[CANCER RESEARCH 50, 1748-1756. March 15. 1990] Cellular and Biochemical Characterization of Thioxanthenes for Reversal of Multidrug Resistance in Human and Murine Cell Lines1

James M. Ford,2 Edward P. Bruggemann, Ira Pastan, Michael M. Gottesman, and William N. Hait1

Departments of Internal Medicine and Pharmacology, Yale University School of Medicine, New Haven, Connecticut 06510 [J. M. F.. W. N. H.], and laboratory of Molecular Biology ¡E.B., I. P., M. M. G.¡,National Cancer Institute, NIH, Bethesda, Maryland 20892

ABSTRACT potent, more specific, and less toxic chemosensitizers for clin ical use. We have previously shown that phenothiazines sensitize multidrug We previously studied the structure-activity relationships of resistant (MDR) cells to chemotherapeutic drugs in a manner related to a series of phenothiazine chemosensitizers, and found that specific structural features, and have identified structurally related thi- oxanthenes with increased anti-MDR activity. We have now studied the specific structural determinants were important for increased structure-activity relationships of 16 thioxanthenes in the human breast anti-MDR activity (4). These included a hydrophobic, tricyclic cancer line MCF-7 Adr". rra/is-Thioxanthene stereoisomers were 2- to ring with a trifluoro substitution at position 2, linked by a 4- 7-fold more potent than cu-thioxanthenes for antagonizing MDR. The carbon alkyl bridge to a tertiary, cationic amino group incor most potent thioxanthenes possessed a halogenated tricyclic ring con porated into a piperazinyl ring. By searching for other com nected by a 3-carbon alkyl bridge to a piperazinyl or piperadinyl side pounds that shared these structural features, we identified the group. The chemosensitizing effects of the lead compound, rra/u-flupen- thioxanthenes as drugs which possess greater activity than thixol, its stereoisomer cis-flupenthixol, its phenothiazine homologue structurally homologous phenothiazines for sensitizing MDR fluphenazine, and the calcium channel blocker verapamil, were further cells to doxorubicin. studied in a series of sensitive and MDR cell lines. rrans-Flupenthixol We have now studied the structure-activity relationships of a caused a greater reversal of cellular resistance to doxorubicin, vinblastine, vincristine, and colchicine in MCF-7 Adr", KB-V1, and P388/DOX MDR series of thioxanthene isomers in a MDR human breast cancer cell line. The finding that frans-thioxanthenes were more potent cells than the other chemosensitizers. In particular, frani-flupenthixol modifiers of doxorubicin resistance in MCF-7 AdrR cells than was 2- to 3-fold more potent for reversing MDR than equimolar concen c/'s-isomers provides the unique opportunity to probe the ster- trations of verapamil. Furthermore, fra/u-flupenthixol fully reversed resistance to doxorubicin, vincristine, and colchicine in MDR MCF-7 eoisomeric specificity of these chemosensitizers against MDR, and NIH 3T3 cells transfected with the md>\ gene. None of these agents as well as against other putative processes responsible for drug altered MDR in a non-P-glycoprotein expressing MCF-7 cell line selected resistance, as a means of better defining their possible mecha- with mitoxantrone, nor in any of the parental cell lines. The stereoselec- nism(s) of action. tive antagonism of the flupenthixol isomers on several putative cellular Accordingly, we compared the effects of the lead compounds, targets was studied to explore the mechanism of their chemosensitizing eis- and frans-flupenthixol, to their phenothiazine structural activity. 1-/.V-and fra/H-flupenthixol were equally active inhibitors of protein kinase C and calmodulin. Both ds- and fra/u-flupenthixol were homologue, fluphenazine, and to the structurally unrelated also potent inhibitors of |'H|a/idopine binding to P-glycoprotein. The chemosensitizer, verapamil, on primary drug resistance, cross- apparent lack of clinical toxicity of rra/u-flupenthixol makes it an attrac resistance, and drug accumulation in several well characterized tive drug for possible use in the modulation of tumor resistance in vivo if cell lines. In addition, the thioxanthene isomers were studied appropriate tissue concentrations can be achieved. for their potency and stereospecificity as inhibitors of CaM and PKC, cellular components potentially important for reversing MDR, and for binding to P-gp. INTRODUCTION

Resistance of tumor cells to chemotherapeutic drugs is con MATERIALS AND METHODS sidered to be a major problem in the clinical treatment of cancer. MDR4 is a form of drug resistance characterized by Cell Lines and Culture. MCF-7 and MCF-7 Adr" human breast decreased cellular sensitivity to a broad range of chemothera cancer cells were maintained in exponential growth in Corning 75-cm2 peutic agents due at least in part to the expression of the mdr\ tissue culture flasks in RPMI 1640 medium supplemented with 5% gene product, P-gp (1). The recent identification of mdr\ fetal bovine serum. The latter cell line was derived by stepwise exposure mRNA and P-gp in human tumors (2) suggests that MDR may to doxorubicin and has been shown to overexpress P-gp and the GSTTr contribute significantly to clinical drug resistance. While a isoenzyme (5, 6). MCF-7/pBC19 cells were derived from a sensitive MCF-7 clone transfected with the mdr\ gene (7). The MCF-7 lines number of pharmacological agents have been shown to partially were a gift from Dr. Kenneth H. Cowan (Medicine Branch, National reverse MDR in vitro (3), there remains a need to identify more Cancer Institute, Bethesda, MD). Received 9/1/89; revised 11/30/89. MCF-7/MITOX cells (from Dr. William S. Dalton, University of The costs of publication of this article were defrayed in part by the payment Arizona Cancer Center, Tucson, AZ), selected for resistance to mitox of page charges. This article must therefore be hereby marked advertisement in antrone, were grown similarly to the other MCF-7 lines, except that accordance with 18 U.S.C. Section 1734 solely to indicate this fact. RPMI 1640 medium was supplemented with 10% fetal bovine serum, 1Supported by the National Cancer Institute (Grants CA43888-03 and POI and cells were continuously grown in 8 x 10~8M mitoxantrone. These CA08341-25) and Grant CH-49507 from the American Cancer Society. W. N. H. is a Burroughs-Wellcome Scholar in Clinical Pharmacology. E. P. B. is cells are cross-resistant to anthracyclines and Vinca alkaloids and supported by a fellowship from the American Cancer Society. display decreased drug accumulation, but do not overexpress P-gp (8). 2Present address: Department of Medicine. S-102C. Stanford University Human epidermal carcinoma KB-3-I cells and the vinblastine-se- Medical School. Stanford, CA 94305. 3To whom requests for reprints should be addressed. lected MDR subline, KB-V1 were grown in monolayer culture at 37°C 4The abbreviations used are: MDR. multidrug resistance: [3H]azidopine, 2,6- in Dulbecco's modified Eagle's medium with 10% fetal bovine serum dimethyl-[4-(2'-trifluoromethyl)phenyl]-l,4-dihydropyridine-3.5-dicarboxylic and L-glutamine. KB-V1 cells were grown in the continuous presence acid, ethyl,)iV-4"-azido[3",5"-3H]ben7-oylaminoethyl)diester;CaM, calmodulin; P- of I Mg/ml vinblastine. These cells overexpress P-gp (9), and possess a gp, P-glycoprotein; PBS, phosphate-buffered saline: PKC. protein kinase C; ICW. mean concentration ±SE that produces 50% inhibition of cell growth compared 100-fold amplification of the mdr\ gene (10). to vehicle-treated controls. Murine leukemic P388 cells and the MDR cell line P388/DOX 1748

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1990 American Association for Cancer Research. REVERSAL OF MDR BY (rani-FLUPENTHIXOI-

(from Dr. Ram Ganapathi, Cleveland Clinic Foundation. Cleveland, produced £10%inhibition of growth. Dose-response curves were cor OH) were grown in suspension in RPMI 1640 medium supplemented rected for the inhibition of cell growth caused by chemosensitizers with 2 mM i.-glutamine, 10% fetal bovine serum, and 10 /¿M2-mercap- alone, and the "fold reversal" of MDR for each drug plus chemosensi- toethanol. tizer combination was calculated as follows: Murine NIH 3T3 fibroblasts and a MDR 3T3 line (3T3/MDR1) transfeeted with a retrovirus expression vector containing a full-length Fold reversal = drug alone complementary DNA from the human mdr\ gene (11) and selected for lCilof 0.5% méthylèneblue(Sigma) in 50% ethanol (w/v) for 30 defined as the amount needed to produce 50% of the maximal activation min at room temperature. Unbound stain was removed by decanting, of phosphodiesterase. The effect of drugs on calmodulin was determined followed by three, 1-liter washes in distilled, deionized water. The plates by their ability to inhibit the activation of phosphodiesterase in the were dried for 12 h and the stained protein was solubilized with 200 p\ presence of 10 units of calmodulin. The specificity of this assay was of sodium jV-lauroyl sarcosine (Fluka, Switzerland) solution [1% (v/v) shown by determining the basal inhibition of phosphodiesterase in the in PBS). The absorbance of each well was determined by spectropho- absence of CaM. tometry at a wavelength of 600 nm with a microculture plate reader Inhibition of Protein Kinase C by Thioxanthenes. PKC was partially (Titertek Model MCC/340) interfaced to an Apple He computer. In purified as previously described (17). Briefly, 1x10* MCF-7 Adr" cells hibition of cell growth was expressed as a percentage of absorbance of were washed once with PBS by centrifugation at 100 x g at 4°C,and vehicle-treated control cultures. IC50was the concentration of drug that the pellet was resuspended in 1.5 ml of 20 mM Tris-HCl buffer con reduced staining (A«»)to50% of vehicle-treated controls. taining 2 mM EDTA, 0.5 mM ethyleneglycol bis(ff-aminoethyl ether)- Inhibition of growth of suspension cells (P388 lines) was determined Ar^VJV'v/V'-tetraacetic acid, 2 mM phenylmethylsulfonyl fluoride, and by using the modified 3-(4,5-dimethylthiazole-2-yl)-2.5-diphenyltetra- 5 mM 2-mercaptoethanol, pH 7.5. The cells were allowed to swell for zolium bromide assay (13). Briefly, 0.5 x IO4P388 cells were plated in 5 min and then gently homogenized with 30 strokes of a Dounce 100-jil aliquots of growth medium into 96-well microtiter plates, and homogenizer. Sucrose was added to a final concentration of 0.33 M and allowed to equilibrate for 2 to 4 h. Drugs diluted in medium were added the homogenate was centrifuged at 100,000 x g for 1 h. The soluble in I()()-/.

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1990 American Association for Cancer Research. REVERSAL OF MDR BY irans-FLUPENTHlXOL reaction mixture were spotted onto P81 cation exchange paper. The little chemosensitizing activity (cis- or trans-162), while several filters were washed 3 times in 75 mM phosphoric acid and counted in thioxanthenes with 3-carbon aliphatic side chains possessed 5 ml of Ecoscint with a Beckman LS 7500 liquid scintillation counter. increased activity (irawî-chlorprothixene,frans-768). It was also Assay conditions were chosen to ensure linearity of the reaction with necessary for cyclic ring side chains substituents to be a distance respect to time of incubation and concentration of tissue. of at least 3 carbons from the thioxanthene ring nucleus for Photoaffmity Labeling of Plasma Membranes from Multidrug Resist ant Cells. P-gp from KB-C1 or KB-V1 MDR cells, selected for resist activity, as demonstrated by trans-7006 (1.6-fold reversal) com ance with colchicine and vinblastine, respectively ( 11), was labeled with pared to fraws-clopenthixol (15.0-fold reversal), otherwise iden the photoactive dihydropyridine calcium channel blocker [3H]azidopine tical molecules except for a 2-carbon versus 3-carbon alkyl (Amersham. Arlington Heights, IL), using modifications of previously bridge. described methods (19). Briefly, 1.0 x 10" cells in 100 ^1 PBS were The most effective thioxanthenes, iraws-clopenthixol, trans- preincubated for l h at 25°Cwith 1 MCi [3H]azidopine (40 Ci/mmol) flupenthixol, and trans-753, produced an approximately 15- in the absence or presence of nonradioactive competing ligands. and then irradiated for 20 min with a UV lamp at 0°C.Photolabeled fold reversal of doxorubicin resistance, and each possessed cyclic piperazinyl or piperadinyl substituents at a distance of 3 membranes were immunoprecipitated with a polyclonal antiserum to P-gp to remove nonspecifically labeled proteins, and analyzed by 8% carbons from the thioxanthene ring, in a trans configuration. Compound 789, similar to clopenthixol, but lacking the exo- sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Drugs and Reagents. The following thioxanthene derivatives were cyclic double bond, also possessed appreciable chemosensitizing activity, caused a 9.7-fold reversal of doxorubicin resistance. generously supplied by Dr. John Hyttel of H. Lundbeck (Copenhagen, Denmark): cis- and /rans-flupenthixol, cis- and /rans-clopenthixol, cis- The thioxanthene isomers displayed a relative lack of potency and «rans-chlorprothixene, compounds N 751, N 753 A, N 753 B, N and lack of stereospecificity as antiproliferative agents (Table 762 A, N 762 B, N 768 A, N 768 B, N 789, N 796 A, N 796 B, and N 1). The IC50s for inhibition of cell growth by the 14 thioxan 7006. Fluphenazine was provided by Dr. S. J. Lucania (E. R. Squibb & thenes ranged from 8 to 40 ßM.There was no consistent Sons), bleomycin (Blenoxane) by Dr. Emmanuel Losada (Bristol Lab stereospecificity. Furthermore, there was no apparent relation oratories, Wallingford, CT), and mitoxantrone (Lederle) purchased ship between potency for inhibition of cell growth and chemo from the Yale-New Haven Hospital Pharmacy Department. Doxorub- sensitizing activity. icin, vincristine, vinblastine sulfate, colchicine, verapamil, adenosine Relative Resistance and Cross-Resistance of Cell Lines to monophosphate, L-phosphatidyl-L-serine, 1,3-diolein, and histone (type III-S) were obtained from Sigma. Myokinase and pyruvate kinase were Cytotoxic Drugs. Table 2 shows the IC50values for the inhibition obtained from Boehringer-Mannheim (Indianapolis, IN) and firefly of cell growth by several different cytotoxic drugs against each luciferin-luciferase was from E. I. du Pont de Nemours & Co. (Wil of the cell lines, and the relative resistance for every drug. The mington, DE). [7-'2P]ATP was obtained from New England Nuclear resistant sublines displayed a typical MDR phenotype, having Research Products. Inc. (Boston, MA). Whatman DE52 ion exchange significant cross-resistance to anthracyclines (doxorubicin, mi cellulose was purchased from Macalaster Bicknell (New Haven, CT). toxantrone), Vinca alkaloids (vincristine, vinblastine), and other P81 cellulose phosphate paper was purchased from Whatman (Hills- natural product drugs (colchicine), but little or no resistance to boro, OR). Other reagents were of analytical grade and were obtained bleomycin. MCF-7/MITOX, P388/DOX, and 3T3/MDR1 cell from commercial sources. lines were more resistant to the drug used for selection (primary Statistical Analysis. Statistical analysis of each dose-response curve resistance), than to the other drugs tested (cross-resistance). was performed by the method of Finney (20). Accordingly, IC50s±SE For example, MCF-7/MITOX cells were 100-fold resistant to for the inhibition of cellular proliferation by drugs alone or in combi nation were determined by linear regression analysis of the logit trans mitoxantrone, but only 50-fold, 20-fold, and 10-fold resistant formed data. The degree of significance of each value of fold reversal to doxorubicin, colchicine, and bleomycin, respectively. MCF- was then determined by using Student's two-tailed t test, and was 7 AdrR cells were 200-fold resistant to the selecting agent, expressed in terms of P values; 95% confidence intervals for the fold doxorubicin, but were even more resistant to colchicine (400- reversal were calculated by Fieller's ratio of means as modified by Bliss fold). Similarly, KB-V1 cells were more resistant to vincristine (21). Student's t test was also used to analyze the significance of (2000-fold) than to the selecting agent vinblastine (500-fold). differences between the fold reversal and IC50sfor inhibition of cellular Reversal of Multidrug Resistance by Chemosensitizers. The proliferation by various drugs. effects of each of the Chemosensitizers on primary and cross- resistance to cytotoxic drugs in sensitive and MDR cells are shown in Table 3. In these studies, we determined IC50s to each RESULTS chemotherapeutic drug in the absence and presence of nontoxic, Effect of Thioxanthene Isomers on Inhibition of Cellular Pro equimolar concentrations of m-flupenthixol, /rans-flupen- liferation and Multidrug Resistance. To study the structure- thixol, fluphenazine, and verapamil, and calculated the fold activity relationships of the thioxanthenes, the antiproliferative reversal of resistance for each combination. None of the Che and chemosensitizing activity of 14 different stereoisomers were mosensitizers had significant activity in potentiating the cyto determined in 200-fold doxorubicin resistant MCF-7 AdrR cells. toxic drugs against the sensitive cell lines, MCF-7, KB-3-1, Table 1 shows the IC50 values for inhibition of cell growth and P388, or NIH 3T3, except for verapamil plus doxorubicin in the fold reversal of doxorubicin resistance caused by equitoxic P388 cells (2.3-fold reversal), and fluphenazine and vinblastine concentrations (2-5 ¿ÕM)ofeach derivative, and demonstrates in MCF-7 cells (2.0-fold reversal). The Chemosensitizers did the stereospecificity of the effect of thioxanthenes on MDR. not alter the primary resistance to mitoxantrone or cross- There was no stereospecific effect on cell growth. Accordingly, resistance to doxorubicin and vinblastine in the non-P-gp- the iraws-isomer of each active pair of thioxanthene chemosen- associated multiple drug resistance cell line, MCF-7/MITOX. sitizers was 2- to 6-fold more effective than the m-isomer for frans-Flupenthixol was the most effective drug for partially the reversal of MDR at equitotoxic concentrations. This rela reversing primary and cross-resistance in P-gp-associated MDR tionship also was true for equimolar concentrations of thio cell lines. In the MCF-7 AdrR, KB-V1, and P388/DOX cell xanthenes (data not shown). lines, mzws-flupenthixol caused an 8- to 42-fold, 20- to 57-fold, Alterations in the length or type of side chain affected anti- and 4- to 7-fold reversal, respectively, of cell resistance to MDR activity. Aliphatic side chains of 2 carbon atoms had doxorubicin, vinblastine, vincristine, and/or colchicine. In these 1750

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1990 American Association for Cancer Research. REVERSAL OF MDR BY rrans-FLUPENTHIXOL

Table 1 Effect of Ihioxanthene stereoisomers on cell growth and mull ¡drugresistance 6 5 4

9

Inhibition of cell growth was determined by exposing MCF-7 Adr* cells to 0-100 UMof each thioxanthene. IC50 is the mean concentration ±SE that produced 50% inhibition of cell growth compard to vehicle-treated controls, as described in "Materials and Methods." To determine the fold reversal, MCF-7 Adr" cells were exposed to 0-100 pM doxorubicin in the presence or absence of thioxanthene at a concentration that alone produced 10% inhibition of cellular growth (2-5 MM).Fold reversal is the IC50 for doxorubicin alone divided by the IC50 for doxorubicin in the presence of thioxanthene: 95% confidence intervals (CI) and P values for comparison of IC5<,sfor inhibition of cell growth by doxorubicin in the absence versus presence of each modifier were derived as described in "Materials and Methods." All values represent the mean of two to three separate experiments; each experiment was done in quadruplicate. Fold reversal Name IC»±SE OiM) ±95% CI

CM-762(B)tram-Ibi ±440 ±0.2° -CI =CH— CH2—N 1.3 ±0.1* CH3 (A)ri'i-chlorprothixeneírartí-chlorprothixene30±620

±213±2 —¿CI = CH—CH2—CH2—N CH

/CH2CH3 c/i-768 (B) 17±3 4.0 ±0.3C -Cl =CH— CH¿—CH2—N rrans-768 (A) 18 ±3 7.2 ±0.6'

cis-796 (A) 28 ±4 1.8 ±0.2" -CF, =CH— CH-f—CH¿—N CH trans-796 (B) 14±3 2.8 ±0.3'

cii-753 (B) 20 ±3 6.7 ±1.8' -Cl =CH— CH¿—CH-Õ—NCH2 froni-753 (A) 40 ±5 15.0 ±2.5'

-CI =CH—CH2—CH2—N NH racemic 751 8±2 1.8 ±0.3''

—¿Cl =CH—CH2-N N—CH2CH2OH trans-1006 13±3 1.6±0.4''

-Cl =CH—CH¿—CH2—NN—CH2CH2OH c/j-clopenthixol 20 ±2 2.6 ±0.4r

franj-clopenthixol 15±2 15.0±2.3'

-CF, :CH—CH¿—CH2—N N—CH2CH2OH cw-flupenthixol 24 ±4 4.8 ±0.6'

rrani-flupenthixol 25 ±4 15.2± 1.9'

-Cl —¿CM;*—CH2—CH¿-NN—CH2CH2OH 789 22 ±5 9.7 ±2.7'

' P, not significant. •¿p

1751

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1990 American Association for Cancer Research. REVERSAL OF MDR BY ira/H-FLUPENTHIXOL

Table 2 Relative resistance of cell lines to cytotoxic drugs penthixol fully reversed doxorubicin and vincristine resistance Inhibition of cell growth was determined by exposing cells to various concen in an MCF-7 line transfected with the mdr\ gene (MCF-7/ trations of doxorubicin (DOX), vinblastine (VLB), vincristine (VCR), colchicine (COLCH), mitoxantrone (MITO), or bleomycin (BLEO) for 48 to 72 h. The pBC19). fra/jj-Flupenthixol was particularly effective as well as relative resistance of each cell line to different drugs was calculated by dividing stereoselective for reversing vincristine resistance, as seen in the 1C.,, of a MDR cell line to a drug by the 1C,,, of its parental drug-sensitive both MCF-7 AdrR and KB-V1 cells. For example, in the latter cell line to the same drug. All values represent the mean of two to three separate experiments with standard errors of less than 5%; each experiment was done in cell line, irans-flupenthixol caused a 10-, 3-, and 4-fold greater quadruplicate. antagonism of vincristine resistance than did fluphenazine, resistanceCell Relative verapamil, and cw-flupenthixol, respectively. In fact, the effects lineMCF-7/DOXMCF-7/MITOXMCF-7/OBC19KB-V1P388/DOX3T3/MDR1DOX20050520010020VLB1003050020VCR200102000COLCH4002016640MITO201002012BLEO11011of fra/is-flupenthixol were stereospecific for reversing MDR in each P-gp-associated MDR cell line, except for 3T3/MDR1, since equimolar concentrations of frawi-flupenthixol caused 1.5- to 4-fold greater antagonism of MDR than its stereoisomer, c/s-flupenthixol. In the 3T/MDR1 cell line, cis- and trans- flupenthixol were equal in their ability to reverse colchicine and doxorubicin resistance. Table 3 Reversal ofmultidrug resistance by Chemosensitizers Potency of Chemosensitizers. The potency of fra/is-flupen- Cells were exposed to various concentrations of doxorubicin (DOX), vinblas tine (VLB), vincristine (VCR), colchicine (COLCH), or mitoxantrone (MITO), thixol and verapamil in MCF-7 AdrR cells was determined by in the absence or presence of c/i-flupenthixol (c/i-FPT), rrans-flupenthixol (trans- performing dose-response curves to doxorubicin in the absence FPT), flupenenazine (FLU), or verapamil (VRP) at concentrations that alone produced < 10% inhibition of cell growth ([CS]). The fold reversal is the IC50for and presence of several different concentrations of the chemo- cytotoxic drug alone divided by the 1C,,, for drug in the presence of chemosensi- tizer, and was derived as described in "Materials and Methods." All values sensitizers. Fig. 1 shows the linear relationship between the represent the mean of 2 to 3 separate experiments with standard errors less than concentration of drug and the chemosensitizing effect for both 5%; each experiment was done in quadruplicate. ira/w-flupenthixol and verapamil. írawí-Flupenthixolwas2- to reversalc/i-FPT1.01.11.51.1725335.0.0.0.1.0.1201520171.41.0531.21.010401336trans-FPT1.01.61.71.015364283101.01.01.01.21.01.0405735201.01.0741.01.010371420VRP1.01.31.01.01333154.0.0.0.7.0.1151830202.31.06ND1.01.02043IS40FLU1.02.01.01.0320121.3.0.0.0.4.0.2106341.71.0231.01.03105133-fold more potent than verapamil in these cells. For example,

CelllineMCF-7MCF-7 (MM)55S5S5553355555555551111S5512512Fold 5 fiM /rans-flupenthixol versus 12 /¿Mverapamil was necessary to cause a 15-fold reversal of MDR. Additivity of Chemosensitizers for Reversing Multidrug Re sistance. Isobologram analysis was performed to determine the interaction between fnms-flupenthixol and verapamil on the Adr"MCF-7/pBC19MCF-7/MITOXKB-3-1KB- cytotoxicity of doxorubicin in MCF-7 AdrR cells. Fig. 2 dem onstrates that the drugs were additive in their effects on sensi tivity to doxorubicin in MCF-7 AdrR cells. Effect of Chemosensitizers on Drug Accumulation. The effect of equimolar concentrations of Chemosensitizers on the accu mulation of doxorubicin in both sensitive and MDR cell lines is shown in Fig. 3. After a 3-h incubation with 10 UMdoxorub icin, MCF-7 AdrR cells accumulated 10-fold less drug than MCF-7-sensitive cells. The presence of 5 ¿IMfrawi-flupenthixol, c/'í-flupenthixol, and verapamil caused a 5-fold, 3-fold, and 4- fold increase in doxorubicin accumulation in MCF-7 AdrR cells, VIP388P388/DOXNIH but caused no change in accumulation in sensitive cells.

40- •¿trans-Flupenthixol 0 Verapamil

30-

3T33T3/MDR1DrugDOXVLBMITOCOLCHDOXVLBVCRCOLCHDOXVCRMITODOXVLBVLBDOXCOLCHVLBVCRDOXCOLCHDOXVLBDOXVLBCOLCHDOXCOLCHCOLCHDOXDOX[CS] i 20- E 2 o

10- three cell lines, fra/w-flupenthixol caused a 2- to 12-fold greater antagonism of MDR than equimolar concentrations of its phe- 4 6 8 10 12 14 16 nothiazine homologue fluphenazine. fra/is-Flupenthixol was up to 3-fold more effective for reversing MDR than the chemosen [chemosensitizer] ( Fig. 1. Potency of chemosensitizing activity for rraru-flupenthixol and vera sitizer verapamil. Against the 3T3/MDR1 cells, a line trans- pamil in MDR cells. MCF-7 Adr* cells were exposed to 0-100 /

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1990 American Association for Cancer Research. REVERSAL OF MDR BY /raw-FLfPENTHIXOL

Table 4 Inhibition of calmodulin und protein kinase C hy chemosensitiiera Antagonism of CaM by chemosensitizers was determined by their ability to inhibit by 50°;the activation of a C'aM-depcndent form of cyclic nucleotide phosphodiesterase. as described in "Materials and Methods." The effect of thioxanthenes on PKC was determined by their ability to inhibit by 50r¿the activity of enzymes partially purified from MCF-7 Adr" cells, as described in "Materials and Methods."

IC, Chemosensitizer CaM PKC £-/.v-Flupenthixol II 38 fran.ï-Flupenthixol 7 28 Fluphenazine 5

Verapamil (uM) Fig. 2. Isobolognim analysis of Ihc chemosensitizing inleraclion between vcrapamil and rran.v-flupenthixol in MCF-7 Adr" cells. Cells were exposed to 0- / 100 /¿Mdoxorubicin plus various combinations of verapamil and /ra/i.v-flupen- thixol 48 h and those concentrations that together caused a 15-fold reversal of MDR were identified (•). . predicted isobole for modifiers which have additive chemoscnsitizing effects. Ten JIMverapamil and 5 UM/ran.s-flupenthixol alone caused a 15-fold reversal. Points, mean value of quadruplicate determina tions which differed by less than 5%. P170-

MCF-7Cells 3000 -i MCF-7/OOXCelte

- - 2000 -

a E 2 S o

Fig. 4. Effect of drugs on binding of |'H]azidopine to plasma membranes from NoCS cis-FPT trans-FPT VRP KB-CI cells. One hundred and 20-fold excess vinblasline. fluphenazine, cis- flupenthixol and rra/is-flupenlhixol were incubated with 250 n\i azidopinc as described in "Materials and Methods." Photolabeled P-gp was immunoprecipi- Chemosensitizer (5 uM) tated and analyzed by sodium dodecyl sulfatc-polyacrylamide gel electrophoresis. Fig. 3. Effect of cquimolar concentrations of chemosensitizers on accumula tion of doxorubicin in sensitive MCF-7 and MCF-7 AdrR cells. Cells were incubated for 3 h with 10 /IM doxorubicin in the absence (No CS) or presence of potent for this effect, while 20- and 100-fold excess fluphena 5 JIM c/J-flupenthixol (cis-FPT)l. fruns-flupenthixol (trans-FPT). or verapamil ( I'RP). Cell-associated doxorubicin (pmol/106 cells) was determined spectrofluo- zine was less effective. Virtually identical results were obtained rometrically as described in "Materials and Methods." Values are means from when KB-V1 cells were used. duplicate determinations with standard errors less than 5«of the means. DISCUSSION Effect of Thioxanthenes on Calmodulin and Protein Kinase C. Since phenothiazines and thioxanthenes are known to antago Certain structural features of phenothiazines appear to be nize CaM, the effect of c/s-flupenthixol, /rans-flupenthixol, and important for the reversal of MDR (4). These include a tricyclic. fluphenazine as inhibitors of a CaM-dependent form of cyclic hydrophobic ring with a trifluoro substitution at C-2, and a nucleotide phosphodiesterase were determined and compared piperazinyl side chain containing a tertiary amino group at a to their chemosensitizing effects. Table 4 demonstrates that the distance of at least 3 carbon atoms from the tricyclic ring. The effects of m-flupenthixol, frans-flupenthixol, and fluphenazine thioxanthenes possessed several of these features and trans- on CaM did not correlate with their effects on MDR. trans- flupenthixol was identified as a potent chemosensitizing agent Flupenthixol was a slightly more potent CaM antagonist than (4). The current studies addressed the structure-activity rela m-flupenthixol (IC50 = 7 UM versus 11 ^M). and both were tionships for the ability of thioxanthenes to reverse MDR slightly less potent than fluphenazine (IC50 = 5 jtivi). (Table 1). As can be seen, certain structural features were cis- and fraws-flupenthixol were also examined for their effect important. For example, thioxanthenes with aliphatic or cyclic on the inhibition of PKC. Table 4 shows that the thioxanthenes side chains of 3 carbons in length were more effective than had similar potency against PKC activity. those with 2-carbon alkyl bridges (compounds 762 and 7006). Inhibition of P-Clycoprotein Photoaffinity Labeling by Che Also, piperazinyl or piperadinyl amino side group substitutions mosensitizers. Fig. 4 demonstrates the effect of chemosensitiz resulted in greater activity than aliphatic side groups (com ers on the binding of ['H]azidopine (250 HM)to P-gp from KB- pound 753, clopenthixol and flupenthixol). The most effective Cl cells (band P170). Twenty- to 100-fold excess vinblastine pair of thioxanthenes, cis- and /ranj-flupenthixol, have a CF., effectively inhibited [''Hjazidopine binding to P-gp; 20- and substitution at position 2 of the thioxanthene ring, resulting in 100-fold excess cis- and fra/w-flupenthixol completely inhibited increased hydrophobicity, and possess a piperazinyl amino side the binding of ['Hjazidopine to P-gp, and appeared equally chain attached to a 3-carbon alkyl bridge. 1753

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1990 American Association for Cancer Research. REVERSAL OF MDR BY mjns-FLl'PENTHIXOL

The spatial orientation of the side chain amino group in xanthenes and verapamil affected doxorubicin accumulation in relation to the tricyclic nucleus was a important determinant of MCF-7 AdrR cells in correlation with their degree of chemosen anti-MDR activity against MCF-7 AdrR cells, but not of anti- sitizing activity, it is likely that these modifiers act through proliferative activity. For every pair of thioxanthene isomers. either direct or indirect inhibition of P-glycoprotein-mediated the rrans-isomer was more effective than the m-isomer for chemotherapeutic drug efflux. reversing MDR, and several, such as frans-flupenthixol, trans- While iranj-flupenthixol was a more potent and effective clopenthixol, and trans-153, were 5- to 7-fold more effective chemosensitizer than m-flupenthixol in each of the in vitro chemosensitizers. Conversely, no consistent stereoisomerism (MCF-7 AdrR, KB-V1) or in vivo (P388/DOX)-derived MDR was seen for the antiproliferative effects of these compounds. cell lines, frans-flupenthixol was equal, or even less effective This suggests that the superior anti-MDR activity of the thi- than m-flupenthixol at antagonizing MDR in the 3T3/MDR1 oxanthenes may be due to specific binding to a receptor with line (Table 3). Since this particular line is resistant by virtue of greater affinity for compounds in the trans configuration, or transfection and expression of an mdr\ gene, it should theoret compounds which are able to assume the trans configuration ically display a "pure" form of P-gp-mediated MDR, allowing (compound 789), and that drugs locked into a cis orientation more precise interpretation of the data. Of note, however, is by a double bond are unable to bind with high affinity, possibly that the mdrl complementary DNA expression vector used was due to steric hindrance. Compound 789, which is identical to derived from a colchicine-selected KB MDR cell line (10) shown clopenthixol except for a single bond instead of an exocyclic by Roninson et al. to possess a mutation in the mdrl gene double bond, allowing free rotation between the piperazinyl side which resulted in a single amino acid change at position 185 of chain and the thioxanthene ring nucleus, was nearly as effective the protein. This mutation was associated with a preferential as fra/js-clopenthixol, and far more effective than m-clopen- increase in resistance to colchicine, possibly due to alterations thixol. in the affinity of P-gp drug-binding sites (28). Since only the Therefore, these structure-activity relationships suggest that 3T3/MDR1 transfected cell line of those used in the present for this class of drugs, compounds with a tertiary amino group studies is known to possess this base change, the apparent loss incorporated into a cyclic ring structure in a particular spatial of stereospecificity of flupenthixol for antagonism of MDR orientation and at a distance of at least 3 carbons from the may also be related to this mutation, thereby changing the hydrophobic, thioxanthenyl ring are optimal for reversing affinity of a drug-binding domain affecting both chemosensitiz MDR. These findings are in agreement with our previous ers and cytotoxic drugs. This may also explain why the che studies of the phenothiazine class of chemosensitizers (4), as mosensitizers were more effective for reversal of resistance to well as those of others with derivatives of indole alkaloids (22). colchicine in this cell line than in any of the other cell lines The clinical properties of the thioxanthenes make them po studied (Table 3). cis- and frans-flupenthixol were equally effec tentially useful for modulating tumor resistance in humans. For tive inhibitors of [-'H]azidopine binding to P-gp from colchicine- instance, it was found that íraní-flupenthixollacked the extra- selected KB-C1 cells (Fig. 4). This P-gp contains the identical pyramidal side effects associated with other clinically used position 185 mutation as does the 3T3/MDR1 cells (28). antipsychotics such as trifluoperazine or fluphenazine in clinical However, the affinity of the thioxanthenes for azidopine-bind- trials of the antipsychotic effect of cis- versus frans-flupenthixol ing sites on P-gp cannot fully explain our results since there (23). Therefore, we chose to further characterize the cellular was no stereoselective displacement of azidopine binding from and biochemical effects of íraní-flupenthixolagainst MDR. KB-VI cells, a line not having the colchicine-induced mutation Several lines of evidence suggested that mz/is-flupenthixol and one which demonstrates enhanced responsiveness to the produced at least part of its effect through interaction with P- trans derivative. gp. First, the chemosensitizing activity of the thioxanthenes Alternatively, cis- and frans-flupenthixol may have equivalent were limited to MDR cell lines that overexpress P-gp. For effects on P-gp in all MDR cell lines, but have stereospecific example, iraws-flupenthixol caused significant reversal of pri effects on additional cellular targets which regulate or modulate mary and cross-resistance in MCF-7 AdrR, KB-V1, P388/DOX, MDR in certain cell lines. This hypothesis could explain the and 3T3/MDR1 cell lines, each known to display decreased loss of stereospecificity of mzHS-flupenthixol in the genetically drug accumulation and an increased expression of P-gp or mdrl manipulated mdrl transfectant, a line which may lack regula mRNA (5, 11, 24-26). However, neither iraws-flupenthixol, nor tory mechanisms or important coinduced gene products com the other chemosensitizers tested, caused any potentiation of mon to the other MDR cell lines. To explore this possibility, drug toxicity in the MCF-7/MITOX multiple drug resistant the effect of the thioxanthenes on several cellular targets which cell line which displays decreased drug accumulation and cross- may have a regulatory role in MDR was examined. Similar to resistance to a typical MDR spectrum of drugs, but does not previous reports (29), cis- and fra/w-flupenthixol were found to express the mdrl gene (8). Furthermore, the chemosensitizers have similar potencies as antagonists of CaM, and were less studied did not significantly modulate the toxicity of drugs nor potent than their phenothiazine homologue, fluphenazine (Ta cause an increased accumulation of doxorubicin in drug-sensi ble 4), a less potent drug against MDR. These data support our tive cell lines. These results suggest that the effects of the previous work demonstrating the lack of correlation between thioxanthenes and verapamil were mediated through a target(s) antagonism of CaM and reversal of MDR by phenothiazines uniquely overexpressed in MDR cells, such as the putative drug (4). efflux pump, P-gp (27). While many thioxanthenes are poor inhibitors of PKC, with The qualitatively similar effects of frawi-fiupenthixol and ICsoS in the millimolar range (30), the effect of cis- or trans- verapamil in each of the sensitive and MDR cell lines studied flupenthixol on the activity of PKC have not been previously suggests a common target for their chemosensitizing effect. studied. Table 4 demonstrates that both flupenthixol isomers Isobologram analysis of their interaction (Fig. 2) showed that were fairly potent inhibitors of the enzyme partially purified mjws-flupenthixol and verapamil were additive in their in vitro from MCF-7 AdrR cells. However, while m//zs-flupenthixol is anti-MDR effect in MCF-7 AdrR cells, although fra/is-flupen- slightly more potent, it seems unlikely that the small difference thixol was more potent (Fig. 1). Furthermore, since the thio (1.4-fold) can adequately explain the differences of thioxan- 1754

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1990 American Association for Cancer Research. REVERSAL OF MDR BY MUU-FLUPENTHIXOL

M. A., Melera, P. W.. Cowan. K. H.. and Goldsmith. M. E. Isolation of thenes in chemosensitizing activity in this cell line. amplified and overexpressed DNA sequences from Adriamycin-resistant The thioxanthenes were found to be potent inhibitors of human breast cancer cells. Cancer Res.. 47: 5141-5148. 1987. ['Hjazidopine binding to P-gp (Fig. 4). This suggests that the 6. Batist. G.. Tulpule. A.. Sinha. B. K.. Katkis. A. G.. Myers. C. E.. and Cowan, K. H. Overexpression of a novel anionicglutathionetransferase in multidrug- putative multidrug transporter itself may be one important resistant human breast cancer cells. J. Biol. Chem.. 261:15544-15549. 1986. target for their chemosensitizing activity. Several investigators 7. Fairchild, C'. R., Moscow, J. A., and Cowan. K. H. Effect of GSTV expression have found that inhibition of ['H]azidopine binding correlates on the pattern of multidrug resistance conferred by transfection of the P- glycoprotein gene. Proc. Am. Assoc. Cancer Res., 30: 2100. 1989. with activity for reversing MDR by chemosensitizers in vitro 8. Taylor. C. W.. and Dalton, W. S. Multiple mechanisms of drug resistance in (31, 32). Further work is in progress to define the precise MCF-7 human breast cancer cells. Proc. Am. Assoc. Cancer Res.. 30: 2109 binding sites of thioxanthenes on P-gp and elucidate the struc . 1989. 9. Ueda. K., Cornwcll, M. M., Gottesman. M. M., Pastan. I.. Roninson. B.. tural specificity of this interaction. Ling. B.. and Riordan, J. R. The mdr\ gene, responsible for multidrug- An additional cellular mechanism which could explain the resistance, codes for P-glycoprotein. Biochem. Biophys. Res. Commun., 141: 956-962. 1986. stereospecific effects of the thioxanthenes is differential drug 10. Roninson. I. B., Chin. J. E., Choi, K., Gros. P.. Housman. D. E., Fojo. A.. accumulation of the isomers. However, we have previously Shen. D.. Gottesman. M. M.. and Pastan. I. Isolation of human mdr DNA studied the cellular accumulation of each flupenthixol stereo sequences amplified in multidrug-resistant KB carcinoma cells. Proc. Nati. isomer in MCF-7 AdrR cells and eliminated the possibility of Acad. Sci. USA. 83: 4538-4532, 1986. 11. Ueda. K.. Cardarelli. C.. Gottesman. M. M., and Pastan. I. Expression of a their selective access to intracellular drug-binding sites (4). full-length cDNA for the human mdr\ gene confers resistance to colchicine, In summary, frans-flupenthixol was a potent and effective doxorubicin, and vinblastine. Proc. Nati. Acad. Sci. USA, 84: 3004-3008, 1987. chemosensitizer in each of the MDR cell lines studied. The 2- 12. Finlay, G. J., Baguley. B. C., and Wilson. W. R. A semiautomated microcul to 3-fold greater potency of /raws-flupenthixol than that of ture method for investigating exponentially growing carcinoma cells. Anal. Biochem., 139: 272-277. 1984. verapamil, along with its lack of known clinical activity or side 13. Carmichael. J.. DeGraff. W. G.. Gazdar. A. F., Minna. J. D., and Mitchell. effects, makes it a potential candidate for clinical use in com J. B. Evaluation of a tetrazolium-based semiautomated colorimetrie assay: bination with doxorubicin or other chemotherapeutic drugs. assessment of chemosensitivity testing. Cancer Res., 47: 936-942, 1987. 14. Berenbaum. M. C. Criteria for analyzing interactions between biologically For example, in vivo studies have demonstrated irans-flupen- active agents. Adv. Cancer Res.. 35: 269-335. 1981. thixol to be a 100- to 1000-fold less potent neuroleptic than 15. Levin, R. M.. and Weiss. B. Mechanism by which psychotropic drugs inhibit c/s-flupenthixol (33). In addition. In vitro studies showed that adenosine cyclic 3',5'-monophosphate phosphodiesterase of brain. Mol. /ra«s-['H]flupenthixol completely lacks specific dopamine re Pharmacol.. 12: 581-589, 1976. 16. Weiss. B.. Lehne. R., and Strada, S. J. A rapid microassay of adenosine ceptor-binding activity, and has little or no a adrenergic, ß 3',5'-monophosphate phosphodiesterase activity. Anal. Biochem.. 45: 222- adrenergic, or 5-HT, blocking activity (34). Studies of the 235. 1972. 17. Palayoor, S. T., Stein. J. M., and Hait, W. N. Inhibition of protein kinasc C clinical pharmacokinetics of frans-flupenthixol have shown that by antineoplastic agents: implications for drug resistance. Biochem. Biophys. the drug undergoes extensive first-pass hepatic metabolism (35). Res. Commun.. 148: 718-725. 1987. 18. Perella, F. W.. Hellmig. B. D., and Diamond, L. Up regulation of the phorbol Furthermore, flupenthixol possesses an apparent half-life of ester receptor-protein kinase C in HL-60 variant cells. Cancer Res.. 46: 567- elimination of 34 to 36 h due to its large volume of distribution 572, 1986. (20 liters/kg) (36). The anti-MDR activity of the thioxanthene 19. Safa, A. R.. Glover. C. J., Sewell, J. L., Meyers, M. B.. Biedler. J. L., and Felsted. R. L. Identification of the multidrug resistance-related membrane metabolites are unknown, although the sulfoxide derivative of glycoprotein as an acceptor for calcium channel blockers. J. Biol. Chem.. phenothiazines remained active (4). Reported peak plasma lev 262:7884-7888, 1987. els of the parent compound following m-flupenthixol treatment 20. Finney. D. J. Statistical Method in Biological Assay. London: Charles Griffin and Co., Ltd.. 1978. (1 mg i.v. or 4 mg p.o.) have been 3-4 ¿(g/liter(7 nivi) (37), 21. Bliss, C. I. Confidence limits for measuring the precision of bioassays. 1000-fold less than concentrations necessary for in vitro che Biometrics. 12: 491-526, 1956. 22. Zamora. J. M., Pearce, H. L., and Beck, W. T. Physical-chemical properties mosensitizing activity. However, it is likely that larger doses of shared by compounds that modulate multidrug resistance in human leukemic frans-flupenthixol may be safely administered to humans due cells. Mol. Pharmacol., 33: 454-462, 1988. to its profound lack of dopaminergic activity. In addition, a 23. Johnstone, E. C.. Crow, T. J., Frith. C. D., Carney, M. W. D., and Price, J. S. Mechanism of the antipsychotic effect in the treatment of acute schizo recent report measuring the steady state levels of cis- and trans- phrenia. Lancet, /: 848-851. 1978. flupenthixol resulting from 4- to 14-mg daily p.o. doses of a 24. Ganapathi. R.. Grabowski. D.. Rouse, W., and Riegler. F. Differential effect of the calmodulin inhibitor trifluoperazine on cellular accumulation, reten 1:1 mixture of both drugs resulted in /rans-flupenthixol levels tion, and cytotoxicity of anthracyclines in doxorubicin (Adriamycin)-resistant of up to 20 nM, with 2-fold greater trans- than c/s-isomer steady P388 mouse leukemia cells. Cancer Res., 44: 5056-5061, 1984. state plasma concentrations (38). Further studies will be re 25. Shen, D.. Cardarelli. C.. Jwang, J., Cornwell. M., Richert. N.. Ishii, S., Pastan. I., and Gottesman, M. M. Multiple drug-resistant human KB carci quired to determine the maximum tolerated in vivo dose and noma cells independently selected for high-level resistance to colchicine. whether or not it will be possible to achieve sufficient tissue Adriamycin, or vinblastine show changes in expression of specific proteins. levels of fra/rs-flupenthixol or any of its potentially active me J. Biol. Chem., 261: 7762-7770. 1986. 26. Fojo. A., Akiyama. S., Gottesman. M. M.. and Pastan, I. Reduced drug tabolites to be capable of producing anti-MDR activity in accumulation in multiply drug-resistant human KB carcinoma cell lines. human tumors. Cancer Res., 45: 3002-3007, 1985. 27. Chen. C. J.. Chin, E., Ueda. K.. Clark. C. P.. Pastan, I., Gottesman. M. M., and Roninson. 1. B. Internal duplication and homology with bacterial trans port proteins in the mdr\ (P-glycoprotein) gene from multidrug resistant REFERENCES human cells. Cell. 47: 381-389, 1986. 28. Choi. K., Chen. C., Kriegler, M., and Robinson. 1. B. An altered pattern of 1. Bradley, G., Juranka. P. F., and Ling. V. Mechanism of multidrug resistance. cross-resistance in multidrug-resistant human cells results from spontaneous Biochim. Biophys. Acta. 948: 87-128. 1988. mutations in the mdr\ (P-glycoprotein) gene. Cell. 53: 519-529, 1988. 2. Goldstein, L. J.. Galski, H., Fojo, A.. Willingham, M.. Lai, S., Gazdar. A.. 29. Norman, J. A., and Drummond, A. H. Inhibition of calcium-dependent Pirker. R., Green. A.. Crist, W.. Brodeur. G. M.. Lieber, M., Cossman. J.. regulator-stimulated phosphodiesterase activity by neuroleptic drugs is un Gottesman, M. M., and Pastan. I. Expression of a multidrug resistance gene related to their clinical efficacy. Mol. Pharmacol., 16: 1089-1094. 1979. in human cancers. J. Nati. Cancer Inst.. 81: 116-124, 1989. 30. Schatzman. R. C.. Wise. B. C., and Kuo, J. F. Phospholipid-sensitivecalcium- 3. Tsuruo. T. Mechanisms of multidrug resistance and implications for therapy. dependent protein kinase: inhibition by anti-psychotic drugs. Biochem. Bio Jpn. J. Cancer Res.. 79: 285-296. 1988. phys. Res. Commun., 98: 669-676. 1981. 4. Ford. J. M., Prozialeck, W. C.. and Hail, W. N. Structural features deter 31. Yang. C. H.. DePinho. S. H.. Greenberger, L. M., Arceci, R. J., and Horwitz. mining activity of phenothiazines and related drugs for inhibition of cell S. B. Progesterone interacts with P-glycoprotein in multidrug-rcsistant cells growth and reversal of multidrug resistance. Mol. Pharmacol.. 35: 105-115, and in the endometrium of gravid uterus. J. Biol. Chem., 264: 782-788, 1989. 1989. 5. Fairchild. C. R.. Ivy. S. P.. Kao-Shan. C.. Whang-Peng. J.. Rosen. M.. Israel. 32. Nogai, I., Kohno. K., Kikuchi. J., Kuwano. M., Akiyama. S.. Kiue, A., 1755

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1990 American Association for Cancer Research. REVERSAL OF MDR BY (ranj-FLUPENTHIXOL Suzuki. K.. Voshida, Y.. Cornwell. M. M., Pastan. I., and Gottesman. M. c/.y(Z)-flupenthixol in Viscoleo". Eur. }. Clin. Pharmacol.. /*: 355-360. M. Analysis of structural features of dihydropyridine analogs needed to 1980. reverse multidrug resistance and to inhibit photoaffinity labeling of P-glyco- -16- Jorgensen, A.. Andersen. }.. Bjorndal, N.. Dencker. S. J., and Lundin. L. protein Biochem Pharmacol jfl- 519-527 1989 Serum concentrations of c/XZ)-flupenthixol and prolactine in chronic schiz- 33. Nielsen. I. M.. Pedersen. V.. Nvmark. M~.Franch. K. F.. Boeck. V.. Fjalland. ophrenic patients treated with flupenthixol and m(Z)-flupenthixol deca- B.. and Christensen, A. V. Comparative pharmacology of flupenthixol and „¿"oate. Psychopharmacology. 77:58-65 1982 some re, erence neurolept,cs.. Acta Pharmacol. , . —¿Toxcol i 33: ,, 353-362.-,-, -i¿-»irt-t-t 1973. 37. Jorgensen, prions A., in and outpatient Fredncson, schizophrenics. O. K. Clopenthixol III. Serum and levels. flupenthixo Acta Psvchiatr. depot 34. Hyttel. J.. Arm. J.. and Bogeso. K. P. Antipsychotic drugs: configurational Scand 279-41-54 1980 stereoisomers. In: D. F. Smith (eds.). CRC Handbook of Stereoisomers: 1g Balani-Gorgia. A. È.Balani, L. P.. Gex-Fabry. M., and Genet, C. Stereose- Drugs in Psychopharmacology. pp. 143-180. Boca Raton. FL: CRC Press. iectjve disposition of flupenthixol: influence on steady-state concentrations Inc. 1984. in schizophrenic patients. Eur. J. Drug Metab. Pharmacokinet.. 12: 123- 35. Jorgensen. A. Pharmacokinetic studies in volunteers of intravenous and oral 128. 1987.

1756

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1990 American Association for Cancer Research. Cellular and Biochemical Characterization of Thioxanthenes for Reversal of Multidrug Resistance in Human and Murine Cell Lines

James M. Ford, Edward P. Bruggemann, Ira Pastan, et al.

Cancer Res 1990;50:1748-1756.

Updated version Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/50/6/1748

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://cancerres.aacrjournals.org/content/50/6/1748. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1990 American Association for Cancer Research.