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Potentiation of the carcinostatic action of azauridine by chloramphenicol
0,015); howe\cr. ;Lsignliicant dccrcasc 111“~LISCIC slgniticant increase in liver to serum AtB ratios (P serum AIR (P c 0.001) occurred. Whether the uptake of ~-anlino~~sub~ltyri~acid serves as a satisfactory model for the transport ofarl amino acids ts unknown. It has recently been observed that its distributi(~it is similar to that of glycine.” In any event, we ha\,e described a very c‘arly estrogen cffcct on the accumulation of thl\ cl-amino acid which corresponds i” time with changes in distribution and metabolic fate of natural amino acids. Although this effect of estrogen is most pronounced in the uterus, there is :I perceptible shift in the distribution of AIB in muscle in a direction opposite to that observed for the uterus.
* Senior Post-doctoral
.a Pre-doctoral
Fellow, U.S. Public Health Servtce. Trainee, U.S. Public Health Service Training (irant Xi-~?1 REFERENCES
I. S. M. KALMAN and M. E. LOMISRO~O,J. f’/xr~tt,. Esp. Tlwrc/p. 131, 265 ( 1X111. 2. M. W. NOALL, T. R. RIGGS, L. M. WALKER and H. N. CFrnlsst:usrN, .Scie/rcr126. 1003 (19.57). 3. M. W. NOALL, Biuchinf. Biopitys. Actu 40, 180 (1960). 4. M. A. TELFER, .4&. Bio&etu. Bioplqx 44, t 11 (1953). 5. G. C. MUELLER. J. CZftr. ~~~~f~~rj/~. 13, 836 (19%). 6. K. L. MANCHESTERand F. G. YOIINC;.&when/. J. 75. 487
~otentiation
of the careinostatic
(IWI).
action of azauridine
by chtoraniphenicol~
CHLORAMPHENICOL,a well-established
antibiotic agent III the chemotherapy of bacterial Infections. has also been shown to depress hematopoiesis in man.l~ y This toxic effect upon bone marrow function led to further i”vestigatton of the possible utility of ~h~oramphentcol as a carcinostatic agent in the treatment of ~xp~rin~ent~~i’i,1. 3 and human “coplasms6 Although ineffective as a carcinostatic agent irt t,ii~), ~~~lor~trnph~~~i~~~~ inhibits the growth of tnammalian cells in culture.: Observations made during the course of treatment with h-azauridine (thu ribonuclcoside of harauracil)H of three patients with leukemia,f suggested that potcntiation of its action by chloramphenicol might have occurred when the antibiotic agent was given for the treatment of intercurrent bacterial infections. Preliminary studies of combinations of these compounds in the chemotherap!, of an experimental neopfasm in “lice have confirmed this impression and the results arc presented m this comtlltlni~atiol~. METHODS Male DBF, hybrid mice, h--8 weeks old and weighing 19-24 g, were used I” :III cxperimenrs. All animals were fed powdered Purina Lab Chow, with and without chlorampherricol:: added 111 concentrations ranging fro”1 0.5 to 2.0 per cent. Azauridine$ was added to the drinking water ol * This invest~~tion was supported, in part, hq a research grant (CY-2817) front ihe Natittnai Cancer institute, U.S. Public Health Service. 7 S. S. Cardoso. : Generously supplied by Parke, Davis and Company. 4 Provided through the Cancer Chemotherapy National Service Center of thu U.S. Public Elcalltt Service (Bethesda, Maryland).
Short communications
253
appropriate
groups of animals in concentrations ranging from 0.15 to 0.5 mg per ml and was available of the L5178Y leukemia was made by removing, from a freshly sacrificed donor mouse, 1 ml of ascites fluid and immediately transferring this to a test tube containing 9 ml of culture medium;O of this dilution of ascitic fluid, 0.1 ml, containing 1.553.0 :i lo8 viable leukemia cells, was injected subcutaneously into the right flank of each mouse, in order to produce a solid neoplasm. In all the experiments the recipient mice were inoculated with the leukemic cells within 15 min after the withdrawal of the latter from the donor mouse. Treatment with azauridinc and chloramphenicol was always begun 24 hr after the subcutaneous inoculation of the neoplastic cells into individually weighed mice. On the day following the final dosage of either azauridine or chloramphenicol, or of both agents, each mouse was weighed, sacrificed, and its tumor was removed and weighed. The average weight of the tumors in each treated group was compared with that of the controls, and the results were recorded in terms of percentage inhibition of neoplastic growth. For experiments in vitro, L5178Y cells were grown in culture, as described by Fischer cr crl.“‘, in the presence or absence of various concentrations of chloramphenicol. ad libirum. Transmission
RESULTS
AND
DISCUSSION
In the studies of the effect of chloramphenicol upon the growth of the L5 178Y cells in citro, concentrations of the antibiotic agent of IO-100 pg per ml of tissue culture medium inhibited the rate of cellular reproduction; the concentration required for 50 per cent inhibition of growth was 1Org per ml of tissue culture medium. In contrast to these results with chloramphenicol in culture, even high doses of the antibiotic agent, when tested for activity with respect to the inhibition of the growth of the lymphoblasts growing as a solid tumor in mice, caused only moderate carcinostasis (Table I). However, in combination with azauridine, chloramphenicol, in doses which by themselves caused no inhibition of growth, potentiated the carcinostatic effect of azauridine (Table 1). Achromycin, an antibiotic with a similar spectrum of antibacterial activity to that of chloramphenicol, failed to increase significantly the inhibition of tumor growth obtained with azauridine alone (Table I).
TABLE
I. EFFECTS
OF AZAURIDINE,
CHLORAMPHENICOL
LYMPHONAS
Change in Treatment*
-
Controls CAP. 0.5 7; AzUR, 0.15 mg/ml AzUR, 0.15 mg/ml. !~CAP, 0.5 “<,
I
Mortality
ACHROMYUN
mt17.7 t24.7
276
124.4
O/IO
121
kl6.2
4.0
Oil0
58
i 8.7
2.0 1.1 0.7
o/20 o/20 0120
~
242 277 174
f 18.6 ;.20.2 : 16.1
o/20
~
79
I_IO.2
~
O/IO
O/IO 0110 o/10 o/10
-
3.0
I.4
I
ON
THE GROWTH
Average tumor weight, mg (+ s.e.) 363 326
2.0
0.5 - 2.0 t 0.5 ..~0.5
.
AND
nr
IN MICE
I
’
body weight, g
Controls CAP, 0.5 ;/, CAP, 1.0% Achromycm, I ,O% AzUR, 0.25 mg/ml AzUR. 0.25 me/ml. r~achrbmycin,? .O“/, AzUR, 0.25 mg/ml, : CAP, 0.5 7;
(L5178Y)
i ~
lmhibition of tumor weight per cent ~
~ 66
,-
841-
67: -I
* For chloramphenicol (CAP) and achromycin, the concentrations given refer to the percentage of the antibiotic agent in the diet, for azauridine (AzUR), the concentrations given refer to those administered in the drinking water. t AzUR vs. AzUR : CAP: P =- 0.001. CAP: I’ < OWl. : AzUR vs. AzUR
254
Short
communications
The mechanism by which chloramphenicol potentlates the effects of arauridmc has not yet been established. It is known that aznuridine, after its intrnccllular cnzymic conversion to azauridinc 5’-phosphate. acts as a compctitivc inhibitor of orotidylic acid decarboxylase. in this manner interfcring with the biosynthesis of pyrimidincs &J FXXO.*‘~lB Evidence has been prcscnted that. in bacteria. chloramphcnicol may exert an inhibitory eKcct primarily upon protein synthesis:‘:‘, If however, it has not been shown that this antibiotic agent can product a similar effect in mammalian cells. On the other hand, as demonstrated in this laboratory’” and clscwhere by IFnllon er (I/..* the rate of conversion of erotic acid. via orotidylic acid, to uridinc nuclcotides by isolated lcukcmic cells from patients treated with azauridinc is generally inhibited at first; subsequently, however, the magnitude of these conversions in increased to Icvcls close to or above those found in ceils obtained before treatment with azauridinc. This increased enz!mic activity may reflect the presence in the ceils of higher levels of the enzyme orotidylic acid decarboxyiase.“’ If thcsc assumptions are correct, chloramphenicol may suppress this attempt on the part of the cells to compensate for the inhibitlon by azauridine 5’-phosphate of the dccarboxylation of orotidylic acid. through the formation of additional orotidylic acid decarboxylasc. and perhaps of enzymes concerned with carlicr steps in the pathwa) of pyrimidine biosynthesis & ,UKO. Regardless of the nicch~lnisiil involved. the potcilti~~tioii of the carcinostatic cl&t of ~~z~~~lri~~inc by chloramphenicol suggests that controlled studies should be carried out with combinations of these two drugs in the treatment of human neoplastic disease. Although the data obtained in mice with this combination show that chloramphcnicol increases the toxicity of azauridine for the host, the situation may not bc comparable to that which obtains in man. since treatment with azauridinc of approximately 50 patients has not disclosed any appreciable toxicity, while in many casts it has favorably influenced the course of certain types of acute leukemia. + In fact. arauridine is a unique agent in man, since even massive doses have failed thus far to inhibit normal liem~~topoiesis,~ a situ~~tion in marked contrast to that which obtains with other anti-cancer agents, or, indeed. with ztzauridinc in such mammalian species as the dog.*’ In conclusion. cvidencc has been presented to indicate that chloramphenicol potcntiates the carcinostatic etl’cct of azauridinc upon L5178Y cells, when grown as lymphomas in mice. as well as the toxicity of nzauridine for this host. A possible mechanism by which this potcntiation is achicbcd has been discussed, and the USC of c~~lora~iph~~iicol in combination with azauridinc in the treatment of neoplastic disease in man is suggested. Pllthough the increased potency of the combin~~tion of azauridine and chloramphcnicoi engenders greater host-toxicity in mice, sclcctivity of action might be obtained in man, since azauridine by itself has cvokcd no significant toxicity in leukemic patients in whom the drug has caused unequivocal evidcncc of objective improvement.
* Personal communication. t These studies, carried out by groups at Yale, the National Cancer Institute, and the Koswell Park Memorial Institute, have been summarized, in part”, lx and full papers descrlbmg these clinical investigations are in preparation. z Postdoctoral support for S. S. Cardoso was provided by a Rcscarch Training Grant (CRTY5012) from the National Cancer Institute, U.S. Public Health Service. $ Present address: Dcp~~rtln~nt of Pliarm~~col[)~y, University of Vermont College of Medicine. Burlington. Vermont. REFERENCES I. 2. 3. 4. 5. 6.
H. R. D. H.
WELCH, C. N. LLWIS and I. KERLAN. Anriht’ot. CAM/C%rt~rotlrer. 4, 607,
1954. Houc~~so~, Laxet 1, 285, 1954. M. GREENBERG and E. M. C-IF., Cmcer Res. Srcppf. 1, 54 (1953). E. SKIPPER and J. R. THUMPSON, Cmxr Rex. Src,u,r~f.2, 151 (19551. H. LEITKE, Cortcei- Res. Suppl. I, 56 (1953). 1. H. KRAKOFF, D. A. KARN~FSKY and J. H. BIJKCH~NAI_, New Eng. J. Med.. 253, 7 1195.5). 6. I. H. KKAKOFF, D. A. KARNOFSKY and J. H. BUKCHENAL, New Enr. J. Mrtl. 253) 7 (19.55). 7. B. DXXU~JEVIC and W. SZYRALSKY. J. EYE. Meal. 112, 509 (1960).
Short communications
255
8. J. J. JAFFE, R. E. HANDSCHUMACHER and A. D. WELCH, Yale J. Viol. Med. 30, 168 (1957). 9. E. E. HALEY, G. A. FISCHER and A. D. WELCH, Cczncer Res. 21, 532 (1961). IO. G. A. FISCHER, Cancer
Res. 19, 372 (1959). Chem. 235, 2917 (1960). C. A. PASTERNAK and R. E. HANDSCHUMACHER, J. Biol. Gem. 234, 2992 (1959). F. GROS, The Nucleic Acids 3, 409 (1960). Academic Press, Inc. T. D. BROCK, Bacterial. Reuiews 25, 32 (1961). S. S. CARDOSO. P. CALABRESI and R. E. HANDSCHUMACHER, Cancer Res. In press. L. H. SMITH, JR., M. SULLIVAN, C. M. HUGLEY, JR., J. C/in. Inties/. 40, 656 (1961). A. D. WELCH, R. E. HANDSCHUMACHER. S. C. FINCH, J. J. JAFFE, S. S. CARDOSO and P. CALABRESI, Cancer Chemother. Rep. 9, 39 (1960). B. G. DELTA and D. PINKEL, Cancer Chemother. Rep. 11, 143 (1961).
11. R. E. HANDSCHUMACHER, J. Biol.
12. 13. 14.
15. 16. 17.
18.
* Senior Post-doctoral
.a Pre-doctoral
Fellow, U.S. Public Health Servtce. Trainee, U.S. Public Health Service Training (irant Xi-~?1 REFERENCES
I. S. M. KALMAN and M. E. LOMISRO~O,J. f’/xr~tt,. Esp. Tlwrc/p. 131, 265 ( 1X111. 2. M. W. NOALL, T. R. RIGGS, L. M. WALKER and H. N. CFrnlsst:usrN, .Scie/rcr126. 1003 (19.57). 3. M. W. NOALL, Biuchinf. Biopitys. Actu 40, 180 (1960). 4. M. A. TELFER, .4&. Bio&etu. Bioplqx 44, t 11 (1953). 5. G. C. MUELLER. J. CZftr. ~~~~f~~rj/~. 13, 836 (19%). 6. K. L. MANCHESTERand F. G. YOIINC;.&when/. J. 75. 487
~otentiation
of the careinostatic
(IWI).
action of azauridine
by chtoraniphenicol~
CHLORAMPHENICOL,a well-established
antibiotic agent III the chemotherapy of bacterial Infections. has also been shown to depress hematopoiesis in man.l~ y This toxic effect upon bone marrow function led to further i”vestigatton of the possible utility of ~h~oramphentcol as a carcinostatic agent in the treatment of ~xp~rin~ent~~i’i,1. 3 and human “coplasms6 Although ineffective as a carcinostatic agent irt t,ii~), ~~~lor~trnph~~~i~~~~ inhibits the growth of tnammalian cells in culture.: Observations made during the course of treatment with h-azauridine (thu ribonuclcoside of harauracil)H of three patients with leukemia,f suggested that potcntiation of its action by chloramphenicol might have occurred when the antibiotic agent was given for the treatment of intercurrent bacterial infections. Preliminary studies of combinations of these compounds in the chemotherap!, of an experimental neopfasm in “lice have confirmed this impression and the results arc presented m this comtlltlni~atiol~. METHODS Male DBF, hybrid mice, h--8 weeks old and weighing 19-24 g, were used I” :III cxperimenrs. All animals were fed powdered Purina Lab Chow, with and without chlorampherricol:: added 111 concentrations ranging fro”1 0.5 to 2.0 per cent. Azauridine$ was added to the drinking water ol * This invest~~tion was supported, in part, hq a research grant (CY-2817) front ihe Natittnai Cancer institute, U.S. Public Health Service. 7 S. S. Cardoso. : Generously supplied by Parke, Davis and Company. 4 Provided through the Cancer Chemotherapy National Service Center of thu U.S. Public Elcalltt Service (Bethesda, Maryland).
Short communications
253
appropriate
groups of animals in concentrations ranging from 0.15 to 0.5 mg per ml and was available of the L5178Y leukemia was made by removing, from a freshly sacrificed donor mouse, 1 ml of ascites fluid and immediately transferring this to a test tube containing 9 ml of culture medium;O of this dilution of ascitic fluid, 0.1 ml, containing 1.553.0 :i lo8 viable leukemia cells, was injected subcutaneously into the right flank of each mouse, in order to produce a solid neoplasm. In all the experiments the recipient mice were inoculated with the leukemic cells within 15 min after the withdrawal of the latter from the donor mouse. Treatment with azauridinc and chloramphenicol was always begun 24 hr after the subcutaneous inoculation of the neoplastic cells into individually weighed mice. On the day following the final dosage of either azauridine or chloramphenicol, or of both agents, each mouse was weighed, sacrificed, and its tumor was removed and weighed. The average weight of the tumors in each treated group was compared with that of the controls, and the results were recorded in terms of percentage inhibition of neoplastic growth. For experiments in vitro, L5178Y cells were grown in culture, as described by Fischer cr crl.“‘, in the presence or absence of various concentrations of chloramphenicol. ad libirum. Transmission
RESULTS
AND
DISCUSSION
In the studies of the effect of chloramphenicol upon the growth of the L5 178Y cells in citro, concentrations of the antibiotic agent of IO-100 pg per ml of tissue culture medium inhibited the rate of cellular reproduction; the concentration required for 50 per cent inhibition of growth was 1Org per ml of tissue culture medium. In contrast to these results with chloramphenicol in culture, even high doses of the antibiotic agent, when tested for activity with respect to the inhibition of the growth of the lymphoblasts growing as a solid tumor in mice, caused only moderate carcinostasis (Table I). However, in combination with azauridine, chloramphenicol, in doses which by themselves caused no inhibition of growth, potentiated the carcinostatic effect of azauridine (Table 1). Achromycin, an antibiotic with a similar spectrum of antibacterial activity to that of chloramphenicol, failed to increase significantly the inhibition of tumor growth obtained with azauridine alone (Table I).
TABLE
I. EFFECTS
OF AZAURIDINE,
CHLORAMPHENICOL
LYMPHONAS
Change in Treatment*
-
Controls CAP. 0.5 7; AzUR, 0.15 mg/ml AzUR, 0.15 mg/ml. !~CAP, 0.5 “<,
I
Mortality
ACHROMYUN
mt17.7 t24.7
276
124.4
O/IO
121
kl6.2
4.0
Oil0
58
i 8.7
2.0 1.1 0.7
o/20 o/20 0120
~
242 277 174
f 18.6 ;.20.2 : 16.1
o/20
~
79
I_IO.2
~
O/IO
O/IO 0110 o/10 o/10
-
3.0
I.4
I
ON
THE GROWTH
Average tumor weight, mg (+ s.e.) 363 326
2.0
0.5 - 2.0 t 0.5 ..~0.5
.
AND
nr
IN MICE
I
’
body weight, g
Controls CAP, 0.5 ;/, CAP, 1.0% Achromycm, I ,O% AzUR, 0.25 mg/ml AzUR. 0.25 me/ml. r~achrbmycin,? .O“/, AzUR, 0.25 mg/ml, : CAP, 0.5 7;
(L5178Y)
i ~
lmhibition of tumor weight per cent ~
~ 66
,-
841-
67: -I
* For chloramphenicol (CAP) and achromycin, the concentrations given refer to the percentage of the antibiotic agent in the diet, for azauridine (AzUR), the concentrations given refer to those administered in the drinking water. t AzUR vs. AzUR : CAP: P =- 0.001. CAP: I’ < OWl. : AzUR vs. AzUR
254
Short
communications
The mechanism by which chloramphenicol potentlates the effects of arauridmc has not yet been established. It is known that aznuridine, after its intrnccllular cnzymic conversion to azauridinc 5’-phosphate. acts as a compctitivc inhibitor of orotidylic acid decarboxylase. in this manner interfcring with the biosynthesis of pyrimidincs &J FXXO.*‘~lB Evidence has been prcscnted that. in bacteria. chloramphcnicol may exert an inhibitory eKcct primarily upon protein synthesis:‘:‘, If however, it has not been shown that this antibiotic agent can product a similar effect in mammalian cells. On the other hand, as demonstrated in this laboratory’” and clscwhere by IFnllon er (I/..* the rate of conversion of erotic acid. via orotidylic acid, to uridinc nuclcotides by isolated lcukcmic cells from patients treated with azauridinc is generally inhibited at first; subsequently, however, the magnitude of these conversions in increased to Icvcls close to or above those found in ceils obtained before treatment with azauridinc. This increased enz!mic activity may reflect the presence in the ceils of higher levels of the enzyme orotidylic acid decarboxyiase.“’ If thcsc assumptions are correct, chloramphenicol may suppress this attempt on the part of the cells to compensate for the inhibitlon by azauridine 5’-phosphate of the dccarboxylation of orotidylic acid. through the formation of additional orotidylic acid decarboxylasc. and perhaps of enzymes concerned with carlicr steps in the pathwa) of pyrimidine biosynthesis & ,UKO. Regardless of the nicch~lnisiil involved. the potcilti~~tioii of the carcinostatic cl&t of ~~z~~~lri~~inc by chloramphenicol suggests that controlled studies should be carried out with combinations of these two drugs in the treatment of human neoplastic disease. Although the data obtained in mice with this combination show that chloramphcnicol increases the toxicity of azauridine for the host, the situation may not bc comparable to that which obtains in man. since treatment with azauridinc of approximately 50 patients has not disclosed any appreciable toxicity, while in many casts it has favorably influenced the course of certain types of acute leukemia. + In fact. arauridine is a unique agent in man, since even massive doses have failed thus far to inhibit normal liem~~topoiesis,~ a situ~~tion in marked contrast to that which obtains with other anti-cancer agents, or, indeed. with ztzauridinc in such mammalian species as the dog.*’ In conclusion. cvidencc has been presented to indicate that chloramphenicol potcntiates the carcinostatic etl’cct of azauridinc upon L5178Y cells, when grown as lymphomas in mice. as well as the toxicity of nzauridine for this host. A possible mechanism by which this potcntiation is achicbcd has been discussed, and the USC of c~~lora~iph~~iicol in combination with azauridinc in the treatment of neoplastic disease in man is suggested. Pllthough the increased potency of the combin~~tion of azauridine and chloramphcnicoi engenders greater host-toxicity in mice, sclcctivity of action might be obtained in man, since azauridine by itself has cvokcd no significant toxicity in leukemic patients in whom the drug has caused unequivocal evidcncc of objective improvement.
* Personal communication. t These studies, carried out by groups at Yale, the National Cancer Institute, and the Koswell Park Memorial Institute, have been summarized, in part”, lx and full papers descrlbmg these clinical investigations are in preparation. z Postdoctoral support for S. S. Cardoso was provided by a Rcscarch Training Grant (CRTY5012) from the National Cancer Institute, U.S. Public Health Service. $ Present address: Dcp~~rtln~nt of Pliarm~~col[)~y, University of Vermont College of Medicine. Burlington. Vermont. REFERENCES I. 2. 3. 4. 5. 6.
H. R. D. H.
WELCH, C. N. LLWIS and I. KERLAN. Anriht’ot. CAM/C%rt~rotlrer. 4, 607,
1954. Houc~~so~, Laxet 1, 285, 1954. M. GREENBERG and E. M. C-IF., Cmcer Res. Srcppf. 1, 54 (1953). E. SKIPPER and J. R. THUMPSON, Cmxr Rex. Src,u,r~f.2, 151 (19551. H. LEITKE, Cortcei- Res. Suppl. I, 56 (1953). 1. H. KRAKOFF, D. A. KARN~FSKY and J. H. BIJKCH~NAI_, New Eng. J. Med.. 253, 7 1195.5). 6. I. H. KKAKOFF, D. A. KARNOFSKY and J. H. BUKCHENAL, New Enr. J. Mrtl. 253) 7 (19.55). 7. B. DXXU~JEVIC and W. SZYRALSKY. J. EYE. Meal. 112, 509 (1960).
Short communications
255
8. J. J. JAFFE, R. E. HANDSCHUMACHER and A. D. WELCH, Yale J. Viol. Med. 30, 168 (1957). 9. E. E. HALEY, G. A. FISCHER and A. D. WELCH, Cczncer Res. 21, 532 (1961). IO. G. A. FISCHER, Cancer
Res. 19, 372 (1959). Chem. 235, 2917 (1960). C. A. PASTERNAK and R. E. HANDSCHUMACHER, J. Biol. Gem. 234, 2992 (1959). F. GROS, The Nucleic Acids 3, 409 (1960). Academic Press, Inc. T. D. BROCK, Bacterial. Reuiews 25, 32 (1961). S. S. CARDOSO. P. CALABRESI and R. E. HANDSCHUMACHER, Cancer Res. In press. L. H. SMITH, JR., M. SULLIVAN, C. M. HUGLEY, JR., J. C/in. Inties/. 40, 656 (1961). A. D. WELCH, R. E. HANDSCHUMACHER. S. C. FINCH, J. J. JAFFE, S. S. CARDOSO and P. CALABRESI, Cancer Chemother. Rep. 9, 39 (1960). B. G. DELTA and D. PINKEL, Cancer Chemother. Rep. 11, 143 (1961).
11. R. E. HANDSCHUMACHER, J. Biol.
12. 13. 14.
15. 16. 17.
18.