-0582 29 3.1.1 CH2B2 C e l l Line 29 3.1.1.1 Toxic i t y of 0582 to Aerobic and Hypoxic CH2B.J C e l l s i n Suspension at 37°C . . 7 29 3.1.1.2 Temperature Dependence of Acute To x i c i t y Under Hypoxic Conditions 32 3.1.1.3 Hypoxic T o x i c i t y Using Prepurified Nitrogen . 33 3.1.2 Tox i c i t y of 0582 i n Aerobic and Hypoxic CHO C e l l s i n Suspension at 37°C . . . . . 36 3.1.3 Toxic i t y of 0582 i n Aerobic and Hypoxic EMT6 C e l l Suspensions 36 3.2 Treatment With 0582 42 3.2.1 Treatment of CH2B„ C e l l s i n Suspension With 0582 . . . 7 42 3.2.2 Treatment of CHO C e l l s i n Suspension With 0582 . , . 49 3.2.3 Treatment of EMT6 C e l l s i n Suspension With 0582 52 3.2.4 Treatment of C e l l P e l l e t s With 0582 . . . 52 3.3 Pretreatment and Post-treatment With 0582 . . . . 61 3.3.1 Pretreatment With 0582 61 v i TABLE OF CONTENTS ( c o n t i n u e d ) 3.3.2 P o s t - t r e a t m e n t W i t h 0582 . . Page 61 4. DISCUSSION 65 4.1 T o x i c i t y o f Ro-07-0582 65 4.2 R a d i o s e n s i t i z a t i o n W i t h Ro-07-0582 70 4.2.1 P r e - and P o s t - t r e a t m e n t 70 4.2.2 R a d i o s e n s i t i z a t i o n i n D i l u t e S u s p e n s i o n . 71 4.2.3 R a d i o s e n s i t i z a t i o n o f C e l l P e l l e t s . . . . 73 4.3 Ro-07-0582 As a P o t e n t i a l R a d i o s e n s i t i z e r . . . . 75 BIBLIOGRAPHY 77 v i i . LIST OF FIGURES F i g u r e T i t l e Page 1 Schematic R e p r e s e n t a t i o n of Proposed 10 Mechanisms of R a d i o s e n s i t i z a t i o n and R a d i o p r o t e c t i o n 2 (a) The G l a s s I r r a d i a t i o n V e s s e l i n which 19 C e l l Suspensions were I r r a d i a t e d (b) The G l a s s V e s s e l f o r C e l l Suspension 19 T o x i c i t y T e s t s 3 The V e s s e l Used f o r C e l l P e l l e t Experiments 25 28 4 S u r v i v a l Curves f o r Hypoxic CH2B„ C e l l s I r r a d i a t e d i n F r e s h or Old Ro-07-0582 5 Percent P l a t i n g E f f i c i e n c y of A e r o b i c CH2B 2 C e l l s as a F u n c t i o n of Incubation Time a t 37°C i n 5, 10, 25 or 50 mM Ro-07-0582 Percent P l a t i n g E f f i c i e n c y of Hypoxic CH2B 2 C e l l s as a F u n c t i o n of Incubation Time at 22.5°C i n 0, 1.0, 5, or 50 mM Ro-07-0582 30 Percent P l a t i n g E f f i c i e n c y of Hypoxic CH2B 2 31 C e l l s as a F u n c t i o n of Incubation Time a t 37°C i n 0, 1.0, 5, 15, or 50 mM Ro-07-0582 34 Percent P l a t i n g E f f i c i e n c y of CH2B 2 C e l l s 35 Gassed w i t h P r e p u r i f i e d N i t r o g e n , Shown as a F u n c t i o n of Incubation Time a t 37°C i n 0, 1, 5, or 50 mM Ro-07-0582 Percent P l a t i n g E f f i c i e n c y of A e r o b i c CHO C e l l s 37 as a F u n c t i o n of Incubation Time at 37°C i n 0, 1.0, 15, or 50 mM Ro-07-0582 10 Percent P l a t i n g E f f i c i e n c y of Hypoxic CHO C e l l s 38 as a F u n c t i o n of Incubation Time a t 37°C i n 0, 1.0, 5, 15, or 50 mM Ro-07-0582 11 Percent Pl a t i n g E f f i c i e n c y of Aerobic EMT6 C e l l s 40 as a Function of Incubation Time at 37°C i n 0, 1.0, 5, 15 or 50 mM Ro-07-0582 v i i i LIST OF FIGURES (continued) F i g u r e T i t l e Page 12 Percent P l a t i n g E f f i c i e n c y of Hypoxic EMT6 41 C e l l s as a F u n c t i o n of Incubation Time a t 37°C i n 0, 1.0, 5, 15, or 50 mM Ro-07-0582 13 The OER f o r CH2B 2 C e l l s 4 3 14 S u r v i v a l Curves f o r CH2B_ C e l l s I r r a d i a t e d 45 a t 22°C i n the Presence of N i t r o g e n , N i t r o g e n P l u s 0.1, 1.0, or 15 mM Ro-07-0582, or Oxygen 15 S e n s i t i z a t i o n and T o x i c i t y of Ro-07-0582 wit h 46 CH2B„ C e l l s i n Suspension as a F u n c t i o n o f Drug C o n c e n t r a t i o n 16 S u r v i v a l Curves f o r CH2B 2 C e l l s I r r a d i a t e d a t 48 22°C i n the Presence of Oxygen or Oxygen Pl u s 100 mM Ro-07-0582 17 S u r v i v a l Curves f o r CHO C e l l s I r r a d i a t e d a t 5 0 22°C i n the Presence of N i t r o g e n , N i t r o g e n P l u s 0.08, 0.6, 3, or 25 mM Ro-07-0582, or Oxygen 18 Ro-07-0582 S e n s i t i z a t i o n o f CHO C e l l s i n 51 Suspension, Shown as a F u n c t i o n of Drug C o n c e n t r a t i o n 19 S u r v i v a l Curves f o r EMT6 C e l l s I r r a d i a t e d a t 53 22°C i n the Presence of N i t r o g e n , N i t r o g e n P l u s 0.09, 0.45, or 2.2 mM Ro-07-0582, or Oxygen 20 Ro-07-0582 S e n s i t i z a t i o n o f EMT6 C e l l s i n 54 Suspension, Shown as a F u n c t i o n of Drug C o n c e n t r a t i o n 21 S u r v i v a l ' Curves f o r CH2B- C e l l s I r r a d i a t e d a t 56 22°C While i n a Suspension Gassed w i t h N i t r o g e n , i n a Suspension Gassed wi t h H u m i d i f i e d N i t r o g e n , or as a C e l l P e l l e t Gassed w i t h H u m i d i f i e d N i t r o g e n 22 S u r v i v a l Curves f o r CHO C e l l s I r r a d i a t e d a t 57 22°C While i n a Suspension Gassed wi t h H u m i d i f i e d N i t r o g e n or as a C e l l P e l l e t Gassed wit h H u m i d i f i e d N i t r o g e n ix LIST OF FIGURES (continued) F i g u r e T i t l e Page 23 S u r v i v a l Curves f o r Hypoxic CH2B„ C e l l s i n 58 P e l l e t Form I r r a d i a t e d a t 2 2°C i n the . Presence of 0, 0.48, or 4.8 mM 0582 24 Ro-07-0582 S e n s i t i z a t i o n of Hypoxic CH2B 2 60 C e l l s i n P e l l e t Form as a F u n c t i o n of C o n c e n t r a t i o n of Ro-07-0582 25 S u r v i v a l Responses f o r CH2B 2 C e l l s (a) 62 I r r a d i a t e d a t 22°C i n the Presence of N i t r o g e n or (b) I r r a d i a t e d F o l l o w i n g Pretreatment f o r 1 Hour a t 22°C With 10 mM Ro-07-0582 26 S u r v i v a l Responses f o r CH2B 2 C e l l s Subjected 64 to a 1 Hour Post-treatment w i t h 0, 10, or 30 mM Ro-07-0582 a t 22°C, F o l l o w i n g I r r a d i a t i o n Under Hypoxic C o n d i t i o n s i n the Absence of 0582 ACKNOWLEDGEMENTS I wish to express my g r a t i t u d e t o Dr. L.D. Skarsgard, under whose s u p e r v i s i o n t h i s p r o j e c t was c a r r i e d out. I would l i k e t o thank Dr. B. P a l c i c f o r h i s h e l p f u l suggestions i n the course o f t h i s work. I am g r a t e f u l t o Dr. C. Gregory f o r s u p p l y i n g the EMT6 c e l l s . I am t h a n k f u l t o my c o l l e a g u e , B i l l Hohman, f o r our many i n f o r m a t i v e and r e f r e s h i n g d i s c u s s i o n s . I am indebted t o Mrs. I. H a r r i s o n and Mrs. R. Dudley f o r t h e i r v a l u a b l e t e c h n i c a l a s s i s t a n c e . And f i n a l l y , s p e c i a l thanks t o P a t t y F i l a n f o r her a s s i s t a n c e , encouragement and gre a t p a t i e n c e d u r i n g the p r o j e c t and f o r her t h o u g h t f u l n e s s i n t y p i n g t h i s t h e s i s . xi 1. INTRODUCTION 1.1 CANCER AND RADIATION THERAPY The e f f e c t i v e n e s s of the treatment of c e r t a i n human tumors by r a d i a t i o n i s b e l i e v e d to be hindered by the e x i s t e n c e of r e l a t i v e l y r a d i o r e s i s t a n t c e l l s w i t h i n the tumors. T h i s r a d i o r e s i s t a n c e i s due to the low oxygen t e n s i o n which a r i s e s i n the tumor i n t e r i o r when i t outgrows the surrounding v a s c u l a r supply. The r e l a t i v e r a d i o r e s i s t a n c e o f c e l l s with a low oxygen t e n s i o n as compared to w e l l oxygenated c e l l s , a phenomenon r e f e r r e d to as the "oxygen e f f e c t " , r e p r e s e n t s a c o n s i d e r a b l e problem to r a d i o t h e r a p y s i n c e the maximum tumor dose i s l i m i t e d by the t o l e r a n c e of the surrounding h e a l t h y , oxygenated t i s s u e . I t i s t h e r e f o r e d e s i r a b l e t o o b t a i n some means of r e d u c i n g the oxygen e f f e c t . A c l i n i c a l approach t o t h i s problem has been the use of h y p e r b a r i c oxygen tanks. In t h i s technique, r a d i o t h e r a p y i s performed while the p a t i e n t i s exposed to oxygen a t 3 atmospheres p r e s s u r e . The aim i s to i n c r e a s e the d i s t a n c e t h a t oxygen d i f f u s e s from the c a p i l l a r i e s and thus improve the oxygenation of hypoxic r e g i o n s of the tumor. However, r e s u l t s of c l i n i c a l t r i a l s so f a r have shown the cure r a t e to be improved on l y s l i g h t l y , so a t t e n t i o n has now s h i f t e d from t h i s approach. 2. The use o f n e u t r o n beams i n c a n c e r t h e r a p y has a l s o grown i n r e c e n t y e a r s . The ma jo r advantage o f n e u t r o n s i s a r educed oxygen e f f e c t r e l a t i v e t o t h a t o f y - r a y s ( B r oe r s e and Ba rend sen , 1 9 6 6 ) . I t has been s ugge s ted by Thoml i n son ( 1 9 7 1 ) t h a t w h i l e n e u t r o n s w i l l p r o b a b l y n o t , i n g e n e r a l , p r o v i d e a l a r g e improvement o v e r x - r a y s o r y - r a y s , t h e r e a r e l i k e l y t o be s p e c i a l s i t u a t i o n s where n e u t r o n s w i l l g r e a t l y i n c r e a s e t h e p r o b a b i l i t y o f c u r e . As w e l l , he s u gge s t s t h a t n e u t r o n s wou ld g e n e r a l l y be o f g r e a t e s t v a l u e a t t he s t a r t o f t h e r a p y when b o t h t h e number and p e r c e n t a g e o f h y p o x i c c e l l s i s maximum. The i n c r e a s e d k i l l i n g o f h y p o x i c c e l l s by n e u t r o n s as compared t o x - r a y s o r y - r a y s wou ld r e s u l t i n an i n c r e a s e d a v a i l a b i l i t y o f oxygen t o t he s u r v i v i n g tumor c e l l s , t hu s s t a r t i n g t h e p r o c e s s o f r e o x y g e n a t i o n . The e x t e n t o f r e o x y g e n a t i o n wou ld d e t e r m i n e t he advantage o f f u r t h e r n e u t r o n doses o ve r x - r a y o r y - r a y d o s e s . A s i d e f r om t h e p o s s i b l e a d v a n t a g e s , t h e expense o f n e u t r o n g e n e r a t o r s i s p r e s e n t l y a l i m i t i n g f a c t o r i n t he a p p l i c a t i o n o f f a s t n e u t r o n s t o c a n c e r r a d i o t h e r a p y . A t h i r d app roach t o r e d u c i n g t he oxygen e f f e c t i s t o f i n d c h e m i c a l r a d i o s e n s i t i z e r s w h i c h i n c r e a s e t h e s e n s i t i v i t y o f h y p o x i c c e l l s t o a l e v e l nea r o r e q u a l t o t h a t o f o xygena ted c e l l s . The c r i t e r i a employed by t ho se s e a r c h i n g f o r c h e m i c a l r a d i o s e n s i t i z e r s a r e : 1) t h e a b i l i t y t o p r e f e r e n t i a l l y s e n s i t i z e h y p o x i c c e l l s t o t he l e t h a l e f f e c t s o f i o n i z i n g r a d i a t i o n , 2) a c c e p t a b l e t o x i c i t y , 3. 3) metabolic s t a b i l i t y , so that the s e n s i t i z e r i s not inactivated p r i o r to i r r a d i a t i o n , 4) suitable c e l l ' c y c l e dependence, so that hypoxic c e l l s which may have stopped at some stage of the c e l l cycle are e f f e c t i v e l y sensitized. 4. 1.2 RADIATION DAMAGE When l i v i n g c e l l s are i r r a d i a t e d w i t h i o n i z i n g r a d i a t i o n such as x-rays or Y-rays, i n t e r a c t i o n s w i t h molecules w i t h i n the c e l l s r e s u l t i n e j e c t i o n of e n e r g e t i c e l e c t r o n s . These e l e c t r o n s q u i c k l y t r a n s f e r t h e i r energy to surrounding molecules through f u r t h e r i o n i z a t i o n s and e x c i t a t i o n s . " D i r e c t e f f e c t " i s the term g i v e n to any i o n i z a t i o n or e x c i t a t i o n which r e s u l t s i n the d i r e c t chemical a l t e r a t i o n (damage) of some c e l l u l a r molecule. The l a r g e water content of the mammalian c e l l accounts f o r 70-80% of the c e l l ' s t o t a l energy a b s o r p t i o n . T h i s energy a b s o r p t i o n r e s u l t s i n the breakage of water molecules i n t o very r e a c t i v e f r e e r a d i c a l s , the most important of which are OH*, H* and e . Some of these f r e e r a d i c a l s r e a c t w i t h aq t a r g e t molecules, r e s u l t i n g i n t a r g e t f r e e r a d i c a l f o r m a t i o n . T h i s type of damage i s s a i d to r e s u l t from i n d i r e c t e f f e c t s . Once the t a r g e t molecule i s damaged, whether by d i r e c t or i n d i r e c t e f f e c t , f u r t h e r r e a c t i o n s may make the a l t e r a t i o n i r r e v e r s i b l e , a process c a l l e d damage f i x a t i o n . Such f i x e d damage may r e s u l t i n c e l l death. A l t e r n a t i v e l y , the t a r g e t molecule may r e v e r t t o i t s o r i g i n a l form by a process r e f e r r e d t o as damage r e p a i r . There has been some c o n t r o v e r s y as to the r e l a t i v e importance of d i r e c t and i n d i r e c t e f f e c t i n c e l l k i l l i n g . Most r e s u l t s i n d i c a t e t h a t i n d i r e c t e f f e c t s predominate. Sanner and 5. P i h l (1969) concluded t h a t about 50% of l e t h a l damage i n t h e i r c o l i B b a c t e r i a l system was the r e s u l t of i n d i r e c t e f f e c t , w h i le E b e r t , Dodd and Nias (1970) found i n d i r e c t e f f e c t s accounted f o r 7 0% of the l e t h a l damage i n mammalian (HeLa) c e l l s . More r e c e n t l y , Chapman and co-workers (1974) suggested, on the b a s i s of r e s u l t s from the use of OH- r a d i c a l scavengers as r a d i o p r o t e c t i v e agents (Chapman e t a l , 1973b), t h a t 6 0-70% of l e t h a l damage to a e r o b i c Chinese hamster c e l l s i s the r e s u l t of f i x a t i o n of i n d i r e c t damage caused by OH-, Qaq~ or H - . I t i s g e n e r a l l y accepted t h a t the OH- r a d i c a l , a s t r o n g o x i d i z i n g agent, i s the most important agent of i n d i r e c t damage i n a e r o b i c c e l l s . Supporting evidence has been p r o v i d e d by Blok e t a l (1967) who looked at i n a c t i v a t i o n and mutation i n an a e r o b i c suspension of 0X17 4 DNA i n the presence of v a r i o u s OH- scavengers; by Roots and Okada (1972) who used the method of a l k a l i n e sucrose g r a d i e n t s (McGrath and W i l l i a m s , 1966) to study the p r o d u c t i o n of s i n g l e s t r a n d breaks i n the DNA of a e r o b i c mammalian c e l l s i r r a d i a t e d d u r i n g exposure to a v a r i e t y of OH- scavengers; and by Chapman e t a l (1973b), whose work u s i n g OH- scavengers as r a d i o p r o t e c t o r s of mammalian c e l l s l e d to the c o n c l u s i o n t h a t approximately 60% of the l e t h a l e f f e c t s of r a d i a t i o n upon a e r o b i c hamster c e l l s i s due t o the i n d i r e c t a c t i o n of OH*. Chapman and h i s co-workers a l s o suggested t h a t ^10% of r a d i a t i o n l e t h a l i t y to a e r o b i c mammalian c e l l s r e s u l t s from the i n d i r e c t a c t i o n of the r e d u c i n g r a d i c a l s e and H-. They suggested t h a t t a r g e t r a d i c a l anions, formed 6 . by the inte r a c t i o n of e with the target molecules, would be quickly repaired by electron transfer to endogenous molecules having a greater electron a f f i n i t y than the target molecule. Studies by Adams et a l (1972) of electron transfer i n ir r a d i a t e d nucleotide solutions provide support for t h i s suggestion. I t i s int e r e s t i n g to note that , which i s considerably electron a f f i n i c , may thus protect target molecules from p o t e n t i a l l y l e t h a l target r a d i c a l anion damage. In summary, Chapman et a l (197 3b) have suggested that i n aerobic c e l l s , about 80% of the i n a c t i v a t i o n i s due to f i x a t i o n of target r a d i c a l s , with about 6 0% r e s u l t i n g from OH-and about 20% from d i r e c t e f f e c t . As well, i t was proposed that about 65% of in a c t i v a t i o n r e s u l t s from f i x a t i o n by oxygen, the remainder r e s u l t i n g from f i x a t i o n by other endogenous substances. Of the remaining 20% of aerobic i n a c t i v a t i o n , these workers suggested that at least a portion could r e s u l t from outright l e t h a l damage in the target molecule by d i r e c t e f f e c t . Chapman and co-workers (1974) reported that under hypoxia approximately 30% of l e t h a l damage to hamster c e l l s i s caused by the i n d i r e c t e f f e c t of OH', compared to about 60% i n aerobic c e l l s . I t i s therefore possible that of the t o t a l b i o l o g i c a l l e t h a l i t y , the portions r e s u l t i n g from d i r e c t e f f e c t s and from the i n d i r e c t e f f e c t s of the reducing species e and H- may be greater under hypoxic conditions than under aerobic conditions. 7. 1.3 MECHANISMS OF RADIOSENSITIZATION Oxygen i s the best known and most e x t e n s i v e l y s t u d i e d r a d i o s e n s i t i z i n g agent. To be e f f e c t i v e a t a l l , oxygen must be prese n t e i t h e r d u r i n g or immediately f o l l o w i n g i r r a d i a t i o n . Shenoy et_ a l (1975) have shown t h a t the a d d i t i o n of oxygen to hypoxic Chinese hamster c e l l s o n l y 5 m i l l i s e c o n d s a f t e r i r r a d i a t i o n y i e l d e d o n l y a s l i g h t i n c r e a s e i n s e n s i t i v i t y . Thus very s h o r t - l i v e d processes are i n v o l v e d i n oxygen s e n s i t i z a t i o n . The o x y g e n - f i x a t i o n h y p othesis (Alexander, 1962) has suggested t h a t t a r g e t f r e e r a d i c a l s , whether from d i r e c t or i n d i r e c t e f f e c t s , r e a c t q u i c k l y w i t h oxygen to form peroxy r a d i c a l s : T- + 0 2 -»• T0 2« T h i s r e a c t i o n was c o n s i d e r e d i r r e v e r s i b l e , thus r e s u l t i n g i n the f i x a t i o n of p o t e n t i a l l y l e t h a l damage. The f i x a t i o n step was proposed t o compete with r e p a i r of the damage v i a hydrogen donation from i n t r a c e l l u l a r r a d i c a l - r e d u c i n g s p e c i e s . I t has been suggested t h a t another p o s s i b l e mechanism of chemical r a d i o s e n s i t i z a t i o n i s the b i n d i n g of i n t r a c e l l u l a r s u l p h y d r y l compounds. Thus the s e n s i t i z i n g a b i l i t y of s e v e r a l s u l p h y d r y l - b i n d i n g compounds, n o t a b l y N-Ethylmaleimide (NEM), has been s t u d i e d . I n t r a c e l l u l a r s u l p h y d r y l compounds have been d e s c r i b e d as r a d i o p r o t e c t i v e substances, r e p a i r i n g damage by hydrogen donation t o t a r g e t f r e e r a d i c a l s (Alexander and Charlesby, 1954) or to o r g a n i c f r e e r a d i c a l s (Loman, Voogd 8. and Blok, 1970). Binding of these sulphydryl compounds would prevent such radioprotection. The importance of i n t r a c e l l u l a r sulphydryl compounds in c e l l s u r v ival has been disputed. Harris, Painter and.Hahn (1969) measured the r a d i o s e n s i t i v i t y of Chinese hamster c e l l s for various concentrations of i n t r a c e l l u l a r non-protein sulphydryl compounds. They concluded that most endogenous non-protein sulphydryl compounds did not produce a radioprotective e f f e c t . In 1969, Mullenger and Omerod proposed a second mechanism by which NEM might s e n s i t i z e ; a reaction with a r a d i o l y t i c species producing an activated molecule which reacts with and damages the target molecule. It was suggested by Adams and Dewey (1963) that the property of r a d i o s e n s i t i z a t i o n i s related to the electron a f f i n i t y of a compound, as most r a d i o s e n s i t i z e r s , including NEM, were known or thought to be electron a f f i n i c . The mechanism i n i t i a l l y proposed was that the s e n s i t i z e r enhanced the i n d i r e c t e f f e c t by capturing e species and releasing them to a supposedly more electron a f f i n i c target molecule. This would have the e f f e c t of prolonging the l i f e of the e r a d i c a l . However, t h i s model does not explain the oxygen e f f e c t because the high electron a f f i n i t y of C^ makes electron transfer from C^ to a target molecule u n l i k e l y . A second model of Adams and Cooke (1969) suggested that electron a f f i n i c s e n s i t i z e r s might act by enhancing the e f f e c t s of d i r e c t damage. Following i o n i z a t i o n of a target molecule, 9. t h e e l e c t r o n was p r o p o s e d t o l o c a l i z e a t some e l e c t r o n a f f i n i c s i t e on t h e m o l e c u l e . E l e c t r o n t r a n s f e r t o a s e n s i t i z e r m o l e c u l e o f g r e a t e r e l e c t r o n a f f i n i t y w o u l d r e d u c e t h e c h a n c e o f r e c o m b i n a t i o n and i n c r e a s e t h e c h a n c e o f d e c a y o f t h e p o s i t i v e t a r g e t i o n t o a f r e e r a d i c a l and s u b s e q u e n t damage f i x a t i o n . A p p l y i n g t h i s mechanism t o o x y g e n s e n s i t i z a t i o n , we h a v e : + ~ e l e c t r o n + • W W >vwv >vwv t r a n s f e r , n ~ c J • i o n i z a t i o n +0» f r e e r a d i c a l I r e c o m b i n a t i o n I n 19 73 Chapman and c o - w o r k e r s f o r m u l a t e d a model t o e x p l a i n b o t h r a d i o s e n s i t i z a t i o n and r a d i o p r o t e c t i o n . The model i s a more g e n e r a l f o r m o f t h e o x y g e n - f i x a t i o n h y p o t h e s i s and s u g g e s t s t h a t t h e r e d o x s t a t e o f t h e c e l l u l a r t a r g e t e n v i r o n m e n t i s t h e c r i t i c a l p r o p e r t y d e t e r m i n i n g t h e l e t h a l e f f e c t s o f r a d i a t i o n . T h i s r e d o x s t a t e d e t e r m i n e s w h e t h e r n e u t r a l t a r g e t r a d i c a l s , o nce f o r m e d , w i l l be r e p a i r e d by r e d u c t i o n o r f i x e d by o x i d a t i o n ( f i g . 1 ) . O n l y n e u t r a l t a r g e t r a d i c a l s a r e c o n s i d e r e d o f i m p o r t a n c e s i n c e , a s p r e v i o u s l y m e n t i o n e d , t h e s e w o r k e r s have s u g g e s t e d t h a t t a r g e t r a d i c a l a n i o n s c a u s e d by e a r e r e p a i r e d by e l e c t r o n t r a n s f e r t o endogenous m o l e c u l e s o f g r e a t e r e l e c t r o n a f f i n i t y . I t was s u g g e s t e d t h a t a r a d i o s e n s i t i z e r c a n a c t e i t h e r : 1) by a d d i n g t o t h e c e l l u l a r p o o l o f r a d i c a l o x i d i z i n g s p e c i e s , 2) by o x i d a t i o n o f t h e endogenous p o o l o f r a d i c a l r e d u c i n g s p e c i e s , 10. DIRECT EFFECT WWW INDIRECT EFFECT VWWV Hop or - H + aq vwwv or WWW Reduction (inactivation) Repair by Oxidation (\e by electron transfer) Fixation by Oxidation ie byadduct formation ancf/br electron transfer Repair by Reduction e.g. SH NADH Inter-radical reactions Oxidation of [p] [P]—radical reducing species -radical oxidizing species GURE 1. Schematic R e p r e s e n t a t i o n of Proposed Mechanisms  of R a d i o s e n s i t i z a t i o n and R a d i o p r o t e c t i o n D i r e c t or i n d i r e c t e f f e c t s on a t a r g e t molecule (WWW) r e s u l t i n t a r g e t r a d i c a l f o r m a t i o n . The mechanisms of s e n s i t i z a t i o n ( (l) , (D ) and p r o t e c t i o n ( (§) , (?) ) are d i s c u s s e d i n s e c t i o n 1.3. or by a combination of these mechanisms. I t was suggested t h a t o x i d a t i o n might occur e i t h e r by e l e c t r o n t r a n s f e r to the more e l e c t r o n a f f i n i c compound or by the b i n d i n g of compounds. R a d i o p r o t e c t o r s would a c t e i t h e r : 3) by adding to the endogenous p o o l of r a d i c a l r e d u c i n g s p e c i e s , thus i n c r e a s i n g the p r o b a b i l i t y of t a r g e t r a d i c a l r e d u c t i o n , or 4) by competing w i t h t a r g e t molecules f o r OH*, H* and other r a d i c a l s , thus i n h i b i t i n g i n d i r e c t damage by these s p e c i e s . These mechanisms are r e p r e s e n t e d s c h e m a t i c a l l y i n f i g . 1. The above model i s capable of e x p l a i n i n g both the oxygen e f f e c t and the r a d i o p r o t e c t i o n and r a d i o s e n s i t i z a t i o n observed with many compounds. I t r e c e i v e s f u r t h e r support from the work of Redpath and W i l l s o n (1973), who used p u l s e r a d i o l y s i s to show t h a t a s c o r b i c a c i d , a r e d u c i n g agent, r e a c t s w i t h and reduces o r g a n i c r a d i c a l s . In p a r t i c u l a r , the presence of a s c o r b i c a c i d e l i m i n a t e d the s e n s i t i z i n g e f f e c t s on S e r r a t i a marcescens by the s t a b l e f r e e r a d i c a l t riacetoneamine-N-oxyl (TAN). T h i s l o s s of s e n s i t i z a t i o n was a t t r i b u t e d t o the r e d u c t i o n of TAN by a s c o r b i c a c i d , which agrees w i t h p r o t e c t i v e mechanism (2) of the above model. 12. 1.4 RADIOSENSITIZERS The f i r s t r e p o r t s of s e l e c t i v e chemical r a d i o s e n s i t i z a t i o n of hypoxic mammalian c e l l s were from Parker, Skarsgard and Emmerson (1966, 1969) u s i n g the o r g a n i c n i t r o x i d e f r e e r a d i c a l t riacetoneamine-N-oxyl (TAN). Many compounds have s i n c e been shown t o possess r a d i o s e n s i t i z i n g a b i l i t y , some b e i n g much more e f f e c t i v e than o t h e r s . The search by Adams and co-workers f o r e l e c t r o n a f f i n i c compounds as s e n s i t i z e r s l e d to the t e s t i n g o f s e v e r a l s u b s t i t u t e d phenones. One of these, para-nitroacetophenone (PNAP) was found to s e n s i t i z e hypoxic mammalian c e l l s in_ v i t r o a t c o n c e n t r a t i o n s i n the s u b - m i l l i m o l a r range (Adams e t al^, 1971; Chapman, Webb and Borsa, 1971). However, t h i s drug f a i l e d t o be u s e f u l i n v i v o because i t s low water s o l u b i l i t y presented d i f f i c u l t i e s i n a d m i n i s t r a t i o n . A more s o l u b l e d e r i v a t i v e of PNAP, N-dimethylparanitro-2-propiophenone (NDPP), has y i e l d e d some s e n s i t i z a t i o n i n v i v o (Denekamp and M i c h a e l , 1972), but on l y f o r drug doses of near l e t h a l v a l u e s . As w e l l , d e s p i t e i t s s e n s i t i z i n g a b i l i t y i n v i t r o , TAN has a l s o been found t o have l i t t l e e f f e c t i n v i v o (Hornsey, 1972). A stimulus t o the search f o r r a d i o s e n s i t i z e r s came w i t h the d i s c o v e r y (Reuvers, Chapman and Borsa, 1972; Chapman e t a l , 1972) t h a t v a r i o u s n i t r o f u r a n d e r i v a t i v e s have e x c e l l e n t r a d i o s e n s i t i z i n g p r o p e r t i e s i n hypoxic Chinese hamster c e l l s a t drug c o n c e n t r a t i o n s which are n o n - t o x i c . Metronidazole, a 13. nitroimidazole, was found to sens i t i z e hypoxic mammalian c e l l s both i n v i t r o (Foster and Willson, 1973; Chapman, Reuvers and Borsa, 1973; P a l c i c , Agnew and Skarsgard, 1974; Asquith et a l , 1974; Thomson and Rauth, 1974) and i n vivo (Begg, Sheldon and Foster, 1974; Denekamp, Michael and Harris, 1974; Rauth and Kaufman, 1975; Denekamp and Harris, 1975). Even though rather high concentrations of metronidazole were required to achieve s e n s i t i z a t i o n , the drug's very low t o x i c i t y and r e l a t i v e l y slow rate of metabolism, both i n animals and i n man, made the res u l t s quite encouraging. The success with metronidazole stimulated the search for other nitroheteroaromatic s e n s i t i z e r s which might se n s i t i z e at lower concentrations while retaining the low t o x i c i t y c h a r a c t e r i s t i c . I n i t i a l tests with another nitroimidazole, Ro-07-0582, have shown t h i s compound to surpass metronidazole as a s e n s i t i z e r of hypoxic mammalian c e l l s i n v i t r o (Asquith et a l , 1974(b); Moore, P a l c i c and Skarsgard, 1975) and i n vivo (Denekamp, Michael and Harris, 1974; Denekamp and Harris, 1975). The purpose of the work described i n t h i s thesis was to study several aspects of r a d i o s e n s i t i z a t i o n by Ro-07-0582 and, as well, the t o x i c i t y of the drug. 2. MATERIALS AND METHODS 2.1 TISSUE CULTURE TECHNIQUES 2.1.1 C e l l L i n e s Three.mammalian c e l l l i n e s have been used i n these experiments. Two l i n e s , CH2B 2 and CHO are Chinese hamster c e l l l i n e s . The CH2B 2 l i n e (Agnew and Skarsgard, 1974) was d e r i v e d from a l i n e CHEF, o r i g i n a l l y d e s c r i b e d by P r e s c o t t and Bender (1963). The CHO l i n e (Hahn and Bagshaw, 1966) i s a s u b l i n e of Chinese hamster ovary c e l l s . The t h i r d c e l l l i n e , EMT6, i s a tumour c e l l l i n e s e l e c t e d i n t i s s u e c u l t u r e from the KHJJ tumour l i n e which, i n t u r n , was d e r i v e d from a primary mammary tumour a r i s i n g i n a BALB/c mouse (Rockwell, Kallman and Fajardo , 1972). 2.1.2 CH2B 2 C e l l C u l t u r e The CH2B 2 c e l l s were grown attached to p l a s t i c i n screw 2 cap f l a s k s of growth area 25 or 75 cm . The growth medium c o n s i s t e d o f MEM F-16 (Grand I s l a n d B i o l o g i c a l Company (GIBCO), Grand I s l a n d , New York) supplemented w i t h 10% f e t a l c a l f serum (GIBCO) and 0.8% p e n i c i l l i n - s t r e p t o m y c i n s o l u t i o n (10,000 u n i t s p e n i c i l l i n and 10,000 meg streptomycin per ml, GIBCO). The c e l l s were incubated under standard c o n d i t i o n s (37°C i n an atmosphere o f 5% C0 2, 95% a i r and 100% h u m i d i t y ) , and had a do u b l i n g time of 12-14 hours. - 14 -15. To maintain the c e l l l i n e i n l o g phase growth ( c e l l s a c t i v e l y growing and d i v i d i n g ) , the c e l l s i n monolayers were s u b c u l t u r e d by t r y p s i n i z a t i o n every 2-4 days. T h i s i n v o l v e d a 5 second wash with 5 ml of 0.1% t r y p s i n ( B a c t o - t r y p s i n , D i f c o L a b o r a t o r i e s , D e t r o i t , Michigan) preheated t o 37°C, f o l l o w e d by an 8 minute treatment w i t h 5 ml t r y p s i n . During the l a s t minute of treatment c e l l s were shaken f r e e o f the s u r f a c e . The t r y p s i n a c t i o n was stopped by immediately adding the t r y p s i n - c e l l suspension t o 10 ml growth medium. The c e l l s were c e n t r i f u g e d 6 minutes a t 50 g, resuspended i n growth medium and counted. 2 For the c e l l l i n e , grown i n 25 cm f l a s k s , a l i q u o t s of 5 5 between 10 and 4 X 10 c e l l s were p l a t e d i n t o f r e s h f l a s k s c o n t a i n i n g 10 ml growth medium. The growth medium was u s u a l l y r e p l a c e d d a i l y and o c c a s i o n a l l y every 2 days. C e l l s f o r experiments were p l a t e d 2 days p r i o r to the experiment a t c 2 1.2 X 10 c e l l s per 75 cm f l a s k c o n t a i n i n g 15 ml growth medium. Fr e s h growth medium was added the f o l l o w i n g day and the c e l l s t r y p s i n i z e d the day of the experiment, u s i n g the technique d e s c r i b e d above. 2.1.3 CHO C e l l C u l t u r e The CHO c e l l s , which have a d o u b l i n g time of 12-13 hours, were grown a t 37°C i n suspension i n sp i n n e r c u l t u r e i n a-medium l a c k i n g n u c l e i c a c i d s (Flow L a b o r a t o r i e s , R o c k v i l l e , Md., Cat. #1F-094C) supplemented w i t h 10% f e t a l c a l f serum and 0.8% p e n i c i l l i n - s t r e p t o m y c i n s o l u t i o n . The c e l l s were maintained i n l o g phase growth ( c e l l c o n c e n t r a t i o n s between 4 5 5 X 10 and 5 X 10 c e l l s / m l ) by r e g u l a r d i l u t i o n w i t h f r e s h medium. 2.1.4 EMT6 C e l l C u l t u r e EMT6 c e l l s , which have a d o u b l i n g time of approximately 2 13 hours, were grown atta c h e d to p l a s t i c i n 25 or 75 cm screw cap f l a s k s . The growth medium c o n s i s t e d of a-medium ( l a c k i n g n u c l e i c acids) supplemented w i t h 15% f e t a l c a l f serum and 0.8% p e n i c i l l i n - s t r e p t o m y c i n s o l u t i o n . The c e l l s were incubated under standard c o n d i t i o n s and t r y p s i n i z e d every 2-4 days by the technique d e s c r i b e d i n s e c t i o n 2.1.2. The growth medium was r e p l a c e d every day or every second day. C e l l s f o r c experiments were p l a t e d 2 days p r i o r to the experiment a t 10 2 c e l l s per 75 cm f l a s k c o n t a i n i n g 15 ml growth medium. The growth medium was r e p l a c e d the day p r i o r to the experiment. 2.2 RADIATION SOURCE I r r a d i a t i o n s were performed wi t h two Co therapy-u n i t s (Theratron Model F and Eldorado Super G, Atomic Energy of Canada L i m i t e d ) , p r o v i d i n g 1.17 and 1.33 MeV gamma r a y s . Dose r a t e s were between 330 and 680 rads/minute. 18. 2.3 CELL SUSPENSION EXPERIMENTS 2.3.1 P r e p a r a t i o n of the C e l l Suspension C e l l s t h a t were to be used f o r t o x i c i t y t e s t s or i r r a d i a t e d i n suspension were o b t a i n e d from l o g phase monolayers i n the case of CH2B 2 and EMT6 c e l l s , or from suspensions of l o g phase CHO c e l l s . F o l l o w i n g CH2B 2 or EMT6 t r y p s i n i z a t i o n (see 2.1.2) or removal of CHO c e l l s from spinner c u l t u r e , the c e l l s were c e n t r i f u g e d and resuspended i n drug t r e a t e d or drug f r e e growth medium. For c e l l suspension s u r v i v a l experiments ( i n v o l v i n g 5 i r r a d i a t i o n ) the c e l l s were resuspended a t 2.5 X 10 c e l l s / m l i n growth medium l a c k i n g sodium b i c a r b o n a t e . The absence of sodium b i c a r b o n a t e h e l d the pH at about 7.2 while g a s s i n g i n the absence of C0 2 (see 2.3.2). A 15-20 ml volume of c e l l suspension was p l a c e d i n s p e c i a l g l a s s i r r a d i a t i o n v e s s e l s ( f i g . 2(a)) and s t i r r e d w i t h a magnetic s t i r r e r . A l l s u r v i v a l experiments were performed a t 22°C. In one s u r v i v a l experiment i t was necessary t o use 10 c e l l s / m l . T h i s experiment i s d e s c r i b e d i n s e c t i o n 3.2.1. 4 For t o x i c i t y t e s t s , c e l l s were resuspended a t 3.2 X 10 c e l l s / m l i n growth medium c o n t a i n i n g sodium b i c a r b o n a t e . The presence of both sodium b i c a r b o n a t e and 5% C0 2 d u r i n g g a s s i n g (see 2.3.2) maintained the pH a t 7.2. A 25-3 0 ml volume of c e l l suspension was p l a c e d i n a 125 ml Erlenmeyer f l a s k ( f i g . 2(b)) and s t i r r e d with a magnetic s t i r r e r . Experiments were 19. ^—Magnetic Stir Bar Ceil Suspension Gas Flow (b) - C e l l Suspension Magnetic Stir Bar FIGURE 2. (a) The G l a s s I r r a d i a t i o n V e s s e l i n which C e l l Suspensions were I r r a d i a t e d N i t r o g e n was flowed a t 0.7 l i t r e s / m i n u t e over a s t i r r e d 15-20 ml suspension of 2.5 X 10^ c e l l s / m l f o r 45 minutes (or longer) p r i o r t o and d u r i n g i r r a d i a t i o n . Samples were o b t a i n e d by removing the stopper b r i e f l y and low e r i n g a p i p e t down the neck of the v e s s e l i n t o the suspension. A l l experiments were performed a t 22°C. (b) The Glass V e s s e l f o r C e l l Suspension T o x i c i t y  T e s t s N i t r o g e n (5% C0 0) was |lowed over a s t i r r e d 25-30 ml suspension of 372 X 10 c e l l s / m l c o n t a i n e d i n a 125 ml Erlenmeyer f l a s k . Samples were o b t a i n e d by removing the s m a l l stopper b r i e f l y and low e r i n g a p i p e t down the g l a s s t u b i n g i n t o the suspension. Experiments were performed a t 22° and 37°C. 20. performed at 22° and 37°C, the flasks being situated i n a temperature-regulated water bath. 2.3.2 Hypoxic and Aerobic Conditions Since c e l l s experience an increased resistance to the l e t h a l e f f e c t s of radiation when they are i r r a d i a t e d i n the absence of oxygen, i t i s most convenient to describe hypoxia as the state i n which radiation resistance i s maximum. In previous work i n t h i s laboratory, Agnew (1972) showed that by gassing a s t i r r e d 15 ml suspension i n an i r r a d i a t i o n vessel with prepurified N 2 (<10 ppm C^) at 0.7 litres/minute, maximum radiation resistance i s attained within 15 minutes. In the c e l l suspension s u r v i v a l experiments reported i n t h i s thesis, hypoxic conditions were achieved by flowing N 2 (Liquid Carbonic, Vancouver) at 0.7 litres/minute over the suspension for 45 minutes (or longer) p r i o r to and during i r r a d i a t i o n . Hypoxic conditions were achieved and maintained during t o x i c i t y tests by continually flowing N 2 + 5% C0 2 (Liquid Carbonic, Vancouver) over the suspensions, with time t=0 corresponding to the st a r t of gassing. The flow rate was maintained at 0.7 litres/minute for 1 hour, then reduced to about 0.4 litres/minute for the remainder of the experiment. As well, the gas was passed through a w a t e r - f i l l e d gas washing bottle before reaching the c e l l suspension. The r e s u l t i n g humidified gas minimized evaporation from the c e l l suspension. One t o x i c i t y test was performed using p r e p u r i f i e d nitrogen (no CO„). The experiment i s described i n section 3.1.1.3. Aerobic c o n d i t i o n s f o r suspension experiments were obtained by f l o w i n g 0 2 (Canadian L i q u i d A i r , Vancouver) i n s t e a d of N^. 2.3.3 P l a t i n g and Colony Formation A l i q u o t s removed from a suspension d u r i n g an experiment were immediately added to normal growth medium, thus d i l u t i n g the drug. The samples were then c e n t r i f u g e d and the c e l l s resuspended i n f r e s h growth medium. The c e l l s were d i l u t e d and p l a t e d i n t o 60 X 15 mm p l a s t i c t i s s u e c u l t u r e p e t r i s (Falcon P l a s t i c s , Oxnard, C a l i f o r n i a , and Lux S c i e n t i f i c , Thousand Oaks, C a l i f o r n i a ) c o n t a i n i n g 6 ml of normal growth 5 medium p l u s , f o r CHO and CH2B 2 c e l l s o n l y , 10 h e a v i l y i r r a d i a t e d (6000 rads under a e r o b i c c o n d i t i o n s ) feeder c e l l s per p e t r i . No EMT6 feeder c e l l s were used because these c e l l s absorb s t a i n , making colony c o u n t i n g d i f f i c u l t . The c e l l s were incubated under standard c o n d i t i o n s , a l l o w i n g s u r v i v i n g c e l l s t o p r o l i f e r a t e and form c o l o n i e s , a colony being d e f i n e d as a t i g h t group of 50 or more c e l l s . The i n c u b a t i o n time amounted to 7 days f o r CHO c e l l s , 8 days f o r CH2B 2 c e l l s , and 9 days f o r EMT6 c e l l s . F o l l o w i n g i n c u b a t i o n , the medium was poured from the p e t r i and the c o l o n i e s s t a i n e d by a 6 minute exposure a t room temperature to a methylene blue s o l u t i o n (2 g / l i t r e ) . 2.3.4 P l a t i n g E f f i c i e n c y and S u r v i v i n g F r a c t i o n Since the experiments r e p o r t e d i n t h i s t h e s i s use colony forming a b i l i t y as the measured end p o i n t , a s u r v i v o r i s d e f i n e d as a c e l l which w i l l form a colony of 50 or more c e l l s w i t h i n the a l l o t t e d i n c u b a t i o n time (see 2.3.3). Even f o r u n i r r a d i a t e d p o p u l a t i o n s , not a l l the c e l l s w i l l form c o l o n i e s , as some w i l l undergo l i m i t e d or no p r o l i f e r a t i o n . Thus the term " p l a t i n g e f f i c i e n c y " w i l l be used i n t o x i c i t y t e s t s to r e p r e s e n t the percentage of the c e l l s p l a t e d (counted by C o u l t e r Counter) which achieve colony s t a t u s . For experiments i n v o l v i n g i r r a d i a t i o n , the r a t i o of the p l a t i n g e f f i c i e n c y of i r r a d i a t e d c e l l s to u n t r e a t e d , or c o n t r o l c e l l s , w i l l be r e f e r r e d to as the " s u r v i v i n g f r a c t i o n " . The s u r v i v i n g f r a c t i o n i s simply a normalized p l a t i n g e f f i c i e n c y , where a l l p o i n t s are normalized to the p l a t i n g e f f i c i e n c y of c o n t r o l c e l l s 2.3.5 S u r v i v a l Curves and Dose M o d i f y i n g F a c t o r s The s u r v i v a l of mammalian c e l l s exposed t o r a d i a t i o n i s g e n e r a l l y shown by means of a s u r v i v a l curve, i n which the l o g of the s u r v i v i n g f r a c t i o n i s p l o t t e d a g a i n s t the dose absorbed. The curves u s u a l l y have a rounded, or "shoulder" r e g i o n i n the range of h i g h s u r v i v i n g f r a c t i o n , f o l l o w e d by an approximately l i n e a r r e g i o n r e p r e s e n t i n g e x p o n e n t i a l s u r v i v a l . A l l s u r v i v a l curves presented i n t h i s t h e s i s are of t h i s form. The r e c i p r o c a l of the s lope of the s t r a i g h t l i n e r e g i o n of a s u r v i v a l curve i s l a b e l l e d D . I f the l i n e a r p o r t i o n i s e x t r a p o l a t e d t o zero dose, the i n t e r c e p t w i t h the o r d i n a t e i s c a l l e d the e x t r a p o l a t i o n number. I f a s e t of s u r v i v a l curves have a s i m i l a r shape, any . two curves can be approximately superimposed by the a p p l i c a t i o n of a s c a l i n g f a c t o r t o the dose v a l u e s . T h i s f a c t o r i s c a l l e d the dose modifying f a c t o r (DMF), and i s a s i n g l e numerical r e p r e s e n t a t i o n of the comparison of two e n t i r e s u r v i v a l curves. Of course,, i f l i n e a r segments are to s h i f t and o v e r l a p completely, a common e x t r a p o l a t i o n number i s r e q u i r e d f o r a l l s u r v i v a l curves. T h i s c o n d i t i o n i s d e s c r i b e d by s a y i n g the cause of the s h i f t between s u r v i v a l curves i s simply "dose modifying". For each of the 3 c e l l l i n e s , the s u r v i v a l curves presented i n t h i s t h e s i s can be adequately d e s c r i b e d by dose m o d i f i c a t i o n . In t h i s t h e s i s the DMF r e p r e s e n t s a comparison of the c e l l s u r v i v a l curve obtained f o r a d r u g - t r e a t e d suspension w i t h the s u r v i v a l curve obtained c o n c u r r e n t l y f o r a d r u g - f r e e suspension. The DMF i s c a l c u l a t e d e i t h e r from the r a t i o of the doses r e q u i r e d to reduce c e l l s u r v i v a l to 0.01 or from the r a t i o of the two D Q v a l u e s . The oxygen enhancement r a t i o (OER) i s d e f i n e d as the DMF obtained when comparing the s u r v i v a l curves of a e r o b i c and hypoxic suspensions (both d r u g - f r e e ) . 2.4 CELL PELLET SURVIVAL EXPERIMENTS 2.4.1 Formation of the C e l l P e l l e t C e l l s were harvested from l o g phase growth (2.1.2, 7 2.1.3) and resuspended at about 10 c e l l s / m l i n normal growth medium l a c k i n g sodium b i c a r b o n a t e and w i t h or without d i s s o l v e d drug. E i g h t ml of suspension was p l a c e d i n t o each of one or more 50 ml c o n i c a l g l a s s c e n t r i f u g e tubes. Hypoxia was achieved by f l o w i n g h u m i d i f i e d N 2 a t 0.7 l i t r e s / m i n u t e over each suspension (see f i g . 3 ( a ) ) , the samples being shaken r e g u l a r l y d u r i n g t h i s p e r i o d . A f t e r a minimum of ten minutes g a s s i n g , the tubes were s e a l e d t o m aintain hypoxia, c e n t r i f u g e d f o r 8 minutes a t 120 g, reconnected to the N 2 flow, and the supernatant removed by a s p i r a t o r (see f i g . 3 ( b ) ) . The N 2 was h u m i d i f i e d by passage through a w a t e r - f i l l e d gas washing b o t t l e , the h u m i d i f i e d gas being r e q u i r e d t o prevent d e h y d r a t i o n of the c e l l p e l l e t . Each c e l l p e l l e t was i r r a d i a t e d w i t h v a r i o u s doses a t times between 15 and 110 minutes a f t e r the end of c e n t r i f u g a t i o n , the t o t a l time from i n i t i a l N 2 flow to i r r a d i a t i o n b e i n g not l e s s than 45 minutes. A l l c e l l p e l l e t experiments were performed a t 22°C. 2.4.2 I r r a d i a t i o n and C e l l S u r v i v a l of P e l l e t Samples Following the r e q u i r e d accumulated doses of r a d i a t i o n , 5 7 samples of the p e l l e t s (5 X 10 -10 c e l l s ) were drawn i n t o the 25. FIGURE 3. The V e s s e l Used f o r C e l l P e l l e t Experiments (a) N i t r o g e n was_flowed a t 0.7 l i t r e s / m i n u t e over an 8 ml suspension of 10 c e l l s / m l f o r a minimum o f 10 minutes. (b) The tubes were then s e a l e d , the v e s s e l c e n t r i f u g e d f o r 8 minutes a t 120 g, the N_ flow r e - e s t a b l i s h e d , and the supernatant removed by an a s p i r a t o r . I r r a d i a t i o n s commenced 45 minutes or more a f t e r the s t a r t of g a s s i n g . P e l l e t -samples were removed by p i p e t (c and d ) . A l l steps were performed i n such a manner as to m a i n t a i n hypoxia. Every c e l l p e l l e t experiment was performed a t 22 C. ends of 1 ml p l a s t i c d i s p o s a b l e p i p e t s (Falcon P l a s t i c s ) , hypoxia being maintained w i t h i n the tube d u r i n g the procedure ( f i g s . 3(c) and ( d ) ) . The samples were resuspended i n 7 ml of normal growth medium a t 22°C, counted, d i l u t e d , and p l a t e d i n t o 15 X 60 mm p l a s t i c p e t r i s c o n t a i n i n g 6 ml of normal growth medium p l u s 10^ h e a v i l y i r r a d i a t e d feeder c e l l s per p e t r i . I n cubation, s t a i n i n g and d e t e r m i n a t i o n of c e l l s u r v i v a l was as d e s c r i b e d i n s e c t i o n s 2.3.3 and 2.3.4. 2.5 Ro-07-0582 The s e n s i t i z e r used i n t h i s work was Ro-07-0582, 1-(2-nitro-l-imidazonyl)-3-methoxy-2-propanol, C 7 H n ° 4 N 3 (M.W. 201.18), s y n t h e s i z e d by Roche Products L t d . To check the s t a b i l i t y of Ro-07-0582 i n s o l u t i o n , a sample of 7 mM drug was d i s s o l v e d i n normal growth medium l a c k i n g sodium b i c a r b o n a t e and s t o r e d a t 2°C i n the absence of l i g h t . A f t e r 41 days the drug was d i l u t e d and used i n a CH2B2 suspension experiment. F i g u r e 4 shows t h a t the s e n s i t i z a t i o n observed w i t h t h i s p r e p a r a t i o n of the drug was e q u i v a l e n t t o t h a t o b t a i n e d w i t h f r e s h p r e p a r a t i o n s . 28. O I— a < CC > > cr CO 001 0001 $ N 2 + 004 mM OLD 0582 0 N 2 + 004 mM FRESH 0582 ® N 2 + 0-4 mM OLD 0582 0-4 mM FRESH 0582 1000 2000 3000 DOSE (racis) FIGURE 4. S u r v i v a l C u r v e s f o r H y p o x i c CH2B C e l l s I r r a d i a t e d i n F r e s h o r O l d Ro-07-0582 D r u g was d i s s o l v e d i n n o r m a l g r o w t h medium l a c k i n g s o d i u m b i c a r b o n a t e a n d s t o r e d 41 d a y s a t 2 C i n t h e a b s e n c e o f l i g h t . The d r u g was t h e n d i l u t e d a n d u s e d i n a CH2B_ s u s p e n s i o n ' e x p e r i m e n t . The " f r e s h " s a m p l e s w e r e p r e p a r e d i m m e d i a t e l y p r i o r t o t h e e x p e r i m e n t . 3. RESULTS 3.1 TOXICITY TESTS WITH Ro-07-0582 In examining the t o x i c i t y of p o t e n t i a l l y useful compounds one i s normally concerned primarily with the toxic e f f e c t s of the compound on normal, aerobic tissue since these are the tissues which one wishes to spare i n the treatment. Thus, pr i o r to the s t a r t of this work, i t was common to measure.toxic ef f e c t s i n aerobic c e l l cultures. The discovery that 0582 exhibits increased t o x i c i t y to hypoxic c e l l s was of in t e r e s t both for a further control of malignant disease and for possible detrimental e f f e c t s upon normally hypoxic body tissues. Therefore the t o x i c i t y of t h i s drug was studied using several c e l l l i n e s under both aerobic and hypoxic conditions. 3.1.1 CH2B2 C e l l Line 3.1.1.1 Toxicity, of 0582 to aerobic and hypoxic CH2B2  c e l l s i n suspension at 37 C Figure 5 shows the res u l t s of experiments i n which aerobic CH2B2 c e l l s i n suspension at 37°C were incubated i n 0582 for various times. Single aliquots were processed and the pl a t i n g e f f i c i e n c y determined by colony forming a b i l i t y . The pl a t i n g e f f i c i e n c i e s were normalized against p l a t i n g e f f i c i e n c i e s of samples taken at the same time from drug-free aerobic solutions. A reduced p l a t i n g e f f i c i e n c y i s observed at 30 . o U J o AEROBIC C H 2 B 2 (37°C) o 0 2 + 5mM 0582 © 0 2 •+ 10mM 0582 0 2 + 25mM 0582 0 2 + 50m'M 0582 I— < u U J CL-IO LZ 1 0 0 h * - A o . A _ « E • B © -A 1 2 3 4 5 6 7 INCUBATION TIME AT 37°C (hours) FIGURE 5. P e r c e n t P l a t i n g E f f i c i e n c y o f A e r o b i c CH2B,, C e l l s  a s a F u n c t i o n o f I n c u b a t i o n Time a t 37 C i n 5, 1 0 , 25 o r 50 mM .Ro-07-0582 3 1 . HYPOXIC CH2B2 (37°C) >-U 0-1- 1 I I _ J I 1 I I I I 1_ 1 2 3 4 5 6 7 8 9 10 INCUBATION TIME AT 37°C (hours) FIGURE 6. P e r c e n t P l a t i n g E f f i c i e n c y o f H y p o x i c CH2B-c e x I s a s a F u n c t i o n o f I n c u b a t i o n Time a t  37°C i n 0, 1.0, 5, 15, o r 50 mM Ro-07-0532 E r r o r b a r s r e p r e s e n t t h e s t a n d a r d d e v i a t i o n b e t w e e n i n d e p e n d e n t measurements, and i n many c a s e s a r e s m a l l e r t h a n t h e p l o t t e d s y m b o l s . 32. 4 h o u r s i n c u b a t i o n i n 25 o r 50 mM 0582, w h e r e a s no t o x i c e f f e c t s a r e s e e n i n t h e 5 a n d 10 mM s a m p l e s . F i g u r e 6 shows t h e p l a t i n g e f f i c i e n c y o f CH2B2 c e l l s p l o t t e d a s a f u n c t i o n o f i n c u b a t i o n t i m e a t 37°C i n Ro-07-0582 u n d e r h y p o x i c c o n d i t i o n s . F o r e a c h p o i n t , two a l i q u o t s o f c e l l s u s p e n s i o n w e r e p r o c e s s e d a n d p l a t e d i n d e p e n d e n t l y . The p o i n t s a n d e r r o r b a r s on f i g . 6 r e p r e s e n t t h e a v e r a g e o f t h e two r e s u l t i n g p l a t i n g e f f i c i e n c i e s ( f r o m a s i n g l e e x p e r i m e n t ) a n d t h e s t a n d a r d d e v i a t i o n , r e s p e c t i v e l y . P o i n t s w i t h o u t e r r o r b a r s h a v e a s t a n d a r d d e v i a t i o n t o o s m a l l t o p l o t . The d r u g i s c o n s i d e r a b l y t o x i c t o c e l l s u n d e r h y p o x i c c o n d i t i o n s . A c o n c e n -t r a t i o n a s l o w a s 1 mM r e d u c e s t h e p l a t i n g e f f i c i e n c y t e n f o l d w i t h i n 7 h o u r s . The h i g h e s t c o n c e n t r a t i o n t e s t e d , 50 mM, showed v e r y l a r g e t o x i c i t y w i t h i n 2 h o u r s . N o t e t h a t t h e r e h a s b e e n no n o r m a l i z a t i o n o f p l a t i n g e f f i c i e n c i e s a g a i n s t t h e d r u g - f r e e c o n t r o l s ( o p e n s q u a r e s ) . C o m p a r i s o n o f f i g s . 5 a n d 6 shows v e r y c l e a r l y a d r a m a t i c d i f f e r e n c e b e t w e e n a e r o b i c a n d h y p o x i c t o x i c i t y o f 0582. F o r e x a m p l e , a 3 h o u r i n c u b a t i o n i n 10 mM 0582 p r o d u c e d no t o x i c e f f e c t s f o r a e r o b i c c e l l s ( f i g . 5 ) , w h i l e f i g . 6 shows t h a t t h e p l a t i n g e f f i c i e n c y o f h y p o x i c c e l l s w o u l d be l e s s t h a n 1 0 % a f t e r s u c h a n i n c u b a t i o n . 3.1.1.2 T e m p e r a t u r e d e p e n d e n c e o f a c u t e t o x i c i t y u n d e r  h y p o x i c c o n d i t i o n s To t e s t f o r t e m p e r a t u r e d e p e n d e n c e o f a c u t e t o x i c i t y u n d e r h y p o x i c c o n d i t i o n s , a n e x p e r i m e n t was p e r f o r m e d w i t h CH2B2 c e l l s incubated i n Ro-07-0582 at 22.5°C. The re s u l t s are shown i n f i g . 7, where each point i s again the average of two independent measurements. Comparison of f i g . 7 and f i g . 6 shows that the hypoxic t o x i c i t y of 0582 i s subs t a n t i a l l y dependent on temperature (note the expanded ordinate scale of f i g . 7) . 3.1.1.3 Hypoxic t o x i c i t y using p r e p u r i f i e d nitrogen To check the possible e f f e c t of trace amounts of 0 2 present i n hypoxic t o x i c i t y t e s t s , an experiment was performed on hypoxic CH2B2 c e l l s i n suspension. Hypoxia was obtained by flowing N 2 containing about 10 ppm 0 2 (measured with a Hersch Oxygen Meter) over the suspension at a rate of 0.7 litres/minute. The 0 2 content was l i k e l y less than that for experiments using N 2 + 5% C0 2, for which measurements of approximately 40 ppm 0 2 have been obtained from similar mixtures. The re s u l t s are plotted on f i g . 8. For each point, two aliquots of c e l l suspension were processed and plated independently. Points without error bars have too small a standard deviation to p l o t . The r e s u l t s of f i g . 8 show the 1 and 5 mM exposures to be more toxic than t h e i r counterparts i n f i g . 6. There i s considerable difference i n the 5 mM curves, i n d i c a t i n g that the hypoxic t o x i c i t y response i s effected by small amounts of remaining 0 2. However, the fa c t that the c e l l k i l l i n g for the 50 mM exposure i n f i g . 8 does not exceed that for f i g . 6 suggests that perhaps these two curves represent the upper l i m i t for toxic e f f e c t s on t h i s c e l l l i n e . 1 A HYPOXIC C H 2 B 2 (22 5°C) ^OmM £ 1 m M •r 50 mM 2 4 6 8 10 12 INCUBAT ION TIME AT 22 - 5°C (hours) 14 FIGURE 7. P e r c e n t P l a t i n g E f f i c i e n c y o f • H y p o x i c C H 2 B 2 C e l l s  a s a F u n c t i o n o f I n c u b a t i o n Time a t 2 2.5 C i n 0, 1.0, 5, o r 50 mM Ro-07-0582 N o t e t h e e x p a n d e d o r d i n a t e s c a l e c o m p a r e d t o f i g . 6. 35. 0-1 • -OmM £ 1 mM 1 5 mM 50 mM 1 2 3 4 5 - 6 7 8 9 10 11 I N C U B A T I O N T I M E A T 3 7 ° C (hours) FIGURE 8. P e r c e n t P l a t i n g E f f i c i e n c y o f C H 2 B 0 C e l l s G a s s e d w i t h P r e p u r i f i e d N i t r o g e n , Shown a s a F u n c t i o n o f I n c u b a t i o n Time a t 37 C i n 0, 1, 5, o r 50 mM Ro-07-0582 The n i t r o g e n c o n t a i n e d a b o u t 10 ppm 0 2* 36. 3.1.2 Toxic i t y of 0582 In Aerobic and Hypoxic CHO C e l l s In  Suspension at 37 C Figure 9 shows the p l a t i n g e f f i c i e n c y of aerobic CHO c e l l s as a function of time of incubation at 37°C i n the presence of Ro-07-0582. For each point, three aliquots of c e l l suspension were processed and plated independently. The points and error bars on f i g . 9 represent the average of the three p l a t i n g e f f i c i e n c i e s and the standard deviation. For comparison, f i g . 10 shows the p l a t i n g e f f i c i e n c y of CHO c e l l s plotted as a function of incubation time at 37°C i n Ro-07-0582 under hypoxic conditions. For each point, two aliquots of c e l l suspension were processed and plated independently. Clearly the hypoxic t o x i c i t y of f i g . 10 i s much greater than the aerobic t o x i c i t y of f i g . 9. For example, 2 hours incubation of CHO c e l l s at 37°C i n 50 mM Ro-07-0582 y i e l d s a pl a t i n g e f f i c i e n c y of 1.2% under hypoxic conditions ( f i g . 10), while the same drug concentration under aerobic conditions produces no detectable t o x i c i t y ( f i g . 9). As well, comparison of f i g s . 6 and 10 shows that 05 82 has a sim i l a r hypoxic k i l l i n g e f f e c t on the CHO and CH2B2 c e l l l i n e s . 3.1.3 Toxic i t y of 0582 In Aerobic and Hypoxic EMT6 C e l l  Suspensions The EMT6 c e l l l i n e was also used for tests of aerobic and hypoxic t o x i c i t y of 0582. As EMT6 i s a mammalian tumor l i n e , the information was a valuable addition to the res u l t s for the CH2B_ and CHO l i n e s . AEROB IC C H O (37°C) y 100 LL LL UJ O Z 50i h-Z Ld 20 CE W Q. 10 JL 1 1 2 3 4 5 6 7 8 I NCUBAT ION T IME AT 37°C (hours) §15 mM 50 mM 9 10 FIGURE 9, P e r c e n t P l a t i n g E f f i c i e n c y o f A e r o b i c C H O C e l 1 s an a F u n c t i o n o f I n c u b a t i o n Time a t 37°C i n 0, 1.0, 15, o r 50 mM Ro-07-0582 N o t e t h e e x p a n d e d o r d i n a t e s c a l e c o m p a r e d t o t h a t o f f i g s . 6 a n d 10. 38. HYPOXIC CHO (37°C) FIGURE 10. P e r c e n t P l a t i n g E f f i c i e n c y o f H y p o x i c CKO C e l l s  as a F u n c t i o n o f I n c u b a t i o n Time a t 37°C i n 0, 1.0, 5, 15, o r 50 mM Ro-07-0582 Figure 11 shows the pl a t i n g e f f i c i e n c y of aerobic EMT6 c e l l s as a function of incubation time i n 0582 at 37°C. Each point i s once more the average of 2 independent measurements. For exposures to 13 hours with drug concentrations of 15 mM or less , there was l i t t l e i f any t o x i c i t y r e l a t i v e to the drug-free control ( s o l i d t r i a n g l e s and s o l i d curve). S l i g h t r e l a t i v e t o x i c i t y was observed for a 22 hour incubation i n 15 mM 0582. Although incubation i n 50 mM 0582 appeared to give large t o x i c i t y , t h i s single experiment provided i n s u f f i c i e n t data to be conclusive. Figure 12 shows the res u l t s for EMT6 c e l l s incubated under hypoxic conditions at 37°C i n the presence or absence of Ro-07-0582. Each point represents the average of two independent p l a t i n g e f f i c i e n c i e s . Comparison of f i g s . 6, 10 and 12 shows that the hypoxic t o x i c i t y i s greater for the EMT6 l i n e than either the C H 2 B 2 or CHO l i n e for incubation i n 15 or 50 mM 0582. However, the 5 mM curves are a l l s i m i l a r , and i t appears from the 1 mM curves that the t o x i c i t y at low concentrations of 0582 i s less for EMT6 than for CH2B2 or CHO c e l l s , e s p e c i a l l y i f one considers the low pl a t i n g e f f i c i e n c y (at long incubation times) of the drug-free EMT6 suspension. 40. 2 4 6 8 10 12 INCUBATION TIME AT 37°C (hours) FIGURE 1 1 . P e r c e n t P l a t i n g E f f i c i e n c y o f A e r o b i c EMT6 C e l l s  a s a F u n c t i o n o f I n c u b a t i o n Time a t 37 C i n 0, 1.0, 5, 15 o r 50 mM Ro-07-0582 N o t e t h e e x p a n d e d o r d i n a t e s c a l e . 41. u z UJ u LZ u. UJ o z Q_ f-z UJ u CE UJ CL 1 2 3 4 5 6 7 8 9 lO 11 12 INCUBATION TIME AT 37°C (hours) FIGURE 12. Percent P l a t i n g E f f i c i e n c y of Hypoxic EMT6 C e l l s  as a Function of Incubation Time at 37 C i n 0, 1.0, 5, 15, or 50 mM Ro-07-0582 3.2 TREATMENT WITH 0582 3.2.1 Treatment of CH2B 2 C e l l s i n Suspension With 0582 In these experiments 0582 was added to the c e l l suspension (maintained at 22°C) just p r i o r to the s t a r t of gassing and dil u t e d immediately following i r r a d i a t i o n by the addition of drug-free medium. In no instance was there t o x i c i t y from exposure to the drug. This was demonstrated by the p l a t i n g e f f i c i e n c i e s of zero dose control samples taken immediately p r i o r to i r r a d i a t i o n . T o x i c i t y would not be expected for reasonable drug concentrations, as f i g . 7 shows that at 22°C there i s n e g l i g i b l e t o x i c i t y even at 50 mM during the f i r s t 2 hours exposure to the drug. Figure 13 shows the su r v i v a l curves of an experiment i n which CH2B2 c e l l s i n a drug-free suspension at 22°C were ir r a d i a t e d i n the presence (squares) or absence (ci r c l e s ) of oxygen, the objective being to accurately determine the OER (see 2.3.5). To enable precise measurement of low s u r v i v a l values, i t was necessary to use suspensions of 10^ CH2B2 ce l l s / m l in t h i s experiment. The s o l i d c i r c l e s are survival values from a suspension which received an i n i t i a l dose of 4100 rads (dose rate 420 rads/min.), thus minimizing the time required to de l i v e r the high doses. This also reduces the enzymatic repair of sublethal damage which might occur over the course of i r r a d i a t i o n . The difference between the hypoxic su r v i v a l curves presumably represents the difference i n the amount of enzymatic repair. 43. 0-0001 1000 2000 3000 4000 5000 D O S E (tads FIGURE 13, The PER f o r C H 2 B ? C e l l s CH2B_ c e l l s w e r e i r r a d i a t e d u n d e r a e r o b i c (open s q u a r e s ) o r h y p o x i c ( c i r c l e s ) c o n d i t i o n s . The s u s p e n s i o n r e p r e s e n t e d by s o l i d c i r c l e s r e c e i v e d an i n i t i a l d o s e o f 4100 r a d s , t h u s m i n i m i z i n g t h e t i m e r e q u i r e d t o d e l i v e r t h e h i g h d o s e s . The OER was a p p r o x i m a t e l y 3 ( s e e 3 . 2 . 1 ) . The straight l i n e segments of the sur v i v a l curves of f i g . 13 were f i t t e d to the data points by the method of least squares. In order to omit data points l y i n g on the "shoulder" of the curve, only points with a su r v i v a l less than or equal to 0.25 were accepted for the least squares f i t . OER values were calculated from the r a t i o of the slopes of the l i n e a r segments. When the hypoxic response was represented by the open c i r c l e s , the calculated OER was 3.07+.04. When open c i r c l e data was used for doses below 4000 rads and closed c i r c l e data for doses above 4000 rads, the OER was 2.84+.06. These values l i e i n the range of OER measurements (2.7 to 3.1) reported by most experimenters using mammalian c e l l s . Figure 14 shows the survival curves for three drug concentrations present during i r r a d i a t i o n of hypoxic C H 2 B 2 c e l l at 22°C. Also included are data obtained the same day with only N 2 or 0 2 present during i r r a d i a t i o n of drug-free suspensions. Clearly the presence of 0582 sensitizes hypoxic c e l l s , the DMF (see 2.3.5) increasing rapidly with drug concentration. The straight l i n e segments of the sur v i v a l curves of f i g . 14 were f i t t e d to the data points by the method of least squares, again using only survival values less than or equal to 0.25. The slopes of the l i n e a r f i t s were used to calculate DMF's of 1.37, 1.85 and 3.37, corresponding to 0.1, 1.0 and 15 mM 0582 respectively. The OER was 3.16. In f i g . 15 are summarized the res u l t s of a large number of s u r v i v a l response measurements sim i l a r to those shown i n 45. Z o \-U < b z > 0-01 > ct: D U) 0001 • N 2 * N 2 + 0-1 mM 0582 * N 2 + 10 mM 0582 * N 2 + 15 mM 0582 00001 1000 2000 D O S E (rads) 3 000 4 0 0 0 FIGURE 14. S u r v i v a l C u r v e s f o r CH2B C e l l s I r r a d i a t e d a t  22 C i n t h e P r e s e n c e o f N i t r o g e n , N i t r o g e n P l u s 0.1, 1.0, o r 15 mM R o - 0 7 - 0 5 8 2 , o r O x y g e n 46. 4-21— i — r i — r i i i ± J L _ l J I L J L 20 40 80 100 001 0-1 1-0 10 100 CONCENTRATION OF Ro-07-0582 (mM) c 0) <J CD Q. * UJ o y  U-111 z o. 60 g N _1 < 01 O FIGURE 15. S e n s i t i z a t i o n and T o x i c i t y of Ro-07-0582 w i t h CH2B 2 C e l l s i n Suspension as a F u n c t i o n of  Drug C o n c e n t r a t i o n © DMF o b t a i n e d under hypoxic c o n d i t i o n s at 2 2 UC; l i m i t s of OER v a l u e s ; A normalized p l a t i n g e f f i c i e n c y a f t e r a 3 hour i n c u b a t i o n a t 37 C under hypoxic c o n d i t i o n s (note the i n v e r t e d o r d i n a t e s c a l e ) ; A normalized p l a t i n g e f f i c i e n c y a f t e r a 6 hour i n c u b a t i o n a t 37°C under a e r o b i c c o n d i t i o n s . Note: The r a t h e r h i g h maximum DMF and OER v a l u e s shown i n t h i s f i g u r e probably r e s u l t from the manner i n which these parameters were c a l c u l a t e d (see s e c t i o n 3.2.1) f i g . 14. DMF's and standard errors are plotted as a function of the concentration of 0582 present during i r r a d i a t i o n under hypoxic conditions. I t can be seen that s e n s i t i z a t i o n occurs for drug concentrations as low as 0.1 mM. A DMF of 2.1 i s attained with 1 mM 0582, while concentrations over 20 mM appear to give a DMF equal to the OER. As well, one experiment ( f i g . 16) showed that addition of 100 mM drug to oxygenated c e l l s did not increase s e n s i t i z a t i o n above that for 0^ alone. The somewhat high OER range shown i n f i g . 15 probably re s u l t s from comparing aerobic responses to hypoxic responses which had been carried to i n s u f f i c i e n t l y high doses to accurately e s t a b l i s h the f i n a l slope (minimum sur v i v a l values were commonly 0.002). When the DMF's are calculated, not from slopes of the survival curves, but rather from the r a t i o of the doses required to reduce su r v i v a l to 0.01, the 0582 DMF approaches a maximum value of approximately 2.8 at 100 mM, whereas the OER calculated i n the same manner i s 2.9 i n these experiments. In order to compare the s e n s i t i z a t i o n and t o x i c i t y of Ro-07-0582, two time-cut t o x i c i t y experiments were ca r r i e d out, one under hypoxic and one under aerobic conditions, using the method described i n section 2. Multiple drug concentrations were tested i n both cases. The t o x i c i t y was measured at 3 hours i n the hypoxic and at 6 hours i n the aerobic suspensions. The r e s u l t s , shown i n f i g . 15, indicate for aerobic c e l l s , that a 6 hour incubation i n concentrations of 0582 up to o I— o < C9 > > ZD CO 0001 0-01 O-OOOIr-1000 2000 3000 DOSE (rads) 4000 FIGURE 16. S u r v i v a l C u r v e s f o r CH2B C e l l s I r r a d i a t e d a t 22 C i n t h e P r e s e n c e o f Oxygen o r Oxygen P l u s 100 mM Ro-07-0582 10 mM produces n e g l i g i b l e loss i n pl a t i n g e f f i c i e n c y although t h i s concentration produces nearly maximum s e n s i t i z a t i o n of hypoxic c e l l s . The toxic e f f e c t of 0582 on hypoxic c e l l s increases sharply for concentrations of 0582 greater than 1.0 mM such that a 3 hour incubation at 10 mM leaves less than 5% of the c e l l s viable. Comparable time-cut t o x i c i t y responses for other incubation times under hypoxia can be constructed from f i g . 6. 3.2.2 Treatment of CHO C e l l s In Suspension With 0582 Figure 17 shows sur v i v a l curves for CHO c e l l s i r r a d i a t e d at 22°C under hypoxic or aerobic conditions. As was done for the CH2B2 l i n e , a least squares f i t was obtained for the l i n e a r portion of each curve. The OER and the DMF values for each drug concentration were determined by comparing the doses which reduced su r v i v a l to 0.01. The DMF's calculated from a number of CHO su r v i v a l curves are shown i n f i g . 18 (closed c i r c l e s ) , and again above 10 mM 0582 the DMF approaches the OER value. Also shown for comparison i n f i g . 18 i s the DMF response of CH2B2 c e l l s (see f i g . 15) calculated not from the slopes of the survival curves but by comparing the doses which reduced survival to 0.01. I t can be seen that when DMF values are calculated i n t h i s way, the maximum DMF and OER approach a common value of approximately 3 for both c e l l l i n e s . 50. * N 2 * N 2 + 008mM 0582 • N 2 + 0-6 mM 0582 _j i I i i 1000 2000 3000 D O S E (rads) FIGURE 17. S u r v i v a l C u r v e s f o r CHO C e l l s I r r a d i a t e d a t  22° C i n t h e P r e s e n c e o f N i t r o g e n , N i t r o g e n  P l u s 0.08, 0.6, 3, o r 25 mM Ro - 0 7 - 0 5 8 2 , o r Oxygen 341 30| 2-6 LL 2-2 • 1-8 14 10 0-6 1 1 — T T O E R R A N G E v j u_L C H O ( s=0 Ol) C H 2 B 2 (S=0 Ol) J u_i J L 001 01 10 10 CONCENTRAT ION O F Ro -07 -0582 (mM) 100 FIGURE 18. Ro-07-0582 S e n s i t i z a t i o n o f CHO C e l l s i n  S u s p e n s i o n , Shown as a F u n c t i o n o f Drug C o n c e n t r a t i o n The 0582 s e n s i t i z a t i o n o f b o t h CHO c e l l s ( c l o s e d c i r c l e s and s o l i d c u r v e ) and CH2B c e l l s ( d a s h e d c u r v e ; e x p e r i m e n t a l p o i n t s n o t shown) i n s u s p e n s i o n a t 22°C i s shown. DMF v a l u e s were c a l c u l a t e d by c o m p a r i n g t h e d o s e s r e q u i r e d t o r e d u c e t h e s u r v i v i n g f r a c t i o n t o 0.01. 3.2.3 Treatment of EMT6 Ce l l s In Suspension With 0582 In f i g . 19 are presented several survival curves for EMT6 c e l l s i r r a d i a t e d at 22°C under aerobic or hypoxic conditions. Least squares f i t s and DMF's were obtained as described i n section 3.2.2. The DMF's calculated from a number of EMT6 sur v i v a l curves are shown i n f i g . 20 (closed c i r c l e s ) , again with the CH2B2 response shown for comparison. The s e n s i t i z a t i o n by 0582 of EMT6 c e l l s i n suspension i s very similar to the s e n s i t i z a t i o n of CH2B2 c e l l s (and CHO, f i g . 18), with drug concentrations of approximately 10 mM and greater producing a DMF approaching the OER. 3.2.4 Treatment of C e l l P e l l e t s With 0582 Cell p e l l e t experiments were desirable as a possible means of checking for c e l l concentration dependent s e n s i t i z a t i o n . An example of the above i s provided by triacetoneamine-N-oxyl (TAN) which produces a much reduced s e n s i t i z a t i o n i n vivo as compared to s e n s i t i z a t i o n achieved i n i n v i t r o t e s t s . Agnew and Skarsgard (1972) observed a reduced s e n s i t i z a t i o n by TAN for CH2B2 c e l l s i n a c e l l p e l l e t as compared to c e l l s i n suspension. A sim i l a r e f f e c t was observed for several other drugs including PNAP (Agnew and Skarsgard, 19 74), and i t was suggested that the c e l l p e l l e t experiment may prove to be a useful indicator of the effectiveness of ra d i o s e n s i t i z e r s i n vivo. © f\J2 * No+0.-09mM 0582 - J 1 1 I _ J . 00001 1000 2000 3000 D O S E (rads) FIGURE 19. S u r v i v a l C u r v e s f o r EMT6 C e l l s I r r a d i a t e d a t  2~2~°C i n t h e P r e s e n c e o f N i t r o g e n , N i t r o g e n  P l u s 0.09, 0.45, o r 2.2 mM R o - 0 7 - 0 5 8 2 , o r  Oxygen LL O 3 4 3 0 2 6 22 18 14 10 0 6 I I I " 1 1 TT O E R R A N G E (S=001) - C H 2 B 2 (S=O0l) I I I - J 1 1 1 J I I L aoi o.i to 10 100 C O N C E N T R A T I O N O F Ro 0 7 0 5 8 2 (mM) FIGURE 20. Ro-07-0582 S e n s i t i z a t i o n o f EMT6 C e l l s i n  S u s p e n s i o n , Shown a s a F u n c t i o n o f D r u g C o n c e n t r a t i o n The 0582 s e n s i t i z a t i o n o f b o t h EMT6 c e l l s ( c l o s e d c i r c l e s and s o l i d c u r v e ) a n d CH2B„ c e l l s ( d a s h e d c u r v e ; e x p e r i m e n t a l p o i n t s n o t shown) i n s u s p e n s i o n a t 22 C i s shown. DMF v a l u e s w e r e c a l c u l a t e d b y c o m p a r i n g t h e d o s e s r e q u i r e d t o r e d u c e t h e s u r v i v i n g f r a c t i o n t o 0.01. Figure 21 presents a comparison of the hypoxic response of drug-free C H 2 B 2 c e l l s i r r a d i a t e d at 22°C i n d i l u t e suspension or i n c e l l p e l l e t form. In order to prevent drying of the c e l l p e l l e t the tsl^ w a s f i r s t humidified by passing through a water-f i l l e d gas washing b o t t l e , a procedure followed i n a l l c e l l p e l l e t experiments. The results of f i g . 21 show the c e l l p e l l e t s u r v i v a l values to be s l i g h t l y but consistently greater than those for c e l l s i n suspension. However, the 3 slopes, calculated from least squares f i t s of s u r v i v a l values of 0.25 or l e s s , are equal to within the standard errors. Similar observations were.obtained using CHO c e l l s i n p e l l e t and suspension ( f i g . 22). I t i s f a i r to conclude from f i g s . 21 and 22 that high c e l l density does not greatly a f f e c t the radiation response of either c e l l l i n e . To check for possible time dependent loss of s e n s i t i z a t i o n of c e l l s i n p e l l e t form, as Agnew and Skarsgard (1974) observed for several drugs, c e l l p e l l e t s were prepared with equal drug concentrations and were i r r a d i a t e d at various times a f t e r formation of the p e l l e t s . The drug concentrations and incubation times were again such that no toxic e f f e c t s were observed upon the zero dose control samples. The res u l t s are presented i n f i g . 23. In t h i s experiment, c e l l p e l l e t s were formed with drug concentrations of 0 (1 p e l l e t ) , 0.48 (3 pel l e t s ) and 4.8 mM (3 p e l l e t s ) . P e l l e t s containing drug were then incubated at 22°C for 18, 50 or 90 minutes before they were ir r a d i a t e d under hypoxic conditions. The r e s u l t i n g s u r v i v a l 5 6 . 1000 2 0 0 0 3 0 0 0 4 0 0 0 D O S E ( r a d s ) FIGURE 21. S u r v i v a l C u r v e s f o r CH2B 2 C e l l s I r r a d i a t e d a t 22 C W h i l e i n a S u s p e n s i o n G a s s e d w i t h N i t r o g e n ,  i n a S u s p e n s i o n G a s s e d w i t h H u m i d i f i e d N i t r o g e n ,  o r as a C e l l P e l l e t G a s s e d w i t h H u m i d i f i e d  N i t r o g e n 57 . •i J 1 1 1 1000 2000 3000 DOSE (rads) FIGURE 22. S u r v i v a l C u r v e s f o r CHO C e l l s I r r a d i a t e d a t  22 C W h i l e i n a S u s p e n s i o n G a s s e d w i t h  H u m i d i f i e d N i t r o g e n o r as a C e l l P o l l e t G a s s e d w i t h H u m i d i f i e d N i t r o g e n oooov 1000 2000 3000 D O S E (rads) FIGURE 2 3 . S u r v i v a l C u r v e s f o r H y p o x i c CH2B^ C e l l s i n  P e l l e t F o rm I r r a d i a t e d a t 22 C i n t h e P r e s e n c e o f 0, 0.48, o r 4.8 mM 0582 To t e s t f o r t i m e - d e p e n d e n t l o s s o f s e n s i t i z a t i o n t h e c e l l p e l l e t s w e r e i n c u b a t e d a t 22 C f o r 18, 50, o r 90 m i n u t e s b e f o r e i r r a d i a t i o n . responses were f i t t e d to a straight l i n e by the method of least squares, again using only su r v i v a l values of 0.25 or les s . These responses show no s i g n i f i c a n t dependence on p e l l e t incubation time. This observation with 0582 i s in sharp contrast with e a r l i e r studies with TAN (Agnew and Skarsgard, 1972), a l l of which demonstrated a rapid loss of s e n s i t i z i n g capacity with p e l l e t incubation time. Survival curves were also obtained for CH2B2 c e l l p e l l e t s exposed to other drug concentrations and these data were pooled with those of f i g . 23. DMF's were calculated from the r a t i o of slopes of the l i n e a r least squares f i t s , and i n f i g . 24 these are plotted with the corresponding standard error. Also shown i n f i g . 24 i s the only r e s u l t for a CHO c e l l p e l l e t , also i r r a d i a t e d at 22°C. The curve, f i t t e d by eye, shows CH2B2 p e l l e t s e n s i t i z a t i o n to be s l i g h t l y less than the s e n s i t i z a t i o n achieved i n d i l u t e suspension ( f i g . 15). However, 0582 concentrations of the order of 20 mM gave a p e l l e t DMF of 2.6, approaching the f u l l OER. T 1 I I | T FIGURE 2 4. Ro-07-0582 S e n s i t i z a t i o n o f H y p o x i c C e l l s i n  P e l l e t Form, a s a F u n c t i o n o f C o n c e n t r a t i o n o f  Ro-07-05 82 B o t h CH2B„ c e l l p e l l e t s ( s o l i d c i r c l e s ) a n d t h e s i n g l e . CHO c e l l p e l l e t ( s o l i d square.) w e r e i r r a d i a t e d a t 2 2. C. 61. 3.3 PRETREATMENT AND POST-TREATMENT WITH 05 82 Because of the pretreatment and post-treatment e f f e c t s observed w i t h TAN by Agnew and Skarsgard (1972), i t was of i n t e r e s t t o determine whether such e f f e c t s e x i s t e d w i t h 0582. 3.3.1 Pretreatment With 0582 CH2B 2 c e l l s suspended a t a c o n c e n t r a t i o n of 2.5 X 10^ c e l l s / m l i n normal growth medium l a c k i n g sodium b i c a r b o n a t e were t r e a t e d f o r 1 hour a t 22°C wi t h 10 mM 0582 under a e r o b i c c o n d i t i o n s . The drug was then removed by twice c e n t r i f u g i n g the suspension and resuspending the c e l l s i n d r u g - f r e e medium l a c k i n g sodium b i c a r b o n a t e . The suspension was p l a c e d i n an i r r a d i a t i o n v e s s e l and, 1 hour a f t e r removal of the drug, i r r a d i a t e d under hypoxic c o n d i t i o n s . F i g u r e 25 shows the r e s u l t s of t h i s experiment. A l s o shown are s u r v i v a l data o b t a i n e d c o n c u r r e n t l y f o r hypoxic CH2B 2 c e l l s i r r a d i a t e d i n dr u g - f r e e medium l a c k i n g sodium b i c a r b o n a t e . W i t h i n experimental accuracy the r e s u l t s show no pretreatment e f f e c t f o r 0582. 3.3.2 Post-Treatment With 0582 5 CH2B 2 c e l l s suspended a t 2.5 X 10 c e l l s / m l were i r r a d i a t e d under hypoxic c o n d i t i o n s . Immediately f o l l o w i n g i r r a d i a t i o n t h r e e a l i q u o t s were removed from the hypoxic suspension and added t o an a e r o b i c s o l u t i o n of 0582 such t h a t the f i n a l drug 6 2 . Z o % LL CD Z > > C£ D (/) 1000 2000 DOSE 3000 4000 (rads) FIGURE 25, S u r v i v a l R e s p o n s e s f o r CH2B,., C e l l s y'a) I r r a d i a t e d  a t 22 C i n t h e P r e s e n c e o f N i t r o g e n o r (b) I r r a d i a t e d F o l l o w i n g P r e t r e a t m e n t f o r 1 H o u r a t 22°C w i t h 10 mM Ro-07-0582 The 0582 was r e m o v e d b y w a s h i n g p r i o r t o i r r a d i a t i o n . c o n c e n t r a t i o n was 0 ( c o n t r o l ) , 10 or 30 mM. A f t e r one hour the c e l l s were removed from the drug by c e n t r i f u g i n g and resuspending i n drug - f r e e medium. The c e l l s were then p l a t e d to determine colony forming a b i l i t y . The suspensions were maintained a t 22°C p r i o r t o , d u r i n g and a f t e r i r r a d i a t i o n . The r e s u l t s , presented i n f i g . 26, show no post-treatment e f f e c t w i t h 0582. 64. z o U < LL O Z 0-1 > 0O1 > D 0) • N 2 © N 2 +1 hour posttreatment in 10 mM 0582 * N2 +1 hour posttreatment in 30 mM 0582 0 0 0 1 0 1000 2 0 0 0 D O S E ( rads ) 3 0 0 0 4000 FIGURE 26. S u r v i v a l R e s p o n s e s f o r C H 2 B 0 C e l l s S u b j e c t e d t o a 1 Hour P o s t - t r e a t m e n t " w i t h 0, 10, o r 30 mM Ro-07-0582 a t 22°C, F o l l o w i n g I r r a d i a t i o n U n d e r H y p o x i c C o n d i t i o n s i n t h e A b s e n c e o f 0582 4. DISCUSSION 4.1 TOXICITY OF Ro-07-0582 Each of the three c e l l l i n e s tested showed that the toxic effects of 0582 i n the absence of i r r a d i a t i o n are quite selec t i v e for hypoxic c e l l s . This can be seen by comparison of f i g . 5 with f i g . 6, f i g . 9 with f i g . 10, and f i g . 11 with f i g . 12. This suggests that the drug may have, i n addition to an impressive r a d i o s e n s i t i z i n g capacity, s i g n i f i c a n t chemo-therapeutic properties. In both cases the e f f e c t s are selective for hypoxic c e l l s . Thus, hypoxic tumour c e l l s , i r r a d i a t e d i n the presence of 0582, would be placed i n a double jeopardy s i t u a t i o n provided that the b i o l o g i c a l h a l f - l i f e of the compound i n the patient was of the order of a few hours. An example of the above may be constructed from our data with the CHO c e l l l i n e . Let us consider the e f f e c t upon aerobic and hypoxic CHO c e l l s subjected to a 4 hour incubation i n 5 mM 0582, with 1000 rads delivered somewhere i n t h i s time i n t e r v a l . Let us also assume that the two e f f e c t s are additive. Figure 18 indicates that hypoxic c e l l s i r r a d i a t e d i n the presence of 5 mM 0582 w i l l experience a DMF of -2.6. From the d e f i n i t i o n of the DMF (see 2.3.5), t h i s implies the sur v i v a l rate w i l l be about the same as for drug-free hypoxic c e l l s exposed to 2600 rads. Figure 17 shows the corresponding surviving f r a c t i o n to be 0.015. Figure 17 also shows that - 65 -1000 rads reduces the s u r v i v i n g f r a c t i o n o f a e r o b i c CHO c e l l s t o 0.005. F u r t h e r , f i g s . 9 and 10 show t h a t a e r o b i c CHO c e l l s w i l l experience no t o x i c i t y w h i le =99% of hypoxic c e l l s w i l l be k i l l e d . The net s u r v i v i n g f r a c t i o n s of o r i g i n a l v i a b l e -3 ~3 c e l l s are 0.15 X 10 f o r hypoxic c e l l s versus 5 X 10 f o r a e r o b i c c e l l s . The net s u r v i v a l of hypoxic c e l l s t r e a t e d as above i s much lower than f o r hypoxic c e l l s i r r a d i a t e d i n the absence of drug, which y i e l d s a DMF of 1.0 and no s e l e c t i v e t o x i c i t y . F i g u r e 17 shows the s u r v i v i n g f r a c t i o n o f d r u g - f r e e hypoxic c e l l s exposed t o 1000 rads t o be 0.4. Comparison of the net s u r v i v i n g f r a c t i o n s i n d i c a t e s t h a t the combination of r a d i o s e n s i t i z a t i o n and t o x i c i t y may be of value as an approach to the hypoxic c e l l problem. There are s e v e r a l p o i n t s which should be d i s c u s s e d a t t h i s stage. F i r s t , the assumption t h a t the e f f e c t s o f t o x i c i t y and r a d i o s e n s i t i z a t i o n are a d d i t i v e i s not u n j u s t , as one would expect any i n t e r a c t i o n of the two would enhance c e l l k i l l i n g and r e s u l t i n reduced s u r v i v a l f o r hypoxic c e l l s , thus improving the e f f e c t i v e n e s s o f the treatment. Secondly, we should c o n s i d e r the e f f e c t i v e n e s s o f i r r a d i a t i o n and t o x i c i t y on c e l l s exposed t o v a r i o u s degrees of oxygenation. T h i s i s necessary because of the d i f f e r e n t oxygen c o n c e n t r a t i o n s t h a t e x i s t i n v a r i o u s r e g i o n s of some tumors. For a s u f f i c i e n t l y l a r g e tumor, one would expect c e l l s t o range from about t o t a l l y hypoxic to a e r o b i c . I f we c o n s i d e r o n l y i r r a d i a t i o n e f f e c t s , the presence of oxygen w i l l r e s u l t i n a lower s u r v i v a l r a t e than t h a t f o r hypoxic c o n d i t i o n s , s i n c e oxygen i s a r a d i o s e n s i t i z e r . Thus r a d i a t i o n treatment would be more e f f e c t i v e on a tumor having c e l l s exposed t o v a r i o u s degrees of oxygenation than on a t o t a l l y hypoxic tumor. I f we now c o n s i d e r only t o x i c e f f e c t s , we might expect t h a t a reduced o v e r a l l t o x i c i t y would be observed f o r c e l l s having a range of 0 2 l e v e l s (such as might be found i n a tumor) as compared t o a group of a c u t e l y hypoxic c e l l s . T h i s i s seen by comparison of f i g s . 6 and 8, which i n d i c a t e s t h a t the hypoxic t o x i c i t y o f 0582 i s a f f e c t e d by f a i r l y s m a l l changes i n 0 2 c o n c e n t r a t i o n i n the c e l l suspension. However, the advantage t o the s e l e c t i v e hypoxic t o x i c i t y e x h i b i t e d by 0582 i s t h a t the more hypoxic the c e l l ' s environment, and t h e r e f o r e the more r a d i o r e s i s t a n t the c e l l , the g r e a t e r the p r o b a b i l i t y of death due to t o x i c e f f e c t s of the drug. Our d i s c o v e r y of d i f f e r e n t i a l t o x i c i t y w i t h 0582 l e d to s i m i l a r o b s e r v a t i o n s w i t h other p o t e n t i a l r a d i o s e n s i t i z e r s . Mohindra and Rauth ( p r i v a t e communication), working w i t h CHO c e l l s , have r e p o r t e d t h a t the t o x i c i t i e s of metronidazole and n i t r o f u r a z o n e e x h i b i t a dependence upon the 0 2 content of the gas f l o w i n g over the c e l l suspensions. F o l l o w i n g a 6 hour i n c u b a t i o n a t 34°C i n 29 mM metronidazole the p l a t i n g e f f i c i e n c y was r e p o r t e d one l o g decreased f o r c e l l s gassed w i t h N 2 c o n t a i n i n g 10 ppm 0 2 ( p l a t i n g e f f i c i e n c y = 5%) as compared to N 2 c o n t a i n i n g 100 ppm 0 2 ( p l a t i n g e f f i c i e n c y = 45%). These o b s e r v a t i o n s are compatible w i t h the o b s e r v a t i o n s (see above and 68. f i g s . 6 and 8) for 0582 and the CH2B2 l i n e . Further, incubation i n 1 mM 0582 for 6 hours with N 2 (=10 ppm 0^, f i g . 8) i s shown to reduce the pl a t i n g e f f i c i e n c y to 3%. Thus i t i s evident that the hypoxic t o x i c i t y displayed by Ro-07-05 82 i s considerably greater than that of metronidazole. This conclusion i s supported by studies of metronidazole performed i n our laboratory (Skarsgard and Harrison, private communication). The greatly increased t o x i c i t y of 0582 under hypoxic conditions could be caused by a metabolic product of reduction of the drug, a reduction proceeding only i n the absence of 0 2. Olive and McCalla (1975) have shown that single-strand breaks are produced i n mammalian c e l l DNA both i n v i t r o and i n vivo as a r e s u l t of nitrofurazone treatment under hypoxic conditions. They also measured the rate of reduction of the drug under various 0 2 l e v e l s and .demonstrated that reduction was eliminated under normal aerobic conditions. Olive and McCalla concluded that the metabolic reduction of nitrofurazone i s required for DNA single-strand break production. Support for t h i s proposal i s provided by the observation that while single-strand breaks were produced i n the DNA of E. c o l i / r by nitrofuran derivatives (including nitrofurazone), no detectable breaks were produced i n an E. c o l i mutant lacking nitrofurazone-reductase a c t i v i t y (McCalla, Reuvers and Kaiser, 1971). Our observation of increased acute t o x i c i t y under hypoxic conditions might therefore be s i m i l a r l y explained by the hypoxic reduction of Ro-07-0582 to form a toxic product. A comparison of f i g s . 6 and 7 shows t h a t the hypoxic t o x i c i t y of 0582 i s s u b s t a n t i a l l y g r e a t e r a t 37°C than a t 22°C, as might be expected i f enzymatic r e d u c t i o n of the drug i s i n v o l v e d . T h i s temperature e f f e c t , however, c o u l d a l s o be due to some secondary m e t a b o l i c event i n v o l v e d i n d e v e l o p i n g the t o x i c e f f e c t . 70. 4.2 RADIOSENSITIZATION WITH Ro-07-0582 4.2.1 Pre- and Post-treatment The absence o f ' p r e - and p o s t ^ i r r a d i a t i o n s e n s i t i z a t i o n (see 3.3.1 and 3.3.2) i n d i c a t e s t h a t 0582 employs a d i f f e r e n t mechanism of s e n s i t i z a t i o n than does TAN, f o r which both p r e -and p o s t - i r r a d i a t i o n s e n s i t i z a t i o n were observed (Agnew and Skarsgard, 1972). I t i s l i k e l y t h a t 0582 s e n s i t i z e s a c c o r d i n g to the e l e c t r o n a f f i n i t y h ypothesis (see 1.3), which i n v o l v e s r e a c t i o n s w i t h s h o r t - l i v e d s p e c i e s , thus s u g g e s t i n g the absence of p r e - and post-treatment s e n s i t i z a t i o n . F u r t h e r support f o r t h i s s uggestion i s p r o v i d e d by the work of W h i l l a n s , Adams, and Neta (1975) who showed, by a study of o n e - e l e c t r o n t r a n s f e r r e a c t i o n s , t h a t the e l e c t r o n a f f i n i t y o f the 2 - n i t r o i m i d a z o l e s t r u c t u r e (e.g. Ro-07-0582) i s g r e a t e r than t h a t of the 5 - n i t r o i m i d a z o l e s t r u c t u r e (e.g. m e t r o n i d a z o l e ) . E l e c t r o n s p i n resonance s t u d i e s supported t h i s o b s e r v a t i o n . Thus the e l e c t r o n a f f i n i t y c o r r e l a t e s w i t h the e f f e c t i v e n e s s of the compound as a s e n s i t i z e r . The p r e - and post-treatment e f f e c t s observed w i t h TAN i n d i c a t e t h a t t h i s s e n s i t i z e r does not s e n s i t i z e l a r g e l y by i n t e r a c t i o n w i t h s h o r t - l i v e d s p e c i e s . I t has been suggested t h a t TAN a c t s by producing s t a b l e u n r e p a i r e d DNA l e s i o n s (Emmerson, 1969; Nakken, S i k k e l a n d and Brustad, 1970; Hohman, P a l c i c and Skarsgard, p r i v a t e communication). Thus, although most a t t e n t i o n i s p r e s e n t l y focused upon e l e c t r o n a f f i n i c compounds, t h i s by no means excludes o t h e r mechanisms of s e n s i t i z a t i o n . 4.2.2 R a d i o s e n s i t i z a t i o n i n D i l u t e Suspension The r e s u l t s of f i g . 16 i n d i c a t e t h a t 0582 demonstrates one of the e s s e n t i a l p r o p e r t i e s of a p o t e n t i a l r a d i o s e n s i t i z e r : s e l e c t i v e s e n s i t i z a t i o n of hypoxic c e l l s . Presence of both 0582 and oxygen d u r i n g i r r a d i a t i o n produces the same s u r v i v a l response as f o r oxygen presence alone. T h i s o b s e r v a t i o n i s e x p l a i n e d by the f a c t t h a t oxygen i s extremely e l e c t r o n a f f i n i c , much more so than any of the e l e c t r o n a f f i n i c r a d i o s e n s i t i z e r s i n c l u d i n g 0582. A c c o r d i n g to the r a d i o s e n s i t i z a t i o n -r a d i o p r o t e c t i o n model of Chapman e t a_l (see 1.3), oxygen and 0582 would compete f o r the f i x a t i o n of damage and the o x i d a t i o n of r a d i c a l r e d u c i n g s p e c i e s . I t i s l i k e l y t h a t under f u l l y a e r o b i c c o n d i t i o n s the oxygen p r e s e n t s a t u r a t e s the f i x a t i o n and o x i d a t i o n p r o c e s s e s . T h e r e f o r e , a d d i t i o n of 0582 r e s u l t s i n no a d d i t i o n a l s e n s i t i z a t i o n . A l t e r n a t i v e l y , the r e l a t i v e l y low e l e c t r o n a f f i n i t y of 0582 compared to oxygen may r e s u l t i n a non-measureable component of l e t h a l i t y under a e r o b i c c o n d i t i o n s . Very s i m i l a r s e n s i t i z a t i o n was achieved when 0582 was p r e s e n t d u r i n g the i r r a d i a t i o n of hypoxic CH2B 2, CHO ( f i g . 18), or EMT6 c e l l l i n e s ( f i g . 20). D e t e c t a b l e s e n s i t i z a t i o n occurs f o r drug c o n c e n t r a t i o n s as low as 0.1 mM, w h i le c o n c e n t r a t i o n s of about 10 mM gave s e n s i t i z a t i o n s i m i l a r to t h a t achieved w i t h oxygen. These r e s u l t s are s i m i l a r t o those o f A s q u i t h e t al_ (1974) who r e p o r t e d the maximum s e n s i t i z a t i o n of 0582 upon Chinese hamster c e l l s ( l i n e V79-GL1) t o y i e l d a DMF of roughly 2.5 a t a drug c o n c e n t r a t i o n of 5 mM. They a l s o r e p o r t e d DMF's of 2.2 a t 1 mM and 1.6 a t 0.4 mM. These val u e s are onl y s l i g h t l y h i g h e r than our DMF values of 1.8 and 1.5 at 1.0 mM and 0.4 mM 0582 r e s p e c t i v e l y , i n CH2B2 c e l l s . The s e n s i t i z a t i o n p r o v i d e d by Ro-07-0582 i s b e t t e r than t h a t o f metronidazole. The maximum DMF (1.8 t o 1.9) f o r metronidazole i s reached a t a c o n c e n t r a t i o n of roughly 10 mM (Asquith e t a l , 1974; Agnew, P a l c i c and Skarsgard, unpublished d a t a ) , whereas a DMF of 1.9 i s a t t a i n e d w i t h 1-2 mM 0582 ( f i g s . 18 and 20). Furthermore, 0582 was observed t o g i v e DMF's g r e a t e r than 1.9, e v e n t u a l l y r e a c h i n g the f u l l OER i n d i l u t e c e l l suspension. It should be mentioned t h a t the p o s s i b i l i t y o f synergism between the t o x i c and r a d i o s e n s i t i z i n g p r o p e r t i e s of Ro-07-0582 should be unimportant i n most of the s e n s i t i z a t i o n s t u d i e s s i n c e the time of exposure t o the drug was g e n e r a l l y 1.5 hours or l e s s . Drug c o n c e n t r a t i o n s l e s s than 50 mM show very l i t t l e t o x i c i t y a t these exposure times, even under hypoxia. Of course, i f the t o x i c and s e n s i t i z i n g e f f e c t s are onl y a d d i t i v e , the c o n t r o l samples would have normalized out the t o x i c e f f e c t s . In c l i n i c a l use of the drug, however, when i t i s l i k e l y t o be pre s e n t i n s i g n i f i c a n t c o n c e n t r a t i o n s f o r many hours, one would 73. expect that both the r a d i o s e n s i t i z i n g capacity and the hypoxic t o x i c i t y of 0582 may be important (see 4.1). 4.2.3 Radiosensitization of C e l l P e l l e t s The s l i g h t r e l a t i v e radioresistance of hypoxic c e l l s i r r a d i a t e d i n c e l l p e l l e t form as compared to hypoxic c e l l s i r r a d i a t e d i n d i l u t e suspension ( f i g s . 21 and 22) i s i n agreement with the observations of Thomson and Rauth (1974), who used c e l l suspensions prepared d i r e c t l y from the KHT fibrosarcoma of C3H mice. When various c e l l densities were ir r a d i a t e d under hypoxic conditions, Thomson and Rauth observed a s l i g h t increase i n su r v i v a l at high c e l l d e n s i t i e s . This was attributed to an increase i n the extrapolation number N rather than a change i n the slope of the curve. Our data appear to support t h i s suggestion, but more precise measurements w i l l be necessary before a firm conclusion can be reached. Figure 24 shows that the 0582 s e n s i t i z a t i o n attained i n c e l l p e l l e t s i s quite comparible with, and perhaps s l i g h t l y less than that attained i n d i l u t e suspension ( f i g s . 15 and 18). Denekamp and Harris have observed with mice that i n j e c t i o n of 1 mg 0582 per gram body weight (which corresponds to 5-7.5 mM 0582 i n the body flui d s ) y i e l d s a DMF of approximately 2 i n two d i f f e r e n t i n vivo assays (Denekamp, Michael and Harris, 1974; Denekamp and Harris, 1975). In our c e l l p e l l e t s , 6 mM 0582 gives a DMF of 2.4 ( f i g . 24). This provides support for the i d e a t h a t the i n v i t r o c e l l p e l l e t system may be u s e f u l as a t e s t system i n t e r m e d i a t e between c o n v e n t i o n a l i n v i t r o and i n v i v o systems (see 3.2.4). F u r t h e r support f o r t h i s s uggestion i s p r o v i d e d by the work of Thomson and Rauth (1974) who showed t h a t NF-167 (a n i t r o f u r a n d e r i v a t i v e ) l o s t the a b i l i t y to s e n s i t i z e hypoxic KHT tumor c e l l s when the c e l l Q c o n c e n t r a t i o n approached 10 c e l l s / m l , w h i l e metronidazole showed l i t t l e l o s s o f s e n s i t i z i n g a b i l i t y . T h i s c o r r e l a t e s w e l l w i t h the observed s e n s i t i z a t i o n of hypoxic KHT c e l l s i n mice by metronidazole and the l a c k of i n v i v o s e n s i t i z a t i o n by NF-167 (Rauth and Kaufman, 1975). 4.3 Ro-07-0582 AS A POTENTIAL RADIOSENSITIZER In section 1.1 there were several properties l i s t e d which are considered desirable for an e f f e c t i v e r a d i o s e n s i t i z e r . The work i n t h i s thesis and the work of others permits us to now consider the attributes of Ro-07-0582 as a po t e n t i a l r a d i o s e n s i t i z e r . Ro-07-0582 d e f i n i t e l y shows the a b i l i t y to p r e f e r e n t i a l l y s e n s i t i z e hypoxic c e l l s to the l e t h a l e f f e c t s of i o n i z i n g radiation (see 4.2.2). The s e n s i t i z a t i o n achieved i n v i t r o i s better than that of metronidazole. Ro-07-0582 has also been shown to sens i t i z e hypoxic c e l l s i n vivo. For drug doses necessary to achieve high lev e l s of se n s i t i z a t i o n the t o x i c i t y of Ro-07-0582 to aerobic c e l l s i s quite acceptable (see 3.2.1 and f i g . 15). The t o x i c i t y to hypoxic c e l l s , however, i s much increased over the t o x i c i t y to aerobic c e l l s , and t h i s may prove to be a useful adjunct to the drug's s e n s i t i z i n g properties i n destroying hypoxic tumor c e l l s . I t has been shown by Asquith et a l (1974b), using Chinese hamster c e l l s , that the s e n s i t i z a t i o n achieved with 0582 i s independent of the position of the c e l l s i n the mitotic cycle. This experiment involved measurement of the DMF by single dose i r r a d i a t i o n s of hypoxic synchronized c e l l s , both i n the presence of 4 mM 0582 and i n drug-free suspensions. Although the s e n s i t i v i t y of the c e l l s changed with position i n the c e l l c y c l e , being most s e n s i t i v e near m i t o s i s and most r e s i s t a n t i n l a t e S-phase, the DMF was constant. The l i m i t e d r e s u l t s of p r e l i m i n a r y serum c o n c e n t r a t i o n measurements i n man i n d i c a t e t h a t 0582 i s absorbed r a p i d l y and i s e l i m i n a t e d from the serum wi t h a h a l f - l i f e of 9-18 hours (Annual Report 1974, Gray L a b o r a t o r y ) . Thus i t i s hoped t h a t Ro-07-0582 has s u f f i c i e n t m e t a b o l i c s t a b i l i t y t o be e f f e c t i v e . As w e l l , s i n g l e doses of up to 10 grams 0582 have been taken o r a l l y , a p p a r e n t l y w i t h few or no s i d e e f f e c t s . T h i s i s very encouraging when the o b s e r v a t i o n s of Denekamp and H a r r i s (1975) are c o n s i d e r e d . 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