PROCEEDINGS
`
`at‘
`
`«.1
`
`Eighty—Third ‘§1___1nual Meeting
`
`of the American Association
`
`for Cancer Research
`
`May 20-23, 1992
`
`San Diego, California
`
`Volume .33
`
`March 1992
`
`Lilly Ex. 2049
`Sandoz v. Lilly IPR2016-00318
`
`

`
`PRECLINICAL PHARMACOLOGY/EXPERIMENTAL THERAPEUTICS
`
`2421
`
`Biological properties of (68)-, (6R)-, and (6R,S)-leucovorirt.
`McGuire, ].]., Russell, C.A., 8: Heltzman, l<.]. Grace Cancer Drug
`Center, Roswell Park Cancer Institute, Buffalo, NY 14263
`(6R,S)-leucovorin (LV) is used to selectively "rescue"> the toxic
`effects of methotrexate (MTX) and to biochemically modulate the
`anti-tumor effects of fluoroplyrlmidines (FPs).
`(65)-LV is
`biologically active, while (6R)- V is assumed to be inert. We
`compared the biological pr
`rtlesof ure fire
`rations of these
`isomers, with particular em
`is on
`eir a ii? to form ol (7-
`Iutam‘yl) metabolites.
`(
`LV was 24000-fol more ab e t
`6R)-L to rotect CCRP-CEM cells from the growth inhibitory
`effects of
`' (65)-LV modulated theGfirowth inhibito
`activity
`of i-‘P5, while at an equivalent levels (
`)-LV did not. 69- and
`(6R,S)-I..V were substrates for human and rat foiylpoly Iutamate
`synthetase (FPGS); (GR)-LV was essentially inactive
`th as a
`substrate and inhibitor of these FPGS. Lack of conversion to
`polyglutamates may thus contribute to the diminished biolo cal
`activity of (6R)-L . After nonenzymatic conversion o l..V
`pre
`rations to their respective 10-formlyltetrah drofolate forms,
`all orms were FPGS substrates; the VI
`of
`e natural isomer
`was only 2-fold hi her“ than that of ‘ate unnatural isomer.
`Stereospecificity of
`S at C-6 of reduced folates is thus
`e
`rofoundl d
`d
`ton the
`e of 1-
`ho
`bstitu t.
`Ken enand
`car
`ti su
`en
`upported y C
`
`2422
`Improved clinical tolerance of loinetrexol with oral folic acid.
`Young, C.W., Currie, V.E Muindi. .l.F., Saltz, l..B., Pisters, K.M.W.,
`Esposito, A..l.. and
`lie. R.W.’, Memorial Sloan-Kettering Cancer
`n .
`-
`lcedntiiggw York.
`10021, and ‘Eli Lilly and Co., lndianapolis,
`Lomettexol (LTX , an inhibitor of GAR transfortnylase that binds
`tightly to cellular olate binding‘protein (FBI? produced severe
`cumu ative toxicity in its initial
`inical trial ( roc. ASCO 9:76. 1990).
`In mice folate depletion increases the toxicity of LTX 100-fold; the
`theta utic index is restored by oral folic acid supplementation (Ptoc
`AAC 32:324. 1991). Nineteen of 53
`tients in our i.v. dose-finding
`trial received LTX in combination wit oral folic acid. -Dose-limiting
`toxicities were thro
`openia and anemia; mucositis and diarrhea
`were also observed. Wit out folic acid, the MTD of LTX was 2.7
`mg[m2 twice weekly x 4 doses, every 28 to 35 days; cumulative
`toxicity was observed. With oral folic acid at 1 mg/day continuously,
`the recommended Phase ll dose is 4 to S mg/m2 twice weekly it 4
`‘
`doses at 28 day intervals; cumulative toxicity is absent. A patient with
`oropharyngea cancer has had a Complete
`es onse of 18 months
`duration: lesser antitumor effects were seen in _ additional patients.
`Concomitant oral folic acid enhances tolerance to the toxic effects of
`lometrexol without eliminatin therapeutic response to the drug.
`This effect could be mediated
`a decrease in enterohepatic
`reabsorption of LTX following its biliary secretion.
`
`2423
`Development of a fluorescence HPDC method for analysis of
`loinetrexol in biologic samples. Muindi, .I.F.,;Cutrie, V.E., Shih, C.‘
`and Young, C.W. Memorial Sloan-Kettering Cancer Center, New
`n .
`-
`'
`IY%l'l(.4l:2Y851lX)21, and ‘Lilly Research Laboratories, Indianapolis,
`Lomettexo 5.10-dideazatetrahydrofolate (LTX . an inhibitor of
`GAR tr
`ormylase that binds ti htly to folate inding proteins
`(FBP). is undergoin clinical trial
`cause of its broad activity
`against trans lant
`turnorsin mice. We are developing a sensitive
`and s eciflc
`IE method for quaiititation of LTX an possible
`meta olites in biologic fluids. Oxidized l0—deaza-pteridines are
`intensely fluorescent but N-10 pteridines and reduced 10-deaza-
`teridines have negligible fluorescence.’ l.TX in plasma or urine is
`reed from FBP by incubation in armnoruum fotmate pH 3, then
`extracted by perchlorate precipitation. The protein-free extract is
`oxidized by exposure to man anese dioxide at 90°C for 1Q minutes
`then chtomatographed on a 18 reverse hase column using
`fluorescence detection with excitation at 30nm and emission at
`425nm. ln human serum and urine the assa is linear between 0 and
`1000 nM; the lower limit of quantitation is
`nM. The within day
`and between day coefficients of variation have been < 10% and <
`has con irmed dru accumu ation and slowed clearance rates
`15% res ectively. Study of patients treated on Days I, 4. 8, and 11
`between Day 1 an Day 11.
`'
`
`2424
`ICI D1804 resistant col Ines. AL Jaeltman. LR. Kelland. M. Brown. W. Gibson.
`rt. Kltnbel. W. Atiame+ and LR. Judson The lnstlute of cancerliasserch. Staten
`U.K. +Dlv.BlotI'ledleal Ros. University of Surrey, Guldlord. U.K
`Three ed lhse. the mouse L1210 letltamh and the huttun CH1 and 41!!
`warlan lines (IO, -10. asand 13nM). were made resistant (step-Mae heremems
`ioeooonu) to the thymidytate synthase (TS) lnhlblot. ICI 0169!. T3 activity was
`raised slgillicantty In the Cl-lt:R the (3.5-told). An ELISA method confirmed a
`raised level of the enzyme protein (-9-fold). Alterexposure to o.1uM ‘H lcl D1894
`(¢u.) tor 24hrs. Oi-l1:F| cells accutndatod normal levels of ’H (~3uM) but whereas
`In the parental cells the polyglutaimte derivatives illllw Dfodomlnated (aim) in
`the G-l1:R eels gluu were the major fractions (70%). The Kl vdusa for gtu. and
`du, were unchanged tor Ts extracted irotn this the (a2nM and 1.2nM
`respectively). Cross-resistance to Fdurd (25-told) suggests that the small elevation
`inTSlevaltnaybeattIa]orm‘achatiiunollGI D1694 tesiatancelnthlstheandtho
`cl'angelnpolygIuemateptolllenIinyaugniet1ttliis(uu,le ~3-foldlessadiveand
`do. and glu. ~2-lcld more active than gu, against T8).
`In the 41M:R eels
`decreased uptaltevlaths reduced-loletecartlet lathe mechanletnotteslstanceto
`ICI Dtwd end alhough potyglutamatlon is reduced as I result of decreased
`uptake the pattern at polyghitanuites Is not slutllcently changed iprodotnlnorlly
`glu“). TheL12to:RlnehasaamaI decreasolntl\a|t\ltlaIvelocltyforI.i:itakeol
`I01 01694 but ls tnalottnechanlstnoi resktanools Istalureto polygluatnatelcl
`D1694. Exposure oi parental and L12to:R cells tor tens to _1uM lcl D1694
`resulted In ~0.35uM wt in both cell lines but only the parental cells iotmed
`polyduamales (~11uM). cross-resuance studies with antlolates of defined
`tmdedactbnconlkmmeproposedmectwlumdteelstancelotfliesecellhes
`
`2425
`Polyglutarnetlon ot the thymldylate eynthnu (TS) Inltlhlter, Icl mesa AL
`Jackman. W. Gbson‘. T.C. Stephens‘ and F.T. Boyle‘. Inst. cancer lies. Stilton.
`UK. *lC| Pltarmaceutleals, Aldetley Park. Ilaccleelleld. Cheshire. UK
`.
`I01 D1694 Isequlnazolhe entlolatethataetsvlalntilbllon oiTS souls
`in clinical study. The potent activity oi the drug against L121o eels (IC..=anM)
`leduetomegoodceIiIatqita1tevIad'neredt.iced-tola1ecamet(liFc) and rapid
`inttaceldar polyglilarnatlon (Jadtnnn at H. Cancer Res. 5125619-6588. I991).
`PolyuutamatlonollclD1694hesbeetittIeasuedh1hehu'nancelIInes.W1L2.
`tymphoblastold (IO,-6nM) and MGF-7 breast (I<_:,,=o.7nM). Csllswore exposed
`to1oonM’H lamest (gtu.)endaI1ttieeeuuu’Hwastoundesgu., Altar
`aomlns -1uM‘l-ldtugoqulvelems wotolotnd lntrecelluhttywlh 1% and eoxgu,
`respectively (d and glu, being the respective malor metabolites). At 4hl8 <1o%
`of the celhlar»
`was git. it both cal lites (W112 - du,-3%. glu,-18$.
`t!U.~2295. ‘Ulla-52%. 9||la"%) ("(7-7 = d||a"995. GU.-40%. Dl|l4"20%.
`du.-22%). By 24 hrs there was -iouu ‘H lnttacelulatly and glu. predominated
`In both cell lines (w1L2 -60% and MGF-1 -55%). WIL2 cells incubated lord hrs
`iolltmodbyzflitshdnig-lteemedlumdld tiotloseaslgtiliicntitattiinointofi-I.
`Ttmpdygltlamationuwoaasamectiamstntotdmgaceumletlonuidretetllui ..
`atidhviawofttnpotomT3kiiI:ltoryactl~AtyoithopolygltIamates(du.
`I0-60nM: giu.- 1nM). we conclude that IGI D1694 acts by metabolism to
`polydmatmtos. 5l.IJp0l‘lll|fl this Is the poorer youth lnhlbllcn of two analogues
`(7-CH, and 2-hl-l,) of I01 D1694 (~150- and some rospectwely) desple being 6
`and 15-fold better T8 lnhbltors
`studies indicate that the 7-CH, compound
`cannot be polyduatnated and that the 2-NH. compound Is pclyduettutad less
`rondllyduoto poorceluhruptake. If0$lfl0lI8l9.|ll.l'Olol.llOthORFC.
`
`2426
`PtmeolIhloIIcID1Dl:enonlItynldyIeueylIlIeohIIiiior.Gatkes.‘.
`Wad J.‘, Flaming A‘. Splers J.’, Smith I1’. Vetwel] J.‘ and Judson I.‘ 1. Inst of
`cancer Res. Sutton. surrey. UK. 2. Rotterdam cancerlnst. Rotterdam, Holland. 3.
`I01 Pha-maeetllcals. Macclssiteld. Cheshka. UK.
`lcl D1694 lsewatersolibletnilnazollnabased tttymldylete synthase ('I'S)lnhltillcr.
`ltsptedocassoi.O88717stiomdunLunnoutactNity.hnmmutdevdopnnnwas
`halted because of liver and kidney toxicities due to poorwetet solubility oepsclnly
`atacidpfl. Thophaaelstudyoilcl D1694begancn27/2/91 attheliloyal
`Marsden Hospital
`(RMI-I) using a constant lv hiuelon over 15 min. 41 3/52
`Patient: live been treated I doses of 0.1. 0.2. 0.4. 0.6. 1.0, 1.6. 2.6 and
`a.5tng/tn’. 27 pts (mm 23. Rotterdam 4; tall, BF: mean age 57: ECOG Ps 0:11,
`1:13. 2:3) have received 51 courses (median 2. range 1-6). Tumour types include
`colon9.aarcoma4. heodand neck4.mesothsllotna3.oteeat2. ovaty1.glloma
`1.tileductt.catchold1ondstomech1. Mueosnlshasbesnseenh4pts(Wt-I0
`grade 1:2. 22). at doses 0.4, as. 1.0 and 1.6tng in‘. One patient had a tall In
`EDTA clearance of 25%l'flUotlOdt:|Be oi0.4mg/ which persisted after stopping
`tiaatrnentatatlmeotgetisraltseddlscaaeprogresdcn. Nosutisequentpttnshad
`atalllntemthinctbnoneptdevelopedgradezanaemhmdleucopeivatmt
`alter a 2nd dose at 2.6mg/m‘. 2 pts (at 1.6 and zsmg/in’) have developed
`asymptomatic abtiotmalnlas of liver lmctlon which roqura further lnvestlfitlon,
`both have progressive disease. Ptnrrmeotthetlc studies show a linear dose/auc
`relationship and clearance conforms to a 3 compartment model. No responses
`haveystbeenobsetvodln24ovaIuabiepts TheM'l’Dhasnotyetbeenreached.
`mhunvtwuuppomdbyvnculeuflneuohcurpalgnutdhtlmunnoeimeah.
`
`PROCEEDINGS OF THE AMERICAN ASSOCIATION FOR CANCER RESEARCH
`VOLUME 33 0 MARCH- 1992
`406
`
`Lilly Ex. 2049
`Sandoz v. Lilly IPR2016-00318