1016 Vol. 6, 1016 1023, March 2000
`
`Clinical Cancer Research
`
`Treatment Regimens Including the Multitargeted Antifolate
`LY231514 in Human Tumor Xenografts
`
`Beverly A. Teicher,1 Victor Chen, Chuan Shih,
`Krishna Menon, Patrick A. Forler, Val G. Phares,
`and Tracy Amsrud
`Lilly Research Laboratories, Lilly Corporate Center, Indianapolis,
`Indiana 46285
`
`ABSTRACT
`The scheduling of antifolate antitumor agents, including
`the new multitargeted autofolate LY231514 (MTA), with 5-
`fluorouracil was explored in the human MX-1 breast carci-
`noma and human H460 and Calu-6 non-small cell lung carci-
`noma xenografts to assess antitumor activity and toxicity (body
`weight loss). Administration of the antifolate (methotrexate,
`MTA, or LY309887) 6 h prior to administration of 5-fluorou-
`racil resulted in additive growth delay of the MX-1 tumor
`when the antifolate was methotrexate or LY309887 and great-
`er-than-additive tumor growth delay (TGD) when the antifo-
`late was MTA. In the H460 tumor, the most effective regimens
`were a M-day course of MTA or LY309887 along with 5-
`fluorouracil administered on the final 5 days. In addition, the
`simultaneous combination of MTA administered daily for 5
`days for 2 weeks with administration of gemcitabine resulted in
`greater-than-additive H460 TGD. MTA was additive with frac-
`tionated radiation therapy in the H460 tumor when the drug
`was administered prior to each radiation fraction. MTA ad-
`ministered along with paclitaxel produced greater-than-addi-
`tive H460 TGD and additive responses along with vinorelbine
`and carboplatin. In the Calu-6 non-small cell lung carcinoma
`xenograft, MTA administered in combination with cisplatin or
`oxaliplatin was highly effective, whereas MTA administered in
`combination with cyclophosphamide, gemcitabine, or doxoru-
`bicin produced additive responses. Administration of MTA
`along with paclitaxel or doxorubicin resulted in additive MX-1
`TGD. Thus, MTA appears to be especially effective in combi-
`nation therapies including 5-fluorouracil or an antitumor plat-
`inum complex.
`
`INTRODUCTION
`MTA2 (N-(4-[2-(2-amino-3,4-dihydro-4-oxo-7H-pyrrolo-
`[2,3-d)-pyrimidin-5-yl)ethyl]-benzoyl]-L-glutamic acid) was dis-
`
`Received 6/8/99; revised 10/26/99; accepted 11/5/99.
`The costs of publication of this article were defrayed in part by the
`payment of page charges. This article must therefore be hereby marked
`advertisement in accordance with 18 U.S.C. Section 1734 solely to
`indicate this fact.
`~ To whom requests for reprints should be addressed. Phone: (317) 276-
`2739; Fax: (317)277-6285; E-mail: teicherbeverlya@1illy.com.
`2 The abbreviations used are: MTA, LY231514; TS, thymidylate syn-
`
`thase; GARFT, glycinamide ribonucleotide formyltransferase; TGD,
`tumor growth delay.
`
`covered 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
`lometrexol, which results in a major shift in activity from the
`inhibition of de novo purine biosynthesis to predominantly the
`inhibition of de novo thymidylate biosynthesis (2-4). MTA is
`an excellent substrate for mammalian folylpolyglutamate syn-
`thase (5) and, in the polyglutamated form with three or more
`glutamyl residues, is a potent inhibitor of the enzymes TS,
`dihydrofolate reductase, and GARFT (2).
`Mice have relatively high concentrations (about 1 p~M) of
`circulating thymidine. Therefore, evaluation of antitumor com-
`pounds with tumors in mice may tend to underpredict both the
`antitumor activity axed toxicity of drugs that inhibit TS as com-
`pared with what may be expected in humans (6, 7). To com-
`pensate for this potential problem in studying antitumor activity
`in mice, MTA was tested using a mutax~t tumor that was thy-
`midine kinase negative, murine lymphoma L5178/TK-/HX, and
`found to be very active (8). MTA was also found to be an
`effective antitumor agent against several human tumor xe-
`nografts 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, feeding a
`low-folate diet axed then repleting the axfimals modulated the
`folate levels in mice by administration of specific doses of folic
`acid (9). Both the antitumor activity and toxicity of MTA could
`
`be modulated in this manner and at certain folate levels, anti-
`tumor activity towaxd specific tumors could be optimized.
`An importax~t component in the development of a new ax~ti-
`cax~cer drug is ax~ understax~ding of its potential for inclusion in
`combination treatment regimens. In recent studies, MTA was tested
`in combination with cisplatin, methotrexate, 5-fluorouracil, pacli-
`
`taxel, docetaxel, doxorubicin, LY309887 [GARFT inhibitor (1, 2)],
`axed fractionated radiation therapy in vivo using the EMT-6 mam-
`maxy caxcinoma, the humax~ HCT 116 colon caxcinoma, axed the
`humax~ H460 non-small cell lung caxcinoma grown as xenografts in
`nude mice (10). Isobologram methodology was used to determine
`the additivity or synergy of the combination regimens. MTA ad-
`ministered with cisplatin, paclitaxel, docetaxel, or fractionated ra-
`diation therapy produced additive to greater thax~ additive tumor
`response by tumor cell survival assay and TGD. Although
`additive tumor response was observed when MTA was adminis-
`tered with methotrexate, synergistic tumor responses were seen
`when MTA was administered with the GARFT inhibitor
`LY309887 or with the topoisomerase I inhibitor irinotecaxL MTA
`was administered in combination with full doses of each ax~ticax~cer
`agent studied, with no evidence of increased toxicity resulting from
`the combination (10).
`In the current studies, MTA was administered alone, in
`combination with standard chemotherapeutic agents, with a
`special focus on scheduling with 5-fluorouracil, gemcitabine,
`and platinum complexes, or with radiation therapy to tumor-
`
`Sandoz Inc. IPR2016-00318
`Sandoz v. Eli Lilly, Exhibit 1104-0001
`
`

`
`Fig. 1 Growth delay of the human H460 non-
`small cell lung carcinoma grown as a xenograft
`in male nude mice after treatment with MTA
`(100 mg/kg) i.p., days (d) 7 13 or days 720;
`methotrexate (0.8 mg/kg) i.p., days 7 13 or
`days 7 20; GARFT inhibitor (LY309887; 30
`mg/kg) i.p., days 7, 10, and 13 or days 7, 10,
`13, 16, and 19; 5-fluorouracil (5-FU; 30 mg/
`kg) i.p., days 7 11 or 16 20 alone or in com-
`binations including an antifolate along with
`5-fiuorouracil administered on days 7 11
`(early) or on days 16 20 (late). Rows, means
`from two independent experiments; bars, SE.
`
`Clinical Cancer Research 1017
`
`MAX. % BODY
`WEIGHT LOSS
`
`~887, d7-13 + early 5-FU
`
`~__~__~ MTX, d7-20 + late 5-FU
`
`~__~__~MTX, d7-13 + early 5-FU
`
`~ MTA, d7-13 + early 5-FU
`
`I-
`Z
`ILl
`
`~
`5-FLUOROURACIL ~ d7-11 (early)
`
`d16-20 (late)
`
`"//’/////////////////////~j--~ d7"13
`
`~ d7-20 MTX
`
`~ d7-13
`
`~ d7-20
`
`~ d7-13
`
`MTA
`
`I
`0 2
`
`I
`4
`
`I
`6
`
`I
`8
`
`I
`I
`I
`I
`I
`I
`I
`1-0 12 14 16 18 20 22 24
`
`TUMOR GROWTH DELAY, Days
`
`5.6
`14.0
`
`12.0
`12.5
`
`8.0
`
`18.0
`
`7.0
`7.0
`
`7.0
`
`1.5
`
`8.0
`3.0
`
`6.0
`5.0
`
`bearing mice to explore the potential interaction of MTA in
`combination anticancer treatment regimens.
`
`MATERIALS AND METHODS
`Drugs
`MTA and LY309887 (GARFT inhibitor) and gemcitabine
`were obtained from Eli Lilly & Co. (2, 11, 12). Cisplatin,
`carboplatin, cyclophosphamide, methotrexate, 5-fluorouracil,
`paclitaxel, docetaxel, vinorelbine, and doxombicin were pur-
`chased from Sigma Chemical Co. (St. Louis, MO). Oxaliplatin
`was purchased from Nescott Fine Chemicals (Santiago, Chile).
`
`Tumors
`The Calu-6 human non-small cell lung adenocarcinoma
`originated from a 61-year old female treated with radiation
`therapy in 1976 (13). The MX-1 breast carcinoma originated as
`a poorly differentiated mammary carcinoma in a 29-year old
`female. Calu-6 cells were purchased from American Type Cul-
`ture Collection (Manassas, VA). The MX-1 breast cnxcinoma
`and H460 human non-small cell lung carcinoma were obtained
`from the National Cancer Institute-Fredrick Cancer Resenxch
`Facility, Division of Cancer Treatment Tumor Repository. Each
`of the tumor cell lines is tumorigenic in nude mice.
`Nude mice, male and female, were purchased from Charles
`River Laboratories (Wilmington, MA) at 5-6 weeks of age.
`
`When the animals were 7-8 weeks of age, they were exposed to
`4.5 Gy of total body radiation delivered using a GammaCell 40
`irradiator (Nordion, Inc., Ottowa, Ontario, Canada). Twenty-
`four h later, MX-1, Calu-6, or H460 tumor cells (5 × 106)
`prepared from a brie of several donor tumors were implanted
`s.c. in a 1:1 mixture of RPMI tissue culture medium and Ma-
`trigel (Collaborative Biomedical Products, Inc., Bedford, MA)
`in a hind leg of the animals. MX-1 tumors grew to 500 mm3 in
`34.7 _+ 2.9 days, Calu-6 tumors grew to 500 mm3 in 19.0 _+ 3.4
`days, and H460 tumors grew to 500 mm3 in 14.0 _+ 0.8 days.
`
`TGD Experiments
`H460 Experiments. Treatments were initiated on day 7
`post-tumor cell implantation, when the H460 tumors were ap-
`proximately 200 mm3 in volume. Animals were treated by with
`MTA (100 mg/kg) i.p. injection on days 7-13 or days 7-20;
`with methotrexate (0.8 mg/kg) by i.p. injection on days 7-13 or
`days 7-20; or with LY309887 (30 mg/kg) by i.p. injection on
`days 7, 10, and 13 or days 7, 10, 13, 16, and 19 alone or along
`with 5-fluorouracil (30 mg/kg) by i.p. injection on days 7-11 or
`16-20. In another experiment, MTA (100 or 150 mg/kg) was
`administered by i.p. injection on days 7-11 and 14-18, on days
`16-22, or on days 16-30 alone or in combination with gemcit-
`abine (60 mg/kg) by i.p. injection on days 7, 10, 13, and 16. In
`a third experiment, MTA (100 mg/kg) was administered by i.p.
`
`Sandoz Inc. IPR2016-00318
`Sandoz v. Eli Lilly, Exhibit 1104-0002
`
`

`
`1018 Treatment Regimens Including MTA in Human Tumors
`
`GEM -> MTA, d16-30
`
`GEM -> MTA, d16-20
`
`MAX. % BODY
`WEIGHT LOSS
`
`25.0
`
`18.0
`
`36,0
`
`19,0
`
`MTA(150)
`td7-11;14-11
`/GEM
`
`MTA(100)/GEM
`d7-11;14-18
`
`3rd d7-16 GEMCITABINE
`
`14.0
`
`~ 100 mg/kg, d16-30
`
`100 mg/kg, d16-20
`
`~ 150 mglkg, d7-11;14-18
`~ 100 mg/kg, d7-11;14-18
`
`MTA
`
`5.0
`
`3.0
`
`12.5
`
`8.0
`
`I
`0 2
`
`I I I I I I I I I
`4 6 8 10 12 14 16 18 20
`
`I I
`22 24
`
`TUMOR GROWTH DELAY, Days
`
`Fig. 2 Growth delay of the human H460 non-
`small cell lung carcinoma grown as a xenograft
`in male nude mice after treatment with MTA
`(100 or 150 mg/kg) i.p., days (d) 7 11 and
`14 18, days 16 22, or days 16 30; gemcita-
`bine (GEM’, 60 mg/kg) i.p., days 7, 10, 13, and
`16 alone or in simultaneous or sequential com-
`binations. Rows, means from two independent
`experiments; bars, SE.
`
`injection on days 7-11 and 14-18 alone or along with oxali-
`platin (12.5 mg/kg) by i.p. injection on day 7; oxaliplatin
`(5 mg/kg) by i.p. injection on days 7 axed 14; cisplatin (10
`m,g/kg) by i.p. injection on day 7; docetaxel (22 mg/kg) by i.v.
`injection on days 8, 12, and 16; paclitaxel (24 mg/kg) by i.v.
`injection on days 8, 10, 12, and 15; vinorelbine (10 mg/kg) by
`i.p. injection on day 8; or caxboplatin (50 mg/kg) by i.p. injec-
`tion on day 8. In the fourth experiment, MTA (100 mg/kg) was
`administered by i.p. injection on days 7-11 and 14-18 or days
`8, 11, 14, and 17 alone or along with fractionated radiation
`therapy (2, 3, or 4 Gy; GammaCell 40, Nordion Inc., Ottawa,
`Ontario, Canada) delivered on days 7-11 and 14-18.
`Calu-6 Experiments. Treatments were initiated on day
`7 post-tumor cell implantation, when the Calu-6 tumors were
`approximately 200 mm3 in volume. Animals were treated
`with MTA (100 mg/kg) administered by i.p. injection on
`days 7-11 and days 14-18 alone or along with cisplatin (10
`mg/kg) by i.p. injection on day 7; cyclophosphamide (125
`mg/kg) by i.p. injection on days 7, 9, and 11; gemcitabine
`(60 mg/kg) by i.p. injection on days 7, 10, 13, and 16;
`doxorubicin (1.75 mg/kg) by i.p. injection on days 7-11;
`oxaliplatin (12.5 mg/kg) by i.p. injection on day 7; or oxali-
`platin (5 mg/kg) by i.p. injection on days 7 and 14.
`MX-1 Experiments. Treatments were initiated on day 7
`post-tumor cell implantation, when the MX-1 tumors were ap-
`proximately 50 mm3 in volume. Animals were treated with
`
`MTA (150 mg/kg) administered by i.p. injection on days 7-11,
`with methotrexate (0.8 mg/kg) administered by i.p. injection on
`days 7-11, with 5-fluorouracil (30 mg/kg) administered by i.p.
`injection on days 7-11, or with combinations of these agents
`administered simultaneously or sequentially, with 6 h between
`drug injections. In ax~other experiment, MTA (100, 150, or 200
`m ,g/kg) was administered by i.p. injection on days 7-11 alone or
`along with paclitaxel (24 mg/kg) administered by i.v. injection
`on days 7, 9, 11, and 13 or along with doxorubicin (1.75 mg/kg)
`administered by i.p. injection on days 7-11.
`The progress of each tumor was measured twice per week
`until it reached a volume of 4000 mm3. Tumor volumes were
`calculated as the volume of a hemi-ellipsoid based on tumor diam-
`eter measurements made using calipers in two dimensions. TGD
`was calculated as the time taken by each individual tumor to reach
`500 mm3 compaxed with the time in the untreated controls. Each
`treatment group included 5 unimals, axed each experiment was done
`twice; therefore the number of unimals per condition was 10. TGD
`times (days) axe the meax~s _+ SE for the treamaent group compaxed
`with those for the control group (14, 15). Toxicity of the treatment
`regimens was assessed using chax~ge in body weight over the
`course of the experiments. Body weights were measured twice per
`week at the same time as tumor diameter measurements.
`All in vivo studies were performed in accordance with NIH
`and American Accreditation Association of Laboratory Animal
`Care guidelines.
`
`Sandoz Inc. IPR2016-00318
`Sandoz v. Eli Lilly, Exhibit 1104-0003
`
`

`
`Table 1 Growth delay of the human H460 non-small cell lung carcinoma grown as a xenograft in male nude mice after treatment with MTA in
`combination with other cytotoxic anticancer therapies
`
`Tumor growth delay (days)
`
`Maximum body weight loss (%)
`
`Clinical Cancer Research 1019
`
`Alone
`
`+ MTA
`
`Alone
`
`MTA (100 mg/kg) d7 11; 14~18~
`Cisplatin (10 mg/kg) d7
`4.0 + 0.3
`Carboplatin (50 mg/kg) d7 2.9 + 0.3
`Oxaliplatin (12.5 mg/kg) d7 5.2 _+ 0.4
`Oxaliplatin (5 mg/kg) d7; 14
`7.7 + 0.7
`Paclitaxel (24 mg/kg) d8, 10, 12, 15
`10.8 + 1.1
`Docetaxel (22 mg/kg) d8, 12, 16
`7.9 + 0.9
`Vinorelbine (10 mg/kg) d7
`5.9 + 0.5
`2 Gy × 10
`10.7 + 0.6
`3 Gy × 10
`13.9 + 1.0
`4 Gy × 10
`23.8 + 1.9
`
`"d, day(s).
`
`+ MTA
`
`3.2 + 0.3
`12.8 + 1.1
`6.6 + 0.5
`6.5 _+ 0.6
`7.9 + 1.0
`14.0 + 1.3
`14.6 + 1.6
`7.6 + 0.8
`12.3 + 0.8
`17.8 + 1.4
`26.7 + 2.2
`
`12.0
`20.0
`8.0
`13.0
`13.0
`27.0
`8.0
`16.0
`16.0
`16.0
`
`8.0
`17.0
`30.0
`15.0
`19.5
`19.0
`36.0
`21.5
`17.0
`17.0
`17.0
`
`MAX. % BODY
`WEIGHT LOSS
`
`0.0
`
`0.0
`
`15.0
`
`0.0
`
`14.5
`
`6.0
`
`15.0
`
`9.0
`
`10.0
`6.0
`
`21.0
`
`10.0
`
`8.0
`
`Fig. 3 Growth delay of the human Calu-6
`non-small cell lung carcinoma xenograft grown
`in female nude mice after treatment with MTA
`(100 mg/kg) i.p., days (d) 7 11 and 14 18;
`cisplatin (10 mg/kg) i.p., day 7; cyclophos-
`phamide (CTX; 125 mg!kg) i.p., days 7, 9, and
`11; gemcitabine (60 mg/kg) i.p., days 7, 10, 13,
`and 16; doxorubicin (1.75 mg/kg) i.p., days
`7 11; oxaliplatin (12.5 mg/kg) i.p., day 7, or
`(5 mg/kg) i.p., days 7 and 14, alone or in
`combinations including MTA. Rows, means of
`two independent experiments; bars, SE.
`
`Z~
`Ua
`
`I-
`
`~ DOXORUBICIN (1.75 mg/kg)d7-11
`
`~////////////,/~ GEMCITABINE (60 mg/kg)d8,11,14,17
`
`CYCLOPHOSPHAMIDE(125
`
`OXALIPLATIN (12.N mg/kg)d7
`
`~ CISPLATIN (10 mg/kg)d7
`
`~MTA (100 mg/kg)d7-11 ;14-18
`
`6 8 10 12 14 16 18 20
`
`22 24
`
`TUMOR GROWTH DELAY, Days
`
`Data Analysis
`For determination of additivity, isobolograms were gener-
`ated for the special case in which the dosage of one agent is held
`constant. This method allowed determination of additive effect
`for different levels of the variable agent (16-20). The radiation
`dose-modifying factor was calculated as the ratio of the radia-
`tion dose required to produce 20 days of TGD in the treated and
`control groups.
`
`Statistical comparisons for the TGD assays were carried
`out with the Duunett multiple comparisons test after a signifi-
`cant effect was found by ANOVA (21, 22).
`
`RESULTS
`
`The doses and schedules for the standard chemotherapeutic
`agents in these studies are standard, widely used regimens for
`each agent. The doses and schedules used for MTA in these
`
`Sandoz Inc. IPR2016-00318
`Sandoz v. Eli Lilly, Exhibit 1104-0004
`
`

`
`1020 Treatment Regimens Including MTA in Human Tumors
`
`MAX, % BODY
`WEIGHT LOSS
`
`~ 25
`
`7.5 18.0
`
`15.0
`
`7.5 11.0 9.5
`
`SIMULTANEOUS
`
`ANTIFOL ->6hr->5FU
`
`a: 15
`
`n- lO
`
`o
`:~ 5
`
`5-FU MTX/5FU MTA/SFU
`
`5-FU MTX->5FU MTA->5FU
`
`TREATMENT GROUP
`
`Fig. 4 Growth delay of the human MX-1 breast
`carcinoma xenograft grown in female nude mice
`after treatment with 5-fiuorouracil (5-FU; 30 mg/
`kg) i.p., days (d) 7 11; MTA (150 mg/kg) i.p.,
`days 7 11; or methotrexate (MTAq 0.8 mg/kg)
`i.p., days 7 11. The antifolate (MTA or MTX)
`was administered simultaneously with each dose
`of 5-fluorouracil, or the antifolate was adminis-
`tered 6 h prior to each dose of 5-fiuorouracil.
`Columns, means of two independent experiments;
`bars, SE.
`
`studies were determined to be optimal for MTA in earlier
`studies (1, 6-8). The human H460 non-small cell hmg carci-
`noma xenograft is a relatively quickly growing tumor tmdergo-
`ing log-linear growth and reaching a volume of about 4000 mm3
`in 36 days post-tumor cell (5 × 106) implantation s.c. in a hind
`leg of male nude mice. Each of the antifolates (MTA, metho-
`trexate, and 309887) produced a duration-dependent TGD in the
`H460 hmg carcinoma (Fig. 1). 5-Fluorouracil was also an active
`antitumor agent against the H460 tumor-producing TGDs that
`were dependent upon the tumor burden at the initiation of
`treatment. For combination regimens, 5-fluorouracil was admin-
`istered on days 7-11 along with each ax~tifolate on days 7-13, or
`5-fluorouracil was administered on days 16-20 along with each
`antifolate in the longer regimen on days 7-20. The most effec-
`tive regimens were the combination of the longer course of
`MTA treatment along with 5-florouracil administration on days
`16-20 axed the combination of the longer course of 309887
`treatment along with 5-fluorouracil administration on days 16-
`20. The least effective combination regimens were those that
`included methotrexate. Administration of 5-fluorouracil eaxly in
`these combination regimens resulted in greater weight loss than
`administering 5-fluorouracil later.
`The antitumor activity of MTA against the H460 tumor is
`dependent upon dose, duration, and tumor burden. In a daily for
`5 days regimen given for 2 weeks, the higher dose of 150 mg/kg
`per dose was more effective thax~ the lower dose of 100 mg/kg
`per dose (Fig. 2). Gemcitabine, administered every 3rd day for
`four doses, was also an active antitumor agent against the H460
`tumor. The simultaneous combination of MTA administered on
`the weekly schedule with administration of gemcitabine resulted
`in greater-thax~-additive TGD of the H460 tumor. However,
`beginning administration of MTA at the completion of the
`gemcitabine regimen resulted in a less effective therapeutic
`regimen. Simultaneous administration of MTA and gemcita-
`bine, although a more effective anticancer regimen, was also a
`
`more toxic regimen as determined by weight loss, especially
`with the higher dose of MTA.
`Antitumor platinum complexes and antitubulin agents axe
`widely used in the treatment of non-small cell hmg carcinoma.
`Combination regimens with MTA axed cisplatin, carboplatin,
`and oxaliplatin were studied. The antitumor platinum complexes
`cisplatin and caxboplatin were administered once on the first day
`of the treatment regimen, axed the ax~titumor platinum complex,
`oxaliplatin, was administered once or once per week for 2
`weeks. Although, among the antitumor platinum complexes
`tested, oxaliplatin produced the greatest TGD in the H460
`xenograft, the combination regimen of MTA and cisplatin pro-
`duced the greatest TGD (P < 0.01; Table 1). Paclitaxel was a
`more effective single agent than docetaxel or vinorelbine against
`the H460 tumor. The combination regimen of MTA with simul-
`tax~eous administration of paclitaxel or docetaxel produced a
`greater TGD than the combination of MTA with vinorelbine.
`Each of the combination regimens was more toxic than the
`single-agent treatments. Cisplatin and oxaliplatin were less toxic
`in combination with MTA than was carboplatin, and paclitaxel
`was less toxic in combination with MTA than was docetaxel.
`Radiation therapy is also widely used for the treatment of
`non-small cell hmg carcinoma. Radiation therapy was delivered
`locally to the H460 xenograft tumor-bearing limb in fractions of
`2, 3, or 4 Gy administered daily for 5 days for 2 weeks.
`Radiation therapy produced increasing TGD with increasing
`dose of radiation (Table 1). The effect of adding treatment with
`MTA to fractionated radiation therapy was investigated. MTA
`administration enhanced the TGD produced by radiation therapy
`and did not add to the toxicity of the radiation therapy.
`The Calu-6 non-small cell hmg carcinoma was grown as a
`s.c. xenograft in female nude mice. MTA was an active antitu-
`mor agent against the Calu-6 tumor. Combinations of MTA with
`the antitumor platinum complexes cisplatin and oxaliplatin were
`tested, with the antitumor platinum complex being administered
`
`Sandoz Inc. IPR2016-00318
`Sandoz v. Eli Lilly, Exhibit 1104-0005
`
`

`
`Clinical Cancer Research 1021
`
`MAX. % BODY
`WEIGHT LOSS
`
`MTA
`
`(200 mg/kg)/DOX
`
`MTA (150 mg/kg)/DOX
`
`MTA (100 mg/kg)/DOX
`
`~ DOXORUBICIN (1.75 mg/kg)d7=11
`
`Fig. 5 Growth delay of the human MX-1
`breast caxcinoma xenograft grown in fe-
`male nude mice after treatment with MTA
`(100, 150, or 200 mg/kg) i.p., days (d)
`7 11; paclitaxel (PAC; 24 mg/kg) i.v., days
`7, 9, 11, and 13; doxorubicin (DOX; 1.75
`mg/kg) i.p., days 7 11, alone or in combi-
`nations including MTA. Rows, means of
`two independent experiments; bars, sSE.
`
`__ MTA(200mg/kg)~
`PAC
`
`MTA (150 mg/kgyPAC
`
`MTA (100 mg/kg)/PAC
`
`PACLITAXEL(24 mg/kg)d7,9,11,13
`
`~ 200 rng/kg, d7oll
`
`150 mg/kg, d7-11
`
`MTA
`
`100 mg/kg, d7-11
`
`0 4
`
`8
`
`12 16 20 24 28 32
`
`36 40
`
`TUMOR GROWTH DELAY, Days
`
`15.0
`
`11.0
`
`9.0
`
`5.0
`
`23.0
`
`21.0
`
`14.0
`
`1.5
`
`10.0
`
`8.5
`
`6.0
`
`simultaneously at the beginning of the MTA treatment course
`(Fig. 3). Treatment regimens including MTA with either cispla-
`tin or oxaliplatin were highly effective, producing greater-than-
`additive TGD of the Calu-6 tumor (P < 0.01 for both cisplatin
`and oxaliplatin combinations). The antitumor alkylating agent
`cyclophosphamide was a highly effective antitumor agent
`against the Calu-6 tumor. The simultaneous combination of
`MTA treatment and cyclophosphamide was additive against the
`Calu-6 tumor. Gemcitabine was also am active single agent
`against the Calu-6 tumor. The simultaneous combination of
`MTA and gemcitabine was additive in TGD. Doxorubicin was
`an active single antitumor agent against the Calu-6 tumor.
`Combination regimens, including MTA with doxorubicin, were
`highly effective treatments against the Calu-6 tumor. Mice bear-
`ing the Calu-6 tumor were less prone to weight loss by the
`combination regimens than mice bearing the H460 tumor; how-
`ever, in each case, the combination regimens were more toxic
`thax~ the single-agent therapies.
`The MX-1 human breast carcinoma was grown as a s.c.
`xenograft in female nude mice. MTA (150 mg/kg) administered
`by i.p. injection on days 7-11 produced a TGD of 3.0 _+ 0.3
`days in the MX-1 tumor. Methotrexate (0.8 mg/kg) administered
`by i.p. injection on days 7-11 produced a TGD of 2.8 _+ 0.3
`days in the MX-1 tumor. 5-Fluorouracil (30 m,g/kg) adminis-
`
`tered by i.p. injection on days 7-11 produced a TGD of 7.5 _+
`0.5 days in the MX-1 tumor (Fig. 4). The simultaneous combi-
`nation of methotrexate and 5-fluorouracil resulted in antagonism
`between the two agents, whereas the simultaneous combination
`of MTA and 5-fluorouracil produced additive TGD in the MX- 1
`tumor. When the administration of methotrexate preceded 5-fluo-
`rouracil treatment, an additive TGD for the combination was
`observed. On the other hand, when the administration of MTA
`preceded 5-fluorouracil treatment, a greater-than-additive TGD
`resulted (P < 0.01). The combination treatment regimen of
`MTA followed by 5-flurorouracil was most effective in maxi-
`mizing tumor response and produced less weight loss than the
`other combination regimens; thus, this sequential regimen had a
`better therapeutic index than the other combination treatments.
`Administration of MTA over a dosage range resulted in
`increasing TGD with increasing dose of MTA in the MX-1
`tumor (Fig. 5). Paclitaxel is a very effective single agent against
`the MX-1 tumor. The simultax~eous combination of MTA and
`paclitaxel resulted in additive TGD of the two ax~titumor agents
`over the dosage range of MTA. Doxorubicin was am active
`antitumor agent against the MX-1 tumor. The simultaneous
`combination of MTA and doxorubicin resulted in additive TGD
`of the two agents, with increasing TGD with increasing dose of
`
`Sandoz Inc. IPR2016-00318
`Sandoz v. Eli Lilly, Exhibit 1104-0006
`
`

`
`1022 Treatment Regimens Including MTA in Human Tumors
`
`MTA. There was increasing weight loss in the paclitaxel and
`doxorubicin regimens as the dose of MTA was increased.
`
`DISCUSSION
`The folate pathway continues to be a tayget for anticancer
`drug development because it is vital to cell survival and appea~s
`to be one of the few aspects of cellular metabolism in which
`there is little or no redundancy. A major challenge in the clinical
`application of antifolates comes in establishing a therapeutic
`index for these agents in tumor cells versus sensitive normal
`tissues such as the bone marrow. One possibility for increasing
`therapeutic potential is to develop treatment regimens in which
`the antifolate is used along with another anticancer agent, re-
`sulting in a greater effect on the tumor cells than on the normal
`tissues. The schedule dependence of in vivo treatment combi-
`nations of methotrexate and 5-fluorouracil were recognized in
`the 1970s using the marine C3H mammary adenocarcinoma,
`where administering the methotrexate 6 h prior to 5-fluoroaracil
`resulted in the greatest tumor response (23). These early studies
`were confirmed and extended to marine colon 38 and several
`human colon carcinoma xenografts, where it was determined
`that administration of the antifolate 4-8 h prior to the 5-
`fluoroaracil maximized tumor cell killing without increasing
`bone marrow toxicity from that of the antifolate alone (24-26).
`In the human MX-1 breast carcinoma xenograft, the same pat-
`tern pertained whether the antifolate was methotrexate or MTA;
`however, the increased tumor response obtained when MTA
`preceded 5-fluoroaracil was much greater than that obtained
`with methotrexate. The pharmacological interaction between
`5-fluoroaracil and an antifolate has been the subject of many
`studies (27). Two biochemical mechanisms have been put for-
`ward to account for the observed additivity. The first relates to
`increased 5-fluoroaracil metabolism to the 5-fluoro-UMP
`through condensation with phosphoribosyl PPi, a metabolite that
`accumulates following the inhibition of de novo purine biosyn-
`thesis. The second relates to enhanced TS inhibition through
`formation of a ternaxy complex consisting of TS, 5-fluoro-
`dUMP and an antifolate. These mechanisms were thought to be
`the origin of the observed potentiation of 5-fluorouracil by
`methotrexate (24, 26) and raltitrexed (28, 29), respectively.
`Given the ability of MTA to inhibit TS, dihydrofolate reductase,
`and GARFT, this ax~tifolate could possibly modulate the activity
`of 5-fluorouracil by both of these mechanisms. However, in this
`regard, direct biochemical ax~alysis has yet to be performed on
`the tumors treated in this study. Nevertheless, the biological
`effect of increased tumor response was clear (Fig. 4). Alterna-
`tive treatment regimens that included an antifolate and 5-
`fluorouracil were tested in the human H460 non-small cell lung
`carcinoma; in these regimens, more extended treatments were
`administered with the antifolate, and 5-fluorouracil was admin-
`istered early or late in the coarse of the antifolate treatment.
`Under these conditions, initiation of the administration of the
`5-fluorouracil 10 days into the 14-day regimen resulted in the
`greatest tumor response with each of the three antifolates
`(Fig. 1).
`A similar scheduling effect was seen with MTA and gem-
`citabine; that is, a greater antitumor effect was obtained when
`
`MTA was given along with gemcitabine than if gemcitabine was
`administered prior to MTA (Fig. 2).
`Gemcitabine has been shown to be a potent radiation
`sensitizer both in vitro and in vivo (30-36), whereas in the
`human H460 non-small cell lung caxcinoma xenograft, MTA
`was additive with radiation (Table 1). MTA has been tested
`previously in combination with radiation therapy against the
`human HCT116 colon carcinoma xenograft axed found to have
`an additive effect with radiation in that tumor (10). MTA also
`had a primaxily additive effective in combination with fraction-
`ated radiation therapy against the human H460 non-small cell
`lung carcinoma. The limitation of the application of this finding
`to clinical trial may be the scheduling of the MTA administra-
`tion along with the radiation therapy, because MTA was admin-
`istered prior to each radiation fraction.
`Combination of MTA with each of the antitumor plati-
`num complexes (cisplatin, carboplatin, and oxaliplatin) re-
`sulted in additive to greater-than-additive tumor response in
`both the H460 and the Calu-6 non-small cell lung carcinoma
`xenografts. In these regimens, the antitumor platinum com-
`plex was administration as a single dose along with the first
`dose of MTA (Table 1 and Fig. 3). It also appears that with
`careful scheduling, MTA can be highly effective when ad-
`ministered along with antitubulin agents, including paclitaxel
`and docetaxel. Although many of the combination regimens
`produced greater weight loss in the animals than did the
`single agents, especially in combinations with 5-fluorouracil,
`increased antitumor activity appeared more important than
`the weight loss.
`From these preclinical in vivo results, it may be concluded
`that MTA can be combined with other anticancer therapies to
`therapeutic advantage. Given the prolonged terminal half-life in
`patients, the scheduling of MTA with other agents could possi-
`bly be adjusted to sequential days without loss of the beneficial
`therapeutic interaction between the agents.
`
`REFERENCES
`1. Shih, C., mad Thornton, D. E. Preclinical pharmacology studies and
`the clinical development of a novel multitargeted antifolate, MTA
`(LY231514). In." A. L. Jackman (ed.), Anticancer Drug Development
`Guide: Antifolate Drugs in Cancer Therapy, pp. 183 201. Totowa, NJ:
`Humana Press Inc., 1998.
`2. Shih, C., Chen, V. C., Gossett, L. S., Gates, S. B., MacKellar,
`W. C., Habeck, L. L., Shackelford, K. A., Mendelsohn, L. G., Soose,
`D. J., Patel, V. F., Andis, S. L., Bewley, J. R., Rayl, E. A., Moroson,
`B. A., Beardsley, G. P., Kohler, W., Ratnam, M., and Schultz, R. M.
`LY231514, a pyrrolo [2,3-d]pyrimidine-based antifolate that inhibits
`multiple folate-requiring enzymes. Cancer Res., 57. 1116 1123,
`1997.
`3. Rinaldi, D. A., Burris, H. A., Dorr, F. A., Woodworth, J. R., Kuhn,
`J. G., Eckardt, J. R., Rodriguez, G., Corso, S. W., Fields, S. M., Langley,
`C., Clark, G., F~xies, D., Lu, P., and Von Hoff, D. D. 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, A. C., Vasey, P. A., Adams, L., Walling, J., Woodworth,
`J. R., Abrahams, T., McCarthy, S., Bailey, N. P., Siddiqui, N., Lind,
`M. J., Calvert, A. H., Twelves, C. J., Cassidy, J., and Kaye, S. B. A
`Phase I and pharmacokinetic study of LY231514, the multitargeted
`antifolate. Clin. Cancer Res., 4. 605 610, 1998.
`5. Habeck, L. L., Mendelsohn, L. G., Shih, C., Taylor, E.