0360.3016192 $5.00 + .OO Copyright G; I992 Pergamon Press plc
??Session F: Thiol Modulation
PROTECTION
G. J. A.
and Protection
BY WR-2721 OF THE TOXICITY INDUCED BY THE COMBINATION OF CISPLATIN AND 5FLUOROURACIL J. PETERS,
M.
VAN
PH.D.,
LAAR,
C. L. VAN
M.Sc, AND
Department
of Oncology,
M. H.
Free University
DER
TRESKES, M.
WILT,
M.Sc.,
M.Sc.,
W.
PINEDO, Hospital,
M.D.,
F. GYERGYAY, J. F. VAN
M.D.,
DER VIJGH,
PH.D.
PH.D.**
PO Box 7057, 1007 MB Amsterdam,
the Netherlands
We evaluated the effects of WR-2721 and its metabolite WR-1065 on in vitro growth inhibition by 5fluorouracil (5FU) and cisplatin (CDDP) and the effect of WR-2721 on in viva toxicity and antitumor effect of SFU and CDDP. In cell culture both WR-2721 and WR-1065 were not able to reverse growth inhibition caused by either 5FU or CDDP. Administration of WR-2721 i.p. at 525 mg/kg to mice resulted in a severe temperature drop to 27’C, at 200 mg/kg hypothermia was less severe. WR-2721 failed to prevent 5FU toxicity, but the maximum tolerated dose of CDDP in the combination with 5FU (at 100 mg/kg) could be increased from 3 to 7 mg/kg. CDDP at 7 mg/kg enhanced leukopenia caused by 5FU at 100 mg/kg to 20% and thrombocytopenia to 40%; WR-2721 reduced leukopenia and prevented thrombocytopenia induced by the combination. Combination of CDDP, 5FU, and WR2721 resulted in an enhanced antitumor activity against the murine colon tumor Colon 26 compared to 5FU alone and to 5FU combined with CDDP at their maximum tolerated dose. 5-fluorouracil, Cisplatinum,
WR-2721 (ethiofos).
The combination of 5FU and CDDP is commonly used in cancer chemotherapy of squamous cell carcinoma of the head and neck (4) and is at least additive (7). For CDDP therapy nephrotoxicity is dose limiting, while for 5FU therapy myelotoxicity is dose limiting depending on schedule. The efficacy of the combination can be improved by scheduling and increasing the modulating CDDP dose. The maximal tolerated dose (MTD) for CDDP in the combination is approximately l/3 of the dose without 5FU. To protect against toxicity induced by an increased CDDP dose, a chemoprotector such as WR272 1 may be used. Here we describe the effects of WR2721 and WR-1065 on the toxicity induced by CDDP and 5FU as single agents and in combination.
INTRODUCTION WR-272
1 (s-2(3-aminopropylamino)ethylphosphorothio
is being used as a radioprotector and as a chemoprotector against cytotoxic effects of several cytostatic agents, such as cisplatin (CDDP) ( 12, 16). The active metabolite of WR-272 1, WR- 1065, is formed by dephosphorylation catalyzed by the plasma membrane enzyme alkaline phosphatase (6). WR-1065 can prevent DNA damage caused by several chemotherapeutic agents (1). In the clinic WR-272 1 has demonstrated to be protective against nephro, neuro-, and ototoxicity induced by CDDP without loss of antitumor activity (2). 5Fluorouracil (5FU) is widely used for treatment of solid tumors, such as advanced colorectal cancer, breast cancer-, and squamous cell carcinoma of the head and neck. 5FU is active through incorporation into RNA and inhibition of thymidylate synthase (TS) by its metabolite FdUMP, mediated by the formation of a ternary complex between FdUMP, 10-methylene-tetrahydrofolate, and TS (11). acid,
ethiofos)
METHODS
AND
MATERIALS
The effects of WR-2721 and WR-1065* on 5FLJ and CDDP toxicity have been tested in vitro on five different cell lines; C26-10, a murine undifferentiated colon ade-
Presented at the Seventh International Conference on Chemical Modifiers of Cancer Treatment, Clearwater, FL, USA, 2-5 February, I99 1. ** Also at the Netherlands Cancer Institute, Amsterdam, The Netherlands. Reprint requests to: G. J. Peters. Supported by a grant of the UICC, Geneva, Switzerland to F. Gyergyay, by grant IKA-88-20 and IKA 87-12 of the Dutch
Cancer Society and by US Biosciences, Blue Bell, PA, USA. G.J. Peters is a recipient of a senior fellowship of the Royal Netherlands Academy of Sciences. Accepted for publication 26 July I99 1. * US Biosciences, West Conshohocken, PA, USA. Present address of F. Gyergyay, National Institute of Oncology, Budapest, Hungary.
785
786
I. J. Radiation
Oncology 0 Biology 0 Physics
nocarcinoma (derived from Colon 26); UM-SCC-1 1B (1 1B), a human squamous cell carcinoma ofthe head and neck; HT-29, a human colon carcinoma; and A2780 and OVCAR3, two human ovarian cancer cell lines. Doubling times varied between 20 and 48 hr. The cells were cultured according to standard conditions (3, 8) in Dulbecco’s Modified Eagles medium supplemented with 5% heat-inactivated fetal bovine serum at 37°C under 5% COz. Drugs were added at day 1 after plating and cells were exposed to drugs continuously for the whole culture period, which was 2 days for C26-10 cells and 3 days for the other cell lines. At day 1 and at the end of the culture periods, the number ofcells was enumerated using the sulforhodamine B (SRB) test (14) which is suitable to quantify not only growth inhibition, but also cell kill (3). Shortly. cells were fixed with trichloroacetic acid, cellular protein was stained with SRB, washed with acetic acid and solubilized with unbuffered Tris. The absorbance was read with an ELISA reader at 540 or 5 10 nm. ICso values (= concentrations that cause 50% growth inhibition) were calculated from the separate growth curves. For the study of the effects of the WR compounds different schedules have been tested: simultaneously and sequential: WR compounds were added 15 min or 24 hr before the drugs, or 15 min or 24 hr after 5FU. CDDP exposure time was either 1 hr or continuously. Evaluation of toxicity of the several drugs (alone and in combination) has been performed in female Balb-c and C57Bl/6 mice as described (9, 10) after weekly i.p. administration. Weight loss was used as a parameter for gastro-intestinal toxicity. Hematologic toxicity of the combination of 5FU and CDDP and/or WR-2721 was evaluated in non-tumor bearing C57Bl/6 mice as described (5, lo), by analysis of leucocyte and thrombocyte counts and hematocrit in weekly blood samples obtained by retroorbital bleeding of the same mouse. The antitumor effect of the combinations was evaluated with the murine colon carcinoma Colon 26 as described (5, 10) using the ratio of the tumor volumes from treated and untreated control animals (T/C values). Values 0.5 < T/C < 1 were considered to represent insensitive; < 0.5, moderately sensitive and < 0.25 sensitive treatment. Values were calculated at several time points after initiation of treatment and maximal values (the lowest) are given. Colon 26 is a tumor causing cachexia to mice resulting in a limited life-span of mice after transplantation of tumors of about 19 days. Treatment was started at day 9 after transplantation. Life-span is calculated from the first day of treatment. The doses of the drugs are defined by their subscripts, so 5FUloo means 100 mg 5FU/kg and CDDP3 means 3 mg CDDP/kg. RESULTS In vitro 5FU and CDDP were used at concentrations that caused a partial growth inhibition. The IC50 values for
Volume
22. Number
4. 1992
5FU were, 1 PM for C26-10 and 5 PM for HT-29 and 1 1B cells: for CDDP, 3 FM for I 1B, 6 PM for C26- IO, 20 PM for HT-29 at continuous exposure: and 5 PM for A2780 and 20 PM for OVCAR3 at 1 hr exposure. In C2610, HT-29 and 11 B, various combinations of CDDP and 5FU have been tested at continuous exposure; however, in this schedule the effects were not more than additive. Different schedules were used for the combination of 5FU and CDDP with the WR compounds. Pretreatment with WR was used since this is the usual way of clinical administration. a short exposure was used because of the short half-life of not only the WR compounds, but also of the cytostatic agents: continuous co-incubation of the WR-compounds with either 5FU or CDDP was performed to enable a maximal interaction between the drugs. WR-272 1 alone did not affect cell growth, but WR1065 caused a minor growth inhibition at 1000 FM (Fig. 1). At continuous co-incubation neither WR-272 1 nor WR-1065 affected the growth inhibition caused by 5FU in C26- 10, HT-29 and 11 B; or by CDDP in C26- IO. HT29, 1 IB. A2780, and OVCAR3 (Fig. 1). When the WR compounds were added before or after 5FU or CDDP no interference of WR with growth inhibition caused by either 5FU or CDDP was observed in either cell line. In viva Initial experiments with WR-272 15Z5revealed a striking decrease in body temperature in Balb/c mice from about 37°C to 32°C after 30 min and to 27°C after 2.5 hr. Recovery lasted longer than 24 hr. Other side effects. such as weakness and atonicity. disappeared slowly. Since the hypothermic effect of WR-272 12oo was less severe (nadir 34°C) this dose was used for all other experiments. WR-272 12oo was administered to Balb/c mice i.p. 30 min before 5FU. Weight loss at 5FUloo, the maximum tolerated dose (MTD) of 5FU alone, was less than 5%, that of 5FUroo and 5FUioo more than 10% (Fig. 2). WR272 12,,o pretreatment did not result in any protective effects, the weight loss due to 5FU treatment was not prevented, and all mice treated at 5FU200 and 5FUXoo died within 20 days, in contrast to mice treated with 5FUIoo with or without WR-272 1. In the combination of CDDP and 5FU we considered 5FU as the active agent and CDDP as the modulating agent, thus 5FU was used at 5FU 10oand the dose of CDDP was escalated. The MTD for the combination was CDDP3 and 5FUloo with a weight loss comparable to that of 5FUitx, alone (Fig. 2). The administration of CDDPS 5 and CDDP, in combination with 5FUloo caused the death of all mice in the second week; mice treated with WR272 1200rCDDP,, and 5FU 1o0survived longer than 15 days and the maximal weight loss diminished (Fig. 2). 5FUioo, CDDP-/ with WR-272 12oowas too toxic for mice bearing Colon 26, while 5FUIoo, CDDPS i with WR-272 lZoo resulted in an acceptable toxicity. The combination of 5FUloo and CDDP7 resulted in a severe leukopenia (Fig. 3). which was followed by a re-
WR-272 1 protection
of Mluorouracil
and cisplatin
HT-29
0 G. J. PETERS et al.
787
UM-SCC-11B
o---___.---___ \ ?? ‘t \
2.00
\\ \\ \
,
.50
\
\
\
*~&__---*A*
1.00
*
‘.
-A-
*&C&f ‘-V-&_-- -----v------
v--_---v,
WR-2721 + SFU
-,A-, WR- 1065 + 5FU
-t
-v-
0.50
.--------+C;
WR-2721
_.o_. WR- 1065
g==-y---m *.%, \
.
0 \
-.-
1
WR-2721 + CDDP
-,V-, WR- 1065 + CDDP -3
Fig. I. Effect of continuous co-incubation (3 days) with WR-272 I and WR- 1065 on growth inhibition of 5FU and CDDP on HT-29 and 1 IB cells. The WR-compounds were tested with and without 5FU (5 FM for HT-29 and I 1B) or CDDP (3 PM for 1 I B and 20 PM for HT-29). The optical density (O.D.) at 540 nm is plotted against the concentration of the WR-compounds. Control growth of cells was comparable to that of cells cultured in the presence of the lowest WR concentration. In case the WR compounds would protect against the cytotoxic effects of either 5FU or CDDP this would have resulted in an increase of O.D. values towards control growth. Data represent one representative experiment out of 3-4, carried out in quadruplicate. SD of the separate values was less than 5%.
bound after 18 days. Addition of WR-2721200 to this combination resulted in a less severe leukopenia and no rebound was observed. The combination of 5FUloo and CDDP3 resulted in a decrease of leucocytes equally to WR-272 12oo, CDDP7 and 5FUroo. 5FUloo alone and CDDP, alone did not cause thrombocytopenia, but in combination thrombocytes decreased to 44% (day 4) followed by an increase to 143%. WR-272 1200prevented this thrombocytopenia and a rebound of 169% occurred.
6FU
CCDP
WR
too
-
200
-
too
7
too
7
*oo
7
100
.too
0
6
Maximal
10
weight
16
20
loss
5FUloo alone caused anemia, but CDDP, did not affect hematocrit. 5FUloo with CDDP, caused a comparable decrease in hematocrit, which could not be prevented by WR-272 12oo. Colon 26 is relatively resistant (T/C > 0.5) against 5FU therapy (5, 10) and therefore suitable for combination studies with 5FU. 5FU,00 treatment resulted in a maximal T/C of 0.6-0.8 and an increase in life-span (ILS) of about 200%. For 5FUloo with CDDP3 the maximal T/C was about 0.4 with an ILS of about 255%. Both the combinations 5FU roO, CDDP, with WR-2721z0,, and 5FU,00, CDDPs.5 with WR-272 12ooresulted in T/C values between 0.2 and 0.3, but the ILS for the CDDP, schedule was only about 150%, while that for the CDDPS.S was more than 300%.
26
1%)
Fig. 2. Effect of WR-272 1 (WR) on the toxicity induced by various combinations with 5FU and CDDP. The numbers left of the Y-axis denote doses in mg/kg: --, not given. Drugs were administered weekly for 2 weeks, and values (means +- SD) represent the maxima1 weight loss (in % of the pretreatment weight of the animals) observed in the first week after treatment. *, weight loss in tumor (colon 26) bearing animals; all other data are from non tumor bearing animals.
Fig. 3. The effect of WR-2721zoo tumor bearing mice treated with CDDP. Values denote the nadirs counts observed after 4 days. C-7
on leucocyte counts in non5FUlo0 in combination with (means & SE) of the blood means CDDP,.
788
I. J. Radiation Oncology 0 Biology 0 Physics
Volume 22, Number
DISCUSSION
comparable to that observed in patients, myelotoxicity being the dose-limiting toxicity for weekly 5FU (11). From our data it is clear that WR-2721 protected against the additional myelotoxicity caused by CDDP, both leukopenia and thrombocytopenia. Interpretation ofthe effects is rather complicated, since the decrease in leuco- and thrombocytes was sometimes followed by a rebound. In order to design an optimal scheduling for clinical application. a more detailed analysis of these effects is warranted. This would include the study of the various proliferation lineages in the ontogeny of the blood cell populations in treated and untreated animals. Initial analysis of the antitumor effect of the combination demonstrated that the antitumor effect of the combination was higher than that of 5FU alone. A complication in these studies was the higher toxicity of the combination in tumor bearing animals compared to nontumor bearing animals requiring a dose reduction of CDDP. The promising data must be extended to other tumor types such as squamous cell carcinoma of the head and neck for which combination therapy of CDDP and 5FU is commonly used. Our data demonstrate that in the combination of CDDP, and 5FU the dose of CDDP can be enhanced, but dose escalation of CDDP should be carefully guided. We conclude that WR-2721 can protect against additional CDDP toxicity in the combination with 5FU, leading to a better efficacy of this combination. However, it is not clear whether in vivo this effect is due to a modulating effect of CDDP on 5FU as has been suggested (13) or an additional effect. The overall benefit, however, can
Evaluation of the potentially protective effects of WR272 I was performed both in vitro and in vivo. Our in vitro data demonstrate that both WR-272 1 and WR- 1065, the dephosphorylated active metabolite, can not reverse or prevent the growth-inhibitory effects of both 5FU and CDDP against tumor cell lines derived from tumors that are treated in the clinic with either 5FU or CDDP or in combination. These data suggest a possible selective effect of WR-272 1. Additional studies with non-tumor cells such as fibroblast lines might enable a possible selective effect in vitro to be demonstrated. Our in vivo experiments were complicated by the severe hypothermia caused by WR-2721 itself. It is not clear what will be the impact of the WR-2721 induced hypothermia, but at a low temperature drug metabolism or pharmacodynamics may be changed, possibly being related to some of the effects described. Comparable effects on body temperature have been described (15) but the mechanisms involved are unclear. Besides the hypothermia, WR-2721szs exhibited some additional toxicity at 5FUloo leading to a decreased survival. In contrast to previous data (16), we could not demonstrate a protective effect against 5FU induced toxicity both WR-272 1.525and WR-272 1200. However, WR-272 1200 did protect against CDDP induced toxicity and prevented lethal effects of CDDP in the combination with 5FU, as we could safely increase the CDDP dose in the presence of WR-272 12oo, but not without WR-272 lzoo pretreatment. Although the use of peripheral blood cell counts for interpretation of bone marrow toxicity has several limitations, this is the usual method for assessment of myeloid toxicity in patients. In addition, the toxicity pattern was
be clearly ascribed 2721.
4, 1992
to the chemoprotective
effect of WR-
REFERENCES 1.
2.
3.
4.
5.
6.
Calabro-Jones. P. M.; Fahey, R. C.; Smoluk, G. D.: Ward, J. F. Alkaline phosphatase promotes radioprotection and accumulation of WR-1065 in V79-171 cells incubated in medium containing WR-272 1. Int. J. Radiat. Biol. 47:2327; 1985. Clover, D.; Kligerman, M. M.; Weler, C.; et al. Clinical trials of WR-272 1 prior to alkylating agent chemotherapy and radiotherapy. Pharmacol. Ther. 39:3-7; 1988. Keepers, Y. P.; Pizao, P. E.; Peters, G. J.: et al. Comparison of the sulforhodamine B protein and tetrazolium assays for in vitro chemosensitivity testing. Eur. J. Cancer 27:897900: 1991. Kish, J. A.; Ensley, J. F.; Jacobs, J.; et a/. A randomized trial of cis-plating (CACP) and .5-fluorouracil (5FU) infusion and CACP + 5FU bolus for recurrent and advanced squamous cell cancer of the head and neck. cancer 56:27402744; 1985. Nadal, J.; Van Groeningen, C. J.; Pinedo, H. M.: Peters, G. J. Schedule-dependency of in vivo modulation of 5-fluorouracil by leucovorin and uridine in murine colon carcinoma. Invest. New Drugs 7: 163-172; 1989. Nakamura, J.; Shaw, L. M.; Brown, D. Q. Hydrolysis of WR-272 1 by mouse liver cell fractions. Radiat. Res. 109: 143-152; 1987.
7. Palmeri, S.; Trave, F.; Russello, 0.; Rustum, Y. M. The role of drug sequence in therapeutic selectivity of the combination of 5-fluorouracil and cis-platinum. Sel. Cancer Ther. 5:169-177; 1989. 8. Peters, G. J.: Laurensse, E.; Leyva, A.; Lankelma, J.; Pinedo, H. M. Sensitivity of human, murine and rat cells to 5-fluorouracil and 5’deoxy-5-fluorouridine in relation to drug metabolizing enzymes. Cancer Res. 46:20-28; 1986. 9. Peters, G. J.; Van Groeningen, C. J.; Laurensse, E.; Lankelma, J.; Leyva, A.; Pinedo, H. M. Uridine-induced hypothermia in mice and rats in relation to plasma and tissue levels of uridine and its metabolites. Cancer Chemother. Pharmacol. 20:101-108; 1987. 10. Peters, G. J.; Van Dijk, J.; Laurensse, E.; ei al. In vitro biochemical and in vivo biological studies of “uridine rescue” of 5-fluorouracil. Brit. J. Cancer 57:259-265; 1988. 1 I. Pinedo. H. M.; Peters, G. J. 5-Fluorouracil: Biochemistry and pharmacology. J. Clin. Oncol. 6:1653-1664; 1988. 12. Piper, J. R.; Stringfellow, C. R., Jr.; Elliot, R. T.; rt al. S2(3-amino-alkyl-amino)ethyldihydrogen phosphorothiates and related compounds as potential antiradiation agents. J. Med. Chem. 12:236-243; 1969. 13. Scanlon, K. J.; Newman, E. M.; Lu, Y.; Priest, D. G. Biochemical basis of cisplatin and 5-fluorouracil synergism in
WR-272 I protection of Sfluorouracil and cisplatin 0 G. J. PETERSc’ta/. human ovarian carcinoma cells. Proc. Natl. Acad. Sci. USA 83:8923-8925; 1986. 14. Skehan, P.; Storeng, R.; Scudiero, D.; et al. New colorimetic cytotoxicity assay for anticancer-drug screening. JNCI 82: 107-I 12; 1990. 15. Travis, E. L.: De Luca, A. M.; Fowler, J. F.; et al. The time
789
course of radioprotection by WR-272 1 in mouse skin. Int. J. Radiat. Oncol. Biol. Phys. 8:843-850; 1982. 16. Wasserman, T. H.; Phillips, E. L.; Ross, G.; Kane, L. J. Differential protection against cytotoxic chemotherapeutic effects on bone marrow CFUs by WR-272 1. Cancer Clin. Trial 4:3-6; 1981.
??Session F: Thiol Modulation
PROTECTION
G. J. A.
and Protection
BY WR-2721 OF THE TOXICITY INDUCED BY THE COMBINATION OF CISPLATIN AND 5FLUOROURACIL J. PETERS,
M.
VAN
PH.D.,
LAAR,
C. L. VAN
M.Sc, AND
Department
of Oncology,
M. H.
Free University
DER
TRESKES, M.
WILT,
M.Sc.,
M.Sc.,
W.
PINEDO, Hospital,
M.D.,
F. GYERGYAY, J. F. VAN
M.D.,
DER VIJGH,
PH.D.
PH.D.**
PO Box 7057, 1007 MB Amsterdam,
the Netherlands
We evaluated the effects of WR-2721 and its metabolite WR-1065 on in vitro growth inhibition by 5fluorouracil (5FU) and cisplatin (CDDP) and the effect of WR-2721 on in viva toxicity and antitumor effect of SFU and CDDP. In cell culture both WR-2721 and WR-1065 were not able to reverse growth inhibition caused by either 5FU or CDDP. Administration of WR-2721 i.p. at 525 mg/kg to mice resulted in a severe temperature drop to 27’C, at 200 mg/kg hypothermia was less severe. WR-2721 failed to prevent 5FU toxicity, but the maximum tolerated dose of CDDP in the combination with 5FU (at 100 mg/kg) could be increased from 3 to 7 mg/kg. CDDP at 7 mg/kg enhanced leukopenia caused by 5FU at 100 mg/kg to 20% and thrombocytopenia to 40%; WR-2721 reduced leukopenia and prevented thrombocytopenia induced by the combination. Combination of CDDP, 5FU, and WR2721 resulted in an enhanced antitumor activity against the murine colon tumor Colon 26 compared to 5FU alone and to 5FU combined with CDDP at their maximum tolerated dose. 5-fluorouracil, Cisplatinum,
WR-2721 (ethiofos).
The combination of 5FU and CDDP is commonly used in cancer chemotherapy of squamous cell carcinoma of the head and neck (4) and is at least additive (7). For CDDP therapy nephrotoxicity is dose limiting, while for 5FU therapy myelotoxicity is dose limiting depending on schedule. The efficacy of the combination can be improved by scheduling and increasing the modulating CDDP dose. The maximal tolerated dose (MTD) for CDDP in the combination is approximately l/3 of the dose without 5FU. To protect against toxicity induced by an increased CDDP dose, a chemoprotector such as WR272 1 may be used. Here we describe the effects of WR2721 and WR-1065 on the toxicity induced by CDDP and 5FU as single agents and in combination.
INTRODUCTION WR-272
1 (s-2(3-aminopropylamino)ethylphosphorothio
is being used as a radioprotector and as a chemoprotector against cytotoxic effects of several cytostatic agents, such as cisplatin (CDDP) ( 12, 16). The active metabolite of WR-272 1, WR- 1065, is formed by dephosphorylation catalyzed by the plasma membrane enzyme alkaline phosphatase (6). WR-1065 can prevent DNA damage caused by several chemotherapeutic agents (1). In the clinic WR-272 1 has demonstrated to be protective against nephro, neuro-, and ototoxicity induced by CDDP without loss of antitumor activity (2). 5Fluorouracil (5FU) is widely used for treatment of solid tumors, such as advanced colorectal cancer, breast cancer-, and squamous cell carcinoma of the head and neck. 5FU is active through incorporation into RNA and inhibition of thymidylate synthase (TS) by its metabolite FdUMP, mediated by the formation of a ternary complex between FdUMP, 10-methylene-tetrahydrofolate, and TS (11). acid,
ethiofos)
METHODS
AND
MATERIALS
The effects of WR-2721 and WR-1065* on 5FLJ and CDDP toxicity have been tested in vitro on five different cell lines; C26-10, a murine undifferentiated colon ade-
Presented at the Seventh International Conference on Chemical Modifiers of Cancer Treatment, Clearwater, FL, USA, 2-5 February, I99 1. ** Also at the Netherlands Cancer Institute, Amsterdam, The Netherlands. Reprint requests to: G. J. Peters. Supported by a grant of the UICC, Geneva, Switzerland to F. Gyergyay, by grant IKA-88-20 and IKA 87-12 of the Dutch
Cancer Society and by US Biosciences, Blue Bell, PA, USA. G.J. Peters is a recipient of a senior fellowship of the Royal Netherlands Academy of Sciences. Accepted for publication 26 July I99 1. * US Biosciences, West Conshohocken, PA, USA. Present address of F. Gyergyay, National Institute of Oncology, Budapest, Hungary.
785
786
I. J. Radiation
Oncology 0 Biology 0 Physics
nocarcinoma (derived from Colon 26); UM-SCC-1 1B (1 1B), a human squamous cell carcinoma ofthe head and neck; HT-29, a human colon carcinoma; and A2780 and OVCAR3, two human ovarian cancer cell lines. Doubling times varied between 20 and 48 hr. The cells were cultured according to standard conditions (3, 8) in Dulbecco’s Modified Eagles medium supplemented with 5% heat-inactivated fetal bovine serum at 37°C under 5% COz. Drugs were added at day 1 after plating and cells were exposed to drugs continuously for the whole culture period, which was 2 days for C26-10 cells and 3 days for the other cell lines. At day 1 and at the end of the culture periods, the number ofcells was enumerated using the sulforhodamine B (SRB) test (14) which is suitable to quantify not only growth inhibition, but also cell kill (3). Shortly. cells were fixed with trichloroacetic acid, cellular protein was stained with SRB, washed with acetic acid and solubilized with unbuffered Tris. The absorbance was read with an ELISA reader at 540 or 5 10 nm. ICso values (= concentrations that cause 50% growth inhibition) were calculated from the separate growth curves. For the study of the effects of the WR compounds different schedules have been tested: simultaneously and sequential: WR compounds were added 15 min or 24 hr before the drugs, or 15 min or 24 hr after 5FU. CDDP exposure time was either 1 hr or continuously. Evaluation of toxicity of the several drugs (alone and in combination) has been performed in female Balb-c and C57Bl/6 mice as described (9, 10) after weekly i.p. administration. Weight loss was used as a parameter for gastro-intestinal toxicity. Hematologic toxicity of the combination of 5FU and CDDP and/or WR-2721 was evaluated in non-tumor bearing C57Bl/6 mice as described (5, lo), by analysis of leucocyte and thrombocyte counts and hematocrit in weekly blood samples obtained by retroorbital bleeding of the same mouse. The antitumor effect of the combinations was evaluated with the murine colon carcinoma Colon 26 as described (5, 10) using the ratio of the tumor volumes from treated and untreated control animals (T/C values). Values 0.5 < T/C < 1 were considered to represent insensitive; < 0.5, moderately sensitive and < 0.25 sensitive treatment. Values were calculated at several time points after initiation of treatment and maximal values (the lowest) are given. Colon 26 is a tumor causing cachexia to mice resulting in a limited life-span of mice after transplantation of tumors of about 19 days. Treatment was started at day 9 after transplantation. Life-span is calculated from the first day of treatment. The doses of the drugs are defined by their subscripts, so 5FUloo means 100 mg 5FU/kg and CDDP3 means 3 mg CDDP/kg. RESULTS In vitro 5FU and CDDP were used at concentrations that caused a partial growth inhibition. The IC50 values for
Volume
22. Number
4. 1992
5FU were, 1 PM for C26-10 and 5 PM for HT-29 and 1 1B cells: for CDDP, 3 FM for I 1B, 6 PM for C26- IO, 20 PM for HT-29 at continuous exposure: and 5 PM for A2780 and 20 PM for OVCAR3 at 1 hr exposure. In C2610, HT-29 and 11 B, various combinations of CDDP and 5FU have been tested at continuous exposure; however, in this schedule the effects were not more than additive. Different schedules were used for the combination of 5FU and CDDP with the WR compounds. Pretreatment with WR was used since this is the usual way of clinical administration. a short exposure was used because of the short half-life of not only the WR compounds, but also of the cytostatic agents: continuous co-incubation of the WR-compounds with either 5FU or CDDP was performed to enable a maximal interaction between the drugs. WR-272 1 alone did not affect cell growth, but WR1065 caused a minor growth inhibition at 1000 FM (Fig. 1). At continuous co-incubation neither WR-272 1 nor WR-1065 affected the growth inhibition caused by 5FU in C26- 10, HT-29 and 11 B; or by CDDP in C26- IO. HT29, 1 IB. A2780, and OVCAR3 (Fig. 1). When the WR compounds were added before or after 5FU or CDDP no interference of WR with growth inhibition caused by either 5FU or CDDP was observed in either cell line. In viva Initial experiments with WR-272 15Z5revealed a striking decrease in body temperature in Balb/c mice from about 37°C to 32°C after 30 min and to 27°C after 2.5 hr. Recovery lasted longer than 24 hr. Other side effects. such as weakness and atonicity. disappeared slowly. Since the hypothermic effect of WR-272 12oo was less severe (nadir 34°C) this dose was used for all other experiments. WR-272 12oo was administered to Balb/c mice i.p. 30 min before 5FU. Weight loss at 5FUloo, the maximum tolerated dose (MTD) of 5FU alone, was less than 5%, that of 5FUroo and 5FUioo more than 10% (Fig. 2). WR272 12,,o pretreatment did not result in any protective effects, the weight loss due to 5FU treatment was not prevented, and all mice treated at 5FU200 and 5FUXoo died within 20 days, in contrast to mice treated with 5FUIoo with or without WR-272 1. In the combination of CDDP and 5FU we considered 5FU as the active agent and CDDP as the modulating agent, thus 5FU was used at 5FU 10oand the dose of CDDP was escalated. The MTD for the combination was CDDP3 and 5FUloo with a weight loss comparable to that of 5FUitx, alone (Fig. 2). The administration of CDDPS 5 and CDDP, in combination with 5FUloo caused the death of all mice in the second week; mice treated with WR272 1200rCDDP,, and 5FU 1o0survived longer than 15 days and the maximal weight loss diminished (Fig. 2). 5FUioo, CDDP-/ with WR-272 12oowas too toxic for mice bearing Colon 26, while 5FUIoo, CDDPS i with WR-272 lZoo resulted in an acceptable toxicity. The combination of 5FUloo and CDDP7 resulted in a severe leukopenia (Fig. 3). which was followed by a re-
WR-272 1 protection
of Mluorouracil
and cisplatin
HT-29
0 G. J. PETERS et al.
787
UM-SCC-11B
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,
.50
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WR-2721 + SFU
-,A-, WR- 1065 + 5FU
-t
-v-
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.--------+C;
WR-2721
_.o_. WR- 1065
g==-y---m *.%, \
.
0 \
-.-
1
WR-2721 + CDDP
-,V-, WR- 1065 + CDDP -3
Fig. I. Effect of continuous co-incubation (3 days) with WR-272 I and WR- 1065 on growth inhibition of 5FU and CDDP on HT-29 and 1 IB cells. The WR-compounds were tested with and without 5FU (5 FM for HT-29 and I 1B) or CDDP (3 PM for 1 I B and 20 PM for HT-29). The optical density (O.D.) at 540 nm is plotted against the concentration of the WR-compounds. Control growth of cells was comparable to that of cells cultured in the presence of the lowest WR concentration. In case the WR compounds would protect against the cytotoxic effects of either 5FU or CDDP this would have resulted in an increase of O.D. values towards control growth. Data represent one representative experiment out of 3-4, carried out in quadruplicate. SD of the separate values was less than 5%.
bound after 18 days. Addition of WR-2721200 to this combination resulted in a less severe leukopenia and no rebound was observed. The combination of 5FUloo and CDDP3 resulted in a decrease of leucocytes equally to WR-272 12oo, CDDP7 and 5FUroo. 5FUloo alone and CDDP, alone did not cause thrombocytopenia, but in combination thrombocytes decreased to 44% (day 4) followed by an increase to 143%. WR-272 1200prevented this thrombocytopenia and a rebound of 169% occurred.
6FU
CCDP
WR
too
-
200
-
too
7
too
7
*oo
7
100
.too
0
6
Maximal
10
weight
16
20
loss
5FUloo alone caused anemia, but CDDP, did not affect hematocrit. 5FUloo with CDDP, caused a comparable decrease in hematocrit, which could not be prevented by WR-272 12oo. Colon 26 is relatively resistant (T/C > 0.5) against 5FU therapy (5, 10) and therefore suitable for combination studies with 5FU. 5FU,00 treatment resulted in a maximal T/C of 0.6-0.8 and an increase in life-span (ILS) of about 200%. For 5FUloo with CDDP3 the maximal T/C was about 0.4 with an ILS of about 255%. Both the combinations 5FU roO, CDDP, with WR-2721z0,, and 5FU,00, CDDPs.5 with WR-272 12ooresulted in T/C values between 0.2 and 0.3, but the ILS for the CDDP, schedule was only about 150%, while that for the CDDPS.S was more than 300%.
26
1%)
Fig. 2. Effect of WR-272 1 (WR) on the toxicity induced by various combinations with 5FU and CDDP. The numbers left of the Y-axis denote doses in mg/kg: --, not given. Drugs were administered weekly for 2 weeks, and values (means +- SD) represent the maxima1 weight loss (in % of the pretreatment weight of the animals) observed in the first week after treatment. *, weight loss in tumor (colon 26) bearing animals; all other data are from non tumor bearing animals.
Fig. 3. The effect of WR-2721zoo tumor bearing mice treated with CDDP. Values denote the nadirs counts observed after 4 days. C-7
on leucocyte counts in non5FUlo0 in combination with (means & SE) of the blood means CDDP,.
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I. J. Radiation Oncology 0 Biology 0 Physics
Volume 22, Number
DISCUSSION
comparable to that observed in patients, myelotoxicity being the dose-limiting toxicity for weekly 5FU (11). From our data it is clear that WR-2721 protected against the additional myelotoxicity caused by CDDP, both leukopenia and thrombocytopenia. Interpretation ofthe effects is rather complicated, since the decrease in leuco- and thrombocytes was sometimes followed by a rebound. In order to design an optimal scheduling for clinical application. a more detailed analysis of these effects is warranted. This would include the study of the various proliferation lineages in the ontogeny of the blood cell populations in treated and untreated animals. Initial analysis of the antitumor effect of the combination demonstrated that the antitumor effect of the combination was higher than that of 5FU alone. A complication in these studies was the higher toxicity of the combination in tumor bearing animals compared to nontumor bearing animals requiring a dose reduction of CDDP. The promising data must be extended to other tumor types such as squamous cell carcinoma of the head and neck for which combination therapy of CDDP and 5FU is commonly used. Our data demonstrate that in the combination of CDDP, and 5FU the dose of CDDP can be enhanced, but dose escalation of CDDP should be carefully guided. We conclude that WR-2721 can protect against additional CDDP toxicity in the combination with 5FU, leading to a better efficacy of this combination. However, it is not clear whether in vivo this effect is due to a modulating effect of CDDP on 5FU as has been suggested (13) or an additional effect. The overall benefit, however, can
Evaluation of the potentially protective effects of WR272 I was performed both in vitro and in vivo. Our in vitro data demonstrate that both WR-272 1 and WR- 1065, the dephosphorylated active metabolite, can not reverse or prevent the growth-inhibitory effects of both 5FU and CDDP against tumor cell lines derived from tumors that are treated in the clinic with either 5FU or CDDP or in combination. These data suggest a possible selective effect of WR-272 1. Additional studies with non-tumor cells such as fibroblast lines might enable a possible selective effect in vitro to be demonstrated. Our in vivo experiments were complicated by the severe hypothermia caused by WR-2721 itself. It is not clear what will be the impact of the WR-2721 induced hypothermia, but at a low temperature drug metabolism or pharmacodynamics may be changed, possibly being related to some of the effects described. Comparable effects on body temperature have been described (15) but the mechanisms involved are unclear. Besides the hypothermia, WR-2721szs exhibited some additional toxicity at 5FUloo leading to a decreased survival. In contrast to previous data (16), we could not demonstrate a protective effect against 5FU induced toxicity both WR-272 1.525and WR-272 1200. However, WR-272 1200 did protect against CDDP induced toxicity and prevented lethal effects of CDDP in the combination with 5FU, as we could safely increase the CDDP dose in the presence of WR-272 12oo, but not without WR-272 lzoo pretreatment. Although the use of peripheral blood cell counts for interpretation of bone marrow toxicity has several limitations, this is the usual method for assessment of myeloid toxicity in patients. In addition, the toxicity pattern was
be clearly ascribed 2721.
4, 1992
to the chemoprotective
effect of WR-
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course of radioprotection by WR-272 1 in mouse skin. Int. J. Radiat. Oncol. Biol. Phys. 8:843-850; 1982. 16. Wasserman, T. H.; Phillips, E. L.; Ross, G.; Kane, L. J. Differential protection against cytotoxic chemotherapeutic effects on bone marrow CFUs by WR-272 1. Cancer Clin. Trial 4:3-6; 1981.
