..
`
`ANTICANCER REsBARCH 18: 3235-3240(1998)
`
`XP-008005757
`
`012
`
`)S9t9
`
`Role of Folic Acid in Modulating the Toxicity and Efficacy of the
`Multitargeted Antifolate, LY231514
`JOHN F. WORZALIA, CHUAN SHlH and RICHARD M SCHULTZ
`
`Cancer Rese4rch Division, Lilly Reseorr:h Laboratories, Eli Lilly and Co., Indianapolis, IN 46285, U.S.A.
`
`)
`
`Abstl'act. We studied the e/ftcts of folic acid on modulating lhe
`toxicity and antitumor efficacy of LY231514. Using several
`human tumor cell lines adapted to growlh in low folate medium,
`folic acid as shown to be 100. to 1000-fold less active than
`·,. olinic acid at proteCting cells from LY231514-induced
`cytotoxicity. The lethality of LY231S14 wa.r eompared in mice
`maintained on standard diet or lo..!!' folate dig. The LDSO
`occumd at~ and 250./old lowsr doles of L¥231514 in DBA/2
`and CD lnu/nu mice, respectively, maintained on low folate diet
`compared to sl4ndard diet. The LS178YJTK-IHX- murine
`lymphoma wn.t much more sensitive to the antitumor action of
`L¥231514 compared to wild type L5178Y-S tumors. For mice
`on low folate diet, LY231514 at 0.3 and 1 mg)kg (qd x 10, i.p.)
`produced 100% inhibition of L5178Yfl'K·IHX· lymphoma
`growth, and significant lethality occurred at c: 3 mg/kg. For mice
`on standard diet, LY231514 produced >95% inhibition of
`tumor growth at 30 to .100 m:/kg, but aU mice died at 800 mglkc.
`Folic acid .wpplementalion was denumstrated to preserve the
`antitumor activity of LY231514 while reducing toxicily. The
`LY231514 may provide a
`cambmation of folic acid with
`meclwnism for enhanced cJinlcal antitumor selectivity.
`
`)
`
`LY231514 is a structurally novel antifolate antlmetabolite that
`possesses
`the unique 6·S-fused pyrrolo[2,3~]pyrimidine
`nucleus (1) instead of the more common 6-6-fused pteridine
`or quinazoline ring structure. The primary mode of antitumor
`activity for LY231514 has previously been ascribed to
`inhibition of thymidylate synthase (TS) (1, 2). However,
`several lines of evidence suggest that multiple enzyme(cid:173)
`inhibitory mechanisms are involved in cytotoxicity, hence the
`acronym MTA multit
`ted antifolate: 1 th
`pattern or A in human leukemia and colon carcinoma
`cell line.<; demonstrate.<; that although TS may be a major site
`
`to: Richllld M. Schultz, cancer Research
`Correspondence
`Division, DC 0546, UUy Research Laboratories, Indianapolis,
`IN 46285, USA Phone (317) 276-5508; fax (317) 277-3652; E.(cid:173)
`mail Schultz_Richard_M@Ully.Com
`
`Key Wolrl.r: LY231514, antitumor activity, antifolate, folic acid.
`
`of action for L Y231S14 at concentrations near the ICSO,
`higher concentrations can lead to inhibition of dihydrofolate
`reductase (DHFR) and/or other enzymes along the purine de
`novo pathway (3); 2) MTA is an excellent substrate for
`folylpolyglutamate synthetase, and the Kt values of the
`pentaglutamate of LY231S14 are 1.3, 7.2, and 65 nM for
`inhibition against TS, DHFR and gl.ycinamide n'bonucleotide
`formyltransferase (GARFr), r
`tlvel 3; 3 intracellular
`and its polyglutamates can
`
`r;
`M. Schultz, unpublished observation);
`1 14 was use
`and 4) early clinical studies demonstrated that patients who
`to respond
`had previously failed
`to ZD1694 and 5-
`fluorouraciUieucovorin treatment responded to LY231514 (4;
`DA Rinaldi, personal communication).
`Several animal studies have indicated that folic acid
`supplementation in combination with antifolate cancer
`therapy can prevent delayed toxicity and enhance the
`therapeutic potential of the GARFF inhibitor lometrcxol (5,
`6) and the TS inhibitor 1843U89 (7). Unexpected delayed
`cumulative toxicity was observed in phase I studies with
`lometrexol,
`thrombocytopenia, anemia, and
`including
`mucositis (8). Additional clinical studies demonstrated the
`pr<ltective effects of tohc acid agamst wmefrexol toxicity in
`humans (9). Morgan and coworkers (10} concluded that n
`daily supplement of 1 mg of folic acid during low-dose
`methotrexate therapy in patients with rheumatoid arthritis
`was useful in lessening toxicity without altering efficacy. In
`the present communication, we investigated the effects of
`folic acid on the antitumor activity and lethality ofLY231514
`in mice.
`
`Materials and Methods
`
`RtiJ(JfflLf. Po lie ac:id, folinic acid (leucovorin), and 3-[ 4,S-dimethylrhlazol-
`2yi)-2.S-diphcnyl tctmzolium bromide (MTI) were p11rchascd from
`Sigma O!emical Co. (St. Louis, MO, USA). 1l1e disodium lialr of
`L Y131514 was li)'nt~cslzcd al Eli Lllly a11d Co. (1).
`
`Cell /Illes. Human CCRF-CEM leukemia cells were obtained from St.
`J\1® Olildren's Research Hllllpital (Memphis, TN, USA). Human
`IGROVl ovariun carcinoma cells were generously suppliccl by Dr.
`
`0250-7005/98 $2.00+ .40
`
`3235
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`ROO!i7S7A
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`ANTICANCER RESEARCH 18: 3235-3240(1998)
`
`Bartou Kamen (Unlv. of Texw; SOuthllleStern Medical Center. DaUas,
`TX, USA). OC3 human c:olon carcinoma cells were ob111ined from Dr.
`Janet Houghton. St. Jude Children's Research Ho~-pital. Humnn KB
`epidennold carcinoma c:clls were purchased from the Amcric:an Type
`Cultun: collection (ATCC, Rockville, MD, USA). The buman IX-lluns
`carcinoma cell line was established Rt l.illy from xenograft tissue. These
`c;ellllnes were adapted to folic acid-free RPMl-1640 medium containing
`L-glutltntine and 2S mM HEPES buCCer (Whiuaker DioprodUCIS,
`Walkersville, MD, USA) and supplemented with 10% dialy2cd fetal calf
`serum (Hyclone Laboratories, Inc. (logan, UT, USA) !tnd 2 nM folinic
`acid. The LSL78Y(l'K-/HX- murine lymphoma coli Une was obtained
`from Ell Lilly Department of Genetic Tnxlcolngy (Greenfield, IN. USA).
`The tumor is a double mutant. deficient in thymidine kinase and
`hypoxanthine phospho ribosyl transCerase. It was cultured In RPMI· 1640 Results
`medium supplemented with 10% hone serum. The L5178Y-S wild type
`lymphoma ceu line was obtained from ATCC and roudnely cultured In
`
`treatment. Twu..<fimensional
`the beginning and eml of drug
`measu~ments (width nnd length) of nil tumors were taken using digital
`electronic calipers interfaced to a microcomputer (12). Tumor weights
`were calculated from these measurements using tbe following formul!t!
`Tumor weight (m&) = tumor length (rum) x tumor width (mm)2h
`
`Percent inhibition or tumor arowth WIIS determined by comparing tltc
`tumor weight in treated groups to that of controls. No group was
`Included
`in the unalysis for therapeutic activity in which deaths
`atlributuble to drug toxicity exceeded 20% of the treated group.
`
`In vitro protective efficJ of folic or folinic acid for the cytotoxic
`F'acher's mediUnl (Whitta~r BioproduciJ. > supp~cmented with lO% \activity of LY231514. We tested the ability of folic and folinic
`•
`•
`horse serum and I mM socltum pyruvate. AU cell hnes were tested and
`found free of mycoplasma contamination by the ATCC.
`actd to protect human carcmoma and leukemia cells from
`LY231514-induced cytotoxicity. Previous studies demon(cid:173)
`strated that the antiproliferative activity of LY231514 for
`In vitro cytatoxicit;y taling. We IJScd a modification of the orlginw MTT
`colorimebie assay described by Masmann (It) to measure cell CCRF-CEM leukaemia cells was completely reversed by the
`of 1
`rln ( OS
`ddi I
`6 ul.A)
`cytotoxicity. Tile bullll!n tumor c;ells (previously adapted to growth in low
`••
`tu••
`folate (2 oM folinic acid) medium) were seeded at 1 x to" ~:ells in 80 td of a
`In a competlt~ve
`eu~vo
`t on
`0.
`to 1
`asslly medium/well in 96-wcll Ottt-bottom ti~ut cultun: plates (Costar, manner (1). This suggested that LY231514 competed wllh
`natural reduced folate cofactors both at transport and
`Cambridge, MA, USA). Assay medium consisted of rolic acid-f"rcc
`Intracellular folate levels and acted as a pure folate
`J.U>Ml-1640 medium supplemented with IO'Jb dialyzed fetal calC serum
`antagonist. In addition we have reported that L Y231514 is
`and 2oM ro!inic acid. WolllA was len blank (100 !LI of growth medium
`!
`.
`.
`without cells). Various levels of Colle or folinic acid (0.1 to 100 liM) were
`•
`added to the wells and incubated for 2 hours prior to addition of prirnanly transported VIa the reduced folate earner (RFC) In
`human cell lines (3). For the current studies, we utilized
`LY231Sl4. LY231SI4 was prepnred In Dulbeao's phosphate-buffered
`tumor cell lines that had been adapted over >4 weekly
`saline (PBS) nl 1 mglrnl, oad a series of two-Cold dilutions were
`$Ub!lequent~ rnnde in PBS. Allquots (In (II) or each eonc:entrntlo~ ~ere passages to growth in low folate (2 nM folinic acid) media.
`addc_d to tnpliente wells. Plates were Incubated for 72 houra at '51 C 111 a Varying concentrations of folic and folinic acid were added to
`humtdlfled atmosphere or S% 002-ln-alr. MTI was dissolved In PBS at
`•
`s mgtml. Following incubation of platc:a, tO Jil of stock MTT solution
`these adapted cells 2 hours pnor to LY231514 exposure. As
`shown in Table I, the sensitivity to LY231514 cytotoxicity
`was added to all wells ofan assay, and the plata! were Incubated at37'C
`(ICso) of low folate medium-adapted ceUs ranged from 3.6
`for two additional bours. Following incubation. liJO.td dimelbyl nalfoxide
`nM (CCRF-CBM leukemia) to 44 nM (IOROVl ovarian
`was added to each well. Following thorough formazan solubilization, the
`b Tty ff, r
`plates were read on II Dynutcch MR600 rc:ndcr, using a test wavden~:lh carcinoma). In addition Table I h
`th
`'d
`8 ows
`e a t 1 o o lC act
`of 570 nm and a reference wavelength of 630 nm.
`•
`and folinic acid
`to modulate
`lhc cytotoxic activity of
`LY2315141n f'tve different human tumor cell lines. Folic acid
`was approximately 100- to lQOO.fold less active than folinic
`acid at protecting cells from LY231514-induced cytotoxicity.
`Folic acid required concentrations of 10 !1M or greater to
`exert significant protection.
`
`Mice. Female CD L nU/nu mice were putcbascd from ChariCi River
`Laboratories (Wilmington, MA. USA). Female DBA/2 mice were
`purchased from Taconic (Germantown, NY, USA). Mice weighed 20 to
`2S grams at the beginning of the studies. Mice Wt:.n: ho~ In
`temperature and bumidity controlled rooma. Mice were fed Dither
`standard labon~tory rodent chaw (Purina Chow #5001) or folic ncid(cid:173)
`dcficicnt diet containing I% suceinylsulfathilm:lle (Purina Cbow
`#S831 C-2): both diets were purclwscd from Ralston Purina Co. (St.
`Louis, MO, USA). The avernge content or folate.~ from natural sources
`in both diet& wD& found tu be 0.03 ppm, whereas the standard diet was
`anal)'lted 10 contain 7.3 ppm of nddod folic acid. It wu csllmatcd tbat
`mice on n lllnndard diet ingested I to 2 mg/kf/day or folates, whDe mice
`on a low folate diet iDgeated 0.1101 to O.OOS mg/kg/day. In some stud ics,
`mice received solubilized fnllc acld once a day by om! savage. Food and
`water wen: provided ad libitum.
`
`In vivo antitumor drug t~:sting. LSI78Y·S and LSi78YtrK·/HX wen:
`estabUshed ond cbai'Rcterlzcd in vivo for tumor growth in syngeneic
`DBA/2 mice. Cells derived fron1 in vitro culture were wubed twice by
`cetttriCugntion {300 g for 10 minutes) In serum·frce medium. Recipient
`DBA/2 mice wen: Jbavcd ~d inowlalcd subcutaneously in the uxiRary
`region with 2 X 1rf> cells in O.S ml serum-free RPMI·1640 medium.
`L Y2J15L4 treatmellt wns administered Lp. on a dally schedule Cor ten
`days and initiated on the day after tumor implant. L.Y23!S,l4 wa&
`dissolved in 0.9% sodium chlori~ solution. All animals w~ weighed Rl
`
`3236
`
`)
`
`Enhanced lethality of L¥231514 to mice with dietary restriction
`of folic acid. Dietary folate deprivation has previously been
`shown to markedly enhance the toxicity of lometrexol (5). To
`assess the importance of dietaty folate in modulating the
`toxicity of LY231514, IDso values were determined in mice
`maintained on standard diet (normal rodent laboratory chow)
`or on a special low folate diet (LFD). LFD mice have been
`shown to be significantly folate deficient in plasma and
`several tissues including liver and implanted tumors (13).
`Mice maintained on LFD
`for
`two weeks before
`intraperitoneal adminstration of LY231514 daily for 10 days
`were extremely sensitive to the toxic effects of LY231514 with
`IDso values of 1.6 and 10 mglkg for CDl nu/nu and DB.A/2
`mice, respectively (Figure 1). In contrast, the LDso values for
`CD1 nu/nu and DBA/2 mice maintained on standard diet
`
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`Worzalla tt al: Folic Acid-Enhanced L Y231S14 Therapeutics
`
`Table I. In vitro proteclilll! tjfectsoffollc or follnlc acid 011 LY23JS14-inducM cytotoxicity.
`
`Relative (-fold) Olange in JCso
`Folie acid cone. In media"
`
`111M
`
`2
`
`Cell line•
`
`ICSO(nM)"
`
`I ORO VI
`
`KB
`
`OCJ
`
`LX· I
`
`CCRF-CEM
`
`44
`
`34
`
`12
`
`4
`
`4
`
`IOJIM
`
`lOOJIM
`
`14
`
`3
`
`3
`
`3
`
`4
`
`2S
`
`17
`
`9
`
`II
`
`22
`
`O.l(.IM
`
`28
`
`2
`
`Folinic acid cone. in media
`lj.!M
`10 j.tM
`
`370
`
`6
`
`105
`
`6
`
`22
`
`>910
`
`78
`
`47
`
`82
`
`130
`
`100 (.1M
`
`>970
`
`>1270
`
`640
`
`1460
`
`4600
`
`1Ctlls were adapted to >4 weekly passages In low folate (2 nM folinic acid) medium.
`bCytotoxicity was determined by MTT assay witb n h exposure to LY231S14. Data represent mean of triplicate determinations.
`°Follc or folinic acid was added two hours prior to L Y231514 addition.
`
`_ _)
`
`were approximately 250- and 60-fold greater, respectively
`than mice on LFD.
`
`Table II. C.Y2JIS14 alllihunor activity again.st LSJ78Y/S wild type and
`L5178Y/TK·IHX·Iymphoma.
`
`Role of folic acid in the antilumor activity of LY231514 against
`the L5178Y murine lymphoma. High circulating thymidine
`levels in mice decrease the efficacy and toxicity of TS
`inhibitors in mice (14, 15). Unless a tumor model which
`cannot salvage thymidine is utilized in mice, only limited
`antitumor effects for specific TS inhibitors have been
`observed. LY231514 treatment (i.p., qd x10) produced
`modest activity against the wild type 15178Y -S murine
`lymphoma (Table II). In contrast, similar treatment of a
`variant of this line, l5178YII'K·/HX-, produced potent tumor
`suppression (100% tumor illhibition on the day following the
`last drug treatment at 30 and 100 mglkg per day) with 11 of 14
`mice tumor-free on day 100 after tumor implantation. This
`tumor is deficient in both thymidine kinase as well as
`hypoxanthine-guanine
`phosphoribosyl
`tl'!lnsferase
`and
`consequently, cannot salvage either thymidine or the purines
`hypoxanthine and guanine. The exquisite sensitivity of the
`L5178Yfi'K·IHX· tumor model to LY231514 treatment
`allowed us to evaluate the effect of low folate diet on tho
`therapeutic activity of this compound. For mice on LFD,
`LY231514 at 0.3 and 1.0 mglkg/day (i.p. qd xlO) produced
`100% inhibition of tumor growth for tumors mea5ured one
`day after the completion of a single course of drug treatment
`(Figure 2). As noted in Figure 1, higher drug levels yielded
`unacceptable toxicity. For mice on LFD tbat received a folate
`supplement of 15 mg./kg/day via oral gavage, significant
`inlubition of tumor growth was noted over a broad dose range
`(10- 1000 .gfdose). Moreover, 100% inhibition of tumor
`growth was observed at 30 to 1000 mg/kf/dose without any
`lethality. This antitumor dose
`response (with
`folate
`supplementation) was virtually identical to that observed for
`mice receiving standard diet. However, the lethality was
`signicantly greater for the mice on standard diet (lethality at
`
`Tumor Dose0
`(ntglkg)
`
`% Tumor [nh. b
`
`fJ Tumor-tree/total
`day llf
`day 100
`
`L5l78Y/S
`
`L5178Y !l'K.-IHX·
`
`10
`
`30
`
`100
`
`10
`
`30
`
`100
`
`0
`
`8
`
`68
`
`90
`
`100
`100
`
`0110
`
`0/10
`
`0/10
`
`on
`sn
`1n
`
`on
`6fT
`
`sn
`
`:LY231SI~wll& administered i.p.on a qd lllO schedule.
`Thmors were measured on the day following the lust drug treatment
`COays represent the number of days since lhempy was initiated.
`
`400 and 800 MHfkg/day of 10% and 100%, rellpectively). Mice
`on standard diet received approximately one-tenth of the
`amount of daily folic acid as the mice on LFD with 15
`mglkg/day supplemental folic acid.
`
`Dbcussloo
`
`The poor predictive value of mouse models for antifolate
`toxicity may be partially due to the fact that standard
`laboratory mouse diets contain high. levels of folic acid.
`Previous data demonstrated that serum and RBC folate levels
`of mice maintained ou a diet fomtulated without added folic
`acid fall to levels considered normal in humans (5, 13). In this
`paper, we demonstrate that mice fed a low folate diet for a
`short period (2 weeks) became 60- to 250-fold more sensitive
`
`_)
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`

`,_
`
`1CIO
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`f 10
`~ eo
`~ 40
`~
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`ANTICANCER RESFARCH 18:3235-3240 (19l18)
`
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`10
`LY231514 (mgJkg per day)
`
`1«10
`
`0 1
`
`0.1
`
`10
`Dnlg Dosage (rn9'kg)
`
`100
`.....
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`
`F'tgUie 1. 71te toxicity of LY231S 14 in mice is Increased by 11 fol4te-d~ficil!lrl
`dJeL DBA{J and CDl rwlnu mice ttlt'tl! fed dlher tr,$/tJndarr:llahorarOI)I dter
`(0 and '\7, rup«tively} or 11 foiG14-tk[.clmt diet for 2 wuk! prior to the
`fir# dou of LY2J lS 14 (• tut4 "', rupectively) 1111d for tht durotlon of tht
`st11dy. Groups of mice (> 10 llltlmaf.slgnmp) 011 uch diet ttlt'tl! given /(}
`daily dtnes of LY23JS14 Lp. at the i11dkated dosu. '17u! data prmnt tll4
`permit kthalirywitftln 3wetluofter lk ltutdoseof LY2J15U.
`
`Figure 2. Antitumor aclivlty of LY2JISU tlrtrtlpy (Lp., qd '14.10) agaiiW
`L5178YITK· JHX· lymphoma for mice on low folore diet with no folate
`MJpp/emmttatimt (0) ond for mice on IDW folalt dkt rhat received IS
`mg/"'J/day dally folate mpp(emelltafion (A). Vertical dashed 1/ne.s reprt~mll
`pr:n:ent ltthaiity iA nrict! on low folate diet with 110 folate supplementation.
`NtJ lethality wa.r tJbmwti In mice that receiwd folate mpplementation.
`
`to the lethality of LY231514 than observed in mice fed
`standard laboratory diet (F'JgUre 1). The antifolate OAR
`intu'bitor,
`lometrexol has pre'lliou y
`een shown
`to
`a.cx:umUJate tn me uvers of folate:aeficienl mice, and this
`accumwatlon was dmunished by the idJJiilliStration of folic
`llCid to these animal& (16). These investigators hypothesized
`that the substantial and unexpected toxicity of lometrexol in
`humans not given concurrent folic acid and in folate-deficient
`mice is due to the sequestration of drug in hepatic tissue, with
`the subsequent slow release of drug to the circulation at
`toxicologically relevant concentrations. The mechanism for
`this accumulation of Jometrexol in liver probably involves
`metabolism to polyglutamate forms by the enzyme folylpoly·
`· y-glutamate synthetase (FPGS). In this regard, Mendelsohn
`and coworkers (6) demonstrated that liver produced the
`greatest response in elevated FPGS to low dietary folate of all
`ti<isues tested. A similar mcclumism probably exists for the
`potentiation of LY231514 toxicity by folate-deficient diet,
`since this compound is an extremely efficient substrate for
`mouse liver FPGS (1). In addition, LY231514 requires
`polyglutamation for cytotoxic potency (3).
`The uptake of natural reduced folate compounds and
`folate analogues into cells appears to involve membrane
`protein receptors of two different classes: a reduced
`folate/methotrexate carrier (RFC), whiclt binds reduced
`folate In the micromolar range, and a high-affinity folate
`binding protein (mFBP), which preferentially binds to
`oxidized folate and other analogs with an affinity <l nM (17).
`Studies uaing a panel ofZR-75·1 human breast sublines with
`differing
`transport properties have demonslrated a
`predominant role for the RFC in intracellular transport of
`
`LY231514 (3). Similarly, we now report that folic acid only
`weakly modulates the c;ytotoxic activity of LY231514 for
`various human leukemia and carcinoma cells adapted to low
`folate conditions (Table 1). Some of these cells (KB and
`lOROVl) have p1eviously been demonstrated to possess
`elevated levels of mFBP (18), further suggesting a minor role
`for mFBP in LY231514 transport.
`LY231514 produced potent antitumor activity against the
`1..5178Y/TK-JHX- lymphoma at lOQ-fold lower dose levels
`(0.3 and 1 m&fkg/day, Figure 2) in LFD mice relative to 30
`and 100 mg/kg (Table II) in mice on standard diet. It is
`interesting to note that the LDso was reduced 3000-fold for
`Jometrexol in I.FD animals, and antitumor activity could not
`be demonstrated even at low dose levels (5). In contrast, the
`shift in both LDso and antitumor activity for mice on LFD
`compared to standard diet were of a similar magnitude
`(approximately lQO-fold) for LY231514. However, LPD
`animals with high
`levels of folate supplementation
`demonstrated decreased lethality to LY231514 compared to
`conventional diet animals, suggesting that folate intake can be
`manipulated to achieve greater therapeutic effects. Oral folic
`acid dramatically decreased the toxicity of LY231514 and
`preserved antitumor activity (albeit at higher dose levels) in
`these mice (Figure 2).
`Previous studies have demonstrated that the multitargeted
`antifolate, LY231514 hns a unique biochemical and
`pharmacological profile. Exciting antitumor activity has been
`observed in phase I and II clinical trials, including responses
`in colon, breast, non-smalJ cell lung and pancreatic cancers.
`More advanced and extensive clinical trials of LY231S14 are
`currently i11 progress. The combination of folic acid with
`
`)
`
`3238
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`...
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`"'
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`I
`./
`
`Worzalla eta/: Folic Acid-Enhanced LY231.514 Therapeutics
`
`LY231514 may provide a mechanism for enhanced clinical
`antitumor selectivity.
`
`Acknowledgements
`
`The authors thank Slleryl Allen. Shcrrl Andis, Pal Furlcr. Pamela
`Rutherford, Tracy Self, and Karla Theobald for their skillful technical
`assistance. We also tbant Dr. Beverly Teicher for help£ul comments
`during the preparation of this manuscript.
`
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`Received May 5, 1998
`Accepted May 22, 1998
`
`BNSDOCIO: <XP ___ 8005757"'-._I_>
`
`3239
`
`NEPTUNE GENERICS 1005-00005
`BNS oaae 5
`
`APOTEX 1 005 - 0005
`
`