MTA (LY231514) in Combination Treatment Regimens
`Using Human Tumor Xenografts and the EMT-6
`Murine Mammary Carcinoma
`
`Beverly A. Teicher, Enrique Alvarez, Pocheng Liu, Ku Lu, Krishna Menon, Jack Dempsey, and Richard M. Schultz
`
`An important component in the development of a
`new anticancer drug is an understanding of its poten-
`tial for inclusion in combination treatment regimens.
`LY231514, a multitargeted antifolate (MTA), was
`tested in combination with cisplatin, methotrexate,
`5-fluorouracil, paclitaxel, docetaxel, doxorubicin,
`LY329201 (a glycinamide ribonucleotide formyltrans-
`ferase [GARFT] inhibitor), and fractionated radiation
`therapy in vivo using EMT-6 mammary carcinoma, hu-
`man HCT 116 colon carcinoma, and human H460 non-
`small cell lung carcinoma grown as xenografts in nude
`mice. Isobologram methodology was used to deter-
`mine the additivity or synergy of the combination
`regimens. I~TA administered with cisplatin, paclitaxel,
`docetaxel, or fractionated radiation therapy produced
`additive to greater than additive tumor response by
`tumor cell survival assay and tumor growth delay.
`While an additive tumor response was observed when
`MTA was administered with methotrexate, synergistic
`tumor responses were seen when MTA was adminis-
`tered with the GARFT inhibitor, LY329201, or with the
`topoisomerase I inhibitor, irinotecan. MTA was admin-
`istered in combination with full doses of each antican-
`cer agent studied, with no evidence of increased toxic-
`ity resulting from the combination.
`Semin Oncol 26 (suppl 6):55-62. Copyright © 1999 by
`W.B. Saunders Company.
`
`N -[4-[2-(2-AMINO-3,4-dihydro-4-oxo-7H-
`
`pyrrolo[2,3 -d]-pyrimidin-5-yl) ethyl]-
`benzo-yl]-L-glutamic acid, LY231514 (MTA), was
`discovered through structure activity relationship
`studies based on the novel antipurine antifolate
`lometrexol.1 MTA contains a pyrrole moiety in
`the place of the tetrahydropyridine ring of lome-
`trexol, which results in a major loss of activity in
`the inhibition of purine biosynthesis and a shift to
`the inhibition of pyrimidine biosynthesis (thymi-
`dylate cycle).2"4 MTA is a substrate for mamma-
`lian folylpolyglutamate synthase5 and is a potent
`inhibitor, especially as the triglutamate, of the
`enzymes
`thymidylate synthase, dihydrofolate reductase,
`glycinamide ribonucleotide formyltransferase
`(GARFF), and aminoimidazole carboxamide ribo-
`nucleotide formyltransferase.6
`In 1984, Jackman et al7 reported that relatively
`high concentrations of circulating thymidine have
`been found in human plasma. Thus, those concen-
`trations found in mice may tend to underpredict
`
`both the antitumor activity and toxicity of
`drugs that inhibit thymidylate synthase compared
`withwhat may be expected in humans.8 MTA
`was a very active antitumor agent against the
`thymidine kinase-negative/hypoxanthine-nega.
`rive murine lymphoma L51784/TK-/HX.9 MTA
`was also found to be an effective antitumor
`agent against several human tumor xenografts
`with normal thymidine kinase levels, including
`the VRC5 colon carcinoma, the GC3 colon
`carcinoma, the BXPC3 pancreatic carcinoma,
`the LX-1 non-small cell lung carcinoma, and
`MX-1 breast carcinoma.1 In several studies, the
`folate levels in mice were modulated by feeding
`a low-folate diet then repleting the animals by
`administration of specific doses of folic acid.*°
`Both the antitumor activity and toxicity of
`MTA could be modulated in this manner, and at
`certain folate levels antitumor activity toward
`specific tumors could be optimized.
`In the current studies, MTA was administered
`alone, in combination with standard chemothera-
`peutic agents, or with radiation therapy to tumor-
`bearing mice to explore the potential interaction
`of MTA in combination anticancer treatment reg-
`imens.
`
`MATERIALS AND METHODS
`
`Drugs
`
`MTA and LY329201 (a GARFT inhibitor) were prepared
`according to published methods and procedures.2,11,12 Cispla-
`tin, methotrexate, 5-flt~orouracil, paclitaxel, and doxorubicin
`were purchased from Sigma Chemical Co (St Louis, MO).
`Irinotecan (CPT-11) was purchased from the Indiana Univer-
`sity Medical School Pharmacy, Indianapolis, IN.
`
`From the Lilly Research Laboratories, Eli Lilly and Company,
`Lilly Corporate Center, Indianapolis, IN.
`Sponsored by Eli Lilly and Company~
`Drs Teicher, Alvarez, Liu, Lu, Menon, Dempsey and Schultz
`are employees of Eli Lilly and Company. Dr Schultz is a stockholder
`of Eli Lilly and Company.
`Address reprint requests to Beverly A. Teicher, PhD, Lilly
`Research Laboratories, Eli Lilly and Company, Lilly Corporate
`Center, Drop Code 0546, Indianapolis, IN 46285.
`Copyright © 1999 by W.B. Saunders Company
`0093 -7754/99/2602-0609510.00/0
`
`Seminars in Ontology, Vol 26, No 2, Suppl 6 (April), 1999: pp 55-62
`
`55
`
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`
`

`
`56
`
`T~tmo~s
`
`The EMT-6 murine mammary carcinoma was an in vivo-in
`~’,itro tumor system. The EMT-6 tumor was carried in BALB/c
`mice (Taconic Farms, Germantown, NY). For the experitnents,
`2 × 106 tumor cells prepared from a brei of several stock tumors
`were implanted subcutaneously into the hind legs of the
`BALB/c mice at 8 to 10 weeks of age,l~a4
`HCT 116 human colon carcinoma originated from a male
`patient in 1979.~s The HCT 116 cell line is tumorigenic in
`nude mice. HCT 116 cells were purchased from ATCC (Rock-
`ville, MD). H460 human non-small cell tung carcinoma was
`obtained from the National Cancer Institute (Bethesda, MD).
`The H460 cell line is tumorigenic in nude mice.
`Nude mice were purchased from Charles River Laboratories
`(Wihnington, MA) at 5 to 6 weeks of age. When the animals
`were 7 to 8 weeks of age they were exposed to 4.5 Gy total body
`radiation delivered using a GammaCel140 irradiator (Nordion,
`Inc, Ottowa, Ontario, Canada). Twenty-four hours later, HCT
`116 or H460 tumor cells (5 × 106) prepared from a brei of
`several stock tumors were implanted subcutaneously in a 1:1
`mixture of RPMI tissue culture media and Matrigel (Collabo-
`rative Biomedical Products, Inc, Bedford, MA). HCT
`tumors grow to 500 mm3 in 19.7 -+ 1.5 days and H460 tumors
`grow to 500 mms in 14.0 -+ 0,8 days.
`
`Tumor Excision Assay
`When the EMT-6 tumors were approximately 100 mm3 in
`volume (8 days after tumor cell implantation), the animals
`were given intraperitoneal injections of various doses of MTA
`(50, 100, 150, or 200 mg/kg) by intraperitoneal injection four
`times (Aivl and PM) over 48 hours alone or in combination with
`cisplatin (10, 20, or 30 mg/kg), methotrexate (1, 5, or 10
`mg/kg), or LY329201 (1, 5, or 10 mg/kg), or with radiation
`therapy (5, 10, 15, or 20 Gy). The second chemotherapeutic
`agents and the radiation therapy were administered with the
`third dose of MTA. Mice were killed 24 hours after treatment
`to allow for full expression of drug cytotoxicity and repair of
`potentially lethal damage. The tumors were excised, and single
`cell suspensions ~vere prepared as described previously33,14,16
`The untreated tumor cell suspensions had a plating efficacy of
`10% to 16%. The results are expressed as the surviving frac-
`tion +- SE of cells from treated groups compared xvith that of
`cells from untreated controls.~3.*~
`
`Bone Marrow Toxicity
`
`Bone marrow cells were taken from the same animals used
`for the tumor excision assay, and the assay for granulocyte/
`macrophage colony-forming units (CFU-GM) was carried out
`as described previously.~3a6 Colonies of at least 50 ceils were
`scored on a Acculi~e colony counter (Fisher, Springfield, NJ).
`The results from three experiments, in ~vhich each group was
`measured in triplicate, were averaged. The results are expressed
`as the surviving fraction +- SE of cells isolated from treated
`animals compared with that of cells isolated from untreated
`animals.
`
`Tumor Growth Delay Experiments
`HCT 116. Treatment was initiated on day 7 after tumor
`cell implantation, ~vhen the HCT 116 tumors were approxi-
`
`TEICHER ET AL
`
`mately 150 mm3 in volume. Animals were treated by intraperi-
`toneal injection with MTA (100 or 125 mg/kg) on days 7
`through 11 and days 14 through J~8, alone or with 5-fluorouracil
`(7.5, 15, or 30 mg/kg ) on days 7 through 11, irinotecan (7.5, 15,
`or 30 mg/kg) on days 7 through 11, or fractionated radiation
`therapy (2, 3, or 4 Gy/fraction) on days 7 through !1 and days
`14 through 18 after tumor cell implantation.
`H460. Treatment was initiated on day 8 after tumor cell
`implantation, when the H460 tumors were approximately 200
`mm3 in volume. Animals were treated by intraperitoneaI in-
`jection with MTA (100 mg/kg) on days 8 through 12 and days
`15 through 19 alone or with 5-fluorouracil (30 mg/kg) by
`intraperitoneal injection on days 8 through 12; cisplatin (10
`mg/kg) by intraperitoneal injection on day 8 or day 15; do-
`cetaxel (22 mg/kg) by intravenous injection on days 8, 10, 12,
`and 15; or doxorubicin. (1.75 mg/kg) by intraperitoneal injec-
`tion on days 8 through 12 after tumor cell implantation. In
`another experiment, MTA (100 mg/kg) was administered by
`intraperitoneal injection on days 8 through 12 alone or with
`5-fluorouracil (30 mg/kg) by intraperitoneal injection on days
`16 through 20 after tumor cell implantation.
`The progress of each tumor was measured three times per
`week until it reached a volume of 2,000 lnm3. Tumor growth
`delay (TGD) was calculated as the time taken by each indi-
`vidual tumor to reach 500 mm3 compared with the time in the
`untreated controls. Each treatment group included five ani-
`mals, and each experiment was repeated three times. Tumor
`growth delay times (days) are the mean values +- SE for the
`treatment group compared with those for the control group.
`
`Data Analysis
`Using the method of Deen and Williams,~7 isobolograms
`were generated for the special case in xvhich the dose of one
`agent is held constant. This method produced envelopes of
`additive effect for different levels of the variable agent and is
`conceptually identical to generating a series of isobolograms
`and replotting the results at a constant dose of one agent on a
`log effect by the dose of the sec0nd-agent coordinate system.
`Dose response curves for each agent were first generated. The
`envelopes of additivity shown were generated from a series of
`isoeffect curves derived from the complete dose response curves
`for each agent. Overall, combinations that produced the de-
`sired effect and that were within the envelope boundaries were
`considered additive. Those displaced to the left were consid-
`ered to be superadditive, while those displaced to the right were
`considered to be subadditive.~8a9 This general approach can be
`extrapolated to the special case in which the level of an agent
`is ’held constant. Under these conditions, an isobologram can
`be derived that plots the expected effect for any level of the
`variable agent, plus the constant agent combinations.2° Exper-
`imentally, this approach is far simpler than classical isobolo-
`gram methodology and readily facilitates determination of ad-
`ditive and nonadditive combinations..3
`Statistical comparisons for the TGD assays were conducted
`with the Dunnett multiple comparisons test after a significant
`effect was found by ANOVA.z*,22
`
`RESULTS
`
`To examine potential interactions ~vith other
`anticancer chemotherapeutic agents and radiation
`
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`
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`
`MTA COMBINATIONS IN IN VITRO MODELS
`
`57
`
`MTA + MTX
`EMT-6 TUMOR EXCISION
`
`4
`
`6
`
`2
`METHOTREXATE DOSE, mg/kg
`
`8
`
`10 12
`
`Z
`
`0.1
`
`Z
`
`0.01
`
`0
`
`therapy, various doses of MTA were administered
`four times over 48 hours to mice bearing the
`EMT-6 mammary carcinoma. Twenty-four hours
`later the tumors were excised and suspensions of
`known numbers of single cells were plated for
`colony formation (Fig 1). MTA killed 50% of the
`EMT-6 tumor cells when the animals received 150
`mg/kg or 200 mg/kg four times. There was no
`toxicity toward the bone marrow CFU-GM with
`these regimens of MTA alone.
`Cisplatin was administered to EMT-6 tumor-
`bearing mice as a single dose of 10, 20, or 30 mg/kg
`in a log-linear manner with increasing dose of the
`drug (Fig 2). One log of tumor cell killing was
`achieved with 17 mgikg of cisplatin. The combi-
`nation of MTA and cisplatin was additive to
`greater than additive. The synergy of the combi-
`nation treatment increased with increasing dose of
`cisplatin. Cisplatin is not very cytotoxic toward
`the bone marrow CFU-GM and combining treat-
`ment of MTA with cisplatin did not increase the
`toxicity toward the bone marrow CFU-GM.
`When administered as a single dose, methotrex-
`ate kills EMT-6 tumor cells in a shallow log-linear
`
`1
`
`z
`
`MTA
`EMT-6 TUMOR EXCISION
`
`0.1
`
`I I I
`150
`50
`100
`MTA Dose, mg/kg
`
`I
`200
`
`Fig I. Survival of EI~T-6 murine mammary tumor cells
`from tumors (0) and bone marrow CFU-GM (©) from animals
`treated with MTA (50, 100, 150, or 200 mg/kg) intraperitone-
`ally four times over 48 hours. The points are the mean values
`of two independent experiments; the bars indicate the SEM.
`
`Fig 2. Survival of EI~T-6 murine mammary tumor cells
`from tumors (0, V) and bone marrow CFU-GM (©, ~7) from
`animals treated with cisplatin (10, 20, or 30 mg/kg) intraperi-
`toneally (0, ©) or with MTA (100 mg/kg) |ntraperltoneally four
`times over 48 hours and cisplatin (10, 20, or 30 mg/kg) intra-
`perltoneally along with the third MTA dose (T, ~). The points
`are the mean values of two independent experiments; the bars
`indicate the SEI~. The shaded area represents the envelope of
`additivity by isobologram analysis.
`
`manner (Fig 3). A single dose of 6 mg/kg of meth-
`otrexate killed 50% of the EMT-6 tumor cells. The
`combination of MTA with methotrexate resulted
`in additive to greater than additive tumor cell
`killing. With the combination treatment regimen,
`50% of EMT-6 tumor cells were killed by 2 mg/kg
`of methotrexate and 90% (one log) of EMT-6
`tumor ceils were killed by 8.5 mg/kg of methotrex-
`ate. The addition of MTA to treatment with
`methotrexate did not increase the cytotoxicity of
`methotrexate toward the bone marrow CFU-GM.
`LY329201 is a potent inhibitor of GARFT. A
`si’ngle dose of 1 mg/kg of LY329201 killed approx-
`imately 1 log of EMT-6 tumor ceils, but there were
`only small increases in the tumor cell killing at the
`higher doses of 5 and 10 mg/kg of LY329201 (Fig
`4). The combination of MTA with LY329201
`produced markedly greater than additive (syner-
`gistic) EMT-6 tumor cell killing across the dosage
`range of LY329201 studied. There was a greater
`
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`
`58
`
`TEICHER ET AL
`
`than 1 log increase in EMT-6 tumor cell killing
`when MTA was combined with LY329201 (5 rag/
`kg) and a greater than 2 log increase in EMT-6
`tumor cell killing when MTA was combined with
`LY329201 (10 mg/kg). Neither LY329201 nor the
`combination of MTA and LY329201 was cyto-
`toxic toward bone marrow CFU-GM.
`Radiation therapy was administered to EMT-6
`tumor-bearing mice in single doses between 5 and
`20 Gy. Radiation killed EMT-6 tumor cells in a
`log-linear dose-dependent n~anner (Fig 5). Treat-
`ment of the animals with MTA and radiation
`therapy produced tumor cell killing that was addi-
`tive for the two treatments.
`The human HCT 116 colon carcinoma was
`selected for the initial study of MTA in combina-
`tion treatments because the HCT 116 tumor is
`
`0.1
`
`Z
`
`0.01
`
`0.001
`
`0.01
`
`FRACTION
`
`MTA + CISPLATIN
`EMT-6 TUMOR EXCISION
`
`0.001
`
`0
`
`5 10 15 20 25 30
`CISPLATIN DOSE, mg/kg
`
`Fig 3. Survival of EHT-6 murine mammary tumor cells
`from tumors (0, Y) and bone marrow CFU-GH (©, ~) from
`animals treated with methotrexate (I, 5, or 10 mg/kg) intra-
`peritoneally (0, ©) or with HTA (I 00 mg/kg) intraperitoneally
`four times over 48 hours and methotrexate (I, 5, or 10 mg/kg)
`intraperitoneally along with the third MTA dose (~r, V). The
`points are the mean values of two independent experiments;
`the bars indicate the SEH. The shaded area represents the
`envelope of additivity by isobologram analysis.
`
`0.0001
`
`0
`
`I : ’ ’ ,’
`2 4 6 8 10
`LY329201 DOSE, mg/kg
`
`12
`
`Fig 4. Survival of EHT-6 murine mammary tumor cells
`from tumors (0, T) and bone marrow CFU-GI~I (O, ~) from
`animals treated with the GARFT inhibitor (LY329201) (I, 5, or
`10 mg/kg)’intraperltoneally (O ,©) or with FITA (100 mg/kg)
`intraperltoneally four times over 48 hours and the GARFT
`inhibitor (I, 5, or 10 mg/kg) intraperitoneally along with the
`third MTA dose (T, V). The points are the mean values of two
`independent experiments; the bars indicate the SEI’I. The
`shaded area represents the envelope of additivity by isobolo-
`gram analysis.
`
`responsive to MTA and because antitumor activ-
`ity of MTA has been observed in patients with
`colon cancer.1,3,4,23,24 Treatment of nude mice
`bearing subcutaneously implanted HCT 116 colon
`tumors with MTA (100 mg/kg) twice daily for 5
`days produced a TGD of 2.7 +- 0.3 days. 5-Flu-
`orouracil administered daily for 5 days produced
`increasing TGD with increasing dose of the drug
`(Fig 6). Treatment with the combination of MTA
`and 5-fluorouracil produced TGD that was addi-
`tive. No toxicity was observed when a full standard
`dose of MTA was administered with a full standard
`dose of 5-fluorouracil.
`Irinotecan adpainistered daily for 5 days pro-
`duced increasing TGD with increasing dose of the
`drug (Fig 7). Treatment of HCT 116 tumor-bear-
`ing animals with MTA and irinotecan resulted in
`greater than additive tumor growth for the two
`drugs, reaching 27 days when the iri~otecan dose
`was 30 mg/kg. No toxicity was observed when a
`
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`
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`
`MTA COMBINATIONS IN IN VITRO MODELS
`
`59
`
`2.8 + 0.3 days. When cisplatin (10 mg/kg) was
`administered as a single dose on the first day of
`treatment with MTA, there was a greater than
`additive TGD; however, when administration of
`cisplatin was delayed to midway through the MTA
`treatment, subadditive TGD resulted. The combi-
`nation of docetaxel and MTA produced greater
`than additive TGD, while the combination of
`paclitaxel and MTA resulted in additive TGD.
`Doxorubicin was not a very active antitumor agent
`against the H460 non-small cell lung carcinoma
`and the combination oi: doxorubicin and MTA
`produced additive TGD.
`AdministraOon of MTA (100 mg/kg) daily for
`14 days to animals bearing the H460 tumor re-
`suited in a TGD of 3.7 days (Table 1). Treatment
`with 5-fluorouracil during the last 5 days of an
`MTA treatment regimen produced additive to
`greater than additive TGD.
`
`<
`r~
`
`0.1
`
`MTA+RADS
`EM~6TUMOR EXCISION
`
`0.01
`
`I I I I
`5
`10 15 20
`RADIATION DOSE, GRAY
`
`Fig 5. Survival of EHT-6 routine mammary tumor cells
`from tumors from animals treated with radiation therapy (5,
`I 0, 15, or 20 Gy) alone (0) or with HTA (I 00 mglkg) intraperi-
`toneally four times over 48 hours and radiation therapy along
`with the third HTA dose (©). The points are the mean values
`of two independent experiments; the bars indicate the SEH.
`The shaded area represents the envelope of additivity by isobo-
`Iogram analysis.
`
`8
`
`7
`
`HCT116 HUMAN COLON CARCINOMA
`
`full standard dose of MTA was administered with
`a full standard dose of irinotecan.
`Fractionated radiation therapy was administered
`locally to the tumor-bearing limb of the nude mice
`carrying human HCT 116 colon carcinoma xeno-
`grafts twice daily for 5 days. Radiation therapy
`delivered in fractions of 2, 3, or 4 Gy produced
`increasing TGD with increasing radiation dose
`(Fig 8). Administration of MTA (100 mg/kg) with
`fractionated radiation resulted in additive TGD
`and administration of MTA (125 mg/kg) with
`fractionated radiation resulted in additive to
`greater than additive TGD. The largest increase in
`TGD observed with the combination of MTA and
`fractionated radiation occurred at the radiation
`dose of 2 Gy, the most clinically relevant dose.
`Combination treatment regimens including
`MTA were also used in nude mice bearing subcu-
`taneously implanted human H460 non-small cell
`lung carcinoma (Table 1). Administration of
`MTA (100 mg/kg) twice daily for 5 days to ani-
`mals bearing the H460 tumor produced a TGD of
`
`I I I I : :
`5 10 15 20 25 30
`5-FluorouracilDose, mg/kg (xS)
`
`Fig 6. Growth delay of human HCT 116 colon carcinoma
`grown as a xenograft in nude mice after treatment with 5-flu-
`orouracil (7.5, 15, or 30 mg/kg) intraperitoneally on days 7 to
`I I after tumor cell implantation alone (0) or along with MTA
`(I 00 mg/kg) intraperitoneally on days 7 to I I and days 14 to 18
`(©). The points are the mean values of two experiments with
`five animals per group per experimpnt; the bars indicate the
`SEH. The shaded area represents the envelope of additivity by
`isobologram analysis.
`
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`
`60
`
`TEICHER ET AL
`
`3O
`
`HCT116 HUMAN COLON CARCINOMA
`MTA (100mg/kg)d7-11;14-18+ Irinotecan
`
`<
`- 20
`
`o ~0
`
`0
`
`0
`
`5 10 15 20 25 30
`
`Irinotecan Dose, mg/kg (x5)
`
`Fig 7. Growth delay of human HCT 116 colon carcinoma
`
`grown as a xenograft in nude mice after treatment with irino-
`
`tecan (7.5, 15, or 30 mg/kg) intraperitoneally on days 7 to I I
`after tumor cell implantation alone (0) or along with HTA (I 00
`
`mglkg) intraperitoneally on days 7 to 1 I and days 14 to 18 (©).
`
`The points are the mean values of two experiments with five
`
`animals per group per experiment; the bars indicate the SEH.
`
`The shaded area represents the envelope of additivity by isobo-
`Iogram analysis.
`
`DISCUSSION
`
`The enzymes involved in the "folate pathway"
`leading to the synthesis of purine and thymidine
`nucleotides represent a vital cellular pathway in
`which little or no redundancy exists, making this
`pathway a prime target for anticancer drug devel-
`opment. Methotrexate, the classic anticancer
`antifolate, has been in clinical use for approxi-
`mately 45 years and has most successfully been
`used in the treatment of leukemia (acute lympho-
`blastic leukemia) and lymphoma (large cell and
`Burkitt’s types).25-27 Methotrexate also has been a
`component of many combination chemotherapy
`regimens for the treatment of solid tumors, espe-
`cially choriocarcinoma, breast cancer, osteogenic
`sarcoma, and head and neck cancer; however, the
`role of methotrexate in these combinations is less
`clear.25"28 The newer antifolates, such as ralti-
`trexed (Tomudex, ZD1694, Zeneca, Macclesfield,
`UK), an inhibitor of the enzyme thymidylate syn-
`
`thase, have focused on the treatment of solid tu-
`mors.
`MTA has been shown to inhibit several en-
`zymes in the folate pathway,1,29 has been investi-
`gated in several phase I clinical trials,3,4,3°’32 and
`has entered phase II clinical trials. An important
`component in the development of a new antican-
`cer drug is an understanding of its potential for
`inclusion in combination treatment regimens. The
`antitumor activity of the combination of MTA
`and cisplatin was additive to greater than additive
`in the EMT-6 mammary carcinoma and the H460
`non-small cell lung carcinoma. MTA antitumor
`activity was also additive to greater than additive
`with the antitumor activity of the taxanes in the
`H460 non-small cell lung carcinoma. In combi-
`nation with radiation therapy, MTA produced ad-
`ditive to greater than additive antitumor activity
`in both the EMT-6 mammary carcinoma and the
`HCT 116 colon carcinoma.
`Although the antimetabolite combinations of
`MTA with methotrexate in the EMT-6 mammary
`carcinoma and MTA with 5-fluorouracil in the
`H460 non-small cell lung carcinoma and HCT
`116 colon carcinoma resulted in additive to
`greater than additive anticancer activity, the corn-
`
`40 T HCT116 HUMAN COLON CARCINOMA
`
`~,
`
`35 f MTA (100or125mg/kg)dT-11;14-18+FRACTIONATED RADIATION
`/
`
`0
`
`2
`3
`1
`RADIATION DOSE, Gray (xlO)
`
`4
`
`Fig 8. Growth delay of human HCT 116 colon carcinoma
`grown as a xenograft in nude mice after treatment with frac-
`tionated radiation therapy (2, 3, or 4 Gy/fraction) on days 7 to
`
`I I and days 14 to 18 after tumor cell implantation alone (0)
`along with HTA (100 mglkg) intraperitoneally on days 7 to I I
`
`and days 14 to 18 before each radiation delivery (©) or along
`
`with HTA (125 mg/kg) intraperitoneally on days 7 to I I and
`
`days 14 to 18 before each radiation delivery (~’). The points are
`
`the mean values of two experiments with five animals per
`
`group per experiment; the bars indicate the SEH.
`
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`

`
`MTA COMBINATIONS IN IN VITRO MODELS
`
`61
`
`the proportion of tumor cells in S phase, as has
`been shown on exposure of HL-60 cells to camp-
`tothecin in cell culture?7 thus increasing the por-
`tion of tumor cells that are susceptible to the
`cytotoxic action of MTA.
`These preclinical in vivo results indicate that
`MTA maintains activity in combination treat-
`ment regimens and can be combined with full
`doses of other anticancer treatments without in-
`creased toxicity. The combinations of MTA with
`irinotecan and docetaxel appear to be especially
`promising.
`
`REFERENCES
`
`1. Shih C, Thornton DE: Preelinical pharmacology studies
`and the clinical development of a novel multitargeted antifo-
`late, MTA (LY231514), in Jackman AL (ed): Anticancer Drug
`Development Guide: Antifolate Drugs in Cancer Therapy.
`Totowa, NJ, Humana Press, 1998, pp 183-201
`2. Shih C, Chen VC, Gossett LS, et ah LY231514, a pyr-
`rolo[2,3-d]pyrimidine-based antifolate that inhibits multiple
`folate-requiring enzymes. Cancer Res 57:1116-1123, 1997
`3. Rinaldi DA, Burris HA, Dorr FA, et ah 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
`4. McDonald AC, Vasey PA, Adams L, et ah A phase I and
`pharmacokinetic study of LY231514, the multitargeted antifo-
`late. Clin Cancer Res 4:605-610, 1998
`5. Habeck LL, Mendelsohn LG, Shih C, et ah Substrate
`specificity of mammalian folylpolyglutamate synthetase for
`5,10-dideazatetrahydrofolate analogs. Mot Pharmacol 48:326-
`333, 1995
`6. Shih C, Chen VJ, Gossett LS, et ah LY231514, a pyr-
`rolo[2,3-d]pyrimidine based antifolate that inhibits multiple
`folate requiring enzymes. Cancer Res 57:1116~1123, 1997
`7. Jackman AL, Taylor GA, Calvert AH, et ah Modulation
`of antimetabolite effects. Effects of thymidine on the efficacy of
`the quinazoline-based thymidylate synthase inhibitor, CB3717.
`Biochem Pharmacol 33:3269-3275, 1984
`8. Jackson RC: Antifolate drugs: Past and future perspec-
`tives, in Jackman AL (ed): Anticancer Drug Development
`Guide: Antifolate Drugs in Cancer Therapy. Totowa, NJ, Hu-
`mana Press, 1998, pp 1-12
`9. Worzalla JF, Self TD, Theobald KS, et ah Effects of folic
`acid on toxicity and antitumor activity of LY231514 multitar-
`geted antifolate (MTA). Proc Am Assoc Cancer Res 38:478,
`1997 (abstr)
`10. Alati T, Worzalla JF, Shlh C, et al: Augmentation of the
`therapeutic activity of lometrexol [(6R)5,10-dideazatetra hy-
`drofolate] by oral folic acid. Cancer Res 56:2331-2335, 1996
`11. Beardsley GP, Mormon BA, Taylor EC, et ah A new
`folate antimetabolite, 5,10-dideaza-5,6,7,8-tetrahydrofolate is a
`potent inhibitor of de novo purine synthesis. J Biol Chem
`264:328-333, 1989
`12. Baldwin SW, Tse A, Gossett LS, et ah Structural fea-
`tures of 5,10-dideaza-5,6,7,8-tetrahydrofolate that determine
`
`Treatment Group
`
`MTA (100 mg/kg), d 8-12, 15-19
`5-Fluorouracil (30 mg/l<g), d 8-12
`MTA + 5FU
`Cisplatin (10 mg/kg), d 8
`MTA + early cisplatin
`Cisplatin (10 mg/kg), d 15
`MTA ÷ late cisplatin
`Docetaxel (22 mg/kg)
`intravenously, d 8, 12, 16
`MTA ÷ docetaxel
`Paclitaxel (24 mg/kg) intravenously,
`d 8, 10, 12, 15
`
`MTA ÷ paclitaxel
`Doxorubicin (I.75 mg/kg), d 8-12
`MTA ÷ doxorubicin
`MTA (100 mg/kg), d 7-20
`5-Fluorouracil (30 mg/kg), d 16-20
`MTA + 5-fluorouracil
`
`Tumor Growth
`Delay (d)
`
`2.8 -+ 0.3
`4.0 -+ 0.3
`7.6 _+ 0.4
`4.0 _+ 0.3
`12.8 ± 0.7*
`8.0 ± 0.6
`7.8 ± 0.3
`
`11.7_+ 1.3
`
`14.1 ± 1.4
`1.0 _+ 0.3
`4.3 _+ 0.3*
`3.7 ± 0.3
`3.8 ± 0.3
`9.3 ± 1.0"
`
`* Significantly different at the 0.05 level from the standard
`drug (ie, that other than MTA) alone by the Dunett multiple
`comparisons test; total 10 animals per point.
`
`bination of MTA and LY329201, a GARFT in-
`hibitor, was markedly synergistic in tumor cell
`killing in the EMT-6 mammary carcinoma. Thus,
`simultaneous potent inhibition of the two en-
`zymes, thymidylate synthase and GARFT, may
`result in a potently lethal metabolic state.
`Irinotecan (CPT-11) is a synthet!c analog of the
`plant alkaloid camptothecin that exerts its antitu-
`mor activity through inhibition on the DNA un-
`winding enzyme topoisomerase 1, resulting in a
`drug enzyme DNA ternary complex and a single
`strand break in the DNA.33,34 Cell culture studies
`have shown that combinations of raltitrexed and
`SN-38, the active metabolite of irinotecan, can
`result in synergistic tumor cell killing.35 The com-
`bination of MTA and irinotecan produced a mark-
`edly synergistic antitumor effect against the hu-
`man HCT 116 colon carcinoma across all the
`doses of irinotecan examined. In general terms,
`this may reflect the fixing of the sublethal damage
`of one of the drugs by the other or may reflect
`enhancement of one of the drug targets by the
`other drug)~ Exposure to irinotecan may increase
`
`Sandoz Inc. IPR2016-00318
`Sandoz v. Eli Lilly, Exhibit 1102-0007
`
`

`
`62
`
`TEICHER ET AL
`
`inhibition of mammalian glycinamide ribonucleotide formyl-
`transferase. Biochemistry 30:1997-2006, 1991
`13. Teicher BA, Holden SA, Eder JP, et ah Preclinical
`studies relating to the use of thiotepa in the high-dine setting
`alone and in combination. Semin Oncol 17:18-32, 1990
`14. Teicher BA, Holden SA, Jacobs JL: Approaches to
`defining the mechanism of enhancement by Fluosol-DA 20%
`with carbogen of melphalan antitumor activity. Cancer Res
`47:513-518, 1987
`15. Brattain MG, Fine WD, Khaled FM, et ah Heterogene-
`ity of malignant cells from a human colonic carcinoma. Cancer
`Res 41:175i-1756, 1981
`16. Teicher BA, Rose CM: Perfluorochemical emulsions can
`increase tumor radiosensitivity. Science 223:934-936, 1984
`17. Deen DF, Williams ME: Isobologram analysis of x-ray-
`BCNU interactions in vitro. Radiat Res 79:483-491, 1979
`18. Steel GG, Peckham MJ: Exploitable mechanisms in
`combined radiotherapy-chemotherapy: The concept of additiv-
`ity. Oncol Biol Phys 15:85-91, 1979
`19. Berenbaum MC: Synergy, additivism and antagonism in
`immunosuppression. A critical review. Clin Exp hmnunol 28:
`1-18, 1977
`20. Dewey WC, Stone LE, Miller HH, et ah Radiosensiti-
`zation with 5-bromodeoxyuridine of Chinese hamster cells x-
`irradiated during different phases of the cell cycle. Radiat Res
`47:672-688, 1971
`21. Teicher BA, Holden SA, Ara G, et ah Cyclooxygenase
`inhibitors: In vitro and in vivo effects on antitumor alkylating
`agents in the EMT-6 murine mammary carcinoma. Int J Oncol
`2:145-153, 1993
`22. Teicher BA, Korbut TT, Menon K, et ah Cyclooxygen-
`ase and lipoxygenase inhibitors as modulators of cancer thera-
`pies. Cancer Chemother Pharmacol 33:515-522, 1994
`23. Takimoto CH: Antifolates in clinical development. Se-
`min Oncol 24:40-51, 1997 (suppl 18)
`24. Brandt DS, Chu E: Future challenges in the clinical
`development of thymidylate synthase inhibitor compounds.
`Oncol Res 9:403-410, 1997
`25. Gorlick R, Bertino JR: Clinical pharmacology and resis-
`tance to dihydrofolate reductase inhibitors, in, Jackman AL
`(ed): Anticancer Drug Development Guide: Antifolate Drugs
`in Cancer Therapy. Totowa, NY, Humana Press, 1998, pp
`37-58
`
`26. Bertino JR, Kamen B, Romanini A: Folate antago-
`nists, in Holland JF, Frei E, Bast RC, et al (eds): Cancer
`Medicine, vol 1. Baltimore, MD, Williams & Wilkins, 1997,
`pp 907-921
`27. Chu E, Allegra CJ: Antifolates, in Chabner BA, Longo
`DL (eds): Cancer Chemotherapy and Biotherapy. Philadelphia,
`PA, Lippincott-Raven, 1996, pp 109-114
`28. Jolivet J, Cowan KH, Curt GA, et ah The pharmacology
`and clinical use of methotrexate. N Engl J Med 309:1094-1104,
`1983
`29. Taylor EC, Kuhn D, Shih C, et ah A dideazatetrahydro-
`folate analogue lacking a chiral center at C.6, N-(4-(2-(2-
`amino-3,4-dihydro-4-oxo-7H-pyrolo(2,3-pyrimidind)-5-yl)
`ethyl)benzoyl)-L-glutamic acid is an inhibitor of thymidylate
`synthase. J Med Chem 35:4450-4454, 1992
`30. Rinaldi DA, Burris HA, Dorr FA, et al: A