Cancer Chemotherapy

3 Biochemical Pharmacology of the Anthracycline Antibiotics N I C H O L A S R.

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Downloaded by EMORY UNIV on March 10, 2016 | http://pubs.acs.org Publication Date: June 1, 1976 | doi: 10.1021/bk-1976-0030.ch003

Biochemistry Section, Baltimore Cancer Research Center, National Cancer Institute, 3100 Wyman Park Dr., Baltimore, M d . 21211

Medical s c i e n t i s t s have a cumulative eighteen year clinical experience w i t h the a n t h r a c y c l i n e a n t i b i o t i c s , adriamycin and daunorubicin. Daunorubicin was introduced i n t o clinical trials in I t a l y by F a r m i t a l i a and i n France by Rhône-Poulenc in 1964. A few years l a t e r , adriamycin was announced by F a r m i t a l i a and started into clinical trials. These trials i n d i c a t e d t h a t daunor u b i c i n was an impressive agent f o r remission i n d u c t i o n i n acute leukemia whereas adriamycin had a wider spectrum of a c t i v i t y a g a i n s t s o l i d tumors as w e l l as leukemias (1,2,3,4,5). There i s little doubt t h a t the a n t h r a c y c l i n e a n t i b i o t i c s i s o l a t e d from Streptomyces have had a major impact on the prospects of cancer chemotherapy, because o f t h e i r degree o f activity f o r i n d u c i n g remission or stopping p r o g r e s s i o n o f malignant growth and t h e i r wide ranging activity a g a i n s t malignancies. However, complicating t h e i r u s e f u l n e s s are the t o x i c s i d e e f f e c t s a s s o c i a t e d with t h e i r a d m i n i s t r a t i o n : myelosuppression, s t o m a t i t i s , nausea, vomiting, a l o p e c i a , e l e c t r o c a r d i o g r a p h i c changes, and a s e r i o u s cumulative dosage r e l a t e d myocardiopathy (6). Although the agents are n e a r l y i d e n t i c a l s t r u c t u r a l l y ( F i g . 1), adriamycin's potency i s about one and one-half times g r e a t e r than daunorubicin in both pharmacologic e f f e c t and i n t o x i c i t y . The only d i f f e r e n c e between the complex molecules is a t the number fourteen carbon p o s i t i o n where adriamycin has an a d d i t i o n a l hydroxyl. Although many s t r u c t u r e s were reviewed e x t e n s i v e l y i n the preceeding d i s c u s s i o n by Dr. Henry, I t h i n k we can reexamine the fundamental s t r u c t u r e from a biochemical view p o i n t . The a n t i b i o t i c molecule is double headed with a hydrophobic end and a h y d r o p h i l i c end. The A, B, and C r e s o n a t i n g r i n g system comprises the hydrophobic end; and the D r i n g , with attached amino sugar are the h y d r o p h i l i c head o f the molecule. In a d d i t i o n to t h i s dual p h y s i c a l c h a r a c t e r i s t i c , the molecules are amphoteric with a b a s i c amino group and the a c i d i c p h e n o l i c hydroxyls of the a n t h r a c y c l i n e r i n g . These p h y s i c a l p r o p e r t i e s p l u s abundance of r e a c t i v e s i t e s o f f e r the p o t e n t i a l f o r numerous i n t e r a c t i o n s with cellular components such as n u c l e i c a c i d s , p r o t e i n s , and lipids.

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Sartorelli; Cancer Chemotherapy ACS Symposium Series; American Chemical Society: Washington, DC, 1976.


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ADRIAMYCI1M

OH

DAUNORUBICIN

H

Figure 1.

Structures of adriamycin and daunorubicin


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Figure 2. Chromosomal damage in human cells induced by daunorubicin (10)

Figure 3.

Kinetics of adriamycin and daunorubicin uptake at 37°C leukemia cells in vitro

in L1210 murine



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The a v a i l a b i l i t y o f b i o t r a n s f o r m a t i o n groups i n c r e a s e s even f u r t h e r the c o m p l i c a t i o n s o f drug d i s p o s i t i o n i n mammals. A c e n t r a l s t r u c t u r a l component of both molecules i s the g l y c o s i d i c bond j o i n i n g the sugar, daunosamine, and the anthrac y c l i n e nucleus. T h i s bond i s very l a b i l e to chemical and enzymatic cleavage; and the s c i s s i o n of the bond i n a c t i v a t e s the compound. For t h i s reason n e i t h e r adriamycin nor daunorubicin are g i v e n o r a l l y s i n c e the g l y c o s i d i c bond i s s p l i t i n the gastrointestinal tract. Both adriamycin and daunorubicin must be g i v e n p a r e n t e r a l l y to be e f f e c t i v e . When adriamycin o r daunorubicin are administered i n t r a venously to animals o r i n humans, the compounds are r a p i d l y absorbed i n t o c e l l s and l o c a l i z e d p r i m a r i l y i n the c e l l nucleus (7,8,9). There i s good anatomical and chemical evidence to i n d i c a t e t h a t the compounds both l o c a l i z e i n the c e l l nucleus and a l s o i n t e r a c t w i t h the nuclear m a t e r i a l . Chromosomal preparat i o n s from human lymphocytes show extensive damage a f t e r daunor u b i c i n treatment ( F i g . 2) (10). The chromosomes show fragmentat i o n , r i n g formation, s p l i t t i n g , s e p a r a t i o n , and other forms of damage. Since both adriamycin and daunorubicin have a unique f l u o r e s c e n c e , they can be d e t e c t e d by f l u o r e s c e n c e microscopy i n cells. With t h i s technique the drug f l u o r e s c e n c e i s l o c a l i z e d a t the n u c l e a r s t r u c t u r e s (8). I t i s remarkable t h a t the seemingly i n s i g n i f i c a n t e x t r a hydroxyl o f adriamycin makes not only a more potent compound, but a l s o a drug w i t h wider spectrum of a c t i v i t y a g a i n s t malignancy. For t h i s reason the drugs have been s t u d i e d and compared. Since the whole animal s t u d i e s are q u i t e complex, we o r i g i n a l l y compared adriamycin and daunorubicin i n mammalian c e l l c u l t u r e c o n t a i n i n g L1210 murine leukemia c e l l s . L1210 c e l l s and other mammalian c e l l l i n e s are i n h i b i t e d more by daunorubicin than by adriamycin. Daunorubicin has a g r e a t e r a c t i v i t y f o r i n h i b i t i n g both DNA and RNA metabolism as seen i n our l a b o r a t o r y and i n others (Table 1) (11,12,13). T h i s , o f course, i s the opposite o f the c l i n i c a l f i n d i n g s and o f f i n d i n g s i n v i v o and was a p e r p l e x i n g o b s e r v a t i o n . The p o s s i b i l i t y remained t h a t the two drugs may be e n t e r i n g the c e l l s at d i f f e r e n t r a t e s , so we compared the accumulation of adriamycin and daunorubicin i n the L1210 c e l l s . Daunorubicin i s taken i n t o the c e l l much more r a p i d l y and to a higher degree than i s adriamycin ( F i g . 3) (11). Therefore the e f f e c t i v e l e v e l of drug i n the c e l l i s much h i g h e r i n the case of daunorubicin. T h i s r e s u l t s i n an apparent s u p e r i o r i t y of daunorubicin over adriamycin a t i n h i b i t i n g n u c l e i c a c i d s y n t h e s i s . However, the s p e c i f i c a c t i v i t i e s o f the amount o f drug i n the c e l l s compared to the amount o f i n h i b i t i o n produced by the drug i n d i c a t e s t h a t adriamycin has a g r e a t e r s p e c i f i c a c t i v i t y than does daunorubicin (11) . A l t e r a t i o n s on the s i d e chain a t the 9 p o s i t i o n have profound e f f e c t s on the p o l a r i t y and s o l u b i l i t y o f the a n t h r a c y c l i n e a n t i biotics. T h i s apparently d i r e c t l y e f f e c t s a b s o r p t i o n of the drug



3

HeLa C e l l

HeLa C e l l

3.

4.

survival

survival

c

L1210 incorporation Uridine-2-14 i n t o RNA

L1210 incorporation Thymidine-methyl H

2.

1.

System

(13)

Kim & Kim

0.03

0.2

Meriwether & Bachur (11)

Meriwether & Bachur (11)

Ref.

DiMarco e t a l (12)

(yM)

0,9

ID 50 μΜ

29 60 73

27 55 74

Drug cone

1.9

12 36 57

16 32 59

Adriamycin Daunorubicin Percent I n h i b i t i o n

In V i t r o I n h i b i t i o n of C e l l A c t i v i t i e s by Adriamycin and Daunorubicin

Table 1



2.

Malony sarcoma v i r u s F r i e n d Leukemia v i r u s Rausher Sarcoma v i r u s

RNA Tumor V i r u s DNA Polymerase

4

E. C o l i - T / L 1 4 1 phage DNA polymerase

67 56 64

22 58 89 98

67 64 66

15 39 72 95

Adr. Daun. % Inhibition

0.066 0,066 0.066

0.011 0.028 0.057 0.114

Drug Cone. (mM)

Chandra e t a l (15)

Goodman e t a l (14)

Ref.

Adriamycin and Daunorubicin I n h i b i t i o n o f DNA Polymerases

Table 2



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i n t o the c e l l . We have compared metabolites and chemical d e r i v a t i v e s o f adriamycin and daunorubicin with changes only on the C9 side chain. Compounds with the lowest " p o l a r i t y " as measured by p a r t i t i o n c o e f f i c i e n t have the h i g h e s t uptake i n t o L1210 c e l l s . There i s a near l i n e a r r e l a t i o n s h i p of p o l a r i t y to drug uptake (Fig. 4 ) . In order to avoid the dynamics of the c e l l membrane, we i n v e s t i g a t e d drug e f f e c t s d i r e c t l y in_ v i t r o on a group of p u r i f i e d DNA polymerases s u p p l i e d by Dr. Maurice Bessman. The polymerases were T4 bacteriophage induced i n E. C o l i . I n h i b i t i o n o f the DNA polymerases was s l i g h t l y g r e a t e r with adriamycin than with daunor u b i c i n (Table 2) (14). From our data and from the data of others (Table 2) (15), i t i s d i f f i c u l t to e x p l a i n why adriamycin has a higher potency i n mammals. The potency o f the two agents i s nearly i d e n t i c a l i n these i n v i t r o DNA polymerase assays. In a d d i t i o n , t h e i r b i n d i n g to DNA i s q u i t e s i m i l a r and the unwinding angle t h a t they produce i s very s i m i l a r . This approach does not e x p l a i n the c l i n i c a l d i f f e r e n c e s seen with adriamycin and daunorubicin. Studies on p u r i f i e d T4 phage induced polymerases uncovered another unpredicted a c t i o n o f the a n t h r a c y c l i n e s which may have s i g n i f i c a n c e i n t h e i r s e l e c t i v e a c t i o n a g a i n s t malignant c e l l s . The p u r i f i e d phage polymerases f a l l i n t o three phenotypic groups, mutagenic, w i l d type, and antimutagenic. The mutagenic s t r a i n s have a high mutation frequency, the antimutagenic s t r a i n s have a low mutation frequency, and the w i l d type f a l l s between with a normal mutation r a t e . We observed that at low drug concentrations the mutagenic polymerases were not i n h i b i t e d but were stimulated (ex., L56) ( F i g . 5) whereas the antimutagenic polymerases (L141) were uniformly i n h i b i t e d as p r e d i c t e d (14). Simultaneously, a 5' exonuclease which i s a p a r t of each enzyme and i s t h e o r i z e d to be the e d i t i n g or e r r o r c o r r e c t i n g system f o r the phage DNA s y n t h e s i s i s uniformly i n h i b i t e d a t a l l concentrations of the drugs. This means t h a t at low drug l e v e l s the mutagenic DNA polymerase i s made even more mutagenic s i n c e the low f i d e l i t y polymerase i s s t i m u l a t e d while the a s s o c i a t e d c o r r e c t i o n system i s i n h i b i t e d . T h i s does not occur i n the antimutagenic enzymes which are uniformly i n h i b i t e d . T h i s may be a clue to how these drugs are s e l e c t i v e f o r leukemic c e l l s . Since the DNA polymerase i n acute leukemia c e l l s i s b e l i e v e d to be mutagenic (16), the drugs may cause the leukemic c e l l s to i n c o r p o r a t e too many e r r o r s i n t o t h e i r DNA to be compatible with s u r v i v a l . As p r e v i o u s l y s t a t e d , a l l of the s t u d i e s i n v e s t i g a t i n g the p h y s i c a l - c h e m i c a l i n t e r a c t i o n s of adriamycin and daunorubicin and the comparative e f f e c t s on c e l l and t i s s u e c u l t u r e i n h i b i t i o n , on enzymes such as DNA polymerase and RNA polymerase, or on b i n d i n g to DNA have shown l i t t l e i f any d i f f e r e n c e between the two compounds. I t i s only i n whole animal s t u d i e s that s i g n i f i c a n t d i f f e r e n c e s between adriamycin and daunorubicin e f f e c t s are e a s i l y d i s c e r n a b l e . T h i s i n d i c a t e s that other u n r e a l i z e d mechanisms are


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1.6

Daunorubicin

1.4-

Ia> ο

1.2 -

to

Ο

Rubidazone Δ/ ο Ε S

0.8


LU

g

Adriamycin 0.6

ο Daunorubicinol

D CD ZD

rr 0.4 û 0.2 • Adriamycinol 1.0

2.0

3.0

4.0

PARTITION COEFFICIENT Figure 4.

Relationship of drug uptake into L1210 cells and n-butanol-water partition coefficient of several anthracycline derivatives



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r e s p o n s i b l e . From t h a t p o i n t of view we can examine other aspects of the biochemical pharmacology of these compounds i n the mammal such as t h e i r metabolic d i s p o s i t i o n . P a t i e n t s t r e a t e d with a d r i a mycin or daunorubicin excrete s i g n i f i c a n t q u a n t i t i e s of the drugs and the metabolites i n the u r i n e (17,18). From these metabolites we have been able t o determine q u a l i t a t i v e l y and q u a n t i t a t i v e l y the metabolic sequences and pathways which the drugs t r a v e r s e (19) . The major metabolic step f o r both adriamycin and daunorubicin i n mammals i s v i a the keto r e d u c t i o n r e a c t i o n (20,21,22) ( F i g . 6 ) . T h i s r e a c t i o n i s c a t a l y z e d by a s o l u b l e aldo-keto reductase found i n a l l c e l l s analyzed. The enzyme r e q u i r e s NADPH as a c o f a c t o r and produces the p h a r m a c o l o g i c a l l y a c t i v e products, d a u n o r u b i c i n o l and adriamycinol r e s p e c t i v e l y from daunorubicin and adriamycin. A f t e r animals are administered adriamycin, the parent drug i s the predominant m a t e r i a l found i n t i s s u e s a t e i g h t hours; but a d r i a mycinol i s a s i g n i f i c a n t m e t a b o l i t e (22). A f t e r daunorubicin a d m i n i s t r a t i o n , d a u n o r u b i c i n o l i s the major drug form found i n t i s s u e s and excreted (22). Since the aldo-keto reductase produces p h a r m a c o l o g i c a l l y a c t i v e m e t a b o l i t e s which d i f f e r from the parent i n p h y s i c a l - c h e m i c a l c h a r a c t e r i s t i c s , t h i s enzyme and i t s l e v e l i n tumor and t i s s u e may help determine the pharmacodynamic e f f e c t s o f these drugs. In studying the t i s s u e d i s t r i b u t i o n o f aldo-keto reductase, we examined human t i s s u e s obtained a t autopsy. A l l human t i s s u e s examined c o n t a i n a c t i v e l e v e l s o f aldo-keto reductase (23). Daunorubicin i s a b e t t e r substrate f o r the a l d o keto reductases i n most animal t i s s u e s and with p u r i f i e d aldo-keto reductase p r e p a r a t i o n s (21,22,23). Both subsequent t o o r concurrent with carbonyl r e d u c t i o n , the a n t h r a c y c l i n e g l y c o s i d e s are metabolized by g l y c o s i d a s e s i n most t i s s u e s (20,24,25). These microsomal enzymes s p l i t the drug or reduced metabolites i n t o aglycones and f r e e amino sugar ( F i g . 7) ( r e a c t i o n s : I -> IV, I I I I I , I I -*• V) . The major g l y c o s i d a s e (I •> IV, II V) i s r e d u c t i v e i n i t s mechanism, r e q u i r e s NADPH f o r a c t i v i t y , produces deoxyaglycones (IV,V) and i s i n h i b i t e d by oxygen (24). As i n the carbonyl r e d u c t i o n , daunorubicin i s the b e t t e r substrate f o r the g l y c o s i d a s e s than i s adriamycin. The aglycone products o f these r e a c t i o n s apparently have no a n t i c a n c e r a c t i v i t y . Since they are l i b e r a t e d i n t r a c e l l u l a r l y , however, these aglycones may have a c t i v i t i e s which are not r e a d i l y apparent and more r e s e a r c h i s necessary to r e s o l v e t h i s q u e s t i o n . The aglycones because o f t h e i r high l i p i d , low water s o l u b i l i t y , are e x c r e t e d p r i n c i p a l l y i n the b i l e ; but must be conjugated f i r s t to i n c r e a s e water s o l u b i l i t y (22,25,26,27,28). P r i o r to conjugation, i n order to o f f e r a more appropriate conjugation s i t e , there i s a 4-0 demethylation o f both daunorubic i n and adriamycin to y i e l d the demethylated aglycone (V + VI) (19). Demethylated aglycones serve as substrate f o r O - s u l f a t i o n and O-beta-glucuronidation y i e l d i n g the 4-0-sulfate conjugate and 4-0-beta glucuronide conjugate (VI VI, VI -> VIII) to be


Sartorelli; Cancer Chemotherapy ACS Symposium Series; American Chemical Society: Washington, DC, 1976. Figure 6.

Aldo-keto reductase reaction


1

5

co

Ci"

S

«s.

3*

Ci

3

δ.

Χ

td > ο


Figure 7.

Pathways for human metabolism of adriamycin (19)


S3


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e x c r e t e d . A l l o f these m a t e r i a l s are found i n human b i l e and human u r i n e (26,27,28). Since the reduced compounds, adriamycinol and d a u n o r u b i c i n o l , show s u b s t a n t i a l a n t i c a n c e r a c t i v i t y , i t i s p o s s i b l e t h a t other m e t a b o l i t e s may have o t h e r types of a c t i v i t y . There are numerous m e t a b o l i t e s w i t h the p o t e n t i a l f o r these substances to be b i o l o g i c a l l y as w e l l as p h a r m a c o l o g i c a l l y a c t i v e . The p o t e n t i a l s are being examined a t the p r e s e n t time. S t u d i e s to date s t i l l have not e x p l a i n e d f u l l y the pharmaco l o g i c d i f f e r e n c e s between adriamycin and d a u n o r u b i c i n . There are major d i f f e r e n c e s i n the d i s p o s i t i o n and metabolism of the agents i n v i v o and i n the p e n e t r a t i o n through c e l l membranes as we have shown. A l s o d i f f e r e n c e s i n t h e i r e f f e c t s on immunosuppression have been r e p o r t e d (29,30). The evidence i s accumulating t o d e f i n e the p r e c i s e mechanisms of a c t i o n o f these drugs. The a n t h r a c y c l i n e a n t i b i o t i c s show promise as cancer chemot h e r a p e u t i c agents. I t i s apparent t h a t both adriamycin and daunorubicin have complex i n t e r a c t i o n s with b i o l o g i c systems. I f e e l t h a t we can look forward to encouraging progress i f we l e a r n and understand more about the b i o l o g i c a l i n t e r a c t i o n s of these agents. Then we may be b e t t e r prepared to design analogs t h a t are s a f e r and more e f f e c t i v e i n t h e i r purpose. LITERATURE CITED 1. B o i r o n , Μ., J a c q u i l l a t , C., W e i l , M., Tanzer, J . , Levy, D., S u l t a n , C., and Bernard, J . : Lancet (1969) 1, 330-333. 2. Wiernik, Ρ.Η. and S e r p i c k , A.A.: Cancer Res. (1972) 32, 2023-2026. 3. Tan, C., Tasaka, Η., Yu, K.P., Murphy, M.L., and Karnovsky, D. Cancer (1967) 20, 333. 4. Bonadonna, G., M o n f a r d i n i , S., Delena, M., F o s s a t i - B e l l a n i , F. and B e r e t t a , G.: Cancer Res. (1970) 30, 2572-2582. 5. Middleman, E., Luce, J . , and F r e i , E.: Cancer (1970) 28, 837-843. 6. Blum, R.H. and C a r t e r , S.K.: Ann. I n t . Med. (1974) 80, 249-259. 7. Bachur, N.R., E g o r i n , M.J., and H i l d e b r a n d , R.C.: Biochem. Med. (1973) 8, 352-361. 8. E g o r i n , M.J., Hildebrand, R.C., Cimino, E.F., and Bachur, N.R.: Cancer Res. (1974) 34, 2243-2245. 9. Silvestrini, R., Gambarucci, C., and Dasdia, T.: Tumori (1970) 56, 137-148. 10. Whang Peng, J . , L e v e n t h a l , B.G., Adamson, J.W., and Perry, S. Cancer (1969) 23, 113-121. 11. Meriwether, W.D. and Bachur, N.R.: Cancer Res.(1972) 32, 1137-1142. 12. DiMarco, Α., Zunino, F., Silvestrini, R., Gambarucci, C., and Gambetta, R.A.: Biochem. Pharmacol. (1971) 20, 1323-1328.


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13. 14. 15. 16. 17. 18. 19.


20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30.

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