9103‘3[udvuoS[1?.II.I11()[lgggv11231o's{12tLI11o[1ods1>.'1odf11101}popizofumoq
`
`
`
`
`
`
`
`“ill-‘R ‘PEU’l'lCS
`.
`.logy and Experimental Therapeutics
`
`
`
`
`
`inhibition of Epidermal Growth Factor Receptor-«Associated
`Tyrosine Ehosphoryiation in Human Carcinomas with
`CE-358,774: Dynamics of Receptor inhibition in Situ and
`Antitumor Eiiects in Athymic Mice‘
`
`VINCENT A. POLLACK, DOUGLAS M. SAVAGE, DEBORAH A. BAKER, KONSTANTINOS E. TSAF’AFill(OS,
`DONALD E. SLOAN, JAMES D.
`l\/IOYER, ELSA G. BARBACCE, LESLIE Fl. PUSTILNM, TERESA A. SMOLAREK,
`JOHN A. DAVIS,
`i\/lADl-lLJF% P. VAEDYA, LEE D. ARNOLD? JOHN L. DOTY, KENNETH K.
`lWATA,3 and
`MECHAEL J.
`l‘v’lC}FilN
`
`Department of Genomics, Targets and Cancer Research, Pfizer Central Research, Groton, Connecticut
`Accepted for publication August 5, 1999
`This paper is available online at http://www.jpet.org
`
`AEQTRAGT
`
`Phospnorylation of tyrosine residues on the epidermal growth
`factor (EGF) receptor (EGFr) is an important early event in signal
`transduction, leading to cell replication for major human carci~
`nomas. CF’~358,774 is a potent and selective inhibitor of the
`E£3iFr tyrosine kinase and produces selective inhibition of EGF—
`mediatecl tumor cell rnitogenesis. To assess the pharmacody—
`namic aspects of E€3iFr
`inhibition, we devised an ex vivo en-
`zyme—linl<ed immunosorbent assay for quantification ot EGFr—
`specific tyrosine phosphorylation in human tumor
`tissue
`specimens obtained from xenogratts growing so. in athymic
`mice. When coupled with pharmacokinetic analyses, this mea-
`surement can be used to describe the extent and duration of
`
`kinase inhibition in vivo. CF’~3.58,774 is an effective, orally ac-
`tive inhibitor of EGFr-specific tyrosine phosphorylation (ED50 =
`
`it has a significant duration of action,
`10 mg/kg, single close).
`producing, on average, a 70% reduction in EGFr~associated
`phosphotyrosine over a 24~h period after a single 100 mg/kg
`dose. inhibition of EGFr phosphotyrosine in an ex vivo assay
`format effectively estimates the potency and degree of inhibi-
`tion cf EGFr—dependent human l_l(3Fi—l_ON—l-lN5 head and neck
`carcinoma tumor growth. Substantial growth inhibition of hu-
`man tumor xenografts was achieved with p.o. doses of the
`compound (EDSC,
`10 mg/Kg dd. tor 20 days). Combination
`chemotherapy with cisplatin produced a significant response
`above that of oisplatin alone with no detectable effects on body
`weight or lethal toxicity. Taken together, these observations
`suggest that CF’~3.58,774 may be useful for the treatment of
`EGFr—clriven human carcinomas.
`
`For the majority of human carcinomas, growth factor re—
`ceptors play an important role in tumorigenesis and progres~
`sion to terminal rlisease states. The epidermal growth factor
`(E(}E‘) receptor (lilGFr) has been implicated in many human
`squamous cell carcinomas (Dianne et al., 1986), such as
`non—small cell lung carcinoma and brain, bladder, breast, and
`ovarian carcinomas (Gru.ll.‘ick,
`l99_‘l_). EGF at picomolar con-
`centrations is mitogenic for cells overexpressing the receptor,
`and antibodies to EGFr abolish EGF-stimulated mitogenesis
`in LlCR~l_;Ol\l~llN5 hearl and neck carcinoma (HN5; lVlooijt,a-
`hedi et
`_‘l_993h,c) and other turner cells (Aboud-Pirak et al.,
`
`Received for publication May 18, 1999.
`1 Portions of this Work were presented at the annual meeting of the Amer-
`ican Associati-on for Cancer Research, April
`l§l97.
`Present address: Department ol'Chemistry, BASE‘ Bioresearch Corp, l00
`Research Dix, W-twcester, MA 01505-4314.
`3 Adclress: DSl Pharnaaceuticals. lnc., 106 Charles Lindbergh l3lVd., Union-
`dalc, NY l,l5-53.
`
`1988; Yonecia et al., 1991a). As an early event in the signal
`transduction process, the ligand transforming growth fac-
`tor—oz or EGF binols to l3lGFr on the surface of tumor cells and
`stimulates: Li) heterorlirnerization and homodimerization oi’
`
`EGFr molecules; 2) interrnolec'ular cross—phosphorylation oi’
`intracytoplasrnic tyrosine residues (EGl3“r autophosplioryla-
`tion; l-ionegger et al., 1989); and 3) activation of the tyrosine
`kinase activity of lilGFr. Apart from binding to the cognate
`ligand, all l{t10‘W11 functions oi’ l3lGFr olejpenci on tyrosine ki-
`nase act,iVi,ty. Foint mu.tations in the kinase oiomain that
`abrogate A’l‘l’ hinoling also abolish li,ganol.-depenolent kinase
`activity and abrogate E€3‘rF/transforming growtli factor~a~.‘in~
`duceoi mritogenesis (ll./loolenaar et al., 3.988). An intact. kinase
`domain
`essential for activation of numerous rlownstream
`
`effectors, inclurling phospholipase £3-7' (ll/la,rgoli,s et al., _‘i_99(l;
`Nishibe et. al., _‘i_99(l; Vlfahl et al., l99ill phosphatidylinosibol
`3—k:inase (Bjorge et al., 1996)), and mitogen-activated. protein
`
`ABBREVEATEGNS: EGF, epidermal growth factor; EGFr, epidermal growth factor receptor; ELISA, enzyme-linked irnmunosorloent assay; HESS,
`Hanks’ balanced salt solution: HN5, LECR-LDl\l—Hl\l5 head and neck carcinoma.
`
`APOTEX EX. 1015-001
`
`

`
`749
`
`Pollack et al.
`
`Vol. 29?
`
`kinase (Ahn et al., 1.990), with the ultimate cellular response
`being DNA synthesis and cell division (Honegger et al.,
`1987). 'l‘ransfection experirnents have shown that l§;GFr over-
`expression alone may lead to constitutive activation of signal
`transduction, leading to uncontrolled niitosis (Di Fiore et al.,
`1987; Velu et al., 1987). The degree of EGFr overexpression
`has been shown to be related to tumorigenicity in some tuinor
`systems (Santon et al., 1986; Velu, 1990). Recent studies of
`biopsy specimens suggest that overexpression of lilGFr is
`associated. with a poor prognosis in bladder (Neal et al., 1985)
`and breast (Sainsbury et al., 1985) carcinomas.
`Despite homology with other tyrosine kinases, selective
`inhibitors have been identified (for a review, see Traxler,
`1998). The EGrFI' tyrosine kinase therefore represents an
`attractive molecular target for pharinacological intervention.
`To monitor the effects of kinase inhibition, the degree of
`EGFr autophosphorylation was examined, because: 1) auto-
`phosphorylation of effector—specii'ic tyrosine residues in—
`creases the velocity‘ of the ltinase reaction (Bertics and Gill,
`1985); 2) autophosphorylation. increases the affinity of the
`EGFr for its substrates, such as phospholipase C-'3/' (Magni et
`al., 1991), allowing these substrates to bind the activated
`receptor (docking site) and thereby become tyrosine phos-
`phorylated; and 3) EG.Fr phosphotyrosine represents the last
`known biochemical event before corninitted steps toward. cel~
`lular division are mediated by downstream effector mecha-
`nisms. For these reasons, we believe quantification of EG=Fr
`autophosphorylation is related to, and characterizes, inhibi-
`tion of the kinase functionality.
`CP~358,’}"?4 is a potent inhibitor of the EGFr tyrosine
`kinase with an lC5O value of 2 nM; CP—358,’774 and its
`analogs have been shown to be direct~acting, reversible,
`ATP-conipetitive inhibitors of l§;GFr tyrosine phosphoryla-
`tion (ll/loyer et al., 1997; Pustilnil: et al., 1997). Specificity
`analysis has indicated. > _‘l.l)()O~.folol. selectivity against other
`tyrosine kinases, such as pp60"‘S‘”“’, pp145“"3i°l, the tyrosine
`kinase activities of the insulin and the insulin—lil<:e growth
`factor-1 receptors; selectivity has been shown against iso-
`lated kinases as well
`in intact cells (ll/loyer et al., 1997).
`Cl’-358,774 inhibits autophosphorylation of the EGFr in a
`variety of EGFr~overeXpressing tumor cells GC50
`20 nM)
`and produces inhibition of mitogenesis, inhibition of tumor
`cell division, and cell cycle arrest. ln some cell types, such
`as l3iFi, CP—358,77-4 induces concentration-dependent ap-
`optosis in vitro.
`Here, we report that CP-358,774 is an effective, orally
`active inhibitor of EGFr tyrosine autophosphorylation. CP-
`358,7’Z’4 can effectively inhibit EGFr tyrosine phosphor;/‘—
`lation in human tumors growing s.c. in athyrnic mice with
`an E1350 value of ll} in g/kg p.o. lt has signi.fica,nt duration
`of action and produces substantial inhibition of human
`EGFr—overeXpressing tumors growing s.c. in athyinic mice.
`Moreover,
`the degree of inhibition of EGFr phosphoty—
`rosine shows good agreeinent with the degree of tumor
`growth inhibition in treated animals. Tlie results of these
`experiments were previously reported at the American
`Association for Cancer Research annual meeting (Pollack
`et al., 1997). The data suggest that (JP-358,774 may be a
`useful new coinpound for therapy of human neoplastic
`diseases.
`
`Materials and Methods
`
`l'l/lice. Three— to 4—weel<—old. female athymic mice (_CD—l nulnul
`were used for human tumor Xenografts. Mice were obtained from
`Charles River Laboratories (Vi/ilmington, MA) and were housed in
`specific pathogen-free conditions, according to the guidelines of the
`American Association for Laboratory Animal Care; all studies were
`carried out with approved institutional experimental animal care
`and use protocols. During these studies, animals were provided
`pelleted food and water ad libitum and kept in a room conditioned at
`70 —‘75"C and 50 to 60% relative humidity with >15 fresh air changes
`per hour. Sentinel heterozygous littermates of the athymic animals
`were monitored routinely (3—weeh intervals) by serological assays
`and were found to be free of exposure to the following agents: murine
`hepatitis virus, ectromelia virus, and Sendai virus. For all studies,
`the mice were allowed to acclimate for 1 to
`days after receipt. of
`shipment; test animals were randomized before cornmcncement of
`treatments and examined twice daily thereal’ter for compound-in-
`duced or tumor~related deaths. Moribiincl. animals were sacrificed. to
`
`red.uce suffering.
`Tnm.or Cell Lines. The l:lN5 cells were obtained from Dr. M. J.
`O’l:iare (liiaddow Labs, Institute of Cancer
`rcli, Sutton, Sur—
`
`rey, UK). All other cells were purchased. from t
`American Type
`Culture Collection (Rockland, MD). All cell lines were free of reovirus
`type 3, pneumonia virus of mice, K—virus, Theiler’s virus, Seiicla,i
`virus, ectromelia virus, and lactate dehydrogenase virus (Microbio-
`logical Associates, Bethesda, l‘v,llJ).
`Cell Culture. Cell lines were passaged by monolayer culture in
`175-C1112 tissue culture flasks (Nunclon; Marsh Biomedical Products,
`Rochester, NY)
`in Dulbecco’s modified Eagle’s medium supple-
`mented with 10% heat~inactivated FBS (llazelton Research Prod-
`ucts, lnc., Lenexa, KS), 300 fig/ml glutamine, 100 U/ml penicillin G,
`100 leg/rnl st.i'epton1y<,in, and 10 gig/ml gentamycin at 3 7°C in a
`humidified 95% air/5% CO2 atmosphere. Routine periodic samples of
`cell culture broths tested negative for Zl/Iycopldsz/no contamination
`(Microbiological Associates). For implantation in vivo,
`the tumor
`cells were harvested from e2«:ponent.ially growing cultures (60———80%
`confluence), detached by light
`trypsinization (O.25% trypsin and
`0.02% EDTA. 1 min), washed in Hanlis’ balanced salt solution
`(HBSEU, resuspended in llBSEl, mixed. with the basement membrane
`preparation Matrigel
`(40234; Collaborative lziiomedical Prod.ucts,
`Bedford, MA), and held in an. ice bath <1 h before injection.
`Che.motherapeu.tan.ts. CP—358,’7'74 [6,'7—bis(2—rnetho>.fy—ethoxy)quina-
`zoline~4—yl]~(3—etliynylphenyl)arnine; l\’lF = C22ll23N3Cl4), a colorless, crys-
`talline, anhydrous compound, was synthesized in our laboratories (Arnold.
`and Sclinur, 1998).
`ln. these stud.i-es, the hydrochloride salt (molecular
`weight = 429.9) was used in all cases, exwpt for that represented in Fig. 7,
`which used the free base (rnolecular weight = 393.4), and the dosage levels
`shown represent the quantity of flee base administered, excluding the
`contribution of the salt. The compound was formulated for i.p. or p.o.
`administration by dis solution ofthe dry powder in a small amount (10% of
`final volume) of dimethyl sulfoxide (DMSOL mixed by vortexing until
`dissolved; during vortexing, sullicient sterile, pyrogen-free pliysiological
`saline (0.15 N NaCl), containing 0.10% (w/V) Pluronic P105 (BASE Wyan-
`dottc, Parsippany, NJ ), was added to produce a hornogeneous line suspen-
`sion. The prepared dosage forms did not produce microbial (X)l()I1'iGS alter
`incubation on brain-heart infusion agar and did not contain endotoxin
`cleterjtahlc by the Limulus amocbocytc lysate
`(Associates of Cape
`Cod. h1c., Woods Hole, MA). Doxorubicin (Adriamycin; Rapid Dissolution
`Forrnu.la)
`purchased from Adria Labs. (Columbus, OH). Cisplatin was
`obtained. as a powder from Sigma Cliemioal Co. (St. Louis, MO). All dosage
`forms were freshly prepared for -eacli day’s treatment. CR3 58,774 and the
`reference agents were dosed according to the optimum tbrmulation, route,
`and regmeiis,
`einpirically derived in previous studies; aggressive dosing
`parameters (single bolus treatments at maximum. tolerated dosage levels)
`were used for maximum antitiimor efficacy of the cytored.uctive agents.
`Test animals were treated between 7 and 9 AM, immediawly alter a 12-h
`dark photoperiod (active phase), to control for variability introduced by
`
`APOTEX EX. 1015-002
`
`
`
`9103‘3[udvuos{12u.mo[lgggv11231o's[1>.I1.Ino[1ods1>.'1odf11101}popizofumoq
`
`
`
`
`
`

`
`1.999
`
`circadian physiological cycles, acoordin g to the methods of Halberg et al.
`(1973).
`EGFr Phosphotyrosine l)eterm.in.ation.s by Era.zyrne~Linked.
`lrmnuno sorbent Assay (ELISA). To determine compound-induced
`inhibition of E-Glrlr-associated tyrosine phosphorylation in human
`tumor explants from athyrnic mice, an ELlSA specific for EGl:"r
`phosphotyrosine was developed. Tumor tissue was harvested at var-
`ious times after dosing (usually 1 h) by careful dissection, immedi-
`ately flash frozen in liquid nitrogen, and then homogenized in buffer
`formulated to prevent further tyrosine phosphorylation as well as
`enzymatic phosphatase activity. A double-antibody El_.lSA provided
`quantitative determinations of the degree of EGFr tyrosine "phos-
`phorylation after specilic capture of EGFr protein.
`Brielly, athymic mice with s.c. tumors (5———10 mm in diameter) were
`euthanized humanely, and tumors were excised with the use of small
`dissecting scissors and mosquito forceps, after which the tumor tis-
`su.e
`immedia.tely flash frozen in liquid nitrogen and stored a.t
`-'70”C before hornogeniza.tion and immunoassay. Tumors were
`weighed, and for each 100 mg of tumor tissue, 1 ml ofice—colcl., sterile
`lysis buffer
`added. Lysis buller contained (per liter) 50 ml of 1 M
`HEPE-S, pll 7.4, buffer, 37.5 ml ofzl M sodium chloride, 0.75 ml of2
`M magnesium chloride, 10 ml ol'100 mM EDTA, 10 ml of glycerol, 10
`ml of Triton X-100, 8 ml of 200 mM sodium orthovanadate, 4.2 g of
`sodium lluoride, 50 /.Lg/ml phenylmethylsulfonyl fluoride, 25 mg of
`soybean trypsin inhibitor, 10 ,u,g/ml leupeptin, and 10 ,u.g/ml aproti—
`nin. Tumors were homogenized. with a Thomas Teflon pestle homog—
`enizer attached to a power drill (or equivalent) and then clarified. by
`centrifugation; the resulting supernatant liquid (800 pl) was trans-
`ferred to microtiter plates in 200~,ul aliquots and maintained at
`----70”CI before
`Appropriate dilutions of tumor homogenates (1:20 —1:40 dilutionsl
`were made in blocking buffer containing (per literl 50 g of bovine
`serum albumin, 10 g of ovalburnin, 0.90% NaCl, and 10 ml\’l
`Tris * l:lCl buffer, pH 7.4. After dilution, 100—p.l aliquots were trans-
`ferred to microtiter wells containing adsorbed monoclonal antibody
`to EGl:"r protein (QlA08; Oncogene Science, Uniondale, NY). The
`plates were then incubated for 30 min at 30°C (or 3 h at room
`temperature) to allow el’licient capture of the ElGFr protein from the
`tumor homogenates. Microtiter Wells were washed six times in a 1:10
`dilution of Plate ‘Nash Concentrate (PN 77
`550; DuPont NEN,
`lrloston,
`l\IlA). To detect phosphotyrosine residues, 100 pl of horse—
`radish peroxicl.ase—conjugated monoclonal antibody specific for phos—
`photyrosine (diluted 1:1000 in blocking buffer) was added to each
`well (FY54 wnjugate, PTO3; Oncogene Science), and plates were
`incubated for 1 h at 30°C. Microtiter wells were then washed
`times in a 1:10 dilution of Plate ‘Nash Concentrate, after which 100
`;.;.l/"well of 3,3',5,5'—tetraInethyll:venzidine substrate was added (50-
`70—04; Kirkegaard and Perry Laboratories, Gaithersburg, MD); color
`development was monitored over 30 min, after which all reactions
`‘N-1"
`e stopped with 100 ;:;.l/well of 0.09 M sulfuric acid. For quantiti-
`
`cation, absorbance was determined at 450 nm with a Bio~Ra(l. (l:ler—
`cules, CA) model 3550 microplate reader. 1:‘.-Glflr phosphotyrosine
`content was calculated after normalization of each sample for total
`protein with a commercial lrit (BCA Protein; Pierce, Rocliford, lhl.
`The absorbance Values for samples from each of the tumor- bearing
`animals (sample size, four mice/treatment groupl were entered into
`a custom Microsoft Excel spreadsheet, where the endpoints (i.e._.
`protein concentrations and phosphotyrosine levels) were calculated.
`ln all
`the 1:‘.-Glflr-a.ssociated tyrosine phosphorylation was ex—
`pressed
`absorbance units/mg total protein. For statistical infer—
`ences, the relationships between groups (i.e., test Versus control
`group) were identified using a computer program for the one—Way
`ANDVA, where the at significance level was assigned at 0.05. P
`Values were determined using Dunnett’s Z statistic. A set of internal
`laboratory standards (i.e.,
`aliquots from previously frozen tissue for
`both treated and control groups) was used to assess the quality and
`reproducibility of the immunoassay; in the course of 5 years’ routine
`
`EGFr inhibition and Antitumor Effects of GP-358,774
`
`T4-l
`
`testing, the results were highly reproducible (ie, the coefficient of
`Variation was <.0.0%).
`EFL-C Determinations of CP~358,.7’74 in Plasma and Tumor
`Tissue. Determination of drug concentration was made by organic
`extraction (acetonitrile) of plasma and tumor samples, followed by
`HPLC. Cl’-3 58,774 in plasma and tumor
`was extracted from
`200411 samples spiked with 100 [Lil of internal standard (UP-292,597;
`
`0.8 n g/',u.l, in ac-—-tonitrile) with 5 ml, of methyl t-butyl ether using an
`Qberbach reciprocating shaker for 10 min. Before extraction, tumor
`tissue
`homogenized in 4 parts deionized. water to 1 part tumor
`specimen (V/m) using an Omni 2000 (Omni International, Gaines—
`ville, VA) tissue hornogenizer. Samples were centrifuged at 3000 rpm
`for 5 min at 22"C using a Jouan centrifuge. The organic layer of each
`sample was transferred to a clean tube, and the methyl z?—butyl ether
`was evaporated to dryness in a Zymarl: Turbo-Vap at 80”C-. All
`samples were reconstituted in 200 _ul of mobile phase consisting of
`70% water a.nd 30% acetonitrile (V/V) brought to pll 2.4 with tritlu—
`oroacetic anhydride (Acros Organics). A 2—liter Volume of mobile
`phase consisted of 1400 ml of l\’lilli—Q deionized water, 600 ml of
`acetonitrile, and 550 ;.;.l of trilluoroacetic anhydride. The analytical
`column was a YMC Basic C-18 (4.6 mm 150 mm, 3 pun). A pump
`(Thermo Separation Products Constametricl-1100) was used to es tab-
`lish a 1.5 ml/rnin llow rate through the column. Cl’-358,774
`detected at 345 nm (AUFS 0.001) using an ultraviolet detector (ll/lil—
`ton Roy Spectro Monitor 3100 variable wavelength detector). The
`retention time for CP—358,'7'74—
`6.5. The lower limit of quantifi-
`C£3t10]’l of the assa.y was 10 ng/ml for plasma and 50 ng/g for tumor
`tissue.
`Tumor Growth lnhibition Studies In Vivo. The tumor growth in—
`hibitory effects ofCP-3 58,774 were measured in young athymic mice bear~
`ing established, palpable (2—4-mm diameterl human HN5 or A43 1 tumors.
`Tumors were induced in the lelt llanl: of 3- to 4-week old athymic
`by
`s.c. injection of 1 X 106 cultured, log phase HN5 or A431 cells in 0.20 ml of
`HESS cxintaining 50% l‘/latrigel. 'l‘uinor
`measured in millimeters
`with Vernier calipers across two diameters three times/week, and the
`tumor volume (mm3l was calculated using the formula: tumor Volume 2
`(lengh >< [widtli]2)"23, according to standard methods (Geran et al., 1972);
`results are expressed as tumor volume (TuV) in IYIII13. To calculate tu-
`mor growth inhibition, the following formula Was used: inhibition 3%) =
`('l‘uGmfl,,m1 — TuGt%)/luG'mUm,, X 100%, Where tumor growth ('l‘uG)
`equals the final tumor size minus the pretreatment tumor size for individ-
`ual treatment groups. This method of tumor implantation provided repro-
`ducible growth in athymic mice, enabling the determination of dose-re-
`sponse effects
`for a Variety of chemotherapeutic agents. For each
`experiment, athymic inioe were randomized. on receipt of shipment and
`again after tumor implantation (ie, before commencement of treatment).
`Data collected from the antitumor studies (e.g., tumor volume) were eval
`uated for statistical significance using one—Way ANOVA (for significant
`antitumor activity, P < .05).
`
`Rescue
`
`inhibition of EGFr Pliosphotyrosine in EH5 Xeno-
`gfrafts. l-lN5 possesses many of the characteristics of EGl3“r-
`dependent squamous cell carcinomas both in Vitro (Mudfla-
`hedi et al., l_993b,c) and in VlV0 (ll/lodjta,hedi et al., l.993a,b).
`ln particular, monoclonal antibodies directed at the EGFr
`can completely block cellular proliferation in Vitro, and for
`this reason, the tumor cell line was selected to evaluate a
`large series of EGFr tyrosine kinase inhibitors. Vfhen adm.‘in~
`istered orally (by garage) or parenterally (i.p.)_, CF’-358,7 71l-
`consistently produced significant, dose—relatecl inhibition of
`HN5 EGFr tyrosine phosphorylation 1 h after dosing (Fig. 1).
`Compared with vehiclatreated controls, a maximum of 80%
`reduction in plies pliotyrosine was observed after’ dosing by
`p.o. or i.p. routes. In several preliminary experiments, the
`
`APOTEX EX. 1015-003
`
`
`
`
`
`9103‘3[udvuos{12u.mo[lgggv11231o's{12tLI11o[1ods1>.'1edf11101}pepsofumoq
`
`
`
`
`
`

`
`
`
`
`
`
`
`
`
`9103‘3[udvuos{12u.mo[lgggv11231o's{12tLI11o[1ods1>.'1edf11101}popizofumoq
`
`742
`
`Pollack et al.
`
`Vol. 291
`
`100
`
`+ CP-358,774 ip
`
`-0- CP-358,774 po
`
`90 i
`so
`
`70 p
`60
`
`so ‘
`
`40 ,
`
`30
`
`2o 2
`
`10
`
`(°/0)
`inhibitionofEGFRPhosphotyrosine
`
`
`O
`
`10 20 30 40 50 60 70 80 90100
`
`Plasma CP-358,774 Conc. (pM)
`Fig. 2. The relationship of plasma CP—358,774 concentration to reduction
`in tumor—associated EGFr phosphotyrosine. At 1 h post—treatment with
`graded doses of 2.9 to 92 mg/kg by either the p.o. or i.p. route, plasma
`CP—358,774 concentrations were determined by HPLC and HN5 tumor-
`associated EGF1‘ phosphotyrosine inhibition by ELISA. The effective
`plasma concentration for 50% inhibition of the target receptor was esti-
`mated at 8 MM (3.1 p.g/ml) and ~12 ,u.M (4.7 p.g/ml) for p.o. and i.p. dosing,
`respectively. These data are representative of three independent experi-
`ments.
`
`reduction relative to vehicle—treated controls). At higher
`plasma concentrations (i.e.,
`l.€)—l.0i) y.l\i, 3.9-3.9 pg/inll, the
`reduction. in EGFr phosphotyrosine ranged from 65 to 75%.
`By interpolation, the effective plasma concentration for 50%
`inhibition of the target receptor was estimated at 8 all/l (3.1
`;.:.g7'rril) and ‘~12 pl.‘-/l (4.7 pg/ml) for p.o. and i.p. dosing,
`respec'tivel_v. ln mouse plasma, 95% of CF-358,774 is bound
`to plasma proteins. Taking these data into account, at 1 h
`after the dose, 50% inhibition of EGFr-associated phospho-
`tyrosine of HN5 tumors occurred at free plasma concentra-
`tions of/1:00 nlvi (160 ng/ml) for p.o. and 600 nlvl (240 ng/inl)
`for i.p. doses of CP—358_.774.
`lluration. of Action of Cl.’-3£i8,?7d. The duration of re-
`duction in EGl3‘r phosphotyrosine alter a single 92 mg/kg
`dose of CP—358_.77-4 was evaluated in the l-ll\lr3 model (Fig. 3).
`After p.o. dosing, significant and substantial inhibition of
`phosphotyrosine ('75- 85%) was observed. for l2 h; re~
`duction Was still measurable (25—-40%). and statistically sig-
`nificant, after 24 h. To a similar degree of efiicacy, parenter-
`ally (i.p.) dosed mice showed substantial inhibition of EGFr
`phosphotyrosine for 12 h; however, no reduction was ob-
`served at 24 h (data not shown). Calculation of the area
`under the curve for reduction in EGFr phosphotyrosine pro-
`vides an estimation. of the overall degree of inhibition over a
`24-h period. Based on the assumption that complete inhibi-
`tion (100%} of EGFr autophosphorylation over a 24-h period
`would produce inhibition of 2400%-l1 (l0()% “coverage"°), p.o.
`dosing elicited.
`inhibition of
`l.69€)%~li
`(70.4% coverage),
`whereas parenteral dosing (i.p.) showed an area under the
`curve of 14299?-h (59.0% coverage).
`‘Within l. h after p.o. dosing, peak plasma and tumor CP~
`358,774 concentrations were reached (124-.6 ,LLl\/l and 54.8
`urnol/lrg, respectively‘). Plasma and tumor concentrations
`then declined rapidly until 6 h, after which concentrations
`
`APOTEX EX. 1015-004
`
`100
`
`90
`
`so E
`70
`
`so
`
`50
`
`40
`
`so
`
`20 t
`10
`
`O
`
`
`
`
`
`inhibitionofEGFRPhosphotyrosine(%)2s.e.m.
`
`
`
`
`-0- CP-358,774 po
`
`—A—- CP-358,774 ip
`
`ED50ip = 9.2 mg/kg
`
`ED50po = 9.9 mg/kg
`
`1
`
`l
`10
`Dosage (mglkg)
`
`2
`
`100
`
`\o/\/°
`/°\/\0
`
`\\
`
`N
`\N
`N2
`
`Fig. 1. a, CP—358,774—mediated inhibition of EGFr—associated phospho-
`tyrosine of HN5 tumor xenografts. Human head and neck carcinomas
`growing s.c. in athymic mice were excised 1 h after dosing either p.o. or
`i.p. with CP—358,7'/'4 formulated in sterile, pyrogen—free 10% DMSO,
`0.85% sodium chloride, and 0.1% Pluronic P105. The tumor EGFr—asso—
`ciated phosphotyrosine was measured by ELISA; the data are a summa-
`tion of 22 (i.p.) and 28 (p.o.) independent experiments. b, CP—358,774 is
`[6,7—bis(2—methoXy—ethoXy)—quinazoline—4—yl]—(3—ethynylphenyl)amine
`(MF : C22H23N3O4, MW 2 393.4).
`
`vehicle (10% DMSO, 0.85% NaCl, and 0.10% Pluronic P105)
`produced. no inhibition. of
`phosphotyrosin.e compared
`with Water or saline treatments.
`
`The data in Fig. 1 are a compilation oi"28 (p.o.) and 22 (i.p.)
`independent experiments, attesting to the reproducible inhi-
`bition by this agent. The effective dose for 59% inhibition. of
`the target receptor’ (ED50) was similar for p.o. and i.p. ad-
`rninistration: 9.9 mg/leg p.o. and 9.2 ing/kg i.p. The minirnal
`effective single dose eliciting statistically significant (P <
`.05) reduction in EGFr~associated phosphotyrosine was
`rngikg (39% reduction) and 2.8 mg/lzg (47% reduction) for the
`p.o. and i.p. routes, respectively. These extrapolated E1350
`values are Within one order of magnitude of the lC5., value
`(20 nM) for the inhibition of l§;GFr phosphotyrosine by CP-
`358,774 in homogeneous populations of HN5 cells growing in
`vitro.
`
`Relationship of Plasma Concentration to EGFr
`Phosphotyrosine inhibition. Figure 2 illustrates the rela-
`tionship of plasma concentration of Cl?-358,774 to inhibition
`of EGFr~associated phosphotyrosine of
`Xenografts. At
`1 h post-treatment with a single dose, plasma concentrations
`of 2 to 10 p_.l‘~/l GP-358,774: (O.79—3.9 itg./ml) were associated
`with a significant reduction in EGFr phosphotyrosine (~ 40%
`
`

`
`
`
`
`
`
`
`9103‘3[advuoS[1?.II.I11()flgcigv11231o's[1>.ILIno[1ods1>.'1odf11101}popcofumoq
`
`EGFr Inhibition and Antitumor Effects of CP-358,774
`
`743
`
`and CP~358,/"774 was not retained in tumors relative to
`plasma (Fig. 3). ln tissue culture, the effects of (I3l’—358,’}’ 74 on
`EGFr phosphotyrosine in l-lN5 cells is freely reversible. Cel-
`lular EGFr phosphotyrosine levels return to levels found in
`untreated cells within minutes of removal of Cl’~358,77+l
`from "the culture medium. Thus there is no clear pharmaco-
`dynarnic explanation for a persistent inhibitory effect of CP-
`358,774 in tumors growing in vivo.
`Antitumor Effects of Cl.’-3£i8,?’?d (HN5). The antitumor
`effec'ts of CP—358_.,’Z’7-4 were deterrnined in the lilGFr—overe2r-
`pressing human HN5 and human A431 epidermoid carcino-
`mas. Both tumors have been shown to be inhibited by mono~
`specific anti~EGFr antibodies in cell culture and in Xenograft
`models (Fan et al., M93;
`ll/lodjtahedi et al., 1993a). Ural
`administration of CP~358,/"774 produced significant dose—re—
`lated antitumor effects against established HN5 growing s.c.
`in athymic mice (ll ig. 4). ‘When ‘test animals were dosed for 20
`consecutive days beginning at 4 days after tumor implanta-
`tion (tumor diameter,
`mm), the minimal effective dose
`for gnilicant antitumor effects was 5.7 m g"l<g/day p.o., using
`one—way ANOVA (P <. .05 with Dunnett’s test). Doses of 11 to
`92 mg/leg/day p.o. produced substantial antitumor effects
`(ie, >5l)% inhibition). During the cou.rse of dosing (days
`4—23 after implantation), tumor-bearing mice treated with
`vehicle alone showed progressive enlargement of turnors;
`spontaneous regressions in vehicle—treated or untreated an-
`imals have not been observed in this niodel.
`
`ln the experiment described above, turnor sizes for CP-
`358,77~i~treated animals were significantly reduced, and this
`inhibitory effect was observed as long as the test animals
`Were being treated. On the cessation of treatment, We have
`found that although tumors gradually enlarge, tumor growth
`rates do not generally equal those of the vehicle controls.
`Using the tumor size measureinents taken only during the
`
`2500
`
`-1-no010c:oc0
`
`Treatment Period
`
`(mm3)
`
`TumorVolume
`100 mg/kg/day po
`
`Time (days) After Implantation
`
`-I- Vehicle Control
`
`125 mg/kg/day po
`
`-0- 1.6 mg/kg/day po
`
`250 mg/kg/day po
`
`—A— 3.1 mg/kg/day po
`
`50.0 mg/kg/day po
`
`-6- 6.2 mg/kg/day po
`
`Fig. 4. Antitumor effects of CP—358,774 p.o. on HN5 Xenografts in athy-
`mic mice. HN5 cells were implanted so. in the flank of athymic mice, and
`after tumors became palpable (2———4—mm diameter, day 4 postimplanta—
`tion), test animals Were treated once daily for 20 consecutive days. Tu-
`mors Were measured across two diameters according to standard meth-
`ods. These data are representative of three independent experiments.
`
`APOTEX EX. 1015-005
`
`1999
`
`
`
`InhibitionofEGFR-Phosphotyrosine(%)
`
`-0- EGFR-PY (% Inhibition)
`
`7140
`
`120
`1oo
`
`0 so
`
`5 so
`
`40
`
`100
`90
`80 :
`7o.
`605
`so
`40
`30'
`2o.
`10’
`
`9 2o ConcentrationofCP-358,774
`
`0
`
`5
`
`10
`
`15
`
`
`20
`25
`
`Time (hr) Post-Dose
`
`
`
`-0- Tumor [CP-358,774] (pmol/kg)
`
`
`Fig. 3. Duration of action of CP—358,774 for inhibition of EGFr phospho-
`tyrosine in HN5 tumors. Athymic mice bearing s.c. bilateral HN5 re-
`ceived a single dose ofCP—358,774 of 92 mg/kg p.o., and at various times
`after the dose, mice were sacrificed and the tumors Were harvested and
`assayed for EGFr phosphotyrosine by ELISA or for CP—358,774 by ex-
`traction and HPLC analysis. These data are representative of three
`independent experiments.
`
`+ Plasma [CP-358,774] (p.M)
`
`remained low, although detectable, for several hours. The
`‘terminal elimination half-life in plasma after p.o. adminis-
`tration could not be determined because plasma concentra~
`tions from 6 to 24 h did not significantly decline; the mean
`tunior half-life after p.o. administration was estimated at
`2.9 h. At 24 h after the dose, plasma and tumor concentra-
`tions were 38 all/I and 4.0 umol/l<:g_. respectively. It is clear
`from Fig. 3 that although plasma and tumor CP~358,'?’}’ .
`concentrations follow similar time courses, the EGl3‘r-associ-
`ated phosphotyrosine reduction does not decline with declin-
`ing plasma and tumor levels and remains at a high level (8€)%
`inhibition) at 12 h after the dose. The reason for this is
`unclear but seems to be a consistent observation for this
`
`compound and related analogs.
`To determine whether inhibition of EGFr tyrosine phos~
`phorylation could lead to decreased expression or increased
`turnover of the suri"ace—bound receptor, tumor homogenates
`were assayed for EGFr protein using a commercial kit (On-
`cogene Science). ln several experiments, EGFr protein con-
`centrations did not change within 24 h of a single dose of
`CP—358_.774 or within 1 h in multiply dosed animals (n
`20
`consecutive daily doses; data not shown). Moreover, it ap-
`peared from our data that in vivo receptor density remained
`relatively constant at 9.4 fmol/pug total protein among several
`experiments. Although we cannot conclude that transient
`changes in receptor density did not occur in these animals, it
`is apparent
`that a prolonged drug—induced reduction in
`EGFr—associated tyrosine phosphorylation could not be eX~
`plained by concurrent. reductions in receptor density. Simi-
`larly, tumor tissue concentrations of Cl’—35£%,774 correlated
`Well with plasma concentrations. In Fig. 3, the use of athymic
`mice bearing bilateral tumors allowed the simultaneous mea-
`surements of EGFr phosphotyrosine, tumor tissue drug con-
`centration, and plasma drug concentrations. On average, the
`peak tumor tissue concentration Was 44- and 29% of the peal:
`circulating plasma levels for p.o. and ip. dosing, respectively,
`
`

`
`744
`
`Pollack et al.
`
`Vol. 291
`
`14°
`
`f(x) = l.368619E+O*x + 1.047061 E+1
`RA2 = 9.160709E-1
`
`120 g
`
`100
`
`so’
`
`60’
`
`4o
`
`20‘
`
`0
`
`10\ 2ll
`
`30
`
`40
`
`50
`
`60
`
`:10
`
`8(l
`
`90 i00
`
`
`
`
`
`TumorGrowthInhibition(%),MultipleDose
`
`
`
`
`
`
`
`
`
`9103‘3[udvuoS[1?.II.I1l()flgggv11231o's{12tuno[1ods1>.'1odfmoi}popizofumoq
`
`
`
`
`
`EGFR-PY inhibition (96) @ 1 hr Post-Dose,