fkNTICANCER RESEARCH 18." 3235-3240 (1998)
`
`Role of Folic Acid in Modulating the Toxicity and Efficacy of the
`Mulfitargeted Antifolate, LY231514
`
`JOHN F. WORZALLA, CHUAN SHIH and RICHARD M. SCHULTZ
`
`Cancer Research Dh,ision, Lilly Research Laboratotqes, Eli Lilly and Co., Indianapolis, IN 46285, U.SM.
`
`Abstract. We studied the effects of folic acid on modulating the
`toxicity and antitumor efficacy of LY231514. Using several
`human tumor cell lines adapted to growth in low folate medium,
`/’olic acid was shown to be 100- to lO00-fold less active than
`j~linic acid at protecting cells fi’om LY231514-induced
`cytotoxicity. The lethality of LY231514 was compared in mice
`maintained on standard diet or low folate diet. The LD50
`occun’ed at 60- and 250-fold lower doses of LY231514 in DBA/2
`and CD1 nu/nu mice, respectively, maintained on low folate diet
`compared to standard diet. The L5178Y/TK-/HX- mut4ne
`lymphoma was much more sensitive to the antitumor action of
`LY231514 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 L5178Y/TK-/HX- lymphoma
`growth, and significant lethality occun’ed at > 3 mg/kg. For mice
`on standard diet, LY231514 produced >95% inhibition of
`am~or growth at 30 to 300 mg/kg, but all mice died at 800 mg/kg.
`Folic acid supplementation was demonstrated to preserve the
`antitumor activity of LY231514 while reducing toxicity. The
`combination of folic acid with LY231514 may provide a
`mechanism for enhanced clinical antitumor selectivity.
`
`LY231514 is a structurally novel antifolate antimetabolite that
`possesses the unique 6-5-fused pyrrolo[2,3-d]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-
`inhibitory mechanisms are involved in cytotoxicity, hence the
`acronym MTA (multitargeted antifolate): 1) the reversal
`pattern for MTA in human leukemia and colon carcinoma
`cell lines demonstrates that although TS may be a major site
`
`Co~7"espondence to: Richard M. Schultz, Cancer Research
`Division, DC 0546, Lilly Research Laboratories, Indianapolis,
`IN 46285, USA. Phone (317) 276-5508; fax (317) 277-3652; E-
`mail Schultz_Richard_M@Lilly.Com
`
`Key Words: LY231514, antitumor activity, antifolate, folic acid.
`
`of action for LY231514 at concentrations near the IC50,
`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 Ki values of the
`pentaglutamate of LY231514 are 1.3, 7.2, and 65 nM for
`inhibition against TS, DHFR and glycinamide ribonucleotide
`formyltransferase (GARFT), respectively (3); 3) intracellular
`concentrations of LY231514 and its polyglutamates can
`exceed 40 ~M in CCRF-CEM cells when ~H-labeled
`LY231514 was used (R.M. Schultz, unpublished observation);
`and 4) early clinical studies demonstrated that patients who
`had previously failed to respond to ZD1694 and 5-
`fluorouracil/leucovorin 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 GARFT 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 folic acid against lometrexol toxicity in
`humans (9). Morgan and coworkers (10) concluded that a
`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 of LY231514
`in mice.
`
`Materials and Methods
`
`Reagents. Folio acid, folinic acid (leucovorin), and 3-[4,5-dimethylthiazol-
`2yl]-2,5-diphenyl tetrazolium bromide (MTr) were purchased from
`Sigma Chemical Co. (St. Louis, MO, USA). The disodium salt of
`LY2315/4 was synthesized at Eli Lilly and Co. (1).
`
`Cel! lines. Human CCRF-CEM leukemia cells were ~htaincd from St.
`Jude Children’s Research Hospital (Memphis, TN, USA). Human
`IGROVI ovarian carcinoma cells were generously supplied by Dr.
`
`0250-7005/98 $2.00+.40
`
`3235
`
`Sandoz Inc.
`Exhibit 1013-0001
`
`JOINT 1013-0001
`
`

`
`ANTICANCER RESEARCH 18:3235-3240 (1998)
`
`Barton Kamen (Univ. of Texas Southwestern Medical Center, Dallas,
`TX, USA). GC3 human colon carcinoma cells were obtained fi’om Dr.
`Janet Houghton, St. Jude Children’s Research Hospital. Human KB
`epidermoid carcinoma cells were purchased from the American Type
`Culture collection (ATCC, Roclcville, MD, USA). The human LX-1 lung
`carcinoma cell line was established at Lilly from xenografl tissue. These
`cell lines were adapted to folic acid-free RPM 1-1640 medium containing
`L-glutamine and 25 hum HEPES buffer (Whittaker Bioproducts,
`Walkersville, MD, USA) and supplemented with 10% dialyzed fetal calf
`serum (Hyclone Laboratories, Inc. (Logan, UT, USA) and 2 nM folinic
`acid. The L5178Y/TK-/HX- murine lymphoma cell line was obtaincd
`from Eli Lilly Department of Genetic Toxicoldgy (Greenfield, IN, USA).
`The tumor is a double mutant, deficient in thymidine kinase and
`hypoxanthine phosphoribosyl nansferase. It was cultured in RPMI-1640
`medium supplemented with 10% horse s+rum. The L5178Y-S wild type
`lymphoma cell line was obtained from ATCC and routinely cultured in
`Fischer’s medium (Whittaker Bioproducts) supplemented with 10%
`horse serum and 1 mM sodium pyruvate. All cell lines were tested and
`found free of mycoplasma contamination by the ATCC.
`
`hi vio’o cytotoxici~y testing. We used a modification of the original MTF
`colorimetric assay described by Mosmann (11) to measure cell
`cytotoxicity. The human tumor cells (previously adapted to growth in low
`folate (2 nM folinic acid) medium) were seeded at I x 104 cells in 80 I11 of
`assay medium/well in 96-well flat-bottom tissue culture plates (Costar,
`Cambridge, MA, USA). Assay medium consisted of folio acid-fiee
`RPMI-1640 medium supplemented with 10% dialyzed fetal calf serum
`and 2 nM folinie acid. Well IA was left blank (100 ~_d of growth medium
`without cells). Various levels of folic or fotinic acid (0.1 to 100 gM) were
`added to the wells and incubated //or 2 hours prior to addition o~
`LY231514. LY231514 was prepared in Dulbecco’s phosphate-buffered
`saline (PBS) at 1 mg/ml, and a series of two-fold dilutions were
`subsequently made in PBS. Aliquots (10 Id) of each concentration were
`added to triplicate wells. Plates were incubated for 72 hours at 37°C in a
`humidified atmosphere of 5% CO~-in-air. MTT was dissolved in PBS at
`5 mg/ml. Following incubation of plates, 10 ~d of stock MTT solution
`was added to all wells of an assay, and the plates were incubated at 37°C
`for two additional hours. Following incubation, 100-~d dimethyl sulfoxide
`was addcd to each well. Following thorough formazan solubilizatinn, the
`plates were read on a Dynatech MR600 reader, using a test wavelength
`of 570 nm and a reference wavelength of 630 nm.
`
`Mice. Female CD 1 nu/nu mice were purchased from Charles River
`Laboratories (Wilmington, MA, USA). Female DBA/2 mice were
`purchased from Taconic (Germantown, NY, USA). Mice weighed 20 to
`25 grams at the beginning of the studies. Mice were housed in
`temperature and humidity controlled rooms. Mice were fed either
`standard laboratory rodent chow (Purina Chow #5001) or folic aci&
`deficient diet containing 1% succinylsulfathiazole (Purina Chow
`#5831C-2); both diets were purchased from Ralston Purina Co. (St.
`Louis, MO, USA). The average content of folates from natural sources
`in both diets was found to be 0.03 ppm, whereas the standard diet was
`analyzed to contain 7.3 ppm of added folio acid. lit was estimated that
`mice on a standard diet ingested 1 to 2 mg/kg/day of folates, while mice
`on a low folate diet ingested 0.001 to 0.008 mg/kg/day. In so~ne studies,
`mice received solubilized fotic acid once a day by oral gavage. Food and
`water were provided ad libitum.
`
`bt vivo antitumor drag testing. L5178Y-S and 1,5178Y/TK-/HX were
`established and characterized in vivo for tumor growth in syngeneic
`DBA/2 mice. Cells derived from in vitro culture were washed twice by
`centrifugation (300 g for l0 minutes) in serum-free medium. Recipient
`DBA!2 mice were shaved and inoculated subcutaueously in the axillary
`region with 2 x ]06 cells’in 0.5 ml serum-free RPMI-1640 medium.
`LY231514 treatment was administered i.p. on a daily schedule for ten
`days and initiated on the day after tumor implant. LY231514 was
`dissolved in 0.9% sodium chloride solution. All animals were weighed at
`
`the beginning and end of drug treatment, T~vo-dimensional
`mm~sure.ments (width and length) of all tumors were taken using digital
`electronic calipers interfaced to a microcomputer (12). Tumor weights
`were calculated from these measurements using the following fi3rmula:
`
`Tumor weight (mg)= tmnor length 0nm) x tumor width (ram)2/2
`
`Percent inhibition of tumor growth was determined by comparing the
`tumor weight in treated groups to that of controls, No group was
`included in the analysis for therapeutic activity in which deaths
`attributable to d~ ug toxicity exceeded 20% of the treated group,
`
`Results
`
`In vitro protective effect of folic or folinic acid for the cytotoxic
`activity of LY231514. We tested the ability of folic and folinic
`acid to protect human carcinoma and leukemia cells from
`LY231514-induced cytotoxicity. Previous studies demon-
`strated that the antiproliferative activity of LY23!514 for
`CCRF-CEM leukaemia cells was completely reversed by the
`addition of leucovorin (0.05 to ~6 gM) in a competitive
`manner (1). This suggested that LY231514 competed with
`natural reduced folate cofactors both at transport and
`intracellular folate levels and acted as a pure folate
`antagonist. In addition, we have reported that LY231514 is
`primarily transported via the reduced folate carrier (RFC) in
`h,uman cell lines (3). For the current studies, we utilized
`tumor cell lines that had been adapted over >4 weekly
`passages to growth in low folate (2 nM folinic acid) media.
`Varying concentrations of folic and folinic acid were added to
`these adapted cells 2 hours prior to LY231514 exposure. As
`shown in Table I, the sensitivity to LY231514 cytotoxicity
`(IC50) of low folate medium-adapted cells ranged from 3.6
`nM (CCRF-CEM leukemia) to 44 nM (IGROV1 ovarian
`carcinoma). In addition, Table I shows the ability of folic acid
`and folinic acid to modulate the cytotoxic activity of
`LY231514 in five different human tumor cell lines. Folic acid
`was approximately 100- to 1000-fold less active than folinic
`acid at protecting cells from LY23]514-induced cytotoxicity.
`Folic acid required concentrations of 10 gM or greater to
`exert significant protection.
`
`Enhanced lethality of LY231514 to mice with dietmy restriction
`of folic acid. Dietary folate deprivation has previously been
`shown to markedly enhance the toxicity of lometrexol (5). To
`assess the importance of dietary folate in modulating the
`toxicity of LY231514, LDs0 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 LY23~514 daily for 10 days
`were extremely sensitive to the toxic effects of LY231514 with
`LDs0 values of 1.6 and 10 mg/kg for CD1 nu/nu and DBA/2
`mice, respectively (Figure 1). In contrast, the LDs0 values for
`CD1 nu/nu and DBA/2 mice maintained on standard diet
`
`3236
`
`Sandoz Inc.
`Exhibit 1013-0002
`
`JOINT 1013-0002
`
`

`
`Worzalla et ak Folic Acid-Enhanced LY231514 Therapeutics
`
`Table I, In vitTv protective effects of jblic or folinic acid on LY231514-induced cytotoxieity,
`
`Relative (-fold) Change in ICs0
`
`Folio acid cone. in mediac
`
`Folinic acid cone. in media
`
`Ce!l linea
`
`[C50 (riM)b
`
`1 gM
`
`10 [~M
`
`100 btM
`
`IGROV1
`
`KB
`
`GC3
`
`LX-I
`
`CCRF-CEM
`
`44
`
`34
`
`12
`
`4
`
`4
`
`1
`
`2
`
`1
`
`1
`
`1
`
`14
`
`3
`
`3
`
`3
`
`4
`
`25
`
`17
`
`9
`
`6
`
`22
`
`0.1
`
`28
`
`1 laM
`
`10 ~M
`
`100 gM
`
`370
`
`6
`
`105
`
`6
`
`22
`
`> 970
`
`78
`
`47
`
`82
`
`130
`
`> 970
`
`> 1270
`
`640
`
`1460
`
`4600
`
`aCells were adapted to >4 weekly passages in low folate (2 nM folinic acid) medium.
`bCylotoxicity was determined by Mq-T assay with 72 h exposure to LY231514. Data represent mean of triplicate determinations.
`CFolic or folinic acid was added two hours prior to LY231514 addition.
`
`were approximately 250- and 60-fold greater, respectively
`than mice on LFD.
`
`Table II. LY231514 antitumor activity against L5178Y/S wiM type and
`L5178Y/TK-/HX-lymphoma.
`
`Role of folic acid in the antitmnor activity of LY231514 against
`the L5178Y mulgne 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 xl0) produced
`modest activity against the wild type L5178Y-S murine
`lymphoma (Table II). In contrast, similar treatment of a
`variant of this line, L5178Y/TK-/HX-, produced potent tumor
`suppression (100% tumor inhibition on the day following the
`last drug treatment at 30 and 100 mg/kg 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
`L5178Y/TK-/HX- 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 mg/kg/day (i.p. qd × 10) 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
`inhibition of tumor growth was noted over a broad dose range
`(10 - 1000 mg/kg/dose). Moreover, 100% inhibition of tumor
`growth was observed at 30 to 1000 mgikg/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 Dosea
`
`% Tumor Inh.b
`
`(mg/kg)
`
`# Tumor-free/total
`day 10c
`
`day 100
`
`,L5178Y/S
`
`I_5178Y/TK-/HX-
`
`10 o 0/10
`
`30
`
`100
`
`8
`
`68
`
`o/10
`
`0/I_0
`
`10 90 0/7 0/7
`
`30
`
`100
`
`100
`
`100
`
`5/7
`
`7/7
`
`6/7
`
`5/7
`
`aLY231514 was administered i.p. on a qd x 10 schedule.
`bTumors were measured on the day following the last drug treatment.
`CDays represent the number of days since therapy was initiated.
`
`400 and 800 mg/kg/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
`mg/kg/day supplemental folic acid.
`
`Discussion
`
`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 folio acid.
`Previous data demonstrated that serum and RBC folate levels
`of mice maintained on a diet formulated without added folio
`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
`
`3237
`
`Sandoz Inc.
`Exhibit 1013-0003
`
`JOINT 1013-0003
`
`

`
`ANTICANCER RESEARCH 18:3235-3240 (1998)
`
`lOO
`
`Q.. 20
`
`I
`/
`/
`/
`/
`
`/
`!
`/
`/
`
`~ 4o
`
`,-lOO
`
`,80
`
`~0 P
`
`o’.~
`
`~o
`~o
`;
`LY231514 (mg/kg per day)
`
`~o~o
`
`o.1 I lO lOO lOOO
`Drug Dosage (m~/kg)
`
`Figure 1. The toxicity of L Y231514 in mice is increased by a folate-deficient
`diet. DBA/2 and CDI nu/nu mice were fed either a standmzt laboratory diet
`(0 and ~/, respectively) or a folate-defieient diet for 2 weeks p~qor to tire
`first dose of LY231514 (@ amt Y, respectively) aud for tire duration of the
`study. Groups of mice (> 10 animals/©’oup) on each diet were given 10
`daily doses of LY231514 i.p. at the indicated doses. The data present the
`percent lethality within 3 weeks after the last dose of LY231514,
`
`Figure 2. Antitamor activity of LY231514 therapy (i.p., qd xlO) against
`L5178Y/TK- /HX- lymphoma for mice on low folate diet with no folate
`supplementation (©) and for mice on low folate diet that received 15
`
`mg/kg/day daily folate supplementation (/k). Ve~¢ical dashed lines represent
`percent lethality in mice on low folate diet with no folate supplementation.
`No letha#ty was obsen,ed in mice that received folate supplementation.
`
`to the lethality of LY231514 than observed in mice fed
`standard laboratory diet (Figure 1). The antifolate GARFT
`inhibitor, lometrexol has previously been sho~vn to
`accumulate in the livers of folate-deficient mice, and this
`accumulation was diminished by the administration 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 lometrexol in liver probably involves
`metabolism to polyglutamate forms by the enzyme folylpoly-
`~/-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
`tissues tested. A similar mechanism 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
`polyglntamation 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 and other analogs with an affinity < 1 nM (17).
`Studies using 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 I). Some of these cells (KB and
`IGROV1) have previously 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
`L5178Y/TK-/HX- lymphoma at 100-fold lower dose levels
`(0.3 and 1 mg/kg/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 LDs0 was reduced 3000-fold for
`lometrexol in LFD animals, and antitumor activity could not
`be demonstrated even at low dose levels (5). In contrast, the
`shift in both LDs0 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
`
`Sandoz Inc.
`Exhibit 1013-0004
`
`JOINT 1013-0004
`
`

`
`Worzalla et al: Folic Acid-Enhanced LY231514 Therapeutics
`
`LY231514 may provide a mechanism for enhanced clinical
`antitumor selectivity.
`
`Acknowledgements
`
`The authors thank Sheryl Allen, Sherri Andis, Pat Forler, 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
`
`l Taylor EC, Kuhnt D, Shih C, Rinzel SM, Grindey GB, Barredo J,
`Jannatipour M and Moran RG: A dideazatetrahydrofolate analogue
`lacking a chiral center at C-6, N-{4-[2-(2-amino-l,7dihydro-4-
`oxopyrrolo [2,3-d]pyrimidine-6-yl)ethyl]benzoyl} glutamic acid, a new
`and potent inhibitor of thymidylate synthase. J Med Chem 35: 4450-
`4454, 1992.
`2 Grindey GB, Shih C, Barnett CJ, Pearce HL, Engelhardt JA, Todd,
`GC, Rinzel SM, Worzalla JF, Gossett LS and Everson TP: LY231514,
`a novel pyrrolopyrimldine antifolate that inhibits thymidylate
`synthase (TS). Proc Am Assoc Cancer Res 33: 411, 1992.
`3 Shih C, Chen VJ, Gossett LS, Gates SB, MacKellar WC, Habeck LL,
`Shackelford ICA, Mendelsohn LG, Soose DJ, Patel VF, Andis SL,
`Bewley JR, Rayl EA, Moroson BA, Beardsley GP, Kohler W,
`Ratnam M and Schultz RM: LY231514, a pyrrolo[2,3-d]pyrimidine-
`based antifolate that inhibits multiple folate-requiring enzymes.
`Cancer Res 57: 1116-1123, 1997.
`4 Rinaldi DA, Burris HA, Dorr FA, Woodworth JR, Kuhn JG, Eckardt
`JR, Rodriguez G, Corso SW, Fields SM, Langley C, Clark G, Faries
`D, Lu P and Van Hoff DD: Initial phase I evaluation of the novel
`thymidylate synthase inhibitor, LY231514, using the modified
`continual reassessment method for dose escalation. J Clin Oncol 13:
`2842- 2850, 1995.
`5 Alati T, Worzalla JF, Shih C, Bewley JR, Lewis S, Moran RG and
`Grindey GB: Augmentation of the therapeutic activity of lometrexol
`[(6-R)5,10-dideazatetrahydrofolate] by oral folio acid. Cancer Res 56:
`2331-2335, 1996.
`6 Mendelsohn LG, Gates SB, Habeck LL, Shackelford ICA, Worzalla J,
`Shih C and Grindey GB: The role of dietary folate in modulation of
`folate receptor expression, folylpolyglutamate synthetase activity and
`the efficacy and toxicity of lometrexol. Advan Enzyme Regu136: 365-
`381, 1996.
`7 Smith GK, Amyx H, Boytos CM, Duch DS, Ferone R and Wilson
`HR: Enhanced antitumor activity for the thymidylate synthase
`inhibitor 1843U89 through decreased host toxicity with oral folio acid.
`Cancer Res 55: 6117-6125, 1995.
`
`8 Ray MS, Muggia FM, Leichman CG, Grunberg SM, Nelson RL,
`Dyke RW and Moran RG: Phase I study of (6R)-5,10-
`dideazatetrahydrofolate: a folate antimetabolite inhibitory to de novo
`purine synthesis. J Natl Cancer Inst 85: 1154-1159, 1993.
`9 Laohavinij S, Wedge SR, Lind MJ, Bailey N, Humphreys A, Proctor
`M, Chapman F, Simmons D, Oakley A, Robson L, Gumbrell L,
`Taylor GA, Thomas HD, Boddy AV, Newell DR and Calvert AH: A
`phase I clinical study of the antipurine antifolate lometrexol
`(DDATHF) given with oral folic acid. Invest New Drugs 14: 325-335,
`1996.
`10 Morgan SL, Baggott JE, Vaughn WH, Young PK, Austin JV,
`I~umdieck CL and Alarcon GS: The effect of folio acid
`supplementation on the toxicity of low-dose methotrexate in patients
`with rheumatoid arthritis. Arthritis Rheum 33: 9-18, 1990.
`11 Mosmann T: Rapid colorimetric assay for cellular growth and
`survival: application to proliferation and cytotoxicity assays. J
`lmmunol Methods 65: 55-63, 1983.
`12 Worzalla JF, Bewley JR and Grindey GB: Automated measurement
`of transplantable solid tumors using digital electronic calipers
`interfaced to a microcomputer. Invest New Drugs 8: 241-251, 1990.
`13 Schmitz JC, Grindey GB, Schultz RM and Priest DG: Impact of
`dietary folic acid on reduced folates in mouse plasma and tissues:
`relationship to dideazatetrahydrofolate sensitivity. Biochem
`Pharmaco148." 319-325, 1992.
`14 Duch DS, Banks SD, Dev IK, Dickerson SJ, Ferone R, Heath LS,
`Humphreys J, Knick V, Pendergast W, Singer S, Smith GK, Waters K
`and Wilson HR: Biochemical and cellular pharmacology of 1843U89,
`a novel benzoquinazoline inhibitor of thymidylate synthase. Cancer
`Res 53: 810-818, 1993.
`15 Jackman AL, Taylor GA, Gibson W, Kimbell R, Brown M, Calvert
`, AH, Judson IR and Hughes LR: ICI D1694, a quinazoline antifolate
`thymidylate synthase inhibitor that is a potent inhibitor of L1210 cell
`growth ht viO’o and in vivo; a new agent for clinical study. Cancer Res
`51: 5579-5586, 1991.
`16 Pohland RC, Alati T, Lantz RJ and Grindey GB: Whole-body
`autoradiographic disposition and plasma pharmacokinetics of 5,10-
`dideazatetrahydrofolic acid in.mice fed folic acid-deficient or regular
`diets. J Pharm Sci 83: 1396-1399, 1994.
`17 Antony AC: The biological chemistry of folate receptors. Blood 79:
`2807-2820, 1992.
`18 Schultz RM, Andis SL, Shackelford KA, Gates SB, Ratnam M,
`Mendelsohn LG, Shih C and Grindey GB: Role of membrane-
`associated folate binding protein in the cytotoxicity of antifolates in
`KB, IGROV1, and LI210A cells. Oncology Res 7: 97-102, 1995.
`
`Received May 5, 1998
`Accepted May 22, 1998
`
`3239
`
`Sandoz Inc.
`Exhibit 1013-0005
`
`JOINT 1013-0005