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Potential use of 52FE porphyrins as tumor scanning agents Thaller, Roy Alan 1981

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POTENTIAL USE OF 5 2pE PORPHYRINS AS TUMOR SCANNING AGENTS by ROY ALAN THALLER B . S c , The U n i v e r s i t y o f B r i t i s h C olumbia, 1978 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES THE FACULTY OF PHARMACEUTICAL SCIENCES DIVISION OF PHARMACEUTICAL CHEMISTRY We a c c e p t t h i s t h e s i s as c o n f o r m i n g t o t h e r e q u i r e d s t a n d a r d THE UNIVERSITY OF BRITISH COLUMBIA September 19 8 0 © Roy A l a n T h a l l e r , 1980 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by his representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of Pharmaceutical Sciences The University of British Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 Date Nov. 10, 1980 -6 ABSTRACT R a d i o i r o n l a b e l l e d p r o p h y r i n s were t e s t e d f o r tumor uptake u s i n g t i s s u e c u l t u r e and animal models. The f o l l o w i n g p o r p h y r i n s were t e s t e d : hematohemin; protohemin; photo-protohemin; 2 - f o r m y l - 4 - v i n y l , 2 - v i n y l - 4 formyl, and 2,4-d i f o r m y l deuterohemin d e r i v a t i v e s ; meso-tetra (4 carbo-xyphenyl) hemin (TCP); tetra-Na-meso-tetra ( 4 - s u l f o n a t o -phenyl) hemin (TPPS); and meso-tetra-(4-N-methylpyridyl) hemin t e t r a i o d i d e (TMPI). 5 2 F e was produced a t TRIUMF by h i g h energy proton s p a l l a t i o n of a n i c k e l t a r g e t . The 5 2 F e was separated from the other s p a l l a t i o n products by s o l v e n t e x t r a c t i o n w i t h methyl i s o b u t y l acetone and i o n exchange chromatography when r e q u i r e d . T i s s u e c u l t u r e s t u d i e s u s i n g P815 mouse tumor c e l l s showed good uptake with protohemin, TCP, or TMPI. Mouse d i s t r i b u t i o n and e x c r e t i o n s t u d i e s i n d i c a t e d t h a t the t a r g e t organ f o r TMPI was the l i v e r (and spleen) and i t s b i o l o g i c a l h a l f -l i f e was 270 days. Animal scans u s i n g r a t s w i t h b r e a s t carcenomas w i t h 5 2 F e l a b e l l e d protohemin, TCP and TMPI showed no tumor uptake a t a l l . The r a d i a t i o n dose to a human was a l s o c a l c u l a t e d . i i i TABLE OF CONTENTS A b s t r a c t i i Table o f Contents i i i L i s t of F i g u r e s i x L i s t of Tables x Acknowledgements x i I I n t r o d u c t i o n 1 II L i t e r a t u r e Review 2 A Tumor S t r u c t u r e and Ph y s i o l o g y 2 B The Role o f Tumor - Imaging Radiopharmaceuticals i n Oncology 5 C Tumor Imaging Radiopharmaceuticals 7 1) M e t a b o l i t e Related Agents 7 a) 1 ^ - A s p a r t i c a c i d 7 b) 1 ^ - C a r b o x y l 1-aminocyclopentanae-c a r b o x y l i c a c i d 7 c) 1 1 C - M e t h y l a t e d polyamine analogs 8 d) 1 8 F - S - f l u r o u r a c i l 8 e) 1 3N-Ammonia 8 f) 1 3N-Glutamine and .l.'3N-Glutamic a c i d 8 g) 7 5 S e - L - s e l e n o m e t h i o n i n e 9 2) R a d i o n u c l i d e s and Other Agents 10 a) A r s e n i c 10 b) Bismuth 10 c) Copper 10 d) Cesium 10 i v e) C o b a l t 11 f) Gold 11 g) Indium 11 h) Mercury 12 i ) Selenium 13 j) Technetium 13 k) Thulium 14 1) T h a l l i u m 14 m) Xenon 15 n) Ytterbium 15 o) G a l l i u m 16 3) R a d i o l a b e l e d Antituman Agents 23 a) Bleomycin 2 3 4) R a d i o i o d i n a t e d Agents 24 a) Chloroquine analogs 24 b) I o d o c h o l e s t e r o l 25 D P o r p h y r i n Nomenclature 25 E P o r p h y r i n Uptake by Tumors 26 F P o r p h y r i n S y n t h e s i s 39 G R a d i o i r o n P r o d u c t i o n 44 1) 5 0cr U,2n) " " F e 47 2) ,5 2cr ( 3He, 3n), 5 2Fe 50 3) 5 5 M n (p,4n) 5 2Fe 52 4) S p a l l a t i o n Reactions 53 H Chemistry and S e p a r a t i o n Methods of I r o n . . .. 54 V. I M e t a l l o p o r p h y r i n S y n t h e s i s 57 I I I M a t e r i a l and Methods 59 A Chemicals 59 1) Po r p h y r i n s 59 2) Chromatography 59 3) R a d i o n u c l i d e s 59 4) Reagents 60 B Instrumentation 60 1) R a d i o a c t i v i t y Measurements 60 a) Gamma Spectrometry 6 0 b) R a d i o n u c l i d e Imaging or Scanning 61 c) D i s t r i b u t i o n S t u d i e s 61 d) E x c r e t i o n S t u d i e s 62 2) A b s o r p t i o n Spectrophotometry 6 2 C P o r p h y r i n S y n t h e s i s 62 1) P r o t o p o r p h y r i n 62 2) P r o t o p o r p h y r i n P i - T e r t i a r y B u t y l E s t e r (DTBE)6 3 3) Photoprotoporphyrin DTBE 65 4) 2-Formyl-4-vinyl d e u t e r o p o r p h y r i n DTBE .. 66 5) 2 - V i n y l - 4 - f o r m y l - d e u t e r o p o r p h y r i n DTBE .. 68 6) 2-Formyl 1 - 4 - v i n y l d e u t e r o p o r p h y r i n f r e e a c i d . . 68 7) 2- V i n y l - 4 - f o r m y l d e u t e r o p o r p h y r i n f r e e a c i d 6 9 8) 2,4-Diformyl d e u t e r o p o r p h y r i n DTBE .. ... 69 v i . 9) 2,4-Diformyl d e u t e r o p o r p h y r i n f r e e a c i d ... 70 10) Photoprotoporphyrin f r e e a c i d . . . . 71 D 5 2 F e P r o d u c t i o n 71 1) Target I r r a d i a t i o n s 71 2) S a f e t y E v a l u a t i o n and AECB L i c e n s e A p p l i c a -t i o n 74 3) Hot C e l l Design and C o n s t r u c t i o n .. .. 78 4) S e l e c t i o n of 5 2 F e Process Chemistry u s i n g 5 9 F e 79 a) Recovery T e s t 79 b) R a d i o n u c l i d e Impurity Determination 8 0 5) High L e v e l 5 2 F e P r o d u c t i o n 81 E M e t a l l o p o r p h y r i n S y n t h e s i s 81 1) 5 9Fe-Hematohemin 81 2) 5 9 Fe-Protohemin 84 3) 5 9Fe-Photoprotohemin 84 4) 5 9 F e - 2 — F o r m y l - 4 - v i n y l deuterohemin .. .. 85 5) 5 9 F e - 2 - V i n y l - 4 - f o r m y l deuterohemin .. .. 86 6) 5 9 F e - 2 , 4 - D i f o r m y l deuterohemin 87 7) 5 9Fe-meso-tetra(4-carboxyphenyl)hemin (TCP) 8 7 8) 5 9 F e - m e s o - t e t r a (4-N-methylpyridyl) hemin t e t r a i o d i d e (TMPI) 88 9) 5 9 F e - t e t r a - N a - m e s o - t e t r a (4-sulfonatophenyl) hemin (TPPS) 8 8 10) N i c k e l S p a l l a t i o n product l a b e l e d meso-tetra (4-N-methy-pyridyl p o r p h y r i n t e t r a i o d i d e 88 v i i . 11) 5 2 Fe-Protohemin 89 12) 5 2 F e - m e s c — t e t r a (4-carboxyphyenyl) hemin 89 13) 5 2 F e - m e s o - t e t r a (4-N-methylpyridyl)hemin  t e t r a i o d i d e 89 F Tumor T i s s u e C u l t u r e Uptake S t u d i e s 8 9 G Animal S t u d i e s 90 1) D i s t r i b u t i o n S t u d i e s ... . . 90 2) S c i n t i g r a p h y 91 3) E x c r e t i o n S t u d i e s 91 IV R e s u l t s and D i s c u s s i o n A P o r p h y r i n S y n t h e s i s 92 1) Hematoporphyrin 92 2) P r o t o p o r p h y r i n .3 92 3) P r o t o p o r p h y r i n DTBE 93 4) Photoprotoporphyrin DTBE Isomer 1 .. .. 94 5) Photoprotoporphyrin DTBE Isomer 2 .. .. 94 6) 2-Formyl-4-vinyl d e u t e r o p o r p h y r i n DTBE .. 95 7) 2-Vinyl-4-Formyl d e u t e r o p o r p h y r i n DTBE .. 95 8) 2-Formyl-4-vinyl d e u t e r o p o r p h y r i n f r e e a c i d 95 9) 2- V i n y l - 4 - f o r m y l d e u t e r o p o r p h y r i n f r e e a c i d 96 10) 2,4-Diformyl d e u t e r o p o r p h y r i n DTBE .. .. 96 11) 2,4-Diformyl d e u t e r o p o r p h y r i n f r e e a c i d 97 12) Photoprotoporphyrin f r e e a c i d 97 B 5 2 F e P r o d u c t i o n 97 1) Target I r r a d i a t i o n s 97 v i i i . 2) S a f e t y E v a l u a t i o n 99 3) S e l e c t i o n of 5 2 F e Process Chemistry u s i n g 5 9 F e 114 a) Recovery T e s t 114 b) R a d i o n u c l i d e Impurity Determination 116 4) High L e v e l 5 2 F e P r o d u c t i o n 118 C M e t a l l o p o r p h y r i n S y n t h e s i s 12 0 1) 5 9Fe-Hematohemin 120 2) 5 9Fe-Protohemin 121 3) 5 9Fe-Photoprotohemin 121 4) 5 9 F e - 2 - F o r m y l - 4 - v i n y l deuterohemin .. .. 123 5) 5 9 F e - 2 - V i n y l - 4 - f o r m y l deuterohemin .. .. 123 6) 5 9 F e - 2 , 4 - D i f o r m y l deuterohemin 123 7) 5 9 F e or 5 2 F e l a b e l e d meso-tetra-(4-carboxylphenyl) hemin or meso-tetra-(4-N-methylpynidyl) hemin t e t r a -i o d i d e on t e t r a - N a - m e s o - t e t r a - ( 4 - s u l f o n a t o -phenyl) hemin 124 D Tumor T i s s u e C u l t u r e Uptake S t u d i e s 125 E Animal S t u d i e s . . . . 131 1) D i s t r i b u t i o n S t u d i e s 131 2) S c i n t i g r a p h y 13 8 3) E x c r e t i o n Study . . 138 F Dosimetry . . . . . . . . . . 141 V C o n c l u s i o n s . . . . . . 147 VI B i b l i o g r a p h y 150 i x . LIST OF FIGURES 1. P o r p h y r i n S t r u c t u r e .. .. .. 27 2. Decay scheme of 5 2 F e .. .. 46 3. P o s s i b l e i s o t o p e p r o d u c t i o n s i t e s a t TRIUMF .. 72 4. BL4A Multisample i r r a d i a t i o n s t a t i o n .. 73 5. BL4A 5 2 F e p r o d u c t i o n f a c i l i t y .. .. 75 6. BLlA 500 MeV i r r a d i a t i o n f a c i l i t y .. .. 76 7. 5 2 F e p r o d u c t i o n hot c e l l breadboard .. 82 8. Dose r a t e vs c o o l i n g p e r i o d .. 105 9. Computer c a l c u l a t e d gamma spectrum I .. 10 6 10. Computer c a l c u l a t e d gamma spectrum I I .. 107 11. Computer c a l c u l a t e d gamma spectrum I I I .. 108 12. D i s t r i b u t i o n c o e f f i c i e n t s vs HC cone. f o r v a r i o u s Fe s e p a r a t i o n methods .. 115 13. Ge(Li) spectrum o f N i s o l u t i o n and product .. 119 14. Tumor t i s s u e c u l t u r e uptake of p r e v i o u s l y l a b e l e d n a t u r a l p o r p h y r i n s .. .. 126 15. Tumor t i s s u e c u l t u r e uptake of n a t u r a l hemins.. 127 16. Tumor t i s s u e c u l t u r e uptake o f a r t i f i c i a l hemins 129 17. Tumor t i s s u e c u l t u r e uptake o f TMPI l a b e l e d w i t h d i f f e r e n t metals .. .. . . 130 18. Tumor t i s s u e c u l t u r e uptake of v a r i o u s metals.. 132 19. Animal d i s t r i b u t i o n of 5 9Fe-TMPI .. .. 133 20. Animal d i s t r i b u t i o n o f 5 9 F e C l .. .. 135 21. Tomographic scan of tumor b e a r i n g r a t s u s i n g 5 2 F e hemins .. .. .. 139 22. E x c r e t i o n curve of 5 9Fe-TMPI .. .. 140 X LIST OF TABLES I. P o r p h y r i n S t r u c t u r e .. 27 IT. R a d i o n u c l i d e s of Iron .. .. 45 I I I . Dose Program C a l c u l a t e d Cross S e c t i o n s .. 100 IV. Dose Program P r o d u c t i o n Rates f o r D i f f e r e n t I r r a d i a t i o n Times .. .. 101 V. Dose Program A c t i v i t i e s f o r V a r i o u s C o o l i n g Times .. .. 102 VI. Gamma Dose Rates .. .. 103 VII . N i S p a l l a t i o n Products .. .. 109 V I I I . M o d i f i e d Gamma Ray Constants .. .. I l l IX. Expected R a d i a t i o n L e v e l from P r o d u c t i o n of 1 mCi of 5 2 F e .. .. 112 X. Comparison of Dose Program to I n i t i a l Experiments 113 XI. 5 2 F e Contamination T e s t s .. .. 117 XII. Animal D i s t r i b u t i o n of 5 9Fe-TMPI .. .. 137 XII I . Animal D i s t r i b u t i o n o f 5 9 F e - c h l o r i d e .. 137 XIV. S Values f o r 5 2 F e .. .. 146 XV. Dose C a l c u l a t i o n s f o r 5 2Fe-TMPI .. .. 146 x i . ACKNOWLEDGEMENT I would l i k e to thank the U n i v e r s i t y o f B r i t i s h Columbia, F a c u l t y o f Graduate S t u d i e s , F a c u l t y of Pharmaceutical S c i e n c e s , TRIUMF, Department of M i c r o b i o l o g y , B r i t i s h Columbia Cancer Re-search Center and Atomic Energy o f Canada L i m i t e d f o r making t h i s t h e s i s p o s s i b l e . I would a l s o l i k e to thank a l l the members of my committee. i I INTRODUCTION In the p a s t , p o r p h y r i n s have been used to d e t e c t e x t e r n a l and g a s t r o i n t e s t i n a l t r a c t tumors i n humans. Exposure of the tumor to u l t r a v i o l e t l i g h t causes the p o r p h y r i n and the tumor to f l u o r e s c e a red l i g h t . Because the tumor has to be exposed to u l t r a v i o l e t l i g h t and observed v i s u a l l y t h i s l i m i t s t h i s type of tumor d e t e c t i n g agent to c e r t a i n a c c e s s i b l e p a r t s of the body. The purpose of t h i s r e s e a r c h was to determine i f r a d i o -a c t i v e l y l a b e l e d p o r p h y r i n s were s t i l l taken up by tumors and i f t h i s r a d i o p h a r m a c e u t i c a l c o u l d be used as a tumor scanning agent i n n u c l e a r medicine. A number of porphyrins were s y n t h e s i z e d , l a b e l e d w i t h r a d i o n u c l i d e s and t e s t e d f o r tumor uptake. The r a d i o n u c l i d e of major i n t e r e s t was r a d i o -a c t i v e i r o n because the i r o n - p o r p h y r i n complex was very s t a b l e both i n v i v o and i n v i t r o . 2 I I LITERATURE REVIEW A Tumor S t r u c t u r e and P h y s i o l o g y A tumor o r neoplasm i s d e f i n e d as an abnormal mass o f t i s s u e , t h e growth o f w h i c h exceeds and i s u n c o o r d i n a t e d w i t h t h a t o f normal t i s s u e . (1) I t can be c l a s s i f i e d as a b e n i g n o r m a l i g -nant tumor a c c o r d i n g t o i t s c l i n i c a l and m o r p h o l o g i c a l f e a t u r e s . A b e n i g n tumor i s a tumor which grows s l o w l y and s t o p s growing when i t reac h e s a c e r t a i n s i z e . I t i s composed o f w e l l -d i f f e r e n t i a t e d mature t i s s u e i m i t a t i n g normal t i s s u e . Death o n l y r e s u l t s i f t h e tumor i n t e r f e r s w i t h a v i t a l o r gan. A m a l i g n a n t tumor u s u a l l y grows v e r y f a s t . I t grows by e x p a n s i o n b u t a l s o by i n f i l t r a t i o n and i n v a s i o n o f s u r r o u n d i n g t i s s u e . I t i s u n e n c a p s u l a t e d and p o o r l y demarcated. However, i t i s p o s s i b l e t h a t a m a l i g n a n t tumor may be e n c a p s u l a t e d and l o c a l i z e d . A m a l i g n a n t tumor tends t o m e t a s t a s i z e and s p r e a d t o o t h e r p a r t s o f t h e body. Removal o f t h e p r i m a r y tumor u s u a l l y r e s u l t s i n t h e r e c u r r e n c e o f a tumor a t t h e same s i t e . N e c r o s i s and u l c e r a t i o n i s v e r y common. Death r e s u l t s from t h i s r a p i d c e l l e x p a n s i o n even i f a v i t a l organ i s not i n v o l v e d . C e l l s a r e u n d i f f e r e n t i a t e d and l a c k adequate m a t u r a t i o n . M e t a s t a s e s from t h e p r i m a r y tumor can spre a d t o o t h e r p a r t s o f 3 t h e body by the l y m p h a t i c s , b l o o d stream and by i m p l a n t a t i o n . M e t a s t i c tumors a r e common i n t h e l u n g s , l i v e r , bones, k i d n e y s , lymph nodes, and a d r e n a l g l a n d s but r a r e i n t h e s p l e e n and s k e l e t a l m u s c l e s . No s i n g l e agent i s r e s p o n s i b l e f o r c a u s i n g c a n c e r and one agent may cause c a n c e r o f one organ w h i l e a n o t h e r agent i s r e q u i r e d t o cause c a n c e r o f a n o t h e r organ. V i r u s e s cause sarcomas i n a n i m a l s but t h e r e i s no d i r e c t e v i d e n c e o f t h i s i n man. Over 90% of a l l c a r c i n o g e n s a r e mutagens. I t i s b e l i e v e d t h a t g r e a t e r t h a n 80% o f a l l c a n c e r s a r e caused by e n v i r o n m e n t a l c a r c i n o g e n s . C h e m i c a l c a r c i n o g e n s a r e a b l e t o a l t e r t h e c e l l ' s DNA o r RNA s t r u c t u r e , r e s u l t i n g i n new o r d i s r u p t e d p r o t e i n p r o d u c t i o n . The new p r o t e i n s may t r a n s f o r m normal c e l l s i n t o c a n c e r c e l l s by a c t i v a t i n g l a t e n t v i r u s e s o r c h a n g i n g t h e c e l l ' s environment (hormonal balance) . R a d i a n t energy ' ( u l t r a — v i o l e t ) and i o n i z i n g r a d i a t i o n may produce c a n c e r by a l t e r i n g the DNA s t r u c t u r e o f a normal c e l l . C a r c i n o g e n e s i s and tumor growth can be d i v i d e d i n t o t h r e e phases: m a l i g n a n t t r a n s f o r m a t i o n , tumor development, and tumor e v o l u t i o n . A t r a n s f o r m e d c e l l c o n t i n u e s t o d i v i d e t o form a c l o n e o f t r a n s -formed c e l l s . I f a c l o n e does not a c q u i r e a b l o o d stream, i t w i l l not c o n t i n u e t o d i v i d e and may d i e . Other f a c t o r s w h i c h w i l l d e t e r m i n e t h e f a t e o f a c l o n e i n c l u d e s t h e body's immune 4 system and c h a l o n e s . The e s t a b l i s h e d c l o n e i s then c o n t r o l l e d by hormones, growth f a c t o r s , and immune system b l o c k i n g f a c t o r s produced by t h e body. The tumor may d i f f e r e n t i a t e i n t o a b e n i g n tumor o r d e d i f f e r e n t i a t e i n t o a m a l i g n a n t tumor. D u r i n g t r a n s f o r m a t i o n , t h e plasma membrane o f t h e c e l l changes c o m p l e t e l y . A l l p r o c e s s e s i n v o l v i n g t h e membrane such as growth, r e g u l a t i o n , m e t a b o l i s m , d i f f e r e n t i a t i o n , e t c . a r e a l t e r e d . The l e v e l s o f c a r b o h y d r a t e , g l y c o p r o t e i n , g l y c o l i p i d , and g l y c o s a -m i n o g l y c a n may i n c r e a s e o r d e c r e a s e depending on t h e t y p e o f c a n c e r . The l e v e l o f complex g i y c o l i p i d s may d e c r e a s e b u t more ar e exposed on t h e c e l l s u r f a c e so t h a t they can i n t e r a c t w i t h a n t i b o d i e s , enzymes, and l e c t i n s . I mportant g l y c o p r o t e i n s r e q u i r e d f o r c o n t r o l and r e g u l a t i o n may a l s o d i s a p p e a r w h i l e new g l y c o -p r o t e i n s p r e v e n t i n g immune k i l l i n g may appear on t h e c e l l s u r f a c e . I n c r e a s e d t r a n s p o r t o f s u g a r s , some amino a c i d s , and phosphates o c c u r i n t r a n s f o r m e d c e l l s . I n c r e a s e d t r a n s p o r t o f sugars i s due t o the i n c r e a s e d maximum v e l o c i t y o f t h e t r a n s p o r t enzymes and not due t o f a s t e r i n s i d e t r a p p i n g by p h o s p h o r y l a t i o n . S m a l l changes i n t r a n s p o r t a c t i v i t y c o u l d d r a m a t i c a l l y a f f e c t c e l l growth when m a r g i n a l n u t r i e n t c o n c e n t r a t i o n s a r e a v a i l a b l e i n t h e s u r r o u n d i n g environment. 5 B The R o l e o f Tumor-Imaging R a d i o p h a r m a c e u t i c a l s i n Oncology-Many t e c h n i q u e s a r e a v a i l a b l e t o d i a g n o s e and l o c a l i z e tumors i n t h e body. These i n c l u d e n u c l e a r t e c h n i q u e s , u l t r a s o u n d , thermography, z e r o a d i o g r a p h y , angiography and o t h e r r a d i o l o g i c a l p r o c e d u r e s . T h i s d i s s e r t a t i o n w i l l l i m i t i t s e l f t o t h e use o f N u c l e a r M e d i c i n e . N u c l e a r m e d i c i n e t e c h n i q u e s a r e f a s t , s e n s i t i v e , n o n i n v a s i v e , and p r o v i d e an i n d i c a t i o n o f tumor and body m e t a b o l i s m . N u c l e a r m e d i c i n e s c a n n i n g p r o c e d u r e s s h o u l d n ot r e p l a c e o t h e r d i a g n o s t i c p r o c e d u r e s , such as p h y s i c a l e x a m i n a t i o n , l a b o r a t o r y t e s t s , and X-ray s t u d i e s . However, i n some i n s t a n c e s they s o u l d be used b e f o r e o t h e r i n v a s i v e t e c h n i q u e s . Tumor s c a n n i n g agents can be d i v i d e d i n t o t h r e e groups: non-s p e c i f i c , l i m i t e d use agen t s ; s p e c i f i c l i m i t e d use a g e n t s ; o r s p e c i f i c g e n e r a l use a g e n t s . N o n s p e c i f i c l i m i t e d use agents i d e n t i f y s p a c e - o c c u p y i n g l e s i o n s or d i s p l a c e m e n t o r a l t e r a t i o n o f normal t i s s u e o n l y . There i s no s p e c i f i c uptake i n tumors. They a r e u s u a l l y l i m i t e d t o one organ o r system. M u l t i p l e scans and o t h e r d i a g n o s t i c p r o c e d u r e s a r e r e q u i r e d t o d e t e r m i n e t h e presence o f a tumor. S p e c i f i c l i m i t e d use agents a r e s p e c i f i c f o r one t y p e o f tumor o r tumors l i m i t e d t o one organ. R a d i o i o d i n e f o r t h y r o i d tumors, i o d o c h o l e s t e r o l f o r a d r e n a l tumors, o r r a d i o l a b e l e d tumor a n t i -6 b o d i e s t o one t y p e o f tumor f i t i n t o t h i s group. S p e c i f i c g e n e r a l use agents s h o u l d be t a k e n up by a l l tumors and not l i m i t e d t o one t y p e o f organ. 6 7 G a - c i t r a t e and 1 1 1 I n -l a b e l e d b l e o m y c i n a r e g e n e r a l use agents but not t o o s p e c i f i c and are t a k e n up by nonmalignant p r o c e s s e s . The c h o i c e o f r a d i o p h a r m a c e u t i c a l and/or r a d i o n u c l i d e t o be used i n n u c l e a r m e d i c i n e i s d e t e r m i n e d by:(2) 1) P h y s i c a l p r o p e r t i e s o f t h e r a d i o n u c l i d e a) Energy and i n t e n s i t y o f photons o r p a r t i c l e s e m i t t e d b) H a l f - l i v e c) P u r i t y d) A v a i l a b i l i t y 2) B i o p h y s i c a l and c h e m i c a l p r o p e r t i e s o f t h e agent a f f e c t i n g d i s t r i b u t i o n and t u r n o v e r i n t h e body. 3); T i s s u e t r a n s m i s s i o n and s c a t t e r i n g a) " Depth of t h e s i t e o f i n t e r e s t b). A b s o r p t i o n c o e f f i c i e n t s o f l o c a l t i s s u e 4) C h a r a c t e r i s t i c s o f t h e imaging system: a\ S e n s i t i v i t y t o l o c a l and d i s t r i b u t e d s o u r c e s b) Energy d i s c r i m i n a t i o n cl I n t r i n s i c r e s o l u t i o n f o r g i v e n photon energy d). R e s o l u t i o n time e l F o c a l d i s t a n c e 7 C Tumor Imaging R a d i o p h a r m a c e u t i c a l s Tumor imaging r a d i o p h a r m a c e u t i c a l s f a l l i n t o t h e f o l l o w i n g c a t a g o r i e s : M e t a b o l i t e R e l a t e d A g e n t s , R a d i o n u c l i d e s and o t h e r Agents, R a d i o l a b e l e d A n t i t u m o r Agents and R a d i o i o d i n a t e d Agents. 1 M e t a b o l i t e R e l a t e d Agents a) ^ C - A s p a r t i c a c i d I n c r e a s e d uptake was obser v e d i n an i m p l a n t e d Walker carcinoma i n t h e t h i g h o f a r a t . (3) Tumor uptake may be due t o i n -c r e a s e d r a t e o f p r o t e i n s y n t h e s i s . b) 1 ^ - C a r b o x y l 1 - a m i n o c y c l o p e n t a n e - c a r b o x y l i c a c i d (ACPC) T h i s agent showed v e r y r a p i d and u n i f o r m b l o o d c l e a r a n c e , o n l y 25% remained i n t h e b l o o d a f t e r f i v e m i n u t e s . U r i n a r y e x c r e t i o n was o n l y 1.1% i n d i c a t i n g t h a t i t was not l o s t by d e c a r b o x y l a t i o n . The organ o f g r e a t e s t uptake was t h e l i v e r , f o l l o w e d by t h e s p l e e n . Other organs such as b l o o d v e s s e l s , h e a r t , s a l i v a r y g l a n d s , nasopharynx, k i d n e y s and b r e a s t s were a l s o imaged. In one c l i n i c a l s t u d y u s i n g s i n g l e photon d e t e c t i o n , more l e s i o n s were d e t e c t e d w i t h t h i s agent t h a n w i t h 6 7 G a - c i t r a t e . (3) However, t h e number o f m a l i g n a n t l e s i o n s was not d e t e r m i n e d . I n f e c t o u s l e s i o n s and a r e a s o f p r e v i o u s s u r g e r y were v i s u a l i z e d f a i n t l y . Based on t a r g e t t o n o n t a r g e t r a t i o s , 6 7 G a was b e t t e r , b u t t h e agent may be u s e f u l i n imaging abdominal l e s i o n s . 8 c) 1 1 C - M e t h y l a t e d p olyamine a n a l o g s I n c r e a s e d uptake was obser v e d i n mouse tumors and t h e p r o s t a t e g l a n d o f a dog was imaged. M e t h y l a t e d p u t r e s c i n e was n o n t o x i c and had lower k i d n e y a c t i v i t y t han m e t h y l a t e d spermine o r s p e r -m i d i n e . (4) d) 1 8 F - S - f l u r o u r a c i l A n i m a l s t u d i e s showed some tumor uptake but a l s o h i g h background l e v e l s . (5^-6) Uptake may be due t o i n c r e a s e d RNA s y n t h e s i s by tumors. e) 1 3N-Ammonia T h i s r a d i o p h a r m a c e u t i c a l was t a k e n up by t h e l i v e r , b r a i n , k i d n e y , h e a r t , s a l i v a r y g l a n d s and b l a d d e r . (7) The v e r y r a p i d b l o o d c l e a r a n c e a l l o w e d v e r y f a s t dynamic s t u d i e s t o be done. (8) Tumor uptake may be due t o i o n exchange o r i t may be i n c o r p -o r a t e d i n t o o t h e r m e t a b o l i t e s . (7) I m p l a n t s o f M o r r i s hepatoma i n t h e f l a n k o f r a t s were w e l l v i s u a l i z e d i n 30-40 m i n u t e s . (9) f ) 13N-<3lutamine and 1 3 N - G l u t a m i c a c i d T i s s u e d i s t r i b u t i o n was s i m i l a r t o 1 3N-ammonia ex c e p t t h a t i t was h e a v i l y c o n c e n t r a t e d i n t h e l i v e r w i t h no c a r d i a c u p t a k e . Tumor uptake v a r i e d from 53% o f l i v e r uptake i n mice w i t h f i b r o s a r c o m a t o 123% o f l i v e r uptake i n mice w i t h polyoma. (10) 9 g) 7 5 S e - L - s e l e n o m e t h i o n i n e Due t o t h e p h y s i c a l p r o p e r t i e s o f 7 5 S e , h i g h energy gamma ra y e m i s s i o n and l o n g T%, o n l y s m a l l doses b a r e l y s u f f i c i e n t f o r r e a s o n a b l e i maging can be g i v e n w i t h o u t e x c e s s i v e p a t i e n t i r r a d i a t i o n . M u l t i p l e window cameras must be used due t o t h e low abundance, m u l t i - e n e r g y photon e m i s s i o n c h a r a c t e r i s t i c s o f 7 5 S e . The m e t a b o l i c a c t i v i t i e s o f m e t h i o n i n e and 7 5 S e - L - s e l e n o -m e t h i o n i n e i s s i m i l a r b ut not i d e n t i c a l . (11) Tumor l o c a l i z a t i o n w i t h t h i s agent was due t o an i n c r e a s e d r a t e o f p r o t e i n s y n t h e s i s (12-13) and DNA s y n t h e s i s . (14) Uptake v i a DNA s y n t h e s i s was assumed b u t not proven. There was some r e l a t i o n s h i p between uptake and v a s c u l a r i t y w i t h l i v e r tumors. (14) However, p a n c r e a t i c tumors h a v i n g a g r e a t e r v a s c u l a r s u p p l y and i n c r e a s e d r a t e o f p r o t e i n s y n t h e s i s accumulated l e s s agent than l e s s d e v e l o p e d tumors. (15) 7 5 S e - L - s e l e n o m e t h i o n i n e was o r g i n a l l y d e v e l o p e d as a p a n c r e a t i c and p a r a t h y r o i d s c a n n i n g r a d i o p h a r m a c e u t i c a l . (16-18) I t was f i r s t used as a tumor s c a n n i n g agent when heavy uptake was seen i n a lymphosarcoma d u r i n g a r o u t i n e p a n c r e a t i c scan. I t a l s o has been used t o di a g n o s e and l o c a l i z e t h e f o l l o w i n g c o n d i t i o n s : n e uroblastoma (19), hepatoma (20), s t a g i n g o f Hodgkin's d i s e a s e (24), lymphoma (13, 21-22), thynoma (23), t h y r o i d l e s i o n s (24) and m e t a s t a t i c melanoma (25). S i n c e i t has poor s p e c i f i c i t y and u n s u i t a b l e gamma r a y e m i s s i o n i t i s no l o n g e r used -as. a g e n e r a l 10 tumor scanning agent. However, i t i s s t i l l used r o u t i n e l y to image the pancreas and p a n c r e a t i c neoplasms which u s u a l l y g i v e r i s e to c o l d spots and r a r e l y hot s p o t s . (26) 2 R a d i o n u c l i d e s and Other Agents a) A r s e n i c 7 4 A s was used to d e t e c t b r a i n tumors but the high energy emmission make imaging d i f f i c u l t and expose the p a t i e n t to high r a d i a t i o n doses. (26) b) Bismuth 2 0 6 B i a c e t a t e gave very high tumor to background r a t i o s but the r a d i a t i o n dose to the kidneys was very h i g h . (27-29) Imaging wi t h c o n v e n t i o n a l d e v i c e s was d i f f i c u l t due to high e n e r g i e s . B i may r e a c t w i t h s u l f h y d r y l groups i n tumor t i s s u e s . (.30) c)[ Copper 6 ' ' C u - c i t r a t e was s i m i l a r t o 5 7Co-bleomycin w i t h l e s s accu-m u l a t i o n i n i n f l a m a t o r y t i s s u e by 24 hours. (31-32) d)_ Cesium The exact mechanism of uptake was not known but was b e l i e v e d to be s i m i l a r t o potassium and rubidium. (33-34) E a r l y uptake was due to the i n c r e a s e d v a s c u l a r i t y of the tumor because 11 i n i t i a l d i s t r i b u t i o n was p r o p o r t i o n a l to f r a c t i o n a l organ blood flow. (.35-37). M e t a b o l i c processes of the tumor may account f o r some of the uptake. (34, 3 8-3 9) High uptake was observed i n the l i v e r and to a l e s s e r e x t e n t i n the kidneys, fundus of the stomach and sple e n . 1 3 1 C s was used to image malignant s u p e r f i c i a l and supra-diaphragmatic tumors. (35) When combined wi t h 1 3 1 i i t was used t o d e t e c t t h y r o i d tumors. (.40-41). 1 2 9Cs has been used to d e t e c t t h y r o i d tumors and to image pulmonary tumors. (42) Increased Cs uptake i n normal t i s s u e made i t d i f f i c u l t t o d e t e c t tumors below the diaphram. Cs was not taken up by t u b e r c u l o s i s l e s i o n s as 6 7 C u was. (42). e) C o b a l t 5 8 C o - c i t r a t e and 5 8Co^bleomycin seem t o have the same tumor s p e c i f i c t y and tumor/normal t i s s u e r a t i o s . (.43) f) Gold 1 9 8 A u - c h l o r i d e bound s t r o n g l y t o p r o t e i n s i n the blood and showed v e r y h i g h uptake by tumors but no c l i n i c a l s t u d i e s have been done. (44) g) Indium -i i i i n - c h l o r i d e showed poorer tumor a f f i n i t y than- Ga but had 12 s u p e r i o r p h y s i c a l p r o p e r t i e s f o r i m a g i n g . (45) However, v e r y l i t t l e was e x c r e t e d i n t o t h e G l o r GU system making abdominal s c a n n i n g e a s i e r . 6 7 G a was c l i n i c a l l y s u p e r i o r and g i v e s b e t t e r tumor t o l i v e r - s p l e e n - m u s c l e and b l o o d r a t i o s . (46-47) C l i n i c a l s t u d i e s have shown 100% (48) and 79% (49) s e n s i t i v i t y b u t t h e agent was n o n s p e c i f i c . However, i t was c o n s i d e r e d t o be a v e r y good b r a i n tumor s c a n n i n g agent t h a t was b e t t e r t h a n b l e o m y c i n , 6 7 G a , a l l p e r t e c h n e t a t e compounds. (50) Recent uses i n c l u d e imaging o f head and neck tumors (51) and s o f t t i s s u e component of bone sarcomas. (52) F a c i a l uptake was lower than 6 7 G a . Tumors near h i g h bone marrow uptake cannot be imaged. No d i f f e r e n c e was o b s e r v e d between 1 1 1 I n - c h l o r i d e and 1 1 1 I n - c i t r a t e i n c l i n i c a l s t u d i e s . (53) A l s o no d i f f e r e n c e between 1 1 1 I n - f l u o r i d e , - a c e t a t e , - l a c t a t e , and -HEDTA was o b s e r v e d i n uptake u s i n g s t e r i l e granulomas. (54) h) Mercury 1 9 7 H g - c h l o r i d e has been used t o d e t e c t f a c e and neck (55-56), b r e a s t (57-58), t h y r o i d (59), b r a i n (60), l u n g (55, 57, 61-67) and k i d n e y tumors. (68) A n i m a l s t u d i e s done on mice u s i n g t r a n s p l a n t e d E h r l i c h a s c i t e s c e l l c a rcinomas suggested t h a t t h i s agent was much b e t t e r t h a n 1 1 1 I n - c h l o r i d e , and 6 7 G a -c i t r a t e . (69-70) However, c l i n i c a l s t u d i e s i n d i c a t e d t h a t t h i s agent was no more s p e c i f i c t h a n any o t h e r g e n e r a l tumor 13 scanning agent. (71) Benign tumors d i d not take up t h i s agent but uptake by inflammatory l e s i o n s was as high as the tumor. (71-73) The very h i g h r a d i a t i o n dose to the kidneys l i m i t e d the use of t h i s agent. 1 9 7Hg-l-mecuri-2-hydroxypropane has been used as a spleen tumor scanning agent. Healthy spleen t i s s u e s e q u e s t r a t e red c e l l s damaged by t h i s agent whereas tumors'do" not.'(74) 1 9 7 H g - c h l o r m e r o d r i n was used to image f a c e and neck (75-76), b r e a s t (75), lung (75), eye (.76), b r a i n (78-79), and kidney (68) ( c o l d spot) tumors. Benign and malignant tumors however, cannot be d i f f e r e n t i a t e d . High r a d i a t i o n dose t o the kidneys and Compton s c a t t e r i n g due to low energy emmission l i m i t e d the u s e f u l n e s s of t h i s agent. Selenium A c l i n i c a l study u s i n g 7 5 S e - s e l e n i t e w i t h a number of h e p a t i c l e s i o n s i n d i c a t e d t h a t t h i s agent was more tumor s p e c i f i c than g o l d or s u l f u r c o l l o i d . Negative scans were r e p o r t e d i n 15 p a t i e n t s with benign l e s i o n s , w h i l e 43 p a t i e n t s with malignant tumors a l l had p o s i t i v e scans. (80) Technetium 9 9mTc-pertechnetate hag been used to image and d e t e c t b r a i n 14 (81-85), t h y r o i d (85-87), b r e a s t (88-89), s a l i v a r y g l a n d (90-92), eye o r b i t a l (93), s p i n a l (94) and e x t r a c r a n i a l (.9 5) tumors. B r a i n imaging was about 80-9 0% r e l i a b l e w i t h about 10% f a l s e n e g a t i v e . (96) Most t h y r o i d tumors d i d not l o c a l i z e b o t h i o d i n e and 9 9 m T c as w e l l as normal t i s s u e ( c o l d s p o t ) . However, some tumors t r a p p e d b u t not o r g a n i f i e d showing i n c r e a s e d p e r t e c h n e t a t e uptake bu t n o t i o d i n e . (85) (.97-98) T h y r o i d m e t a s t a s e s may not be imaged e a r l y w i t h t h i s agent, t h e r e f o r e i o d i n e must be used. Imaging of b r e a s t c a n c e r w i t h t h i s agent has low r e l i a b i l i t y and h i g h r a t e o f f a l s e n e g a t i v e s (20%). W a r t h i n ' s tumors d f t h e s a l i v a r y g l a n d produce hot s p o t s w h i l e o t h e r tumor t y p e s produce c o l d s p o t s . Scanning ~ was o f no d i a g n o s t i c v a l u e b u t h e l p e d l o c a l i z e t h e tumor e x a c t l y b e f o r e s u r g e r y . k); Thulium 1 7 0Tm i s v e r y s i m i l a r t o 1 5 9 Y b . I t i s not produced c o m m e r c i a l l y . 9 9mTc compounds have r e p l a c e d t h i s agent f o r bone s c a n n i n g b u t i t may s t i l l be o f v a l u e f o r s o f t - t i s s u e tumor s c a n n i n g . 1) T h a l l i u m 2 0 1 T l ^ c h l o r i d e d i d not show any tumor uptake u s i n g a n i m a l s t u d i e s . Lung c a n c e r was f i r s t d e t e c t e d w i t h t h i s agent d u r i n g r o u t i n e m y o c a r d i a l i m a g i n g . A c l i n i c a l s t u d y i n v o l v i n g 15 p a t i e n t s showed t h a t i t was not s u p e r i o r t o 6 7 G a - c i t r a t e 15 ( 2 0 1 T l 73.3% p o s i t i v e and 6 7 G a 75% p o s i t i v e ) . 2 0 l r r l was used f o r d e t e c t i n g neck and c h e s t tumors (99) and Hodgkin's lymphoma (100). The advantages o f t h i s agent over 6 7 G a were t h a t scans c o u l d be done as e a r l y as 5 - 10 minutes a f t e r i n j e c t i o n and i t d i d not c o n c e n t r a t e i n bone o r bone marrow, making tumor imaging of t h e mediastinum e a s i e r . The d i s a d v a n t a g e s were t h a t i t c o n c e n t r a t e d i n abdominal organs l i k e 6 7Ga and t h e low energy o f t h e mercury X-rays made deep s e a t e d tumor imaging d i f f i c u l t . m) Xenon F a t t y neoplasms and l i p o s a r c o m a s were d e t e c t e d u s i n g t h i s l i p i d s o l u b l e i n e r t gas. One c l i n i c a l s t u d y i n v o l v i n g 3 p a t i e n t s w i t h r e c u r r e n t l i p o s a r c o m a s showed t h a t t h e tumor c o u l d be imaged a f t e r r e b r e a t h i n g 1 3 3 X e f o r 5 m i n u t e s . (101) n). Y t t e r b i u m Of a l l t h e r a d i o l a n t h a n i d e s o n l y 1 7 0Tm, 1 6 9 Y b and 1 7 7 L u b i n d s t r o n g l y t o plasma p r o t e i n s and have tumor a f f i n i t y . H i g h e r l a n t h a n i d e s were t a k e n up by t h e bone, w h i l e l ower l a n t h a n i d e s were t a k e n up by t h e RE system. (102-104) Uptake o f 1 6 9 Y b -c i t r a t e by normal t i s s u e was much lower than t h a t o f 6 7 G a -c i t r a t e . Bone uptake was about two t i m e s h i g h e r than 6 7 G a . 16 C a r r i e r i n the p r e p a r a t i o n reduced tumor a f f i n i t y and i n c r e a s e d the background. In a c l i n i c a l study of over 400 p a t i e n t s i n 10 h o s p i t a l s the o v e r a l l p o s i t i v e r a t e was 65.3% (33.3-89.5%) and the f a l s e p o s i t i v e r a t e was 29.2% (0-62.5%). The p o s i t i v e r a t e a l s o v a r i e d by anotamical r e g i o n - e x t r e m i t e s and p e l v i c area 100%, head and neck 78.5%, lung 77.8% and abdomen 48.3%. (105-106) o) G a l l i u m 6 7 G a - c i t r a t e i s the most common ge n e r a l tumor scanning agent used i n n u c l e a r medicine today. I n j e c t e d 6 7 G a - c i t r a t e binds to t r a n s f e r r i n and l e s s t i g h t l y to h a p t o g l o b i n , albumin, and l e u k o c y t e s . The tumor takes up the t r a n s f e r r i n - 6 7 G a complex. Uptake due to the accumulation of l a b e l e d lymphocytes, plasma c e l l s , g r a n u l o c y t e s , and macrophages around or i n the tumor does not account f o r the h i g h uptake. (107) Once the 6 7 G a i s i n s i d e the c e l l i t binds to g a l l i u m b i n d i n g granules (GBC), which are lysomal i n nature. (108) T h i s was confirmed by other people (109-110) u s i n g s u b c e l l u l a r f r a c t i o n a t i o n techniques. A microsomal f r a c t i o n which binds Ga has a l s o been d i s c o v e r e d . ( I l l ) Normal l i v e r takes up Ga. S t u d i e s done have i n d i c a t e d t h a t i t binds to l i v e r lysosmes. However, when the p r o t e i n t h a t bound the Ga was i s o l a t e d from the lysosomes i t turned out to 17 be t r a n s f e r r i n . T h e r e f o r e , l i v e r uptake was due t o e n d o c y t o s i s o f t h e G a - t r a n s f e r r i n complex. (112) I t i s n o t known i f tumors c o n t a i n more lysosomes o r have h i g h e r e n d o c y t o s i s a c t i v i t y t h a n normal c e l l s . There i s some form o f r e l a t i o n s h i p between 6 7Ga uptake and the r a t e o f DNA s y n t h e s e s . (113) I t i s not known i f 6 7 G a b i n d s o r i n t e r a c t s w i t h DNA. Uptake by e x p e r i m e n t a l i n f l a m m a t o r y l e s i o n s was due t o d i r e c t uptake o f t h e 6 7 G a by t h e b a c t e r i a i n t h e l e s i o n s (114) o r by 6 7 G a l a b e l e d p o l y m o r p h o n u c l e a r l e u k o c y t e s (PMN). (115) PMN's had a h i g h e r a f f i n i t y f o r 6 7 G a than lymphocytes, w h i l e r e d b l o o d c e l l s had no a f f i n i t y . However, o n l y 20% o f t h e t o t a l uptake was a c c o u n t e d f o r by d i r e c t uptake by b a c t e r i a and PMN's; the r e m a i n i n g 80% was i n a s o l u b l e f r a c t i o n . The b i o l o g i c a l d i s t r i b u t i o n depends on t h e time t h e scan i s t a k e n a f t e r i n j e c t i o n and t h e age o f the p a t i e n t . The r e n a l c o r t e x t a k e s up t h e h i g h e s t amount a f t e r i n j e c t i o n . A f t e r t h e f i r s t 24 hours t h e Ga s h i f t s from t h e r e n a l c o r t e x t o t h e bone and lymph nodes. A f t e r t h e f i r s t week i t s h i f t s from the bone and lymph nodes t o t h e l i v e r and s p l e e n . C h i l d r e n have i n c r e a s e d b l o o d f l o w t o t h e e p i p h y s e a l p l a t e a r e a s 18 o f growing bone w i t h a s y m m e t r i c a l r e l a t i v e i n c r e a s e d uptake i n t h e s e r e g i o n s ; and i n c r e a s e d thymic and s p l e n i c u p t a k e . (116) About one t h i r d o f t h e dose i s e x c r e t e d d u r i n g t h e f i r s t week a f t e r i n j e c t i o n . About 25% o f the dose i s e x c r e t e d by t h e k i d n e y s d u r i n g t h e f i r s t 24 h o u r s . About 10% i s e x c r e t e d v i a t h e GI t r a c t d u r i n g t h e f i r s t week. The r e m a i n i n g 6 5% a f t e r t h e f i r s t week i s d i s t r i b u t e d t h r o u g h out t h e body. W i t h i n 4 8 t o 72 hours a f t e r i n j e c t i o n about 5% o f the dose c o n c e n t r a t e s i n the l i v e r , 1% i n t h e s p l e e n , 2% i n t h e k i d n e y s , and 24% i n t h e s k e l e t o n i n c l u d i n g bone marrow. The a d r e n a l g l a n d , bowel, and l u n g c o n c e n t r a t e a f a i r amount. Uptake by m uscle, b r a i n , f a t , b l o o d and s k i n i s low. (117). The g e n e r a l i n d i c a t i o n ^ f o r a 6 7 G a scan i s ( i ) A d j u n c t t o t h e d i a g n o s i s o f s u s p e c t e d p r i m a r y o r m e t a s t a t i c m a l i g n a n c y , p a r t i c u -l a r l y b r o n c h o g e n i c c a r c i n o m a , H o d g k i n 1 s d i s e a s e , and c e r t a i n lymphomas. ( i i ) S t a g i n g o f a p p r o p r i a t e d m a l i g n a n c e s . Scans may be o f a d j u n c t i v e v a l u e , o r may a i d i n the p l a n n i n g o f laparotomy, lymphangiography, o r o t h e r s t a g i n g p r o c e d u r e s . I t i s p a r t i c u l a r l y v a l u a b l e f o r s t a g i n g d i s e a s e i n p a t i e n t s f o r whom i n v a s i v e p r o c e d u r e s a r e c o n t r a d i c t e d . I t a l s o may be u s e f u l i n l o c a t i n g m e t a s t a s e s i n s i t e s n ot e a s i l y examined by i n v a s i v e methods ( s k u l l e t c . ) . ( i i i ) F o l l o w up o f p a t i e n t s who have r e c e i v e d s u r g e r y , r a d i a t i o n t h e r a p y o r chemotherapy f o r m a l i g n a n t 19 d i s e a s e s t h a t can be imaged b e f o r e therapy, and long term n o n i n v a s i v e f o l l o w up to ensure a g a i n s t asymptomatic r e c u r -rence of tumors. (iv) Search f o r o c c u l t malignancy when the p a t i e n t presents symptoms su g g e s t i v e of n e o p l a s t i c d i s e a s e but without demonstrable or c o n f i r m a b l e d i s e a s e by ot h e r methods, (v) H e l p i n g i n d i a g n o s t i c d i f f e r e n t a t i o n between c e r e b r a l v a s c u l a r l e s i o n s and b r a i n tumors. (118) 6 7 G a i s known to accumulate i n the f o l l o w i n g benign or non-malignant p r o c e s s e s : angioimmunoblastic lymphadenopathy (AILD) (119-121), my e l o f i b r o u s (120) , secondary s y p h i l i s of the myo-cardium (122), p a r a t h y r o i d adenoma (123), p a n c r e a t i t i s (124), pseudocysts of the pancreas (125), l i v e r benign h e p a t i c adenoma ( f o c a l nodular h y p e r p l a s i a ) (126-127), l i v e r a c tinomycosis (128), p o s t o p e r a t i v e i n t f a a b d o m i n M abscesses (129-131), Crohn's d i s e a s e (132), a c t i v e u l c e r a t i v e c o l i t i s (133), p e r i t o n i t i s (134), other Gl inflammatory f o c i (135), o s t e o m y l i t i s (136), c e l l u l i t i s (136), c e r e b r a l i n f a r c t i o n s , m y o c a r d i a l i n f r a c t i o n s , f r a c t u r e s , Paget's d i s e a s e , p y e l o n e p h r i t i s , pneumonia, a c t i v e t u b e r c u l o s i s , a c t i v e s a r c o i d o s i s , s i a l o a d e n i t i s , g a s t r i t i s , s u r g i c a l wounds, rheuma-t o i d a r t h r i t i s , l a c t a t i n g b r e a s t , gynecomastia, and other s i t e s of b a c t e r i a l or mycotic i n f e c t i o n or inflammation. (138) " G a -ses : c i t r a t e does not benign neoplasms, accumulate i n the f o l l o w i n g benign proces-c y s t i c d i s e a s e of b r e a s t , l i v e r , or 20 t h y r o i d , c i r r h o s i s , hemangioma, i n a c t i v e t u b e r c u l o s i s r e a c t i v e lymphadenopathy, c e r e b r o v a s c u l a r a c c i d e n t and e n c e p h a l i t i s . (13 8) 6 7 G a - c i t r a t e i s g r e a t e s t value i n d e t e c t i n g bronchogenic carcinomas i r r e s p e c t i v e of c e l l type. The s e n s i t i v i t y f o r d e t e c t i n g lung cancer i n one study i n v o l v i n g 489 s t u d i e s was 93%. (137) 6 7 G a scanning i s u s e f u l i n p r e v e n t i n g unnecessary thoractomies and d e t e c t i n g disseminated forms which cannot be cured by surgery. A ne g a t i v e scan w i t h n e g a t i v e r a d i o l o g i c a l , c l i n i c a l , and c y t o l o g i c a l examination r u l e out the p o s s i b i l i t y of a tumor. 6 7 G a i s u s e f u l i n s t a g i n g of Hodgkin's d i s e a s e , e s p e c i a l l y i n p a t i e n t s f o r whom lymphangiography i s c o n t r a d i c t e d . The u s e f u l -ness f o r m o n i t o r i n g therapy i s not known. The scan i s of g r e a t value i n f o l l o w i n g p a t i e n t s who are asymptomatic. E a r l y s t u d i e s i n d i c a t e d t h a t the s e n s i t i v i t y f o r d e t e c t i n g Hodgkin's d i s e a s e was 76%. (139) T h i s was l a t e r confirmed by other s t u d i e s , 90% (140), 88% (141), and 87% (142). 6 7 G a i s l e s s s e n s i t i v e i n d e t e c t i n g non-Hodgkin's lymphomas than Hodgkin's d i s e a s e . S e n s i t i v i t y v a r i e s on the type of lymphoma and anatomical r e g i o n s . In one study (142.) w i t h 167 cases of untreated lyphomas, 78% had one or more p o s i t i v e s i t e s on the scan. Only 51% of the h i s t o l o g i c a l l y proven s i t e s were p o s i t i v e . 21 A negative scan does not r u l e out the d i s e a s e . S i t e s were dete c t e d on the scan which were not d e t e c t e d by other methods. A p o s i t i v e scan i n d i c a t e s d i s e a s e but a n e g a t i v e scan does not r u l e out the d i s e a s e . 6 7Ga i s u s e f u l i n d e t e c t i n g tumors more s p e c i f i c a l l y than a r o u t i n e b r a i n scan. A 6 7 G a b r a i n scan should be done i f : (.143-147) Uptake on c o n v e n t i o n a l scan i s e q u i v o c a l . F o l l o w up scanning a p a t i e n t r e c o v e r i n g from craniotomy. Scanning p a t i e n t s with a primary neoplasm t h a t m e t a s t a s i z e s f r e q u e n t l y t o be b r a i n . H e l p i n g i n d i a g n o s i t i c d e f f e r e n t i a t i o n between c e r e b r a l v a s c u l a r l e s i o n s and b r a i n tumors. Uptake by i n f a r c t s lower than tumor uptake or not at a l l (.143), but one study d i d get i n c r e a s e d up-take. (14 4)1 T h i s may have been due to c a r r i e r Ga i n the prepar-a t i o n . Scans are of no v a l u e i n the i n i t i a l d i a g n o s i s of acute leukemia but are h e l p f u l i n f o l l o w i n g therapy.and r e c u r r e n c e s of f o c a l involvement. (14 8) In p a t i e n t s w i t h b r e a s t carcinoma undergoing therapy the p o s i t i v e r a t e was 54% and 14 out of 21 (.67%) i n p a t i e n t s not undergoing therapy. C14 9) Routine l i v e r and bone scans are more s e n s i t i v e than 6 7 G a scans i n d e t e c t i n g secondary tumors. The scan may be of some v a l u e i n d e t e c t i n g m e d i a s t i n a l involvement but the scan i s not used r o u t i n e l y . (150) 22 The 6 7 G a scan i s very important i n the s t a g i n g of t e s t i c u l a r tumors. T e s t i c u l a r tumors are s i m i l a r to lymphomas and u s u a l l y asymptomatic. T o t a l bowel c l e a n s i n g i s important to o b t a i n good images. One study showed a 4 3/4 6 (94%) d e t e c t i o n r a t e w i t h no f a l s e p o s i t i v e s and 3/21 (14%) f a l s e n e g a t i v e s . (151) 6 7 G a scans are of no v a l u e i n d e t e c t i n g p r o s t a t i c carcinomas, p r o s t a t i c bone metastases, bladder tumors or u r e t h r a tumors. Kidney tumor d e t e c t i o n r a t e was 32% w i t h a very h i g h i n c i d e n c e f a l s e p o s i t i v e s (77%). (152) 6 7 G a scanning i s u s e f u l f o r head and neck tumors with a d e t e c t -a b i l i t y r a t e of 87% and 7% f a l s e p o s i t i v e r a t e (squamous c e l l carcinoma), however on l y 77% of secondary s i t e s were d e t e c t e d (.80). P a t i e n t s r e c e i v i n g r a d i a t i o n therapy produced a high l e v e l of f a l s e n e g a t i v e scans. (153-154) 6 7 G a u s u a l l y d e t e c t s primary and to a l e s s e r extend, secondary bone tumors, but the bone scan i s much more s e n s i t i v e . (155) Carcinomas of the t h y r o i d and subacute t h y r o i d i t i s accumulate 6 7 G a w h i l e benign tumors do not. Scanning i s of l i t t l e d i a g n o s t i c v a l u e . (156-157) 23 3 R a d i o l a b e l e d Antitumor Agents a) Bleomycin Bleomycin i s an o n c o s t a t i c p o l y p e p t i d e produced by the organism Streptomyces v e r i t i c i l l u s . Normally copper i s bound to the alpha-amino group and the carbamoyl group of the beta-amino-abanylamide r e s i d u e . The copper i o n can be d i s p l a c e d by other metal i o n s . I t had been l a b e l e d with 5 7 C o (158-159) (162-164), 6 7Cu (158 , 165-167)., 6 2 Zn (184), l l x I n (.158-159, 154, 168-169), 6 7Ga (163), 9 9 m T c 0-58-159, 162 168, 170), 5 1 C r (159 , 171), 2 3 9Np (172), 2 3 7U (1721, 1 1 1 0 La (172), 1 5 3Sm (172), 1 9 5 m P t (1731 and 2 0 3 P b . D i v a l e n t c a t i o n s of Cu, Ni , Co and Zn are most s t a b l e i n v i t r o . However onl y Co i s s t a b l e i n v i v o . (.158-161,1741 5 7Co-bleomycin was used to image tumors of the lung, b r a i n , stomach, oesophagus, and pancreas. (.175-179) Gliomas were a l s o d i f f e r e n t i a t e d from other b r a i n tumors u s i n g t h i s agent. T h i s agent a l s o l o c a l i z e s i n a b s c e s s e s a n d inflammatory l e s i o n s l i k e 6 7 G a - c i t r a t e . S t u d i e s done u s i n g t h i s agent and 5 7 C o i n experimental tumor models have showed no d i f f e r e n c e between the two i n tumor uptake. (.43) However, s u b c e l l u l a r d i s t r i b u t i o n studies- i n d i c a t e d t h a t 5 7 C o - c h l o r i d e and 5 7 C o T - b l e o m y c i n bound to d i f f e r e n t p a r t i c l e s w h i l e 1 1 1 I n - c h l o r i d e and 1 1 1 I n - b l e o m y c i n bound to the same p a r t i c l e . Bleomycin a l s o bound to DNA (180) causing s t r a n d s c i s s i o n u n c o i l i n g of the h e l i x and d e c r e a s i n g 24 i t s m e l t i n g p o i n t . (181-182) 9 9 m T c - b l e o m y c i n was s i m i l a r t o 1 1 1 In and 5 7 C o - l a b e l e d bleomycin complexes. Adenocarcinomas were w e l l imaged but malignant lymphomas were not, due t o the high b l o o d and abdominal a c t i v i t y . (183) T h i s i n d i c a t e d t h a t the 9 9 m T c d i s s o c i a t e d o f f the bleomycin. 1 1 1 I n - b l e o m y c i n i s the o n l y commercially a v a i l a b l e l a b e l e d bleomycin. I t i s u n s t a b l e i n the body, Cu ions displace*' • the 1 1 1 In ions from the molecule. (184) Free 1 1 1 In binds to plasma p r o t e i n s . There was no advantage i n u s i n g t h i s agent'or 6 7 G a . Probably s i m i l a r r e s u l t s w i l l be obtained i f 1 1 1 In was i n j e c t e d }• by i t s e l f . There was lower abdominal uptake with t h i s agent t h a t 6 7 G a a l l o w i n g e a s i e r d e t e c t i o n of p e l v i c tumors. Uptake s t u d i e s u s i n g 1 2 3 I - b l e o m y c i n have shown t h a t i t has higher tumor/blood and t u m o r / l i v e r r a t i o s than 6 7 G a or 5 7 C o -bleomycin i n mice. (162) 4 R a d i o i o d i n a t e d Agents a) C h l o r o q u i n e analogs Chloroquine i s a drug used to t r e a t m a l a r i a . The r a d i o i o d i n a t e d analog of c h l o r o q u i n e (NM115) was found to c o n c e n t r a t e i n dermal (185-187) and o c u l a r (188-189) melanotic melanoma. C l i n i c a l 25 use was l i m i t e d by the high uptake i n the lungs and e x c r e t i o n from l i v e r v i a b i l e i n t o the G l t r a c t . One i n t e r e s t i n g use of t h i s agent was i n u s i n g u l t r a s o u n d f i r s t t o l o c a l i z e the tumor and measuring uptake w i t h a d e t e c t o r to determine i f the tumor was a melanoma. (190) A new analog may be used i n the f u t u r e because i t i s more s p e c i f i c . (191) b) I o d o c h o l e s t e r o l R a d i o i o d i n a t e c h o l e s t e r o l , an organ s p e c i f i c tumor scanning agent, concentrates,, i n the a d r e n a l gland. The agent was shown to be most h e l p f u l in:(192) D i f f e r e n t i a t i n g Cushing's syndrome from h y p e r f u n c t i o n i n g a d r e n a l c o r t i c a l adenoma, l a t e r a l i z i n g aldosteronomas. D e t e c t i n g post-adrenalectomy remnants i n p a t i e n t s w i t h p e r s i s t e n t c o r i s o l excess, d i a g n o s i n g androqen-s e c r e t i n g and C o r t i s o l excess syndromes b e f o r e c o n v e n t i o n a l methods and d i a g n o s i n g c o r t i c a l carcinomas i n p a t i e n t s whose adr e n a l v e i n cannot be c a t h e t e r i z e d or are h y p e r s e n s i t i v e to c o n t r a s t medium. D P o r p h y r i n Nomenclature Porphyrins are r e l a t e d t o the fundamental s t r u c t u r e of porphin, which c o n s i s t s of f o u r p y r r o l e - l i k e r i n g s l i n k e d by f o u r CH groups or methane; b r i d g e s i n a r i n g system, C 2 0 H 1 4 N 4 ( F i g . 1). The p o r p h y r i n s t r u c t u r e c o n t a i n s a c e n t r a l 16-membered r i n g formed from 12 carbon and 4 n i t r o g e n atoms c o n t r i b u t e d by 4 p y r r o l r i n g s . The p o s i t i o n s a s s i g n e d t o t h e a l t e r n a t i n g d ouble and s i n g l e bonds of p o r p h y r i n a r e a r b i t a r y due t o resonance s t r u c t u r e s . By t h e a d d i t i o n o f v a r i o u s s i d e groups d i f f e r e n t t y p e s o f p o r p h y r i n s can be o b t a i n e d (Table I ) . There a r e two systems of n o m e n c l a t u r e , one t h e F i s c h e r approach and a more updated approach from th e Commission on Nomenclature o f B i o l o g i c a l C h e m i s t r y . (193) Only th e l a t t e r approach w i l l be d i s c u s s e d . The updated approach uses a new r i n g numbering scheme but r e t a i n s t h e t r i v a l names of most p o r p h y r i n s r a t h e r than t h e new c h e m i c a l name. T r i v a l names ar e s t i l l used f o r i r o n complexes o r p o r p h y r i n s . These a r e shown below: Heme--An i r o n p o r p h y r i n complex Ferroheme-An i r o n ( I I ) p o r p h y r i n complex Ferriheme--An i r o n ( I I I ) p o r p h y r i n complex Hemochrome-^A l o w - s p i n i r o n p o r p h y r i n complex w i t h one o r more s t r o n g f i e l d a x i a l l i g a n d s (eg p y r i d i n e ) Hemin-—A c h l o r o ( p o r p h y r i n a t o ) i r o n ( I I I ) complex Hematin-An h y d r o x o ( p o r p h y r i n a t o ) i r o n ( I I I ) complex Canhydro o r u-oxo dimers)" E P o r p h y r i n Uptake by Tumors The f i r s t r e p o r t e d uptake o f p o r p h y r i n s by tumors was i n 1942. (194) T h i s was l a t e r c o n f i r m e d by o t h e r p e o p l e s t u d y i n g t h e c a r c i n o g e n i c a c t i o n o f b o t h p o r p h y r i n s and m e t h y l c h o l a n t h r e n e i n mice. (195) 27 TABLE 1 PORPHYRIN STRUCTURES Positions 2 3 7 8 12 13 17 18 Hematoporphyrin M HE M HE M P P M Protoporphyrin M V M V. M P P M Photoprotoporphyrin M. V M OH FE M. P P M M OH FE M Y M P P M 2- Formyi- k- viny 1 Deuteroporphyrin M F M V M P P M 2-Vinyl-if- formyi Deuteroporphyrin M V M F M P P M 2,4-Diformyl Deuteroporphyrin M F M F M P P M 5 10 15 20 me so-1 e t ra (if-N-methylpyridyl) porphine t e t r a I MP MP MP Mp meso-tetra(if-car-oxyphenyl) porphine CP CP CP CP t e tra-Na-meso-1e t r a (4-sulfantophenyl) porphine SP SP SP SP cont. 28 TABLE 1 CONT. Where: M = - C H 3 (methyl) F = -COH (formyl) HE = -CHOHCH3 (hydroxyethyl) P = -CH2CH2C00H V = -CH=CH2 FE = =CHO (formylethylidine) 29 Hematoporphyrin i n j e c t e d IP conc e n t r a t e d i n subcutaneous sarcomas causing the tumor to f l u o r e s c e a red c o l o u r under u l t r a - v i o l e t l i g h t . A f u r t h e r study done by the same group - u s i n g normal and tumor b e a r i n g animals ( i n c l u d i n g methylcholanthrene induced s p i n d l e c e l l fibrosarcomas and rhabdomyosarcomas; t r a n s p l a n t e d mammary adinocarcinomas, fibrosarcomas; and spontaneous mammary carcinomas) has shown t h a t hematoporphyrin, p r o t o p o r p h y r i n , mesoporphyrin, coproporphyrin, and z i n c hematoporphyrin were a l l taken up by the tumors. (196) A l s o other nontumor s i t e s i n c l u d i n g i n j u r y s i t e s , p l a c e n t a , d e v e l o p i n g embryos and lymph nodes (197) accumulated some p o r p h y r i n s . Another more d e t a i l e d study a l s o showed t h a t m e t a l l o -p o r p h y r i n s were taken up by tumors. (198) The f i r s t c l i n i c a l study u s i n g hematoporphyrin i n 1953 showed no tumor uptake. (199) I n j e c t e d doses were kept low ( l e s s than 120mg/ p a t i e n t ) due to the r e p o r t e d t o x i c i t y of hematoporphyrin. (200) The t o x i c i t y was due to the phenol i n the product not the agent i t s e l f . D e f i n i t e tumor uptake was r e p o r t e d u s i n g p h e n o l - f r e e hematoporphyrin a t doses of 500-1000mg/patient w i t h no t o x i c i t y . (.201) The p o r p h y r i n was i n f u s e d over a p e r i o d o f 3-12 hours. Squamous c e l l carcinoma of the tongue and pe n i s ; adenocarcinoma ( s i g n o i d c o l o n , rectum, ascending c o l o n ) ; o l f a c t o r y - g r o v e meningiona; and carcinoma of the b r e a s t a l l showed p o s i t i v e porphy^' r i n uptake. 30 Adenocarcinoma of the p r o s t r a t e , ependynoma of the c e r v i c a l c o r d , and f i b r o t i c abscesses of the b r e a s t d i d not take up the p o r p h y r i n . They r e p o r t e d t h a t t h i s technique was very u s e f u l i n d e t e c t i n g tumors and v i s u a l i z i n g them d u r i n g removal surgery. Q u a n t i t a t i v e s t u d i e s u s i n g animals were done l a t e r u s i n g hemato-p o r p h y r i n . (203) P o r p h y r i n s f o l l o w i n g i n j e c t i o n were c h e m i c a l l y separated from the tumor and muscle t i s s u e and assayed by both f l u o r o m e t r i c and s p e c t r o p h o t o m e t r y procedures. Only the tumor of the r a t (Walker carcinoma-sarcoma) and the Harderian gland of the eye (.203) showed p o s i t i v e uptake by f l u o r e s c e n c e a t autopsy. T o t a l uptake by muscle t i s s u e was zero by f l u o r e s c e n c e w h i l e hematopor--p h y r i n uptake i n c r e a s e d to lO'jjg/g a t 24 hours and decreased again to 2yg/g a t 120 hours a f t e r i n j e c t i o n . I n c r e a s i n g the i n j e c t e d hematoporphyrin dose i n c r e a s e d tumor uptake up to a maximum of 8 0 mg hematoporphyrin per animal. Higher doses d i d not i n c r e a s e tumor uptake. A f t e r hematoporphyrin a d m i n i s t r a t i o n both p r o t o p o r p h y r i n and d e u t e r o p o r p h y r i n were found i n the tumor t i s s u e . I t was assumed t h a t hematoporphyrin was converted to the other p o r p h y r i n s i n the tumor r a t h e r than b e f o r e i t entered the tumor. T h i s was suggested because 1"*C i n c o r p o r a t e d i n t o the carbon of the hydroxymethyl group of hematoporphyrin dimethyl e s t e r incubated w i t h bone marrow c e l l s o nly 5% was found i n p r o t o p o r p h y r i n and 15% i n carbon d i o x i d e . (204) Another i n t e r e s t i n g theory to e x p l a i n tumor uptake- by porphyrins 31 was t h a t o n l y human tumor t i s s u e c o n t a i n a p h o s p h o l i p i d m a t e r i a l c a l l e d " m a l i g n o l i p i n " w h i c h s p e c i f i c a l l y bound t h e p o r p h y r i n . (205) Due t o t h e i n c r e a s i n g use o f hem a t o p o r p h y r i n i t s t o x i c i t y had t o be s t u d i e d . The MLDioo f o r w h i t e mice was 0.3mg/g body w e i g h t f o r crude h e m a t o p o r p h y r i n and 0.15mg/g f o r t h e d e r i v a t i v e (see b e l o w ) . A l s o t h e d e r i v a t i v e was shown t o be a b e t t e r p h o t o s e n s i t i z e r than the c rude h e m a t o p o r p h y r i n . C o n t r a r y t o o t h e r p e o p l e , o t h e r drugs i n c l u d i n g p r o t o p o r p h y r i n d i d not m o d i f y i t s photodynamic a c t i o n . (206) Commercial h e m a t o p o r p h y r i n was u s u a l l y c o n t a m i n a t e d w i t h o t h e r p o r p h y r i n s . (207) A hem a t o p o r p h y r i n d e r i v a t i v e was p r e p a r e d by" d i s s o l v i n g t h e crude m i x t u r e i n g l a c i a l a c e t i c a c i d : c o n c e n t r a t e d s u l f u r i c a c i d ( 1 : 15), f i l t e r i n g , and p r e c i p i t a t i n g t h e d e r i v a t i v e by n e u t r a l i z a t i o n w i t h 15 t o 20 volums o f 3% sodium a c e t a t e . The d e r i v a t i v e was a b e t t e r tumor l o c a l i z i n g agent t h a n t h e crude mix-t u r e w i t h l ower t o x i c i t y l i m i t s and p a t i e n t d o s e s . There was a l s o m i n i m a l a c c u m u l a t i o n i n l y m p h a t i c t i s s u e , b e n i g n fibroadenomas and f r e s h woulds p r o b a b l y due t o serum c o n c e n t r a t i o n i n t h e a r e a . The d e r i v a t i v e was not t a k e n up by granulomatous l e s i o n s nor d i d i t pass t h r o u g h t h e p l a c e n t a l b a r r i e r t o t h e f e t u s . However, t h e u t e r u s and membrane d i d show f l u o r e s c e n c e . (208) The t r u e c h e m i c a l s t r u c t u r e o f t h e d e r i v a t i v e i s s t i l l n o t known tod a y . 32 Lipsori c ontinued to do c l i n i c a l s t u d i e s with the hematoporphyrin d e r i v a t i v e . (209) The dose was 2mg/kg of body weight. The d e r i v a t i v e was i n j e c t e d i n t o the rubber t u b i n g used f o r intravenous i n f u s i o n of 5% glucose because of the burning s e n s a t i o n when i n j e c t e d ' d i r e c t l y i n t o the v e i n . F i f t e e n p a t i e n t s were used i n the c l i n i c a l t r i a l . Tumors were observed by e i t h e r bronchoscopy or esophagoscopy u s i n g u l t r a - v i o l e t l i g h t . I f the l i g h t was of s u f f i c i e n t i n t e n s i t y to reach the tumor there were no f a l s e p o s i t i v e s or f a l s e n e g a t i v e s . In three cases not enough l i g h t reached the tumor. F l u o r e s c e n c e was not i n f l u e n c e d by the c e l l type of the malignant l e s i o n . The o n l y adverse s i d e e f f e c t was a minor degree of p h o t o s e n s i t i v i t y e x h i b i t e d by one p a t i e n t who d i s r e g a r d e d i n s t r u c t i o n s a g a i n s t immediate exposure to d i r e c t s u n l i g h t . The p h o t o r e a c t i o n l a s t e d a few days and caused o n l y s l i g h t d i s c o m f o r t to the p a t i e n t . An u n n a t u r a l p o r p h y r i n , t e t r a p h e n y l p o r p h i n e s u l f o n a t e (TPPS). was s t u d i e d by Winkelman. (210). TPPS was found to be more h i g h l y c o n c e n t r a t e d i n Walker carcinosarcoma than any other t i s s u e of the r a t ; 10X more than hematoporphyrin. At autopsy there was b r i l l i a n t red f l u o r e s c e n c e i n the tumor, b r i g h t r e d f l u o r e s c e n c e d i s t r i b u t e d i r r e g u l a r y i n the lung, and f a i n t r e d f l u o r e s c e n c e i n the lymph nodes and pancreas. No f l u o r e s c e n c e was apparent i n the l i v e r , kidney or s p l e e n . Q u a n t i t a t i v e a n a l y s i s u s i n g f l u o r e s c e n c e and a b s o r p t i o n spectrophotometry showed c o n s i d e r a b l e amounts i n the 33 l i v e r , kidney, and sp l e e n ; f o l l o w e d by lung. However, s m a l l amounts were prese n t i n a l l t i s s u e s assayed ( t o t a l 18) . Skin and muscle always had the l e a s t amount. Maximum uptake o c c u r r e d at 24-48 hours and remained h i g h u n t i l the t h i r d day. Organ uptake at 6-120 hours f o r other t i s s u e s was the same but l i v e r showed e a r l y high accumulation. As the dose i n c r e a s e d from 1 t o 7 5mg/animal so d i d the tumor and t i s s u e uptake. The r a t i o of TPPS content of the l i v e r and kidney t o tumor was h i g h e s t i n animals r e c e i v i n g the l a r g e s t doses and the lowest i n those r e c e i v i n g l e s s than 10mg/ animal. Animals r e c e i v i n g 75mg e i t h e r d i e d d u r i n g i n j e c t i o n or developed profound muscular weakness and tachypaea and d i e d 6-10 hours l a t e r . Serium e l e c t r o p h o r e s i s of animals r e c e i v i n g TPPS showed t h a t TPPS migrated with the albumin f r a c t i o n mainly and some wit h the g l o b u l i n f r a c t i o n . TPPS behaved l i k e b i l i r u b i n but not l i k e other p o r p h y r i n s (uroporphyrin, p r o t o p o r p h y r i n , or hematoporphyrin). (211) I t was a l s o shown t h a t the Walker carcinosarcoma of the r a t c o n t a i n s some endogenous p o r p h y r i n . The h i g h e s t c o n c e n t r a t i o n was i n the n e c r o t i c area of the tumor f o l l o w e d by the sub-capsular area, and then the v i a b l e r e g i o n . TPPS a l s o accumulated h i g h e s t i n the n e c r o t i c t i s s u e f o l l o w e d by the v i a b l e area, and then the sub-c a p s u l a r area. I t was assumed t h a t the endogenous p o r p h y r i n was removed from c i r c u l a t i o n r a t h e r than i n s i t u breakdown of heme. V i s u a l o b s e r v a t i o n of the tumor i n d i c a t e d b r i g h t e s t f l u o r e s c e n c e 34 i n the p e a r l y white n e c r o t i c f o c i and no f l u o r e s c e n c e i n the c e n t r a l hemorrhagic n e c r o t i c area. Although l i v e r , s p l e e n , and kidney e x h i b i t e d no red f l u o r e s c e n c e sometimes they c o n t a i n e d more TPPS than the tumor. (212) The e a r l i e r c l i n i c a l study by L i p s o n was expanded to i n c l u d e 35 more p a t i e n t s . (213) The dose of the hematoporphyrin d e r i v a t i v e was reduced from 2 to 1.5mg/kg. Both bronchoscopy and esophago-scopy were very u s e f u l i n d e t e c t i n g areas which were negative d u r i n g X-ray, v i s u a l , and b i o p s y s t u d i e s which then l a t e r developed i n t o malignant tumors. The o n l y problem was t h a t f l u o r e s c e n c e c o u l d not be observed i f the l i g h t d i d not reach the tumor or i f the tumor was obscured by b l o o d . They recommended the procedure when the c l i n i c a l p i c t u r e suggests a malignant neoplasma but endoscopy f a i l e d t o r e v e a l i t : where a t y p i c a l or cancer c e l l s were found i n the sputum but the tumor c o u l d not be d e t e c t e d by roentgenograms; or i n cases of i n o p e r a b i l i t y because the l e s i o n was near v i t a l s t r u c t u r e s as demonstrated by other techniques. The technique has been extended to other areas of the body i n c l u d i n g the c e r v i x , vagina, t r a c h e o b r o n c i a l t r e e , esophagus, peritoneum, and rectum. (214) T h i r t y - t h r e e of 35 primary carcinomas of the c e r v i x and vagina were d e t e c t e d by f l u o r e s c e n c e w i t h the hemato-p o r p h y r i n d e r i v a t i v e . F l u o r e s c e n t endoscopy d e t e c t e d 32 malicrnant l e s i o n s out of 34 cases. 35 The hematoporphyrin d e r i v a t i v e was used to d e t e c t n e o p l a s t i c t i s s u e i n the mouth, pharynx, and l a r y n x . (215) Twenty-nine of 40 p a t i e n t s had epidermoid carcinoma a l l of which were p o s i t i v e w i t h hemato-po r p h y r i n d e r i v a t i v e . Both benign neoplasms (3 cases) and c h r o n i c inflammation (4 cases) d i d not show any f l u o r e s c e n c e w i t h the agent. The technique was very u s e f u l i n showing areas which were malignant when b i o p s i e d w h i l e previous b i o p s i e s were n e g a t i v e . They recommended t h a t b e f o r e the agent be used r o u t i n e l y t h a t i t s s i d e e f f e c t s must be s t u d i e d c a r e f u l l y . Hematoporphyrin decreases p l a t e l e t adhesiveness and aggregation causing c e l l s to be i n c a p a b l e of s u p p o r t i n g the c l o t r e a c t i o n . (216) P a t i e n t s should a l s o r e c e i v e c a r e f u l fundoscopic examination before and a f t e r the study because of the h i g h uptake of p o r p h y r i n s by the eye and i t s photodynamic behavior. (217) Sanderson reviews most of the development of u s i n g hematoporphyrin as a d i a g n o s t i c t o o l to 1972. (218) They b e l i e v e d t h a t the f l u o -r e s c e n t technique was not the u l t i m a t e method f o r e a r l y l o c a l i z a t i o n of lung cancer but i t had p o t e n t i a l i n l o c a l i z i n g i n s i t u and e a r l y i n v a s i v e bronchogenic carcinoma. The photodynamic a c t i o n of hematoporphyrin has been used to d e s t r o y tumors both i n v i v o or i n v i t r o although no c l i n i c a l s t u d i e s have been done. These p h o t o - o x i d i a t i o n r e a c t i o n s appear to i n v o l v e the 36 the p r o d u c t i o n o f e l e c t r o n i c a l l y e x c i t e d m e t a s t a b l e m o l e c u l a r oxygen ( s i n g l e t oxygen) as a h i g h l y r e a c t i v e and t o x i c s u b s t a n c e . (129) Glioma c e l l s i n c u l t u r e and subcutaneous tumors i n r a t s were k i l l e d by h e m atoporphyrin and l i g h t . However i n v i v o tumor k i l l i n g was n o t complete r e s u l t i n g i n many s m a l l e r tumors due t o the f a c t t h a t not a l l c e l l s were exposed t o the l i g h t o r took up t h e h e m a t o p o r p h y r i n . (220) Over 300 tumors o f v a r i o u s mouse o r r a t t y p e s were s t u d i e d u s i n g p h o t o r a d i a t i o n t h e r a p y and hemato-p o r p h y r i n . (221) A dose o f 2-5mg/kg f o r mice and 10-15mg/kg f o r r a t s was used. The h i g h e s t t u m o r / l i v e r r a t i o o f 4 was o b t a i n e d i n 24 hours w i t h a tumor c o n c e n t r a t i o n o f 40yg/g. F l u o r e s c e n c e m i c r o -scopy i n d i c a t e d d i s c r e t e c y t o p l a s m i c uptake by tumor c e l l s and not by the n u c l e u s . The i n v i t r o h e m a t o p o r p h y r i n - l i g h t s u r v i v a l c u r v e was s i m i l a r t o an X-ray s u r v i v a l c u r v e but X-rays were a t l e a s t a f a c t o r o f 10,000 more e f f e c t i v e . In v i v o a l l tumors r e g r e s s e d t o a n o n p a l p a b l e mass w i t h i n a few days a f t e r t r e a t m e n t b u t o n l y 48% were c u r e d (.6 months w i t h o u t tumor r e c u r r e n c e ) . Lack o f a c u r e was more a d e f i c i e n c y i n t h e mechanism o f t h e t e c h n i q u e r a t h e r than t r e a t m e n t f a i l u r e . T h i s was due t o i n a d e q u a t e l i g h t i n t e n s i t y and/or p o r p h y r i n c o n c e n t r a t i o n i n a l l a r e a s o f t h e tumor. Complete d e s t r u c t i o n o f 2-3 cm t h i c k tumors was p o s s i b l e a f t e r t h r e e or f o u r exposures t o t h e l i g h t . The photodynamic d e s t r u c t i v e n a t u r e o f h e m a t o p o r p h y r i n was e l i m i n a t e d by a s p e c i f i c s i n g l e t oxygen t r a p p i n g agent 1 , 2 - d i p h e n y l i s o b e n z o f u r a n . (222) 37 There has been much i n t e r e s t i n t h e use o f hematoporphyrin i n t h e photochemotherapy o f b r a i n tumors because i t does not pass t h r o u g h the normal b l o o d - b r a i n b a r r i e r (223) and r e d l i g h t i s a b l e t o pass t h r o u g h t h e s k u l l o f mammals. (224) L i g h t and t h i s agent were a b l e t o k i l l g l i o m a c e l l s i n c u l t u r e i n l e s s than 8 minutes and g l i o m a s i n r a t s i n about 40 m i n u t e s . (225) A dose o f 20mg/kg was used. The e x a c t n a t u r e o f t h e photodynamic r e a c t i o n o f hematoporphyrin on the c e l l i s n o t known but i t may mod i f y the DNA s t r u c t u r e . (226) Hematoporphyrin ( g r e a t e r than 5 x 10 - I fM) and l i g h t r e s u l t s i n the s e l e c t i v e d e g r a d a t i o n o f the guanine m o i e t y . DNA t r e a t e d t h i s way e x h i b i t e d l o w e r e d s e d i m e n t a t i o n c o e f f i c i e n t s , lowered t e m p e r a t u r e h e l i x - c o i l t r a n s i t i o n s and i n c r e a s e d buoyant d e n s i t y v a l u e s c o n s i s t e n t w i t h s i n g l e - c h a i n s c i s s i o n s and t h e g e n e r a t i o n o f s i n g l y - s t r a n d e d r e g i o n s . C o n c e n t r a t i o n s o f hematoporphyrin l e s s t h a n 2.5 x 10" h M r e s u l t e d i n a b i p o l y m e r e x h i b i t i n g a l l t h e above p h y s i c a l p r o p e r t i e s e x c e p t a h i g h e r s e d i m e n t a t i o n r a t e and t h e DNA was a g g r e g a t e d . Of the f o u r deoxynucLeosides o n l y deoxyguanosine was d e s t r o y e d . Another i n t e r e s t i n g f e a t u r e o f p o r p h y r i n s t h a t has been shown was t h a t they produced dose dependent m o d i f i c a t i o n s o f r a d i a t i o n e f f e c t s i n mammalian t i s s u e . T h i s s u g g e s t e d t h a t p o r p h y r i n s may be u s e f u l as tumor s e l e c t i v e r a d i a t i o n s e n s i t i z e r s i n r a d i o t h e r a p y . Hemato-p o r p h y r i n was t h e f i r s t one t e s t e d and produced r a d i a t i o n s e n s i t i -z a t i o n . (227-231) Other p e o p l e d e s c r i b e d b o t h a r a d i o p r o t e c t i v e and 38 r a d i o s e n s i t i z a t i o n a c t i o n o f h e m a t o p o r p h y r i n . (232) The use o f b o t h n a t u r a l and s y n t h e t i c p o r p h y r i n s o f t e n gave c o n t r a d i c t o r y r e s u l t s p r o b a b l y due t o t h e use o f impure p r e p a r a t i o n s . The N i and Zn m e t a l c h e l a t e o f m e s o - t e t r a ( p - c a r b o x y p h e n y l ) p o r p h i n e was e f f e c t i v e as a r a d i a t i o n s e n s i t i z e r a t c o n c e n t r a t i o n s as low as 10" 9M. The Zn p o r p h y r i n was e q u a l l y e f f e c t i v e when added i m m e d i a t e l y a f t e r i r r a d i a t i o n o r p a r t i a l l y e f f e c t i v e when added 90 minutes p o s t i r r a d i a t i o n i n d i c a t i n g p o s s i b l e i n t e r f e r e n c e on r e p a i r mechanisms. (233) I t was proposed t h a t the d i f f e r e n c e i n s e n s i t i v i t y o f b i o -l o g i c a l l y a c t i v e NDA t o gamma i r r a d i a t i o n under a e r o b i c oxygen and a n a e r o b i c c o n d i t i o n s was due t o o r g a n o m e t a l l i c complexes. M e t a l l o -p o r p h y r i n mimic t h e s e o r g a n o m e t a l l i c compounds w i t h r e g a r d t o t h e oxygen e f f e c t on DNA i n v i t r o w h i l e normal p o r p h y r i n s ( p r o t o p o r p h y r i n and c o p r o p o r p h y r i a d i d n o t . The oxygen e f f e c t on DNA by hemin c o u l d be e l i m i n a t e d by a d d i n g a h i g h c o n c e n t r a t i o n o f phosphate o r EDTA. (.234) The i n c o r p o r a t i o n o f a m e t a l i n t o t h e p o r p h y r i n d i d not d e s t r o y i t s tumor s e e k i n g p r o p e r t y . (235) T h i s a l l o w e d one t o use r a d i o -a c t i v e m e t a l s and p o r p h y r i n s as tumor imaging agents i n n u c l e a r m e d i c i n e . 6 4 C u - p r o t o p o r p h y r i n was shown t o c o n c e n t r a t e i n mouse tumors d e s p i t e i t s l a c k o f good tumor t o l i v e r , b l o o d , o r muscle r a t i o s . (236) However, human s t u d i e s showed no tumor uptake of t h i s agent. (237) In the use o f 5 7Co-hematoporph-y-ri-n-, tumor b e a r i n g 39 a n i m a l s appeared t o accumulate about h a l f o f t h e amount o f r a d i o -a c t i v i t y • i n t h e l i v e r , k i d n e y , and s p l e e n ( l i v e r / t u m o r r a t i o was 4.7). Twenty f o u r hours a f t e r i n j e c t i o n tumor c o n c e n t r a t i o n was h i g h e r t h a n b l o o d o r muscle but o n l y 1/5 o f l i v e r . High c o n c e n t r a t i o n i n t h e l i v e r and s p l e e n was due t o t h e m e t a b o l i s m of t h e agent. (239) 5 7 C o - h e m a t o p o r p h y r i n was e x c r e t e d m o s t l y t h r o u g h u r i n e w h i l e 6 ^ C u - p r o t o p o r p h y r i n was e l i m i n a t e d m a i n l y t h r o u g h f e c e s . I t was p o s s i b l e t h a t t h e 5 7 C o compound was s t a b l e i n v i v o and t h a t t h e 6 1*Cu compound was not r e s u l t i n g i n c o l l o i d a l p a r t i c l e s . They c o n c l u d e d t h a t 5 7 C o - h e m a t o p o r p h y r i n may have a p o t e n t i a l v a l u e f o r tumor d e t e c t i o n but the tumors must not be l o c a t e d near t h e l i v e r , s p l e e n , and RE system. F P o r p h y r i n S y n t h e s i s In the p a s t p o r p h y r i n s y n t h e s i s and s e p a r a t i o n was v e r y d i f f i c u l t because no s t a n d a r d methods were a v a i l a b l e and workers'were r e l u c t a n t t o p u b l i s h e x a c t d e t a i l s . The work done by D i N e l l o (240) and D i N e l l o and Chang (241) has g r e a t l y e l i m i n a t e d t h e above problems. S e p a r a t i o n o f n a t u r a l p o r p h y r i n s i n t h e d i c a r b o x y l i c a c i d form i s n ot p o s s i b l e because they a r e too p o l a r . They a r e s o l u b l e i n ^ p y r i d i n e , d i m e t h y l s u l f o x i d e , and p o t a s s i u m h y d r o x i d e ( c a r b o x y l i c a c i d groups go t o c a r b o x y l a t e a n i o n s ) . A c i d c o n d i t i o n s make them s o l u b l e i n methanol as d i p r o t o n a t e d p o r p h y r i n d i c a t i o n s . S e p a r a t i o n 40 on s i l i c a g e l (242) or c e l l u l o s e (243) p o s s i b l e . The standard method i s to prepare the p r o p h y r i n e s t e r from the f r e e a c i d f o l l o w e d by chemical m o d i f i c a t i o n s and chromatography ( s i l i c a g e l or alumina). The p u r i f i e d p o r p h y r i n e s t e r can e i t h e r be metalated, p u r i f i e d , and h y d r o l y z e d or h y d r o l y z e d , metalated and p u r i f i e d . The former method i s p r e f e r e d because alumina or s i l i c a g e l chromatography i s e a s i e r than c e l i t e chromatography. The a c i d h y d r o l y s i s i s a simple procedure and minimizes i m p u r i t i e s but p o r p h y r i n s w i t h a c i d and base l a b i l e s i d e chains may become hydrated ( v i n y l groups on protoporphyrin) (244). Conventional dimethyl e s t e r s must be d e e s t e r i f i e d u s i n g a h y d r a t i o n r e a c t i o n which c o u l d modify the o r i g i n a l p o r p h y r i n s t r u c t u r e . A l s o hydro-genation r e a c t i o n s c o u l d modify the p o r p h y r i n s t u r c t u r e . The t e r t i a r y b u t y l e s t e r e l i m i n a t e s the above two problems because i t can be d e e s t e r i f i e d v i a an e l i m i n a t i o n r e a c t i o n w i t h r e s u l t s i n the p r o d u c t i o n of i s o b u t y l e n e and water need not be p r e s e n t d u r i n g the r e a c t i o n . Most n a t u r a l p o r p h y r i n s need not be s y n t h e s i z e d from p y r r o l e s but can be m o d i f i e d from an e x i s t i n g p o r p h y r i n s t r u c t u r e . Hematopor-p h y r i n i s the u s u a l s t a r t i n g m a t e r i a l because i t i s the cheapest source of p o r p h y r i n . I t can a l s o be prepared by t r e a t i n g blood w i t h s u l f u r i c a c i d which removes the i r o n from the hemin and hydrates the v i n y l s i d e chains to h y d r o x y e t h y l groups. (246-247) The common 41 l a b o r a t o r y p r o c e d u r e i s t o t r e a t hemin w i t h HBr i n a c e t i c a c i d and decompose w i t h w a t e r t o g e t hematoporphyrin o r methanol t o get h e m a t o p o r p h y r i n d i m e t h y l e s t e r . Commercial h e m a t o p o r p h y r i n i s r e l a t i v e l y impure and c o n t a i n s s u b s t a n t i a l amounts o f mono-h y d r o x y e t h y l m o n o v i n y l d e u t e r p o r p h y r i n (two isomers) and s m a l l amounts o f p r o t o p o r p h y r i n . (246-247) Diazomethane can be used t o e s t e r i f y h e m a t o p o r p h y r i n b u t n o t hematohemin and p u r i f i e d by the method o f Caughey. (248) I r o n i n s e r t i o n i s b e s t done a t a low tem p e r a t u r e t o p r e v e n t de-h y d r a t i o n o f t h e a - h y d r o x y e t h y l groups u s i n g t h e f e r r o u s s u l f a t e -a c e t i c a c i d method. (249-251) P r o t o p o r p h y r i n i s t o be p r e p a r e d by t h e removal o f i r o n from protohemin. (244) Other methods i n v o l v e t h e p r o d u c t i o n o f t h e d i m e t h y l e s t e r and chromatography. (248, 252) Both methods a r e t e d i o u s and r e s u l t i n low\ y i e l d s . However, p r o t o p o r p h y r i n can be p r e p a r e d from h e m a t o p o r p h y r i n d i h y d r o c h l o r i d e by r e f l u x i n g i n DMF which r e s u l t s i n i n s t a n t a n e o u s and q u a n t i t a t i v e d e h y d r a t i o n . (240, 241, 253) The r e s u l t i n g p r o t o p o r p h y r i n :lias"' a p u r i t y e q u a l t o o r b e t t e r than t h e b e s t c o m m e r c i a l l y a v a i l a b l e compound by t h e TLC method o f E l l f o l k and S i e v e r s . (254) However, impure h e m a t o p o r p h y r i n d i H C l can produce c o n t a m i n a t e d p r o t o p o r p h y r i n . (241) Protohemin cannot be e s t e r i f i e d w i t h diazomethane. Protohemin o r 42 i t s e s t e r s a r e not l i g h t s e n s i t i v e b u t p r o t o p o r p h y r i n and i t s d e r i v a t i v e s a r e l i g h t s e n s i t i v e . P r o t o p o r p h y r i n r e a c t s w i t h l i g h t and oxygen t o g i v e p h o t o p r o t o p o r p h y r i n or 1 ( 3 ) h y d r o x y - 2 ( 4 ) d e v i n y l -2 ( 4 ) f o r m y l e t h y l i d i n e p r o t o p o r p h y r i n . (255-257) The d i t e r t i a r y b u t y l e s t e r (DTBE) o f p r o t o p o r p h y r i n can be syn-t h e s i z e d v i a t h e a c i d c h l o r i d e method (240) m o d i f i e d from th e method o f Schwartz (259) and chromatographed on s i l i c a g e l w i t h c h l o r o f o r m : e t h e r (.100:1). D e e s t e r i f i c a t i o n o f p r o t o p o r p h y r i n DTBE can be done by b u b b l i n g anhydrous HC1 through a methylene c h l o r i d e s o l u t i o n o r by r e f l u x i n g i n the same s o l v e n t c o n t a i n i n g t r i f l u o r o a c e t i c a c i d (240) (TFA). R e f l u x i n g i n TFA r e s u l t s i n t h e g r o s s d e c o m p o s i t i o n o f hemins i f t h e y a r e used i n s t e a d o f p o r p h y r i n s . The s p i r o g r a p h i s p o r p h y r i n , 2 - f o r m y l - 4 - v i n y l d e u t e r o p o r p h y r i n and t h e i s o s p i r o g r a p h i s p o r p h y r i n , 2 - v i n y l - 4 - f o r m y l d e u t e r o p o r p h y r i n can be s y n t h e s i z e d from p r o t o p o r p h y r i n e s t e r s v i a t h e p h o t o p r o t o p o r p h y r i n i somers (258) o r d i r e c t l y from permanganate o x i d a t i o n o f p r o t o p o r -p h y r i n e s t e r s . (.260) The I n h o f f e n (.258) method i n v o l v e d the c o n v e r s i o n o f p r o t o p o r p h y r i n d i m e t h y l e s t e r t o t h e two p h o t o p r o t o -p o r p h y r i n d i m e t h y l e s t e r s . These two e s t e r s were then e a s i l y s e p a r a t e d by column chromatography on s i l i c a g e l . The i s o s p i r o - ^ g r a p h i s p o r p h y r i n r e l a t e d c h l o r i n comes o f f f i r s t , f o l l o w e d by t h e s p i r o g r a p h i s ( n a t u r a l ) p o r p h y r i n r e l a t e d c h l o r i n . I s o m e r i c c h l o r i n s a r e f r e q u e n t l y more e a s i l y s e p a r a t e d than isomer p o r p h y r i n s because 43 the d e v i a t i o n from p l a n a r i t y i n d u c e d by c h l o r i n f o r m a t i o n m a g n i f i e d the d i f f e r e n c e s i n the i n t e r a c t i o n between t h e isomers and chromato-g r a p h i c a d s o r b e n t s . (240) The pure isomers o f p h o t o p r o t o p o r p h y r i n e s t e r s were reduced w i t h b o r o h y d r i d e and t r e a t e d w i t h a c i d f o l l o w e d by h y d r a t i o n t o g i v e c i s - d i o l p o r p h y r i n e s t e r s . The c i s - d i o l p o r -p h y r i n e s t e r s were c l e a v e d when t r e a t e d w i t h p e r i o d a t e t o g i v e the s p i r o g r a p h i s and i s o s p i r o g r a p h i s p o r p h y r i n e s t e r s . Many workers had problems w i t h the p e r i o d a t e c l e a v a g e because o f i n c o r r e c t r e a c t i o n c o n d i t i o n s and benzene as a s o l v e n t , d i o x a n e i s a b e t t e r s o l v e n t . (261-262) The method o f A s a k u r a and Sona (261) u s i n g permanganate o x i d a t i o n o f p r o t o p o r p h y r i n e s t e r r e s u l t e d i n t h e d i r e c t p r o d u c t i o n o f mixed s p i r o g r a p h i s and i s o p i r o g r a p h i s p o r p h r i n e s t e r s and d i f c r m y l d e u t e r o p o r p h y r i n e s t e r . The p o r p h y r i n e s t e r s were s e p a r a t e d from the d e u t e r o p o r p h y r i n e s t e r by column chromatography. The s p i r o -g r a p h i s and i s o s p i r o g r a p h i s p o r p h y r i n e s t e r must be s e p a r a t e d by t h i c k l a y e r chromatography. The m o d i f i c a t i o n o f I n h o f f e n ' s p r o c e d u r e was b o t h more c o n v e n i e n t on a l a r g e s c a l e and gave s u p e r i o r y i e l d s (and can be used w i t h p r o t o p o r p h y r i n DTBE). (239-240) D i f o r m y l d e u t e r o p o r p h y r i n can be p r e p a r e d from t h e p r o t o p o r p h y r i n e s t e r by permanganate o x i d a t i o n and al u m i n a chromatography w i t h 1 , 2 - d i c h l o r o e t h a n e : c h l o r o f orm (.2:1) as an e l u e n t . (248)., The 44 a d d i t i o n o f magnesium s u l f a t e d u r i n g o x i d a t i o n p r e v e n t e d t h e d i f o r m y l compound from b e i n g f u r t h e r o x i d i z e d t o t h e d i c a r b o x y l compound. I n s t e a d o f a l u m i n a , s i l i c a g e l w i t h c h l o r o f o r m : e t h e r (100:1) u n t i l t h e p r o t o p o r p h y r i n e s t e r i s e l u t e d f o l l o w e d by 50:1 of t h e same s o l v e n t system c o u l d be used t o s e p a r a t e t h e DTBE's. (240-241) However, t h e monoformyl m o n o v i n y l and d i f o r m y l compounds w i l l have t o be rechromatographed. The two f o r m y l groups on d i -fo r m y l d e u t e r o h e m i n a r e v e r y s t r o n g e l e c t r o n w i t h d r a w i n g groups. G R a d i o i r o n P r o d u c t i o n Of a l l t h e r a d i s o t o p e s o f i r o n o n l y 5 2 F e •:' i s * s u i t a b l e t o be used i n n u c l e a r m e d i c i n e based on h a l f - l i f e and gamma photon energy e m i s s i o n . (Table I I ) 5 2 F e decays by p o s i t r o n e m i s s i o n y i e l d i n g a n n i h i l a t i o n r a d i a t i o n o f 511 KeV s u i t a b l e f o r imaging w i t h t h e newer p o s i t r o n cameras o r ECAT s c a n n e r s . Both methods o f decay r e s u l t i n t h e p r o d u c t i o n o f 5 2 m M n (Th = 21m) and a gamma r a y o f 165KeV. 5 2 m M n decays d i r e c t l y t o s t a b l e 5 2 C r (98%) o r v i a 5 2Mn (Th = 5.7d) (2%) . ( F i g . 2) 5 2 F e due t o i t s s h o r t h a l f - l i f e {Th =8.2h) must be made l o c a l l y when r e q u i r e d , o n l y 5 5 F e and' 5 9 F e a r e a v a i l a b l e c o m m e r c i a l l y . I n v i v o h e m a t o l o g i c a l s t u d i e s were done w i t h 5-10 u C i / p a t i e n t o r bone marrow im a g i n g w i t h l O O u C i / p a t i e n t o f 5 2 F e . (265) The r a d i a t i o n dose t o bone marrow was 2.5 ra d s f o r 5 2 F e compared t o 50 rads w i t h 5 9 F e . (.266) Nuclide TABLE II RADIONUCLIDES OF IRON T£ Decay Gamma Energy 4 9 F e 0 . 0 8 8 B +, EC 5 2 p e 8 . 2 h B +, EC: 169 KeV 5 3 F e 2 .53 m 7 0 1 1328 5 3 * F e 8 .53 * B " » E C 3 7 8 5 5 F e 2.7 y EC No gamma 5 9 F e kk.G d B- 1099 1292 6 0 F e 1 0 5 y B" 58.6 27 61 ^ -i R" 1200 b i F e 6.1 m 1020 300 46 744 K»VJ 933 K.V 92 % P* J " % EC 1434 K.V M C r (STABLE) F i g . 2. Decay Scheme of 5 2 F e (From Ref. 273) S e v e r a l methods c o u l d be used t o produce 5 2 F e . These i n c l u d e t h e f o l l o w i n g : 5°Cr(a,2n) 5 2Fe, 5 2 C r ( 3 H e , 3 n ) 5 2 F e , 5 3 M n ( p , 4 n ) 5 2 F e and s p a l l a t i o n r e a c t i o n s . 1) : 5 0 C r ( a , 2 n ) 5 2Fe 5 2 F e was f i r s t produced by bombarding n a t u r a l chromium w i t h 30 MeV a l p h a p a r t i c l e s v i a t h e 5 0 C r ( a , 2 n ) 5 2 F e n u c l e a r r e a c t i o n . (266) High y i e l d s were r e p o r t e d b u t no numbers were s t a t e d . A l s o 5 5 F e was produced by t h e 5 3 C r ( a , 2 n ) 5 5 F e and 5 2 C r ( a , n ) 5 5 F e n u c l e a r r e -a c t i o n s depending on the p a r t i c l e energy and t a r g e t t h i c k n e s s used. Simple dose c a l c u l a t i o n s were a l s o done. Three y C i o f 5 2 F e would g i v e a r a d i a t i o n dose t o t h e b l o o d o f 15 mr. C o n t a m i n a t i o n o f 5 2 F e w i t h 5 5 F e (600 d e f f e c t i v e Th) would i n c r e a s e t h i s v a l u e from 15 t o 200 mr. The r a d i a t i o n dose from c o m m e r c i a l l y a v a i l a b l e 5 9 F e was 300 mr. T h e r e f o r e , i n o r d e r t o keep t h e r a d i a t i o n dose down 5 2 F e must not be c o n t a m i n a t e d w i t h 5 5 F e . E x c i t a t i o n f u n c t i o n s t u d i e s have shown t h a t the t h r e s h o l d f o r the 5°Cr(a,2n) 5 2Fe n u c l e a r r e a c t i o n was near 24 MeV and t h e peak above 70 MeV f o r t h i c k (20 MeV) t a r g e t s . (267) A h i g h p u r i t y n a t u r a l chromium t a r g e t ( 5 0 C r 4.3%, 5 2 C r ( 8 3 . 8 % ) , 5 3 C r 9.6%, " C r 2.7%, l e s s t han 100 ppm Fe) compressed out o f powder was used. Improved h e a t c o n d u c t i v i t y a l l o w e d h i g h e r beam c u r r e n t s t o be used. Chromium e l e c t r o p l a t e d copper f o i l was not used due t o t h e t h i n e s s o f t h e l a y e r and i n c r e a s e d r a d i o a c t i v i t y from t h e copper. A t h r e e hour 48 bombardment-of 6 5 MeV a l p h a p a r t i c l e s a t 12 uA beam c u r r e n t produced about 100-200 y C i o f 5 2 F e . A y i e l d o f 150 y C i o f 5 2 F e a t t h e end of c h e m i c a l s e p a r a t i o n r e q u i r e d a beam c u r r e n t o f 4 0 yA/hr. The ^ p r o d u c t i o n r a t e was 8.5 y C i / y A h r / g o f Cr t a r g e t w i t h a s p e c i f i c y a c t i v i t y o f 0.5 y C i 5 2 F e / y g Fe. About 5-6% o f 5 5 F e was produced v i a 5 2 C r ( a , n ) 5 5 F e r e a c t i o n and 0.05% 5 2Mn. The y i e l d c o u l d have been improved by u s i n g e n r i c h e d 5 0 C r t a r g e t m a t e r i a l i n s t e a d o f n a t u r a l chromium. The f i n a l p r o d u c t c o n t a i n e d 1.0 yg o f Cr, 300 yg of Fe, and 1.0 yg o f Mn. The t a r g e t and h o l d e r 3 hours a f t e r EOB had a r a d i a t i o n f i e l d o f 5 R/hr a t 12 i n . A l l c h e m i c a l o p e r a t i o n s were done r e m o t e l y and b e h i n d 2 i n c h e s o f l e a d . The t a r g e t was d i s s o l v e d i n c o n c e n t r a t e d HC1 f o r 1 hour and r e f l u x e d f o r an o t h e r hour t o reduce t h e h y d r o c h l o r i c a c i d (HC1) from 12N t o 7N. C a r r i e r F e + + 12 yg) and M n + + (4 yg) were th e n added and t h e m i x t u r e o x i d i z e d by n i t r i c a c i d . The m i x t u r e was then c o o l e d t o 10° b e f o r e e x t r a c t -i o n . The 5 2 F e was e x t r a c t e d i n t o p r e - c o o l e d d i - i s o p r o p y l e t h e r two t i m e s . The ether- l a y e r was washed s i x t i m e s w i t h 8N HC1. The i r o n was back e x t r a c t e d t h r e e t i m e s i n t o w a t e r . The 5 2 F e C l 3 was then c o n v e r t e d t o t h e c i t r a t e form by e v a p o r a t i n g t o near d r y n e s s under n i t r o g e n and a d d i n g oxygen f r e e d i s t i l l e d w a t e r , a s c o r b i c a c i d ( r e d u c i n g agent 1 0 - 2 mM) and sodium c i t r a t e . The s o l u t i o n was made n e u t r a l , i s o t o n i c , and s t e r i l e b e f o r e b e i n g used. T o t a l p r o c e s s i n g time was 6-7 hou r s . P r o c e s s time was reduced t o 3-4 hours by an automated system. (268) 49 An i n f i n i t l y t h i c k chromium p l a t e d t a r g e t was bombarded by 30 MeV alpha p a r t i c l e s w i t h beam c u r r e n t s up to 300 uA and a p r o d u c t i o n r a t e of 50 y C i 5 2Fe/100 uAh a t EOB. Using a p r o p o r t i o n i n g pump the d i s s o l v e d t a r g e t was mixed i n a s p i r a l g l a s s tube (phase mixer) and e x t r a c t e d i n an U-tube (phase s e p a r a t o r ) . O v e r a l l e x t r a c t i o n e f f i c i e n c y dropped from 99% to 85% w i t h t h i s system. Large amounts of 5 5 F e were a l s o produced l i m i t i n g the time a f t e r EOB when i t c o u l d be used. T h i s was the f i r s t time c a r r i e r f r e e 5 2 F e was produced. In another experiment a copper t a r g e t p l a t e d w i t h n i c k e l and then p l a t e d w i t h h i g h p u r i t y chromium was bombarded wi t h 30 MeV alpha p a r t i c l e s at 500 uA. (269) The y i e l d of 5 2 F e was 3.3 yCi/uAhr however, l a r g e amount;.'1- of 5 5 F e (14%) was produced at EOB. The f i n a l product c o n t a i n e d 5 yg of Cr and Ni and 2 0 yg of Fe. The chromium l a y e r was d i s s o l v e d e l e c t r o l y t i c a l l y (0.003 A/cm ) i n HCl i n l e s s t h a t 20 minutes. The s o l u t i o n was then b o i l e d . Unheated s o l u t i o n o n l y had an e x t r a c t i o n e f f i c i e n c y of l e s s than 25%. A d d i t i o n of hydrogen peroxide i n c r e a s e d t h i s to about 80%. Heating the s o l u t i o n and e x t r a c t i n g i t w h ile s t i l l hot i n c r e a s e d t h i s to g r e a t e r than 80% to 98%. C o o l i n g the b o i l e d s o l u t i o n decreased the e x t r a c t i o n e f f i c i e n c y to 70-80%. B o i l i n g caused the i r o n to-be o x i d i z e d from F e + + to F e + + + and changed the c o n c e n t r a t i o n of s e v e r a l chromium (.III) c h l o r i d e isomers known to e x i s t i n a c i d media. The e x t r a c t i o n with d i - i s o p r o p h y ether was done i n a s p e c i a l swan-50 necked v e s s e l . The back e x t r a c t i o n (98-100% e f f i c i e n c y ) was done i n a c e n t r i f u g e tube. The aqueous l a y e r was b o i l e d t o d r y n e s s and r e c o n s i t u t e d w i t h i s o t o n i c (3.8%) sodium c i t r a t e . The e n t i r e p r o c e s s time was about 1.5 ho u r s . E n r i c h e d 5 0 C r t a r g e t s were used a t BNL but found t o be i m p r a c t i c a l due t o t h e h i g h c o s t o f t h e i s o t o p e . R e p r o c e s s i n g was time con-suming and i n v o l v e d l o s s e s o f t h e v e r y e x p e n s i v e i s o t o p e . The p r o d u c t i o n rate,was 10.5 uCi/uAh w i t h a 39 MeV a l p h a beam o n l y 35% of t h e t h e o r e t i c a l p r o d u c t i o n r a t e . The 5 °Cr'(a, 2n) 5 2 F e r e a c t i o n r e q u i r e d a l p h a p a r t i c l e s w i t h e n e r g i e s g r e a t e r t h a n 65 MeV t o i n c r e a s e t h e y i e l d and d e c r e a s e 5 5 F e p r o d u c t i o n . The problem w i t h the r e a c t i o n was t h a t v e r y few p a r t i c l e a c c e l e r a t o r s c o u l d a t t a i n t h i s h i g h energy and n a t u r a l chromium c o n t a i n s l e s s than 4.3% 5 0 C r . 2) 5 2 C r ( 3 H e , 3 n ) 5 2 F e The 5 2 C r ( 3 H e , 3 n ) 5 2 F e r e a c t i o n was f i r s t used a t BNL. (270) The e x c i t a t i o n f u n c t i o n f o r 5 2 F e p r o d u c t i o n peaked a t about 30-40 MeV w i t h a w i d t h o f about 15 MeV and a c r o s s - s e c t i o n o f 5-10 m i l l i b a r n s (mb). The e x c i t a t i o n c u r v e f o r d i r e c t 5 2 m M n and 5 2Mn p r o d u c t i o n had s i m i l a r shapes w i t h c r o s s - s e c t i o n s o f 70 mb and 200 mb r e s p e c t i v e l y . N a t u r a l chromium ( 8 3 . 7 % 5 2 C r ) was bomabrded w i t h 4 5.5 MeV 3He p a r t i c l e s w i t h an average y i e l d o f 50 uCi/uAh ( t h i n t a r g e t — 0.56 mm). Only 0.001% 5 5 F e was produced compared t o 5-15% w i t h the 5 2 C r (.a, 3n) 5 2 F e r e a c t i o n . The 5 2 F e was s e p a r a t e d from t h e t a r g e t 51 by s o l v e n t e x t r a c t i o n w i t h d i - i s o p r o p y l e t h e r (1-5 yg) Fe and Mn c a r r i e r added. S i m i l a r s t u d i e s a t a lower beam energy were done l a t e r . (271) N a t u r a l chromium was e l e c t r o p l a t e d onto copper p l a t e s . The t a r g e t was bombarded w i t h 3He p a r t i c l e s a t 23 MeV. The y i e l d was 0.7 yCi/u7Ah and 0.3% 5 5Fe. The copper b a c k i n g was removed w i t h 10 M n i t r i c a c i d , a n d t h e chromium t a r g e t d i s s o l v e d i n H C l . The 5 2 F e was s e p a r a t e d from t h e chromium t a r g e t by e i t h e r d i - i s o p r o p y l e t h e r s o l v e n t e x t r a c t i o n o r i o n exchange chromatography. The 12 M HCl s o l u t i o n was passed t h r o u g h a 150 mm x 13 mm d i a m e t e r column o f Bio-Rad AGlx8 C l ~ form 100-200 mesh a n i o n exchange r e s i n . Washing the column w i t h 12 M HCl (12 5 mis) removed a l l t h e i m p u r i t -i e s . F i f t y mis o f 1 M HCl were used t o e l u t e t h e 5 2 F e from the column. S i m i l a r e x p e r i m e n t s a t 3 5 MeV 3He, 80 uA beam on 0.01 i n t h i c k n a t u r a l chromium t a r g e t produced 15 y C i o f 5 2 F e / u A h r . (272) The t a r g e t had a r a d i a t i o n l e v e l o f 50 R/hr a t 4 i n a f t e r s e v e r a l hours bombardment. The t a r g e t was d i s s o l v e d i n 9 M H C l . C o n c e n t r a t e d n i t r i c a c i d and 20 ygs o f F e + + + were added t o o x i d i z e t h e 5 2 F e . The s o l u t i o n was passed t h r o u g h a Dowex A - l r e s i n column ( 1 cm x 10 cm) a t a f l o w r a t e o f 3 ml/min. Washing w i t h 50 ml o f 9 M HCl removed t h e chromium and manganese, w h i l e 2 7 ml o f 2 M HCl removed any r e s i d u a l g a l i u m . The 5 2 F e was th e n e l u t e d w i t h 0.1 M H C l . The s o l u t i o n was e v a p o r a t e d f o u r t i m e s t o d r y n e s s and a s o l u t i o n o f 10 mg a s c o r b i c a c i d and 6% soidum c i t r a t e was added. P r o c e s s time o f 1.5-2 hours w i t h a c h e m i c a l y i e l d o f 85-9 5% was o b t a i n e d . The 5 2 C r ( 3 H e , 3 n ) 5 2 F e r e a c t i o n t a k e s advantage t h a t 5 2 C r i s 84% o f n a t u r a l chromium so e n r i c h e d t a r g e t s d i d not have t o be used. However, i t has t h e same problem as t h e f i r s t r e a c t i o n t h a t few machines can a c c e l e r a t e 3He p a r t i c l e s t o 45-50 MeV. 3) 5 5 M n ( p , 4 n ) 5 2 F e Manganese d i o x i d e o f h i g h p u r i t y grade was bombarded w i t h 6 5 MeV p r o t o n s a t 0.5 uA f o r 1 hour. The t a r g e t l e s s h o l d e r had a r a d i a -t i o n r e a d i n g o f 500 mR/hr a t 1 f t 1 hr a f t e r EOB. The p r o d u c t i o n r a t e was 160 uCi/uAh ( s p e c i f i c a c t i v i t y 1.2-1.3 uCi / u g F e ) . The 5 5 F e l e v e l was not measured but c a l c u l a t e d t o be l e s s than 3%. The i r o n was p u r i f i e d by i o n exchange chromatography (Dowex 1x8 100-200 mesh 1 cm " x 12 cm long) . The s o l u t i o n w i t h 5 yg i r o n c a r r i e r was l o a d e d i n 6 N HC1. Washing w i t h 6 N HC1 removed the Mn, Na, Cr and A l . The i r o n was e l u t e d w i t h 0.5 N HC1. Chromatography was r e p e a t e d a second t i m e . The p r o c e s s i n g t i m e was 2 hours and 70-80% e f f i c i e n t . The f i n a l p r o d u c t c o n t a i n e d 60-65 yg o f i r o n due t o the i m p u r i t y i n manganese d i o x i d e t a r g e t m a t e r i a l . \212) Both p r e v i o u s n u c l e a r r e a c t i o n s r e q u i r e h i g h e n e r g i e s and have low c r o s s s e c t i o n s due t o the c r o s s i n g o f a d o u b l e l y charged p a r t i c l e 53 across the Coloumb b a r r i e r of the t a r g e t n u c l e u s . The 5 5Mn(p,4n) 5 2 F e r e a c t i o n does not have t h i s problem T h i s i s an i d e a l r e a c t i o n because the o n l y s t a b l e " i s o t o p e o f manganese i s 5 5Mn (100% abundant). Again the 55 MeV proton beam energy i s too h i g h f o r n e a r l y a l l m e d i c a l c y c l o t r o n s . An e x c i t a t i o n f u n c t i o n measur-i n g both 5 2 F e and 5 5 F e c r o s s s e c t i o n s and p r o d u c t i o n r a t e s must be done from 0-100 MeV b e f o r e t h i s n u c l e a r r e a c t i o n can be used to produce 5 2 F e r o u t i n e l y . 4) S p a l l a t i o n R eactions Bombardment of t a r g e t s w i t h h i g h energy proton beams of 100 MeV or g r e a t e r cause fragmentation of the primary t a r g e t n u c l e i r e s u l t i n g i n the p r o d u c t i o n of new n u c l e i . One to two protons or neutrons are knocked out, the remaining nucleus b o i l s o f f f u r t h e r neutrons r e s u l t i n g i n p r o t o n - r i c h i s o t o p e s . U n l i k e low energy n u c l e a r r e a c t i o n s , s p a l l a t i o n r e a c t i o n s are v e r y n o n s p e c i f i c . The d e s i r e d f i n a l product (10-15 mass u n i t s lower) must be separated c h e m i c a l l y from ot h e r r a d i o e l e m e n t s . Another problem i s t h a t n e i g h b o r i n g r a d i o i s o t o p e s may i n t e r f e r e w i t h the d e s i r e d one. High energy proton (588 MeV) s p a l l a t i o n of Mn, Co, N i and Cu 7 54 t a r g e t s has been s t u d i e d . (274) The cr o s s s e c t i o n s f o r 5 2 F e pr o d u c t i o n are Mn 0.066 mb, Co 0.152 mb, Ni 1.35 mb, and Cu 0.148 mb. Other c r o s s s e c t i o n s have been r e p o r t e d i n the l i t e r a t u r e — Co 0.76 mb (277); Cu 0.70 mb (275) and 0.25 mb (276). The n i c k e l t a r g e t was used f o r a l l f u r t h e r experiments. The i r r a d i a t e d t a r g e t contained the f o l l o w i n g r a d i o n u c l i d e s kBCr, 5 7 C o , 5 6 N i , 5 7 N i , 5 2Mn, 5 6Co, 5 5 C o , " 8V, kkSc, 5 2 mMn and 5 2 F e . The t a r g e t was d i s s o l v e d i n hot c o n c e n t r a t e d n i t r i c a c i d , taken to dryness, r e d i s s o l v e d i n 6 N HC1 and e x t r a c t e d w i t h d i - i s o p r o p y l e t h e r . The r e s u l t i n g ether s o l u t i o n c o n t a i n e d o n l y 5 2 F e and i t s daughter 5 2 mMn. A f t e r 8.4 day decay of t h i s s o l u t i o n hhSc, ^ m S c , k7Sc, I t 8V, 5 5Fe and 5 9 F e were de t e c t e d . The % a c t i v i t y a t EOB was ^ S c 0.02%, kl*mSc 0.11%, 4 7 S c 0.01%, * BV 0.14%, 5 5 F e 3.3% and 5 9 F e 0.16%. S t a b l e N i had a low d i s t r i b u t i o n c o e f f i c i e n t of l e s s than 4 x 10" 4 so only 0.04% s t a b l e Ni was i n the f i n a l product. A 4 g/cm 2 t a r g e t at 6 00 MeV and 500 uA of proton beam c u r r e n t would y i e l d 350 mCi of 5 2 F e / h . Up to 95% of the 5 2 F e c o u l d be separated with contaminants l e s s than 0.5% t o t a l and 3.3% 5 5 F e . I f t h i s 64 g target•-was 7 used f o r 100 p a t i e n t doses 0.4 mg of s t a b l e n i c k e l would be admin i s t e r e d per p a t i e n t . Another e x t r a c t i o n would reduce t h i s to 0.2 ng. H Chemistry and Se p a r a t i o n Methods of Iron Iron d i s s o l v e s i n HC1, s u l f u r i c a c i d and d i l u t e phosphoric a c i d to form f e r r o u s s a l t s . N i t r i c , p e r c h l o r i c , bromic, and i o d i c a l s o y i e l d f e r r o u s s a l t s i f the a c i d s are d i l u t e and the r e a c t i o n occurs 55 a t room t e m p e r a t u r e . C o n c e n t r a t e d a c i d o r h e a t i n g g i v e s f e r r i c s a l t s . I n s o l u b l e o x i d e f i l m s a r e formed w i t h c o n c e n t r a t e d n i t r i c , i o d i c and p e r c h l o r i c a c i d s and slow down the r e a c t i o n . F e r r o u s i r o n i s q u a n t i t a t i v e l y o x i d i z e d t o f e r r i c i r o n by s t r o n g o x i d i z -i n g a g e n t s - n i t r i c a c i d , hydrogen p e r o x i d e , d i c h r o m a t e , and p e r -manganate. F e r r i c i r o n i s q u a n t i t a t i v e l y reduced by stannous c h l o r i d e , h y d r o x y l a m i n e H C l , h y d r o q u i n i n e , and i r o n and H 2S. F e r r i c i r o n can be e x t r a c t e d from HCl by many o r g a n i c s o l v e n t s . E x t r a c t i o n w i t h e t h y l e t h e r i s w e l l s u i t e d f o r f e r r i c i r o n c oncentrations^rom-. 10" 4 t o 10 - 1M. (278-279) The h i g h e r the f e r r i c i o n c o n c e n t r a t i o n t h e g r e a t e r i s t h e d i s t r i b u t i o n c o e f f i c i e n t , t h e maximum i s a t 6N H C l . I s o p r o p y l e t h e r p r o v i d e s a more q u a n t i t -a t i v e e x t r a c t i o n w i t h l e s s s e n s i t i v i t y t o a c i d c o n c e n t r a t i o n a t i t s maximum (7.5-8.ON HCl) (280). I s o p r o p y l e t h e r has a g r e a t e r t h a n 96% e x t r a c t i o n e f f i c i e n c y w i t h 1 mg o f i r o n as compared w i t h 80% f o r e t h e r . F e r r o u s i r o n i s n o t e x t r a c t e d w h i l e f e r i c i r o n i s e x t r a c t e d as t h e s o l v a t e d m o l e c u l e (H+FeCl^ -) •2_k (281-282) . I s o p r o p y l e t h e r o r e t h y l e t h e r can be used t o s e p a r a t e Fe from Cu, Co, Mn, N i , A l , C r , Zn, V ( I V ) , T i and s u l f a t e b u t not from V ( V ) , Sb (V) , Ga(.III). , T e ( I I I ) , Mo or phosphate. U s i n g hydrobromic o r n i t r i c a c i d i n s t e a d o f h y d r o c h l o r i c a c i d g r e a t l y reduces e x t r a c t i o n e f f i c i e n c y . (283-284) Amyl a c e t a t e has been used t o s e p a r a t e f e r r i c i r o n from Mo and Sn 56 b u t n o t from V. (285) Ketones have been s t u d i e d and were more e f f i c i e n t t h a n e t h e r s o r e s t e r s . The d i s t r i b u t i o n c o e f f i c i e n t between i r o n ( f e r r i c ) and i s o b u t y l m e t h y l ketone and 5.5-7N HC1 i s a t l e a s t 1000 and may a l l o w b e t t e r s e p a r a t i o n from V. (286) E x t r a c t i o n w i t h d i k e t o n e s such as a c e t y l a c e t o n e i n x y l e n e , carbon t e t r a c h l o r i d e o r c h l o r o f o r m ; o r 2 - t h e n o y l t r i f l u o r o a c e t o n e (TTA) i n benzene has been used t o s e p a r a t e f e r r i c i r o n from o t h e r elements. (287-290) Other elements (Cu, Mn, Mo, T r , V, Z r , Be, Ga) which co-e x t r a c t w i t h i r o n ; t h e amount depends on t h e pH o f e x t r a c t i o n . TTA p e r m i t s e x t r a c t i o n a t a lower pH t h a n a c e t y l a c e t o n e b u t e q u i l i b r i u m i s a t t a i n e d v e r y s l o w l y . The advantage of TTA i s t h a t t h e o r g a n i c r e s i d u e can be e a s i l y removed by h e a t i n g . C u p f e r r o n i n c h l o r o f o r m , e t h e r , e t h e r and benzene, o r e t h y l a c e t a t e i s i d e a l t o s e p a r a t e i r o n from s o l u t i o n s c o n t a i n i n g l a r g e amounts o f Ca and phosphate but Ga, S b ( I I I ) , T i ( I V ) , S n ( I V ) , Z r , V ( V ) , U ( I V ) , Mo(VI) and Cu may c o e x t r a c t w i t h IN HC1 o r s u l f u r i c a c i d . (291-292) Very l i t t l e work has been done i n t h e e x t r a c t i o n o f f e r r o u s i r o n . The f o l l w i n g compounds have been s t u d i e d : (.293) F e r r o u s i r o n can be e x t r a c t e d w i t h b a t h o p h e n a n t h r o l i n e i n i s o a m y l a l c o h o l o r n - h e x y l a l c o h o l a t pH 4; p h e n y l - 2 - p y r i d y l ketone i n i s o a m y l a l c o h o l o r c h l o r o f o r m from IM NaOH; t r i p y r i d y l - s - t r i a z i n e i n n i t r o b e n z e n e a t pH 4 w i t h p e r c h l o r a t e o r i o d i d e ; i s o n i t r o s a c e t o p h e n o n e i n c h l o r o f o r m 57 at pH 8-9 (294); p o l y e t h e r s (295); or t r i - n - b u t y l phosphate. (296) F e r r i c i r o n i s s t r o n g l y adsorbed onto anion exchange r e s i n s from concentrated HCl a c i d s o l u t i o n s . (297) Other elements are not adsorbed s t r o n g l y on the r e s i n and these elements can be removed with d e c r e a s i n g HCl c o n c e n t r a t i o n s . Iron i s e l u t e d o f f - the, column: wit h d i l u t e HCl l e s s than IN or water. Ferrous and f e r r i c i r o n can be separated by paper chromatography as c h l o r i d e or t a r t r a t e complexes, (298) c h l o r i d e complexes i n ether-methanol-HCl-water s o l v e n t (299) or butanol-12N HCl (85:15) (300); or a c e t a t e complexes i n n - b u t a n o l - e t h a n o l - a c e t i c acid-water (40:25:25:35) (301) as a s o l v e n t . I M e t a l l o p o r p h y r i n S y n t h e s i s There are t h r e e g e n e r a l methods f o r l a b e l i n g p o r p h y r i n s : 1) A c e t a t e method--lt has been used f o r V, Mn, Fe, Co and N i . Thr protons of the p o r p h y r i n to be metalated ate t r a n s f e r e d to the a c e t a t e . I t can be used f o r a l l d i v a l e n t metal ions except those u n s t a b l e i n a c e t i c a c i d and some t r i v a l e n t i o n s . 2) P y r i d i n e m e t h o d — I t can be used f o r m e t a l l o p o r p h y r i n s very l a b i l e toweard a c i d . T h i s r e a c t i o n cannot be used w i t h metal ions with high charge because p y r i d i n e forms complexes w i t h the metal c a r r i e r . 3) Dimethylformamide method--Dimethylformamide (weakly c o o r d i n a t i n g h i g h - b o i l i n g oxygen-donor solvent) has been used to do 68 d i f f e r e n t m e t a l a t i o n s however, anhydrous metal h a l i d e s may be r e q u i r e d . (302) 58 In the past, the c o n c e n t r a t i o n of the metal has been much l a r g e r than t h a t of the p o r p h y r i n . T h i s was not p o s s i b l e i f c a r r i e r - f r e e r a d i o n u c l i d e s were used to l a b e l the p o r p h y r i n . E i t h e r c a r r i e r had to be added d e c r e a s i n g s p e c i f i c a c t i v i t y or a new m e t a l a t i o n procedure had to be developed. Porphyrins have been l a b e l e d with 1 1 1 In by the a c e t i c a c i d method i n 2 hours. However, C l ions s t r o n g l y i n t e r f e r e w h i l e other metals i n the r a d i o n u c l i d e a l s o i n t e r f e r e . (303) 59 I I I M a t e r i a l and Methods A Chemicals 1) P o r p h y r i n s Hematoporphyrin d i h d r o c h l o r i d e was obtained from ICN Pharmaceuticals, C l e v e l a n d , Ohio or Sigma Chemical Co., St. L o u i s , Mo. Meso-tetra(4-carboxyphenyl) porphine, meso-t e t r a (4-N-methylpyridyl) porphine t e t r a i o d i d e , and t e t r a -Na-meso-tetra(4-sulfantophenyl) porphine (12 hydrate) were obt a i n e d from Strem Chemicals Inc., Newburyport, Ma. 2) Chromatography S i l i c a g e l f o r column chromatography (Woelm a c t i v i t y I, 70-150 mesh, c a t a l o g number 402747) was obtained from ICN Pharm-a c e u t i c a l s , C l e v e l a n d , Ohio. Polyamide f o r t h i n l a y e r and column chromatography was obtained from Brinkmann Instruments Westbury, N.Y. Dowex AG 1-X8 anion exchange r e s i n and b a s i c alumina AGIO were o b t a i n e d from Bio-Rad L a b o r a t o r i e s , Richmond Ca. 3) R a d i o n u c l i d e s 5 9 F e - f e r r i c c h l o r i d e was obtained from Amersham Corp., A r l i n g t o n Heights, 111. 6 7 G a - c i t r a t e was o b t a i n e d from New England Nuclear, Boston Ma. eo 4) Reagents A l l o t h e r c h e m i c a l s were o f r e a g e n t grade o r b e t t e r . D i -chloromethane, methanol, and t - b u t a n o l were d r i e d u s i n g e i t h e r c a l c i u m h y d r i d e o r sodium m e t a l . B I n s t r u m e n t a t i o n 1) R a d i o a c t i v i t y Measurements a)'Gamma S p e c t r o m e t r y F o r s i n g l e r a d i o n u c l i d e measurements t h e AECL Canb e r r a S e r i e s 30 M u l t i c h a n n e l A n a l y z e r w i t h a 2 i n x 2 i n h i g h r e s o l u t i o n N a l ( T i ) d e t e c t o r (Canberra I n d u s t r i e s , M e r i d e n , Ca) was used. F o r f i n a l 5 2 F e p r o d u c t a c t i v i t y d e t e r m i n a t i o n , a r a d i o i s o t o p e dose c a l i b r a t o r ( C a p i n t e c I n c . , M o n t v a l e , NJ) was used. Gamma s p e c t r o s c o p y was done e i t h e r on t h e TRIM system, S a f e t y system, AECL/TRIUMF O r t e c 7044 M i n i c o m p u t e r Based Data A q u i s i -t i o n and A n a l y s i s system (EG & G O r t e c I n c . , Oak Ri d g e , TN), No v a t r a c k O r t e c 7044 M i n i c o m p u t e r Based Data A q u i s i t i o n and A n a l y s i s system o r AECL N u c l e a r Data ND660 M u l t i c h a n n e l A n a l y z e r System ( N u c l e a r Data I n c . , Schaumburg, 111). Both the O r t e c o r N u c l e a r Data systems were equipped w i t h a 99.5 cc G e ( L i ) c o a x i a l d e t e c t o r ( e f f i c i e n c y 18.2%) (EG & G O r t e c , Oak Ri d g e , TN). o r a 50 cc Ge Phyge p l a n a r d e t e c t o r (Aptec N u c l e a r I n c . , L e w i s t o n , NY). 61 The system was c a l i b r a t e d u s i n g l i q u i d s t a n d a r d s i n a 1.0 ml s t a n d a r d AECL spectrum v i a l u s i n g 1 3 7 C s , 5 7 C o , 6 0 C o , 1 3 3 B a , 5 l tMn, and 2 2 N a (New England N u c l e a r , B o s t o n , Ma.) The system was a l s o c a l i b r a t e d u s i n g Amersham (Amersham Corp., A r l i n g t o n H e i g h t s , 111.) o r NEN p o i n t s o u r c e s . A l l s t a n d a r d s were t r a c e a b l e t o NBS. b) R a d i o n u c l i d e Imaging o r Scann i n g A n i m a l scans were done a t t h e Vancouver G e n e r a l H o s p i t a l u s i n g e i t h e r t h e S e a r l e Pho/Con tomographic scanner w i t h a h i g h energy c o l l i m a t o r o r S e a r l e LFOV s t a n d a r d gamma gamera w i t h a medium energy c o l l i m a t o r ( S e a r l e R a d i o g r a p h i c s , Des P l a i n e s , 111.). The Pho/Con was s e t on b o t h the gamma r a y and on the 511 KeV peak and summed t o g e t h e r . ..,c)_ D i s t r i b u t i o n S t u d i e s An automated gamma c o u n t e r w i t h a 3 i n c h w e l l t y p e sodium i o d i d e T l ( a c t i v a t e d ) d e t e c t o r was used t o count t h e a n i m a l t i s s u e ( N u c l e a r C h i c a g o , Des P l a i n e s ) R a d i o a c t i v e samples were p l a c e d i n c o u n t i n g tubes (15.6 x 125 mm) (Amersham S e a r l e , Des P l a i n e s ) and counted i n 4 p i d e t e c t i o n geometry. The gamma c o u n t e r was c a l i b r a t e d u s i n g a J 3 7 C s s o u r c e (Amersham-S e a r l e , Des P l a i n e s ) U s i n g t h e photopeak o f 662 KeV, t h e f i n e and h i g h v o l t a g e c o n t r o l s were a d j u s t e d u n t i l "the photopeak 62 f e l l i n t h e 662nd d i v i s i o n . The a t t e n u a t o r was s e t a t 16, h i g h v o l t a g e a t 800, f i n e v o l t a g e a t 8 f o r a range f o r 0-2 MeV. d) E x c r e t i o n S t u d i e s F o r whole-body e x c r e t i o n s t u d i e s , a n i m a l s were counted i n a gamma c o u n t e r equipped w i t h two t h r e e i n c h N a l c r y s t a l s (Tubor, N u c l e a r C h i c a g o , Des P l a i n e s ) . The machine was c a l i b r a t e d w i t h a 1 3 7 C s s o u r c e . The c o u r s e h i g h v o l t a g e was s e t a t 1000, f i n e h i g h v o l t a g e a t 3 7 and the a t t e n u a t o r *f a t 8 f o r 0-2 MeV range. 2) A b s o r p t i o n S p e c t r o p h o t o m e t r y A b s o r p t i o n s p e c t r o p h o t o m e t r y was done on the Beckman Model 25 r e c o r d i n g s p e c t r o p h o t o m e t e r w i t h a s o l v e n t b l a n k (Beckman In s t r u m e n t s I n c . , I r v i n e , Ca.) C P o r p h y r i n S y n t h e s i s 1) P r o t o p o r p h y r i n C o m m e r c i a l l y a v a i l a b l e h e m a t o p o r p h y r i n d i - H C l was checked f o r p u r i t y by s i l i c a g e l t h i n l a y e r chromatography ( t i c ) w i t h benzene: m e t h a n o l : f o r m i c a c i d (BMF) (110:30:1) and v i s i b l e s p e c t r o p h o t o m e t r y . F i v e hundred and s i x t y ml o f DMF were b r o u g h t t o a b o i l w i t h s t i r r i n g and a v i g o r o u s stream of n i t r o g e n . 63 With vi g o r o u s s t i r r i n g , 6.2 g of hematoporphyrin d i h y d r o c h l o r i d e i n a s m a l l beaker was lowered i n t o the f l a s k c o n t a i n i n g the DMF. A f t e r 30 seconds to allow f o r the hematoporphyrin to d i s s o l v e and dehydrate, the s o l u t i o n was q u i c k l y c o o l e d to 40-50° i n a hot water bath, then a tap water bath and f i n a l l y an i c e bath. The s o l u t i o n was p l a c e d i n a 1 1RB f l a s k and the DMF removed by r o t a r y e v a p o r a t i o n a t 40-50° and a vacuum pump. The t a r y r e s i d u e was d i s s o l v e d i n minimal 90-100% formic a c i d . The p r o t o p o r p h y r i n was p r e c i p i t a t e d with d i e t h y l e t h e r . The p r e c i p i t a t e was f i l t e r e d , washed wit h ether u n t i l the f i l t r a t e was s l i g h t l y c o l o r e d , sucked dry, washed wit h water, sucked dry, washed wit h e t h e r , and d r i e d to constant weight over KOH. The protoporphyrin, was'checked f o r p u r i t y by v i s i b l e s p e c t r o -photometry and s i l i c a g e l t i c with BMF (110:30:1). The p r o t o -p o r p h y r i n was s t o r e d i n the dark and i n a t i g h t l y c l o s e d c o n t a i n e r to prevent r e a c t i o n w i t h l i g h t and oxygen to form p h o t o p r o t o p o r h y r i n . Because p r o t o p o r p h y r i n and i t s e s t e r s were extremely l i g h t s e n s i t i v e a l l subsequent experiments with t h i s compound were done under subdued l i g h t . 2). P r o t o p o r p h y r i n D i - T e r t i a r y B u t y l E s t e r (DTBE) F i v e hundred mg of p r o t o p o r p h y r i n were d i s s o l v e d i n 50 ml of dry dichloromethane, heated to r e f l u x i n an o i l bath w i t h s t i r -r i n g and under n i t r o g e n . Two and a h a l f ml of o x a y l c h l o r i d e 64 were added v e r y c a r e f u l l y w i t h a d r o p p i n g f u n n e l . The r e a c t i o n was n o t o v e r l y v i g o r o u s i f the d i c h l o r o m e t h a n e was d r y . R e f l u x i n g f o r 15 minutes a l l o w e d the complete f o r m a t i o n of t h e a c i d c h l o r i d e . The e x c e s s o x a y l c h l o r i d e and d i c h l o r o -methane were removed by d i s t i l l a t i o n and vacuum d i s t i l l a t i o n t o remove the l a s t few m i l l i l i t e r s o f s o l u t i o n , b o t h a t l e s s than 60° t o o b t a i n maximum y i e l d s . The r e s i d u e a c i d c h l o r i d e was d i s s o l v e d i n m i n i m a l d i c h l o r o m e t h a n e and a g a i n removed by d i s t i l l a t i o n . T h i s was done t o remove any r e s i d u a l o x a y l c h l o r i d e a f t e r t h e f i r s t d i s t i l l a t i o n . The d r y a c i d c h l o r i d e was r e d i s s o l v e d i n 30 ml o f d r y d i c h l o r o m e t h a n e and h e a t e d t o r e f l u x w i t h a condenser f i t t e d w i t h a d r y i n g t u b e . Three and t h r e e t e n t h s ml o f d r y t e r t i a r y b u t y l a l c o h o l were added, r e f l u x e d f o r 45 m i n u t e s , f o l l o w e d by a n o t h e r 3.3 ml o f d r y t e r t i a r y b u t y l a l c o h o l . A f t e r 4 5 minutes o f a d d i t i o n a l r e f l u x i n g e xcess t e r t i a r y b u t y l a l c o h o l and d i c h l o r o m e t h a n e was removed by r o t a r y e v a p o r a t i o n . The r e s i d u e was d i s s o l v e d i n 66 ml o f c h l o r o f o r m . S a t u r a t e d sodium b i c a r b o n a t e s o l u t i o n was added w i t h v i g o r o u s s t i r r i n g u n t i l c a rbon d i o x i d e e v o l u t i o n s t o p p e d . T h i r t y - t h r e e ml o f w a t e r were added t o t h e c h l o r o f o r m s o l u t i o n and t h e l a y e r s s e p a r a t e d i n a s e p a r a t o r y f u n n e l . The o r g a n i c l a y e r was washed w i t h w ater t h r e e t i m e s and d r i e d o v e r sodium s u l f a t e . S o l v e n t was t h e n removed by r o t a r y e v a p o r a t i o n i n vacuo. The crude p r o t o p o r p h y r i n DTBE was p u r i f i e d by column chromato-graphy on s i l i c a g e l grade IV. The r e s i d u e was d i s s o l v e d i n m i n i m a l c h l o r o f o r m and added t o a 150 g column ( 2 cm d i a m e t e r x 50 cm long) o f s i l i c a g e l . The column was e l u t e d w i t h c h l o r o f o r m : e t h e r (100:1) . The f i r s t band, a brown c o l o r , e l u t e d was some unknown compound. The second band (re d c o l o r ) was t h e p r o t o p o r p h y r i n DTBE. The r e d band was e v a p o r a t e d t o dr y n e s s i n vacuo, and was c r y s t a l l i z e d from benzene (lml/10'Omg) .' The mother l i q u o r s were c o n c e n t r a t e d and a second c r o p o f c r y s t a l s was o b t a i n e d . The p u r i t y o f the p r o t o p o r p h y r i n DTBE was checked by v i s i b l e s p e c t r o p h o t o m e t r y , t i c u s i n g c h l o r o f o r m , and m e l t i n g p o i n t d e t e r m i n a t i o n . 3) P h o t o p r o t o p o r p h y r i n DTBE Isomer 1: l - h y d r o x y - 2 - d e s v i n y l - 2 - f o r m y l e t h y l i d i n e p r o t o p o r p h y r i n DTBE (.Br r i n g r e a c t e d ) Isomer 2: 3 - h y d r o x y - 4 - d e s v i n y l - 4 - f o r m y l e t h a l i d i n e p r o t o p o r p h y r i n DTBE (A r i n g r e a c t e d ) F i v e hundred mg o f p r o t o p o r p h y r i n DTBE were d i s s o l v e d i n 250 ml o f d i c h l o r o m e t h a n e c o n t a i n i n g 10% p y r i d i n e . The s o l u t i o n was exposed t o d i r e c t s u n l i g h t f o r 1-3 days o r mercury vapor lamp f o r 3-4 hours u n t i l t h e amount o f u n r e a c t e d p r o t o p o r p h y r i n DTBE 66 remained constant by t i c a n a l y s i s u s i n g c h l o r o f o r m . The mixture was reduced t o 100 ml, washed with water, IN HCl twice, 5% sodium b i c a r b o n a t e s o l u t i o n , and water again. The s o l u t i o n was d r i e d over sodium s u l f a t e and taken to dryness by r o t a r y e v a p o r a t i o n i n vacuo. The crude p h o t o p r o t o p o r p h y r i n DTBE was r e d i s s o l v e d i n minimal dichloromethane and chromatographed on 100 g of s i l i c a g e l grade IV (2 cm x 50 cm column), e q u i l i b r a t e d and e l u t e d w i t h dichloromethane: ether (20:1). The f i r s t band (red) was the unreacted p r o t o p o r p h y r i n DTBE. The second band (green) was the B r i n g r e a c t e d photoprotoporphyrin DTBE isomer (Isomer 2 ) . The t h i r d band (green) was a mixture of both isomers and was rechromatographed on 70 g s i l i c a g e l t o separate the isomers. The f o u r t h band (green) was the A r i n g r e a c t e d photoproto-p o r p h y r i n DTBE isomer (Isomer 1.) Each isomer f r a c t i o n was combined and taken to dryness and c r y s t a l l i z e d from chloroform-methanol. P u r i t y was checked by a b s o r b t i o n spectrophotometry and m e l t i n g p o i n t a n a l y s i s . 4). 2-Formyl-4-vinyl d e u t e r o p o r p h y r i n DTBE S p i r o g r a p h i s ( n a t u r a l p o r p h y r i n DTBE F i f t y mg of the A r i n g r e a c t e d photoprotoporphyrin DTBE (Isomer 2) were d i s s o l v e d i n 25 ml of dry dichloromethane. 67 A s o l u t i o n c o n s i s t i n g of 50 mg of sodium borohydride i n 2 ml of dry methanol was added and the r e a c t i o n mixture s t i r r e d f o r 1 hour a t room temperature. The c o l o r of the s o l u t i o n changed from green to red-brown. A f t e r 1 hour t i c with dichloromethane-ether (20:1) as a s o l v e n t was done to check f o r r e a c t i o n completeness. The green photoprotoporphyrin band was completely r e p l a c e d by the gray a l c o h o l band. The red band near the s o l v e n t f r o n t a l s o appeared (unknown compound). A c e t i c a c i d was then added dropwise u n t i l hydrogen e v o l u t i o n ceased and excess sodium borohydride was d e s t r o y e d . The mixture was poured i n t o 100 ml of water. The s o l u t i o n was e x t r a c t e d w i t h 100 ml of c h l o r o f o r m and the o r g a n i c l a y e r washed wi t h 5% sodium b i c a r b o n a t e water, and d r i e d over sodium s u l f a t e and taken to dryness by r o t a r y e v a p o r a t i o n . The brown-green r e s i d u e was d i s s o l v e d i n 50 ml of dioxane. A s o l u t i o n c o n s i s t i n g of 100 mg of sodium i o d a t e i n 1 ml of water was added f o l l o w e d r a p i d l y by 0.2 ml of c o n c e n t r a t e d s u l f u r i c a c i d . The r e a c t i o n was allowed to go to completion f o r 4 hours at room temperature w i t h continous s t i r r i n g , a f t e r which i t was poured i n t o 150 ml of 5% sodium c h l o r i d e s o l u t i o n and e x t r a c t e d with 50 ml of d i c h l o r o -methane or c h l o r o f o r m . The o r g a n i c l a y e r was washed wi t h 5% sodium b i c a r b o n a t e s o l u t i o n , water, and d r i e d over sodium s u l f a t e and taken to dryness by r o t a r y e v a p o r a t i o n . The r e d -p u r p l e r e s i d u e was d i s s o l v e d i n minimal c h l o r o f o r m and chromato-graphed on 20 g s i l i c a g e l grave IV with chloroform: methanol 68 (20:1) o r c h l o r o f o r m : e t h e r (20:1) as t h e e l u e n t . The f i r s t r e d band t o come o f f was t h e s p i r o g r a p h i s p o r p h y r i n DTBE. The second band w h i c h came o f f much l a t e r c o u l d be t h e mono t e r t i a r y b u t y l e s t e r . The monoformyl m o n o v i n y l d e u t e r o p o r p h y r i n f r a c t i o n was t a k e n t o d r y n e s s by r o t a r y e v a p o r a t i o n and c r y s t a l l i z e d from c h l o r o f o r m - p e t r o l e u m e t h e r (30-60°). P u r i t y was checked by a b s o r p t i o n s p e c t r o p h o t o m e t r y i n c h l o r o f o r m . 5) 2 - V i n y l - 4 - f o r m y l d e u t e r o p o r p h y r i n DTBE I s o s p i r o g r a p h i s p o r p h y r i n DTBE T h i s p r o c e d u r e was s i m i l a r t o 2 - F o r m y l - 4 - v i n y l d e u t e r o p o r p h y r i n DTBE e x c e p t t h a t t h e B r i n g r e a c t e d p h o t o p r o t o p o r p h y r i n DTBE (Isomer 1) was used i n s t e a d o f t h e A r i n g r e a c t e d p h o t o p r o t o -p o r p h y r i n DTBE (Isomer 2 ) . 6) 2 - F o r m y l - 4 - v i n y l d e u t e r o p o r p h y r i n f r e e a c i d T w e n t y - f i v e mg o f t h e 2 - f o r m y l - 4 - v i n y l d e u t e r o p o r p h y r i n DTBE was d i s s o l v e d i n 10 ml o f d r y d i c h l o r o m e t h a n e c o n t a i n i n g 10% t r i f l u o r o a c e t i c a c i d . The s o l u t i o n was r e f l u x e d f o r 4 hours i n an i n e r t atmosphere ( n i t r o g e n ) . The p r o g r e s s o f t h e r e a c t i o n was m o n i t o r e d by two t i c systems: 1) benzene-methanol (110:17) DTBE Rf = 0.7, monoester Rf = 0.5 and f r e e a c i d Rf = 0; and 2) BMF (110:17:1) DTBE R f = 0.8, monoester R f = 0.7 and f r e e a c i d Rf = 0.5. When t h e r e a c t i o n was complete t h e d i c h l o r o -69 methane. The p o r p h y r i n f r e e a c i d was p r e c i p i t a t e d by adding 1 drop of t r i e t h y l a m i n e t o the s o l u t i o n . The p r e c i p i t a t e was f i l t e r e d , a i r d r i e d , washed with dichloromethane, and d r i e d to constant weight. P u r i t y was checked by t i c a n a l y s i s u s i n g the same two s o l v e n t systems and by a b s o r p t i o n spectrophotometry. 7) 2 - V i n y l - 4 - f o r m y l d e u t e r o p o r p h y r i n f r e e a c i d A: s i m i l a r procedure to the 2-Formyl-4-vinyl d e u t e r o p o r p h y r i n f r e e a c i d except 2 - v i n y l - 4 - f o r m y l d e u t e r o p o r p h y r i n DTBE was used i n s t e a d of 2 - f o r m y l - 4 - v i n y l d e u t e r o p o r p h y r i n DTBE. 8) 2,4-diformyl d e u t e r o p o r p h y r i n DTBE One hundred mg of p r o t o p o r p h y r i n DTBE were d i s s o l v e d i n 10 ml of acetone and heated to r e f l u x . A s o l u t i o n c o n t a i n i n g 200 mg of magnesium s u l f a t e and 100 mg of potassium permanganate i n 50 ml of water was added dropwise to the DTBE s o l u t i o n f o r 4 5 minutes. The r e a c t i o n mixture was co o l e d to room temperature and f i l t e r e d . The f i l t r a t e was poured i n t o 1 1 of water and e x t r a c t e d with c h l o r o f o r m — 1 0 0 ml twich, 50 ml and them 30 ml. The c h l o r o f o r m from each e x t r a c t i o n was combined, d r i e d over sodium s u l f a t e , and taken t o dryness by r o t a r y e v a p o r a t i o n . The r e s i d u e was d i s s o l v e d i n minimal c h l o r o f o r m and chromato-graphed on 20 g of s i l i c a g e l grade IV e q u i l i b r a t e d i n c h l o r o -form: ether (100:1). The column was e l u t e d with chloroform: ether (100:1) u n t i l the f i r s t band (red) came o f f . When a l l 70 the unreacted p r o t o p o r p h y r i n DTBE was o f f the column the e l u t i n g s o l v e n t system was changed to chloroform: ether (50:1). The next r e d band t o come o f f was the monoformyi monovinyl deutero-p o r p h y r i n DTBE mixed isomer f r a c t i o n . T h i s was f o l l o w e d by a mixed f r a c t i o n o f the monovinyl monoformyl de u t e r o p o r p h y r i n DTBE mixed isomers and the d i f o r m y l d e u t e r o p o r p h y r i n DTBE. The l a s t band t o come o f f was the d e s i r e d product d i f o r m y l deuteropor-p h y r i n DTBE. The mixed f r a c t i o n was rechromatographed on 15 g of s i l i c a g e l grade IV wit h ch l o r o f o r m : ether (50:1) as the s o l v e n t . The pure f r a c t i o n s of d i f o r m y l d e u t e r o p o r p h y r i n were combined , taken to dryness and c r y s t a l l i z e d from c h l o r o f o r m -methanol. P u r i t y was checked by m e l t i n g p o i n t a n a l y s i s and a b s o r p t i o n spectrophotometry. D i f o r m y l d e u t e r o p o r p h y r i n f r e e a c i d Ten mg of d i f o r m y l d e u t e r o p o r p h y r i n DTBE were d i s s o l v e d i n 5 ml of dry dichloromethane. Anhydrous HCl gas was bubbled through the s o l u t i o n f o r about 6-10 hours. The progress of the r e a c t i o n was monitored by the same two t i c systems used i n the d e e s t e r i -f i c a t i o n of monoformyl monovinyl d e u t e r o p o r p h y r i n DTBE. A f t e r completion o f the r e a c t i o n the s o l u t i o n was evaporated to dry-ness. The f r e e a c i d r e s i d u e was d i s s o l v e d i n 3 mis of p y r i d i n e . A d d i t i o n o f 5 ml of hot a c e t i c a c i d r e s u l t e d i n the immediate c r y s t a l l i z a t i o n of the d i f o r m y l d e u t e r o p o r p h y r i n . P u r i t y was checked by t i c a n a l y s i s and a b s o r p t i o n spectrophotometry. 71 10) P h o t o p r o t o p o r p h y r i n f r e e a c i d One hundred mg o f p r o t o p o r p h y r i n were d i s s o l v e d i n 100 ml o f p y r i d i n e . The s o l u t i o n was exposed t o l i g h t as d e s c r i b e d f o r p h o t o p r o t o p o r p h y r i n DTBE. The p y r i d i n e was removed by r o t a r y e v a p o r a t i o n . The r e s i d u e was d i s s o l v e d i n 90-100% f o r m i c a c i d and an e q u a l volume o f p y r i d i n e was added. The crude photo-p r o t o p o r p h y r i n was p r e c i p i t a t e d by a d d i n g e t h e r t o t h e s o l u t i o n . The p r e c i p i t a t e was f i l t e r e d and washed. T i c a n a l y s i s u s i n g BMF (110:17:1) was done on t h e crude p r o d u c t . D 5 2 F e P r o d u c t i o n 1) T a r g e t I r r a d i a t i o n s The TRIUMF c y c l o t r o n a t the U n i v e r s i t y o f B r i t i s h Columbia was used t o i r r a d i a t e 0.625 i n d i a m e t e r , 4.5 g n i c k e l t a r g e t s f o r the p r o d u c t i o n o f 5 2 F e . The TRIUMF c y c l o t r o n i s unique because i t s s p e c i a l l y shaped magnet a c c e l e r a t e s a c o n s t a n t stream o f p r o t o n p a r t i c l e s and t h e use o f H~ i o n a l l o w s m u l t i p l e beams o f d i f f e r e n t e n e r g i e s and c u r r e n t s t o be e x t r a c t e d . There a r e t h r e e p o s s i b l e i s o t o p e p r o d u c t i o n f a c i l i t i e s a t TRIUMF ( F i g . 3 ) . The beam l i n e 4A o p e n - a i r m u l t i s a m p l e i r r a d i a t i o n s t a t i o n ( F i g . 4 ) was used t o i r r a d i a t e n i c k e l t a r g e t s f o r low l e v e l - t r a c e r and s a f e t y e v a l u a t i o n s t u d i e s . The i r r a d i a t i o n f a c i l i t y c o n s i s t e d o f t h r e e s t r i n g e r s w h i c h moved t a r g e t s i n and o u t o f an open a i r Future Neutron Therapy Facilities BL4A(W h42M Isotope Production Cyclotron Vjnferim Radioisotope Laboratory -H" ION SOURCE H" POLARIZED ION SOURCE Thermal Neutron Facility •-Neutron Activation Analysis L-IOI/F F i g 3. P o s s i b l e I s o t o p e P r o d u c t i o n S i t e s a t TRIUMF-. Jk 1. DUMP -A i i i . 10 PLATE SEM CHAMBER SL0 SLO 3 STRINGERS x 10 TARGETS EACH SYN SYN F i g . 4. BL4A Multisample i r r a d i a t i o n s t a t i o n . 74 chamber where t h e p r o t o n beam passed t h r o u g h . N i c k e l t a r g e t s were g l u e d onto an aluminium f o i l b a c k i n g s u p p o r t e d i n an aluminium t a r g e t h o l d e r . For h i g h e r l e v e l p r o d u c t i o n o f 5 2 F e a new t a r g e t system u p s t r e a d from the 4A m u l t i s a m p l e i r r a d i a t i o n f a c i l i t y was d e s i g e n d , b u i l t , and i n s t a l l e d ( F i g . 5 ) . The t a r g e t had beam windows on b o t h s i d e s o f t h e t a r g e t h o l d e r t o p r o v i d e complete containment o f t h e t a r g e t . F o r h i g h l e v e l p r o d u c t i o n o f Fe t a r g e t s were i r r a d i a t e d i n the 50 0 MeV i s o t o p e p r o d u c t i o n f a c i l i t y by t h e A p p l i e d Program Group a t TRIUMF ( F i g . 6 ) . 2) S a f e t y E v a l u a t i o n and AECB L i c e n s e A p p l i c a t i o n B e f o r e t a r g e t s c o u l d be i r r a d i a t e d a d e t a i l e d s a f e t y e v a l u a t i o n r e g a r d i n g t y p e s o f r a d i o e l e m e n t s produced, a c t i v i t y l e v e l s , r a d i a t i o n l e v e l s , e t c . had t o be done. A l s o , b e f o r e t a r g e t s c o u l d be c h e m i c a l l y s e p a r a t e d , d e t a i l e d c a l c u l a t i o n showing e x p e c t e d r a d i a t i o n f i e l d s , r a d i a t i o n e x p o s u r e , amount o f l e a d s h i e l d i n g r e q u i r e d , e t c . had t o be done. A l l f a c i l i t i e s and p r o c e d u r e s had t o be approved by t h e TRIUMF S a f e t y Group (TSG), TRIUMF S a f e t y A d v i s o r y Committee (TSAC) and Atomic Energy o f Canada C o n t r o l Board (AECB). The Simon F r a s e r U n i v e r s i t y Dose program and i n i t i a l low l e v e l e x p e r i m e n t s were used f o r t h e s e c a l c u l a t i o n s (304). The Dose 75 F i g 5. BL4A 5 2 F e P r o d u c t i o n F a c i l i t y . 76 77 program c a l c u l a t e d c r o s s s e c t i o n s i n m i l l i b a r n s (mb) f o r the va r i o u s radioelements produced v i a the Rudstam (305) or Si l b e r b e r g - T a s o (306) formulae and determined y i e l d s f o r a given proton c u r r e n t assuming one gram of t a r g e t m a t e r i a l and one uA of beam c u r r e n t . The program allowed one to use experimental c r o s s s e c t i o n values i f they were known i n s t e a d c a l c u l a t i n g them. In a d d i t i o n the f o l l o w i n g were c a l c u a l t e d : the c o n t r i b u t i o n to the gamma dose r a t e i n mrad, source s t r e n g t h i n mCi, the c o n t r i b u t i o n to the beta dose r a t e , the c o n t r i b u t i o n t o the danger parameter, and the c o n t r i b u t i o n to the gamma spectrum. The output c o n t a i n e d the f o l l o w i n g : the t a r g e t date; t o t a l c r o s s s e c t i o n f o r each i s o t o p e ; and f o r each combination of i r r a d i a t i o n and c o o l i n g times the f o l l o w i n g were c a l c u l a t e d : t o t a l number of gammas, t o t a l number of betas, the gamma dose i n mrad/hr a t 1 m, the beta dose r a t e , the danger parameter and gamma spectrum; l i s t of s t a b l e n u c l e i produced and a l i s t of alpha e m i t t e r s . In the i n i t i a l experiments used f o r the s a f e t y e v a l u a t i o n , n i c k e l t a r g e t s were i r r a d i a t e d i n the 4A open-air multisample i r r a d i a t i o n f a c i l i t y . I r r a d i a t e d t a r g e t s were d i s s o l v e d i n concentrated n i t r i c a c i d , evaporated to dryness and r e d i s s o l v e d i n 8N HCl. The 5 2 F e was e x t r a c t e d from the h y d r o c h l o r i c a c i d s o l u t i o n i n t o d i - i s o p r o p y l ether and back e x t r a c t e d i n t o water. A l i q u o t s were taken b e f o r e and a f t e r s o l v e n t e x t r a c t i o n and 78 counted f o r r a d i o a c t i v i t y u s i n g a G e ( L i ) d e t e c t o r as d e s c r i b e d p r e v i o u s l y . Based on t h e e f f i c i e n c y c u r v e o f t h e d e t e c t o r , cpms were c o n v e r t e d t o y C i v a l u e s . The s p e c i f i c gamma r a y c o n s t a n t f o r each r a d i o n u c l i d e was c a l c u l a t e d w i t h no l e a d s h i e l d i n g and v a r i o u s t h i c k n e s s e s o f s h i e l d i n g . (307) Hot C e l l D e s i g n and C o n s t r u c t i o n A h o t c e l l was d e s i g n e d and b u i l t i n t h e Interm R a d i o i s o t o p e L a b o r a t o r y a t TRIUMF t o p e r m i t t h e s a f e h a n d l i n g o f i r r a d i a t e d n i c k e l t a r g e t s and t o a l l o w the 5 2 F e t o be s e p a r a t e d from t h e t a r g e t . (308-310) The h o t c e l l c o n s i s t e d o f a s t a i n l e s s - s t e e l fume hood ( l i n e r ) (30 i n X 60 i n w o r k i n g s u r f a c e ) s u r r ounded by 4 i n c h e s o f l e a d on a l l s i d e s and bottom, and 2 i n c h e s o f l e a d on t o p . Over 1200 2 i n c h X 4.5 i n c h X 16 (22 l b s ) l e a d b r i c k s were used f o r s h i e l d i n g . A 16 i n c h X 16 i n c h X 2 i n c h l e a d g l a s s window ( d e n s i t y 6.2 g/cc) a l l o w e d o b s e r v a t i o n o f t h e fume hood c o n t e n t s . The window a c t u a l l y c o n s i s t e d o f 8 s h i g l e 8 i n c h X 8 i n c h X 2 i n c h p i e c e s o f l e a d g l a s s . The h o t c e l l was equipped w i t h two T r u - M o t i o n M i n i - M a n i p M a s t e r S l a v e m a n i p u l a t o r s (PaR Programmed and Remote Systems C o r p o r a t i o n , S t . P a u l , M i n n ) . Access f o r two 9 i n c h d i a m e t e r l e a d p i g s c o n t a i n i n g t h e i r r a d -i a t e d t a r g e t was p r o v i d e d by two e n t r y / e x i t p o r t s w i t h r a i s e d l i p s on t h e bottom o f t h e h o t c e l l . When t h e hot c e l l was i n use, t h e s e p o r t s were s e a l e d b u t u n s h i e l d e d . Lead p i g s were 79 r a i s e d o r l o w e r e d by two 12 v o l t e l e c t r i c h o i s t s ' mounted on top and o u t s i d e o f t h e fume hood and hot c e l l . R o u t i n e c h e m i c a l equipment was p l a c e d i n o r removed from t h e hot c e l l t h r o u g h t h e l e a d s h i e l d e d "Pass-Thru" on t h e l e f t hand s i d e o f t h e hot c e l l . L a r g e r i t e m s were e i t h e r l o a d e d or removed from the hot c e l l t h r o u g h t h e two bottom e n t r y / e x i t p o r t s o r t h e removable l e a d g l a s s window. 4) S e l e c t i o n o f 5 2 F e P r o c e s s C h e m i s t r y u s i n g 5 9 F e a) Recovery T e s t s A column (15 mm d i a m e t e r X 12 cm) o f Dowex 1-X8 100-200 mesh i o n exchange r e s i n was p r e p a r e d and washed w i t h 20 ml o f 8N HC l . A 25 ml s o l u t i o n c o n t a i n i n g 10 g o f n i c k e l a c e t a t e , 30 drops o f 30% hydrogen p e r o x i d e and 0.1 ml o f 5 9 F e c i t r a t e (0.6 y C i ) was added t o t h e column. The column was washed w i t h 3 bed volumes o f 8N H C l . The 5 9 F e was e l u t e d o f f w i t h 2 X 15 ml f r a c t i o n s o f 0.5N H C l . A l i q u o t s o f t h e s t a r t i n g s o l u t i o n , washing s o l u t i o n , and f i n a l p r o d u c t were counted f o r r a d i o a c t i v i t y . The above p r o c e d u r e was r e p e a t e d u s i n g d i f f e r e n t HCl concen-t r a t i o n s d u r i n g t h e l o a d i n g o f t h e column (0, 2, 4, 8, 10, and 12N H C l ) . The same 2 5 ml s o l u t i o n as above was p r e p a r e d . The s o l u t i o n was e x t r a c t e d w i t h 20 ml o f d i - i s o p r o p y l e ther.- The e t h e r was 8 0 washed w i t h 2 0 ml o f 8N HC1 and t h e i r o n back e x t r a c t e d i n t o 10 ml o f wa t e r . Each f r a c t i o n was counted f o r r a d i o a c t i v i t y . The above experiment was r e p e a t e d u s i n g v a r i o u s HC1 concen-t r a t i o n s (0, 2, 4, 6, 8, 10, 12N HC1). The above ex p e r i m e n t was a l s o r e p e a t e d u s i n g m e t h y l i s o b u t y l ketone i n s t e a d o f d i -i s o p r o p r o p y l e t h e r . R a d i o n u c l i d e I m p u r i t y D e t e r m i n a t i o n An i r r a d i a t e d n i c k e l t a r g e t was d i s s o l v e d i n 30 ml o f h o t c o n c e n t r a t e d n i t r i c a c i d and t h e n i t r i c a c i d was e v a p o r a t e d t o d r y n e s s . The r e s i d u e was r e d i s s o l v e d i n 25 ml o f 8N HC1. A f i v e ml f r a c t i o n was assay e d f o r r a d i o a c t i v i t y . A n o t h e r 5 ml f r a c t i o n was e x t r a c t e d w i t h 5 ml o f d i - i s o p r o p y l e t h e r , washed w i t h 5 ml o f 8N HC1, and back e x t r a c t e d i n t o 5 ml o f w a t e r . Each f r a c t i o n was assay e d f o r r a d i o a c t i v i t y . A n o t h e r 5 ml f r a c t i o n was e x t r a c t e d w i t h m e t h y l i s o b u t y l ketone i n s t e a d o f d i - i s o p r o p y l e t h e r . Another 15 ml f r a c t i o n was passed t h r o u g h a Dowex 1-X8 i o n exchange r e s i n column (15 mm d i a m e t e r X 12 cm) The column was washed w i t h 3 bed volumes o f 8N HC1. The i r o n was e l u t e d o f f w i t h 0.5N HC1 made up t o 15 ml a g a i n w i t h c o n c e n t r a t e d HC1 and water t o make a 8N HC1 s o l u t i o n . F i v e ml of t h e 15 ml s o l u t i o n was counted f o r r a d i o a c t i v i t y . A nother 5 ml f r a c t i o n was e x t r a c t e d w i t h d i - i s o p r o p y l e t h e r and a n o t h e r 5 ml f r a c t i o n w i t h m e t h y l i s o b u t y l ketone as d e s c r i b e d above. Each f r a c t i o n was decayed.and counted f o r r a d i o a c t i v i t y . 81 •5) High L e v e l 5 2 F e P r o d u c t i o n . A b r e a d - b o a r d s e t up c o n t a i n i n g t h e g l a s s w a r e f o r t h e n e c e s s a r y c h e m i c a l o p e r a t i o n s f o r use i n t h e h o t c e l l was d e s i g n e d (Fig.7) The i r r a d i a t e d n i c k e l t a r g e t was d i s s o l v e d i n a m i x t u r e o f 30 ml of c o n c e n t r a t e d n i t r i c a c i d and 30 ml o f c o n c e n t r a t e d HCl w i t h h e a t . A f t e r d i s s o l u t i o n t h e s o l u t i o n was e v a p o r a t e d t o d r y n e s s and r e d i s s o l v e d i n 20 ml o f 8N H C l . Twenty ml o f m e t h y l i s o b u t y l ketone were added HCl t a r g e t s o l u t i o n and mixed. A f t e r phase s e p a r a t i o n , t h e bottom i n o r g a n i c l a y e r was d i s c a r d e d . Ten ml o f d i s t i l l e d w a t e r were added and mixed. The bottom l a y e r c o n t a i n i n g t h e 5 2 F e p r o d u c t was d i s p e n s e d i n t o b o t t l e s . The p r o d u c t was swipe t e s t e d and removed from the h o t c e l l t o a fume hood. I t was h e a t e d t o b o i l i n g t o d r i v e o f f any r e s i d u a l s o l v e n t , c o o l e d and a d j u s t e d t o pH 7-8 w i t h e i t h e r HCl o r NaOH. An a l i q u o t was removed t o be checked f o r p u r i t y u s i n g a G e ( L i ) d e t e c t o r . T o t a l a c t i v i t y was d e t e r m i n e d by a dose c a l i b r a t o r . A f t e r each s e p a r a t i o n o f 5 2 F e the g l a s s w a r e was r i n s e d w i t h d i s t i l l e d w a t e r . E M e t a l l o p o r p h y r i n S y n t h e s i s 1) 5 9Fe-Hematohemin The f e r r o u s s u l f a t e o r c h l o r i d e method was used (244). Ten mg o f h e m a t o p o r p h y r i n were d i s s o l v e d i n a s o l u t i o n c o n t a i n i n g 0.5 ml o f p y r i d i n e , 10 ml o f g l a c i a l a c e t i c a c i d and 10 mg o f F i g q 7 . Fe Production Hot c e l l Breadboard. 83 a s c o r b i c a c i d i n a pear-shaped or long necked f l a s k . N i t r o g e n was bubbled through the s o l u t i o n w i t h the a i d of a gas i n l e t tube. The s o l u t i o n was heated i n an o i l bath t o 80°. The 5 9 F e (100 yCi) as the f e r r i c c h l o r i d e or c i t r a t e was added to the mixture and the temperature r a i s e d to 90°. A f t e r 5 minutes, 1.0 ml of s a t u r a t e d f e r r o u s c h l o r i d e or s u l f a t e was added. A f t e r an a d d i t i o n a l 5 minutes a t 90°, the n i t r o g e n was turned o f f and the s o l u t i o n allowed to c o o l to room temperature. A i r was then passed through the s o l u t i o n f o r 30 seconds. This s o l u t i o n was then added to 10 ml of d i s t i l l e d water and 20 ml of c h l o r o f o r m . A f t e r e x t r a c t i o n the o r g a n i c l a y e r was washed wit h 0.2M HCl and then w i t h d i s t i l l e d water s e v e r a l times. The hemin was e x t r a c t e d out of the o r g a n i c l a y e r i n t o 5 ml of 1.0N NaOH. The s o l u t i o n was ad j u s t e d to pH 7-8 with about 5 ml of 1.ON HCl.. The progress of the r e a c t i o n was checked by paper chromatography with e t h a n o l (Hemin R^=0, 5 9 F e Rf=l) and a b s o r p t i o n spectrophotometry. Each f r a c t i o n was a l s o counted f o r r a d i o a c t i v i t y to determine r e a c t i o n y i e l d s . The f i n a l product was checked by a b s o r p t i o n spectrophotometry, paper chromatography as above and t i c u s i n g n - b u t a n o l - w a t e r - a c e t i c a c i d (50:1.5:1.4) (protohemin Rf=0.47, hematohemin R^=0.40) and hexane-n-propanol-acetic a c i d (10:5:1.5) (hematohemin R^=0.13 protohemin R f=0.28) and polyamide chromatography u s i n g methanol-a c e t i c a c i d (100:2)(hematohemin R f=0.69, protohemin R^-0.34). 84 2) 5 9 F e - P r o t o h e m i n T h i s p r o c e d u r e was s i m i l a r t o hematohemin e x c e p t t h a t p r o t o -p o r p h y r i n was used i n s t e a d o f h e m a t o p o r p h y r i n . The p r o d u c t was t h e n c r y s t a l l i z e d from c h l o r o f o r m / m e t h a n o l . The same q u a l i t y c o n t r o l p r o c e d u r e s were done on protohemin as hemato-hemin (see above) '. 3) 5 9 F e - P h o t o p r o t o h e m i n S e v e n t y - f i v e mg o f p h o t o p r o t o p o r p h y r i n were d i s s o l v e d i n 10 ml o f DMF. The s o l u t i o n was heated t o 100° under a n i t r o g e n atmosphere. Fe i n the form o f f e r r i c c h l o r i d e o r c i t r a t e (100 uCi) was added. A f t e r 15 minutes o f c o n s t a n t s t i r r i n g , 20 gm o f f e r r i c c h l o r i d e were added. A f t e r an a d d i t i o n a l 30 minutes o f h e a t i n g and s t i r r i n g t h e s o l u t i o n was c o o l e d t o room temp e r a t u r e and t h e DMF removed by r o t a r y e v a p o r a t i o n . The hemin r e s i d u e was d i s s o l v e d i n m i n i m a l p y r i d i n e and an e q u a l amount o f 90-100% f o r m i c a c i d was added. The hemin was p r e c i p i t -a t e d by t h e a d d i t i o n o f e t h e r . The p r e c i p i t a t e was f i l t e r e d and washed w i t h e t h e r , a i r d r i e d , washed w i t h w a t e r , a i r d r i e d , washed w i t h e t h e r a g a i n , and then d r i e d t o c o n s t a n t w e i g h t . The c r u d e photohemin was p u r i f i e d by b o t h t h i c k l a y e r and t h i n l a y e r chromatography. F i f t y mg o f c r u d e photohemin were d i s s o l v e d i n m i n i m a l p y r i d i n e c o n t a i n i n g 5% o f 90-100% f o r m i c a c i d . T h i s s o l u t i o n was a p p l i e d t o a 30 cm X 30 cm t h i c k s i l i c a g e l p l a t e . 85 The p l a t e was d e v e l o p e d on BMF (110:30:1). The green photo-p r o t o h e m i n band (m i d d l e band) was s c r a p e d o f f , p u l v e r i z e d , and the hemin e l u t e d w i t h methanol. The s o l u t i o n was reduced t o 2 ml and 2 5 ml o f p e t r o l e u m e t h e r were added. The hemin was p r e c i p i t a t e d , f i l t e r e d , and washed as f o r t h e crude photo-protohemin as above. I t was a g a i n p u r i f i e d on s i l i c a t i c p l a t e as above e x c e p t a f t e r chromatography, t h e methanol s o l u t i o n was t a k e n t o d r y n e s s . The p r u i f i e d p hotoprotohemin was d i s -s o l v e d i n 5 mis o f 1.0N NaOH and t h e pH a d j u s t e d t o pH 7-8 w i t h about 5 mis o f 1.0N H C l . The p r o g r e s s o f t h e r e a c t i o n and p u r i f i c a t i o n , and p u r i t y o f the f i n a l p r o d u c t was m o n i t o r e d by paper chromatography w i t h e t h a n o l , a b s o r p t i o n s p e c t r o p h o t o -metry, t i c u s i n g BMF (110:30:1) and polyamide t i c u s i n g BMF and m e t h a n o l - a c e t i c a c i d (100:2). 4) 5 9 F e - 2 - f o r m y l - 4 - v i n y l deuterohemin T h i r t y mg o f 2 - f o r m y l - 4 - v i n y l d e t e r o p o r p h y r i n were added t o 10 ml o f DMF a t r e l u x under n i t r o g e n . 5 5 9 F e as the f e r r i c c h l o r i d e o r c i t r a t e was added f o l l o w e d by 15 mg o f f e r r i c c h l o r i d e 15 minutes l a t e r . The p r o g r e s s o f the r e a c t i o n was f o l l o w e d by paper chromatography as p r e v i o u s l y d e s c r i b e d and a b s o r p t i o n s p e c t r o p h o t o m e t r y . When the r e a c t i o n was complete (45-47min) t h e m i x t u r e was t a k e n t o dr y n e s s by r o t a r y e v a p o r a -t i o n . The crude hemin was p r e c i p i t a t e d by e t h e r , washed and p u r i f i e d by t h i c k l a y e r s i l i c a g e l chromatography as p r e v i o u s l y 86 d e s c r i b e d f o r 5 9 F e - P h o t o p r o t o h e m i n . The p u r i f i e d hemin was e x t r a c t e d from the s i l i c a g e l by BMF (110:30:1). The s o l v e n t m i x t u r e was s l o w l y removed by r o t a r y e v a p o r a t i o n u n t i l the p r o d u c t p r e c i p i t a t e d . The p r e c i p i t a t e was washed w i t h benzene and a i r d r i e d . The p u r i f i e d hemin was c r y s t a l l i z e d by d i s s o l v i n g i t i n 1.5 ml o f p y r i d i n e and 3 ml o f c h l o r o f o r m added. The s o l u t i o n was f i l t e r e d t h r o u g h g l a s s wool and t h e wool washed w i t h two 1.35 ml a l i q u o t s o f p y r i d i n e -c h l o r o f o r m (35:100). The f i l t r a t e and washings were combined, h e a t e d t o b o i l i n g and 25 ml o f b o i l i n g a c e t i c a c i d were added. Then 0.3 5 ml o f c o n c e n t r a t e d HC1 were added and t h e m i x t u r e h e a t e d t o b o i l i n g a g a i n . The s o l u t i o n was a l l o w e d t o s t a n d f o r 24-48 hours t o complete c r y s t a l l i z a t i o n . The hemin was r e -c o v e r e d by s u c t i o n f i l t r a t i o n . The 2 - f o r m y l - 4 - v i n y l d e u t e r o -hemin was d i s s o l v e d i n 5 ml o f 1.0N NaOH and the p H ' a d j u s t e d t o pH 7-8 w i t h about 5 ml o f 1.0N HC1. The p u r i t y was checked by a b s o r p t i o n s e p c t r o p h o t o m e t r y and s i l i c a g e l and p o l y m i d e t i c as d e s c r i b e d p r e v i o u s l y f o r 5 9 F e - p h o t o p r o t o h e m i n . 5) 5 9 F e - 2 - v i n y l - 4 - f o r m y l deuterohemin T h i s p r o c e d u r e was s i m i l a r t o 2 - f o r m y l - 4 - v i n y l deuterohemin e x c e p t t h a t 2 - v i n y l - 4 - f o r m y l d e u t e r o p o r p h y r i n was used i n s t e a d o f 2 - f o r m y l - 4 - v i n y l d e u t e r o p o r p h y r i n . 87 6) 5 9 F e - 2 , 4 - d i f o r m y l deuterohemin T h i r t y mg of d i f o r m y l d e u t e r o p o r p h y r i n were metalated u s i n g the f e r r i c c h l o r i d e method ( r e f l u x i n g i n DMF) as p r e v i o u s l y d e s c r i b e d . The r e s u l t i n g r e s i d u e was d i s s o l v e d i n minimal methanol. The a d d i t i o n . o f water caused the crude product to p r e c i p i t a t e . The p r e c i p i t a t e was a i r d r i e d , washed wi t h ether, and a i r d r i e d a g a i n . The crude hemin was d i s s o l v e d i n 5 ml of p y r i d i n e c o n t a i n i n g 10% a c e t i c a c i d . T h i s s o l u t i o n was s t r e a k e d out on a t h i c k l a y e r s i l i c a g e l p l a t e and d r i e d . The preloaded s i l i c a g e l was scraped o f f , p u l v e r i z e d , and suspended i n BMF (110:30:1). The s l u r r y was poured onto a 25 g polyamide column e q u i l i b r a t e d w i t h the same s o l v e n t system. The column was e l u t e d w i t h BMF (110:30:1) u n t i l the minor band was e l u t e d . The hemin eas e l u t e d o f f the column wi t h BMF (110:70:1). The band was taken to dryness and c r y s t a l l i z e d and prepared as d e s c r i b e d f o r 2-formy-4-vinyl deuterohemin. 7) 5 9Fe-meso-tetra(4-carboxyphenyl) hemin (TCP) Ten mg of meso-tetra(4-carboxyphenyl) porphine were d i s s o l v e d i n 10 ml of g l a c i a l a c e t i c a c i d c o n t a i n i n g 10 mg a s c o r b i c a c i d and 10 mg of sodium a c e t a t e . The s o l u t i o n was heated to r e f l u x under n i t r o g e n . 5 9 F e i n the form of f e r r i c c h l o r i d e was added and r e f l u x e d f o r 30 minutes. Then, 1 ml of s a t u r a t e d f e r r o u s c h l o r i d e s o l u t i o n was added and r e f l u x e d f o r an a d d i t i o n a l 30 minutes. The n i t r o g e n was turned o f f and the s o l u t i o n c o o l e d to 88 room temperature. A i r was passed through the s o l u t i o n f o r 1-2 minutes. F i n a l l y , the s o l u t i o n was ad j u s t e d to pH 7-8 w i t h sodium b i c a r b o n a t e . The % tag was determined u s i n g paper chroma-tography as d e s c r i b e d p r e v i o u s l y . 8) 5 9 F e - m e s o - t e t r a ( 4 - N - m e t h y l p y r i d y l ) hemin t e t r a i o d i d e T h i s procedure was s i m i l a r to 5 9Fe-meso-tetra(4-carboxyphenyl) hemin except t h a t meso-tetra(4-N-methylpyridyl) porphine t e t r a -i o d i d e was used i n s t e a d o f meso-tetra(4-carboxyphenyl) porphine. 9) 5 9 F e - t e t r a - N a - m e s o - t e t r a ( 4 - s u l f o n a t o p h e n y l ) hemin (TPPS) This procedure was s i m i l a r to 5 9Fe-meso-tetra(4-carboxyphenyl) hemin except t h a t tetra-Na-meso-tetra(4-sulfonatophenyl) porphine (12 hydrate) was used i n s t e a d o f meso-tetra(4-carboxyphenyl) porphine. 10) N i c k e l s p a l l a t i o n product l a b e l e d meso-tetra(4-N-methyl-pyridyl) p o r p h y r i n t e t r a i o d i d e  The f e r r i c c h l o r i d e method ( r e f l u x i n g i n DMF) was used as d e s c r i b e d p r e v i o u s l y except n i c k e l s p a l l a t i o n HC1 s o l u t i o n a f t e r n i t r i c e v a p o r a t i o n was used i n s t e a d o f 5 9 F e . A f t e r l e a b e l l i n g , the DMF s o l u t i o n was evaporated to dryness and resuspended i n water or 1.0N NaOH. The pH was a d j u s t e d to pH 7-8 u s i n g e i t h e r HC1 or NaOH. 89 11) 5 2Fe-Protohemin-This procedure was s i m i l a r to 5 9Fe-protohemin except t h a t 5 2 F e f e r r i c c h l o r i d e was used i n s t e a d of 5 9 F e f e r r i c c h l o r i d e . 12) 5 2Fe-meso-tetra(4-carboxyphenyl) hemin This procedure was s i m i l a r to 5 9Fe-meso-tetra(4-carboxyphenyl) hemin except t h a t 5 2 F e f e r r i c c h l o r i d e was used i n s t e a d of 5 9 F e f e r r i c c h l o r i d e . A simpl e r procedure was a l s o used where the s o l u t i o n was heated to b o i l i n g not under n i t r o g e n and the a s c o r b i c a c i d and f e r r o u s c h l o r i d e were not added. 1 3 ) 5 2 F e - m e s o - t e t r a ( 4 - N - m e t h y l p y r i d y l ) hemin t e t r a i o d i d e T h i s procedure was s i m i l a r to 5 2Fe-meso-tetra(4-carboxyphenyl) hemin except meso-tetra(4-N-methylpyridyl) porphine t e t r a i o d i d e was used i n s t e a d o f meso-tetra(4-carboxyphenyl) porphine. :• F. Tumor T i s s u e C u l t u r e ! Uptake Studi e s P815 tumor c e l l s were grown f o r s e v e r a l days b e f o r e use i n RMPI 1640 t i s s u e c u l t u r e medium. A 1/10 d i l u t i o n o f c e l l s i n RMPI 1640 t i s s u e c u l t u r e medium was done b e f o r e uptake s t u d i e s were done. F i v e ml of t h i s s o l u t i o n were dispensed i n t o 60 mm x 15 mm t i s s u e c u l t u r e d i s h e s . A f t e r 1 hour o f i n c u b a t i o n , 0.5 ml of 5 9 F e l a b e l e d hemin ( s p e c i f i c a c t i v i t y 10 yCi/mg hemin; hemin c o n c e n t r a t i o n 0.5mg hemin/dish) or r a d i o n u c l i d e were added t o a l l but two p e t r i 90 d i s h e s . A 0.5 ml sample o f c e l l s u s p e n s i o n a t t h e s t a r t and end of the e x p e r i m e n t was t a k e n and counted f o r a c t i v i t y . A t s e l e c t e d time i n t e r v a l s (0, 1, 3, 6, 12, 24, 48, 72 hours) a 0.5 ml sample was t a k e n from each d i s h and f i l t e r e d t h r o u g h a m i l l i p o r e f i l t e r . The c e l l s were washed w i t h 5 ml o f PBS and counted f o r r a d i o -a c t i v i t y . A l s o a t s e l e c t e d t ime i n t e r v a l s as s t a t e d above, a 0.5 ml sample was t a k e n from t h e two n o n - r a d i o a c t i v e t i s s u e c u l t u r e d i s h e s and counted f o r c e l l number u s i n g a c o u l t e r c o u n t e r . A l l r e s u l t s o f uptake were c o r r e c t e d f o r v a r y i n g amounts o f r a d i o -a c t i v i t y and c e l l numbers d u r i n g t h e c o u r s e o f u p t a k e . Each time p o i n t p e r hemin o r r a d i o n u c l i d e was done i n d u p l i c a t e and t h e whole experiment was r e p e a t e d f i v e t i m e s . A l l hemins and r a d i o n u c l i d e s were t e s t e d t o g e t h e r per experiment t o r u l e o ut any d i f f e r e n c e s between i n d i v i d u a l hemins o r r a d i o n u c l i d e s . G A n i m a l S t u d i e s 1) D i s t r i b u t i o n S t u d i e s Male CDI Swiss mice w e i g h i n g 30-40 g were i n j e c t e d IV ( t a i l v e i n ) e i t h e r w i t h 5 9 F e C l 3 o r 5 9Fe-TMPI a t l e s s than 0.1 mg p o r p h y r i n / a n i m a l i n 0.1 ml'. A t s e l e c t e d t i m e i n t e r v a l s (0, 0.5, 1, 3, 6, 12, 24, and 48 hours) a f t e r i n j e c t i o n , a n i m a l s were p l a c e d i n e t h e r u n t i l comatose and d e c a p i t a t e d . The f o l l o w i n g organs were counted f o r r a d i o a c t i v i t y : b l o o d , l i v e r , s p l e e n , k i d n e y , bone, eyes and l u n g . A t l e a s t f i v e a n i m a l s 91 were used per time p o i n t . A n i m a l s were g i v e n f o o d and w a t e r ad l i b i u m . 2) . S c i n t i g r a p h y 5 2Fe-TMPI a t a dose o f 10 mg p o r p h y r i n / a n i m a l i n l e s s t h a n 0.5 ml was i n j e c t e d LV (ear v e i n ) i n t o normal New Zealand male w h i t e r a b b i t s w e i g h i n g 2-2.5 k g . A n i m a l s were scanned s e v e r a l hours a f t e r i n j e c t i o n as p r e v i o u s l y d e s c r i b e d . Tumor b e a r i n g r a t s w i t h b r e a s t c a r c i n o m a on neck o r s i d e r e g i o n s and some w i t h p r o s t a t e c a r c i n o m a were i n j e c t e d w i t h 5 2 F e - c h l o r i d e ( I I I ) , 5 2 F e - p r o t o h e m i n , 5 2Fe-TMPI o r 5 2Fe-TCP (IV t a i l v e i n <5 mg p o r p h y r i n / a n i m a l , <0.5 m l ) . The a n i m a l s were scanned t h r e e hours and 24 hours a f t e r i n j e c t i o n . 3) E x c r e t i o n S t u d i e s Male CDI Swiss mice w e i g h i n g 25-25 g were i n j e c t e d IV ( t a i l v e i n ) w i t h 0.1 mg p o r p h y r i n / a n i m a l i n 0.1 ml. A t s e l e c t e d time i n t e r v a l s (.1, 2, 3, 6, 7, 8, 9, 10, 2 0 days) a f t e r i n j e c t i o n a n i m a l s were cou n t e d f o r r a d i o a c t i v i t y as p r e v i o u s l y d e s c r i b e d , (whole body c o u n t e r ) . 92 IV RESULTS AND DISCUSSION A. P o r p h y r i n S y n t h e s i s 1) Hematoporphyrin The a b s o r p t i o n spectrum i n p y r i d i n e i n d i c a t e d t h a t the hematoporphyrin was pure. A b s o r p t i o n Spectrum: P y r i d i n e , . . . 402 500 - 570 596 J Amax(obs) 402 499.5 532 569.2 596 623 Amax(lit) T h i n l a y e r chromatography showed t h a t a very s m a l l amount of p r o t o p o r p h y r i n was prese n t which chromatographed f a r ahead of the hematoporphyrin. A spot between hematoporphyrin and p r o t o p o r p h y r i n which was monohydroxyethyl monovinyl d e u t e r o p o r p h y r i n was a l s o p r e s e n t . Note: A l l l i t e r a t u r e v a l v e s f o r a b s o r p t i o n s p e c t r o -photometry and m e l t i n g p o i n t s here from r e f e r -ences 240 or 241. 2) P r o t o p o r p h y r i n The y i e l d o f the r e a c t i o n was 80-90%. T h i s c o u l d have been improved i f a b e t t e r method was a v a i l a b l e to scrape o f f a l l the p r e c i p i t a t e and not l o s e i t because of i t s s o l u b i l i t y i n eth e r . The s a t u r a t e d p r o t o p o r p h y r i n ether s o l u t i o n was saved and used when the r e a c t i o n was repeated. 93 The v i s i b l e a b s o r p t i o n spectrum i n p y r i d i n e and t i c a n a l y s i s i n d i c a t e d t h a t the p r o t o p o r p h y r i n was not contaminated w i t h hematoporphyrin. A b s o r p t i o n Spectrum: P y r i d i n e , , . . 409 506 541 576 - 631 2 xmax(obs) ...... 409 506 541 576 605 631 Xmax(lit) 3) P r o t o p o r p h y r i n DTBE A l l t h r e e a n a l y t i c a l techniques i n d i c a t e d t h a t the p r o t o -p o r p h y r i n DTBE was pure. A b s o r p t i o n Spectrum: Chloroform , , . 406 506 541 576 - 631 Amax(obs) 406.5 506 540.5 575.5 603 630 Xmax(lit) M e l t i n g P o i n t : Obs 229-230° L i t 228-229° (dec) T h i n l a y e r chromatography i n c h l o r o f o r m ( p r o t o p o r p h y r i n R f = 0 , p r o t o p o r p h y r i n DTBE R^=l) showed no d e t e c t a b l e amounts of p r o t o p o r p h y r i n f r e e a c i d . I f the s i l i c a g e l column was overloaded or too s m a l l , the brown band tended to run i n t o the p r o t o p o r p h y r i n DTBE band and a c l e a n s e p a r a t i o n c o u l d not be o b t a i n e d . The r e s u l t 94 / o f t h i s was t h a t t h e y e i l d was much h i g h e r t h a n t h e t r u e a c t u a l y i e l d . The y i e l d o f t h e r e a c t i o n was 65%. 4) P h o t o p r o t o p o r p h y r i n DTBE Isomer 1 (A r i n g r e a c t e d ) The y i e l d o f t h e r e a c t i o n was 18%. The isomer was pure as d e t e r m i n e d by a b s o r p t i o n s p e c t r o p h o t o m e t r y and m e l t i n g p o i n t a n a l y s i s . A b s o r p t i o n Spectrum: C h l o r o f o r m , , . . 387 436 500 565 613 671 Xmax(obs) X m a x ( l i t ) M e l t i n g P o i n t : Obs 226-228 L i t 227-229 436 500 565 613 671 5) P h o t o p r o t o p o r p h y r i n DTBE Isomer 2 (B r i n g r e a c t e d ) The y i e l d o f t h e r e a c t i o n was 2 0%. The compound was pure, A b s o r p t i o n Spectrum: C h l o r o f o r m , , . 422 - 567 609 669 Xmax(obs) 422 500 565 608 668 X m a x ( l i t ) M e l t i n g P o i n t : Obs 226-228° L i t 226-228° 95 6) 2-Formyl-4-vinyl d e u t e r o p o r p h y r i n DTBE The y i e l d o f the r e a c t i o n was 55%. A b s o r p t i o n s p e c t r o -photometry i n d i c a t e d t h a t the compound was pure. A b s o r p t i o n Spectrum: Chloroform . , . . 420 519 558 583 641 Xmax(obs) . , , 420 518.5 559 584 642 Xmax(obs) 7) 2 - V i n y l - 4 - f o r m y l d e u t e r o p o r p h y r i n DTBE The y i e l d was 60% and the p o r p h y r i n was pure. A b s o r p t i o n Spectrum: Chloroform , , . , 420 519 558 583 .641 Xmax(obs) Xmax(lit) 420 518.5 559 584 642 8) 2-Formyl-4-vinyl d e u t e r o p o r p h y r i n f r e e a c i d T h i n l a y e r chromatography a n a l y s i s i n d i c a t e d t h a t o n l y the f r e e a c i d was prese n t and none of the either. S p e c t r o -photometry showed t h a t the product d i d not change or was not contaminated. The r e a c t i o n y i e l d was 98%. A b s o r p t i o n Spectrum: Chloroform , , , . 420 519 558 583 641 Xmax(obs) Xmax(lit) 420 518.5 559 584 642 96 9) 2 - V i n y l - 4 - f o r m y l d e u r e r o p o r p h y r i n f r e e a c i d No e s t e r s were found i n the f r e e a c i d product by t i c a n a l y s i s , The r e a c t i o n y i e l d was 85%. Ab s o r p t i o n Spectrum: Chloroform , , , . 420 519 558 583 641 Xmax(obs) 420 518.5 559 584 642 Xmax(lit) 10) 2,4-Diformyl d e u t e r o p o r p h y r i n DTBE The r e a c t i o n y i e l d was 10%. The product was pure by a b s o r p t i o n spectrophotometry and m e l t i n g p o i n t a n a l y s i s . A b s o r p t i o n Spectrum: Chloroform , . . . 437 527 563 595 650 Xmax(obs) Xmax(lit) 436 526 562 595 650.5 M e l t i n g Point: Obs 227-228° L i t 228-229° (dec) 97 11) 2,4-Diformyl d e u t e r o p o r p h y r i n f r e e a c i d Thin l a y e r chromatography a n a l y s i s of the product showed t h a t i t was f r e e of the monoester and d i e s t e r . The r e a c t i o n y i e l d was 98%. The a b s o r p t i o n spectrum was normal. A b s o r p t i o n Spectrum: P y r i d i n e , , , . 436 525 562 594 649 Xmax(obs) 4 3 6 5 2 5 561 593.5 649 Xmax(lit) 12) P hotoprotoporphyrin f r e e a c i d (mixed isomers) T h i n l a y e r chromatography a n a l y s i s showed t h a t the crude photoprotoporphyrin c o n t a i n e d about 5% of unreacted pro-t o p o r p h y r i n and other minor i m p u r i t i e s . B. 5 2 F e P r o d u c t i o n 1) Target I r r a d i a t i o n s The o p e n - a i r multisample i r r a d i a t i o n f a c i l i t y i n beam l i n e 4A was l i m i t e d to beam c u r r e n t s l e s s than 250 nA and t o t a l a c t i v i t y / t a r g e t t o s e v e r a l hundred m i c r o c u r i e s . There was no containment of r a d i o a c t i v e m a t e r i a l i f a t a r g e t was to de-l e t e or explode. Open-air o p e r a t i o n a l s o h i g h l y a c t i v a t e d surrounding a i r i n the beam l i n e t u n n e l . The removal of a h i g h l y r a d i o a c t i v e t a r g e t from the f a c i l i t y had to be done by hand and p l a c e d i n t o a l e a d t r a n s p o r t p i g . I t was _ d i f f i c u l t to s h i e l d the above procedure and reduce r a d i a t i o n 98 exposure t o the o p e r a t o r . The aluminium backing o f the t a r g e t h o l d e r was a l s o very r a d i o a c t i v e and g r e a t l y i n -creased the r a d i a t i o n f i e l d . The 5 2 F e i r r a d i a t i o n f a c i l i t y reduced the h a n d l i n g o f the i r r a d i a t e d t a r g e t , e l i m i n a t e d the a i r a c t i v a t i o n , and allowed higher beam c u r r e n t s to be run. The use of beam l i n e 4A had s e v e r a l problems. I t was v i r t u a l l y i m p o s s i b l e to run p a r a s i t i c to another experiment. Other t a r g e t s up stream from the i r r a d i a t i o n f a c i l i t i e s caused the beam spot s i z e to be i n c r e a s e d a t the i r r a d i a -t i o n f a c i l i t i e s . T h i s r e s u l t e d i n running o n l y 100-200 nA i n order not to t r i p the beam. Another problem w i t h running p a r a s i t i c was t h a t e n t r y i n t o the beam l i n e to lo a d or remove an i r r a d i a t e d t a r g e t was c o n t r o l l e d by the prime user o f the beam l i n e . Many times, access was delayed f o r many hours making i t i m p o s s i b l e t o set up any type of schedule of when the f i n a l product would be ready. General r a d i a t i o n f i e l d s i n the beam l i n e area ranged from 50 mr/h to over 3 R/h near the i r r a d i a t i o n f a c i l i t i e s . The use of the 500 MeV i r r a d i a t i o n f a c i l i t y e l i m i n a t e d the above problems. Although the f a c i l i t y was running p a r a s i t i c i t was no problem to i r r a d i a t e t a r g e t s . The t r a n s p o r t p i g 99 used f o r the 5 2 F e i r r a d i a t i o n f a c i l i t y d i d not f i t i n t o the 500 MeV hot c e l l . T h e r e f o r e a t a r g e t t r a n s f e r had to be done i n the AECL hot c e l l . 2) S a f e t y E v a l u a t i o n The c r o s s s e c t i o n s f o r 500 MeV proton s p a l l a t i o n c a l c u l a t e d by the Dose program showed t h a t the c r o s s s e c t i o n f o r 5 2 F e p r o d u c t i o n was low compared to the other major contaminants (Table I I I ) . A l s o many r a d i o n u c l i d e s were produced ranging from Z=17 to Z=28 and A=34 to A=61. Changing the proton energy from 200 to 500 MeV d i d not change the c r o s s s e c t i o n v a l u e s . P r o d u c t i o n r a t e s f o r v a r i o u s r a d i o n u c l i d e s f o r a 1 g N i t a r g e t were c a l c u l a t e d f o r v a r i o u s i r r a d i a t i o n times (Table I V ) . The s h o r t e r the i r r a d i a t i o n time the lower the a c t i v i t y o f the contaminants i n c l u d i n g 5 5 F e and 5 9 F e . The one hour i r r a d i a t i o n time was i m p r a c t i c a l because not enough beam c u r r e n t was a v a i l a b l e on beam l i n e 4A. I t would be p o s s i b l e on the 500 MeV i r r a d i a t i o n f a c i l i t y p r o v i d e d the t a r g e t s i z e was i n c r e a s e d from 0.625 i n to 4 i n . i n diameter. T h i s was not p r a c t i c a l because the i n c r e a s e d t a r g e t s i z e would not f i t i n t o the r e a c t i o n v e s s e l and l a r g e volumes of a c i d s would be r e q u i r e d to d i s s o l v e the t a r g e t . Based on the r a d i o n u c l i d e a c t i v i t y (Table V) and gamma dose r a t e at 1 m (Table VI) both v a l u e s c o u l d be reduced by one-half i f a 1 h decay p e r i o d f o l l o w e d 12 h of i r r a d i a t i o n . R o u t i n e l y 100 TABLE III DOSE PROGRAM CALCULATED CROSS SECTIONS Radionuclide X-section (mb) Radionuclide 6 1 C 0 6 0Co 3 7 . 1 5 2 F e 3 9 . 3 5 1 Cr 5 9 F e 0 . 6 8 1 5 1 Mn 5 S C o 4 3 . 0 5 0 y 5 7Mn 0.421 ^So 3 8 . 1 6 2 . 4 ^Cr 5 6Mn 1.34 * C o 3 4 . 1 4 8 v 2 5 . 2 for 0 . 1 6 8 i | 7 T i 6 0 . 8 ^ 6Sc 1 8 . 2 4 6 ^ 5 V 2 4 . 2 4 5 T i ^Co 1.73 ^ S e 5 5Mn 4 6 . 5 5 3 F e 8 . 5 6 ^ 2Sc 5 2Mn 2 3 . 9 ^ C l X-section (mb) 2.35 27.8 8.52 7.89 0.0174 19.7 6.7 0.0842 15.9 2.0 6.54 1.43 1 .23 5 . 0 4 5.57 2.84 5.76 0.573 101 TABLE IV DOSE PROGRAM PRODUCTION RATES FOR DIFFERENT IRRADIATION TIMES Irr a d i a t i o n time (hr) Cooling time (hr) Radionuclide 61 60 59 58 57 Co Co Fe Co 56 56 55 Co 5 6Mn Co Ni Fe ?5co 5 2Mn 51 cr 5 1 Mn ^ S c <*9V ^Cr 4 8 v ^ 8 C r 44 43 Sc 0.674 0.0371 0 . 0 0 0 2 4 0 . 8 1 8 0 . 0 0 7 3 5 2 . 0 4 0 . 4 3 2 0 . 0 2 2 2 0.206 0 . 0 0 3 1 7 1 .83 0.141 2 . 9 3 0 . 3 0 8 0 . 0 6 1 8 3 . 6 6 0 . 0 0 7 5 5 0 . 0 0 3 8 2 . 8 4 0.00227 0 . 0 4 9 0 . 1 0 2 0 . 7 1 9 0.711 12 0 A c t i v i t y (mCi) 2 . 9 9 2 . 0 2 0 . 0 0 3 7 . 2 0 0 . 0 9 9 2 2 . 3 2 . 3 0.271 2.41 0 . 0 4 1 1.86 15 .37 2 4 . 5 6 2 . 6 5 0 . 7 7 3 15.21 0 . 0 3 0 3 0 . 0 4 7 5 12 .2 0 . 0 2 5 0 . 5 9 7 1.07 4 . 8 3 4 .61 24 1 1.96 0 . 0 3 8 4 0 . 0 0 5 7 9 . 4 5 0 . 2 2 8 3 9 . 3 1 .83 0 . 5 5 5 4 . 6 6 0 . 0 8 7 2.91 0 . 1 1 5 8 . 7 7 3 . 3 3 1.56 6 . 1 4 0 . 0 1 4 6 0 . 0 9 6 8 4 . 5 2 0 . 0 4 3 8 1 .20 5 . 2 0 4 . 3 2 102 TABLE V DOSE PROGRAM ACTIVITIES FOR VARIOUS COOLING TIMES Irra d i a t i o n Time 12 hrs Cooling Time (hrs) Radionuclide 61 60 59 58, 57 Co Co Fe Co Co 57™ 56 56 56 Ni Mn Co 'Ni 55 55 53 52 Fe Co Fe Mn 5 2 F e 51 Cr 5 1Mn ^ S c 4 9 v 49, 7Cr Sc 48 * 8 C r ^ S c 0 1 12 84 A c t i v i t y (mCi) 2 . 9 9 1.97 0.0191 2 . 0 2 0 . 0 3 8 7 0 . 0 0 0 3 6 0 . 0 0 0 3 6 0 . 0 0 3 0 . 0 0 3 0 . 0 0 2 8 3 0 . 0 0 2 6 4 7 . 2 0 6 . 6 8 2 . 9 8 0 . 1 0 9 0 . 0 9 9 0 . 1 0 2 0 . 1 2 5 0 . 2 1 5 2 2 . 3 2 1 . 9 17.7 0 . 8 7 3 2 . 3 0 1.86 0 . 0 9 1 7 -0.271 0.271 0 . 2 8 0 0 . 3 5 5 2.41 2 . 4 0 2.78 1.08 0 . 0 4 1 0 .041 0 . 0 4 5 8 0 . 5 4 4 18 .6 17 .9 11.7 0 . 0 2 8 8 15.37 0 . 1 1 5 - -2 4 . 6 6 . 6 8 1.34 0 . 6 3 5 2 . 6 5 2 . 4 4 0.971 -0.773 0 . 7 8 4 0.781 0.633 15.21 6 . 1 4 0 . 0 0 0 2 9 -0.0303 0 . 0 1 4 7 - -0 . 0 4 7 5 0 .481 0 . 0 4 8 6 -12 .2 4 . 5 2 - 0 . 0 4 7 8 0 . 0 2 5 0 . 0 2 4 9 0 . 0 2 0 1 0 . 0 0 1 7 6 0 . 5 9 7 0 . 5 9 9 0.603 0 . 4 9 2 1.07 1.04 0 . 7 4 5 0.00670 4 . 8 3 4 . 2 3 1.13 -4.61 3 . 8 6 0.551 -103 TABLE VI GAMMA DOSE RATES (MRAD/HR AT IM) Irra d i a t i o n time (hrs) 1 12 12 Cooling time (hrs) 1 0 1 Gamma dose rate 14.5 131 67 I r r a d i a t i o n time (hrs) 12 12 12 Cooling time (hrs) 3 12 24 Gamma dose rate 50 .6 3 2 . 9 2 4 . 8 I r r a d i a t i o n time (hrs) 12 12 24 Cooling time (hrs) 48 84 1 Gamma dose rate 8 . 2 6 4 . 2 6 9 9 . 8 I r r a d i a t i o n time (hrs) 24 24 Cooling time (hrs) 12 24 Gamma dose rate 57.6 4 4 . 4 104 the t a r g e t was allowed to decay a t l e a s t 1 h i n the beam l i n e b e f o r e being removed. The gamma dose r a t e dropped o f f f a i r l y f a s t w i t h time, thus g r e a t l y r e d u c i n g r a d i a t i o n h e a l t h p h y s i c s problems w i t h waste d i s p o s a l ( F i g . 8). The r a d i o a c t i v e waste was allowed to decay 20-100 days b e f o r e being removed from the hot c e l l . A lthough the dose r a t e decreased very r a p i d l y w i t h time the number of gamma rays per energy d i d not (Fi g . 9 , 10, 11). The computer c a l c u l a t e d gamma spectrum f o r v a r i o u s c o o l i n g times showed t h a t most of the gammas were of high energy and a c t u a l l y t h e i r numbers i n c r e a s e d as the c o o l i n g p e r i o d i n -creased. The number of hig h energy gammas was a problem i n s h i e l d i n g the hot c e l l and t r a n s p o r t f l a s k . Based on i n i t i a l experiments, high energy proton s p a l l a t i o n of n i c k e l produced a l a r g e number of. r a d i o n u c l i d e s from Z = 21 to Z = 28 and A=43 to A=61 (Table VI3) . Most of the r a d i o -n u c l i d e s produced emitted h i g h energy gamma rays from 800 KeV to 1.5 MeV. The Th ranged from 3.7 6 min to 4 8 y r with the average around a few days. The f o l l o w i n g r a d i o n u c l i d e s were always d e t e c t e d kkSc,k8V, 4 8 C r , 5 2 mMn, 5 2Mn, 5 2 F e , 5 5 C o , 5 7 C o , 5 6 N i and 5 7 N i . S p e c i f i c gamma ray constants f o r the r a d i o n u c l i d e s and 105 our =3 2:0. in cc E C O 1 ° . (Um. c o w o IRRflD. 12 HRS ENERGY (MEV) 500 BEAM (MU-RMP) 1.000 WEIGHT (G) 1.000 NICKEL RT 500 MEV 0 1 . COOLING PERIOD IN DRYS ceo Ql/I, 0=0 a: cr cc CEO I o. OCtfl UJ o z CE O S IRRRD. 12 HRS ENERGY (MEV) 500 BERM (MU-RMP) 1.000 NICKEL RT 500 MEV 10-* 6° COOLING PERIOD IN DRYS F i g 8. Dose r a t e vs. c o o l i n g p e r i o d . 106 NICKEL RT 500 MAXIMUM ENERGY 500.00 NEV IRRADIATION 12.00 HRS COOLING 0.000 HRS MEV tn >-C E . CCOj j o CC UJr-C Q O ; 2 3 Lfl In In - 0 . 0 0 1.00 2.00 3.00 U.00 ENERGY IN MEV 5.00 6.00 7.00 F i g 9. Computer c a l c u l a t e d gamma spectrum I 107 tn l i c r « cc UJr-03O, 2 3 f l O H * - 0 . 0 0 NICKEL AT 500 MEV MAXIMUM ENERGY 500.00 MEV IRRADIATION 12.00 HRS COOLING 1.000 HRS —, , W | w w — w - i * »• 1 1.00 2.00 3.00 4.00 S.00 ENERGY IN MEV 6.00 7.00 P i g 10. Computer c a l c u l a t e d gamma spectrum I I 108 553 NICKEL RT 500 MAXIMUM ENERGY 500.00 MEV IRRADIATION 0.50 ORYS COOLING 7.000 ORYS MEV fl tn >-C C o ; 5 3 c r • O • CC U J . o - * — -o.oo 1.00 2.00 * - « r 3.00 ENERGY 4.00 IN MEV 5.00 6.00 ~7.oo F i g 11. Computer c a l c u l a t e d gamma spectrum I I I 109 TABLE VII NI SPALLATION PRODUCTS Radionuclide Decay Mode T i «SC B +, EC 3 . 9 ^ m S c IT, EC 2 . 4 Bt 3 . 9 *Bo B" 3 . 4 ^ 8Sc B" 1.8 ^ T i EC 48 4 8 v B +, EC 16 B" 3 . 8 4 8 C r EC 23 ^ C r B~ 42 5 1 c r EC: 28 B +, IT, EC 21 5 2Mn B +, EC 5 . 7 B +, EC. 8 . 2 5 5 F e EC 2 . 6 5 Q F e B" 4 5 . 1 5 5 C o B +, EC 18 5 6 C o B +, EC. 77 5?Co EC 270 6 1C0 B" 1.7 5 6 N i EC 6.1 5 7 N i B +, EC 36 Gamma Ray Energy (KeV) h 2 2 0 , 370 d 271 h 1157 d 159 d 1312 y 6 6 , 78 d 9 8 3 , 1312 m 1433, 1331 h 3 1 0 , 116 m 90 .7 d 320 m 1437 d 7 4 4 , 8 4 7 , 9 3 8 , 1434 h 165 y MN X-rays d 1292, 1099 h 9 3 0 , 1410 d 8 4 7 , 1238 d 122, 136 h 67 .4 d 163, 812 h 1377, 129 110 m o d i f i e d gamma ray cons t a n t s f o r v a r i o u s t h i c k n e s s e s of le a d s h i e l d i n g were c a l c u l a t e d (Table V I I I ) . Some r a d i o -n u c l i d e s had very high gamma ray constants which were not reduced s i g n i f i c a n t l y by the l e a d s h i e l d i n g due to h i g h energy gamma r a y s . Based on 10-12 hrs i r r a d i a t i o n and 1 hr of c o o l i n g , 30 times more s p a l l a t i o n products than 5 2 F e were produced (-Table IX) . The major products were 5 7 N i (14X) , 5 2 l t W (4X) , ^ S c (3.5X), 5 2Mn : (1.7X) and 5 6 N i (1.2X). To produce one mCi of 5 2 F e (30 mCi s p a l l a t i o n products) the t a r g e t had a r a d i a t i o n f i e l d of 27 mR/h a t 1 m. F i v e cm of l e a d would reduce t h i s to 1.41 mR/h a t lm, 7.5 cm of l e a d t o 0.229 mR/h a t lm, and 10 cm of l e a d to 0.0709 mR/h a t lm (Table I X ) . The m a j o r i t y of r a d i a t i o n came, from 5 7 N i , 5 2 m M n , 5 6 N i , ^ S c , 5 2Mn, 5 6 C o and L f 8 S c . Comparison o f the i n i t i a l experiments to the Dose program showed both c a l c u l a t e d s i m i l a r gamma dose r a t e s but d i f f e r -ent a c t i v i t i e s f o r the d i f f e r e n t r a d i o n u c l i d e s ( T a b l e X). T h i s was due to the d i f f e r e n c e on how the Dose program c a l c u l a t e d c r o s s s e c t i o n s from a c t u a l experimental c r o s s s e c t i o n s and the gamma ray constants used. I l l TABLE VIII MODIFIED GAMMA RAY CONSTANTS Gamma Ray Constants (RAr/Ci) cm Lead Radionuclide 0 5cm 7.5cm 10cm **Sc 0.578 2.60 X IO" 2 4 . 18 X 10-3 6.35 X 1 0 - * * 7Sc 0.0556 0 0 0 * 8Sc 1.67 2 .01 X i o - 2 . 2.67 X 10-3 2 .52 X * 8V 1.56 5.90 X 10"*2 8.90 X 10-3 1.84 X 10-3 i f 8 C r 1.77 5.91 X 1 0 " 2 8.91 X 10-3 1.85 X 10-3 5 2 mMn 0.734 3.27 X 1 0 ~ 2 5.21 X 10-3 8 .10 X IO"* 52Mn 0.799 2.20 X 1 0 ~ 2 9.91 X 1 0 " * 1.07 X IO"* 5 2Fe 0 .818 3 . 2 8 X 1 0 " 2 5.22 X 10-3 8.13 X IO"* 55co 0.586 1.47 X i o - 2 2.32 X 10-3 6.17 X IO"* 5 6 C o 1.76 1.34 X IO" 1 3.76 X 10" 2 1.17 X 1 0 " 2 57 Co 0.09 0 i o - 1 0 0 5 6 N i 2.26 1.55 X 4.24 X i o - 2 1 .29 X 1 0 " 2 5 7 N i 0.83 4.99 X 1 0 " 2 1.10 X 1 0 " 2 2.50 X 10-3 112 TABLE IX EXPECTED RADIATION LEVEL FROM PRODUCTION OF, 1 MCI OF 5 2 F e mR/hr at 1 m cm lead Z A c t i v i t y 0 5cm 7.5cm 10cm 5 2Fe 1 mCi 0 . 8 1 8 3 . 2 8 X 10" 2 5 . 2 2 X 10-3 8 . 1 3 X ^*Sc 3 . 4 9 2 . 0 2 9 . 0 7 X 10" 2 1.46 X 1 0 " 2 2 . 2 8 X 10-3 *?Sc 0 . 1 7 0 0 . 0 0 9 5 0 0 0 * 8Sc 0 . 7 5 0 1 .25 1.51 X 10" 2 2 . 0 0 X 10-3 1 .89 X IO"* 4 8 f * 8 C r 0 . 5 2 4 0 . 8 1 7 3 . 0 9 X 10" •2 4 . 6 6 X 10-3 9 . 6 4 X io-* 0 . 3 3 0 0 . 5 8 4 1.95 X 10" «2 2 . 9 4 X 10-3 6.11 X io-* 5 2 m M n 4.06 2 . 9 8 1.33 X 10" 1 2 . 1 2 X 1 0 " 2 3 . 2 9 X 10-3 5 2Mn 1.74 1.39 3 . 8 3 X 10" •2 1.72 X io-3 1.86 X IO"* 5 5Co 1 .00 0 . 5 8 6 1.47 X 10" 2 2 . 3 2 X 10-3 6 . 17 X IO"* 0 . 7 8 0 1.37 1 .05 X 10" 1 2 . 9 3 X i o - 2 9 . 1 3 X 10-3 5 7Co 0 . 1 3 5 0 . 0 1 2 2 0 1 0 0 5 6 N i 1.24 2 . 8 0 1 .92 X 10" 5 . 2 6 X 1 0 " 2 1 .60 X 1 0 " 2 5 7 N i 14.7 1 2 . 2 7 . 3 4 X 10" 1 1.62 X 1 0 " 1 3 . 6 8 X i o - 2 Sum 3 0 . 1 26 .8 1.41 2 . 2 9 X i o - 1 7 . 0 9 X i o - 2 TABLE X COMPARISON OF DOSE PROGRAM TO INITIAL EXPERIMENTS Radionuclide Dose Program I n i t i a l Experiments A c t i v i t y 5 2 F e 1 mCi 1 mCi **Sc 1 .82 3 . 4 9 * 7Sc - 0.70 * 8Sc 0 . 0 0 9 4 0 . 7 5 0 4 8 V 0 . 2 2 5 0 . 5 2 5 0 . 3 3 0 5 2 mMn 3 . 0 0 4.06 5 2Mn 9 . 2 7 1.74 5 5Co 7 . 0 2 1 .00 5 6 C o 0 . 1 0 2 0.78 5 7Co 0 . 0 3 7 4 0 . 1 3 5 5 6 N i 0 . 1 0 2 1.24 5 7 N i 8 . 4 2 14.7 * 8 C r 0 . 4 0 4 Sum 3 1 . 4 3 0 . 1 Gamma Dose Rate Gamma dose rate 2 5 . 3 (mR/hr at 1 m) 26.8 114 S e l e c t i o n o f 5 2 F e Process Chemistry u s i n g 5 9 F e a) Recovery T e s t s When the 5 9 F e was passed through the column 99.4% of the a c t i v i t y stuck to the r e s i n . Some of the N i a l s o stuck to the r e s i n . No 5 9 F e was d e t e c t e d i n the column washes. The N i zone s l o w l y moved down the column but a l s o broad-ened to s e v e r a l cm. With 0.5 N HC£ e l u t i o n , the N i band s t a r t e d to move down the column w i t h the s o l v e n t f r o n t . A f t e r the major N i band was e l u t e d o f f the column, 7,9% of the a c t i v i t y was i n 1-2 ml of s o l u t i o n . One hundred per cent o f the a c t i v i t y was i n 7.5 ml of s o l u t i o n . Repeating of t h i s procedure a t d i f f e r e n t HC£ con c e n t r a -t i o n s showed t h a t most of the i r o n I I I stuck to the column between 8 M and 12 M HC£ with the peak around 9.5 M ( F i g . 1 2 ) . The d i s t r i b u t i o n c o e f f i c i e n t was 10"*. Removal of the i r o n from the column was p o s s i b l e w i t h any HC£ c o n c e n t r a t i o n l e s s than 1 M of HC£ down to o r d i n a r y d i s t i l l e d water. The d i - i s o p r o p y l ether e x t r a c t i o n e f f i c i e n c y ( o v e r a l l ) was 97.5% wit h 8 N HC£ . The forward e x t r a c t i o n e f f i c i e n c y was 99.4% while the back e x t r a c t i o n e f f i c i e n c y was 98.1%. The maximum d i s t r i b u t i o n c o e f f i c i e n t of 100 o c c u r r e d bet-ween 5.5 and 7 M HC£ c o n c e n t r a t i o n ( F i g . 12). 115 J I I I I L F i g 12. D i s t r i b u t i o n c o e f f i c i e n t s vs HC£ cone f o r v a r i o u s Fe separ a t i o n , methods' 116 \ b) R a d i o n u c l i d e Impurity Determination In g e n e r a l the e x t r a c t i o n method with d i - i s o p r o p y l ether or methyl i s o b u t y l ketone was b e t t e r than the i o n exchange column (Table X I ) . Valves i n the t a b l e were expressed as p u r i f i c a t i o n r a t i o s . A s o l u t i o n of N i s p a l l a t i o n products would have a value of 10,000. A l l r a d i o n u c l i d e s r e g a r d -l e s s o f a c t i v i t y were normalized to t h i s v a l v e . The problem w i t h the column was t h a t other r a d i o n u c l i d e s ( 1 + 6Sc, 5 1 C r , 51tMn, and e s p e c i a l l y 5 6Co) came o f f the column very c l o s e to the 5 2 F e and i t was not always p o s s i b l e to keep them separate d u r i n g e l u t i o n . However i t was p o s s i b l e to e l i m i n a t e a l l the 4 8 V and 5 6 N i by e i t h e r s o l v e n t e x t r a c t i o n or i o n exchange chromatography. A l -though the 5 6 N i e l u t e d very c l o s e to the 5 2 F e i t c o u l d be separated from the 5 2 F e due to i t s i n t e n s e green c o l o r . There was no d i f f e r e n c e i n s e p a r a t i n g t + 6 Sc u s i n g methyl i s o b u t y l ketone or d i - i s o p r o p y l ether e x t r a c t i o n . How-ever both i o n exchange chromatography and s o l v e n t e x t r a c -t i o n were needed to completely e l i m i n a t e a l l the k6Sc i n the f i n a l product. Methyl i s o b u t y l ketone e x t r a c t i o n reduced the 5 1 C r concen-t r a t i o n by a f a c t o r of 2 as compared w i t h d i - i s o p r o p y l ether e x t r a c t i o n . Again both s o l v e n t e x t r a c t i o n and i o n exchange chromatography were r e q u i r e d to completely 117 TABLE XI 52 FE CONTAMINATION TESTS P u r i f i c a t i o n Factors* P u r i f i c a t i o n Method (X 10"*) Radionuclide MIKB DIPE Column 46 48. Sc V 51 Cr 54 Mn 56 56. Co Ni 1 0 . 0 1 0 . 5 135 0 0 0 0 2 . 9 7 5 . 8 3 102 4 . 8 1 4 . 3 5 2 0 . 8 . 3 . 8 4 2 . 2 2 5290 0 0 Column & MIKB 0 2 . 2 4 1 .98 0 Column & DIPE 0 0 0 5.65 2 . 1 8 0 No P u r i f i c a t i o n 1 0 , 0 0 0 (Ni s p a l l a t i o n solution) • P u r i f i c a t i o n F a c t o r — R a t i o of Ni sp a l l a t i o n solution to that of the p u r i f i e d solution of Fe-52. Value of unpurified Ni s p a l l a t i o n solution = 1 0 , 0 0 0 . A l l values: normalized to th i s value. 118 e l i m i n a t e a l l the 5 1 C r i n the f i n a l product. There was no d i f f e r e n c e i n e i t h e r methyl i s o b u t y l ketone or d i -i s o p r o p y l ether e x t r a c t i o n i n the l e v e l of 5 1 +Mn. Column io n exchange chromatography and methyl i s o b u t y l ketone e x t r a c t i o n reduced t h i s l e v e l by a f a c t o r o f two. D i -i s o p r o p y l e t h e r e x t r a c t i o n was b e t t e r than methyl i s o b u t y l ketone i n re d u c i n g the 5 6 C o l e v e l by a f a c t o r of two. Based on t h i s data i t was decided to use s o l v e n t e x t r a c -t i o n w i t h methyl i s o b u t y l ketone f i r s t f o l l o w e d by i o n exchange column chromatography and e l u t e d with water when r e q u i r e d t o p u r i f y the 5 2 F e . The s o l v e n t e x t r a c t i o n step reduced many of the r a d i o n u c l i d e s which overloaded the i o n exchange column. The i o n exchange step allowed the f i n a l product to be conce n t r a t e d i n a very s m a l l volume. High L e v e l 5 2 F e P r o d u c t i o n The t o t a l p r o c e s s i n g time was from 3 to 4 hrs with the eva p o r a t i o n step and c o o l i n g steps t a k i n g the l o n g e s t . The product was c l e a n as determined by gamma spectrometry ( F i g . 13). The r a d i a t i o n f i e l d f o r the p r o c e s s i n g o f a N i t a r g e t r e s u l t i n g i n 1.6 mCi of 5 2 F e a t the end of s e p a r a t i o n was 1.5 mR/h c o n t a c t through the l e a d g l a s s window. Only 0.1 mR/h c o n t a c t was measured through 4 i n of l e a d s h i e l d i n g . 119 E Ch o 1 l f 3 * 8 11 A. N i S o l u t i o n a f t e r E x t r a c t i o n 1 F i n a l -^Fe Product 0 400 800 12003 Gamma Ray Energy (KeV) 1600 A! Energy Radionuclide A Energy Radioi 1 112 * S C r 10 847 2 126 5 7 N i 11 932 5 5 C o 3 158 5 6 N i 12 948 ^ 8 V 4 271 5 6 N i 13 1157 <*Sc 5 308 * S C r 14 1241 6 477 5 6 N i 15 1313 ^ 8 V 7 511 P o s i t r o n 16 1376 5 7 N i 8 745 5 2Mn 17 1409 9 812 5 6 N ± 18 1437 5 2 m M n B Energy Radionuclide 1 169 2 511 P o s i t r o n 3 1437 5 2 m M n F i g , 13 Ge(Li) Spectrum of N i S o l u t i o n and Product. 120 The r a d i a t i o n f i e l d a t the o p e r a t o r p o s i t i o n a t window l e v e l was 0.25 mR/h. Ten mR/h c o n t a c t on top and 2 0 mR/h c o n t a c t on the s i d e s of the l e a d t r a n s p o r t p i g were measured. C. M e t a l l o p o r p h y r i n S y n t h e s i s 5 9 Fe-Hematohemin A f t e r the r e a c t i o n p e r i o d , paper chromatography i n d i c a t e d t h a t 45% of the 5 9 F e went i n t o the p o r p h y r i n . During the f i r s t e x t r a c t i o n 55% of the a c t i v i t y went i n t o the c h l o r o -form l a y e r . T h i s was due to incomplete e x t r a c t i o n from the r e a c t i o n mixture i n t o the o r g a n i c l a y e r . E x t r a c t i o n c o u l d have been improved by u s i n g l a r g e r volumes of c h l o -roform but i t was not i n c r e a s e d due to the problems a s s o c i a -ted w i t h h a n d l i n g l a r g e r volumes of r a d i o a c t i v e s o l u t i o n s . During the HC£ wash and water wash 8% and 16% r e s p e c t i v e l y unreacted 5 9 F e was removed. The % t a g determined by count-in g a l l f r a c t i o n s was 55%. Paper chromatography of the f i n a l product i n d i c a t e d t h a t the tag was g r e a t e r than 99%. Any unreacted 5 9 F e was r e -moved i n the e x t r a c t i o n s t e p s . A b s o r p t i o n spectrophotometry of the product i n p y r i d i n e reduced w i t h sodium d i t h i o n i t e showed t h a t the product was pure. 121 Ab s o r p t i o n Spectrum: P y r i d i n e , , , . 410 519 550 2 Xmax(obs) ,, ... 409 519 549 Xmax(lit) T h i n l a y e r chromatography and polyamide chromatography i n d i c a t e d t h a t the product was pure and d i d not decompose du r i n g m e t a l a t i o n . 2) 5 9 Fe-Protohemin The % t a g as determined by paper chromatography was 6 0%. The % t a g determined by co u n t i n g a l l f r a c t i o n s was 65%. The above v a l u e s r e f e r to the m e t a l a t i o n r e a c t i o n o n l y . A f t e r s o l v e n t e x t r a c t i o n , paper chromatography i n d i c a t e d t h a t the %tag was g r e a t e r than 99%. The hemochrome a b s o r p t i o n spectrophotometry i n 4 M p y r i -dine and 0.2 N KOH a l s o showed t h a t the product was pure. A b s o r p t i o n Spectrum: Hemochrome , , i_ \ 420 526 557 Xmax (obs) ,, . . . 419 526 557 Xmax(lit) 3) 5 9Fe-Photoprotohemin F i r s t the f e r r o u s s u l f a t e or c h l o r i d e method was used to metalate the p o r p h y r i n . Although paper chromatography i n d i c a t e d a good % tag s e v e r a l problems were encountered. 122 F i r s t , the photoprotohemin had a low s o l u b i l i t y i n c h l o r o -form. I n c r e a s i n g the volume of c h l o r o f o r m to 50 ml or higher s t i l l r e s u l t e d i n a l a r g e l o s s of the product. Second, d u r i n g HC£ e x t r a c t i o n (wash) a heavy p r e c i p i t a t e formed a t the s o l v e n t i n t e r f a c e . T h i r d , d u r i n g the water wash, a very heavy p r e c i p i t a t e formed a t the s o l v e n t i n t e r -f a c e . During the e x t r a c t i o n procedure 76% of the product was l o s t . A b s o r p t i o n spectrophotometry of the crude photoprotohemin (and t i c ) showed t h a t i t was contaminated w i t h protohemin r e s u l t i n g from the p r o t o p o r p h y r i n i n the photoprotoporphy-r i n and a decomposition product of photoprotohemin r e -s u l t i n g from the m e t a l a t i o n r e a c t i o n . The decomposition c o u l d have been reduced by u s i n g the f e r r o u s s u l f a t e method r a t h e r than the f e r r i c c h l o r i d e method but a new p u r i f i c a t i o n procedure would have to be designed. The f i r s t p u r i f i c a t i o n step was always r e q u i r e d . The second step was o n l y r e q u i r e d to remove the remaining protohemin i f i t i n t e r f e r e d i n the uptake s t u d i e s . A b s o r p t i o n spectrophotometry ( p y r i d i n e and 2% formic acid) and t i c i n d i c a t e d t h a t the product was pure. The %tag of the f i n a l product was g r e a t e r than 99% 123 Ab s o r p t i o n Spectrum: Hemochrome , , , N 416 624 Xmax(obs) / i -4.x 4 1 7 625 Xmax(lit) 5 9 F e - 2 - f o r m y l - 4 - v i n y l .deuterohemin Again, d u r i n g the m e t a l a t i o n r e a c t i o n , some of the product decomposed and the p u r i f i c a t i o n step was r e q u i r e d to remove t h i s contaminate. A b s o r p t i o n spectrophotometry (4 M p y r i d i n e 0.2 ISFKOH) i n d i c a t e d t h a t the product was pure. The %tag of the f i n a l product was g r e a t e r than 99%. Ab s o r p t i o n Spectrum: Hemochrome , # r_ \ 434 538 582 Xmax(obs) Xmax(lit) 434 538 582 5 9 F e - 2 - v i n y l - 4 - f o r m y l deuterohemin R e s u l t s s i m i l a r to those f o r 5 9 F e - f o r m y l - 4 - v i n y l deuterohemin were o b t a i n e d . 5 9 F e - 2 , 4-Diformyl deuterohemin Again the p u r i f i c a t i o n step was r e q u i r e d to remove the m e t a l a t i o n r e a c t i o n decomposition products. The c r y s t a l -l i z a t i o n step was very important to remove a c e t a l formation d u r i n g column chromatography and/or s o l v e n t e v a p o r a t i o n . A b s o r p t i o n spectrophotometry and t i c i n d i c a t e d t h a t the 124 product was pure. The %tag was g r e a t e r than 9 9%. Ab s o r p t i o n Spectrum: Hemochrome , , . 452 550 587 max(obs) ,, . . . 452 550 587 ma x ( l i t ) 7) 5 9 F e or 5 2 F e l a b e l l e d meso-tetra (4-carboxyphenyl) hemin, meso-tetra (4-N-methylpyridyl) hemin t e t r a i o d i d e or tetra-Na-meso-tetra (4-sulfonatophenyl) hemin The f e r r o u s s u l f a t e m e t a l a t i o n procedure was t r i e d . The problem w i t h i t was t h a t the hemins were so water s o l u b l e t h a t they c o u l d not be t r a n s f e r r e d i n t o an o r g a n i c s o l v e n t . There was very l i t t l e t r a n s f e r i n t o c h l o r o f o r m . However there was more t r a n s f e r i n t o dichloromethane but i t would s t i l l have r e q u i r e d a l a r g e volume of s o l v e n t f o r complete t r a n s f e r . There was no t r a n s f e r i n t o 4-methyl-2-pentanone, ether, or d i - i s o p r o p y l e t h e r . S o l v e n t s where the hemins were s o l u b l e , DMF or p y r i d i n e , however were m i s c i b l e w i t h water. The f e r r i c c h l o r i d e m e t a l a t i o n procedure a l s o worked but any r e s i d u a l DMF c o u l d have caused problems i n the uptake s t u d i e s . T h e r e f o r e , the sodium a c e t a t e - a c e t i c a c i d method was used. The %tag determined by paper chromatography was from 90-100%. 125 D. Tumor T i s s u e C u l t u r e Uptake S t u d i e s The m a j o r i t y of hemins and some of the r a d i o n u c l i d e s showed an i n i t i a l uptake f o l l o w e d by a d e c l i n e phase which again was f o l l o w e d by an uptake phase u s u a l l y h i g h e r than the f i r s t uptake phase. Of the two r a d i o a c t i v e l a b e l l e d p o r p h y r i n s used b e f o r e , protohemin showed b e t t e r uptake than hematohemin ( F i g . 14). Hematohemin uptake was 18.3% a t 1 h, dropping to 10.6% a t 24 h- ' and sl o w l y i n c r e a s i n g to 12.4% a t 72 h . P r o t o h e m i n had a very high i n i t i a l uptake of 63.7% a t 3 h , 'followed by a d e c l i n e to 30% a t 6 h f o l l o w e d by another r a p i d uptake to 87% a t 24 h. Of the n a t u r a l p o r p h y r i n s t r i e d the deuterohemin d e r i v a t i v e s showed the best e a r l y uptake ( F i g . 15). The deuterohemin d e r i v a t i v e s showed r a p i d uptake of 69% a t 10 h. f o l l o w e d by a slow i n c r e a s e i n uptake to 100% a t 72 h. The 2-formyl-4 - v i n y l , 2 - v i n y l - 4 - f o r m y l , and the 2,4-diformyl deuterohemins had s i m i l a r uptake and were shown as one curve. Photoproto-hemin showed r a p i d i n c r e a s e to 37% at 3 h but was f o l l o w e d by a d e c l i n e phase, 20% a t 10 h , and again f o l l o w e d by an i n c r e a s e to 100% at 72 h:. 100 r 80 a 60 £ 40 20 0 u i i I— 1 6 10 24 o — o HEMATO • — • PROTO Time (hr) 72 t o ON F i g 14. Tumor t i s s u e c u l t u r e uptake of p r e v i o u s l y l a b e l l e d n a t u r a l p o r p h y r i n s . F i g 15. Tumor t i s s u e c u l t u r e uptake of n a t u r a l hemins. 128 Of the a r t i f i c i a l hemins t e s t e d , meso-tetra (4-carboxy-phenyl) hemin (CP) showed very r a p i d uptake to 60% a t 3 h: f o l l o w e d by a slow d e c l i n e to 30% a t 72 h.' ( F i g . 1 6 ) . T e t r a -na-meso-tetra (4 sulfonatophenyl) hemin (TPPS) had a s i m i l a r low uptake curve as hematoporphyrin. Meso-tetra (4-N-methyl p y r i d y l ) hemin t e t r a i o d i d e (TMPI) a l s o showed v e r y r a p i d i n i t i a l uptake a t TPPS (60% a t 1 h") but decreased to 42% at 10 h. and then i n c r e a s e d to 100% a t 72 h. . ( F i g 17) . Due to the s h o r t Th of the 5 2 F e , o n l y hemins w i t h r a p i d i n i t i a l uptake were i n v e s t i g a t e d f u r t h e r . These i n c l u d e d the f o l l o w i n g : protohemin, TCP, and TMPI. Other hemins t h a t showed good uptake a t 24 to 7 2 h c o u l d not be used because a very l a r g e amount of 5 2 F e would have to be g i v e n r e s u l t i n g i n a very l a r g e r a d i a t i o n dose to the p a t i e n t . I f o ther r a d i o n u c l i d e s were going to be used, TMPI would be the b e s t c h o i c e because i t shows very h i g h i n i t i a l uptake which was i d e a l f o r s h o r t - l i v e d r a d i o n u c l i d e s and uptake continued up to 10 0% a t 7 2 h which i s i d e a l f o r l o n g e r -l i v e d r a d i o n u c l i d e s . TMPI was l a b e l l e d w i t h 5 9 F e (as d e s c r i b e d above), Co, Cr, and Mn. Of the f o u r , Fe-TMPI was the b e s t ( F i g . 17). Co-TMPI showed no uptake u n t i l 3 hrs and was 21% a t 6 h, ., d e c r e a s i n g to 18% a t 10 h'., Both Cr-TMPI and Mn-TMPI showed no uptake a t 10 h..;, 100 r 80 h o_oTCP •—•TPPS o •| 60 a ^ 40 20 0 H I I L _ 1 6 10 24 Time (hr) 72 to W3 F i g 16. Tumor t i s s u e c u l t u r e uptake of a r t i f i c i a l hemins. I-J / II I I I 1 6 10 24 o-• -A-•o FE TMPI -•CO TMPI CR M M TMPI _J 72 Time (hr) 17. Tumor t i s s u e c u l t u r e uptake o f TMPI l a b e l l e d w i t h d i f f e r e n t mejta-Ls. 131 Uptake of the r a d i o n u c l i d e s by themselves was very d i f f e r e n t from t h a t o f the l a b e l l e d p o r p h y r i n ( F i g . 1 8 ) . Both 5 9 F e and 6 7 G a had s i m i l a r uptake p a t t e r n s . 6 7 G a showed maximal uptake of 2% a t 1 h f o l l o w e d by a d e c l i n e to 0.3% at 3 h. and a ve r y slow uptake t o 1.4% a t 72 h.'. 5 9 F e was taken up to 13% a t 1 h , dropped to 2.1% a t 3 h and showed slow uptake to 25% a t 72 h. . Both 1 + 6 S c and 5hMn showed no uptake un-t i l 3 h.; but 4 6 S c was taken up to 49% and 5kRn to 32% at 6 h: , k8V, 5 6 C o , and 5 1 C r showed no uptake a t 10 h.. E. Animal S t u d i e s D i s t r i b u t i o n S t u d i e s The t a r g e t organs of the 5 9 F e - l a b e l l e d TMPI were the l i v e r and spleen ( F i g 19). The l i v e r continued to take up the compound throughout the study. The % dose/g organ f o r the l i v e r a t 48 h was 28.6%. Uptake by the spleen i n c r e a s e d d r a m a t i c a l l y and peaked a t 12 h to 22.7% dose/g organ and slo w l y decreased to 10.6% dose/g organ a t 48 h. Lung ( F i g 20), kidney and eye (Table XII) remained r e l a t i v e l y c o nstant throughout the study - lung 2.5% dose/g organ; kidney 4.7% dose/g organ and eyes 0.5% dose/g organ. Blood was r e l a t i v e l y c o nstant a t 2.8% dose/ml u n t i l 24 h but i n -creased to 7.6% a t 47 h. Bone slowly i n c r e a s e d from 1.6% 100 80 •J 60 Q. Z) ^ 40 20 0 o — o FE — SC — MM •—• GA — V CO CR 5-i ii i ' l_ 1 6 10 24 Time (hr) to _J 72 F i g 18. Tumor t i s s u e c u l t u r e uptake of v a r i o u s metals. —• Liver — o Spleen •—• Lung o I I I I 1 6 12 24 Time (hr) 48 ' F i g 19. Animal d i s t r i b u t i o n o f 5 9 F e TMPI 134 dose/g-organ a t 6 hr to 4.9% dose/g a t 48 h. Although no d i s t r i b u t i o n s t u d i e s have been done wi t h t h i s p o r p h y r i n or other F e - l a b e l l e d p o r p h y r i n s , o t h e r p o r p h y r i n s a l s o showed hi g h l i v e r uptake. TPPS was found i n c o n s i d e r -a b l e amounts i n the l i v e r , s p l e e n , kidney and lung (210), although o n l y the tumor e x h i b i t e d red f l u o r e s c e n c e . The l i v e r , spleen and kidney c o n t a i n e d more TPPS than the tumor (212) 6 ^ C u - p r o t o p o r p h y r i n was shown to c o n c e n t r a t e i n mouse tumors wi t h poor tumor to l i v e r , blood and muscle r a t i o s (236). C l i n i c a l s t u d i e s show no tumor uptake a t a l l (237). 5 7Co-hematoporphyrin showed s i m i l a r d i s t r i b u t i o n i n nromal and tumor-bearing animals although the tumor d i d show some accumulation (238). Most of the r a d i o a c t i v i t y was i n the l i v e r , kidney and spleen. Twenty fo u r hours a f t e r i n j e c t i o n tumor uptake was higher than blood or muscle, but o n l y 1/5 o f l i v e r uptake. The l i v e r was the t a r g e t organ f o r these p o r p h y r i n because the l i v e r i s the n a t u r a l s i t e of normal p o r p h y r i n and b i l e a c i d metabolism. P o r p h y r i n s are broken down i n t o b i l e a c i d s i n the l i v e r . The d i s t r i b u t i o n of the 5 9 F e CI3 was completely d i f f e r e n t from t h a t of the 5 9 F e - l a b e l l e d TMPI i n d i c a t i n g t h a t the 5 9 F e was attached to the TMPI and not f r e e ( F i g . 20 Table •I % Dose/gm Organ sex 136 X I I I ) . The 5 9 F e - c h l o r i d e was s t r o n g l y taken up by the lung. The % dose/g organ was 67.3% a t time 0; i n c r e a s e d to 74.5% dose/g organ a t 1 h and decreased d r a m a t i c a l l y to 22% dose/g organ a t 6 h. Lung uptake remained r e l a t i v e l y c o nstant a t 18% dose/g organ from 6 to 4 8 h. Spleen uptake i n c r e a s e d to 19% dose/g organ a t 6 h and remained r e l a t i v e l y c o nstant f o r the r e s t of the study. L i v e r uptake remained r e l a t i v e l y constant from 0 to 48 h at 15% dose/g organ. Bone i n c r e a s e d to 3.4% dose/g organ at 12 h and remained constant t h e r e a f t e r . Both kidney (15% dose/g and eye (0.2% dose/g)remained constant throughout the study. Blood was slowly i n c r e a s i n g from 1.4% dose/ml at 0.5 h to 11% dose/ml a t 4 8 h.. No s t u d i e s have been done wi t h 5 9 F e - c h l o r i d e but w i t h 5 9 F e -c i t r a t e . The 5 9 F e - c i t r a t e showed hi g h uptake by the spleen, marrow and blood w i t h low uptake by other s o f t t i s s u e s (311). Blood uptake was 19% dose/g a t 72 h and s p l e e n uptake was ' 117% dose/g a t 7 h. Lung uptake was not t e s t e d f o r . 5 9 F e -c i t r a t e showed no a f f i n i t y f o r tumor t i s s u e as 6 7 G a - c i t r a t e d i d . During the d i s t r i b u t i o n study 30%, (8/40) of the animals d i e d d u r i n g i n j e c t i o n i f the dose was given too f a s t . Redu-c i n g the dose by 1/2 or i n j e c t i n g v ery slowly prevented t h i s . Animals went i n t o muscular spasms and d i e d w i t h i n a few minutes. Animals who s u r v i v e d the i n j e c t i o n were h e a l t h y and TABLE XII ANIMAL DISTRIBUTION OF 7FE-TMPT %Dose/Gm Organ (Mean ± SEM) Time(h) Blood Kidneys Bone Eyes 0 6.57 * 0.642 6.25 ± 1.95 0.730 ± 0.218 0.330 ± 0.115 0.5 1.65 ± 0.240 4.76 ± 1.47 1.91 ± 0.840 0.437 * 0.289 1 1.76 ± 0.667 5.29 * 1.36 1.01 ± 0.316 1.02 ± 0.408 3 2.73 ± 0.870 3.56 ± 0.632 0.331 ± 0.133 0.521 ± 0.409 6 2.63 * 0.396 2.37 ± 0.480 1.57 ± 0.678 1.19 ± 0.639 12 2.75 * 0.455 4.33 * 0.565 1.92 ± 0.147 0.686 ± 0.247 24 2.72 ± 0.596 4.67 * 0.450 2.75 ± 0.309 0.578 ± 0.209 48 7.61 ± 0.897 5.30 ± 1.60 4.90 * 0.466 0.415 ± 0.298 TABLE X I I I ANIMAL DISTRIBUTION OF 5 9FE-CHLORIDE %Dose/Gm Organ (Mean ± SEM) Time(h) Blood Kidneys Bone Eyes 0 3.56 0.722 2.60 0.574 0.570 ± 0.142 0.125 ± 0.0725 0.5 1.39 ± 0.194 0.86 ± 0.079 0.295 * 0.039 0.109 ± 0.032 1 1.64 0.146 1.53 0.143 0.593 * 0.121 0.115 * 0.037 3 1.75 0.407 1.59 ± 0.124 0.618 *. 0.201 0.132 * 0.038 6 2.72 0.404 1.62 ± 0.382 2.86 ± 0.431 0.289 ± 0.072 12 4.53 ± 0.696 1.50 ± 0.167 3.47 ± 0.786 0.128 ± 0.056 24 6.77 ± 0.687 1.56 0.071 3.42 ± 0.374 0.280 ± 0.050 48 10.8 ± 0.694 1.76 ± 0.178 3.42 ± 0.405 0.162 ± 0.056 138 normal 4 8h a f t e r i n j e c t i o n . I n j e c t i n g the p o r p h y r i n too f a s t over loads the animal and was r e l a t e d to c o n c e n t r a t i o n P a t i e n t s g i v e n hematoporphyrin experienced severe burning, when i n j e c t e d d i r e c t l y i n t o the v e i n (209). I n f u s i o n over a long p e r i o d w i t h glucose caused no d i s c o m f o r t to the p a t i e n t . S c i n t i g r a p h y S t u d i e s i n normal r a b b i t s showed t h a t a l l the 5 2Fe-TMPI l o c a l i z e d i n the l i v e r . S t u d i e s u s i n g 5 2Fe-TMPI, 5 2 F e -TCP, and 5 2Fe-Protohemin with tumor b e a r i n g r a t s showed no tumor l o c a l i z a t i o n a t 3 h and 24 h a f t e r i n j e c t i o n (Fig.21) A l l of the a c t i v i t y went to the l i v e r . 5 2 F e - c h l o r i d e III) d i d c o n c e n t r a t e i n the tumor and RE system. E x c r e t i o n Study The whole body e x c r e t i o n curve showed t h a t the b i o l o g i c a l h a l f - l i f e of 5 9Fe-TMPI was extremely long ( F i g . 22) The e f f e c t i v e h a l f l i f e can be c a l c u l a t e d from : t h ef f t ^ b i o x trsphy t % b i o + tJsphys where t^b i o t^phy 270d 8.2h 6480 h t h e r e f o r e tfcef f ^8. 2h TUMOR 1 PROTO 2 TCP 3 FE 4 TMPI F i g 21. Tomographic scan of tumor b e a r i n g r a t s u s i n g 5 2 F e hemins " U O CQ ' c o E 0 0 oo O o \ 1 00 <32©-0-80 60 40 20 0 o- •o-o-o-o-o- •o- •o o J I I I L 2 4 6 8 10 Time (days) _ J 20 - J F i g 22. E x c r e t i o n curve of 5 9Fe-TMPI 141 I t appears t h a t the 5 9Fe-TMPI was taken up by the l i v e r but cannot be metabolized and thus cannot be e x c r e t e d from the body too r e a d i l y . Again 30% (3/10) of the animals d i e d d u r i n g i n j e c t i o n when giv e n the dose too f a s t . Reducing the dose by 1/2 on i n j e c t i n g very slowly prevented t h i s . Mice 1 week a f t e r i n j e c t i o n showed some s i g n s of muscular weakness and h a i r l o s s but were normal a f t e r 2 weeks. F. Dosimetry The r a d i a t i o n dose from 1 mCi of 5 2Fe-TMPI to the whole body, l i v e r , s pleen, kidney and lung were c a l c u l a t e d . The f o l l o w -i n g assumptions were made f o r the dose c a l c u l a t i o n s : 1) The d i s t r i b u t i o n and e x c r e t i o n data determined i n mice a l s o a p p l i e d to humans. 2) The d i s t r i b u t i o n and b i o l o g i c a l h a l f - l i f e o f 5 2Fe-TMPI w i l l be the same as 5 9Fe-TMPI 3) The e f f e c t i v e th was 8.2 h 4) Since organ uptake was i n c r e a s i n g or remained constant d u r i n g the f i r s t t^phy or 8.2 h of the d i s t r i b u t i o n study, an e f f e c t i v e th of 8.2 h was used f o r every organ. 5) The maximum organ uptake i n the f i r s t 8.2 h: o f the d i s t r i b u t i o n study were : 142 Organ Max % Dose/G.' Organ Max % Dose L i v e r 14.4 27 Spleen 18.6 2.32 Kidney 6.25 3.13 Lung 3.33 0.732 The absorbed doses were c a l c u l a t e d u s i n g the f o l l o w i n g equation proposed by Loevinger (312) and m o d i f i e d by Snyder e t a l (313) D ( r ^ r 2 ) = A 2 Z,Ai <|>i (r^-<-r2) rads = A 2 i A i $i(r1-«-r2) rads where: _ D ( r j ^ r 2 ) ~ T h e mean absorbed dose to a t a r g e t organ r]_ from a r a d i o n u c l i d e u n i f o r m l y i n source organ r 2 (rads) A2 = The cumulated a c t i v i t y i n source organ r 2 (vCi-h) m-^  = The mass of t a r g e t organ r ^ (g) A i = The e q u i l i b r i u m dose constant f o r r a d i a -t i o n type i = 1,2,3.. wit h f r a c t i o n a l frequency n^ per d i s i n t e g r a t i o n and a mean energy i n MeV = 2.13 n. E. (g-rads) 143 <J>i (+^ r^) = The absorbed dose f r a c t i o n o f energy f o r t a r g e t organ r ^ f o r p a r t i c l e s i emitted i n source organ r 2 ^Hr^^r^) = The s p e c i f i c absorbed f r a c t i o n of energy f o r t a r g e t organ r ^ f o r par-t i c l e s i emitted i n source organ The above equation can be s i m p l i f i e d : D (r± + r2) = A 2S (r± - r 2 ) where S ( r ^ ^ r 2 ) _ £ j_Ai $ i ( r 1 ^ r 2 ) S v a l u e s were o b t a i n e d from Snyder e t a l (313) Table XIV) Since t h e r e are g e n e r a l l y a number of source organs, the t o t a l average dose t o t a r g e t organ r ^ i s D ( r ^ = | D ( r x * r 2 ) y * = 2 A 2 S ( r l " r 2 ) where: A, = J ^ Aoe ->t a) Whole Body Assuming uniform d i s t r i b u t i o n i n the whole body and 69,880 g standard body weight the whole body dose was c a l c u l a t e d as f o l l o w s : A W B = AoE 1.44 ;t he£f « hj 144 w h e r e <*hj = t h e i n i t i a l v a l u e o f i t h e x p o n e n t i a l c o m p o n e n t o f r a d i o n u c l i d e t h a t a p p e a r s i n t h e s o u r c e . = (.1000) (.1.44) (8.2) = 11800 y C i - h D T B = A W B S ( T B . T B ) = (11800) ( 1 . 4 x l O _ S ) 0.1652 r a d / m C i w h e r e S (TB-«-TB) = 1 . 4 x l 0 ~ s r a d / y C i - h C u m u l a t e d A c t i v i t y T h e f o l l o w i n g v a l u e s w e r e c a l c u l a t e d f r o m t h e d i s t r i -b u t i o n d a t a a n d t h e e f f e c t i v e <\, A = 11800 y C i - h WB A L = A o Z l . 4 4 t % e f f <*hj = (0.27) (1000) (1.44) (8.2) A L = 3188 u C i - h A s = 274 y C i - h A k = 370 y C i - h A L u = 8 6.4 y C i - h Mean d o s e L i v e r - 'v D =E A 2 S ( r 1 ^ r 2 ) 145 DL = Awb S(I^WB) + A L S(L^L) + A s S(L«-S) + A k S(L^K) + A L S(L«-Lu) = 11,800(1.5xl0 _ s) +. 3188(3.8xl0~ 4) + 2 7 4 ( 6 x l 0 - 6 ) + 370(2.2xl0- 5) + 86.4 (3.8xl0~ 4) = 1.4 3 rads/mCi Spleen D s = A W B S (S+-WB) + A L S (S+-L) + A s S(S+-S) + A kS(Sf-K) + A L u S ( S ^ L u ) = 1.06 rads/mCi ^ <\, % Kidney D k = A W B S (K«-WB) + A L. S (K+-L) + A s S(K«-S) a. a. + A k S (K+-K) + A L u S (K^-Lu) = 0.924 rads/mCi Lung DT = AWB S(LU^-WB) + A L S(LU^-L) + A S S(LU^S) + A K S (Lu-6-K) + A L U S(Lu^Lu) = 0.2 71 rads/mCi Table XV summarizes the dose val u e s c a l c u l a t e d . 146 TABLE XIV S VALUES FOR ^ F E (RAD/UCT-H) Source Organ Whole Body L i v e r Spleen Kidney Lung Target Organ L i v e r 1.6 X IO"? 3.8 X 1 0 ' * 6.0 X 10~ 6 2.2 X 10~ 5 1.4 x i d * * 5 Spleen 1.6 X IO"? 5.6 X 1 0 " 6 3.1 X 10~ 3 4.7 X 10~ 5 1.2 X 10*"5 Kidney 1.6 X 1 0 - 5 2.1 X 10~ 5 4.8 X IO**5 1.8 X 10~ 3 5.7 X 10"*6 Lung 1.5 X 1 0 - 5 1.4 X 10~ 5 1.2 X 10"* 5 5.2 X 10~ 6 5.1 X 1 0 ' * From Ref. 313 TABLE XV DOSE CALCULATIONS FOR 5 2FE-TMPI Organ Whole Body L i v e r Spleen Kidney Lung Dose (rad/mCi) 0.165 1.43 1.06 0.924 0.271 147 V CONCLUSIONS No problems were encountered i n the s y n t h e s i s of the v a r i o u s p o r p h y r i n s . The o n l y disadvantage being t h a t some r e a c t i o n y i e l d s were l o w , r e s u l t i n g i n the use of l a r g e q u a n t i t i e s of s t a r t i n g m a t e r i a l s . Q u a l i t y c o n t r o l of the d e s i r e d product was very simple because a b s o r p t i o n spectrophotometry and/or t h i n l a y e r chromatography was used. The 500 MeV i r r a d i a t i o n f a c i l i t y was the b e s t f a c i l i t y to i r r a d i a t e t a r g e t s because e n t r y i n t o the beamline was not r e q u i r e d . A l s o t a r g e t s c o u l d be i r r a d i a t e d a t any time w i t h -out r e q u e s t i n g beam time. 5 2 F e produced by hi g h energy proton s p a l l a t i o n and sepa-r a t i o n by methyl i s o b u t y l ketone was pure as determined by Ge(Li) spectroscopy. The amounts of 5 5 F e and 5 9 F e i n the 5 2 F e l i m i t e d the c l i n i c a l use of the 5 2 F e but the r a d i a t i o n dose to the p a t i e n t from 5 5 F e contaminated 5 2 F e was s t i l l lower than the 5 9 F e . The d i s s o l v i n g o f the t a r g e t and e v a p o r a t i o n of the n i t r i c a c i d were the most time con-suming s t e p s . M e t a l l o p o r p h y r i n s y n t h e s i s procedures had to be m o d i f i e d when c a r r i e r - f r e e r a d i o n u c l i d e s were used to l a b e l the p o r p h y r i n s . Most procedures d i d remove any unreacted 148 p o r p h y r i n and r a d i o n u c l i d e . However, t h i s was not p o s s i b l e w i t h the a r t i f i c i a l hemins. These were so water s o l u b l e t h a t they were imp o s s i b l e to t r a n s f e r i n t o an o r g a n i c s o l v e n t . I f these p o r p h y r i n s are going to be used i n the f u t u r e , b e t t e r s e p a r a t i o n techniques must be developed. TMPI, TCP and protohemin showed hi g h i n i t i a l uptake u s i n g t i s s u e c u l t u r e techniques w i t h mouse tumor c e l l s (P815). Both p o r p h y r i n s used i n d e t e c t i n g tumors showed poor uptake. TMPI l a b e l l e d w i t h other r a d i o n u c l i d e s a l s o showed poor uptake along w i t h the r a d i o n u c l i d e s by themselves. Normal mice d i s t r i b u t i o n s t u d i e s u s i n g 5 9Fe-TMPI showed t h a t the t a r g e t organ f o r t h i s agent was the l i v e r and sp l e e n . 5 2 I n i t i a l animal scans u s i n g Fe-TMPI and normal r a b b i t s showed t h a t i t a l l l o c a l i z e d i n the l i v e r s t u d i e s u s i n g 5 2Fe-TMPI, 5 2Fe-TCP and 5 2 F e - p r o t o h e m i n with tumor b e a r i n g r a t s showed no l o c a l i z a t i o n a t a l l . A l l of the above agents went to the l i v e r . However 5 2 F e - c h l o r i d e d i d c o n c e n t r a t e i n the tumor and RE system. I t appears from t h i s study, the more un n a t u r a l the porphy-r i n s t r u c t u r e t h e b e t t e r the tumor and l i v e r uptake. I f l i v e r uptake i n the animal c o u l d be reduced, the porphy-r i n may go to the tumor. I t was assumed t h a t the v e r y un-149 n a t u r a l s t r u c t u r e of TMPI would show high tumor uptake and low l i v e r uptake. Based on t h i s study, a l l p o r p h y r i n s t e s t e d were not s u i t a b l e to be used as a tumor scanning agent i n n u c l e a r medicine. More work has to be done on the mechanism of p o r p h y r i n up-take and metabolism i n both tumor and l i v e r t i s s u e . A l a r g e number of 5 2 F e l a b e l l e d p o r p h y r i n may have to be screened to f i n d one which has h i g h tumor and low l i v e r uptake. 150 VI BIBLIOGRAPHY 1. Anderson W.A.D. and S c o t t i T.M., Synopsis of Pathology, S a i n t L o u i s , C V . Mosby Company, 1976 p.345, 2. McCready w i t h Radioact. IAEA , Vienna: 3. 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