[CANCERRESEARCH55,830-835, February15,19951

ABSTRACT

A-84441, a potent new antitumor quinolone, was active in vitro and invivo against murine and human tumors. A-84441, a prodrug, was compa

rable in potency to the parent compound with an IC@ range of 0.03—0.49pg/mI againsta panelof murine and human tumor cell lines. The parentcompound bound mammalian DNA in a magnesium-dependentmannerand causedinhibitionof DNA and RNA synthesis.A-84441producedasignificant increasedlife span and cures in three lines of i.p. Implantedmurine tumors. A-84441 was active against sevenof nine solid tumorsincludings.c.murine tumorsand human tumor xenografts.The cornpound appeared to be more active when administered i.v. compared to i.p.

injection. Antitumor efficacy was little effected by treatment schedule,although multiple divided dosing was generally more effective than singledose treatment. A-84441 was over 10-fold-more active against murineleukemiccellsthanagainstnormalmurinebonemarrowcells.The acutetoxicity of A-84441foUowingsingleor multiple dosingrangedbetween11and 50 mg/kgdependenton scheduleof administrationwhengivenbyi.v. or i.p. route. The agent had no apparent toxicity or efficacy whenadministeredp.o.

INTRODUCTION

A numberof cancerchemotherapeuticagentsinteractwith DNA bymultiple mechanisms and cause diverse cytotoxic lesions (1). Thesestructurally diverse agents include doxorubicin, m-amsacrine, mitoxantrone, bleomycin, actinomycin D, and cis-platinum. DNA-activecompounds can bind the grooves of the B-form of DNA (mithramycinand distamycin A), intercalate (doxorubicin and ellipticine), bisintercalate (acridines and echinomycin), covalently bind (anthramycin andmitomycin C), or form coordination complexes (cis-platinum). Manycompounds, however, show complex interactions (2—4),such as intercalation with binding in both the major and minor grooves (nogalomycin) or binding followed by slower base-specific alkylation (pluramycins) or strand breakage (CC-1065). In addition to directdamaging effects on DNA, a number of DNA-active agentsinhibit theactivity of topoisomerase II (5). Compounds either stabilize the covalent DNA-protein intermediate in the relaxation reaction (doxorubicin, ellipticine, and m-amsacrine) or block catalytic activity(merberone and fostriecin) (6, 7).

Considerable effort has been focused on understanding DNA-druginteractions with the intent of improving the selectivity of knowndrugs and discovering unique DNA-binding agents. These effortsinclude diverse approachessuch as modeling the DNA-binding pharmacophore of topoisomerase II inhibitors (8), synthesis of CC-i065analogues that retain antitumor activity but have less toxicity (9),increasing the sequence selectivity of binding and empirical screening

for new agents (10).A relatively new approachto the rational designof DNA-active

anticancer agents is based on the quinolone class of antibacterials.Quinolones represent one of the first classesof antibacterials whosemechanism of action is inhibition of bacterial topoisomerase II (DNAgyrase) (11, 12). Norfioxacin and ofloxacin selectively inhibit bacte

Received 7/6/94; accepted 12/8/94.The costsof publicationof this articleweredefrayedin partby thepaymentof page

charges. This article must therefore be hereby marked advertisement in accordance with18U.S.C.Section1734solely to indicatethis fact.

I To whom requests for reprints should be addressed, at Abbott Laboratories, Depart

ment4PR,Building AP9A, Abbott Park,IL 60064-3500.

rial topoisomerase II and kill Gram-negative and Gram-positive bacteria, yet do not show significant cytotoxicity toward eukaryote cells(i3). However, some newer fluoroquinolones were found to be cytotoxic to Chinese hamster ovary cells (13). While inhibition of mammalian topoisomerase may be a liability for an antibacterial agent,quinolones with activity against the mammalian enzyme may providenew approachesto antitumor drug design (14).

Yamashita et a!. (15) evaluated 90 quinolones for ability to inhibitmammaliantopoisomeraseII activity andcorrelatedbiochemicalactivity with in vivo P388 or B16 antitumor activity. In another study,Chu et a!. (16) reportedpreliminarydatathatshowedthat quinoloneshalogenatedat C-6 and C-8 with a cyclopropyl at N-i ofthe quinoloneskeleton exhibited antitumor activity. Several compounds of thisseries have demonstrated in vitro and in vivo antitumor activity. Oneof this series, A-8444i, a norvaline prodrug of the parent compoundA-83669 (Fig. i), showed promising preliminary activity and hassubsequently been evaluated in substantial detail. In this report, wedescribe the DNA binding properties, cytotoxicity, and spectrum of invivo antitumor activity of the tetracyclic antitumor quinoloneA-84441.

MATERIALS AND METHODS

Synthesis of A-84441. A double cyclization synthetic route was used tosynthesizeA-84441 (17). The synthesisof A-84441 hasbeendescribedindetailpreviously(16).Theparentcompoundwaspreparedandevaluatedasaracemate. The prodrug was a diastereometric mixture.

In Vitro Evaluation of Antitumor Compounds. The in vitro potency ofexperimental compounds and control drugs was determined using a colorimetnc assayto assesscell cytotoxicity as described previously (16). Briefly, tumorcell lines were maintained in RPM! 1640 plus 10% FCS. Experimental andcontrol drugs were dissolved in ethanol, DMSO, water, or the solvent recommendedby themanufacturer,andaddedto thetumorcellsin 96-wellmicrotiterplates.The cells wereexposedto drugsfor 3 days.Cell viability wasdetermined by @m'2dye reduction using absorbance at 470 nm. The IC50 wasdetermined to be the drug concentration that produced 50% growth inhibition.

DNA-binding Studies. DNA (type I calf thymus; Sigma) was dissolved ataconcentrationof 2 mg/ml in Dulbecco'sPBSanddialyzed(cellulosedialysistubing,molecularweightcutoff of 12,000—14,000;SpectraPor,Los Angeles,CA) overnightat 4°Cagainsta 1,000-foldvolumeof thesamebuffer.P388murine leukemia cells obtained from the American Type Culture Collectionweremaintainedin RPM! 1640supplementedwith 10%fetalbovineserum.Toassessthe antiproliferativeeffectsof the parentcompoundA-83669againstP388cells, 200-ml cultures(96-well tissueculture plates;Nunc, Roskilde,Denmark) were established at 1 X 10' cells/mi in the presence or absence ofcalf thymus DNA (200 @.&Wml)for 48 h. After 48-h exposures, P388 cells were

enumeratedusingtheMTT assaydescribedabove.The absorbancespectraof A-83669 in the presenceand absenceof calf

thymusDNA wasmonitoredat300—800nmwith a Perkin-ElmerLambda38UV/visible spectrophotometer(Perkin Elmer Cetus,Norwalk, Cl'). A peak,which centeredat 390 nm, was observedthat decreasedin the presenceofexcessDNA and10mMMg2@.Significantshiftsin absorbancemaximaor thepresenceof an isobesticpoint werenot observed.The effectsof calf thymusDNA, RNA (type IV calf liver, Sigma), ATP, and BSA (type V, Sigma) on theabsorbanceofA-83669weredeterminedasfollows.To asolutionof A-836696(initial concentrationwas 100g.tg/mI)in a cuvet,portionsof a solutionof theappropriate agent (2 mg/ml stock solutions) were added, the resulting sample

2 The abbreviations used are: MTF, 3-(4,5@methyl@azol-2-yl)-2,5-diphenyltetrazo.

hum bromide;% ILS, percentageof increasein life span;CFU. colony-formingunits.

830

Biological Characterization of a Novel Antitumor Quinolone

Jacob J. Clement,1 Neal Burros, Kenneth Jarvis, Daniel T. W. Chu, Joseph Swiniarski, and Jeffrey Alder

Abbott Laboratories, Abbott Park, Illinois @fJf$54.35@j('J(J. J. C., N. B., K. J., D. T. W. C., J. A.), and Arthur D. Little, Inc., Cambridge, Massachusetts 02140-2390 (J. S.)

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Drug A-549 lung HT-29 colon HCT-8 colon MCF-7breastA-84441

0.49 0.18 0.130.29A-836690.23 0.11 0.110.25Doxorubicin0.09 0.13 0.050.30Etoposide1.35 2.70 1.272.43Mitomycin

C 0.03 0.08 0.050.26cis-Platinum1.73 4.26 1.563.33Murine

tumorsB16F1O

Lewis lung P388P388/DOXA-84441

0.03 0.06 0.030.27A-836690.02 0.06 0.020.14Doxorubicin0.003 0.02 0.0042.06Etoposide0.09 0.18 0.0092.66Mitomycin

C 0.03 0.02 0.0070.13cis-Platinum0.87 0.78 0.020.06Table

2 ActivityofA-84441againstsystemicmurinetumorsaTreatmentTumor

Site of routeand Best CuresDosetypeinjection schedule % ILS (%)(mg/kg)P388

i.p. i.p. BID, Dl-9 85 1010.0i.p.QD, D1-5 10115.6i.p.QD, D1-5 73 1015.6P388/ADR

i.p. i.p. BID, Dl-9 5010.0i.p.QD, D1-5 278.0M5076

i.p. i.p. BID, D1-9 95 3010.0i.p.QD, Dl-9 99 105.0i.p.Q4Dx3 59 1020.0i.p.04DX3 5410.0i.p.04DX3 3810.0C26

i.p. i.p. QD, Dl-9 92 4010.0i.p.Q4Dx3 6920.0i.p.Q4Dx3 42 1020.0P388

iv. iv. Q4Dx3 5620.0B16

iv. iv. Q4DX3 1319.6Lewis

lung iv. iv. O4Dx3 31 40.0

CHARACTERIZATIONOF A NOVEL ANTITUMOR QUINOLONE

obtainedfrom Arthur D. Little, Inc. P388/DOXdoxorubicin-resistantsubline,G38 colon carcinomas,B16 melanoma,B16F1Osubline,Pan02 pancreatictumor, CX-1 human colon carcinomas, and the MX-1 human mammarycarcinomas were obtained from the National Cancer Institute (Bethesda, MD).

In Vivo Evaluations of Antitumor Compounds. P388, P388/DOX (doxorubicin resistant) and M5076 tumor cells were harvested from ascites fluid andinjectedi.p. at 106cells/mouse.P388andP388/DOXtumor cells weremoeulated into CDF1 mice, and M5076 tumor cells were injected into BDF1 mice.A 1:50(w/v) breiof C26 tumorwasproducedusinga tissuehomogenizer(‘Fekmar,Cincinnati, OH) and 0.5 ml of the cell suspension was injected i.p.into CDF1 mice. All drugs were delivered i.p. against i.p. tumors. Drugefficacy for i.p. tumorswas basedon the % ILS of treatedversusuntreatedmice and on apparent cures at 30 days (P388 and P388/DOX) or 60 days

(M5076andC26).For i.v. inoculations,P388andB16tumorcellswereharvestedfrom ascites

fluid, andLewis lungwaspreparedby homogenizingsolid tumor tissuefrommice.B16(106cells/mouse)andLewislung(5.0—5.8X 10@cells/mouse)wereinjected i.v. into BDF1 mice and P388 cells (106 cells/mouse) were injectedi.v. into CDF1mice.Drug efficacy for i.v. tumorswasbasedon the% ILS oftreatedversusuntreatedmice,andon curesbasedon 30—day(P388)or 60-day(Lewis lung; B16) survival rates.

For solid tumor models, M5076, C26, B16, and Lewis lung were allprepared from solid tumor tissue. Breis were produced by homogenization ofthesolid tissue.A 1:10(w/v) brei wasmadewith M5076,B16,or Lewis lung

Table1 In vitro IC50values(pg/mi)againsthumanand murine tumor cell lines

Human tumors

A-83669

a Each entry represents a complete experiment including 3—4dose groups of the testagent,a positivecontrol, and a groupof untreatedtumor-bearingmice.

831

N@@á

SH A-8444l

Fig. 1. Structure of A-83669 and 84441, quinobenoxazine antitumor agents.

was mixed, and the absorbance at 390 nm was recorded within 3 mm. Raw

absorbance values were corrected for the dilution, and percentage change wascalculated from the initial absorbance. Appropriate buffers, as indicated in thetext, were used to determinethe effects of metal ions on the binding ofA-83669to macromolecules.

Incorporation Studies. The effects of agentson the apparentratesofprotein,RNA, andDNA synthesiswereassessedby measuringcellular incorporationof tritiated leucine,uridine,andthymidinefrom culturefluids. P388cultureswereestablishedin 24-well microtiterplates(2 X 10@cells/ml;2-micultures), gassed with 5% CO2 in air, and incubated at 37°Cfor 1 h in thepresenceor absenceof A-83669at theconcentrationsindicatedin thetext.Toinitiate incorporations, 20 ,d of Dulbecco's PBS (0.8% NaCl-0.22%Na2HPO4-7 H20-0.02% KH2PO4-0.02% KCL; w/w) containing [3H]leucine (2mCi/mi), [3H]uridine (1 mCi/mI), or [3H]thymidine (1 mCi/mi) were added.After 1 h, the cell suspensionwas removedand diluted with 10 ml ofDulbecco's PBS. Cells were collected on 0.45-mm nitrocelluiose filters (Mulipore, Bedford,MA) by vacuumfiltration, andmacromoleculeswereprecipitatedby the additionof cold 5% trichloroaceticacid. Filters were extractedthree times with 5 ml of cold 5% trichloroacetic acid, washed with 5 ml ofwater,andrinsedwith 5 ml of 70%ethanol.Filtersweretransferredto 20-miscintillation vials and 12 ml of scintillation fluor (Aquasol; NEN ResearchProducts, Boston, MA) were added/vial. Radioactivity was determined byliquid scintillation spectrometry.Quenchingwas similar in all samplesasdetermined by the ratio of counts in a narrow tritium channel and a wide ‘4Cchannel.Tritiated compoundswere obtainedfrom Amershamand were thehighestspecificactivity available.

Animals. Mice wereobtainedfrom HarlanSprague-Dawley,Indianapolis,IN. FemaleBALB/c X DBA/2F1(CD2F1beige,hereafterreferredto asCDF1),andC57BL16X DBA/2F1(B6D2F1/HSdblack,hereafterreferredto asBDF1)mice were housed 10 animals/cage on bedding and given free access to foodandwater.Hsd:athymicnude-nu(hereafterreferredto as“nude―)werehoused10/cage in sterilized barrier cages and were given free accessto sterilized foodand water.

Tumor Cells. P388 leukemia, M5076 reticulum cell sarcoma, C26, Lewislung carcinoma,HT-29, HCF-8, MCF-7, and MBA-9812 tumor cells were

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Table4 Activity ofA-84441 againsthuman tumorxenograftsTreatmentroute

andDoseBestCuresTumorschedule(mg/kg)%TWV'(%)CX-!

colon i.p.QD,D2-112079MBA-9812mammary i.p.QD,D2-l12076LX-1

lung i.p. OD, D2-l12043MX-1mammary iv.Q4Dx3408430HT29colon iv.Q4Dx34021a

@ tumor weight inhibition.

Table5 IV Scheduledependencyfor A-84441againsts.c.M5076tumorsTotalDose/lnj.°

dose Days to Delay toSchedule (mg/kg) (mg/kg) % Inhibitions' 1.6g 1.6g(days)QD,

Dl 20.0 20 43 27.34.6Q8Hx2,Dl 20.0 40 56 28.76.0O4Hx3,Dl 13.33 40 80 32.09.3QD,

D1-9 20.0 180 80 31.08.3Q8Hx2,D1-9 5.0 90 31 26.23.5Q4Hx3,D1-9 3.3 90 54 29.77.0Q4Dx4

20.0 80 83 32.59.8Q8Hx2,Q4Dx4 10.0 80 68 28.65.9Q4Hx3,Q4Dx4 3.3 40 43 27.14.4Untreated

NAC NA (0) 22.7NAa

Inj., injection.

Tumor weight inhibition when untreated controls equaled 1.6g.CNA, not applicable.Table

6 Acutetoxicity (W@ and LD,@,)ofA-84441 inmiceTreatment

LD50LD10Routeschedule (mgIkg/inj.'@)(mg/kg/nj.)iv.

Dl 50.027.2D1-522.710.2Q4Dx334.019.6i.p.

DI 49.821.9D1-911.29.4Q4Dx335.320.9P.O.

Dl>400a

Inj.,injection.

CHARACTERIZATIONOF A NOVEL AN11TUMORQUINOLONE

Table 3 Activity ofA-84441 against murine solid s.c. tumor? removed, stored in Bouin's fixative, and CFUs per spleen were determined.This assaydeterminedA-84441potencyversusleukemicandstemcells.

RESULTS

In Vifro Potency of A-84441. A-8444i, the parent A-83669, andfour anticancer drugs were assayed against four human and fourmurine tumor lines using a IC50 end point to assessin vitro potency.Each potency determination was conducted at least three times.A-8444i was generally more potent than etoposide or cis-platinum,and lesspotentthandoxorubicinor mitomycinC (Table1).The IC50valuesfor A-84441 were lessthan 1 @.aWmlagainsthumantumorlinesand generally less than 0.1 @agMagainst murine tumor lines. Thedoxorubicin subline of the P388 leukemia was sensitive to bothquinolonesbut lessso than the parent line. Overall, the activity ofA-8444i was similar to that of the parent compound. The R and Sforms of the parent compound had nearly identical in vitro cytotoxicactivity (data not shown). In the National Cancer Institute in vitropanelof 50 humantumor lines,A-8444i hadbroadspectrumactivityagainst melanoma, ovarian, renal, small, and non-small cell lung,colon, leukemic, and central nervoussystemtumor cell lines. The50% tumor growth inhibition values were less than 1 p.g/ml againstthese cell lines (data not shown).

Efficacy of A-84441 against Systemic Tumors. A-84441 administered i.p. was active against four i.p. implanted tumor lines asmeasuredby % ILS and apparenttumor cures(Table 2). Treatmentwith A-84441 at maximum tolerated doses was associated with a

73—101%ILS against P388, and a 27—50%ILS against the doxorubicin-resistant P388 line (P388/DOX). Treatment of M5076 and C26tumors with A-8444i at maximum tolerated doses was associatedwith 30 and 40% cures. ILS values varied with treatment schedulewith greater efficacy seen with daily and twice daily dosing ascompared to an intermittent treatment schedule.

Treatment (i.v.) of i.v. P388 leukemia, B16 melanoma, and Lewislung carcinoma with A-8444i was marginally effective. The greatestefficacy was demonstratedagainstP388 where treatmentwas associated with a 56% 11.5. Less activity was demonstrated against i.v.

TreatmentTumorrouteand

scheduleBest % VWI―Dose(mg/kg)M5076i.p.BID, D1-9

i.p.Q2Dx6i.p. Dli.p. QD, D7-16iv. QD, D1-5iv. Q4Dx3iv. Dlp.o. QD, Dl-968

3741418499481810.0

10.020.010.010.040.050.0

20.0C26i.p.BID, D1-9

i.p. QD, D1-9i.p. Q4Dx3iv. QD, D1-5iv. Q4Dx3iv. Dlp.o. QD, D1-962

8561879558

—1810.0

10.010.024.040.057.0

20.0Lewislungi.p. QD, D1-9

i.v. 04DX365 592.540.0G38i.p.BID,D1-9410.0B16i.p.BID,D1-9675.0Pan

02i.p. BID, D1-9712.5

a @chentry represents a complete experiment including 3—4dose groups of the testagent,a positivecontrolgroup,anda groupof untreatedtumor-bearingmice.

b@ tumor weight inhibition.

tissue and 0.5 ml was injected s.c. into BDF1 mice. For C26, a 1:50 w/v breiwasmadeand0.5 ml was injecteds.c. into CDF1mice.For Pan02 or 08tumor, 1-mm2 trocars were prepared from solid tumor tissue and injected s.c.

Drug efficacy for solid tumors was based on tumor weight inhibition or delayin tumor regrowthto reacha specifiedmass(1.0 or 1.6 g). Tumor weightinhibition (TWI) was calculated from the ratio of the mean tumor mass fromtreated(7) versuscontrol (C) animalsby the formula TWI 1-(T/C). Allsolid tumors were measured in two orthogonal directions by vernier calipers2—3times/week. Tumor mass (length X width) was calculated as (L X W2)/2.

For xenograft solid tumor models, HT29, and MX-1 inocula were preparedby homogenizationof solidtumortissue.A 1:10w/v breiwasmadewith HT29tumor tissue and 0.5 ml was injected s.c. into nude mice. A 1:8 w/v brei wasmadewith MX-1 tumor tissueand0.5 ml was injecteds.c. into nudemice.CX-!, LX-1, and MBA-9812 were preparedas 1-mm2trocars and wereinjecteds.c.into nudemice.Drug efficacywith s.c.xenografttumor inoculations was based on percentage of inhibition of mean tumor mass in treatedmice when control mean tumor mass was approximately 0.5 g (percentageinhibition).

Dosing. A-84441wasdissolvedin sterile indictablewater(Abbott Laboratones) and was administered in a volume of 0.5 ml i.p. or i.v. A-84441 wasadministeredondifferentschedulesasdetailedin the“Results―textandtables.

BoneMarrow Assay. A-84441wasadministeredi.v. on day0 to normalCDF1 mice. On day 1, femoral bone marrow cells were harvested, counted, and

injectediv. into lethally irradiatedmice.Onday8, thespleenswereremovedandstoredin Bouin's fixative andcounted.SplenicnoduleCRIs wereusedasindicators of stem cell viability after exposure to A-84441. Based on thefemoralcell count,thequantityof cells injected,andtheCFU spleniccounts,theviable CFUsper femurof mice treatedwith A-8444wascalculated.

In a separatetrial, 1 X 106 L1210 leukemia cells were injected i.v. intoCDF1miceonday0. Onday3, A-84441wasadministeredi.v. asabove.Onday 4, femoralbonemarrowcells wereharvested,counted,andinjectediv.into lethally irradiatedmiceasdescribedabove.On day 10,thespleenswere

832

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CHARACrERIZA11ONOF A NOVEL AN11TUMORQUINOLONE

Acute Toxicity Studies. The LD@ and@ values for A-8444iin mice were similar for i.v. and i.p. dosing on several schedules

___________ (Table6).ThesingledoseLD50valueswere50.0and49.8mgfkgbyi.v. and i.p. routes, respectively. With multiple dosing, the LD50valuesfor daily dosingdecreasedfor both i.v. and i.p. administration.Although the total administered dose was greater For five daily i.v.doses,the LD50 value was 22.7 mg/kgday, while nine daily i.p. dosesyieldedan LD50 value of ii.2 mg/kg-day. No mortality was observedfollowing p.o. dosingup to 400 mg/kg.

Bone Marrow Studies. In normal mice administered A-8444i ona Q4Hx3 schedule at i.v. dosesup to 50 mg/kg, normal bone marrowcolony forming units were reduced by less than one log (Fig. 2).Similar doses of A-84441 to leukemic mice produced nearly a three

20 30 40 50 log reduction in leukemic colony forming units. A similar differential

Dose(mg/kg) was produced in matched sets of normal and leukemic mice admin

istered A-8444i as a single bolus dose.Mechanism of Action Studies. Mechanism of action studies were

conductedwith the parentcompound,A-83669, which is formed inhuman or rat plasma within 60 s from the hydrolysis of the norvalineprodrug, A-84441. These studies concentrated on the binding proper

ties of the compounds and the effects on macromolecular synthesis.The in vitro potency of A-83669 against P388 cells was reduced bythe additionof exogenouscalf thymusDNA at 200 tag/mi (Fig. 3A).Bindingto mammalianDNA wasdependenton thepresenceof iO m@i

—0--- Normal.—.-— Leukemic

I.001

0 10

100 200 300

lA.83669l. ILg/mI

Fig.2. Fractionof CFU survivingperfemurin irradiatedmiceinjectedwithfemoralbonemarrowcells from normalandleukemicmiceadministeredthreeiv. doses(Q4Hx3)of A-84441.

injected Lewis lung tumors. No curative activity was seen in i.v.tumor models.

Efficacy of A-84441 against Murine Solid Tumors A-84441was evaluatedin six murinesolid tumor models.Table 3 summarizesthe optimal responsesof a thesetumors from a number of separateexperiments, each experiment consisting of 3—4dose levels of testagent,an untreatedtumor-bearinganimal group,and a groupof micetreated with a positive control compound specific for the tumor model.The M5076 reticulumcell sarcomaandC26 coloncarcinomamodelswere used to evaluateA-8444i administeredby variouscombinationsof route, dose,and schedule.The Lewis lung, 08 colon carcinoma,B16 melanoma, and Pan 02 pancreatic tumors were evaluated on amore limited basisto determinethe spectrumof activity of A-84441in experimental models. Treatment (i.v.) of M5076 and C26 tumorsappeared to be more effective than i.p. treatment. Definitive analysisof routeof administrationdependencywas complicatedby the lack ofsimilar compound administration schedulesfor i.v. and i.p. treatments.Administration i.v. of A-8444i on a Q4Dx3 schedule produced a 99and 95% ll@Sin M5076 and C26 tumors. Five daily i.v. treatmentsproducedan 84 and87% ILS in thesametumortypes.Using 50% ILSasa criterionfor antitumoractivity, A-8444i was active in five of thesix tumors tested. The C38 colon carcinoma was the only refractory

solid tumor found in this study. No cures were associated withtreatmentwith A-84441 againstthesemurine tumors.

Efficacy of A-84441 against Human Tumor XenograftsA-8444i was evaluated in several experiments for activity againsthumantumor xenograftsin nudemice. The activity of the compoundagainst five xenografts is summarized in Table 4. Treatment(i.p.) with20 mg/kg A-8444i on a QD, D2—iischeduleproduceda 79 and76%ILS in the CX-i colon tumor and MBA-98i2 mammary tumor,respectively. The compound produced less than a 50% ILS in theDc-i tumor model.Treatment(i.v.) with A-84441on a days1,5, and9 schedulewas associatedwith curativeactivity in the humanMX-ixenograft model. Treatment with A-8444i was not active against theHT29 colon tumor.

Schedule Dependency Studies. The s.c. M5076 tumor system wasused to more fully evaluate the schedule dependency of A-8444iactivity. The compound was given on a day 1, day 9, or intermittentlyon either a daily, twice daily, or threetimesdaily schedule(Table 5).In this model, the efficacy of i.v. administrationwas not stronglyscheduledependent.For dosingregimensextendingbeyondone day,single daily dosing was slightly more effective than multiple dailydosing. For day 1 only treatment three times/day dosing was moreeffective than a single dose.

A

8

I0

[A-83669l. @Lg/m1

B

400 500

Fig. 3. A, exogenousDNA reducesthe antiproliferative potency of A-83669. P388cultures were established (10' cells/mi; 200 pi) in medium with A-83669 at the concentrationsindicatedin the presence(V) or absence(0) of 200 paJml calf thymusDNA.After 48-h incubations, cell proliferation was determined by reduction of MTF. Resultsrepresent percentageof inhibition normalized to untreated cultures (200 @tg/miexogenousDNA hadnoeffectontheproliferationof P388cellsundertheseconditions).B, bindingof A-83669 to DNA is Me@ dependent.The effectsof DNA on the absorbanceofA-83669at 390runin thepresenceJ) or absence(0) of 10matMe@. DNA (2 mg/mI)andA-83669stocksweremadein Dulbecco'sPBSwithorwithout10mistMg2@.DNAwasaddedto a covetcontaining100pg/mIA-83669(initialconcentration),thesolutionwas mixed, and the absorbancewas recorded. The initial absorbances(mean ±SD; n 3)ofA-83669 in thepresence(0.563±O.019)orabsence(O.567±0.015)ofmagnesiumionwerenotsignificantlydifferent.Resultsindicatethepercentageof changein absorbanceobservedaftercorrectionfor the changein volume.

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CHARACFERIZA11ONOF A NOVEL ANTITUMOR QUINOLONE

and RNA synthesis with lesser effects on protein synthesis. Thispattern of inhibition was similar to that observed after exposure toactinomycin D, which is considered an inhibitor of RNA synthesis(18).

Mechanismof action, cytotoxicity, and in vivo studieswere conducted using the racemic mixture of the compounds. While definitivemechanism of action studies have not been conducted with the chiralforms of these agents, the in vitro cytotoxicity of the chiral forms aresimilar (data not shown). Additional mechanism studies are ongoing.

in vivo therapy with A-8444i was curative, or was associated withsubstantial increases in ILS against i.p. murine tumors. Efficacyagainst murine tumors injected i.v. was limited to increased life span.The i.v. modelsare more challengingin that multiple foci of tumorgrowth are initially produced by this inoculation. A-8444i was associatedwith significantdelaysin solidtumorgrowth in severalmurinetumor models. A-8444i therapy was associated with significant activity against human tumor xenografts. Curative activity was associated with A-8444i therapy against MX-i tumors, while delays in

400 500 tumor growth were observed in some of the other xenografts.The in vitro potency data for A-8444i was not particularly valuable

for predicting in vivo activity. For example, the in vitro potency ofA-8444i was identical against murine Bi6 and P388 tumor lines.However, A-8444i was more active againstP388 than againstB16tumor cells injectedi.v. Similarly, the in vitro potenciesof A-8444iwere generally less versushuman tumor cell lines than against murinetumor lines, but in vivo activity appeared similar between humantumor xenograftsand murine solid tumors.

Quinolones as a class have demonstrated potential as antitumoragentsbut precise mechanismsare not firmly established. Most quinolonesact astype II topoisomerasepoisonsthroughstabilizationof thebacterial or mammalian enzyme-DNA cleaveable complex (14), withantibacterial quinolones acting as poisons to bacterial topoisomeraseII (19). Some quinoloneshave shown interactionswith mammaliantopoisomerase I and II (20). The antibacterial quinoiones ciprofloxacin, ofloxacin, and norfioxacin demonstrate slight interaction with

EC

C0@'

V0

01,,@0CeVen

.C0

0

-5

-10

-15

-20

-250 100 200 300

[A-83669], @.tg/m1

Fig. 4. Effects of DNA, RNA, protein, and AlP on the absorbanceofA-83669 at 390run. Calf thymus DNA (s), calf liver RNA (El), BSA (0), and AlT (•)were added to a100 @sg/mlsolution of A-62176 in Dulbecco's PBS supplemented with 10 mo@iMg@@asdescribed in Fig. 3B.

Mg2@(Fig. 3B). A-83669 interactedwith mammalianDNA, but notRNA, protein, or AlT as determined by changes in absorbancefollowing incubation with the different compounds (Fig. 4). A-83669inhibited cellular DNA and RNA synthesis but had less effects onprotein synthesis based on incorporation of 3H-labeled precursors(Fig. 5).

DISCUSSION

A-84441 is a promising new antitumor quinolone with potent invitro activity against human and murine tumor cells. The prodrug isconverted to the parent compound in less than 60 s in human or ratplasma (data not shown). in vivo efficacy was observed againstseveral systemic and solid tumor models in mice. Scheduling trialsestablished i.v. dosing as more effective than i.p. dosing with onlymodest schedule dependency. Mechanism of action studies demonstrated Mg2@-dependent binding of A-83669, the parent compound,to DNA and inhibition of DNA and RNA synthesis. The quinoloneproduced selective in vivo inhibition of leukemic cells over normalbone marrow cells.

The in vitro potency of A-84441 is similar to that of several other

therapeutics, including doxorubicin, etoposide, and cis-platinum. Inthe National Cancer Institute tumor panel, A-8444i demonstratedbroad activity against human tumor cells including lung, colon, andbreast types. Similarly, the compound was active in vitro against themurine tumor cells used in animal studies. A-84441 had an IC50 of0.27 @g/mlagainst doxorubicin-resistant P388, suggesting partialsensitivity of the multidrug resistancephenotype relative to the parentline.

Mechanism of action studies show that the parent compound,A-83669, binds to DNA in a magnesium-dependentmanner. Bindingwas specific for DNA since RNA, protein, or AlP did not reduceabsorbanceof A-83669. Cellular incorporation studieswere consistentwith a DNA-active agent. Inhibition of macromolecular synthesisdoes not provide conclusive evidence of mechanism, but patterns ofinhibition of DNA, RNA, and protein synthesis often correlate withthe general cytotoxic actions of the agent. A-83669 inhibited DNA

10 1 0.1

100

80

@60.@

@40

20

0

[A-83669], jig/mi

Fig.5. Effectsof A-83669on whole-cellDNA, RNA, andprotein synthesis.P388cultures(2 x i0@cells/mi;2-mi cultures)wereestablishedin mediumin thepresenceofA-83669attheconcentrationsindicated.After1 h, incorporationof radiolabeledprecursors was initiated and cells were collected on nitrocellulose filters after an additional 1 hof incubation.Filters were extractedwith 5% trichloroaceticacid, and radioactivityincorporatedwas determinedby liquid scintillation counting. Results representthepercentageof inhibition(mean±SD;n = 3;noerrorbarsrepresentsthemeanofasingleexperiment) of incorporation normalized to untreated controls ([3H)thymidine incorporated= 32,800;[3Hjuridine = 10,150;[3H]leucine= 3,230).

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CHARACIERIZATION OF A NOVEL ANTITUMOR QUINOLONE

mammalian topoisomerase (21), although these antibacterials are allmore selectivefor Escherichiacoli topoisomerase(22). Other quinolones has also shown more substantial mammalian topoisomerasepoison activity (23—25).Kohlbrenner et a!. (25), for example, demonstrated that several isothiazole-quinolones interact with mammaliantopoisomerase type II as poisons and had potent in vitro activity.However, these compoundshad poor activity versus murine solidtumors. In contrast, treatment with A-84441 was associated with invivo efficacy versusboth humanandmurinesolid tumors.The parentcompound,A-83669, binds to DNA as describedabove and inhibitstopoisomerase activity (26). This compound, however, does not act asa mammalian topoisomerasepoisonsbut as an inhibitor of the catslytic activity of theenzyme(26). This propertymakesA-83669 anditsprodrug A-84441 mechanistically unique among quinolones reportedto date. The biological activity reported here for A-84441 and titspotentially novel mechanism of action suggest a promising newdirection for antitumor drug discovery.

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1995;55:830-835. Cancer Res   Jacob J. Clement, Neal Burres, Kenneth Jarvis, et al.   Biological Characterization of a Novel Antitumor Quinolone

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