Leukemia (2002) 16, 1808–1817 2002 Nature Publishing Group All rights reserved 0887-6924/02 $25.00 www.nature.com/leu Schedule-dependent synergism and antagonism between methotrexate and cytarabine against human leukemia cell lines in vitro M Akutsu1, Y Furukawa2, S Tsunoda1, T Izumi1,3, K Ohmine1,3 and Y Kano1 1Division of Medical Oncology, Tochigi Cancer Center, Tochigi, Japan; 2Division of Stem Cell Regulation, Jichi Medical School, Tochigi, Japan; and 3Division of Hematology, Jichi Medical School, Tochigi, Japan Methotrexate (MTX) and cytarabine have been widely used for istic effects.16,18,23 The sequential exposure to both agents also the treatment of acute leukemias and lymphomas for over 30 produced conflicting results.15,17,18,20,21,23,24 No well-designed years. However, the optimal schedule of this combination is yet to be determined and a variety of schedules of the combination clinical randomized trial of this combination has been has been used. We studied the cytotoxic effects of MTX and available. cytarabine in combination against human leukemia cell lines at In experimental combination studies, a great deal of con- various schedules in vitro. The effects of the combinations at fusion has arisen because dose–response curves or survivals the concentration of drug that produced 80% cell growth inhi- for anticancer agents are variable and different investigators 80 bition (IC ) were analyzed using the isobologram method of have disagreed as to what constitutes an ‘expected’ level of Steel and Peckham. Simultaneous exposure to MTX and cytara- 25,26 bine for 3 days produced antagonistic effects in human T cell effect when two agents are combined. Although dose- leukemia, MOLT-3 and CCRF-CEM, B cell leukemia, BALL-1, response curves and survivals of anticancer agents are Burkitt’s lymphoma, Daudi, promyelocytic leukemia, HL-60 and important factors in the analysis of the effects of drug combi- Philadelphia chromosome-positive leukemia, K-562 cells. Sim- nations, many studies lack full dose–response data for each ultaneous exposure to MTX and cytarabine for 24 h produced drug, either alone or in combination. antagonistic effects, sequential exposure to MTX for 24 h fol- In in vivo studies, synergism (therapeutic synergism)is often lowed by cytarabine for 24 h produced synergistic effects, and the reverse sequence produced additive effects in both CCRF- defined as if the combinations produce a better response than CEM and HL-60 cells. Sequential exposure to MTX for 24 h fol- either drug alone, with acceptable toxicity. This definition is lowed by cytarabine for 3 days also produced synergistic a quite different concept from true synergism, which means effects in MOLT-3 cells. Cell cycle analysis supported these that the effect of combination is more than expected, when observations. Our findings suggest that the simultaneous combined.27 In in vitro studies, the classical isobologram28 administration of MTX and cytarabine is not appropriate and and fractional product method29 have often been used, which the sequential administration of MTX followed by cytarabine may be the optimal schedule of this combination. assume linear–dose response curves for each drug and combi- Leukemia (2002) 16, 1808–1817. doi:10.1038/sj.leu.2402573 nations, which is seldom the case. The median effect Keywords: methotrexate; cytarabine; synergism; antagonism; iso- method30 requires rigid sigmoid dose–response curves, based bologram upon the Michaels-Menten and Hill model. However, cyto- toxic mechanisms of anticancer agents are multifactorial and dose–response curves are often not sigmoid and regression Introduction analyses are difficult or forced. We use the isobologram method of Steel and Peckham,31 because this method can be Methotrexate (MTX)and cytarabine have been widely used used to calculate additive interaction of most combinations, for the treatment of acute leukemias and lymphomas for over irrespective of the shapes of the dose–response curves of the 30 years. However, the optimal schedule of the combination agents and whether they have independent or overlapping of MTX and cytarabine is yet to be determined. In regimens damage. including MTX and cytarabine, the schedules of administering To clarify the optimal schedule of the combination of MTX MTX and cytarabine have been variable. In general, MTX and and cytarabine, we studied the effects of simultaneous and cytarabine have been used separately.1–3 However, in some sequential exposure to MTX and cytarabine in human regimens, MTX and cytarabine have been administered simul- leukemia cell lines in vitro using the isobologram of Steel and taneously (on the same day), eg as triple combinations for cen- Peckham. The present findings clearly demonstrated marked tral nervous system (CNS)prophylaxis and CNS leukemia, 4,5 antagonistic effects in simultaneous exposure to MTX and PROMACE-cytaBOM6,7 and AMOPLACE8 for non-Hodgkin’s cytarabine and marked synergistic effects in sequential lymphoma, POG8602 and EORTC58881 for pediatric acute exposure to MTX followed by cytarabine, suggesting that the lymphoblastic leukemia (ALL)9,10 and sequentially on different optimal schedule of this combination may be the sequential days, eg Hyper-CVAD for ALL and CHOD/BVAM for administration of MTX followed by cytarabine, but not the lymphoma.11–14 simultaneous administration of these agents. Experimental findings for the combination of MTX and cyta- rabine, most of which were obtained more than 20 years ago, are variable (Table 1). In both in vitro and in vivo experiments, Materials and methods the simultaneous exposure to MTX and cytarabine produced conflicting results:15–24 synergistic effects15,19–22 and antagon- Cell lines Experiments were conducted with the T cell ALL lines MOLT- 3 and CCRF-CEM, the B cell ALL line BALL-1,32 Burkitt’s lym- Correspondence: Y Kano, Division of Medical Oncology, Tochigi Cancer Center, Yonan 4-9-13, Utsunomiya, Tochigi, 320-0834, Japan; phoma line Daudi, the Philadelphia chromosome-positive Fax: 011–81–28–658–5488 leukemia line K-562, and the acute promyelocytic leukemia Received 27 November 2001; accepted 12 March 2002 line HL-60. CCRF-CEM, HL-60, K-562, MOLT-3 cells were Table 1 Previous experimental studies for the combination of methotrexate (M)and cytarabine (C) System Cell line Schedule Evaluation Effect Ref. In vitro L1210 simultaneous: M + C classical isobologram antagonism 16 (cell growth L5178Y simultaneous: M + C classical isobologram antagonism 18 inhibition) L1210 simultaneous: M + C (5 h) fractional product concept synergism 21 ND simultaneous: M + C ND increased dCPT 22 3924A simultaneous: M + C (72 h) ND antagonism 23 8999R simultaneous: M + C (72 h) ND synergism 23 L5178Y sequential: M (2 h) → C (1 h) fractional product concept synergism 17 L1210 sequential: M (3 h) → C (2 h) fractional product concept antagonism 21 L5178Y sequential and simultaneous: M (6 h) → M + C (42 h) classical isobologram antagonism 18 L1210 sequential and simultaneous: M (3 h) → M + C (2 h) fractional product concept synergism 21 3924A sequential and simultaneous: M (4 h) → M + C (68 h) ND antagonism 23 8999R sequential and simultaneous: M (4 h) → (M + C) (68 h) ND synergism 23 M Akutsu MTX and ara-C in combination HL-60 sequential and simultaneous: M (2 h) → M + C (2 h) ND synergism 24 L5178Y sequential and simultaneous: C (6 h) → M + C (42 h) classical isobologram additive 18 et al 3924A sequential and simultaneous: C (4 h) → M + C (68 h) ND antagonism 23 8999R sequential and simultaneous: C (4 h) → M + C (68 h) ND antagonism 23 (colony N1S1, 3924 sequential and simultaneous: M (4 h) → M + C (6 h) fractional product concept antagonism 23 formation) BF5, 8999F sequential and simultaneous: M (4 h) → M + C (6 h) fractional product concept synergism 23 In vivo L1210 simultaneous: M + C survival time antagonism 15 TLX/5 simultaneous: M + C survival time antagonism 18 L1210 simultaneous: M + C survival time synergism 19 L1210 simultaneous: M + C fractional product concept synergism 20 TLX/5 sequential: M → C survival time additive 18 L1210 sequential: M → C fractional product concept synergism 20 L1210 sequential: C → M fractional product concept synergism 20 ND, not described. Leukemia 1809 MTX and ara-C in combination M Akutsu et al 1810 obtained from the Health Science Research Resources Bank MTT assay (Osaka, Japan). BALL-1 and Daudi cells were obtained from RIKEN Cell Bank (Tsukuba, Japan). All cell lines were cultured Cell growth inhibition was determined by a 3-(4,5-dimethyl- in RPMI1640 medium (GIBCO, Grand Island, NY, USA)sup- thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.33 plemented with 10% heat-inactivated fetal calf serum Fifty microliters of MTT (1 mg/ml)was added to each well. (GIBCO), 50 units/ml penicillin and 5 ␮g/ml streptomycin at Following incubation for 4 h at 37°C, culture plates were cen- ° 37 C in a humidified chamber with 5% CO2 in air. trifuged at low speed and the supernatant was removed. Dimethyl sulfoxide (150 ␮l)was then added and the plates were shaken to solubilize the MTT-formazan product. Drugs Absorbance at 570 nm was measured with a Titertek multi- scan. For the background control, control (no drug), each MTX and cytarabine were obtained from Lederle Japan drug, or drug combinations, the four intermediate data among (Tokyo, Japan), and Nihon Shinyaku (Tokyo, Japan), respect- the eight data were used for the analysis, and the two highest ively. Drugs were dissolved in and diluted with culture and the two lowest data were discarded. We established a medium. linear relation between the MTT assay and cell number within the range of the experiments shown. Cell growth inhibition by the combination of MTX and cytarabine Isobologram Eight plates were prepared for one combined drug test. Dose–response interactions between MTX and cytarabine at the point of IC80 were evaluated using the isobologram method of Steel and Peckham.31 The theoretical basis of the Protocol 1: simultaneous exposure to MTX and cytarabine for isobologram method and the procedure for making the isobol- 3 days: All six leukemia cell lines were used for the ogram has been described in detail.34,35 Based on the dose– experiments.