Cancer Chemother Pharmacol (1989) 25:161 - 166 ancer hemotherapyand harmacology 9 Springer-Verlag 1989 Influence of scheduling on two-drug combinations of alkylating agents in vivo* Beverly A. Teicher, Sylvia A. Holden, Steven M. Jones, J. Paul Eder, and Terence S. Herman Dana-Farber Cancer Institute, 44 Binney Street, Boston MA 02115, USA Summary. The effects of schedule and sequence on the sur- vival of EMT6 tumor cell and bone marrow (CFU-GM) obtained after treatment using combinations of cyclophos- phamide (CTX) and thiotEPA or melphalan (L-PAM) were examined and analyzed by isobologram methodol- ogy. On a single-injection schedule, when CTX and thio- tEPA were given simultaneously or thiotEPA was given prior to CTX, the result was slightly greater than additive tumor-cell kill. However, when CTX preceded thiotEPA by 4 h, there was less than additive cell kill. When the in- terval between the administration of the two drugs was 8 h, both sequences of the drugs produced greater than addi- tive tumor-cell kill. Simultaneous administration of CTX and thiotEPA on a multiple-injection schedule resulted in sub-additive tumor-cell kill. On the multiple-injection schedule, extending the interval between injections of CTX and thiotEPA to 4 and 8 h resulted in increasing tumor-cell kill. With the 4- and 8-h intervals, no significant sequence-dependent difference in tumor-cell kill was ob- tained. The results of CTX and L-PAM combinations paralleled those of CTX and thiotEPA. Bone marrow (CFU-GM) survival was used as a representative normal tissue with which to compare tumor-cell survival after each treatment to obtain a measure of therapeutic effect. The trends for the ratios of bone marrow: tumor cell survival were the same for the treatment sequences of CTX with thiotEPA or L-PAM; however, greater magnitudes of dif- ferential tumor-cell kill were obtained with CTX L-PAM combinations. Using this measure, the greatest therapeutic effectiveness was seen with single-dose L-PAM or thio- tEPA followed 4 h later by CTX and with CTX given as a single or as multiple doses followed 8 h later by L-PAM or thiotEPA. Such data from tumor-model systems may be useful in the development of more effective alkylating agent regimens for use in the clinic. * This work was supported by grant PO1-37859 from the Nation- al Cancer Institute, a grant from Lederle Co., Pearl River NY, and a grant from the Mathers Foundation Abbreviations: L-PAM, melphalan, L-phenylalanine mustard; thiotEPA, N,NIN~l-triethylenethiophosphoramide; CTX, cyclo- phosphamide; PBS, phosphate buffered saline; FBS, fetal bovine serum; DME, Dulbecco's minimal essential medium; CFU-GM, granuIocyte-macrophage colony forming units. Offprint requests to: B. A. Teicher Introduction In developing effective chemotherapeutic regimens using very high drug doses, questions concerning scheduling and sequencing are critical [6, 8, 12, 19-22, 28]. We have pre- viously examined the issue of alkylating agent dose [7, 26] and the effect of scheduling on the therapeutic effect of alkylating agents given singly [27] in tumor-model systems. In this report, we focused on the effect of scheduling on two-drug combinations, specifically, thiotEPA with cyclophosphamide (CTX) and melphalan (L-PAM) with CTX in vivo using EMT6 tumor-cell survival and the granulocyte-macrophage progenitor colony-forming abili- ty of bone marrow as endpoints. Materials and methods Drugs. CTX and thiotEPA were obtained from the Dana- Farber Cancer Institute pharmacy. L-PAM was purchased as the pure powder from Sigma Chemical Co. (St. Louis, Mo). For in vivo testing, drugs were freshly prepared just prior to their use, with PBS as the final diluent. Tumor line, The EMT6 murine mammary carcinoma is an in vivo-in vitro tumor system [15- 18]. The tumor was car- ried in BALB/c mice (Taconic Farms, Germantown, NY). For the experiments, 2 x 106 tumor cells prepared from a brei of several stock tumors were implanted i.m. into the legs of 8- to 10-week-old BALB/c mice. Tumor excision assay. For each experiment, two tumors were implanted per mouse and two animals were treated at each dose level; therefore, four tumors were pooled at each point. When the tumors had reached approximately 100 mm 3 in volume (about 1 week after tumor-cell implan- tation), the drugs were given as single doses by i.p. injec- tion (0.2 ml) or as three doses by i.p. injection at 4.5-h intervals over 9 h. Combinations of CTX and thiotEPA or L-PAM were injected i.p. on the various schedules described in Fig. 1-3. Mice were sacrificed 24 h after treatment to enable the full expression of drug cytotoxicity and repair of potentially lethal damage and then soaked in 95% ethanol. The tumors were excised and single-cell suspensions were prepared as previously described [25]. The untreated tumor-cell suspensions had a plating ef- ficiency of 8%- 12%. Results are expressed as the surviving fraction (_+SE) of cells from treated groups compared