(CANCER RKSKARCII 49. 690I-61«)?. December 15. 1989] Reversal by Cefoperazone of Resistance to Etoposide, Doxorubicin, and Vinblastine in Multidrug Resistant Human Sarcoma Cells'

Michael P. Gosland, Bert L. Lum, and Branimir I. Sikic2 Oncology Division. Department of Medicine, Stanford I'niversity School of Medicine, Stanford, California 94305

ABSTRACT MDR can be reversed or modulated by many drugs, including verapamil, trifluoperazine, quinidine. reserpine, and cyclospor- The cephalosporins are a family of semisynthetic antibiotics, some ine A (8-18). These agents share some or all of the features of of which have structural features associated with substrates for the known substrates of P-gp, including lipophilicity, a planar multidrug transporter, P-glycoprotein. The activity of a series of six cephalosporins in reversing multidrug resistance (MDK) was examined polycyclic stereochemistry, and weak basicity. The mechanism in MDK variants (Do cells) of the human sarcoma line MES-SA. DxS of modulation in most cases is thought to be competitive cells express high levels of the mdr\ gene product P-glycoprotein and inhibition of drug efflux. are 25- to 30-fold resistant to doxorubicin (DOX), etoposide (VP-16), Verapamil and trifluoperazine have been used in clinical trials and vinblastine (VBL). Cytotoxicity was measured by the M'lT assay. to reverse MDR (19-24). A major problem with these trials Cefoperazone (1.0 HIM) was the most effective modulator of MDK, has been toxicity of the modulators (heart block by verapamil lowering the IC«for VP-16 by 29-fold (29x), for VBL by 16x, and for and depression of the central nervous system by trifluopera DOX by 14x. Ceftriaxone at 1.0 HIMproduced lOx modulation of VP-16 zine), at drug concentrations below those which reverse MDR cytotoxicity, 8x for DOX, and 2x for VBL. The reversal of resistance in vitro. The ability of these agents to modulate MDR is was concentration dependent, decreasing to 4x and 5x, respectively, for DOX with 0.25 HIMcefopcra/.one and ceftriaxone. No modulation of unrelated to their primary pharmacological action, since their cytotoxicity was observed in the parental MES-SA cells, which do not ability to reverse drug resistance does not correlate with their express mdr\. Cefazolin, cefotetan, ccphradine, and ceftazidime were properties of calcium channel blocking or calmodulin inhibition ineffective, producing less than 5x modulation of DOX at 1.0 HIM.Among (25-31). these cephalosporins, cefopcra/.one and ceftriaxone were the most highly Some of the cephalosporin antibiotics (Fig. 1) share structural protein bound in the media (30 and 52%), and the most lipid soluble, characteristics with agents known to modulate MDR, and are with octanol/water partitioning coefficients of —¿0.49and—¿0.60.Varying considerably less toxic in vivo. In this study, we assessed the the serum concentration in medium from 5 to 50% had less than a two ability of a series of cephalosporin antibiotics to modulate MDR fold effect on the modulation of MDK by ceftriaxone. The ability to and enhance drug accumulation in human sarcoma cells, and reverse MDK among these agents is associated with lipid solubility, high identified important physicochemical characteristics for mod protein binding, a polycyclic planar geometry, and the presence of the piperà /ine group in ccfoperazone. These data and the potential for ulating activity. achieving high tissue concentrations indicate that Cefoperazone merits further study as a modulator of MDR. MATERIALS AND METHODS

INTRODUCTION Reagents. The sources of the drugs used in these experiments are as follows: DOX (Adriamycin), Adria Laboratories, Columbus. OH; VBL The typical MDR' phenotype includes cross-resistance to (Velban), Eli Lilly Co., Indianapolis, IN; VP-16 (Vepesid), Bristol anthracyclines. Vinca alkaloids, podophyllotoxins, and other Myers Co., Wallingford. CT: verapamil (Isoptin). Knoll Pharmaceuti cytotoxic compounds, and increased expression of a membrane cals. Whippany, NJ; cephradine (Velosef), E. R. Squibb and Sons Inc., protein termed P-gp (1-3). The proposed mechanism of resist Princeton. NJ: Cefoperazone (Cefobid), Roerig, New V'ork, NY; cefo ance is a decrease in the accumulation and retention of the tetan (Cefotan), Stuart Pharmaceuticals, Wilmington. DE; ceftriaxone xenobiotics by P-gp, functioning as an active efflux pump. (Rocephin), Roche Laboratories, Nutley, NJ; ceftazidime (Tazidime) Evidence supporting this mechanism includes the association and ccfazolin (Kefzol). Eli Lilly Co.. Indianapolis, IN. The MTT salt and other chemicals unless specified were obtained from Sigma Chem of MDR with increased expression of the mdr\ gene which ical Co. (St. Louis, MO). All reagents were initially reconstituted in encodes P-gp, the demonstration of direct binding of transport Dulbecco's phosphate-buffered saline (GIBCO, Grand Island, NY) and substrates to P-gp by photoaffinity labelling, and transfer of the final dilutions were made in McCoy's 5A medium (GIBCO) supple MDR phenotype by transfection of the mdr\ gene (1-6). The mented with penicillin (100 fjg/ml), streptomycin (100 ^g/ml), insulin demonstration of increased mdr\ expression in some normal (5 fig/ml), and I0rr newborn calf serum (GIBCO). High-performance tissues and human cancers supports the hypotheses that P-gp liquid Chromatograph) grade I-octanol was purchased from Aldrich is involved both in normal detoxification of various cytotoxins Chemical Co., Milwaukee, WI. and in resistance of some cancers to chemotherapy (7). Cell Culture. The cells used in these experiments were the human sarcoma cell line MES-SA and its doxorubicin-selected MDR variant Received 6/16/89: revised 9/7/89: accepted 9/18/89. Dx5 (32, 33). Monolayer cultures of MES-SA and DxS cells were The cosls of publication of this article were defrayed in part b\ the payment grown in tissue culture flasks (Corning Glass Works, Corning, NY) of page charges. This article must therefore be hereby marked advertisement in containing McCoy's 5A medium and 10rc newborn calf serum. Cells accordance with 18 I'.S.C. Section 1734 solely to indicate this fact. 1Supported by American Cancer Society Grant CH-41 1 and NIH (¡rantCA- were maintained at 37°Cin a humidified atmosphere containing 5% 49605. CO2 and subcultured every 5 to 7 days. ! Recipient of a Faculty Development Award in Clinical Pharmacology from MIT Cytotoxicity Assay. Cells grown in 75-cnr tissue flasks were the Pharmaceutical Manufacturers Association Foundation. To whom requests for reprints should be addressed, at Oncology Division. Room M-211. Depart harvested with 0.06 M EDTA, and cell number and viability was ment of Medicine. Stanford l'ni\ersity Medical Center. Stanford. C'A 94305- determined by a hemocytometer and trypan blue dye exclusion. Cells 5306. were seeded in 96-well plates (Falcon, Becton Dickinson Co., Lincoln "The abbreviations used are: MDR. multidrug resistance; DOX, doxorubicin; Park, NJ) at 8 x IO1 cells per well and allowed to grow for 24 h at ICW, the drug concentration resulting in 50'< inhibition of MIT dye formation: 37°C.The cells were then treated with drugs. DOX. VBL. and VP-16 MTT. 3-(4.5-dimethylthiaTOl-2-yD-2.5-diphenyltetra/olium bromide: log,,,?, the were used at concentrations from 3.0 x IO'1*to 3.0 x IO"6 M and the partition coefficient or log ratio of the concentration of drug in octanol versan aqueous medium: P-gp. P-glycoprotein; VBL, \inblastine; VP-16. etoposide. cephalosporin antibiotics at concentrations ranging from 0.25 to 1.0 6901 Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1989 American Association for Cancer Research. MULTIDRUG RESISTANCE AND CEFOPERAZONE

on a LKB Biochrom spectrophotometer and compared to standard curves. The percentage of drug protein bound was determined by the formula: 100 x (total drug concentration-free drug concentration)/total drug concentration. Accumulation Studies with |'I I]Vinblast inc. MES-SA and Dx5 cells CONII C---CONII--, CEFOPERA20N1-: were harvested with 0.06 M EDTA, washed twice in Hank's balanced salt solution with 10% newborn calf serum, and resuspended at a cell concentration of 2.0 x IO6cells/ml in McCoy's 5A medium with 10% newborn calf serum and 20 mm of HEPES buffer (Sigma Chemical Company, St. Louis. MO). Both the MES-SA and Dx5 cells were exposed to ['Hjvinblastine (specific activity, 23 Ci/mmol; Amersham International, UK) at a concentration of 1.25 u\\ alone and in the NH presence of 1.0 mivi of cefoperazone. Cells were incubated for 60 min in a water bath at 37°Cwith shaking. Then 0.5-ml aliquots (containing ŒI-TRIAXONK IO6cells) were removed layered on silicone oil (Versilube F50; General Electric Co., Waterford, NY) in microcentrifuge tubes, and centrifuged at 12,000 x g for 1 min to remove the cells from drug-containing medium (2). Following the aspiration of the medium and oil, the tube tips containing the cell pellets were cut off and the cells were solubilized in 0.2 N NaOH. The tube contents were then neutralized, scintillation CEFOTETAN fluor was added, and radioactivity was counted. Statistical Analyses. Correlations of lipid solubility and protein bind ing with MDR modulation were performed by linear regression. A coefficient of variation was determined for each drug concentration on each plate (four wells per drug concentration) and standard deviations were determined from each IC50of DOX with the various modulators. Each experiment was performed at least three times.

RESULTS

Modulating of Cytotoxicity. The strain of Dx5 cells in these CEFAZOLIN experiments was 30-fold resistant to DOX compared to the parental drug-sensitive MES-SA cell line. They were also ap proximately 30-fold cross-resistant to both etoposide and vin- Fig. 1. Chemical structures of the ccphalosporins. blastine. As a positive control for modulation of MDR, vera- pamil (6 //M) lowered the 1C5(,of DOX from 3 x IO'7 M to 1.3 mM. Verapamil was used as a positive control for modulation at 6.0 x 10'* M in Dx5 cells, nearly complete reversal of resistance. /iM. Cells were incubated for 72 h with the cytotoxic agent (DOX, VBL, The IC«,ofDOX in MES-SA cells was 1.0 x IO"8 M. or VP-16) and the modulating drug. Then the medium was removed The structures of the six cephalosporins used in these exper from each well, and 100 p\ of fresh medium and 10 /¿IofMTT dye (5 iments are shown in Fig. 1. Among these, cefoperazone (1.0 mg/ml in phosphate-buffered saline stock solution) were added to each mivi) was the most effective modulator of MDR, lowering the well. Cells were incubated with MTT for 6 h, after which blue formazan IC50 for VP-16 from 10 x 10~" M to 3.4 x 10~7M (modulation crystals were dissolved in 0.1 N HCI in 2-propanol (100 ^1 per well). ratio of 29x); the 1C«,forDOX from 3 x 10~7M to 2.2 x 10~8 Within 30 min after dissolving the crystals, each plate was read on a M (14x); and the 1C,,, for VBL from 5.3 x IO'8 M to 3.3 x 10~9 Dynatech MR580 Microelisa reader with a test wavelength of 570 nm and a reference wavelength of 630 nm (34-36). Each drug concentration M (16x), Tables 1-3. Cefoperazone completely reversed resist was assayed in quadruplicate. The ICW(the drug concentration resulting ance to VP-16 (Table 2), and reduced the IC5(Iof VBL and in 50% inhibition of MTT dye formation, compared to untreated DOX in Dx5 cells to a level only two-fold higher than the controls) was determined directly from semilogarithmic dose-response sensitive parental line. curves. Ceftriaxone at 1.0 mivi produced lOx modulation of VP-16 Octanol-VVater Partitioning Studies. Five of the cephalosporin anti cytotoxicity, 8x for DOX, and 2x for VBL. The reversal of biotics (cefoperazone, ceftriaxone, cephradine, cefazolin, and cefotetan) were initially reconstituted in phosphate-buffered saline (pH 7.4) to a concentration of I x IO"* M. Then 3 ml of this solution was added to Table I Modulation of resistance to f>O\ in DxS cells by cephalosporin antibiotics 3 ml of high-performance liquid chromatography grade I-octanol and vigorously mixed, and the phases were allowed to separate. Drug (mM)Control.Cephalosporin DOX.(nM)°300 ratio*U6487S4332 concentrations were determined using an LKB Biochrom spectropho- aloneCefoperazoneDOX ±8222 tometer to measure optical density at a wavelength of 272 nm in the .0)Cefoperazone( 1 ±950 aqueous phase before and after octanol-water partitioning. Reference (0.5)Cefoperazone ±1275 solutions were also prepared for each antibiotic at concentrations from (0.25)Ceftriaxone(I.O)Ceftriaxone ±738 1 x 10 6 to 1 x 10~4 M, which provided a linear standard curve. The ±1045 (0.5)Ceftriaxone ±1055 partition coefficient. log10P,was determined by dividing the concentra (0.25)Cefazolin ±2085 tion of drug in the octanol phase by that in the aqueous phase (37). (1.0)Ceftazidime ±101 Protein Binding Studies. Three ml of each cephalosporin (at a final (1.0)Cephradine 10 ±15120± concentration of 1.0 x 10~5 in McCoy's 5A medium, pH 7.4) were (1.0)Cefotetan 20130 placed in Centricon-30 microconcentrator tubes (Amicon, Danvers, (1.0)ICM. ±30Modulation " Each value represents the mean ±standard deviation of at least three MA) and centrifuged at 1500 x g for 45 min at room temperature. determinations. Medium without drug was used as a blank for reading the optical h The modulation ratio is defined as the ratio of the IC5Ufor DOX alone versus density of the ultrafiltrate in each tube. Samples were read at 272 nm the IC50in the presence of the modulating agent. 6902 Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1989 American Association for Cancer Research. MU.IÕDRl'C; RESISTANCE AND CEFOPERAZONE

Table 2 Modulation of resistance lo VP-16 in D.\5 cells by cefoperazone and 100 ceftriaxone

ratio*291241051 (IHM)Control,Cephalosporin M)10.00.340.862.401.042.157.85l.Ox0.050.040.140.090.070.49Modulation-n. (n 80- aloneCefopera/oneVP- 16 .0)Cefoperazone( 1 (0.5)Cefopera/one 60 - (0.25)Ceftriaxone .0)Ceftriaxone( 1 0.5Ccftriaxone oc 0.25I* 3 40 - " Each value represents the mean ±standard deviation of at least three determinations. *The modulation ratio is defined as the ratio of the IC50 for VP-16 alone versus the IC50in the presence of the modulating agent. 20-

Table 3 Modulation of resistance to V'BLin I)x5 cells by cefopera:one and ceftriaxone .01 10 100 1000 (nM)°53 (m\i)Control.Cephalosporin ratio*1C74222 aloneCefoperazoneVBL ±4.33.3 ETOPOSIDE (uM) .0)Cefoperazone( 1 ±0.27.9 Fig. 3. Modulation by cefoperazone of the cytotoxicity of etoposide in Dx5 (0.5)C'efopera/one ±0.215 (0.25)Ceftriaxone cells. CFP, cefoperazone. G, no CFP; »,1.0 HIMCFP: •¿0.5mM CFP; O, 0.25 ±7.026 1.0)Ceftriaxone( ±1.429 mM CFP. 0.5Ceftriaxone ±1.434 0.251C*,, ±1.5Modulation 0 Each value represents the mean ±standard deviation of at least three 100 determinations. * The modulation ratio is defined as the ratio of the IC»for VBL alone versus the 1Câ„¢inthe presence of the modulating agent.

100 60 -

80 - OC 3 en 40 -

60 - 20 -

¡ 40 - OT

.0001 001 .01 20 VINBLASTINE (uM) Fig. 4. Modulation by cefoperazone of the cytotoxicity of vinblastine in Dx5 cells. CFP, cefoperazone. D. no CFP; »,1.0 mM CFP; (•).0.5 mM CFP; 0.0.25 mM CFP. 001 01 10 100

DOXORUBICIN (uM) Table 4 Physicochemical properties of Cephalosporin antibiotics DrugCefoperazone Proteinbound*30%52% Fig. 2. Modulation by cefoperazone of the cytoloxicity of doxorubicin in Dx5 cells. CFP. cefoperazonc. D. no CFP: ».1.0 BM CFP; I 0.5 mM CFP; O. 0.25 HIMCFP. Ceftriaxone -0.60 Cefotetan —¿12 22% Cefazolin -OÃŒ90 15%8% resistance was dependent on the concentrations of cefoperazone CephradineLog,„P°-0.49-1.5îî and ceftriaxone over the range of 0.25-1.0 mM, Tables 1-3, °As determined by oclanol-water partitioning (see "Materials and Methods"). and Figs. 2-4. No modulation of cytotoxicity was observed in * Determined in McCoy's 5A medium containing 10rínewborn calf serum. the parental MES-SA cells, which do not express mdr\ (data not shown). Cefazolin, cephradine, cefotetan, and ceftazidime calf serum. Table 4. Ceftriaxone was 52% protein bound, were relatively ineffective as modulators for DOX cytotoxicity, whereas cefoperazone was 30% bound. The correlation of per and were not tested with VBL and VP-16 (Table 1). centage protein binding in medium by the various agents and Octanol-Water Partitioning Studies. Cefoperazone was the their ability to modulate MDR was poor (r- = 0.181). most lipid soluble of the cephalosporins, with a partition coef Changing the protein concentration in the medium did not ficient (logioP) of —¿0.49atpH 7.4, Table 4. Ceftriaxone was markedly alter modulating activity. Increasing the protein con the next most lipid soluble agent with a log,0P of—0.60. There centration in the medium from 10% to 50% only slightly was a highly significant correlation between lipid solubility and decreased modulation of DOX by ceftriaxone, with the IC5o MDR modulation of these five agents (H = 0.881). increasing from 3 x 10~" to 5 x 10~" M. Protein Binding and Effects of Serum Concentration on Mod Cellular Accumulation of Vinblastine. The effect of 1.0 mM ulation of MDR. Cefoperazone and ceftriaxone were also the cefoperazone on the accumulation of ['H]vinblastine in MES most highly protein bound agents in medium with 10% newborn SA and Dx5 cells is presented in Table 5. Cefoperazone did not 6903 Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1989 American Association for Cancer Research. MULTIDRUG RESISTANCE AND CEFOPERAZONE Table 5 Effect of cefoperazone (CFP) on vinblastine accumulation in drug- sensilive MES-SA versus multidrug resistant Dx5 cells solubility has also been shown to be important in the modula Cells were incubated for 60 min at 37"C with 2 >IM[3H]vinblastine, with and tion of MDR by other classes of drugs such as analogues of without the presence of 1.0 ITIMCFP. reserpine, quinoline, and dipyridamole (29-31). Because of Cell line (-) CFP (+) CFP concern that plasma protein binding in vivo may lessen the MES-SA 43.0 ±1.4" 42.0 ±0.7 efficacy of modulation, we varied the serum concentration from Dx5 20.1 ±0.5 43.2 ±3.5* 5 to 50%. Only a modest (two-fold) decrease in modulation °Expressed as nanomolesof [3H|vinblastine per IO6cells. Values represent the was seen with the 10-fold increase in protein concentration. mean ±standard deviation, n = 3. " P< 0.05, (-) CFP versus (+) CFP. The relative efficacy of cefoperazone as a modulator of MDR may be related to its /V-ethylpiperazine group, which is unique among these cephalosporin antibiotics (Fig. 1). This structural affect drug accumulation in the MES-SA cells, which do not characteristic is shared with trifluoperazine and prochlorpera- express detectable levels of P-gp. However, it restored vinblas zine, two phenothiazines which modulate MDR (15, 25, 31, tine accumulation in the Dx5 cells to the same level as in the 38). Other phenothiazones which lack this chemical moiety, sensitive control cells. such as thioridazine, were not effective modulators (25, 31). The importance of this piperazine group is also suggested by DISCUSSION its presence in dilazep, a verapamil analogue, which is a more potent modulator than its parent compound (40). In addition, The search for safe and effective modulators of MDR is of the antihistamine prenylamine does not modulate MDR, unlike importance for several reasons. First, there is increasing evi cinnarzine, a compound different only in the addition of a dence that the mdr\ gene is expressed in many human cancers, piperazine group (30). These examples support an important and is likely to contribute to failure of chemotherapy in those role for the piperazine moiety in binding to P-gp. cancers (1, 3, 7). Second, P-gp is a multidrug transporter whose In summary, we have shown that the cephalosporin antibiotic substrates are not restricted to cytotoxins, with many different cefoperazone is an effective modulator of MDR in vitro. This kinds of agents capable of reversing resistance in preclinical modulation occurs at drug concentrations which may be achiev models (1-3, 8-18, 25-31, 37-42). Structure-activity relation able without major toxicity in patients (43-45). P-gp has been ships for modulation of MDR have been studied in several shown to be present at high levels in several normal tissues, classes of drugs, and some general principles governing the and is likely to be important in the normal disposition of MDR- binding and export of compounds by P-gp are emerging (10, related cytotoxins (7). Further studies should be performed on 12, 27-31, 37-41). Finally, initial clinical trials with two of the the efficacy of cefoperazone in vivo in preclinical models as well most potent modulators, verapamil and trifluoperazine, have as its effects on the pharmacokinetics and toxicity of anticancer been limited by severe toxicities of the modulators at plasma agents involved in MDR. concentrations below those necessary to reverse resistance based on preclinical experiments (19-24). ACKNOWLEDGMENTS The unacceptable toxicity of verapamil and trifluoperazine at concentrations required to modulate MDR in patients is not We thank our colleagues, in particular D. H-M. Lau, J. M. Berry, surprising, since these drugs are primarily used for their actions J-P. Marie, K. L. Ross, N. A. Brophy, A. D. Lewis, and M. N. Ehsan, in cardiac conduction and the central nervous system, respec for their discussions and review of the manuscript. tively. 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6905 Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1989 American Association for Cancer Research. Reversal by Cefoperazone of Resistance to Etoposide, Doxorubicin, and Vinblastine in Multidrug Resistant Human Sarcoma Cells

Michael P. Gosland, Bert L. Lum and Branimir I. Sikic

Cancer Res 1989;49:6901-6905.

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