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Speaking of DD, how many negative Nellies grasp this? Abstract. Background/Aim: Multidrug resistance poses aserious challenge in cancer therapy. To address this problem,we designed and synthesized Adva-27a, a novel non-esterGEM-difluorinated C-glycoside derivative of podophyllotoxin.Materials and Methods: Adva-27a activity was evaluated in avariety of assays including inhibition of topoisomerase IIa,cytotoxic activity in drug-sensitive and drug-resistant cancercell lines, metabolic stability in human liver microsomes andpharmacokinetic properties in rats. Results: Adva-27a exhibiteddose-dependent human topoisomerase IIa inhibitory activityand dose-dependent growth inhibitory activity in several drug-sensitive and two multidrug-resistant cancer cell lines. In themultidrug-resistant cell lines, MCF-7/MDR (breast cancer) andH69AR (small-cell lung cancer), Adva-27a was significantlymore potent than etoposide. The metabolic stability of Adva-27a in human liver microsomes and its pharmacokineticproperties in rats were better than those of etoposide.Conclusion: Our studies have identified Adva-27a as a noveltopoisomerase II inhibitor with superior cytotoxic activityagainst multidrug-resistant human cancer cells and moredesirable pharmacokinetic properties than etoposide.Etoposide (VP-16), which is a derivative of podophyllotoxin(Figure 1A and B), is widely used in human cancerchemotherapy, i.e.in small-cell lung cancer and testicularcancers (1, 13). It acts as a topoisomerase II “poison” bystabilizing the cleavage complex formed between the enzymeand its DNA substrate, leading to the inhibition of relegation,accumulation of chromosomal breaks and triggers cell death(23). However, similar to many other chemotherapeuticagents, the clinical utility of etoposide has been hamperedby the lack of activity of the drug on certain tumor types,particularly multidrug resistant tumors (reviewed in (13)). Infact, etoposide has been found to be a substrate of multidrugefflux transporters, such as P-glycoprotein (P-gp) (2, 12). Several strategies have emerged to design and synthesizeanalogs of etoposide or podophyllotoxin with improvedpharmaceutical and clinical activities (13, 31). These includeglycosylated etoposide derivatives, such as teniposide,phosphorylated etoposide, such as etopophos, and non-glycosylated podophyllotoxin derivatives containing nitrogen,such as GL331 (14, 15) and TOP-53 (26). Interestingly,teniposide and etopophos appear to be less toxic and arecurrently used in clinics, though they show little improvementof activity on drug resistant tumors compared to etoposide(13, 31). Furthermore, the non-glycoside, non-etherderivatives GL331 and TOP-53 have shown improved activityagainst topoisomerase II although still limited activity againstmultidrug resistant cells (7, 14, 16, 17, 26).In the search for novel etoposide analogs with improvedefficacy and pharmacokinetic properties, which can overcomemultidrug resistance of human cancer cells, we designed andsynthesized Adva-27a, a non-ester, nitrogen-containingglycoside analog of etoposide (Figure 1C). This studydescribes the evaluation of Adva-27a using a battery ofbiochemical, cell biological, and pharmacological analyses.Compared to etoposide, Adva-27a was found to besubstantially more potent against two multidrug-resistanthuman cancer cell lines, and has a better metabolic stabilityand pharmacokinetic properties than etoposide. Taken4423Correspondence to: Dr. Abderrazzak Merzouki, Institute ofBiomedical Engineering, Department of Chemical Engineering,École Polytechnique, PO Box 6079, Station Centre-ville, Montréal,Québec, Canada, H3C 3A7. Tel: [phone], ext 4799, Fax: [phone], e-mail: [e-mail] Key Words: Adva-27a, topoisomerase II, cancer, multidrug-resistant,multidrug efflux transporters.ANTICANCER RESEARCH 32: 4423-4432 (2012)Adva-27a, a Novel Podophyllotoxin Derivative Found to BeEffective against Multidrug Resistant Human Cancer CellsABDERRAZZAK MERZOUKI1, MICHAEL D. BUSCHMANN1, MYRIAM JEAN1, REBECCA S. YOUNG2, SI LIAO2, SUSANNAH GAL2, ZUOMEI LI3and STEVE N. SLILATY41Institute of Biomedical Engineering, Department of Chemical Engineering, École Polytechnique, Montreal, QC, Canada;2Department of Biological Sciences, Binghamton University (State University of New York),Binghamton, NY, U.S.A.;3Amplia PharmaTek Inc., Montreal, QC, Canada;4Sunshine Biopharma Inc., Montreal, QC, Canada0250-7005/2012 $2.00+.40
together, these results suggest that further development ofAdva-27a is warranted as a novel topoisomerase II inhibitorfor use in human cancer therapy with multidrug-resistanttumors.Materials and MethodsSynthesis of Adva-27a. Synthesis of Adva-27a (a-D-gluco-3-octulopyranose, 1, 2-dideoxy-2, 2-difluoro-1, [[(5S, 5aS, 8aR, 9R)-5, 5a,6,8,8a,9-hexahydro-8-oxo-9-(3,4,5-trimethyoxyphenyl) furo[3’,4’:6,7] naphtha[2,3-d]-1,3-dooxol-5-yl]amino]) was performedby TFChem (Val de Reuil, France). The details of the synthesis wereas described for molecule 32a in US Patent ApplicationUS20090318675A1. The molecular mass of Adva-27a is 655.59Dalton. The chemical structure of Adva-27a is shown in Figure 1C.Topoisomerase II enzyme inhibition assay. Analysis of the activityof human topoisomerase IIa was done using the HumanTopoisomerase II DNA Decatenation Assay Kit Plus (Catalognumber HDD96KE) from Profoldin Inc. (Hudson, Massachusetts,USA). The assay uses a spin column to separate concatenated fromdecatenated DNA. Briefly, Adva-27a or etoposide (MP BiomedicalLLC, Ohio, USA) was dissolved in 100% DMSO to make a 100 mMstock solution. Stock solutions were further diluted in DMSO to 10mM, followed by 2-fold serial dilutions. The final concentration ofAdva-27a or etoposide was in the range of 0.781 µM to 200 µM. Theactivity of human topoisomerase IIa to catalyze the production ofdecatenated DNA from concatenated DNA (provided in the kit) wasmonitored in 96-well assay plates following the manufacturer’sinstructions. The reaction mixture (50 mM Tris-HCl, pH 8.0, 125mM NaCl, 0.5 mM EDTA, 10 mM MgCl2, 6.5 µg/mL concatenatedDNA, 1 mM ATP and 5 U/mL recombinant human topoisomeraseIIa enzyme) with various drug amounts was incubated at ambienttemperature for 20 min and loaded onto filter plates. After filtration,plates were washed (10 mM Tris-HCl, pH 7.5 and 10 mM NaCl). Ineach well of the receiver plate containing decatenated DNA, SYBRGreen II dye (provided with the kit) was added and fluorescence ona microplate reader was monitored at 535 nm using an excitationwavelength of 485 nm.Cell culture. Human prostate carcinoma cell line, PC-3, human non-small cell lung carcinoma cell line, A549, and multi-drug-resistanthuman small-cell lung cancer cell line, H69AR, were obtained fromthe American Type Culture Collection (ATCC, Manassas, Virginia,USA). All cell lines were cultured in a 37 °C CO2incubator. PC-3cells were cultured in RPMI-1640 media with 10% FBS, while A549cells were cultured in DMEM media with 10% FBS. Glutamine (2mM), penicillin (100 I.U.) and streptomycin (100 µg/mL) were addedto the media. The breast cancer cell lines, MCF-7 and MCF-7/MDR,the latter being a doxorubicin-resistant derivative of the former (30),were grown in RPMI-1640 supplemented with L-glutamine andHEPES (ATCC), 0.1% insulin (Sigma Aldrich, St. Louis, MO, USA),10% fetal bovine serum (Thermo Scientific, Logan, UT, USA) andpenicillin/streptomycin (5000 U/mL and 5000 µg/mL, respectively)(Lonza Biowhittaker, Walkersville, MD, USA) at 37 °C in 5% CO2with the MCF-7/MDR cells maintained in media containing 1 µMdoxorubicin. One week prior to starting the drug treatment, the MCF-7/MDR cells were transferred to media without doxorubicin. Cells ofthe human multidrug-resistant small-cell lung cancer cell line,H69AR, which was derived from the parental cell line, NCI-H69 (19)were cultured in RPMI-1640 media with L-glutamine (LonzaBiowhittaker, Walkersville, MD, USA). For the H69AR cells, 20%FBS was added. Penicillin (100 I.U.) and streptomycin (100 µg/mL),HEPES buffer (10 mM) and sodium pyruvate (1 mM) were added tothe media. Cells were split twice per week in T75 tissue culture flasksusing 0.25% trypsin.Cytotoxicity assay in human cancer cell lines. Two differentmethods were used to analyze the cytotoxicity of the drugs. PC-3cells and A549 cells were plated at 4000 cells/well and 3000cells/well, respectively while H69AR cells were plated at 6500cells/well in 96-well tissue culture plates in triplicates. Twenty-fourh post-plating, cells were treated with DMSO, or Adva-27a oretoposide in a serial dilution. In assays with PC3 and A549 cells,the final concentration of DMSO was 1% for all wells treated withAdva-27a or etoposide. In assays with H69AR cells, the finalconcentration of DMSO was 2.5%. At 68 h post-treatment, 20 µLof Alamar Blue (Invitrogen Canada Inc., Burlington, Ontario,Canada) were added to each well, and 4 h later, the plates were readon a microtiter plate reader (SpectraMax 190, Molecular Devices) atANTICANCER RESEARCH 32: 4423-4432 (2012)4424Figure 1. Chemical structures of podophyllotoxin (A), etoposide (B) and Adva-27a (C).
two wavelengths (570/600 nm). The percentage of reduction ofAlamar Blue was calculated using the following formula assuggested by the manufacturer:% reduction of Alamar Blue=(117216A570-80586A600)/(155677A’600-14652A’570)*100Where A is the absorbance at 570 nm or 600 nm of test wells and A’is the absorbance at 570 nm or 600 nm of negative control wells(media-only, no cells with Alamar Blue). The cell growth % wascalculated as follows:% Growth=(% of reduction of Alamar Blue in a treated well)/(themean of % of reduction of DMSO-treated wells)*100For analysis of the cytotoxicity in the MCF-7 and MCF-7/MDRcells, 6000 cells were plated per well in 24-well plates. The cellswere allowed to incubate for 18-24 hours, then DMSO (0.5%) orAdva-27a or etoposide (Sigma Aldrich, St. Louis, MO, USA) wereadded at various concentrations. The cells were then monitored forcell viability 1-9 days later by adding Alamar Blue (10% of media)and reading the fluorescence at 2 and 4 h in a fluorescencemicrotiter plate reader (Cytofluor II Biosearch, Bedford, MA, USA)with excitation filter at 530/25 and emission filter at 620/40 and again of 48. Growth curves data were calculated as % fluorescencerelative to the value detected in comparable cells in the presence ofDMSO-only. For statistical analysis, unpaired Student’s t-test wasused to compare the group treated with a drugvs. the group treatedwith DMSO. Standard errors of the mean (SEM) were used torepresent the variation of the data.Quantitative PCR (qPCR) analysis of mdr1 mRNA in MCF-7/MDRcells. RNA extraction was performed using the RNA XS®extractionkit from Macherey-Nagel Inc. (Bethlehem, PA, USA) according tothe manufacturer’s protocol. Total RNA was quantified and RNAintegrity was evaluated by the ratio of 28S/18S ribosomal RNAusing the Agilent BioAnalyzer 2100 (Agilent Technologies,Mississauga, ON, Canada), following the manufacturer’s protocol. Atotal of 1 µg purified RNA was reverse-transcribed in a final volumeof 20 µL using the First Strand cDNA Transcriptor Kit (RocheDiagnostics, Laval, Quebec, Canada) with oligo-dT primers asrecommended by the supplier. For mdr1(or ABCB1) mRNAexpression level, the primers and probes were TaqMan GeneExpression Assays-on-Demand products from Applied Biosystems.Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used asinternal control. The ABI PRISM®7900HT Sequence DetectionSystem (Applied Biosystems) was used to detect the amplificationlevel and was programmed with an initial step of 3 min at 95 °C,followed by 45 cycles of 5 sec at 95 °C and 30 sec at 60 °C. Allreactions were performed in triplicate and the average values of Ct(threshold cycle) were used for quantification. GAPDH was used asendogenous control. The relative quantification of target genes wasdetermined using the 2–??CTmethod. Briefly, the Ct values of targetgenes were normalized to an endogenous control gene (endogenouscontrol) [?CT=Ct (target gene) – Ct (endogenous control)] andcompared with a calibrator: ??CT=?Ct target – ?Ct calibrator.Relative expression (RQ) was calculated using the SequenceDetection System 2.2.2 software using the RQ=2-??CTformula.The measurements data were collected and expressed as meanvalues±standard deviation (s.d.) and were analyzed with theStatistica 9.0 software (STATSOFT; Statistica, Tulsa, OK, USA).Statistical significance was determined by one-way ANOVAfollowed by Tukey post hoc test. Differences were considered highlysignificant at **p<0.01.Western blot analysis of P-gp.The level of the P-gp protein in bothMCF-7 and MCF-7/MDR cell lines was assessed using western blotanalysis. Cells (2×106) were harvested by centrifugation and washedtwice with PBS, and then lysed in 200 mL RIPA buffer (50 mMTris–HCl, pH7.4, 1% NP-40, 50 mM NaCl, 0.1% SDS, 1% Na-deoxycholic acid, 1 mM sodium orthovanadate, 2 mM PMSF) for30 min on ice with gentle shacking. After centrifugation at 12,000×g, 4 °C for 10 min, the total protein concentration was determinedby the BCA protein assay with BSA as standard. An aliquot ofprotein (30 µg) was separated by 5% SDS-PAGE and thentransferred to a PVDF membrane. The membrane was blocked for 2h at room temperature with 5% non-fat dry milk in PBS containing0.05% Tween-20 (PBST), then, the membranes were incubated withprimary a antibody anti-P-gp (CALBIOCHEM, Anti-P-GlycoproteinMouse mAb C219, Cat#517310) at 1:5000 dilutions in 5% non-fatmilk overnight at 4 °C, then the membranes were washed with PBSTfor three times, and incubated with the secondary rabbit anti-mouseIgG-HRP at 1:10000 dilution for 1 h at room temperature. Finally,the signals were visualized using enhanced chemiluminescencedetection (Thermo Scientific, Rockford, IL).Human liver microsomal clearance assay.Adva-27a and etoposide(at a final concentration of 1 µM) were incubated in duplicates with0.3 mg/mL human liver microsomes (Invitrogen) at 37 °C. Twocontrol compounds were tested at the same concentration: warfarinas a low-metabolized compound and verapamil as a high-metabolized compound (5, 10). The reaction contained microsomalproteins in 100 mM potassium phosphate, 2 mM NADPH, 3 mMMgCl2, pH 7.4. A control was run for each test agent omittingNADPH, to detect for NADPH-free metabolism. At the indicatedtimes (0, 10, 20, 30 and 60 min), an aliquot was removed from eachreaction and mixed with an equal volume of ice-cold Stop Solution(acetonitrile containing 1 µM propanol). The stopped reactions wereincubated at least 10 min at –20 °C. The samples were centrifuged toremove precipitated proteins (3500 rpm, 20 min at roomtemperature) and the supernatants were analyzed by LC-MS/MS(liquid chromatography-tandem mass spectrometry) using anAgilent 6410 mass spectrometer, coupled with an Agilent 1200HPLC (high pressure liquid chromatography) and a CTC PALchilled autosampler. After separation on a C18 reverse phase HPLCcolumn (Agilent) using an acetonitrile-water gradient system, peakswere analyzed by mass spectrometry (MS) using ESI ionization inMRM mode. Data were converted to % drug remaining by dividingthe current amount by the time zero concentration value and werefitted to a first-order decay model to determine half-life (T1/2).Intrinsic clearance (CLint) was calculated from the T1/2andmicrosomal protein concentration using the formula:CLint=ln(2)/(T1/2[microsomal protein concentration])Pharmacokinetic analysis of Adva-27a and etoposide in rats. Adva-27a or etoposide was weighed in an airtight container and dissolvedin 40% HP-ß-CD by vortexing, followed by sonication andhomogenization until a uniform stock solution of 1.0 mg/mL wasobtained. The pH value of the stock solution was 7.0. The dose usedfor injection was 2.5 mg/kg. Five-to six-week-old male Sprague-Dawley Rats (Vital River Laboratory Animal Technology Co. Ltd.,Beijing, China) were used. The body weight of rats was between240 and 255 g at the beginning of dosing. Animals were examinedfor their general health condition and acclimatized for 5 days priorto compound administration. Animals were housed 3 per cage withMerzouki et al: New Epipodophyllotoxin Effective in Multidrug Resistance4425
an automatic watering system. Cage card and tail masking was usedto identify the rats. The animal room environment was controlled(temperature 20-25 °C, relative humidity 40-70%, and 12 h light/darkcycle with fresh air change). A standard certified commercial rodentchow (Beijing Keaoxieli) irradiated by Co-60 and sterile water wereprovided to the animals ad libitum. Animals were fasted overnightprior to dosing. Adva-27a or etoposide was given under one singlebolus injectionvia the jugular vein. Blood (300 µL at each timepoint) was collected at 5, 15, 30 min, and 1, 2, 4, 6, 8 and 24 h post-injection. After each blood collection, 0.3 mL saline was giventhrough a cannulated tube in the carotid artery. Food was returned 2h post-dosing. The animals were monitored up to 2 h post-injectionand again at 24 h post-injection. Blood samples were collected fromcarotid artery into a tube containing EDTA-K2 (2.5%) (BeijingChemical Reagent Company, Beijing, China) and were centrifugedat ×2000 g for 5 min at 4°C. Obtained plasma was transferred intoa polyethylene microcentrifuge tube and stored at –80 °C untilanalysis. At the end of the study, all rats were euthanized. Fordetection of etoposide in rat plasma, LC-MS/MS was used. Thechromatographic system consisted of an Agilent 1200 Series LCSystem and a GRACE Alltima HP C18 column (5 µm, 2.1×50 mm)was connected to an AB API 4000 tandem mass spectrometer. Datawere acquiredvia the multiple reactions monitoring (MRM) system.The MS/MS ion transitions were monitored at m/z of 589.2/229.0for etoposide. A gradient HPLC method was employed for theseparation. Mobile phase A consisted of 0.1% formic acid and 10%acetonitrile in water, and mobile phase B consisted of 0.1% formicacid in 100% acetonitrile. The gradient profile was as follows(minute, %A/%B): 0, 95/5; 0.1, 95/5; 0.2, 5/95; 1.8, 5/95; 1.9, 95/5;3.0, 95/5. The flow rate was 0.4 mL/min with an injection volume of10 µL. Plasma concentrations were analyzed using a non-compartmental method. Plasma concentration of Adva-27a wasanalyzed by LC-MS/MS, similarly to the method for etoposideexcept that the MS/MS ion transitions were monitored at m/z of656.3/397.3 and the gradient profile was shown as follows (minute,%A/%B): 0, 90/10; 0.1, 90/10; 0.2, 5/95; 1.4, 5/95; 1.5, 90/10; 2.6,90/10.ResultsDose-dependent inhibition of topoisomerase IIa in vitro. Anew derivative of etoposide, Adva-27a, was designed andsynthesized (Figure 1C). Adva-27a was found to induce adose-dependent inhibition of recombinant humantopoisomerase IIa activity in vitro, as monitored by theproduction of decatenated DNA (Figure 2). The IC50 ofAdva-27a for inhibition of topoisomerase IIa wasdetermined to be 13.7 µM. Under the same experimentalconditions, etoposide was confirmed to be a topoisomeraseIIa inhibitor with an IC50of 4.0 µM. Compared to etoposide,the replacement on the C4 position with an aminoalkyl chainand on the E4’ position with a methoxyl group in Adva-27aresulted in a ~3-fold reduction in enzyme inhibitory activityin vitro.Growth inhibition of drug-sensitive and multidrug-resistantbreast cancer cells. We analyzed the growth inhibitoryactivity of Adva-27a compared to etoposide in the drug-sensitive breast cancer cell line, MCF-7, and in a multidrug-resistant variant of MCF-7 derived by doxorubicin selection,MCF-7/MDR. With the drug-sensitive MCF-7 cell line, theconcentration-dependent growth inhibitory activity of Adva-27a was similar to that of etoposide starting atapproximately 20 µM (Figure 3A). Similarly, the time-dependence study performed at 100 µM Adva-27a oretoposide yielded nearly superimposable curves (Figure 3B).With the MCF-7/MDR cell line however, quite the oppositewas observed. In these cells, etoposide even at 100 µM hadlittle effect on cell growth, while the new compound, Adva-27a, caused a dramatic reduction in cell growth starting at aconcentration between 20 and 30 µM (Figure 3A). Theresults of the time-dependence study were also dramatic interms of the much greater inhibitory effect of Adva-27a onthe MCF-7/MDR cells relative to etoposide (Figure 3B).With etoposide, some MCF-7/MDR cell growth inhibitionwas observed between days 2 and 5, but after that, the cellsquickly recovered and regrew to the same level as theDMSO-treated control population. This apparent cell growthslowdown and recovery phenomenon was not seen withAdva-27a. Instead, Adva-27a limited cell growth veryeffectively starting at day 3 and continued the inhibitoryeffect progressively through day 9 (Figure 3B). We furtherstudied the mechanisms underlying the sensitivity of theMCF-7/MDR cells to Adva-27a by analyzing the level ofexpression of the mdr1gene using qPCR and the P-gpprotein using western blotting. The mdr1gene whichencodes for P-gp was the first ABC transporter identified tobe overexpressed in breast cancer cell lines displayingmultidrug resistance (28). P-gp is a broad spectrum effluxANTICANCER RESEARCH 32: 4423-4432 (2012)4426Figure 2. Dose-dependent inhibition of recombinant humantopoisomerase IIa by Adva-27a or etoposide in vitro. Adva-27a oretoposide in the range of 0.781 µM to 200 µM was used in humantopoisomerase IIa activity assay, as described in the methods section.The production of decatenated DNA from concatenated DNA wasmonitored by fluorescence in a 96-well plate providing the percentage ofenzyme inhibition data shown.
pump and is known to be involved in the transport of manydrugs including doxorubicin and etoposide (28). Both mdr1mRNA and P-gp protein levels in MCF-7/MDR cells weresignificantly higher than in MCF-7 cells (Figure 4). Morespecifically, the expression level of mdr1mRNA was 29-times greater in MCF-7/MDR than in MCF-7. Based on theMerzouki et al: New Epipodophyllotoxin Effective in Multidrug Resistance4427Figure 3. Dose- and time-dependent cytotoxic activity of Adva-27a andetoposide in MCF-7 and MCF-7/MDR breast cancer cells. MCF-7 orMCF-7/MDR cells were cultured in the presence of differentconcentrations of Adva-27a or etoposide and the cells were assayed formetabolic activity using the fluorescence of Alamar Blue, as describedin the methods section. The fluorescence relative to that detected in 1%DMSO-treated control cells is plotted versus the concentration of thecompound after 7 days of treatment (panel A) and versus the days oftreatment with 100 µM of the compounds (panel B). The data arerepresentative of an experiment performed 3 times.Figure 4. Levels of mdr1 mRNA and P-gp protein in MCF-7 and MCF-7/MDR cells. (A) Total RNA extraction was performed on MCF-7 andMCF-&/MDR cells. Total RNA was quantified and RNA integritymeasured using the Agilent BioAnalyzer 2100 (Agilent Technologies,Mississauga, ON). (nt)=Nucleotide, L=standard ladder, the green bandis a lower marker, which allows sample alignment and permitscomparison for RIN calculation. RIN=RNA integrity number, is analgorithm-based numbering system that calculates-RNA integrity with10 being the most intact and 1 being fully-degraded. (B) mRNA level ofmdr1 was detected with real-time PCR; **p<0.01. (C) Protein levels ofP-gp in MCF-7 and MCF-7/MDR cell lines were assessed by westernblot analysis using 30 µg total protein from each cell line.
fact that Adva-27a is very effective at killing the MCF-7/MDR cells, it appears that this compound is nottransported by this efflux protein.Growth inhibition of multidrug-resistant small-cell lungcancer cells. To investigate if Adva-27a could also overcomemultidrug resistance in another human cancer cell line of adifferent cancer type and tissue origin, we performedcytotoxicity studies using the human small-cell lung cancercell line, H69AR. This multidrug resistant cell line wasderived from the parental cell line, NCI-H69 by usingAdriamycin (doxorubicin) selection (19). Figure 5 shows thatAdva-27a can reduce the growth of H69AR cells moresignificantly than etoposide starting from 50 µM (p<0.01).The doxorubicin-selected H69AR cells used in this study areknown to overexpress ABCC1 (MRP1), one of the threemost frequently encountered members the ATP-bindingcassette family of drug transporters involved in multidrugresistance (4, 9). It is interesting to observe that Adva-27acan overcome multidrug resistance in cancer cells associatedwith different ABC transporters. Cytotoxicity of Adva-27a in other cancer cells. We alsoanalyzed the growth inhibitory activity of Adva-27a,compared to etoposide in PC-3, a prostate cancer cell line,and A549, a non-small-cell lung cancer cell line. Adva-27aexhibited dose-dependent growth inhibitory activity in PC-3and A549 cells following 72 h of incubation (Figure 6A andB). In PC-3 cells, the growth inhibitory activity of Adva-27aappeared to be dose-dependent but weaker than that ofetoposide with IC50’s of 3 µM and 42 µM, for etoposide andAdva-27a, respectively (Figure 6A). In the A549 cell line, thegrowth-inhibitory activity of Adva-27a was also dose-dependent and largely similar to that of etoposide (Figure6B). Adva-27a had previously been shown to inhibit thegrowth of other cancer cell lines including KB (pharyngealcancer), SF-268 (brain cancer), HL-60 (leukemia) and HT-29(colon cancer) (US Patent Application: US20090318675A1).ANTICANCER RESEARCH 32: 4423-4432 (2012)4428Figure 5. Anti-proliferative activity of Adva-27a and etoposide at equalmolar concentrations in the multidrug-resistant human small-cell lungcancer cell line, H69AR, following a 72-hour incubation. The AlamarBlue reduction was determined using absorbance at 570 and 600 nmand used as an indicator of cell growth, as described in the methodssection. “**”, p<0.01 compared to the values of etoposide at equalmolar concentration.Figure 6.Dose-dependent anti-proliferative activity of Adva-27a andetoposide in the PC-3 prostate cancer cell line (A) and the A549 lungcancer cell line (B) following a 72-hour incubation. The Alamar Bluereduction was determined using absorbance at 570 and 600 nm andused as an indicator of cell growth, as described in the methods section.
Metabolic stability of Adva-27a in human liver microsomesin vitro.Non-renal clearance of etoposide accounts for 60%of elimination of etoposide in humans (8). Though themetabolism of etoposide is not fully elucidated, it is knownthat the NADPH-dependent cytochrome P450system and theNADPH-independent glucuronidation process are partlyinvolved in the human non-renal clearance of etoposide (22,27). In the present study, we analyzed the metabolic stabilityof Adva-27a and etoposide in vitro using human livermicrosomes. Similar to etoposide, Adva-27a was found tohave a desirable medium-low clearance rate in human livermicrosomes in vitro via both NADPH-dependent andNADPH-independent mechanisms (Table I). The estimatedT1/2of Adva-27a was 38.7 min when NADPH was addedand 54 min without the addition of NADPH. For etoposide,the estimated T1/2with and without NADPH was 35.8 and48 min, respectively (Table I). Under the same experimentalconditions, verapamil, a high clearance control, exhibited anestimated T1/2 of ~14 min in the presence of NADPH and aT1/2 of >200 min without NADPH. In contrast, warfarin, alow clearance control, had a clearance rate in the presenceof NADPH of more than 200 min (Table I).Pharmacokinetic properties of Adva-27a and etoposide.Anew analog of etoposide with increased plasmaconcentration and slower clearance may lead to improvedefficacy of cancer treatment. The pharmacokineticproperties of etoposide in rats had previously been analyzedusing a simple HPLC-based method with a detection limitof 10 ng/mL of plasma. For the present study, we developeda more sensitive method of bioanalysis using LC-MS/MSwith a detection limit for etoposide is 1 ng/mL (seeMaterials and Methods). Using this 10-fold more sensitivedetection technique, we tested whether Adva-27a had animproved pharmacokinetic profile compared to etoposide inrats under similar experimental conditions. Adva-27a andetoposide were administered to rats at 2.5 mg/kg by asingle intravenous (i.v.) bolus injection and the plasmaconcentration of the drugs was analyzed over time (Figure7). The pharmacokinetic parameters of Adva-27a andetoposide were determined by non-compartmental modelingand are presented in Table II. After i.v.administration, bothcompounds were cleared from the plasma in abi-phasicmode and had a similar terminal plasma half-life (T1/2) of1.13-1.16 h and a mean plasma resident time (MRT) of0.27-0.37 h. Interestingly, however, Adva-27a produced amean initial plasma concentration (C0) of 8779 ng/mL,which was approximately 2.8-times the value for etoposide(3183 ng/mL) (Table II). When converted to molarconcentration, the value for Adva-27a was found to beapproximately 2.5- times that of etoposide. The area underthe plasma concentration-versus-time curve (AUCinf) forAdva-27a (2211 hr*ng/mL) was approximately 2.4-timesthat of etoposide (933 hr*ng/mL), while the plasmaclearance rate (CL) for Adva-27a (19.0 mL/min/kg) was2.4-fold lower than that for etoposide (44.7 mL/min/kg)(Table II). On the other hand, etoposide had a largervolume of distribution at steady state (Vss) in plasma (1.04L/kg) than that of Adva-27a (0.323 L/kg) but both valueswere far greater than the plasma volume of 0.03 L/kg forrats, suggesting the distribution of both compounds incompartments other than plasma, namely tissues and organs(Table II).Merzouki et al: New Epipodophyllotoxin Effective in Multidrug Resistance4429Table I. Microsomal intrinsic clearance (CLint) and half-life (T1/2) ofAdva-27a and etoposide in human liver microsomes in vitro. With NADPHWithout NADPHCLint T1/2, CLint T1/2, (µL/min*mg)min(µL/min*mg)minAdva-27a6038.74354Etoposide6435.84848Verapamil16813.73>200Warfarin1>200NDNDND, Not determined.Figure 7. Time-dependent change in plasma concentration of Adva-27a(open diamond) or etoposide (filled square) in rats after a singleintravenous bolus injection at a dose of 2.5 mg/kg. Mean values andstandard error of mean (SEM) are presented (n=3 for each compound).The large variation at 6 h was represented by the rats treated with Adva-27a (concentration 1.22 ng/mL with SEM of 1.13 ng/mL).
DiscussionWe have designed and synthesized a new epipodophyllotoxinwhich bears the laboratory name Adva-27a. The structure ofAdva-27a differs from that of the most commonly knownepipodophyllotoxin, etoposide, in that it has an amino linkedGEM-difluoro glycoside at the C4 position and a methoxylgroup at the E4’ position (Figure 1). The latter appears to beresponsible for the observed ~3-fold reduction intopoisomerase II inhibitory activity compared to etoposide,as the C4 substitutions have previously been shown not to beinvolved in the enzyme inhibition function (3, 21, 29).According to recent reports, the glycoside moiety ofetoposide rests in a spacious binding pocket of thetopoisomerase with relatively few interactions with theenzyme (3, 21, 29). Thus, the C4 substitution in Adva-27ashould generally be allowed and is not likely responsible forour observed reduction in topoisomerase IIa inhibitoryactivity. We suspect that the substitution of the E4’ hydroxylgroup with a methoxyl group in Adva-27a may have resultedin the observed reduction in topoisomerase II inhibitoryactivity. As reported in the literature, the hydroxyl group onthe E4’ position imparts crucial hydrogen bonding and Vander Waals interactions between the E ring and certain aminoacid side chains of the topoisomerase II enzyme (3, 21, 29).Interestingly however, the C4 and E4’ changes in Adva-27a have resulted in a remarkable cytotoxic activity againstmultidrug-resistant breast and lung cancer cells (Figures 3and 5). At present, the precise mechanism by which thesesubstitutions have allowed Adva-27a to overcome multidrugresistance in these cells is not known. However, it appearedthat Adva-27a is able to evade recognition by both P-gp(overexpressed in MCF-7/MDR cells) and MRP-1 (H69ARcells). It is also possible that Adva-27a may be able to evadeBCRP, as it is likely that this drug resistance protein is alsoexpressed to some extent in both MCF-7/MDR and H69AR(4, 6, 28). To date, it is still unknown what drives the abilityof a drug to evade recognition by the ATP-binding cassettefamily of proteins. The apparent ability of Adva-27a to evadethese efflux pumps will allow it to be used as a mono-therapy without any requirement for the addition ofinhibitors of the ATP-binding cassette family of proteins. Thesensitivity of different cell lines and cancers to etoposide iswell-known and is consistent with the cell killing observed inthis work (14, 18, 20, 25, 26). The fact that Adva-27a isequally potent in killing drug-sensitive human cancer cells(MCF-7, A549, PC3, KB, SF-268, HL-60 and HT-29), butmore effective than etoposide in killing multidrug-resistantcancer cells (MCF-7/MDR and H69AR) implies that Adva-27a could form the basis of a new therapy to overcomemultidrug resistance in human cancer. It would be interestingto test directly whether Adva-27a is a weaker substrate of thedrug transporters, P-gp, MRP1 and BCRP, compared toetoposide.In addition to cytotoxic effects, we studied the metabolicstability of Adva-27a in human liver microsomes in vitro.Our results showed that the clearance rate of Adva-27a inhuman liver microsomes is similar to that of etoposide andinvolves both NADPH-dependent and NADPH-independentpathways (Table I). The NADPH-dependent cytochromeP450family of enzymes comprises the major enzymesinvolved in drug metabolism, accounting for approximately75% of all drug clearance reactions (11). Many drugs mayincrease or decrease the activity of the cytochrome P450isozymes. In addition, the level of cytochrome P450expression varies across individuals due to genetic variationsdepending on ethnic backgrounds (24). These are a majorsource of adverse drug interactions and general toxicity (11,ANTICANCER RESEARCH 32: 4423-4432 (2012)4430Table II. Pharmacokinetic parameters of Adva-27a and etoposide in rat plasma.Animal T1/2C0AUClastAUCinfAUCExtr VzVss CL MRT #(h)(ng/mL)(h*ng/mL) (h*ng/mL)(%)(L/kg)(L/kg) (mL/min/kg)(h)11.095659198519870.0851.980.38521.00.30Adva-27a21.309276211321160.1172.210.31119.70.2530.9911404252725290.0721.420.27216.50.27Mean1.138779220822110.0911.870.32319.10.28SD0.1629042832830.0230.410.0572.30.02CV%13.833.112.812.825.021.918.012.28.641.1631069059060.1044.631.07046.00.379Etoposide51.2827159399400.1424.901.07044.30.38661.0337279529540.2073.890.96743.70.354Mean1.1631839329330.1514.471.03644.70.373SD0.1351024250.0520.520.0591.20.017CV%10.8162.582.6334.511.75.72.74.7T1/2, Half-life; C0, initial plasma concentration; AUC, area under the curve; Vz, volume of distribution; Vss, volume of distribution at steady state;CL, intrinsic clearance; MRT, mean plasma resident time; SD, standard deviation; CV, co-efficient of variation.
24). It remains to be determined whether the cytochromeP450-mediated metabolism of Adva-27a will be differentfrom that of etoposide, considering that Adva-27a has anextra O-methyl group at the E4’ position (Figure 1).Finally, we analyzed the pharmacokinetic properties in ratsof Adva-27a and etoposide under similar experimentalconditions. Collectively, the results indicate that Adva-27a hadmuch better plasma accumulation and slower plasma clearancerate in rats compared to etoposide. Since Adva-27a had notonly a lower clearance but also a lower plasma distribution thanetoposide, we speculate that Adva-27a may have distributedinto tissues and organs to a greater extent than etoposide. It willbe interesting to compare the tissue distribution of the twocompounds by direct measurements. The difference in tissuedistribution may help to target Adva-27a to other tumor types,which are resistant to etoposide. Another analog of etoposide,TOP-53, was found to have superior anti-tumor activity in non-small cell lung cancer, a tumor type resistant to etoposide,partly due to its superior tissue distribution in xenograft models(26). If the larger area under the curve (AUC) and higherplasma concentration of Adva-27a in rats, and its superiorgrowth-inhibitory activity towards multidrug-resistant cancersin vitro relative to etoposide can be extrapolated to humans, itmay be that Adva-27a has greater clinical efficacy. Overall, our investigations indicate that Adva-27a is ableto kill multidrug-resistant breast cancer cells in vitro and hasmore desirable metabolic stability and pharmacokineticproperties in vitro and in vivothan etoposide. 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