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zyxwvutsrqp zy zyxwv 51 Scand J Haematol Suppl 44, Vol 34, 1986 CELLULAR PHARMACOLOGY AND OPTIMAL THERAPEUTIC CONCENTRATIONS OF 1-@-0- ARABINOFURANOSYLCYTOSINE 5'-TRIPHOSPHATE I N LEUKEMIC BLASTS DURING TREATMENT OF REFRACTORY LEUKEMIA WITH HIGH-DOSE SINE 1-8-D-ARABINOFURANOSYLCYTO- W i l l i a m P l u n k e t t l , Stephen Iacoboni2. Michael J . Keating2 Department o f Chemotherapy Research1 Department o f Hematology2 The U n i v e r s i t y o f Texas M.D. Anderson H o s p i t a l and Tumor I n s t i t u t e a t Houston, Houston, Texas 77030, USA. ABSTRACT The pharmacology o f 1-8-D-arabinofuranosylcytosine 5 ' - t r i p h o s p h a t e (ara-CTP) has been s t u d i e d i n t h e c i r c u l a t i n g leukemic b l a s t s o f p a t i e n t s w i t h r e f r a c t o r y leukemia d u r i n g t h e r a p y w i t h h i g h doses o f s i n g l e - a g e n t ara-C. C e l l u l a r ara-CTP was analyzed by high-pressure l i q u i d chromatography. The median t r o u g h c o n c e n t r a t i o n s o f ara-CTP i n b l a s t s o f p a t i e n t s who responded t o i n t e r m i t t e n t high-dose ara-C ( 3 g/m2 x 4-12 doses) was 196 vM, whereas 75 VM was t h e l o w e s t t r o u g h ara-CTP c o n c e n t r a t i o n t h a t d i s c r i m i n a t e d between complete r e m i s s i o n and t r e a t m e n t f a i l u r e (p=0.03). The median steady s t a t e ara-CTP c o n c e n t r a t i o n s i n t h e b l a s t s o f p a t i e n t s who responded t o a h i g h dose continuous i n f u s i o n p r o t o c o l ( 2 doses o f 3 g/m2 e v e r y 12 h r f o l l o w e d by a continuous i n f u s i o n o f 3303000 m g / d x 4 d) were c l u s t e r e d between 79 and 206 vM. The values o f t r e a t m e n t f a i l u r e s were p r e d o m i n a n t l y o u t s i d e t h i s range (p<0.005). These r e s u l t s suggest t h a t t h e o p t i m a l c o n c e n t r a t i o n range f o r i n t r a c e l l u l a r ara-CTP i n t h e t r e a t m e n t o f r e f r a c t o r y leukemia may be 75 t o 200 pM. INTRODUCTION The most e f f e c t i v e d r u g f o r t h e s u c c e s s f u l t r e a t m e n t o f a d u l t a c u t e myelogenous leukemia is 1-p-arabinofuranosylcytosine (ara-C) (1). Over t h e years, i n v i t r o s t u d i e s have c o n t r i b u t e d t o a s u b s t a n t i a l understand i n g o f t h e c e l l u l a r metabolism o f ara-C and t o t h e mode o f a c t i o n o f i t s active CTP) metabolite (2-4). 1-8-D-arabinofuranosylcytosine However, 5'-triphosphate (ara- r e l a t i v e l y l i t t l e information exists t h a t relates p a t i e n t response t o t h e b i o c h e m i c a l pharmacology o f ara-CTP i n t h e leuke- Key words: ara-CTP, high-dose ara-C, i n d i v i d u a l i z e d therapy, leukemia. Running t i t l e : C e l l u l a r Pharmacology o f ara-CTP refractory 52 mic c e l l s d u r i n g d r u g a d m i n i s t r a t i o n (5). Recent a p p l i c a t i o n o f h i g h - p r e s s u r e l i q u i d chromatographic procedures t o t h i s problem (6,7) and t h e t h e r a p e u t i c use o f ara-C doses 30 t i m e s g r e a t e r t h a n t h o s e a d m i n i s t e r e d i n c o n v e n t i o n a l c o m b i n a t i o n chemotherapy have f a c i l i t a t e d t h e i n v e s t i g a t i o n o f t h e i n t r a c e l l u l a r metabolism o f ara-CTP i n tumor c e l l s (8,9). The g o a l o f t h e p r e s e n t s t u d y was t o g a i n an u n d e r s t a n d i n g o f t h e r e l a t i o n s h i p between t h e c e l l u l a r metabolism o f t h e a c t i v e f o r m t o ara-C and t h e c l i n i c a l response t o ara-C t h e r a p y . I n t h e process, we hoped t o i d e n t i f y a range o f ara-CTP c o n c e n t r a t i o n s i n leukemic c e l l s t h a t i s a s s o c i a t e d w i t h p o s i t i v e c l i n i c a l response. I t i s w i d e l y r e c o g n i z e d t h a t t h e a c t i v e m e t a b o l i t e o f ara-C i s t h e t r i - phosphate, ara-CTP (10.11). Ara-CTP appears t o have a d i r e c t e f f e c t on zy zyxwvut DNA s y n t h e s i s because i t competes w i t h dCTP i n t h e i n h i b i t i o n o f DNA polymerase (11,12) and i s i n c o r p o r a t e d i n t o DNA (13). These a c t i o n s a r e strongly associated w i t h t h e loss o f clonogenicity o f c e l l s i n c u l t u r e ( 1 4 ) . I n a d d i t i o n ara-CTP i s known t o p a r t i c i p a t e i n aspects o f i n t e r m e - .005 .004.003 - E 0 a3 .001 hl I - OI zyxwv 0 0 C L1 m In .004 %I araCTP J I n a .003 .002 .oo 1 0 10 20 Minutes 30 Fig. 1. High-pressure liquid chromatographic separation of nucleoside triphosphates. A. Standard compounds. CTP, 1.85 nmol; ara-CTP, 1.80 nmol; UTP 1.70 nmol; ATP, 2.10 nmol; GTP, 1.90 nmol. B. An e x t r a c t o f 1x107 p e r i p h e r a l leukemic b l a s t s f r o m a p a t i e n t w i t h r e f r a c t o r y acute myelogenous leukemia sampled 12 h r a f t e r i n f u s i o n o f 3 g/m2 ara-C. The mean c e l l volume was 0.31 pL. Calculated c e l l u l a r concentrations of nuc 1eos i d e CTP. 310 vM; triphosphates: ara-CTP, 250 pM; UTP, 1.290 vM; GTP, 790 pM. zyxwvu 53 d i a r y m e t a b o l i s m t h a t i n v o l v e p h o s p h o l i p i d s (15) and g l y c o p r o t e i n s ( 1 6 ) . These a c t i o n s may e x p l a i n t h e t o x i c i t y o f ara-C i n v o l v e d i n DNA s y n t h e s i s . However, t o c e l l s t h a t are not a s i n g l e mechanism o f c y t o t o x i c i t y f o r ara-CTP has n o t been i n d e n t i f i e d . and i t i s l i k e l y t h a t s e v e r a l t o x i c a c t i o n s c o n t r i b u t e t o c e l l death. F o r t h a t reason, we have chosen t o f o cus on t h e c e l l u l a r m e t a b o l i s m o f ara-CTP i n leukemia c e l l s d u r i n g t h e r a PY. RESULTS AND DISCUSSION C i r c u l a t i n g leukemic b l a s t s f r o m p a t i e n t s w i t h r e f r a c t o r y a c u t e l e u k e mia were f r a c t i o n a t e d by F i c o l l - H y p a q u e d e n s i t y c e n t r i f u g a t i o n . A f t e r t h e c e l l s were counted and t h e i r mean volume was determined, c e l l u l a r n u c l e o t i d e s were e x t r a c t e d w i t h p e r c h l o r i c a c i d and s e p a r a t e d by anion-exchange h i g h - p r e s s u r e l i q u i d chromatography ( 1 7 ) . F i g u r e 1 A shows t h e s e p a r a t i o n o f a m i x t u r e o f r e f e r e n c e compounds. Using t h i s procedure, t h e l o w e r li- m i t o f d e t e c t i o n o f ara-CTP i s a p p r o x i m a t e l y 25 pmol. A s i m i l a r f r a c t i o n a t i o n o f t h e n u c l e o s i d e t r i p h o s p h a t e s i n an e x t r a c t o f p e r i p h e r a l leuke- m i c b l a s t s i s o l a t e d f r o m t h e b l o o d o f a p a t i e n t r e c e i v i n g high-dose ara-C t h e r a p y i s shown i n f i g u r e IB. T h i s chromatogram demonstrates t h a t even 3 g/m2 12 hours a f t e r i n f u s i o n o f z of ara-C, ara-CTP remains a m a j o r n u c l e o t i d e i n t h e leukemic c e l l s o f t h i s p a t i e n t . S e r i a l sampling o f p e r i p h e r a l b l o o d d u r i n g and f o l l o w i n g ara-C infu- zyxwvu zyxwvutsr zyxwvut s i o n s gave a p h a r m a c o k i n e t i c p r o f i l e o f ara-CTP m e t a b o l i s m i n leukemic 1000 500 5 &l- o 200 -: ,$<100 -- 4 # A ,**\ 50 \'\ -..*. A ' -2, \ F i g . 2. Accumulation and r e t e n t i o n o f ara-CTP by c i r c u l a t i n g leukemic b l a s t s o f f i v e p a t i e n t s w i t h r e f r a c t o r y acute myelogenous leukemia a f t e r a (3 2-hr infusion of ara-C g/m2). Cellular concentrat i o n s of ara-CTP were d e t e r m i '-+ ned f o r each p a t i e n t by t h e h i g h - p r e s s u r e 1 i q u i d chromatog r a p h i c method shown i n f i g u r e 1. Those i n d i v i d u a l s i n d i c a t e d by open symbols and s o l i d l i n e s a c h i e v e d complete r e m i s s i o n i n , response t o high-dose ara-C 12 t h e r a p y . R e s i s t a n t p a t i e n t s a r e i n d i c a t e d by c l o s e d symbols and dashed l i n e s . -.. $ -z 20 - 10 $ 5 zyxwvutsrq zy =- 2 1 -++ 9 ' / 0 . a \ A I I jaZ3-l 4 8 Hours kk. \;'. '. zyxwvu zyx 54 c e l l s (Fig. I t i s c l e a r t h a t t h e r e i s g r e a t d i v e r s i t y among p a t i e n t s 2). w i t h r e s p e c t t o t h e a b i l i t y o f t h e c i r c u l a t i n g leukemic b l a s t s t o accumul a t e and t o r e t a i n ara-CTP. More d e t a i l e d s t u d i e s have demonstrated good c o r r e l a t i o n s between t h e ara-CTP c o n c e n t r a t i o n s i n c i r c u l a t i n g b l a s t s and t h o s e i n t h e marrow b l a s t s f r o m t h e same p a t i e n t (18). . I n a d d i t i o n , it has been p o s s i b l e t o demonstrate t h a t t h e c e l l u l a r pharmacokinetics o f ara-CTP i n t h e c i r c u l a t i n g leukemic b l a s t s o f p a t i e n t s w i t h r e c u r r e n t zy zyxwvutsrq a c u t e leukemia were s t r o n g l y c o r r e l a t e d w i t h c l i n i c a l response t o h i g h dose ara-C t h e r a p y (19). Although t h e peak c o n c e n t r a t i o n s o f ara-CTP t h e s e response c a t e g o r i e s were n o t s i g n i f i c a n t l y d i f f e r e n t , in s t r o n g asso- c i a t i o n s were found between response and t h e r a t e o f ara-CTP e l i m i n a t i o n , t h e t r o u g h ara-CTP c o n c e n t r a t i o n , and t h e area under t h e ara-CTP accumul a t i o n and e l i m i n a t i o n c u r v e . I n p a r t i c u l a r , t h o s e p a t i e n t s whose c e l l s r e t a i n e d ara-CTP more e f f e c t i v e l y , which a l l o w e d f o r g r e a t e r i n t r a c e l l u l a r exposure t o t h e t o x i c m e t a b o l i c , had a s i g n i f i c a n t l y g r e a t e r chance o f a c h i e v i n g a complete r e m i s s i o n t h a n d i d t h o s e p a t i e n t s whose c e l l s d i d n o t r e t a i n ara-CTP exposure t o ara-CTP (19,20). findings. zy as e f f e c t i v e l y and t h e r e f o r e had a r e l a t i v e l y b r i e f The d a t a presented i n f i g u r e 2 r e f l e c t t h e s e zyxwv zyxwvutsrq ’“7 The t r o u g h c o n c e n t r a t i o n o f ara-CTP, 0 ,0°1 * O 0tI O 0 0, 8 O Complete Remission .. 0 Failure t h a t amount p r e s e n t i n leukemic Fig.3. Relationship between t h e response o f p a t i ents w i t h r e f r a c t o r y acute leukemia to intermittent high-dose ara-C t h e r a p y and t h e trough concentration o f in circulating ara-CTP blasts 12 h r a f t e r infusion o f t h e f i r s t dose o f ara-C. The l o w e s t c e l l u l a r ara-CTP c o n c e n t r a t i o n t h a t marked a d i s t i n c t i o n between compl e t e r e m i s s i o n and t r e a t ment failure (resistant disease plus supportive care f a i l u r e ) a t a s i g n i f i c a n t l e v e l (p=0.03) was 75 pM, which i s i n d i c a t e d by t h e dashed l i n e . zyxwvut zyxwvuts zyxwv 55 c e l l s j u s t b e f o r e t h e n e x t dose o f ara-C i n t h e i n t e r m i t t e n t dose r e g i - men. It i s p o s s i b l e t h a t t h e lowest trough ara-CTP c o n c e n t r a t i o n t h a t i s associated w i t h t h e attainment o f complete remission might serve as an i n d i c a t o r o f t h e lower l i m i t o f t h e m e t a b o l i t e associated w i t h c l i n i c a l response. F i g u r e 3 i s a r e p r e s e n t a t i o n o f t h e t r o u g h ara-CTP concentra- t i o n s i n 52 p a t i e n t s w i t h relapsed acute leukemia who were t r e a t e d w i t h i n t e r m i t t e n t high-dose ara-C doses) (20). ( 3 g/m2 over 2 hr, every 12 h r f o r 4-12 P a t i e n t s who achieved a complete remission had a median t r o u g h ara-CTP c o n c e n t r a t i o n o f 196 vM. The t r e a t m e n t - f a i l u r e category i n c l u d e s i n d i v i d u a l s who achieved marrow hypoplasia b u t f a i l e d t o regene- r a t e a normal marrow, and those who d i e d w i t h a p l a s t i c marrows as w e l l as p a t i e n t s w i t h r e s i s t a n t disease. The lowest trough ara-CTP c o n c e n t r a t i o n t h a t was s i g n i f i c a n t l y d i f f e r e n t f o r p a t i e n t s who responded and those f o r whom treatment f a i l e d was 7 5 VM (p=0.03). Because t h e t r o u g h i s t h e lowest c e l l u l a r ara-CTP c o n c e n t r a t i o n experienced by t h e c e l l s d u r i n g t h e course o f therapy, i t suggests a p o t e n t i a l lower l i m i t o f ara-CTP asso- c i a t e d w i t h response t o i n t e r m i t t e n t high-dose ara-C therapy. As an ex- t e n s i o n o f t h i s reasoning, t h e median value f o r p a t i e n t s w i t h r e s i s t a n t disease, 23 pM, must be viewed as being inadequate t o achieve d e s i r e d therapeutic results. A subsequent group p a t i e n t s who had disease c h a r a c t e r i s t i c s s i m i l a r t o t h e p a t i e n t s t r e a t e d w i t h i n t e r m i t t e n t high-dose ara-C, were t r e a t e d w i t h zyxwvutsrqpon 40 L ara-C by continuous i n f u s i o n . Each p a t i e n t ’ s dose was determined by t h e a b i l i t y o f t h e c i r c u l a t i n g b l a s t s o f t h a t p a t i e n t t o accumulate and r e t a i n 7005 i A A ui c u) Q 5 .-t “ k v g A A 300 1 w A 200 200 al c 0 Ga 100 75 D Q al Responae F i g . 4. Steady s t a t e ara-CTP concentrations in 1eukemic b l a s t s d u r i n g pharmacologically directed continuous infusion ara-C therapy. Patients w i t h e i t h e r relapsed acute leukemia (n=25) o r chronic myelogenous leukemia i n b l a s t c r i s i s (n=13) i n i t i a l l y received an i n f u s i o n o f 3 g/m2 over 2 h r . The area under t h e ara-CTP accumulation and r e t e n t i o n curve was d e t e r mined i n an o n - l i n e fashion. T h i s i n f o r m a t i o n was used t o c a l c u l a t e a continuous i n f u s i o n dose r a t e o f ara-C t h a t would m a i n t a i n t h e d e s i r e d steady s t a t e c o n c e n t r a t i o n o f ara-CTP (19) * zyxwv zyxwvut 56 ara-CTP a f t e r a t e s t dose o f 3 g/m 2 (19,21). The area under t h e c u r v e f o r c e l l u l a r a c c u m u l a t i o n and e l i m i n i a t i o n o f ara-CTP was used t o c a l c u - l a t e a dose o f ara-C t o be i n f u s e d c o n t i n u o u s l y o v e r 96 h r t h a t was p r o jected as necessary t o m a i n t a i n t h e level of ara-CTP i n circulating b l a s t s a t g r e a t e r t h a n 75 IM d u r i n g t h e i n f u s i o n (19,21). The median c o n c e n t r a t i o n o f ara-CTP was 75 pM. The steady s t a t e a r a - CTP c o n c e n t r a t i o n s t h a t were achieved exceeded t h e c a l c u l a t e d t a r g e t l e - v e l s by a mean o f 3.3 +- 1.8-fold. It i s l i k e l y t h a t t h i s was due t o s a t u r a t i o n o f t h e r a t e o f ara-CTP accumulation d u r i n g t h e b o l u s i n f u s i o n , l e a d i n g t o an u n d e r e s t i m a t i o n o f t h e a b i l i t y o f t h e c e l l s t o a n a b o l i z e ara-C ( 2 2 ) . Pharmacologic e v a l u a t i o n o f t h e p a t i e n t s t r e a t e d on t h i s p r o t o c o l presented t h e o p p o r t u n i t y t o compare t h e steady s t a t e concentrat i o n s o f ara-CTP d u r i n g continuous i n f u s i o n w i t h t h e response o f each p a t i e n t ( F i g . 4). There was a s i g n i f i c a n t c l u s t e r i n g o f steady s t a t e ara-CTP values f o r t h e p a t i e n t s who achieved complete r e m i s s i o n between 79 VM and 206 IM (10 o f 17). We have d e s i g n a t e d t h i s as t h e " t h e r a p e u t i c range". rast, o n l y 2 o f 21 p a t i e n t s whose steady s t a t e ara-CTP I n cont- concentrations f e l l o u t s i d e o f t h i s range achieved a complete r e m i s s i o n (p<0.005). The c e l l s o f t h e p a t i e n t s w i t h r e s i s t a n t disease appear t o have been e i t h e r i n c a p a b l e o f m a i n t a i n i n g ara-CTP l e v e l s up t o t h e t h e r a p e u t i c range o r f a i l e d t o respond, d e s p i t e c e l l u l a r ara-CTP concentrations of hundred micromolar. The f o r m e r group may have been r e s i s t a n t t o ara-C t h e r a p y because o f an i n a b i l i t y t o accumulate ara-CTP. several W h i l e t h i s charac- t e r i z e s o n l y a m i n o r p o r t i o n o f t h e p a t i e n t group s t u d i e d (see a l s o 19), t h e i r tumor c e l l s may have biochemical p r o p e r t i e s s i m i l a r t o t h e " k i n a s e d e f i c i e n t " c e l l s . Kinase d e f i c i e n c y i s t h e most common f o r m o f ara-C r e s i s t a n c e seen i n e x p e r i m e n t a l systems (23,24). The p a t i e n t s w i t h r e s i s - t a n t disease i n t h e h i g h steady s t a t e ara-CTP group m i g h t have f a i l e d f o r reasons r e l a t e d t o u n f a v o r a b l e c e l l u l a r k i n e t i c s o r because o f an insens i t i v i t y o f DNA s y n t h e t i c processes t o t h e a c t i v e m e t a b o l i t e . The leukemic c e l l s o f p a t i e n t s who experienced a p r o f o u n d a n t i l e u k e m i c e f f e c t b u t who d i e d w i t h a p l a s t i c marrow had a wide d i s t r i b u t i o n o f steady s t a t e ara-CTP concentrations. causes, pharmacologic While these i n d i v i d u a l s d i e d from a v a r i e t y o f r e s u l t s *suggest t h a t t h e p o r t i o n o f t h i s group whose steady s t a t e ara-CTP l e v e l s were w i t h i n t h e t h e r a p e u t i c range m i g h t have achieved r e m i s s i o n had t h e i r s u p p o r t i v e c a r e been s u c c e s s f u l . A d i f f e r e n t c o h o r t w i t h e x t r e m e l y h i g h steady s t a t e ara-CTP would n o t have b e n e f i t e d f r o m more i n t e n s i v e t h e r a p y . values p r o b a b l y Conversely, t h e i r 57 c l i n i c a l course m i g h t have r e s u l t e d i n remission r a t h e r than a p l a s t i c death had t h e y received a lower dose o f ara-C. I n conclusion, s i g n i f i c a n t r e l a t i o n s h i p s e x i s t between pharmacokinetics o f c e l l u l a r ara-CTP and p a t i e n t response t o s i n g l e - d r u g ara-C therapy f o r zyxwv zyx r e f r a c t o r y leukemia. A c o r r e l a t i o n between t h e c l i n i c a l response w i t h t h e minimum t r o u g h ara-CTP concentrations d u r i n g i n t e r m i t t e n t high-dose ara-C therapy and t h e lowest values associated w i t h response d u r i n g continuous i n f u s i o n t r e a t m e n t i n d i c a t e s t h a t 75 lib! ara-CTP i s a lower l i m i t f o r e f f e c t i v e treatment. Median ara-CTP t r o u g h values f o r p a t i e n t s who responded t o i n t e r m i t t e n t high-dose ara-C (196 pM) and t h e upper bounds f o r responders t o continuous i n f u s i o n therapy i n d i c a t e t h a t 200 yM may be t h e upper l i m i t o f t h e t h e r a p e u t i c range. Recognition o f a l i k e l y t h e r a p e u t i c range o f t h e ara-CTP c o n c e n t r a t i o n i n leukemia c e l l s provides an o b j e c t i v e t a r g e t f o r t h e design o f f u t u r e chemotherapeutic regimens. I n d i v i d u a l i z e d therapy based on t h e c e l l u l a r pharmacology o f ara-CTP i s a p o s s i b l e approach t o a c h i e v i n g t h e r a p e u t i c c e l l u l a r drug concentrations (19,Zl). ACKNOWLEDGEMENTS This work was supported i n p a r t by g r a n t s CA28153 and CA32839 from t h e N a t i o n a l Cancer I n s t i t u t e , DHHS. The authors g r a t e f u l l y acknowledge t h e e x c e l l e n t t e c h n i c a l assistance and d e d i c a t i o n o f Theresa Adams. Sherri Chubb, Rodney C r o f t , and B i l l i e Nowak. REFERENCES 1. Keatlng MJ, McCredie KB. Bodey GP. Smith TL, Gehan E. F r e i r e i c h EJ. Improved prospects f o r long-term s u r v i v a l i n a d u l t s w i t h acute myelogenous leukemia. 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(P815) or Biochem Pharmacol 1982;31:3831-7. Address f o r correspondence: W i l l i a m P l u n k e t t , Ph.D. Dept o f Chemotherapy Research The U n i v e r s i t y o f Texas M.D. Anderson Hospi t a l and Tumor I n s t i t u t e a t Houston 6723 8 e r t n e r Avenue Houston, Texas 77030. USA