(cancer research 37, 1666-1672, june 1977] antitumor … · (cancer research 37, 1666-1672, june...

(CANCER RESEARCH 37, 1666-1672, June 1977]

SUMMARY

Chartmeusin has exhibited significant therapeutic activityagainst three experimental mouse tumors (ascitic P388,Li 210 leukemia, and Bi 6 melanoma) when tumor cells wereinoculated i.p. and drug was administered i.p. In furthertesting against P388 leukemia, no activity was observedwhen drug was administered p.o., s.c., or i.v.

Chartneusin was very slowly absorbed from the small intestine, thus explaining the lack of activity when administered p.o. When given i.p., the drug precipitated in thepenitoneal cavity and was slowly absorbed over several hr.The strong activity observed by this route was related to theprolonged and intimate contact of drug with tumor cells inthe penitoneal cavity. Upon s.c. administration, extensiveprecipitation occurred. Subsequent dissolution and absorption from the injection site were very slow, andmeasured plasma and tissue levels were quite low. Biliaryexcretion of chartmeusin, the predominant route of elimination, was very rapid, with 80 to 100% of the dose appearingas unchanged drug in the bile within 6 hr after i.v. administration. Rapid biliary excretion after i.v. administration wasreflected in a rapid decline in plasma and tissue concentrations to levels (shown by in vitro cell kill experiments) lessthan those necessary to kiII.P388 cells. When the bile ductsof i.v.-dosed leukemic mice were ligated, therapeutic activity was observed, confirming that the physiological disposition of chartneusin exerts a major influence on its themapeutic utility.

INTRODUCTION

Chartneusin is a Streptomyces-pmoduced antibiotic whichwas originally reported in 1953 (7). Upon its discovery, thebiological activity of chantreusin was investigated in a vanety of screening systems at The Upjohn Company. It wasalso submitted to the National Cancer Institute and theSloan-Kettering Institute for antitumor testing in the systems then in use. Except for a limited spectrum of antimicrobial activity (2), little on no activity was observed in anyscreen, and antitumom testing was suspended in 1957. The

I This study was supported in part by Contract NO1-CM-43753 with the

Division of Cancer Treatment, NIH, Department of Health, Education andWelfare.

2 To whom requests for reprints should be addressed.

Received November 5, 1976; accepted March 2, 1977.

chemical structure of chartmeusin was elucidated and published in 1964 (3, 12) (Chart 1). Recently, a reevaluation ofolder natural products in new in vivo screening systems hasrevealed that chartmeusin has significant chemotherapeuticactivity in mice against the P388 and L1210 leukernias andthe relatively refractory B16 melanoma. Biochemical studies have revealed that chartreusin binds to DNA and inhibitsRNA and DNA synthesis (8). This report documents theantitumor activity of chartreusin in experimental mouse tumon systems and presents drug disposition and kinetic cellkill data that explain the dependence of activity on the routeof drug administration.

MATERIALS AND METHODS

Chartreusln. Chartneusin was isolated and purified fromfermentations carried out at The Upjohn Company, Kalarnazoo, Mich. Chemical identity and purity were determined byIA, UV, nuclear magnetic resonance, and mass spectralmethods and elemental analysis.

Formulation for Administration to Animals. Chartreusinsolutionsfori.p.,p.o.,ors.c.administrationtoratsonmicewere prepared by dissolving crystals in 0.01 NNaOH at 3 to 4mg/mI, adjusting the pH to 9.2 to 9.5, and bringing todesired volume with 0.2 M Na2HPO4(pH 9.2). At this pH, amaximum solubility of 2.0 mg/mI could be achieved. Solutions for i.v. administration were prepared at S mg/mb in20% N,N-dimethylacetamide:0 .2 M Na2HPO4(v/v).

Chartreusin suspensions for i.p. or s.c. administrationwere prepared with the use of a Potter-Elvehjem type tissuehomogenizer (Tni-R Instruments, Rockville Center, N. Y.) ineither 0.9% NaCI solution:0.1% Tween 80 or 0.3% hydnoxypropylcellubose:0.9% NaCI solution:O.1% Tween 80.

Chemotherapy Studies. The animals used in chemotherapy experiments were C57BL/6 x DBA/2 F1 (hereaftercalled B6D2F,) mice, 6 to 8 weeks of age (18 to 25 g).Female mice were used in the B16 melanoma studies; malemice were used in all others. Groups of 6 to 10 mice werehoused in plastic cages and given pelleted feed and waterad libitum. L1210 and P388 leukemias were maintained bycontinuous i.p. passage in syngeneic DBA/2 female mice.B16 melanoma and Lewis lung carcinoma were carried s.c.in syngeneic C57BL/6 male mice. Standardized protocols ofthe Drug Research and Development Program, NationalCancer Institute, were followed for continuous passage ofthe tumor and for implantations of tumors into B6D2F, micefor chemotherapy experiments (4). L1210 and P388 leuke

1666 CANCERRESEARCHVOL. 37

Antitumor Activity and Preliminary Drug Disposition Studieson Chartreusin (NSC 5159)1

J_ Patrick McGovren,2 Gary L. Neil, Sherl L. Crampton, Maxine I. Robinson, and John D. Douros

CancerResearchUnit, TheUpjohnCompany,Kalamazoo,Michigan49001(J. P. M., G. L. N., S. L. C., M. I. R.Jand NationalCancerInstitute,NIH,SilverSpring, Maryland20910(J. D. 0.)

on June 21, 2021. © 1977 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

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Chartreusin Antitumor Activity and Disposition

sues were dissected and stored frozen until assay: kidneys,spleen, liver, small intestine, heart, lung, thyrnus, musclefrom beg, and brain. Blood was centrifuged to obtainplasma, which was frozen until assay.

In vitro liver metabolism was investigated by incubatingchartmeusin (100 and 200 pg/mI) with the supemnatant froma mouse liver homogenate centrifuged at 9000 x g and aNADPH-genemating system (9).

Assay for Chartreusin. Bile and urine samples were assayed undiluted or diluted with 0.2 M Na2HPO4by applying80-pi aliquots to wells formed in Sarcina lutea-seeded agamtrays. After incubation (37Â°,14 to 20 hn), the diameters of thezones of growth inhibition were measured. These zoneswere compared with those obtained with chartmeusin standards prepared in drug-free bile and urine at the samedilution and applied to each agamtray.

Tissue and fecal samples were diluted in pH 7, 0.1 Msodium phosphate buffer and homogenized with a TekmarTissumizem (TekmamCo., Cincinnati, Ohio).

Plasma, tissue, and fecal samples were assayed by extracting chartreusin into chloroform, evaporating the solvent, reconstituting the extracted material in 0.01 N NaOH,and applying to Sarcina lutea-seeded agar trays as descnibed above. Standards were prepared by the addition ofknown quantities of chartreusin to drug-free tissue homogenates for each tissue analyzed and extracted in the samemanner as described above. A separate tissue standardcurve was applied to each agamtray.

The specificity of the microbiological assay for unchanged chartreusin was evaluated by TLC3 and bioautogmaphy.Aliquots of whole bile and urine collected for 6 hrfrom matsdosed i.v. with chartmeusin were extracted withchloroform. The concentrated extract was applied to flexible plastic-backed TLC plates (Bakerflex silica gel lB-F; J. T.Baker Chemical Co., Phillipsburg, N. J.). The plates weredeveloped in chloroform :methanol:water (85:14:1). Separated bands were visualized by their fluorescence under 360nm UV. The silica gel surfaces of the plates were thenplaced in contact with S. Iutea-seeded agamtrays for 30 mmand removed. The trays were incubated, and zones of inhibition corresponding to the separated bands were observed. Aliquots of bile and urine were then applied to silicagel-coated glass TLC plates (Uniplate; Analtech, Inc. , Newark, Del.) which were developed as before. Bands werevisualized and scraped off the glass plate. The separatedbioactive materials were eluted from the silica gel and assayed microbiobogicably. Aliquots of whole bile and urinewere also assayed to determine total bioactivity. Bile andurine from untreated animals were processed in the samefashion.

Cell Kill Studies. In studies of P388 cell kill kinetics, cellswere maintained in culture in Roswell Park Memorial Institute Medium 1634 supplemented with fetal calf serum (5%),NaHCO3 (0.75 mg/mI), penicillin (0.1 mg/mI), and streptomycin (0.05 mg/mI), as described previously (1). In thismedium, the cells grew exponentially with a doubling timeof about 12 hr. Chartreusin was dissolved and then diluted

3 The abbreviations used are: TLC, thin-layer chromatography; ILS, in.

crease in life-span.

D-FUCOSE

D- DIGITALOSE

Chart 1. Structure of chartreusin.

mias were implanted i.p. at 10@and 106 cells/mouse, mespectively. In some experiments, P388 leukemia was inocubated i.v. into the lateral tail vein at 10@cells/mouse. B16melanoma was implanted i.p. (0.25 ml of a 1:5 w/v tumorbrei). Lewis lung carcinoma was implanted s.c. (106 viablecells), as previously described (10).

Absorption, Distribution, Metabolism, and ExcretionStudies. Male Sprague-Dawley rats (160 to 300 g) and maleB6D2F, mice (18 to 25 g) were used in all disposition studies.

Oral absorption was studied by administering p.o. dosesof 50 mg/kg by gavage to bile duct-cannulated rats andmeasuring the cumulative amount of chartreusin appearingin the bile, feces, and urine at intervals to 48 hr. Afterdosing, matswere placed in special restrainer cages (Stoelting Co., Chicago, III.), which were modified with devices toallow separate collection of urine and feces.

Biliary excretion studies were conducted in bile ductcannulated rats. Animals were anesthetized with sodiumpentobarbital (50 mg/kg i.p.) and bile ducts were cannubated with PE1O tubing (Intramedic polyethylene tubing;Clay Adams, Pamsippany, N. J.). Abdominal incisions wereclosed with animal wound clips (Autoclips; Clay Adams).Body temperature was maintained by directing heat lampsonto the rats. Bile was collected into tamed plastic tubeswhich were reweighed at the end of a collection intervaland frozen until assay.

Absorption of chartreusin after i.p. administration wasstudied by administering doses of 10 and SOmg/kg to bileduct-cannulated rats. The rats received the same volume ofchartreusin solution at each dose level (as 0.4 or 2.0 mg/mI)to duplicate the conditions of the therapy studies in mice inwhich a constant volume was administered to each animalat each dose. The rats were kept unconscious and lying ontheir backs for the 1st 6 hr of the experiment so that drugwould not leak out through the abdominal incision. After 6hr, the rats were placed in restrainer cages and allowed toregain consciousness. Bile was collected at intervals to 48hr after administration.

Metabolism cages (having open mesh flooring with devices to separate urine from feces) were used to studyurinary and fecal excretion in rats and mice.

The time course of tissue distribution of chartreusin wasmeasured inmice afters.c.administrationand inratsafteri.v. administration. At selected intervals after administration, groups of animals were anesthetized with ether ormethoxyflurane, blood was withdrawn from the inferiorvena cava into heparinized syringes, and the following tis

JUNE 1977 1667



J.P.McGovren etal.

with medium prior to the addition of cells. At the end of drugexposure, cells were centrifuged and washed to removedrug and then suspended in medium to determine cloningefficiency. Cell survival was determined by cloning in softagar (5) and has been described previously (1). In the calculation of percentage survivals, the control samples (no drugtreatment) were normalized to 100% survival. The cloningefficiency of control cells was 60 to 90%. The relative standamddeviation in determining cell survival was about 15%.

Chemotherapy Study In Bile Duct-ligated Mice. Thechemothenapeutic activity of chartreusin was investigatedin P388 leukemic mice which had ligated bile ducts toprevent elimination of chartmeusin in the bile. The ductswere ligated with silk suture and the abdominal incisionswere closed with animal wound clips. Three or 4 days aftersurgery, the mice were inoculated i.v. with 106 P388 cells.Chartneusin treatment (i.v.) was begun on the day afterinoculation. One group (28 mice) was dosed with 25 mg/kgi.v. for 4 days. The other group (15 mice) was dosed withvehicle (20% N,N-dimethylacetamide/0.2 M Na2HPO4).Control studies were performed in which normal (nonligated)animals were inoculated i.v. with P388 leukemia and dosedi.v. with chartreusin (25 mg/kg) or with vehicle on Days 1 to4.

RESULTS

Chemotherapeutic Activity. When administered i.p. tomice inoculated i.p. with P388 on L1210 leukemia on B16melanoma, chartneusin displayed significant activity over awide dose mange(Tables 1 and 2). A maximum increase inlife-span of 131% was observed in P388 leukemic mice at adose of 50 mg/kg. Significant therapeutic activity (over25% ILS) was observed against P388 leukemia over a rangeof 0.8 to 100 mg/kg. In the P388 testing, similar results wereobtained whether solutions or suspensions were administered.

Significant activity was also exhibited in L1210 leukemicmice over a dose range of 10 to 80 mg/kg (46% maximumILS at 10 mg/kg).

In B16 melanoma testing, 83 to 99% ILS was obtained atdoses of 25, SO,and 100 mg/kg, and 3 cures were observed.

No activity was observed against s.c.-implanted Lewislung tumor over a dose range of 12.5 to 200 mg/kg administeredi.p.(datanotpresented).

In further testing against P388 leukemia, no activity wasobserved when tumor cells were inoculated i.p. and drugwas given p.o., s.c., or iv. Also, no activity was seen whenP388 was inoculated iv. and drug was injected i.p. on i.v.(data not presented).

Cell-Kill Studies. The in vitro P388 cell-kill studies mevealed that relatively high chartmeusin concentrations mustbe maintained for long times in order to kill tumor cells.Chart 2 shows that 5 @gchartreusin per ml for 24 hr killed99% of the cells. However, 60% of the cells survived a 6-hrexposure at that same concentration. Similar results wereobtained in L1210 cell-kill kinetic studies and will be thesubject of a future report (B. K. Bhuyan and M. Robinson,unpublished results).

Assay for Chartreusln. The sensitivityof the microbiobogicab assay (10 @g/rnl)was adequate to measure drug con

centrations in bile and urine. However, plasma and tissuelevels were below this value, and it was necessary to extractrelatively large volumes of pooled plasma ontissue homogenates to amplify the sensitivity. Because of extensive bilianyexcretion, feces contained high levels of chartmeusin. However, extractions were necessary to eliminate interferingmaterials.

Experiments were conducted to determine the specificityofthe microbiological assay for chartmeusin. When relativelylarge volumes of bile or urine from chartreusin-tmeated matsor mice were extracted and applied to TLC plates, one bandin addition to chartreusin, which was not present in bile onurine from untreated animals, could be observed under 360nm light after development. The band traveled more slowlythan chartreusin under these conditions, indicating that itwas more polar. Bioautogmaphy indicated that the cornpound also inhibited the growth of S. Iutea , but whenchartneusin and the polar metabolite were ebuted from theTLC sombent and assayed, unchanged chartreusin accounted for virtually all of the total bioactivity present inthe original fluids. The quantity of the ebuted metabolitewas below assay sensitivity limits, suggesting minimalmetabolism of chartmeusin.

The microbiological method was very reproducible. Standard curves of log chartneusin concentration versus diameten of the zone of growth inhibition gave linear regressioncorrelation coefficients of 0.95 or greater for all tissues andfluids.

In Vitro Liver Metabolism. No decline in total bioactivitywas observed when chartreusin was incubated with the9000 x g supernatant from a mouse liver homogenate prepamation.

Bibiary Excretion. The most striking feature of the studiesof chartreusin disposition was the rapid and extensive bilianyelimination of unchangeddrug. Chart 3 showsthat, withdoses of S and 10 mg/kg i.v.,80 to 100% was eliminatedinthe bile oven6 hr. The previously mentioned studies of assayspecificity indicated that chartmeusin was not being significantly metabolized before bibiary excretion. When thesedata are plotted as bibiary excretion nate versus time (Chart4), a peak is noted shortly after dosing, followed by a biphasic decline with a terminal 1st-order half-life of about 1hr. The limited tissue distribution data discussed belowsuggest that the decline in biliary excretion mateparalleledthe rapid decline in drug concentrations in tissues.

Tissue Distribution. Following i.v. dosing, tissue concentrations rapidly declined to levels less than those necessaryto kill P388 cells. Chart 5 shows liver and small intestineconcentrations in bile duct-cannulated (bile-diverted) ratsafter a 10 mg/kg i.v. dose. Levels in all tissues except livenand small intestine were less than 1 @g/gby 1 hr afteradministration. Liven concentrations were initially high butdeclined to 1 @g/gby 4 hr. Because the rats used in thisexperiment were bile diverted, the measured chartreusinrepresented the actual tissue concentration rather thandrug excreted in bile. The levels in small intestine did notdecline in parallel with the other tissues. No chartreusin wasdetected in brain (assay sensitivity was approximately 0.5

@g/g).The rate of decline of chartneusin in tissues after iv.dosing roughly paralleled the decline in biliamy excretion




Effect of i.p.-administered chartreusinon the life-span of mice inoculated i.p. with P388leukemiaSurvival

timeWt(days)Dose

(mg/kg/i.p. inSchedulechangesjection)(Days)Vehicle(g)MedianRangeILS(%)Untreated

controlb2.19.48-132001-9SuspensioncToxic3.43â€”4Toxic100â€”2.716.0

10-217050â€”2.120.816â€”22121250.320.818-2212112.51.219.716-221096.30.616.315-19733.10.915.013-16591.60.514.0

13-1748Untreatedcontrol2.19.48-13501-9Solutionâ€•â€”1.521.8

12â€”2513125â€”0.320.314-2511512.5â€”0.119.3

18-251056.30.118.816-201003.10.315.012-16591.6â€”0.313.713-14450.80.812.39-14300.40.711.4

11-1221Untreatedcontrol1.79.31281,5,and9Suspensionc0.917.0

11â€”238264â€”0.416.313-237532â€”1.116.011-1772160.116.1

13-177380.514.011-1650

Effect of i.p.-administered chartreusin on the life-span of mice inoculated i.p.leukemia and 816 melanomawith

L1210Tumorwt

Survivaltime (days)Doseâ€•(mg/kg/i.p. in- changeâ€•

jection) (g) Mean RangeILS(%)L1210Untreated

controlc 0.7 9.580 â€”2.6 12.7 10-1740 â€”0.7 13.0 11-1620 0.1 12.8 12-1510 0.2 13.8 10-18

5 â€”0.5 11.0 10-122.5 0.3 11.7 10-161.25 0.6 9.3 9-1033

3735d

4616

23B16Untreated

control â€”0.8 20.5100 â€”2.9 38.0 7-54

50 â€”1.7 40.8 8-5525 â€”0.3 37.7 17-4512.5 â€”0.1 30.0 21-3685@

9983C46


Table 1

a Average weight change per mouse between Days 1 and 5.6 Untreated controls received injections of the respective vehicles.C Chartreusin was suspended in 0.9% NaCI solution: 0.3% hydroxypropylcellulose: 0.1% Tween

80 or 0.9%NaClsolution: 0.1%Tween80.d Chartreusin was dissolved in 0.01 N NaOH at 4 mg/mI, the pH was adjusted to 9.2 to 9.5, and the

solution was brought to the desiredvolume with 0.2 M Na2HPO4.

Table 2

a Doses were administered, Days 1 to 9, as suspensions in 0.9% NaCI solution: 0.1%

Tween80.b Average weight change per mouse between Days 1 and 5.

C Untreated controls received injections of the vehicle.

d One cure (survival to Day 60) was observed.

e Two curves (survival to Day 60) at 100 mg/kg and 1 cure at 25 mg/kg were observed.

matewith a 1st-order half-life of about 1 hr.Absorption Studies. Knowledge of the extent of biliary

excretion after i.v. dosing was used to determine the extentof p.o. absorption. Two bile duct-cannulated rats receivedSO mg/kg p.o. , and the amount of drug in the bile was

measured at intervals. Each rat excreted a total of 6% of thedose in the bile in 72 hr. An additional 0.1% appeared in theurine in each mat, indicating that the total extent of p.o.absorption was only 7 to 8%. The feces collected over 72 hrafter drug administration contained 53 and 19% of the dose,

1669JUNE 1977



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Chart 2. Survival of P388 cells after exposure to chartreusin. Followingdifferent periods of exposure at 2.5 or 5 pg/mI, aliquots of cells wereremoved and washed , and the cell survival was determined

@O

SO

U'

C.,

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3TIME (KR)

Chart 4. Rate of chartreusin excretion in bile of rats after 5 and 10 mg/kgiv. dosing. Points, mean Â±S.D. of data from 3 to 6 rats.

30

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4 5 6

SO

20

. LIVER0 SM. INTESTINE

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TIME (HOURS)Chart 5. Chartreusin concentrations in liver and small (SM.) intestine of

bile-diverted rats after 10 mg/kg i.v. dosing. Symbols, mean Â±S.D. of 2 to 5animals.

quantities of drug present, reflecting chartmeusin excretedin bile as well as drug actually in the tissue.

After i.p. administration to mats and mice, chartmeusinprecipitated extensively from solution and was slowly absorbed . In an attempt to quantitate the rate of absorption ofdrug from the penitoneabcavity, the appearance of chartreusin in the bile after i.p. administration (10 or SOmg/kg) wasmeasured in bile duct-cannulated matsfor 48 hr after dosing.The rats were dissected after 48 hr, and the penitoneal cavitywas examined for unabsombedchartreusin. No drug wasobserved inthe animalsthatreceived10 mg/kg, butdeposits of chartreusin were clearly visible adhering to the abdominab viscera in rats that received 50 mg/kg. The resultsare plotted (Chart 6) as the percentage of the administereddose appearing in the bile as a function of the time afteradministration. The chart also shows the percentage of a10-mg/kg i.v. dose appearing in the bile as a reference forthe situation in which absorption is considered to be instantaneous. Because biliamyelimination of chartmeusin was sorapid and extensive after i.v. administration, the mateof drugexcretion in bile after i.p. administration reflected the overall rate at which chartreusin dissolved and was absorbedfrom the penitoneab cavity into the general circulation.

+10.g/k@.+@ mIkg.iv.

1 2 3 4 5 S

TIME(HR)Chart 3. Cumulative percentage of 5 and 10 mg/kg iv. doses of chartreu

sin appearing in the bile of rats. Points, mean Â±S.D. of data from 3 to 6 rats.

indicating that much of the drug was still in the gastrointestinal tract. The volume of feces excreted by the animals waslow, indicating poor gastrointestinal function, probably as aresult of surgical trauma.

The extensive biliary excretion of chartmeusin suggestedthat enterohepatic circulation of drug would be possible.However, the very slow p.o. absorption suggested that theamount of drug involved in enterohepatic recycling wouldbe insignificant. This was verified by administering i.v.doses to matsand mice and measuring cumulative urinaryand fecal excretion. Urine contained 5% of the dose, and75% was recovered in the feces in 48 hr.

The s.c. administration of 225 mg/kg to mice resulted inextensive precipitation and very slow dissolution of drug atthe injection site. On the basis of observation of the size ofthe deposit of chartreusin remaining at the injection site,very little drug was absorbed up to 96 hr after administration. Tissue and plasma levels were measured at intervals to12 hr after dosing. The levels were below assay sensitivity(approximately 0.5 @g/g)in most tissues. In the tissues inwhich drug could be detected, the levels were similar ateach time point. Plasma levels were 1 to 2 @tg/ml,livercontained 20 to 40 @g/g,and kidney bevelswere 2 to 3 @g/g.Assays of segments of small intestine showed significant


44@

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oLIGATION + TUMOR + DRUG

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6 12 15 24 30 36 42 45TIME (KR)

Chart 6. Cumulative percentage of 10 and 50 mg/kg i.p. doses and 10 mg/kg i.v. dose appearing in bile of rats. Points, mean of 3 to 4 animals.

Both groups excreted chartreusin at a much slower matethan ratsdosed i.v.,indicatingthatredissolutionand absorption were rate controlling in the overall disposition.After14 hr,85% ofthe10mg/kg dose had beeneliminated,and bile concentrations were too low to be measured. Theabsorption process was considerably slower in the 50 mg/kg group, as evidenced by the lower extent excreted at eachtime. A similar phenomenon occurring in i.p.-dosed mice inchemotherapy studies could explain the excellent doseresponse relationship observed with i.p.-inoculated P388leukemia. The drug-cell contact time in the penitoneal cavitywould be disproportionately increased with increasingdose.

Chemotherapeutic Activity In Bile Duct-ligated Mice.When chartreusin was administered i.v. to i.v.-inoculatedP388 beukemicmice that had ligated bile ducts, therapeuticactivity was observed (Chart 7). The data are presented assurvival curves for 4 treatment groups: (a) leukernic, vehicle-treated; (b) beukernic, chartreusin-treated; (c) ligated,leukemic, vehicle-treated; and (d) ligated, leukemic, chartreusin-treated. Almost one-half of the ligated, leukemic,chartreusin-treated mice died by Day 5, an indication oftoxicity resulting from chartreusin, since the mice dosedonly with the vehicle survived past Day 4. After Day 5, thesurvival curve leveled off, and by Day 13, although all of theligated control mice were dead, 36% of the treated animalsremained alive. The remaining mice died between 13 and 19days, with the exception of 2 mice which survived to Day 30.Normal (nonligated) P388 leukemic mice died between Days8 and 10, and i.v. chartreusin treatment did not increasetheirlife-span.

DISCUSSION

Based on the impressive activity displayed by chartreusinin 3 experimental mouse tumor systems, extensive expenimental therapeutic evaluation , biochemical charactenization, and drug disposition studies were undertaken. Biochemical studies showed that chartreusin is an effectivecytotoxic agent and suggested its mechanism of action (8).However, the therapeutic studies revealed an extreme dependence of activity on the route of drug administration.Chartreusin increased the life-span of P388 beukemic mice

2 4 6 8 10 12 14 16 iS 20 30DAYSAFTERINOCULATION

Chart 7. Effect of bile duct ligation on the survival of v-inoculated leukemic mice dosed iv. with chartreusin. Mice were inoculated with 10' P388cells iv. Treated mice were dosed i.v. on Days 1 to 4 with chartreusin, 25 mgIkg or vehicle.

only when tumor cells were inoculated i.p. and drug wasadministered i.p. Studies of chartreusin disposition andcell-kill kinetics have explained this behavior.

Killing of P388 cells in vitro by chartneusin requires a longduration of exposure and/or a high exposure concentration. In therapy studies, chartreusin was quite effectivewhen both tumor cells and drug were given i.p. because ofextensive precipitation and slow dissolution of drug in thepemitoneabcavity. The tumor cells remaining in the cavitywere exposed for relatively prolonged periods to the highlocal concentration of chartreusin. An observation thattends to support this interpretation is that moribund micethat had been inoculated i.p. with P388 cells and dosed i.p.on Days 1 to 9 with chartneusin had very little withdrawableascites (

J. P. McGovren et al.

4. Geran, R. I., Greenberg, N. H., Macdonald, M. M., Schumacher, A. M.,and Abbot, B. J. Protocols for Screening Chemical Agents and NaturalProducts against Animal Tumors and Other Biological Systems. CancerChemotherapy Rept., 3: (Part 3) 1-103, 1972.

5. Himmelfarb, P., Thayer, P. 5., and Martin, H. Growth of Colonies ofMurine Leukemia L1210 in Vitro. Cancer Chemotherapy Rept., 51: 451-454, 1967.

6. Ho, D. H. W., and Freireich, E. J Clinical Pharmacology of Arabinosylcytosine. In: A. C. Sartorelli and D. G. Johns (eds.), Antineoplastic andImmunosuppressive Agents, Part 2, pp. 257-271 . New York: SpringerVerlag, 1975.

7. Leach, B. E., Calhoun, K. M., Johnson, L. E., Teeters, C. M., andJackson, W. G. Chartreusin, a New Antibiotic Produced by Streptomyceschartreusis, a New Species. J. Am. Chem. Soc., 75: 4011-4012, 1953.

8. Li, L. H., and Clark, T. A. Cytotoxicity and Biochemical Effects of Chartreusin (NSC-5159) on Mouse Leukemia. Proc. Am. Assoc. Cancer Res.,17:96, 1976.

9. Lubawy, W. C., Kostenbauder, H. B., and Stavchansky, S. A. CrossInhibition of Drug Metabolism by Drug Metabolites; Increase of Zoxazolamine Paralysis Time by the Major Metabolite of Diphenylhydantoin.Res. Commun. Chem. Pathol. Pharmacol., 8: 75-82, 1974.

10. Ovejera, A. A., Johnson, R. K., and Goldin, A. Growth Characteristicsand Chemotherapeutic Response of Intravenously Implanted Lewis LungCarcinoma. Cancer Chemotherapy Rept., 5: (Part 2) 111-125, 1975.

11. Paul, M. A., and Dave, C. A Simple Method for Long-Term Drug Infusionin Mice: Evaluation of Guanazole as a Model. Proc. Soc. Exptl. Biol.Med., 148: 118-122, 1975.

12. Simonitsch, E., Eisenhuth, W., Stamm, 0. A., and Schmid, H. TheStructure of Chartreusin. I. HeIv. Chim. Acta, 47: 1459â€”1475,1964.

We are currently attempting to conduct such studies intumorous mice (ii). If this effort is successful, it wouldprovide impetus for further development of chartmeusin.

ACKNOWLEDGMENTS

The authors gratefully acknowledge Dr. L. J. Hanka for his assistance indeveloping microbial assays; C. L. Blowers, T. F. DeKoning, and R. Garza fortheir excellent assistance in conducting P388 chemotherapy studies; theSouthern Research Institute, Birmingham, Ala., for performing B16 melanosarcoma studies; the Mason Research Institute, Worcester, Mass. , for conducting L1210 leukemia studies; and Kay Franz for her patient assistanceduring the preparation of this manuscript.

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1977;37:1666-1672. Cancer Res J. Patrick McGovren, Gary L. Neil, Sheri L. Crampton, et al. Chartreusin (NSC 5159)Antitumor Activity and Preliminary Drug Disposition Studies on

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