from fermentation products of Streptomyces sandaensis No.6897 in our research laboratories (13, 14). It has a novelchemical structure: hydoxylamine function whose hydoxy groupconstructs a hemiketal moiety in its intramolecular structure.FR900482 was found to show antitumor activity more potentthan or equivalent to MMC against transplantable experimentaltumors in mice (14, 15), and its hematotoxic and myelosuppressive effects were weaker than those of MMC in mice (16).Especially, the compound only slightly decreased peripheralplatelets in mice (16). These characteristics led us to search forderivatives with stronger antitumor activity than the parentcompound and with still weaker side effects, and we havechemically synthesized and tested many derivatives ofFR900482 for cytotoxicity against tumor cells in vitro. Fromthese, we selected a triacetyl derivative, FK973, for furtherstudies on in vivo and in vitro antitumor activity.
FK973 was shown in this study to have more potent antitumor activities than MMC against various murine tumors implanted in mice and against human tumors xenografted in nudemice. The new derivative showed myelosuppressive effect inmice, but was also less myelosuppressive than MMC in mice.FK973 is therefore considered to be the first candidate amongthe dihydrobenzoxazine derivatives for clinical use.
MATERIALS AND METHODS
Drags
FK973 was prepared in our research laboratories. The chemicalstructures of FK973 (previously called FR66973), and FR900482, theoriginal lead in our series, are shown in Fig. 1. MMC and AtJ)R werepurchased from Kyowa Hakko Kogyo Co. Ltd., Tokyo, Japan, CDDPfrom Sigma, Co. Ltd., St. Louis, MO, and CPM and VCR fromShionogi & Co. Ltd., Osaka, Japan. In in vivo antituinor test, FK973,was dissolved in and diluted with 10% polyoxyethylated (60 mol)hydrogenated caster oil in saline. The other drugs were dissolved in ordiluted with saline. The solutions were given i.p. or i.v. in a volume of10mI/kg bodyweight. Saline or 10%polyoxyethylated(60 mol) hydrogenated castor oil in saline solution was given to the control animals.In the in vitro culture test and hematotoxicity test, all the drugs weredissolved in or diluted with culture medium supplementedwith 10%FBS (HyClone Laboratories, Logan, UT).
In Vitro Tests
P388 cells were maintained and treated in suspension in RPMI 1640medium (Nissui Seiyaku Co., Ltd. Tokyo, Japan) supplemented with10%FBS, penicillin (50 units/ml)-streptomycin (50 @igJml)(Flow Laboratories, North Ryde, Australia), and 2-mercaptoethanol(5 x iO@M).L1210 cells were maintained and treated in suspensionin RPMI 1640medium supplemented with 10% FBS and penicillin (50 units/mI)-streptomycin (50 @aJml).B16 and Colon 38 cells were maintained andtreated in adherence in RPMI 1640 medium supplemented with 10%FBS and penicillin (50 units/ml)-streptomycin (50 g&g/ml).LLC cellswere maintained and treated in adherence in Dulbecco's modified EagleMedium (Nissui Seiyaku Co., Ltd., Tokyo, Japan) supplemented with10% FBS, penicillin (50 units/nil)-streptomycin (50 @sgJm1),and 2-mercaptoethanol (5 x iO@M).CCRF-CEM cells were maintained andtreated in suspension in RPMI 1640 medium supplemented with 10%
ABSTRACT
FK973, a new, substituted dihydrobenzoxazine (l1-acetyl-8-carbsmo
deca-2,4,6-trien-6,9-diyl diacetate), was obtained by chemical modifieslion of a novel antibiotic which was belated from the fermentationproducts of Strepton,ycessaadaeasisNo. 6897. FK973 had cytotoxiceffects against in Pure cultured human and marine tumor cells. FK973in doses of 0.032-5.6 mgJkg(i.p.) had stronger antitumor activities andhigher chemotberapeutic ratio than mytomycin C against such marineascitic tumors as P388 and L1210 leukemia, B16 melanoma, M5076reticulum cell sarcomaof ovarian origin, Colon 26 carcinoma, Ehrlichcarcinoma, and M11134 bepatonsa. In tests against marine and humansolid tumors Implanted s.c. in normal mice and nude mice, respectively,FK973 (I.v.) inhibited growth of marine tumors (M5076 sarcoma, Colon38 carcinoma,B16 melanoma,and Lewis lu.g carcinoma) by 66-100%sad human tumors (LX-1 lung, MX-1 mammary, and SC-6 stomachcaxc1no@ by 84-99%. In studies with drug-resistant P388 leukemia,FK973 was also effectiveagainst vincristlne-resistantP388, moderatelyeffectiveagainst mltomycinC (MMC)- and adriasnydn-reslstant P388,and partially effective against cyclopbosphamide-resistant P388 cells inmice. Leukopenic effects of FK973 and MMC in mice were comparableat doses whichgave antitumoractivityalmost equally.FK973 had noeffect on the numbers of platelets and red blood cells, whereas MMCmarkedlydecreased both. FK973decreasedthe numbersofcolony formlug units in spleen and in culture and the effect was less than that ofMMC. Therefore, FK973 may give weaker myelosuppression thanMMC. The results suggest that FK973 will be a beneficial drug for thetreatmentof cancer.
INTRODUCFION
Antitumor chemotherapeutic drugs such as MMC2 and ADRhave potent antitumor activities in animals and humans (1—5).The drugs have been widely used in the treatment of variousneoplastic diseases (1—3).However, one of their major sideeffects is myelosuppression expressed as leukopenia or thrombopenia (1—3,6—8),and this limits their clinical usefulness (1—3). Efforts have been made to develop compounds with strongerantitumor activity and weaker myelosuppression (9—12).
FR900482, a substituted dihydrobenzoxazine, was isolated
Received7/8/87; revised 11/12/87; accepted12/2/87.The costsof publicationof this articleweredefrayedin part by the payment
of pagecharges.Thisarticlemustthereforebe herebymarkedadvertisementinaccordancewith 18U.S.C.Section1734solelyto indicatethis fact.
a To whom requests for reprints should be addressed.2 ‘p,@ abbreviations used are: MMC, mitomycin C; P1(973, ll-acetyl-8-
Evaluation of AntitUmOr Effects against Solid Tumors. In the experiments on th@irinesolid tumors, M5076 cells (1 x l0@)were implanteds.c. in B@C3F1mice. Fragments (2 x 2 x 2 mm) of Colon 38, B16 cells(5 x l0@),and LLC cells (1 x 106) were implanted s.c. in BDF1 mice.All the tumors were implanted into the left flank of mice. In all theexperiments, 20 and 10 mice were used in the control and drug treatedgroup, respectively. Tumor weight, as derived from caliper measurements ofthe length and width oftumors, was calculated by the formula:Tumor weight (mg) = 1/2X a X b2,where a represents the length and brepresents the width (mm).
Drug efficacyagainst murine solid tumors was based on the percentage ofmean tumor weight ofthe treated group (1') to that ofthe controlgroup (C).
Growth inhibition (%)
— (1 — Mean tumor weight T\@ 100
“Meantumorweight,C)
H@OCO O1,OCONH,
FK973 FR900482
Fig. 1. Structures of P1(973and FR900482.
FBS and penicillin (50 units/ml)-streptomycin (50 @sg/ml).PC1O andMKN 45 cells were maintained and treated in adherence in RPMI 1640medium supplementedwith 10% FBS and penicillin (50 units/mI)-streptomycin (50 @g/ml).The cells were grown in 5% CO2-95% airatmosphere at 3TC.
For the assessment of cytotoxicity, twofold serial dilutions of drugwere prepared in 96-well .iicroplates (Falcon 3072; Becton DickinsonLabware, Oxnard, CA) containing 100 Mlof full medium. P388 andL1210 cells (2 x 10@),B16, LLC, Colon 38, and PC1Ocells (1 x l0@'),and CCRF-CEM and MKN45 cells (4 x 10k)in 100 Mlofinedium werethen added and incubated in the microplates. The cells were incubatedfor 48 h. Viability ofP388, Ll210, and CCRF-CEM cells was assessedmicroscopically with trypan blue penetration as an indicator of celldeath, and the percentage of survival of the cells was calculated. Forthe adherent cells, the medium was aspirated, and the plates were fixedand stained with 0.5% crystal violet in 20% methanol. The dye wasextracted with 100 @slof glycine hydrochloride buffer solution 10.1Mglycine hydrochloride buffer (pH 3.0):ethanol, 7:3J. Cell lysis wasmeasured with a Titertek MUItiSkan autoreader (Flow Laboratories,McLean, VA) set for absorption at 540 nm. The percentage of cytotoxicity was calculated by the formula:
Cytotoxicity(%)
= (1 —mean A (drug-treated)/mean A (nontreated)) x 100
(C)
In the experiments on human solid tumors, fragments (2 x 2 x 2mm) of LX-l, MX-l, and SC-6-JCK were implanted s.c. in BALB/cnu/nu mice.All the tumorswereimplantedinto the left flank of mice.Four or five mice were used per group. The initial and fmal tumorweights were calculated on the first injection day (just before injection)and the last evaluation day, respectively. The chan@esin mean tumorweightare shown for each group ofmice. Change in mean tumor weight= mean tumor weight (final)/mean tumor weight (initial).
Drug efficacy was expressed as the percentage of mean tumor weightof the treated group (T) to that of the control group (C).
Growth inhibition (%)
- (@ - Change in mean tumor weight T\@ 100
—“ Change in mean tumor weight, C)
(D)
Activity Criteria. The criteria for activity in in vivo tumor modelswere estimated according to a modification of the method used by theNational Cancer Institute (4, 9). We used two criteria described in eachtable, and definedthe small and largevalues in T/C or l-T/C (%)asmoderate and good activities, respectively.
Hematotoxicity
BloodCell Counts. The drugs were given i.v.4o mice. The peripheralblood was obtained from the orbital vein. WBC, RBC, and PLT werecounted automatically with a blood automatic i*alyzer (TechniconHemalog 8/90 model; Technicon Instrument Co., Ltd., Tarray Town,NY)
PreparationofBMC. The mice weresacrificedby cervical dislocation.Both ends of the femurs were cut out aseptically. The BMC were thenflushed out with a syringe with a 26-gauge needle into Hank? solutionand pooled from five mice. A portion of the pooled BMC was stainedwith 0.1% Turke solution, and the viable nucleated cells were countedmicroscopically in a hematocytometer.
Measurement of CPU-s. CFU-s were assayed according to a modification of the method of Till and McCulloch (17). Briefly, BMC (1 xi0@cells) were given i.v. into recipient mice iimdiated (750R) with aX-ray irradiator (Hitachi X-ray Irradiator MB@-1505R Hitachi Mcdico Co., Ltd., Tokyo, Japan) at a rate of 30 rads/inin. On day 7, thespleen of the recipient mice were removed and fixed with Bouin'ssolution, and the spleen colonies were counted under a dissectionmicroscope.
Measurement of CFU-c. CFU-c were measured according to a modification of the method of Pike et a!. (18). Briefly, 5 x 10@BMC in oneml of a-minimum essential medium (Flow Laboratories, McLean, VA)supplemented with 0.7% methylcellulose, 16% horse serum (FlowLaboratories, McLean, VA), and penicillin (40 units/ml)-streptomycin(40 gigJml) were plated in 35-mm diameter culture dishes (CorningGlass Works, Corning, NY). Sixteen % of L-cell-conditioned mediumwas used as a source of colony-stimulating factor. Triplicate cultures
(A)
where mean A represents averageabsorbance. Triplicate wellswereusedin the experiments.
In Vi,'o Tests
4&nim*lS. Female mice ofBDF1 (C57BL/6 x DBA/2), CDF, (BALB/C X DBA/2), B@CF1 (C57BL/6 x C3H), DBA/2, C57BL/6, C3H/HeN
and ICR strains were purchased from Charles River Japan Inc., Atsugi,Japan. Male mice ofBALB/c nu/nu strain were purchased from CLEAJapan Inc., Tokyo, Japan.
Tumors. P388 and L1210 were maintained i.p. by serial passage inDBA/2 mice. B16, LLC, and Colon 38 were maintained s.c. by serialpassage in C57BL/6 mice. M5076 was maintained i.p. by serial passagein C57BL/6 mice. Colon 26 was maintained s.c. by serial passage inBALB/c mice. MH134 was maintained i.p. by serial passage in C3H/HeN mice. Ehrlich was maintained i.p. by serial passage in ICR mice.P388/MMC, P388/CPM, P388/VCR, and P388/ADR were mainmIned i.p. by serial passage in CDFI mice. LX-1, MX-1, and SC-6-JCK were maintained s.c. by serial passagein BALB/c nu/nu mice.
EvaluationofAntitumor Effectsagainst Murine ASCItiCTumors. P388cells (1 x 10'), L1210 cells (1 x I0@),Colon 26 bre (1:100 brei/0.5ml), and P388/MMC, P388/CPM, P388/VCR, and P388/ADR cells(1 x 10') were inoculated i.p. in CDF, mice. B16 brei (1:10 brei/0.5ml) was inoculated i.p. in BDF1 mice. Ehrlich cells (1 x 10') andMH134 cells (1 x 10') were inoculated i.p. in ICR and C3H/HeN mice,respectively. In the tests with P388, P388/MMC, P388/CPM, P388/VCR, and P388/ADR, 12 and six mice were used in the control anddrug treated groups, respectively. In other tumor tests, 20 and 10 micewere used in the control and drug treated groups, respectively.
Drug efficacy against the ascitic tumors was assessed as a percentageof MST of treated group (7) to that of the control group (C).
were made for each cell suspension, and incubated at 3TC in 5% CO2in humidified air. The colonies were grown for 7 daysand counted.
RESULTS
Cytotoxicity against in Vitro Cultured Tumor Cells. The cytotoxic effects ofFK973 were examined against in vitro culturedmurine and human tumor cells. The results are shown in Table1. FK973 showed strong cytotoxicity against the murine andhuman tumor cell lines tested. In general, the cytotoxicity ofFK973 was stronger than that of MMC and CDDP, and wasweaker than that of ADR.
Antitumor Effects on Murine Ascitic Tumors in Mice. Theantitumor effects ofFK973 against seven kinds ofmouse ascitictumors were examined and compared with those of antitumordrugs such as MMC, ADR, and CDDP. The tumors wereinoculated i.p. to mice on Day 0. As shown in Table 2, thedrugs were given i.p. to mice once a day for 5 days (Days 1—5)in the test on P388, and once a day for 9 days (Days 1—9)inthe tests on L1210 and B16. FK973, MMC, ADR, and CDDPdose-dependently prolonged the life of mice bearing P388: thehighest MST ratio (%) were @243for FK973, 200 for MMC,
@255for ADR, and @222for CDDP. FK973 in doses of 0.01to 5.6 mg/kg showed positive activity, that is, MST ratios (%)of 120 or higher. MMC, ADR, and CDDP were active in dosesof0.1 to 1.8 mg/kg, 0.1 to 3.2 mg/kg, and 1.0 to 3.2 mg/kg,respectively. The effective dose range of FK973 was thereforethe widest of the four drugs. Against Ll2lO tumor, FK973,MMC, ADR, and CDDP also showed antitumor effects inmice. FK973 had the MST ratio (%) of 163 and had positiveactivity over the widest dose range (0.1—5.6mg/kg). AgainstB16, all four drugs were again effective, and FK973 and ADRhad almost the same maximum MST ratios of>375 and >353,respectively, in comparison with 176 for MMC and 185 forCDDP. The effective dose range of FK973 (0.032—5.6mg/kg)was again the widest of the four drugs.
In the experiments shown in Table 3, the drugs were giveni.p. to mice once a day for several days. FK973 was activeagainst the four kinds of tumors tested, and its antitumor effectwas stronger than that of MMC against M5076, Ehrlich, andMH134. The drug showed activity comparable to MMC againstColon26.Theeffectivedoserangesof FK973werewiderthanthose of MMC against all the tumors tested.
Antitumor Effects on Murine Solid Tumors in Mice. To compare the antitumor effects of the drugs against solid tumors,M5076, Colon 38, B16 and LLC were implanted s.c. to miceon Day 0. The drugs were given i.v. to mice once a day on Days1, 4, 7, and 10, and the tumor weights were measured on Day17, 18, or 21. The results are shown in Table 4. FK973, MMC,ADR, and CDDP inhibited tumor growth by more than 58%in the experiment on M5076. Against Colon 38, FK973, MMC,and ADR markedly inhibited growth, but CDDP had no effect.In the tests on B16, the tumor weights were markedly decreasedby treatment with FK973 in doses of 3.2 and 10 mg/kg. MMC
in a dose of 3.2 mg/kg, ADR in doses of 3.2 and 5.6 mg/kg,and CDDP in a dose of 5.6 mg/kg were also active against thetumor. In the experiment on LLC, the four drugs showedmoderate antitumor activity.
Antitumor Effects on Human Xenograft Tumors in Mice. Theexperiments were performed to evaluate the antitumor effectsofFK973 against three kinds ofhuman tumors, LX-l, MX-l,and SC-6, implanted s.c. in BALB/c nu/nu mice. Tumorweights were measured about 21 days after tumor implantation.The results are shown in Table 5. Against LX-1, FK973 indoses of 5.6 and 10 mg/kg, MMC in a dose of 3.2 mg/kg, andCDDP in a dose of 10 mg/kg inhibited tumor growth by morethan 80%, whereas ADR had no effect. In the experiment onMX-1, FK973, MMC, and CDDP completely inhibited tumorgrowth in a wide range of doses, but ADR was effective only ata dose of 10 mg/kg, which seems to be the maximum tolerabledose. In the test on SC-6, FK973 showed an antitumor effect,but the other drugs did not.
Antitumor Effects on Drug-resistant P388 in Mice. Table 6shows the antitumor effects ofFK973 against tumor cells crossresistant to other chemotherapeutic drugs in mice. FK973 wasactive against P388 tumor cells which were either sensitive orresistant to MMC, whereas MMC was not active against MMCresistant P388. The life of mice bearing CPM-resistant P388was prolonged only by the highest doses of FK973 and MMC,and CPM was not effective at any dose against CPM-resistantP388. FK973 in a dose of 10 mg/kg was equally active againstCPM-resistant and nonresistant P388 cells. MMC in a dose of5.6 mg/kg was less active against CPM-resistant P388 thanagainst the nonresistant cells. FK973 and MMC were alsoeffective in vincristine (VCR) and ADR-resistant models.
Hematotoxic Effects in Mice. The hematotoxicity of equivalently effective i.v. doses of FK973 and MMC was compared inmice. When the drugs were given once, FK973 in i.v. doses upto 10 mg/kg scarcely affected the PLT count, whereas MMCin a dose of 5.6 mg/kg caused a decrease on day 7 (Fig. 24).FK973 and MMC markedly decreased the number of WBC,with the lowest count obtained on day 3; the count returned tonormal on day 7 in the animals dosed with 1.0 mg/kg of FK973and on day 14 in those dosed with 3.2 mg/kg of FK973 or 1.0mg/kg of MMC (Fig. 2B). FK973 had no effect on the numberof RBC, but MMC caused a slight reduction (Fig. 2C).
When FK973 in doses up to 10 mg/kg or MMC in doses upto 3.2 mg/kg was given three times to mice (Days 0, 3, and 6),FK973 had no effects on the number of PLT at any dose, butMMC in doses of 1.0 and 3.2 mg/kg markedly decreased thenumberof PLT, withthelowestcountobtainedonday13,andthe PLT count did not return to the control level in animalstreated with 3.2 mg/kg ofMMC (Fig. 3A). Both the compoundssimilarly decreased the number of WBC (Fig. 3B). The numberof RBC was decreased slightly on day 9 after injections ofFK973 in a dose of 10 mg/kg or MMC in a dose of 1.0 mg/kg,and markedly on dayS 13 and 20 after injections of MMC in adose of 3.2 mg/kg (Fig. 3C).
TableI C@toxicityofantitumordrugsonvariouscelllinesin vitroCellswereincubatedwith drugsat 37'C for 48h in 5%CO@-95%air incubator.
Table 2 Antitwnoreffects ofFK973 and the referencedrugs on marine ascitictwnovsTumorcells were inoculated i.p. to mice on Day 0. The drugs were given ip. to mice once a day for 5 days (Days 1-5) in the P388 test, and for 9 days (1)ays 1-9)
in the L1210andB16tests.MSTwasmeasured.Micewereusedin groupsofsix (12in thecontrol)in the P388test,andin groupsof 10(20in thecontrol)intheL1210andB16tests.Criterin+, @120and++, i75forp388,and+, @l25and++, i50forL1210andB16.P388
Table 3 Antitwnor effectsofFK973 and the referencedrugs on marine ascitictumorsTumorcells were inoculated i.p. to mice on Day 0. The drugs were given i.p. to mice once a day on Days 1, 5, 9, and 13 in the M5076 test, on Days 1 and 5 intheColon
26 test, and Days 1, 4, and 7 in the Ehrlich and MH134 tests. MST was measured.Micewere used in groups of 10 (20 in the control). Criterin +, l25and++,150 for M5076 and MH134, +, 130 and ++, @I50 for Colon 26, and +, @l20 and ++, @175forEhrlich.M5076
aNumbersin parentheses,numberofsurvivorsonDay30in P388andL1210tests,andonDay60 in B16test.C To; a T/Cvalue of@86% indicates toxicity.
a Q4D, every 4 daysQ3D, every 3 days.bNumbersin parentheses,numberofaurvivorsonDay60 in theM5076,Colon26andEhrlichtests,andon Day67 in theMH134test.C To; a T/Cvalue of@86% indicates toxicity.
Effects on CFU-s and CFU-c. Fig. 4 shows the time course of on Day 7, and the recovery was faster than that in the MMCpercentage of change in the CFU-s and CFU-c of BMC after treated animals.single i.v. injection ofFK973 or MMC. FK973 and MMC dosedependently decreased the numbers ofCFU-s and CFU-c, with DISCUSSIONthe maximum decrease on the next day of injection. The effect
ofFK973 was weaker than that ofMMC, and at 1.0 mg/kg the In the present study, FK973 was cytotoxic against in vitrocompound scarcely affected CFU-s and CFU-c counts. More- cultured tumor cells, and was active against marine asciticover, the numbers of CFU-s and CFU-c in the mice treated tumors, and murmne and human solid tumors in mice. MMC,with 10 mg/kg ofFK973 gradually returned to the control level ADR, and CDDP widely used with good therapeutic efficacy
Table 4 Antitumoreffecty ofFK973 and the referencedrugs on marine solidtumorsTumorcellswereimplanteds.c.to miceon Day 0. The drugsweregiveni.v. to miceoncea dayon Days 1, 4, 7, and 10.Tumor weightsweremeasuredon Day 21
in the M5076 andColon 38 tests,on Day 18 in the B16 test,andon Day 17 in the LLC test.Mean tumorweightswerecalculated.Mice wereusedin groupsof 10.Criteria:+, @58;++, @90.M5076
Table S Antitumoreffects ofFK973 and the referencedrugs on human tumorxenogn@ftedin nudemiceTumorcells were implanted s.c@to mice on Day 0. The drugs were given i.v. to mice three times at 4-day intervals beginning around Day 10. Tumor weightwasmeasuredon around Day 21. Mice were used in groups offour or five. Ctheria +, @804*,@90.LX-1
(s.c.-Lv.) MX-l (s.c.-i.v.) SC-6(s.c.-i.v.)DoseQ4Da, 3 times Q4D, 3 times Q4D, 3 times
Drug (mg/kg) l-T/C(%) Activity l-T/C(%) Activity 1-T/C(%)ActivityFK973
3 days.aTox,asurvivalrateof<65% on theevaluationdayindicatestoxicity.
a Q4D, every 4 days.bFK973 was dissolved in and diluted with salinefordosesup to 10 mg/kg, and in 10% HCO6Ofora doseofl8 mg/kg.C Tox, survival rate of<65% on the evaluation day indicates toxicity.
Fig. 2. Effects ofa single injection ofFK973 or MMC on PLT(A), WBC (B),andRBC(C) countsin theperipheralblood.Thedrugsweregiveni.v. to miceon Day 0. The counts were taken at various times after treatment. The results areexpressedaspercentageof controLMice wereusedin groupsof five for eachexperiment0, FK9731 maJkg @,FK9733.2maJkg0, FK97310mg/kg•,MMC 1 mg/kg; A, MMC 3.2 maJkg , MMC 5.6 mgJkg bars, SE.
FK973 MMC
r79 13 20
AFK973 MMC
D•yssft•, njsctèon
B@ MMC1401
Days sit., nI.ctêon
Fig. 4. Effects ofFK973 or MMC on the number ofCFU-s (A) and CFU.c(B) of bone marrow cells per femur. The drug was given @v.to mice on Day 0.The numbersofCFU-s and CFU-c ofbone marrow cells per femur wereexaminedat varioustimesafter treatment.The resultsare expressedas percentageofcontroLMicewereusedin groupsof fivefor eachexperiment.0, FK9731maJkg; t@,FX973 3.2 maJkg 0, FK973 10 maJkg •,MMC I mg/kg A, MMC 3.2mg/kg U, MMC 5.6 mg/kg.
also showed promising antitumor activities against murine andhuman tumors. Murine and human tumors such as P388,L1210, B16, LX-l, and MX-1, which were used in this study,seem to give positive correlation between animal and clinicaleffects of chemotherapeutic agents (4, 5, 19, 20). The fact thatFK973 was more active than or similarly active to MMC, ADR,and CDDP shows that FK973 might be a useful antitumorcompound with a relatively broad spectrum in humans.
The effectivedoserangesof FK973 againstmurinetumorswere much wider than those of MMC and CDDP and waswider than or equivalent to that of ADR. The compoundtherefore might be relatively safer than MMC, ADR, or CDDP.
FK973 was active against MMC-, VCR-, and ADR-resistantP388, but only partially effective against CPM-resistant P388.We have no explanation for this activity and can only speculatethat FK973 has a different mode of action and a differentantitumor spectrum from MMC, VCR, and ADR.
Most of the antitumor chemotherapeutic drugs induce hematotoxicity in humans (1—3,6—8).FK973 and MMC decreased the number of peripheral WBC in mice almost equally,but their effects on the number of PLT differed, with nodecrease noted in the animals treated with FK973. We havealready shown that FR900482, the parent compound, had noeffect on PLT count in mice (16), so we postulate here that theFR900482 derivatives might all behave similarly in regard toPLT. We also studied the effects of FK973 on hematopoieticstem cells and granulocytic-macrophagic precursors by messuring the numbers of CFU-s and CFU-c, respectively, of BMCin mice to assess its myelosuppressive activity, and found thatthe compound is less myelosuppressive than MMC. One of themain limiting factors of antitumor chemotherapeutics is hematotoxicity, and leukopenia and thrombocytopenia are themost frequent and serious side effects in cancer chemotherapy(1—3,6—8).We believe that FK973 might not cause thrombocytopenia, although leukopenia would arise in clinical treatment, and that the weaker hematotoxicity of FK973 might bean important advantage in clinical application.
1171
i 100
I,.@ so
U
20
@1 3 7 14 01
Days afterinjection
0
C0U
@ 50
0@-@07913 20
B
0
C0U0
0
C0U
0
Days after the first inject ion
Fig. 3. Effectsofthree doses of FK973 or MMC on PLT (A), WBC (B), andRBC (C) counts in the peripheral blood. The drugs were given Lv. to mice onDays 0, 3, and 6. The counts were taken at various times after treatment. Theresultsareexpressedaspercentageof controLMicewereusedin groupsof fiveforeachexperiment.O,FK9731mg/kgt@.,FK9733.2maJkgD, FK973lOmg/kg; •,MMC 0.32 mg/kg A, MMC 1 mg/kg , MMC 3.2 mg/kg.
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FK973.A NEW ANTITUMOR AGENT
Against in vitro cultured tumor cells, FK973 was more cytotoxic than the parent compound (14). In in vivo tests, FK973was more active than or similar to FR900482 in activity againstmurine tumors implanted in mice (15). The effective dose rangeofFK973 against murine ascitic tumors, especially B16, in miceseems to be wider than that ofFR900482 (15). The hematotoxicand myelosuppressive effects of FK973 were similar to those ofFR900482 in mice (16). FK973 is a triacetylated derivative ofFR900482, and the latter was not developed owing to stabilityproblems (14). We expect that FK973 would be better absorbedin the cells than FR900482 because the compound is morelipophilic than its parent. This lipophilic nature of FK973should allow a wider therapeutic ratio in antitumor activitiesthan that ofFR900482. The hematotoxic and myelosuppressiveeffects of FK973 were, however, almost the same as those ofFR900482, and we suggest that the sensitivities between tumorcells, and BMC and blood cells to FK973 differ partially. Insummary, our fmdings show that FK973 would be a uniqueantitumor agent with promising antitumor activity, some broadspectrum of activity, some wide therapeutic range, less crossresistance to other drugs and less hematotoxicity and myelosuppression.
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