ANTICANCER REsEARCH 18: 3235-3240(1998)
`
`XP-008005757
`
`012
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`
`Role of Folic Acid in Modulating the Toxicity and Efficacy of the
`Multitargeted Antifolate, LY231514
`JOHN' F. WORZALLA, CHUAN SH1H and RICHARD M. SCHULTZ
`
`Cancer Rese4rch Division, lilly Research Laboratories, Eli Lilly and Co., Indianapolis, IN 46285, U.S.A.
`
`)
`
`Abstract. We studied the effects of folic acid on modulating lhe
`toxicity and antitumor efficacy of LY231514. Using seveml
`human tumor cell lines culapted to growth in low folate medium,
`folic acid as shown to be 100- to 1000-fold less active than
`·,. olinlc acid at proteCting cells from LY231SI4-induced
`cytDtoxicily. The lethality of LY231S14 was compared in mice
`maintained on standard diet or low folate dret. The LDSO
`occutf'8d at 60- and 250-fold lower doses of L¥231514 in DBA/2
`and CD 1 nu/nu mice, respectively, maintained on low folate diet
`compared to sl41tdllrd diet. The LS178YITK·IHX- murine
`lymphoma wa.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 mglkg (qd x 10, i.p.)
`produced 100% inhibition of L5178Yfl'K·IHX· lymphoma
`growth, and significant lethality ocaured at a:: 3 mg/kg. For mice
`on standard diet, LY231514 produced >95% inhibition of
`tumor growth at 30 to JOO mglkg. but aU mice died at 800 mg/kg.
`Folic acid .wpplementalion was demonstrated to preserve the
`antitumor activity of LY231514 while reducing toxicily. The
`combmotion of folic acid with
`LY231514 TIUlY provide a
`mechanism for enhanced clinJcol onlitumor selectivity.
`
`)
`
`LY231514 is a structurally novel antifolate antlmeta.bolite that
`possesses
`the unique 6·5-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 arc involved in cytotoxicity, hence the
`acronym MTA multit
`ted antifolate: 1
`th
`A in human leukemia and colon carcinoma
`pattern or
`cell lines demonstrates that although TS may be a major site
`
`to: Richard M. Schultz, Cancer Research
`Cornspondence
`Division, DC 0546, UUy Research Laboratories. Indianapolis,
`IN 46285, USA Phone (317) 276-5508; fax (317) 271-3652; E.(cid:173)
`mail Schultz_Richard_M@Ully.Com
`
`Key Wonl.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 14 values of the
`pentaglutamate of LY231514 are 1.3, 7.2, and 65 nM for
`inhibition against TS, DHFR and gl.ycinamide n'bonucleotide
`formyltransferase (GARFr), r
`tivel 3 ; 3 intracellular
`and its polyglutamates can
`
`1 14 was use
`M. Schultz, unpublished observation);
`and 4) early clinical studies demonstrated that patients who
`had previously failed to respond
`to ZD1694 and 5-
`fluorouraciUleucovorin 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 GARFI' inhibitor lometrcxol (5,
`6) and the TS inhibitor 1843U89 (7). Unexpected delayed
`cumulative toxicity was observed in phase I studies with
`lometrexol,
`including
`thrombocytopenia, anemia, and
`mucositis (8). Additional clinical studies demonstrated the
`protective effects of fohc acid agamst iometrexol toxtaty m
`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
`
`RtagenLr. Folic a<.id, folinic acid (JeuCOYOrin ), and 3-( 4,S-dimethylthiazol-
`2yi)-2.S-diphenyl tctrazolium bromide (MTI'} were p11rchased from
`Sigma O!cmieal Co. (St. Louis, MO, USA). 11te disodlum salt of
`LY131S14 wauynt~cslzcd at Eli Ully and Co. (1).
`
`Cell lutes. Human CCRF-CEM leukemia cells were obtained from St.
`Jude Otildren's Research Hospital {Memphis, TN, USA). Human
`IGROVl ovariun carcinoma cells -re generously supplied by Dr.
`
`0250-7005/98 $2.00+.40
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`3235
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`Rl10!i7S7A
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`Wockhardt Exhibit 1005 - 1
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`ANTICANCER RESEARCH 18: 3235-3240(1998)
`
`Barton Kamen (Unlv. of Texw; SOuthwestern Medical Center. DaUas,
`TX, USA). GCJ human colon carcinoma cells were obtained from Dr.
`Janet Houghton. St. Jude Children's Rcscarc:h Ho~'Pital. Humnn KB
`epidennold carcinoma cells were purchased from the American Type
`Culture collection (ATCC, Rockville, MD. USA). The burnan LX-I lung
`carcinoma cell line was established Rt l.illy from xenograft tissue. These
`cell lines were adapted to folic acid-Cree RPMl-1640 medium containing
`L-glutantine and 2S mM HEPES buffer (Whitlaker Dioproducu,
`Walkersville, MD, USA) and supplemented with 10% dialy21:d fetal calf
`serum (Hyclone Laboratories, Inc. (Logan. UT, USA) and 2 nM folinic
`acid. The LS178Y/TK·IHX· murine lymphoma cell line was obtained
`from Ell Ully Department of Genetic Toxicology (Greenfield, IN, USA).
`The tumor is a double mutant. deficient in thymidine kinase and
`hypoxanthine phosphorlbosyl transferase. lt was cultured In RPMI-1640 Results
`medium supplemented with 10% hone sci'\Ul1. The LSI78Y-8 wild type
`lymphoma cell line was obtained from ATCC nod roudnely cultured In
`
`treatment. 1\vu..<Jimensional
`eml of drug
`the beginning 11nd
`mcasu~ments (width and length) of all tumora were taken using digital
`electronic calipers interlaced to a microcomputer (12). Tumot weights
`were calc:u!Dted from these measurements using the following formula:
`Tumor weight (mg) = tumor length (mm) x tumor width (mm)2h
`
`Percent inhibition of tumor growth WIIS determined by comparing the
`tumor weight in treated groups to that of controls. No group was
`included in the unalysis for therapeulic activity in which deaths
`o.tlributllble to drug toxicity exceeded 20% of the treated group.
`
`In vitro protective effecJ of folic or folinic acid for the cytotoxic
`F'JSCher's medium (Whitt11~r Bioproducls. ). supp!emented with lO% \activity of LY231514. We tested the ability of folic and folinic
`
`horse serum and I mM sochum pyruvate. All cell Imes 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 cytotoxicity tating. We used a modifiution of the original MTT
`colorimebic assay described by Mosmann (It) to measure cell CCRF-CEM leukaemia cells was completely reversed by the
`addition of leucovorin (0.05 to 16 .. u) In a competitive
`cytotoxicity. Til~ ~u~n tumo~ cells (previously adapted 10 ~thin low
`Colnte (2 nM foluuc 11ctd) med1um) were seeded nt 1 x 10" ccUs m 80 (ll of
`•
`.,....
`•
`asslly mediumlwt:ll in 96-wcll o11r-bottom til611l: cultun: plates (Custar,
`roaMer (1). This suggested that LY231514 competed wtth
`natural reduced folate cofactors both at transport and
`Cambridge, MA, USA). Assay medium consisted of folic acid-free
`RPMI-1640 medium supplemented wiU1 10% dialyzed fetal calC serum
`intracellular folate levels and acted as a pure folate
`antagonist. In addition we have reported that L Y231514 is
`and 2 nM folinic acid. WclllA wus left blnnk (tOO jiJ of growth medium
`!
`.
`.
`'I
`rim
`without cells). Various levels of folic or folinic acid (0.1 to 100 jiM) were
`added to the wells and incubated for 2 hours prior to addition of P an Y transported VIa the reduced folate earner (RFC) tn
`human cell lines (3). For the current studies, we utilized
`LY231Sl4. LY231S14 was prepared In Dulbecco's phosphate-buffered
`saline (PBS) nt 1 mglrnl, ond a series of two-Cold dilutions were
`tumor cell lines that had been adapted over >4 weekly
`$Ub!lequent~ made In PBS. Aliquots (10 111) or eac:h eoncenrrntlo? ~ere passages to growth in low folate (2 nM folinic acid) media.
`added to tnpl!cnte wells. Pl11tes were Incubated for 72 houra at '51 C tn 11 Varying concentrations of folic and folinic acid were added to
`humidified atmosphere or S% 002-fn-alr. MTI was dissolved In PBS at
`.
`these adapted cells 2 hours pnor to LY231514 exposure. As
`5 mgtml. Following incubation of plates, 10
`jiJ of stock M'l'T solution
`shown in Table I, the sensitivity to LY231514 cytotoxicity
`was added to all wells of an assay, and the plates were incubated at37'C
`for two additional hours. Following incubation. lllO-td dimethyl sulfoxide
`(ICso) of low folate medium-adapted ceUs ranged from 3.6
`nM (CCRF-CEM leukemia) to 44 nM (IOROVl ovarian
`was added to each well. Followi11g thorough formazan solubilization, the
`plates were rclld on 8 Dynutec:h MR600 tc:~~der, using a test wavelength
`carcinoma). In addition Table I shoWli the ability of folic acid
`of 570 nm and a reference wavelength of 630 nm.
`, ,
`.
`•
`•
`.
`and fohmc ac1d
`to modulate !he cytotoXIc activity of
`LY231514 in f'tve different human tumor cell lines. Folic acid
`was approximately 100. to l()(J().fold less active than folinic
`acid at protecting cells from LY231514-induced cytotoxicity.
`Folic acid required concentrations of 10 11M or greater to
`exert significant protection.
`
`Mice. Female CD I nU/nu mic:e were purchased from C.'harlca River
`Laboratories (Wilmington, MA, USA). Female DBA/2 mice were
`purchased from Taconic (Gennantown, NY, USA). Mice weighed 20 to
`2S grams at the beginning of the studies. Mice were hoii:ICd In
`temperature and bumidity controlled roolll8. Mice were fed Dither
`standard labot11tol)' rodent chaw (Purina Chow #5001) or folic acid·
`deficient diet containing
`I% suecinylsulfnthiazole (Purina Chow
`#S831C·2): both diets wen: purchusc\1 from Ralston Purina Co. (St.
`Louis, MO, USA). The avernge content of folate.~ from nnturlll sources
`in both diet& wn& found tu be 0.03 ppm, whereas the standard diet was
`analyzed to contain 7:3 ppm of ndded folic ncid. It was estimated that
`mice on n lllnndard diet ingested I to 2 mgllqr/dny or folntes, whDe mice
`on a low folate diet ingested 0.1101 to O.OOS mg/kg/dey. In some studies,
`mice received solubilized fnllc acid once a day by ornt gavage. Food and
`water were provided ad libitum.
`
`In vivo antitumor drug t~Siing. LSI78Y-S nnd LSI78Y/TK·/HX were
`estabUslled ond cbamcterlmd In vivo for tumor growth in syngeneic
`DBA/2 mice. Cells derived front in vitro culture were wubed twice by
`centrifugation {300 g for 10 minutes) in serum-free medium. Recipient
`DBA/2 mice were shaved and inoculated subcutaneously in the uxiDary
`region with 2 X trf> cells "in O.S ml serum-free RPM1-1640 medium.
`L Y231514 tre11tment was administered i.p. on a daily schedule Cot ten
`days nnd initiated on the day after tumor implant. LY2315.14 wu
`dissolved in 0.9% sodium chloride solution. All animals wete weighed at
`
`3236
`
`)
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`Enhanced lethality of LY231514 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).
`two weeks before
`Mice maintained on LFD
`for
`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 COl nu/nu and DB.A/2
`mice, respectively (Figure 1). In contrast, the LDso values for
`COl nu/nu and DBA/2 mice maintained on standard diet
`
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`
`Worzalla tt al: Folic Acid-Enhanced LY231514 Therapeutics
`
`Table I. In vitro proteclilll! q[ectsoffollc or follnlc acid 011 LY23J514-indllctd C}fotoxicity.
`
`Relative (-fold) Olange in JCso
`Folic acid cone:. In media0
`
`Folinic: acid eonc. in media
`
`111M
`
`2
`
`Cell line•
`
`ICSO(nM)b
`
`!ORO VI
`
`KB
`
`OC3
`
`LX-1
`
`CCRF-CEM
`
`44
`
`34
`
`12
`
`4
`
`4
`
`IO!IM
`
`100JIM
`
`O.lJ.LM
`
`1j.!M
`
`14
`
`3
`
`3
`
`3
`
`4
`
`2S
`
`17
`
`9
`
`6
`
`22
`
`28
`
`2
`
`370
`
`6
`
`105
`
`6
`
`22
`
`JOJ,LM
`
`>910
`
`78
`
`47
`
`82
`
`130
`
`100 p.M
`
`>970
`
`>1270
`
`640
`
`1460
`
`4600
`
`•cells were adapted to >4 weekly passages In low folate (2 nM folinic acid) medium.
`bCytotoxicity was determined by M'IT assay with n b exposure to LY231S14. Data represent mean of triplicate determinations.
`°Folic 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. LY2JIS14 alllihtmor activity again.st LSJ78Y/S wild type and
`LS178Y/TK-IHX·Iymphoma.
`
`Role of folic acid in the antitumor activity of L¥231514 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 xlO) produced
`modest activity against the wild type 15178Y-S mudne
`lymphoma (Table II). In contrast, similar treatment of a
`variant of this line, l5178Y/I'K·/HX-, produced potent tumor
`suppression (100% tumor inhibition 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
`transferase
`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 the
`therapeutic activity of this compound. For mice on LFD,
`LY231514 at 0.3 and 1.0 roNday (i.p. qd xlO) produced
`100% inhibition of tumor growth for tumors measured 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 that 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 msfkg!dose). Moreover, 100% inhibition of tumor
`growth was observed at 30 to 1000 mg/lcf/dose without any
`response (with
`lethality. This antitumor dose
`folate
`supplementation) was virtually identical tn that observed for
`mice receiving standard diet. However, the lethality was
`signicantly greater for the mice on standard diet (lethality at
`
`Tumor Dose0
`(mglkg)
`
`% Thmor [nh. b
`
`fl Tumor-tree/total
`day llf
`day 100
`
`LSI78Y/S
`
`L5178Yil'K-/HX·
`
`10
`
`30
`
`100
`
`10
`
`30
`100
`
`0
`
`8
`
`68
`
`90
`
`100
`
`100
`
`0/10
`
`0/10
`
`0/10
`
`017
`sn
`7n
`
`on
`6fT
`
`sn
`
`:LY231514 wa& administered i.p. on a qd 1110 schedule.
`Thmors were mea8111'ed on the day following the lust drug treatment
`C:Oays represent the number of days since lhempy was initiated.
`
`400 and 800 rr11fkf/day of 10% and 100%, respectively). 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.
`
`Discussion
`
`The poor predictive value of mouse models for antifolate
`tnxlcity 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 formulated 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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`BNSDOCID: <XP __ 8005757 A .... _l_>
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`ANTICANCER RESFARCH 18:3235-3240 (1998)
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`LY231514 (mgJkg per day)
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`Dnlg Dosage (m&Jkg)
`
`F'tgUie 1. 71te toxicity of LY231S 14 in mice is Increased by a foiDte-dejicitmt
`diet. DBA/2 and CDI twlnu mice ltlm fed either tr,$/andanllaboratory diet
`(0 and "il. resp«tillely) or Cl folCI~-tkfiCimt diet for 2 week8 prior to the
`fir# do.se of LY2J IS 14 <• tllld "', rupeetively) alld for the dumtlon of tht
`st11dy. Groups of mice (> 10 OJtimalslgnmp) 011 each t:h'et tvm given 10
`daily doses of Ll'Z31Sl4 Lp. at the i11dlcated doses. T1u! data present the
`permit kthCI/irywitftln Jwetluafter 1M ltutdoseof L'IZJ15U.
`
`Figure 2. Antilumor aclivlry of l.Y2JISU tlttrtl.py (Lp., qd 'I< 10) again.ll
`L5178YfTK· JHX- lymphoma for mice on low folore diet with 110 folote
`.s~~pplemettJation (0) alld for mice on low folaM dkt rhat rec:tived 15
`mg//qJ/diiJ dally folate mpp(emelltation (A). Vertical dashed line.s reprewll
`pm:cnt lethality In mice on low foltzte diet with 110 folate supplementation.
`NQ lethality was obmved In mice that rect:ived folate supplementatWn.
`
`to the lethality of LY231514 than observed in mice fed
`standard laboratory diet (F'.gure 1). The antifolate GAREf
`lometrexol has previously been shown
`intu'bitor,
`to
`a.cx:umUJate tn me hvers of folate:aeficienl mice, and this
`accumwatlon was dmurushed by the aammistratton of folic
`acid to these animals (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 meclumism probably exist~ for the
`potentiation of LY231514 toxicity by folate-deficient diet,
`since this compound is an extremely efficient subslnlte 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), which binds reduced
`folate In the micromolar range, and a high-affinity folate
`binding protein (mFBP), which preferentially binds to
`oxidized folate lllld other analogs with an affinity <1 nM (17).
`Studies uaing a panel of ZR-75-1 human breast sublines with
`differing
`transport properties have demonstrated a
`predominant role for the RFC in intracellular transport of
`
`LY231514 (3). Similarly, we now report that folic acid only
`weakly modulates the cytotoxic activity of LY231514 for
`various human leukemia and carcinoma cells adapted to low
`folate conditions (Table 1). Some of these cells (KB and
`IOROVl) have previously been demonstrated to possess
`elevated levels of mFBP (18), further suggesting a minor role
`for mFBP in LY231514 transport.
`LY231S14 produced potent antitumor activity against the
`L5178Y/TK.-JHX- lymphoma at lOQ-fold lower dose levels
`(0.3 and 1 m~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 lFD 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 100-fold) for LY231514. However, LFD
`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 has 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-small cell lung and pancreatic cancers.
`More advanced and extensive clinical trials of LY231514 are
`currently in progress. The combination of folic acid with
`
`3238
`
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`Rntl!'i7S7A
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`RN~ n:>n.,. .4
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`Wockhardt Exhibit 1005 - 4
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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 Sheryl Alleo, Shcrrl Andis, Pal Furlcr. Pamela
`Rutherford, Tracy Self, and Karla Theobald for their skillful technical
`assistance. We also thank Dr. Beverly Teicher for helpful comments
`during the preparation of this manuscript.
`·
`
`References
`
`Taylor BC, Kuhnt D, Shih C, Rlnzel SM, Orlndcy GB, Barreda J,
`lannatipour M and Moran RO: A dideazatctrahydro£olate nnalogue
`Jacking a chiral center at C-6, N-{4-(2-(2-amlno-1,7dihydro:.4-
`oxopyrrolo [2,3-d)pyrlmidlne-6-yl)ethyl)benzoyl} glul!mic acid, a new
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`
`Received May 5, 1998
`Accepted May 22, 1998
`
`BNSDOCIO: <XP ___ 8005757A_I_>
`
`3239
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`BNS oaae 5
`
`Wockhardt Exhibit 1005 - 5