Canter Trmtnzent Rrvizuxr {I990} 17, lO9~l 17
`
`In vitro and in viva evaluation of US-NCI .5 Q t:
`compounds in human tuxnor xenografts
`.
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`xiv
`Heinz-Herbert Fiebig,* Dietmar P. Be:-ger,* Bernd R. Winterhalthiwhad 0’
`Jacqueline Plowmanf
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`*DepartmerzI of Internal 4-Medicine, Uniz:ers1'l): ofFm'Imrg, fiug5ielIer.rlrzu5r 55, 1)~7800
`Freibwg, I".R.G., and 'fI)evelo/Jmenlal T/zerapeulics Prrigram, 1)ivi.ti0n
`(farmer Trmtment,
`Naliorstzl (.}271(er Inxlliute, Britfzes/In ./MD 2(}8.92_. I/'..5‘..-l.
`
`Introduction
`
`The search for new drugs with antineoplastic activity or analogs of established cytostatic
`8
`Y
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`dru s with inctreaserl etliv;-ac and reduced toxicity was the ma'or ob’:-ctivc nfUS-National
`(lancer Institute screening p1”0_]CClS. Between l975 and 1985 novel agents were tested in a
`‘compound-oriented’ screening system based on initial in viva testing in the mouse leukemia
`P388 and subsequent studies in a panel offivc murine and three human tumor xenografts
`(10%. This screenin
`ro ram was successful
`rimaril ’ in idexltifvin
`com ounds with
`,
`S P
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`clinical activl: ‘ a ainst leukemias and lvm humus r’l6‘.
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`Since 1985, the National Cancer Institute ('N(,‘l'} has develo ed a new ‘dnseasooricnted’
`l P
`E
`gs
`}
`P
`at ) math to dru} screenin
`based on human rumor cell
`line )an(*ls re resemative of
`partrcular tumor types (2). The objertive of this type. olstirer-rnirrg is to idemily compounds
`which exert selective effects on particular tumor types and I0 lbllow-up these leads in rizro
`utilizing cell lines previously shown to be sensitive.
`We have tested 28 com )0unds which dis la ed a(:ti\-‘it’ in the old or the new NC!
`I
`P
`Y
`3
`primary screen in a combined 17: vzlra/‘m zrivo secondary screen using human tumor xeno-
`grafts. First, large scale tests were performed in the clonogeuic assay. Only the most
`sensitive tumors were subscq uently studied in nude mice, where the in who pharmacological
`behariorrr ofa drug is considered,
`
`Methods
`
`Our in zzifw and in aim test procedure has been described recently [6—8';~. Human tumors
`established in serial passage in nude mice were used for all experiments. The human
`origin ofthc tumors was confirmed by isoenzymatic and immunohistochemical methods.
`Tumor models were selected from a panel of 220 well characterized, regularly growing
`xenografts (9).
`New compounds were studied in vitro for anticancer activity in human tumor xeuografts,
`human bone marrow (CFU—GM) and in the leukemia P388 using a modification of the
`0305-~7372."9(ll2&f4l()9+09 SO3.(}(li0
`1990 Academic Press Limited
`
`109
`JTX 168 Par Pharm., Inc.
`Case lPR2016-00084
`
`Novartls v. Breckenridge,
`Roxane, Par, C.A. Nos.
`14-1043, 14-1196, 14-1289
`
`Exhibit 1034
`
`Par Pharm., Inc. v. Novartis AG
`
`Ex. 1034-0001
`
`Novartis v. Breckenridge,
`Roxane, Par, C.A. Nos.
`14-1043, 14-1196, 14-1289
`
`JTX 168
`
`Ex. 1034-0001
`
`

`
`H0
`
`H.-H. FIEBIG ET AL.
`
`clonogenic assay as described by Hamburger & Salmon (1 I}. The most sensitive tumors
`were subsequently studied in Lrivo. Prirnary in Liitra screening was done in {our highly
`sensitive xenografts (small cell and large cell ofthe lung, breast and SIOfl1'clCl1:_l, two resistant
`xenografts as well as the P388. Secondary in vitro screening was performed in a total of M
`responsive and six resistant human tumor xenografts and in two to four marrow specimens.
`Compounds with a greater or similar effect on tumor cells in comparison to human bone
`marrow were subsequently studied in visa in the two most sensitive xenografts transplanted
`subcutaneously into nude mice. The comparison of in mftro/in trivo activity enabled assess-
`ment ofthe relevant in zzitro dose based on in vino pharmacological behavior ofa compound.
`if remission or at least no change was observed in viva, the new compound undergoes
`disease—oriented testing usually in 40-60 xenografts. Drugs were applied by continuous
`exposure until the end of the experiment. A compound was considered active, ifit reduced
`colony formation oftreated {T} groups to 30% or less of the control
`group value.
`For in ma experiments 6-8-weel-;—0ld female zitliymic nude mice of NMRI genetic
`background were used. Tumor slices averaging 33 ><3><0_5~I mm in diameter were
`implanted subcutaneously into both flanks ofthe animals. Treatment was started after 2--
`6 weeks when the median tumor diameter was 6—7 mm. The antitumor effect was evaluated
`
`in non-regressing tumors after 3~l weeks. Data
`tumor regression.
`following maximal
`evaluation was performed using specifically designed software. Relative tumor size [R'l‘S}
`values were calculated for each single tumor by dividing the tumor size day X by the
`tumor size day 0 at the time ofrandomization. Median RTS values were used for further
`evaluation. Tumor doubling time (Di) of test and control groups was defined as the
`period required to reach a relative tumor size of 200%. The efiect of treatment was
`classified as complete remission {RTS on day 2l or 28 < 10% of initial value}, partial
`remission rfl l—50‘j4',), minimal regression (51-75?/(,), no change (76—l24:“trc;'.,j or progression
`2 lQI'7",-ii, A tumor was considered to be sensitive, ilregression or no change was achieved.
`Additionally, tumor inhibition was evaluated in contparing the relative tumor size of
`treated with the control group. The specific growth delay {SG ') was calculated with
`regard to the tumor doubling time (UT) as described by Steel (17).
`
`Results and discussion
`
`Twenty-eight compounds of interest which have emerged from NC! primary screening
`were tested in vitro and in vino. A summary of the activity in human tumor xenografts in
`the clonogenie assay and in nude mice is given in Table 1. Results for hepsulfam, 4-
`ipomeanol, oxanthrazole, penclomedine, pyrazine diazohydroxide and raparnyein are
`given in detail below.
`
`Hepsulfam ( NSC-32.9680}
`
`The l,7-heptanediol—bis—sulfarnate (Figure 1) was synthesized in an attempt to improve
`the antitumor efficacy ofbusulfan through introduction ofa more polar leaving group.
`Hepsulfam showed a broader preclinical aeti
`'ty than busulfan in the NCI in viva screening
`systems. Schedule dependency studies determined a single i.p. bolus injection as the most
`effective administrzttion method. Cross-resistance of melphalan and cisplatimresistant
`P388—sublines to liepsullarri was observed in z;ia~o (18).
`Hepsulfam and busulfan were tested simultaneously in human solid tumor xenografts in
`
`NPC02232985
`
`Ex. 1034-0002
`
`

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`Ex. 1034-0003
`
`

`
`. FIEBIG ET AL.
`
`/CHzCH;~ NH—CHzCH;-OH
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`
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`thlpomeanol (NSC 349438)
`
`Penclomadirse (NSC—338720)
`
`N
`
`-No
`
`(/1\N N:.:N-—-OH
`
`Pyrazine Dsazohydroxide
`(NSC-365456)
`
`Ropamycin (NSC-226080)
`
`FfL'un> 1. Chemical structures ofsclcrrcd US-NCI mmpounds.
`
`zxirro and in Z7‘!-Zll(J4 In the clonogenic assay, both compounds showed broad spectrum activity
`and :1 similar response profile. Howcvcer, given the same dose level all pg/ml at continuous
`drug exposure {Table 2] , hepsulfam was active in 6/19 xenografts {32‘j/0) whereas busulfan
`reduced colony growth to a T1/C €
`30% in 2210 tumors (‘20%).
`In zrivo both compounds were tested against the large cell lung cancer xcnograft LXFL
`52‘). At 21 dose level of I50 mg/kgiday given day l i.p‘, busulfan therapy resulted in ‘no
`change’ on day ‘ll. Hepsulfam-treated tumors regressed completely on day 21 and did
`not regrow within the observation period of 70 days (Table 4). Further tests will be
`performed with this compound.
`
`NPC02232987
`
`Ex. 1034-0004
`
`

`
`E\"1‘&l,l.l.-\TlOl‘C OF US-~NCl
`
`(I().\1l-’(JifI\’1JS
`
`Table 2.
`
`In zvitro effect of hepsulfam (NSC-329680) vs. busulfan
`
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`Activity of selected US-NCI compounds in human tumor xenugrafzs in the clonogenic
`assay in vitro
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`4-lpanuezzrrm’ { ;\7SC— 349438’)
`
`21 pulmonary ioxin bioemtivalcd through a lung cytochrome
`lj» L9
`4~Ipumca11ol (Figure-.
`P450 patlwcay. Because ofits assumed lung specificity, lpomeanul is being developed for
`clinical trial by the Lung Cancer Drug Discovery Project of the NC] (5)
`lpomcanol was tested in human tumor xenografts of different histologies in the clone-
`genic asszty. At a dose level of 10 ugfml ipumeanol was active in 4/4]
`tumors (f9‘f,»’,:,},
`namely 4/5 sma1l—c¢=.ll carcinomas of the lung {Table 3). Bronchogcnic carcinomas oflargc-
`cell in = 4), squamous—ccll
`= I) or adenocarcinoma (n 2 4} subtypes or tumors ofother
`histologics did not respond to ipnmeanol, At the high dose of 100 lzgfml,
`ipomeazml
`inhibited Colony?‘ formation in 10,143 xenografts tested (26%). In viva, ipomcanol was tested
`in three small-cell lung carcinomas Previously shown to be sensitive in z:z'lra. The maximally
`
`NPC02232988
`
`Ex. 1034-0005
`
`

`
`ll-4
`
`H.-H. FIEBIG ET AL.
`
`tolerated dose was determined as 12.5 mg/kg/day given on days l—4 i.p. All tumors treated
`showed progressive growth indicating only limited client of ipomeanol in Live (Table 4).
`Further studies will be conducted using a weekly i.v. schedule.
`
`Oxam/zrazole ('./VSC-349174]
`
`Oxanthrazole {Figure l) is one of a series of 5-fiaminoalkyljaminol-substituted ?1n[l]l"B~
`[l,9—cdjpyrazol-6(2H)0ues [anthrapyrazolesl synthesized by the Warner Lambert Co. It
`showed broad spectrum activity in various murine and human tumor models and was less
`cardiotoxic than doxorubicin in a cultured fetal mouse heart model (4).
`Oxanthrazole was highly active in human tumor xenografts in the clonogenic assay in
`zritra. At a dose level ofO.3 pg/ml, 9/43 tumors (21%) were responsive without toxicity to
`human bone marrow (Table 3). At
`I pg/ml tumor colony formation was inhibited in
`20/48 xenografts, however; marginal bone-marrow toxicity was observed.
`In tumor-
`bearing nude mice oxanlhrazole was active in two xenografis at a maximally tolerated
`dose of 60 mg,/kg/day given on days i, 8 and L3 i.p. It effected a minor regression in a
`gastric cancer {CXF 97} and a large—cell lung carcinoma (LXFI. 529}. Five xenografts
`showed progressive growth and did not respond to oxanthrazole in viva (Table 4). OX2tl'l-
`thrazolc has completed clinical Phase l trials in the United States and has started Phase
`II evaluation.
`
`Periclovriedirze 4’ NSC- 338 7 20 j
`
`The synthetic oz—picoline-derivative penclomedine (Figure 1), 3,5-dichloro-2,4,-dirnethoxy-
`6~(trichloromethyl)pyridine, was active in the P388 prescreen. Intraperitoneal
`treat-
`ment caused tumor regressions in the subcutaneously implanted CD8Fl and 3/lXl
`in
`the subrcnal capsule assay. In non—brcast tumor models only moderate activity was ob-
`served iy/I4»). Resistance studies indicated cr0ss—resistanee with Melphalan and Collateral
`sensitivity with /‘tmsacrinc U2).
`As penclomedine needs bioactivation, the compound was tested only in tum0r~bearing
`nude mice in viva {Table 4}. The maximally tolerated dose was determined as L50 mg/'kg,~’
`day given on days 1, 4 and 7 i.p. In the breast cancer xenograft MAXF 401, penclomcdine
`etlected a complete remission on day 35. No regrowth was noted until the end of the
`experiment on day lO5. Activity was retained when penclomedine was given orally at a
`dose level of 300 nigjkgfday' given on days 1, 4, 7 i.p. The large—cell lung cancer LXFL
`529 was resistant to penclomedine and grew progressively.
`
`P}ra.«:ine rliazri/gydruxide, PZDH ( NSC~361456)
`
`Pyrazine-2-diazc-hydroxide (Figure 1} is a product ofa congener development program
`based on pyridine-2-diazohydroxide initiated by the Drug Synthesis and Chemistry Branch
`of the NCI. lts antitumor activity was observed initially in the P388 prescreen. Broad
`spectrum activity was observed in various in viva models including advanced-stage and
`metastatic disease if I3}, PZDH may act via alkylation and needs bioactivation. Evaluation
`against leukemia sublincs with acquired resistance to standard clinical drugs demonstrated
`(truss-resistance with Melphalan. The in vilro eytotoxieity of PZDH is increased under acid
`and hypoxic Conditions, which may favor selective toxicity to solid tumors in vivo (3).
`Pyrazine diazohydroxide was tested in three human tumor xenografts in nude mice
`
`NPC02232989
`
`Ex. 1034-0006
`
`

`
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`NPC0223299O
`
`Ex. 1034-0007
`
`

`
`H6
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`ll.-H. FIEBIG E7’
`
`(‘liable 4;. The maximum tolerated dose was 100 mgfkg/clay given on days 1, 8 and l5
`i.p. A high antineoplastic efiicacy was observed in all tumors tested. In the gastric camrr
`GXF 97, a partial remission was achieved, the breast cancer model .\-lAXl" 401 regressed
`completely 28 days alter the start of therapy. In the large—cell lung cancer LXFL 529, a
`minor regression was observed. Overall, PZDH exerted high antineoplrtstitt activity in zsizro.
`Phase I clinical trials should commence in the USA. by mid-1990.
`
`Ra/Jarrgyrin. {.VS(.‘—226080)
`
`The ni;u:roz;ycliC antibiotic rapamycin (Figure l), was originally selected for its activity
`against the so implanted CD81’! murine mammary tumor and i.r. implanted murinr:
`cpenclymoblastoma. It was developed by Ayerst Laboratories and inhibits DNA synthesis
`hy iiitertereiire with thymidine incorporation I541»).
`ln human tumor xt‘.ri<)gz".1l'ts,
`l‘I5.p211’1’l)'ClI1 showed 21 high cytotoxic potency in the lfl()!l()-
`genie 2tSS2l_\‘ in zzilro (‘Table 2]. At £1 dose level of 0.001 pg/‘ml,
`l~l_,-"41 tumors {3«l".;,t were
`responsive: however, consiclerable bone-marrow toxicity was also noted.
`in m'a=0, rapamyrin was tested in seven xenografts ofdillerent liistologies <f'I}1hle 4). The
`mzixitnuin tolerated dose was determined as 100 mg;'l<;gf(lay given on days l—4 i.p. Urug-
`induced mortality was observed generally about ‘2~3 weeks after drug application. A minor
`regre.~;sioit {relative tumor size 74% at day 21) was achieved in an adenocarcinoma of the
`lireast. Five tumors showed ‘no change’ with ‘.1 meclian duration of 28 days, a small-cell
`lung cancer xcnograft grew progressively.
`Further development of rapamycin was stopped due to solubility problems and toxicity
`assotrizitcd with the cremophor used in the experimental formulation. lVl'cl_l0I‘ emphasis is
`plactrcl on the (l(‘\'ClOpII1C1l[0ll1CW analogs ofiaparnyriii.
`
`References
`
`l)lowman.J., Ra,mpal,‘_]. B., Szititvy, /\.. Haugwitz, R. D. & Namyanan, V. l...
`_ Baker, 17_ (1.. Hand, E.
`(1987; Syntliesis
`rhemiczxl stability, and precliniwl antitumor activity of pymzine tliazohydroxide, sodiurn
`salt (NSC 361456}. Anti-Carirer Drug Des. 2: 297- 309.
`l.., M:tyo,_}. G. &
`. Boyd, M. R., Sl1fM“m£Il~i(‘F, R. H., Alley, M. (1., Scxirliero, D. A, Monk.=.. A., Fine, 1).
`Chabner, B. A. {I989} Dmg development. In: Roth,_]. Al, Ruckdc-schcl,_}. C. 8: Weiscnburger, '1'. ll. eds,
`Tlmracit Oncology, Vol. 7. Philadelphia: W. B. Sauilders. Chapter 51.
`'. Brodlijehrtr._}.
`l.. Moore,
`l).
`Mcldcr, l). (.'.. VA-’ill<t*., ’l'.J. 8: Powis, (J. {H188} In who cyttitoxicit}-' of
`pyrazinc-2~di:i2ohydroxidt-: Sp(;*(illl(‘,lty' for h\,=poxit' cells and efleets 0l‘l11i(‘!'0S0t1‘lBl (‘0ll‘ll.‘Ul):iliOll. Inuit.
`.l.’ru,-
`i')n.tgs G: 3~9.
`. Bruome. M. C.,johnson, R. K., Silveira, D. M. & Wodinsky, I. H983; Biochemical and biological cflects
`nltw0l1niqt1€' antimmor 2}.n(ll)l()llt‘.St raparnyt-in {NSC 226080‘) and machcrin ll {NSC 330500). Proc. Am
`Ass. Cancer Res. 24: 321.
`l\rl(.'l.l‘lIl(1l'C, l. L... Grieshaber. C. 19.. Clialmer, B. A.
`'. Christian. M. (L, Wines, R. F... lxylancl-_]otiL‘s, B..
`& Boyd, M. R. H989‘; ll-Ipomranol: a novel investigational new drug for lung ("«lI\Ct‘f. j. Natl Ckzrwr /71.31.
`81: H33 H43.
`. Fichig, H. H. (1987) Feasibility and usefulness ofhumztn tumor xenogralts in scrondary screening. lnrert.
`:\’eu Drugs 5: .78.
`;’l987l Colony assay with human
`Hcnss, H. 8; Lohr, G. W.
`. Yiehig. H. H.. Schmid, J. R., Bieser, W.
`tumor xenografts, murine tumors and human bone marrow. Potential for anticancer drug tl(‘Vt‘l(IpI|if’I'1l.
`Eur.
`(,‘1mrr*r Clin. Orarol. 23: 937~-94-8.
`, Hebig), H. I-L, Winterh;tlter_. B. R., Berger, D. I’. & Lohr, G. V-‘V. U988) Combined in viva {clonogrnic assay‘,
`
`NPCO2232991
`
`Ex. 1034-0008
`
`

`
`EVALUATION OF US-NCI COMPOUNDS
`
`ll?
`
`and in viva test procedure with human tumor xcnografts for anticancer drug development. Proc. Am. Ass.
`Cancer R95. 29: 493.
`. Firbig. ll. H. Wimcrhalrcr, B. R., Berger, D. l’., Lbhr, G. W. (1939) Combined in z'i1rafintviv0 test procedure
`with human tumor xcuografus for anticancer drug development. Stralzlerrlher On/cal. 165: 522-524.
`. Goldin, A. & Vcnditti,J. V1. H980) The new NC! screen and its implications for clinical evaluations. Remit
`Remit: (faricrr RP)‘. 70: 5320.
`. Hamburger, A. W. & Salmon, 5. E. (19777 Primary bivassay of human tumor stem cells. Science l97: 46l—
`-M3.
`. Harrison, 5. D._]r., Plr,waman._]., Dylrcs. D._]., VVand. W’. R. 8: Griswold, D. P._]r. U989) Treatment mutt’
`and schedule dependence and cross~rcsistancc of pcnclumedinc (NSC 338720) in preclinical models. I’roc.
`Am. An: Canm Res. 30: 577.
`. Harrison, 3. D. jr., Plowman, J., Dykcs, D. J., Wand, W. R. & Griswold, D. P. jr. (1990) Schedule
`clrpcmlcnce, activity against natural metastases, and cr0ss~rcsisLance of pyrazinc diazohydroxide {sodium
`salt, NSC 361456) in preclinical models in viva. Cancrr Clmnother. Pimrmatai. 25: 425-9.
`. Pluw'man_._]., Harrison, S. 1)., Dykcs. D. j., Paul], K. I)., Naraynnan, V. L., Tobol. H. K., Mar1in,j. &
`Griswold. D. P.
`(1939): Prccliitical nutitumor .1c.tivit)' of an 1-Picoline derivative, Perxclornetlim: [NSC
`333720), on human and murinfl tumors. Cancer Rey. 49:
`l90‘3 I913.
`. Plrm-rnati,,]. & Jacksuit. R. C. (1985) :\nthrapyraz0les. a new class ofimcrcalating agents with high~lcvel,
`broad spectrum activity against murine tumors. Cancer Res. 45: 553'2~5539.
`. S!aqut*1._ M.j.. Byar, D. P., Green,
`B. & Ruzvncwtsig, ;\-I. (1983) Clinical predictivily oftransplamablc
`tumor systrms in the st:lc'(‘liun ufncw drugs for solid tumun: rationale for a threc~sLagc strategy. Came! Trmt.
`Rep. 67: 753465.
`. Stccl, G. C., Cuurttriiay, V. D. & Peckhani, M._]. {I982} The immune-suppressed mouse as an altrrnatiw
`host for heremrransplantalion. In: Fogh,J., ed. The Nude Moms in Expat-inienlal and Clinical Rmarclz, V01. 2,
`pp. 207 227. New York: Academic Press.
`. Wand, W. 11.. Plowman,j., P-uull, K. D., i\‘arayanan, V. L., Bailey, D. M., Harrison, S. D._]., Dykes, D.
`j., Luster, W. R... Jr. & Griswold, I). P., _]r. {I988} Preclinical antitumor activity of hcpsulfam {NSC
`329680). Prmr. Am. A5505. Cmtrer RM. 29: 333.
`
`NPC02232992
`
`Ex. 1034-0009