Effects of Vincristine in Combination with Methotrexate and Other Antitumor Agents in Human Acute Lymphoblastic Leukemia Cells in Culture1

[CANCER RESEARCH 48, 351-356, January 15, 1988) Effects of Vincristine in Combination with Methotrexate and Other Antitumor Agents in Human Acute Lymphoblastic Leukemia Cells in Culture1

Yasuhiko Kano, Takao Ohnuma,2 Tsuyoshi Okano, and James F. Holland

Department ofNeoplastic Diseases and Cancer Center, Mount Sinai School of Medicine, One Gustave Levy Place, New York, New York 10029

ABSTRACT dose MTX was incorporated into clinical protocols (8, 9). In further studies, however, other investigators failed to demon The effects of vincristine (VCR) in combination with methotrexate strate the therapeutic synergism of the VCR-MTX combination (MTX) and other antitumor agents were evaluated by cell growth inhi when simultaneously or sequentially administered in this order bition assay using a human acute lymphoblastic leukemia cell line (MOLT-3). The data were analyzed with the aid of an isobologram using using the same L1210 system (2, 10, 11). Therefore, the ther apeutic advantage of the VCR-MTX combination is at best the concept of an envelope of additivity (G. G. Steel and M. J. Peckman, Int. J. RadiÃ¢t.Oncol., 5: 85-91, 1979). Simultaneous exposure of VCR controversial. To evaluate the effect of drug combinations in both in vivo and MTX produced subadditive or mutually protective interactions. Se and I'Mvitro studies, the concepts of synergy, additivity, and quential exposure to VCR first followed immediately by MTX produced similar interactions. When the interval of VCR exposure first and then antagonism have been widely used. However, the concept of MTX was increased from 0 to 3,8, and 24 h, the inhibition of cell growth additivity which is the basis of the definition of synergy and moved from protection and subadditiv ity to additivity only. The reversed antagonism has been used in a less precise manner. Workers order of exposure to the 2 drugs produced an entirely different picture. have simply added the effects of the constituents used sepa Thus, when the interval of MTX exposure first followed by VCR in rately. Synergy, additivity, and antagonism were then deemed creased from 0 to 3,8, and 24 h, the inhibitory effects of the combination changed progressively from the area of subadditivity to the area of to be present if the effect of the drug combinations were, supraadditiviry. When these data were evaluated using median effect plot respectively, more than equal to or less than the sum of the analyses (T-C. Chou and P. Talalay. In: New Avenues in Developmental effects of the constituents. If the dose-response curves of two Cancer Chemotherapy, pp. 36-64. Orlando, FL: Academic Press, 1987), agents follow closely to first-order kinetics, the drug combina strongly synergistic interaction of this sequence at space intervals was tion can be evaluated by this method. However, dose-response confirmed. These data show that the synergistic effects were produced curves do not always follow first-order kinetics, and in such only when MTX was followed 8 or 24 h later by VCR. Other schedules cases the method described above is not suitable for the evalu were only additive or even antagonistic. ation of the effect of drug combinations (12-16). To solve this Simultaneous exposure of VCR with daunorubicin, 1-0-D-arabinofur- problem, Steel (14, 15) proposed a method for analyzing the anosylcytosine, or bleomycin also had subadditive and protective effects. effects of combined radiotherapy-chemotherapy with nonlinear VCR, followed by daunorubicin with the interval of 24 h and vice versa, was again subadditive and protective. VCR, followed by I-/i-n-arabino- dose-response curves. We applied this method for the evalua tion of the effects of the VCR combination with MTX and furanosylcytosine with the interval of 24 h and vice versa, was again other antitumor agents I'Mvitro. subadditive or additive only. Simultaneous and continuous exposures of VCR with vinblastine or L-asparaginase were only marginally supraad After the present work was completed (17), we came across ditive. a newer analysis for the combined effects of 2 drugs, median effect plot analysis, developed by Chou and Talalay and increas ingly used in recent years (18-20). A portion of our data were INTRODUCTION reanalyzed using this new concept. The combination of VCR3 with MTX or other antitumor agents has been widely used for the treatment of various types MATERIALS AND METHODS of malignant diseases in humans. The administration of VCR prior to or together with MTX enhanced the steady level of Cell Line intraeellular MTX and decreased the efflux of MTX in animal A human acute lymphoblastic leukemia cell line, MOLT-3 (21), was and human tumor cells (1-6). VCR was also reported to en maintained as a suspension in culture flasks (No. 3024; Falcon Plastics, hance intraeellular polyglutamate formation of MTX (7). These Oxnard, CA) containing RPMI-1640 medium (GIBCO, Grand Island, biochemical observations favor the administration of VCR prior NY) supplemented with 10% heat-inactivated FBS (GIBCO) and anti to or simultaneously with MTX in patients with cancer. Indeed, biotics [penicillin (100 lU/ml) and streptomycin (100 Â¿tg/ml);Charles therapeutic synergism was reported with this combination for Pfizer, Brooklyn, NY]. the treatment of mice bearing LI 210 leukemia (6). On the basis of these observations, administration of VCR prior to high- Chemotherapeutic Agents Received9/29/86;revised8/17/87;accepted10/15/87. VCR sulfate and VLB sulfate were obtained from Eli Lilly, Indian The costs of publication of this article were defrayed in part by the payment apolis, IN; MTX, Lederle Laboratories, Pearl River, NY; DNR, Far- of page charges. This article must therefore be hereby marked advertisement in mitalia, Milan, Italy; ara-C, Upjohn, Kalamazoo, MI; BLEO sulfate, accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Bristol Laboratories, Syracuse, NY; ASN-ase, Merck Sharp & Dohme, 1Supported in part by USPHS Grant 15936 from the National Cancer Insti West Point, PA; and TMQ through the National Cancer Institute, tute, Bcthcsda, MD; by the Iwama Memorial Sony International Fellowship; by the T. J. Marteli Memorial Foundation for Cancer and Leukemia Research, Inc., Bethesda, MD. New York, NY; by the United Leukemia Fund, Inc., New York, NY; and by the Chemotherapy Foundation, New York, NY. Cell Growth Inhibition by Combined Chemotherapeutic Agents 1To whom requests for reprints should be addressed. 3The abbreviations used are: VCR, vincristine; VLB, vinblastine; MTX, meth Biological effects of the drugs were evaluated by cell growth inhibi otrexate; DNR, daunorubicin; ara-C, 1-ÃÃ-D-arabinofuranosylcytosine;BLEO, tion assay (22, 23). Logarithmically growing cells were harvested from bleomycin; ASN-ase, L-asparaginase; TMQ, 2,4-diamino-5-methyI-6-(3',4',5'- the medium and resuspended to a final concentration of 1 x IO5cells/ trimethoxyanilino)methyl quinazoline; FBS, fetal bovine serum; CI, combination index; IDso, drug concentration producing 80% inhibition of cell growth on Day ml of fresh medium. 3 as compared to drug-free control. For the continuous mode of exposure to 2 drugs, cell suspensions 351

The total area enclosed by these 3 lines conceivably represents additive response or an envelope of additivity (Fig. 1). With combinations of graded doses of Drug A and a chosen dose of Drug B, a single dose-response curve can be drawn. When the experi mental IDÂ»,,concentration in this combination falls left of the envelope (Fig. 1, Point Pa), the 2 drugs have supraadditive interaction. When the experimental data point is within the envelope, this combination is considered to be noninteractive (additive) (Fig. 1, Point Pb). When this point is in the area right of the envelope, but within the square produced by 1.0 of the ordinate and abscissa, the drugs have subadditive interac tion (Fig. 1, Point Pc). When the point is outside the square, both drugs are considered to be mutually protective (Fig. 1, Point Pd). We find that isoeffect curves had a reasonable amount of variation from experiment to experiment when repeated. We chose to present a O 0.2 0.4 0.6 08 1.2 representative isobologram after confirmation that, in repeated exper DOSE OF A iments, individual combination data points were reproducibly in the same area of supraaddit iviiy, additivity, or subadditivity irrespective of Fig. 1. An envelope of additivity is constructed from the dose-response curve of 2 drugs (.-*and fi). If experimental data points Pa, Pb, Pc, and I'd fall in the the envelope being lean or obese. area specified in the diagram, the 2 drug interactions can be classified as supraad Median Effect Plot Analyses ditive, additive, subadditive, and mutually protective, respectively (14, IS). The combined effects of 2 drugs in terms of synergism, summation, were dispensed (1 ml) into individual wells of a 24-well tissue culture or antagonism can also be analyzed by the median effect plot (18-20). plate with lid (No. 3047; Falcon). Each drug solution at different If the observed effect of 2 drugs acting simultaneously is larger or concentrations was added (10 ÃŸ\)to individual wells containing cell smaller than that calculated from the product expression, it is assumed suspensions. The plates were then incubated in a humidified atmosphere that synergism or antagonism, respectively, has occurred. This method of 95% air/5% CO2 at 37Â°Cfor 72 h. involves plotting dose-effect curves for each drug and for one or more For the short-term mode of exposure, the cell suspensions were multiple-dilution combinations of the drugs using the "median effect" dispensed into tissue culture tubes (No. 3033; Falcon) and exposed to equation 2 drugs with various concentrations in 95% air/5% CO2 at 37"C for 5 h. At the end of incubation, the cells were washed twice with fresh fa/fu = (D/Dmr medium with 2% FBS and were resuspended to the same volume of the medium with 10% FBS. The cell suspensions were dispensed (1 ml) where D is the dose, Dm is the dose required for 50% effect (e.g., 50% into individual wells of the 24-well plate and incubated under the same inhibition of cell growth at 72 h as compared to drug-free control), fa conditions for 72 h. is the fraction affected by the dose I), fit is the fraction unaffected, and For sequential short-term exposure of 2 drugs, the cell suspensions m is a coefficient signifying the sigmoidicity of the dose-effect curve. were dispensed into tissue culture tubes and exposed for 5 h, washed The dose-effect curve was plotted using a logarithmic conversion of twice, and resuspended in fresh medium plus 10% FBS. The cell this equation which determines the m and />â€ž,values.Based on the suspensions were then incubated for 0, 3, 8, and 24 h. At the end of slope of the dose-effect curves, it can be decided whether the agents the incubation period, the cells were exposed to the second drug for 5 have mutually exclusive effects (e.g., similar mode of action) or mutually h, washed twice, and resuspended to the same volume of RPMI-1640 nonexclusive effects (e.g., independent mode of action). A CI was then medium with 10% FBS. The cell suspensions were again incubated determined using the equation further for 72 h. Viable cell growth was determined by the trypan blue exclusion method. The dose-response curve was plotted on a semilog CI = (Ph. (Ph. scale as a percentage of control, the cell number of which was obtained (A), (A)2 from samples with no drug exposure, but processed simultaneously. The experiments were carried out in duplicate and repeated twice or where (D*)i is the dose of Drug 1 required to produce x% effect alone. (/i,), for x% cell kill can be determined by drawing a least-square more. regression line on a computer graphic system (19). (/)), is the dose of Isobologram Analyses that drug required to produce the same x% effect in combination with (D)2. Similarly, (Â£>J2isthe dose of Drug 2 required to produce x% Analyses of drug interactions were performed by constructing "an effect alone, and (O)2 is the dose required to produce the same effect in envelope of additivity" (14, 15) on an isobologram as described previ combination. If the drugs are mutually exclusive, then a is 0; if mutually ously (22). Based on available dose-response curves, we analyzed the nonexclusive, then a is 1. When CI = 1, the interaction is considered combined effect of 2 drugs at the point of IDgo-Three isoeffect curves additive; when CI < 1, synergism is indicated, and when CI > 1, were drawn as follows. antagonism is indicated. Using this method, we analyzed the data Mode I Line. When the dose of Drug A is chosen there remains an presented in Fig. 3 below. increment in effect to be produced by Drug B. If 2 drugs were to act independently, the addition is performed by taking the increment in RESULTS doses, starting from zero, that give log survivals which add up to ID80. Mode II (A) Line. When the dose of Drug A is chosen, an isoeffect VCR-MTX Combination. For the simultaneous and continu curve can also be calculated by taking the dose increment of Drug B ous exposure of cells to VCR and MTX, the data points fell on that gives the required contribution to the total effect up to the limit, the right side of the envelope (Fig. 2A). Certain data points in this case, ID8(>. were in the area of subadditivity, and others were in the area of Mode II (B) Line. Similarly, when the dose of Drug B is chosen, an protection. These observations can be interpreted to show that isoeffect curve can be calculated by taking the dose increment of Drug A that gives the required contribution to 11)Â«0. simultaneous exposure to VCR and MTX produced subadditive When the dose-response curve of Drug A follows first-order kinetics, or mutually protective interactions. The combination of VCR Mode II (B) line will be identical to Mode I line, and vice versa. When exposure for 5 h followed by continuous exposure to MTX both drugs follow first-order kinetics all three isoeffect lines will con (Fig. 2B) or simultaneous 5-h exposures to VCR and MTX verge to make a straight line connecting 1.0 of the ordinate and abscissa. (Fig. 2(7) produced similar interactions, subadditivity and pro- 352

0 0 02 04 06 08 10 12 14 0 02 04 06 08 IO 12 O 02 04 06 08 IO 12 14 Fig. 2. Isobologram of VCR in combina DOSE OF VCR tion with MTX.. I. continuous and simultane 0 E F G ous exposure to VCR and MTX; B, short-term exposure (5 h) to VCR followed by continuous exposure to MTX; C, short-term, simultane ous exposure to VCR and MTX; D, short-term exposure to VCR followed by short-term ex posure to MTX without interval; /â€¢.,sameas in l> except with a 3-h interval; /-'. same as in D except with an 8-h interval; G, same as in D except with a 24-h interval; H, short-term ex posure to MTX followed by short-term expo sure to VCR without interval; /, same as in H except with a 3-h interval; J, same as in H except with an 8-h interval; K, same as in H except with a 24-h interval.

02 04 06 08 IO O 02 04 06 08 IO O 02 04 06 08 IO O 02 04 06 08 IO DOSE OF VCR

100 60 Fig. 3. Dose-response curves in VCR- MTX combination. A, short-term (5 h) simul 3 40 taneous exposure to VCR and MTX; B, short- oto uj Ã®20 term exposure to MTX followed by short-term -Â¡T) exposure to VCR with a 3-h interval; C, short- term exposure to MTX followed by short-term Ilio _\ exposure to VCR with a 24-h interval. MTX H 6 concentrations (M) for each symbol are shown in ,-(. VCR concentrations (M) in the abscissa SU 4 are to be divided by Id1'to give the actual value l\ studied. _ o

O IO 20 30 O IO 20 O IO 20 30 VINCRISTINE (xlO~9M) Â»IO'9M) VINCRISTINE ( VINCRISTINE ( tection. Sequential exposure of 2 drugs, however, produced posure to VCR and DNR produced subadditive effects (Fig. interval-dependent changes in the 2-drug interaction. Thus, 4A). The effects of simultaneous 5-h exposure to the 2 drugs when the cells were exposed to VCR for 5 h, followed by MTX were subadditive or mutually protective (Fig. 4Ã„).Sequential for 5 h, the data moved from the areas of subadditivity and exposure to 2 drugs with a 24-h interval produced essentially protection at the 0-h interval (Fig. 2D) into the envelope of identical interactions whether VCR preceded DNR or vice versa additivity with the interval of 3, 8, and 24 h (Fig. 2, E, F, and (Fig. 4, Cando). G, respectively). The interval-related changes in the 2-drug VCR-ara-C Combination. The simultaneous continuous or interaction were more pronounced when sequential exposure to simultaneous 5-h exposure to VCR and ara-C resulted in sub- the 2 drugs was reversed. Thus, when cells were exposed to additive or protective interaction (Fig. 4, E and F). Five-h MTX for 5 h, immediately followed by exposure to VCR for 5 exposure to VCR followed by 5-h exposure to ara-C with the h, the data points were in the area of subadditivity (Fig. 2H). interval of 24 h produced the combination data points within With the interval increasing to 3 and to 8 h, the data points the envelope of additivity (Fig. 4G). The reversed order of moved into the envelope of additivity (Fig. 21) and then into exposure in similar experiments moved the data points into the the area of supraadditivity (Fig. 27), respectively. The supraad area of marginal subadditivity, not far from additivity (Fig. 411). ditive interaction was further enhanced when the interval was VCR Combination with TMQ, VLB, ASN-ase, or BLEO. All extended to 24 h (Fig. 2K). the studies were done as simultaneous and continuous exposure Examples of the dose-response curve, which were the basis to 2 drugs. VCR combinations with TMQ (Fig. 5A) or BLEO for constructing an isobologram of subadditivity (Fig. 2C), (Fig. 5D) were subadditive and protective, while VCR combi additivity (Fig. 21), and supraadditivity (Fig. 2K), are shown in nations with VLB (Fig. SB) and ASN-ase (Fig. 5C) tended to Fig. 3, A, B, and C, respectively. be supraadditive, albeit, again not far from the area of additivity. VCR-DNR Combination. Simultaneous and continuous ex Median Effect Plot Analysis. In order to evaluate the rela- 353

Fig. 4. Isobologram of VCR in combina tion with DNR or ara-C. A, continuous and simultaneous exposure to VCR and DNR; B, simultaneous short-term (5 h) exposure to VCR and DNR; C, short-term exposure to VCR followed by short-term exposure to DNR with a 24-h interval; D, short-term exposure to DNR followed by short-term exposure to VCR 0 0.2 04 06 08 10 0 02 04 06 O8 10 12 O 02 04 06 08 IO 12 with 24-h interval; Â£,continuous and simul DOSE OF VCR taneous exposure to VCR and ara-C; F, short- F G H term simultaneous exposure to VCR and ara C; C, short-term exposure to VCR followed by short-term exposure to ara-C with a 24-h in terval; H, short-term exposure to ara-C fol lowed by short-term exposure to VCR with a 24-h interval.

"O O2 04 06 08 IO 12 1.4 O 02 04 06 08 IO 02 04 06 08 IO O 02 04 06 08 IO DOSEOF VCR

Fig. 5. Isobologram of VCR in combina tion with TMQ, VLB, ASN-ase, or BLEO. A, continuous and simultaneous exposure to VCR and TMQ; B, continuous and simulta neous exposure to VCR and VLB; C, contin uous and simultaneous exposure to VCR and ASN-ase; /'. continuous and simultaneous ex posure to VCR and BLEO. 02 04 06 08 IO O 02 04 06 08 10 0 02 04 06 08 10 12 DOSE OF VCR

tionship between data obtained from Steel's isobologram and correspond to a more common terminology of synergism, sum those from median effect plot analysis, we analyzed the raw mation, antagonism, and strong antagonism, respectively, used data of Fig. 3 for the determination of the CI. The results are by Chou. Since analytical methods and definitions used by these shown in Table 1. Table 1, upper section, shows that, when 2 2 workers are different, however, results obtained by these 2 drugs are added simultaneously, their cell kill effects had mainly methods do not necessarily fall within each respective category. antagonistic interactions, especially at low-MTX concentra Accordingly, we used each word as defined and used by the 2 tions (note the area where data values are underlined). At high- respective workers. We observed that data obtained by the 2 MTX concentrations, effects were additive or slightly synergis- methods gave a reasonable degree of proximity in judging tic (note the area where data values are in bold face). Since at synergism, summation, and antagonism. fa of 0.80 level (IDgo) CI values are often >1, the median effect Our studies show that the inhibitory effects of VCR in plot analysis gave reasonable proximity to subadditive isobol combination with MTX are schedule dependent. Exposure to ogram data obtained at IDÂ»,,.When sequential drug exposures VCR prior to or together with MTX resulted in subadditive with a 3-h interval were made in an order of MTX followed by and protective interactions (antagonism), indicating that the VCR, drug combinations became mild to moderately synergistic effects of the 2 drugs in combination are less than those ob in most of the data values (Table 1, center section), and when tained when either drug is used alone. Only the sequence of the interval of the 2 drugs was extended to 24 h, the 2 drug MTX exposure first followed by VCR, with sufficient intervals, interactions became strongly synergistic (many data with CI was supraadditive (synergistic). Progressively stronger syner values smaller than 0.5 in Table 1, center section). It is of note gism of this sequence with increasing interval was confirmed again that, in Table 1, lower section, the CI values in the range by the median effect plot analysis. Our in vitro observations are offa = 0.8 level were in the vicinity of 1.0 (additive interaction), consistent with reports by Chello et al. that delay of VCR and in Table 1, lower section, the CI values were much smaller, administration for 24 h after MTX did produce increased indicative of progressively stronger synergistic interaction with survival in mice bearing LI210 leukemia (10, 11). the increased interval between the 2 drug exposures. As the biochemical rationale of the VCR-MTX combination, Thus, in comparing data in Fig. 1C versus Table 1, upper VCR-induced enhancement of steady-state levels of intracellu- section, Fig. 27 versus Table 1, center section, and Fig. 2K versus lar MTX, decreased MTX efflux, and increased intracellular Table 1, lower section,v/e find that Steel's subadditivity, additiv- MTX poly glutamai ion were described (1-7). These reports are ity, and supraadditivity correspond closely to Chou's antago in contrast to observations made by Bruckner et al. who re nism, summation, and synergism, respectively. ported suppressive effects of VCR on MTX-induced inhibition of DNA synthesis (24). Our data are consistent with the findings DISCUSSION of Bruckner et al. The reasons why exposure to MTX followed by VCR pro Linguistically speaking, supraadditivity, additivity, subaddi duced synergistic interaction cannot readily be explained. No tivity, and mutually protective interaction used by Steel should biochemical data are available as to whether MTX enhances 354

Table I Median effect plot analysis of data presented in Fig. 3 Data values for the combination are presented as fa (fractional inhibition). CI for mutually nonexclusive assumption, and CI for mutually exclusive assumption, numbered in parentheses in this order. VCRMTY(xlO-*M) Simultaneous exposure to MTX and (X 10-'M)3 10 0 50.59 0.20.1 0 0.690.55 0.12Â°~337(2~7f3)1.00.10 0.651.13 1.36(1.07) 0.40 07J9~~2.42(1.77) (1.31)~T"66 ~T82(1.29)

0!982*~0.854(0.834)0.9850.812(0.776)0.9850.986(0.877)3(1.47) 0^9-T727(1.08)103.0 0.70 07721.43(1.13)0.881.04(0.893)0.9381.11(0.998)VCR07780^541.23 1.15(0.990) (0.949)0.90 0.870.960(0.876)300.86 0.910.954 1.08(0.808) (0.885)0.962 0.92 0.9251.26(1.18)20.390.371.39(T721)0302.08(1.51)0.9620.672 0.805(0.608) (0.698)100.850.801.57(T755)o~53^T611.46)133(1.15)0752TT1.31(TÃ–6)0.9670.971(0.81)300.980.371.19(1.19)079~71.13(1.12)

hMTX VCRMTY(xlO-*M) . (XlO-'M)50.600.600.940(0.916)0.770.676(0.631)0.780.878(0.767)0.870.773(0.663)0.891.23(TÃ•Ã•Ã•)100.790.810.960(0.955)0.880.729(0.711)0.890.758(0.714)0.940.560(0.522)0.950.663(0.586)300.960.970.893(0.892)0.9170.846(0.844)0.980.663(0.659)0.9870.514(0.503)0.9880.514(0.503)24 00

00.1 0.081.0

0.483.0

0.5210

0.7830

0.8120.200.301.04(0.918)0.501.14(0.927)0.601.42(1.14)o^TS1.13(0.969)0.871.18(TUS)VCR

kMTX VCRMTY(xlO-'M) .

00.1 0.201.0

0.303.0 17(T61)0.790.396(0.363)0.860.427(0.391)0.970.105(0.103)VCR(xlO-'M)50.400.590.862(0.838)0.700.835(0.754)0.880.412(0.393)0.930.327(0.311)0.970.202(0.195)100.770.860.752(0.750)0.950.415(0.414)Â«.9650.344(OJ42)300.970.9860.614(0.614)

0.6010

0.7230

0.8620.18OJ70.735(0.662)0.402

" Numbers with underscore, antagonistic interactions. * Numbers in bold face, synergistic interactions.

VCR uptake and/or binding of VCR to microtubules. ditive interaction of VCR-ASN-ase in vitro, this combination We find simultaneous exposure of VCR-MTX, VCR-ara-C, was reported to be clinically more neurotoxic (27). Possible and VCR-BLEO, all of which are a part of clinically active explanations for the discrepancy between in vivo and in vitro multidrug combinations, to be not more than additive in vitro. data include: (a) target cells are different in each experimental The VCR-DNR combination was shown to be subadditive and system; (b) data from in vivo experiments take into account mutually protective in vitro, whereas the same combination was both therapeutic and toxic effects, while data from in vitro reported to be therapeutically synergistic in mice bearing LI210 experiments at the cellular level give effects that will be either leukemia and in rats bearing L5222 leukemia (25, 26). Further therapeutic or toxic depending upon the target cells used; (c) more, while our study tends to show that somewhat supraad pharmacokinetic values are different between in vivo and in 355

Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1988 American Association for Cancer Research. VCR AND MTX IN COMBINATION //V VITRO M. High-dose methotrexate with citrovorum factor in osteogenic sarcoma. vitro conditions; (d) different methods are used for analyzing Progress Report II. Cancer Treat. Rep., 61: 675-679, 1977. drug combinations (28, 29); and (e) different definitions of 10. Chello, P. L., and Sirotnak, F. M. Increased schedule-dependent synergism synergism and antagonism are used. of vindesine versus vincristine in combination with methotrexate against L1210 leukemia. Cancer Treat. Res., 65: 1049-1053, 1981. We find clinically active drug combinations do not necessarily 11. Chello, P. L., Sirotnak, F. M., Dorick, D. M.. and Moccio, D. M. Schedule- produce supraadditivity or synergism in vitro. Conversely, syn- dependent synergism of methotrexate and vincristine against murine 11210 leukemia. Cancer Treat. Rep., 63: 1889-1894, 1979. ergistic combination in vitro may not always be therapeutically 12. Berenbaum, M. C. Synergy, additivism, and antagonism in immunosuppres- more efficacious. Nevertheless, our in vitro observations of sion, a critical view. Clin. Exp. Inumimi]., 28: 1-18, 1977. supraadditivity (synergism) with the sequence of MTX followed 13. Carter, W. H., Wampler, G. L., Stablein, D. M., and Campbell, E. D. Drug activity and therapeutic synergism in cancer treatment. Cancer Res., 42: by VCR, supported by experimental animal data (10, 11), favor 2963-2971. 1982. the exploration of this sequence for the treatment of VCR- and 14. Steel, G. G. Terminology in the description of drug-radiation interaction. MTX-sensitive human neoplasms. Similarly, VCR plus VLB Int. J. RadiÃ¢t.Oncol., J: 1145-1150. 1979. 15. Steel, G. G., and Peckman, M. J. Exploitable mechanisms in combined may at least be additive when combined. Neuropathy is the radiotherapy-chemotherapy: the concept of additivity. Int. J. Radial. Oncol.. most frequent and dose-limiting toxicity of VCR, whereas the 5:85-91.1979. 16. Valeriote, F., and Lin, H. Synergistic interaction of anticancer agents: a dose-limiting toxicity of VBL is myelosuppression. The com cellular perspective. Cancer Chemother. Rep. Part I, 59: 895-900, 1975. bination of VCR and VLB is tempting whether it lessens the 17. Kano, Y., Ohnuma, T., Holland, J. F., and Goldsmith. M. A. Schedule- neurotoxicity or enhances therapeutic efficacy in the treatment dependent synergism and antagonism of methotrexate (MTX) and vincristine (VCR) combinations in vitro. Proc. Am. Assoc. Cancer Res., 24: 270, 1983. of patients with Vinca alkaloid-sensitive neoplasms. 18. Chou, T-C., and Talalay, P. Quantitative analysis of dose-effects relation ships: the combined effect of multiple drugs on enzyme inhibitors. Adv. Enzyme. Regul., 22: 27-55, 1984. 19. Chou. J., and Chou, T-C. 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Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1988 American Association for Cancer Research. Effects of Vincristine in Combination with Methotrexate and Other Antitumor Agents in Human Acute Lymphoblastic Leukemia Cells in Culture

Yasuhiko Kano, Takao Ohnuma, Tsuyoshi Okano, et al.

Cancer Res 1988;48:351-356.

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