Aq_({AmWican Associationfor CancerResearch
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`NNUAL MEETING OF THE
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`Li AMERICAN ASSOCIATION
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`FOR CANCER RESEARCH
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`93TdAnhual Meeting
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`April 6-10, 2002 San Francisco, Cali omia
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`Volume 43 A March 2002
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`In _IoInt eponsorsfiip
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`flee
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`ef Medicine of the UnIversit}e of Soethern rCalirfo1-nia
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`The premier meeting for cancer research in the post-genomic era W
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`'
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`Featuring the latest developments in basic, translational, and clinical cant-?e‘r research
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`Par Pharm., Inc.
`Exhibit 1030
`Page 001
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`Par Pharm., Inc.
`Exhibit 1030
`Page 001
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`

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`Proceedings of the
`American Association
`for Cancer Research
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`AMERICAN ASSOCIATION FOR CANCER RESEARCH, INC.
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`Officers
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`President
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`. .Waun Ki Hong
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`President—Elect
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`. .Susan Band Horwitz
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`. .Bayard D. Clarkson
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`Treasurer
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`Past President
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`. .Tom Curran
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`Chief Executive Officer .
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`. .Margaret Poti
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`Board of Directors
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`Until 2002
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`Until 2003
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`Until 2004
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`Mina Bissell
`Michael B. Kastan
`Edison T. Liu
`Frank Rauscher, 111
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`Frederick R. Appelbaum
`Michaele C. Christian
`Ronald A. DePinho
`Lorraine Gudas
`Tyler E. Jacks
`William G. Nelson, V
`George F. Vande Wotide
`Peter K. Vogt
`Barbara L. Weber
`Geoffrey M. Wahl
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`lnc. (AACR), Public Ledger Building, Suite 826, 150 S.
`Address inquiries to the Office of the American Association for Cancer Research,
`Independence Mall \West, Philadelphia, PA 191065483 [Telephone-: (215) 4409500; Fax: (215) /1/10/9315].
`
`The Pmmedingr /Jft/rIeAr»m‘z'rzz12Arroriationfir C/mm” Rtarzrzzn‘/7 is printed for the AACR by Cadtnus Professional Communications. Linthicum, MD
`210902908 and is included in the membership dues for active and associate members. Volume 43 of the 1’roL'eedz'ng.r of‘:/7eA17ze17'r/zn Ar:oc.'z'zztizmfiJr
`Cancer Rcsmrr/7 (ISSN O197~O16X) succeeds Volume 42 of the ]’rarrm’z'rzgr aft/ac Anzerizzm Ari'az'iz1ti07z fin‘ Cancer Remzrc/7. The Pruzwa/z'n_g: inay be
`obtained at a price of $55.00 through registration at the Annual Meeting of the American Association for Cancer Research, April 6-10, 2002, or
`ordered by writing to: AACR Subscription Office, PO. Box 11806, Birmingham, AL 35202 [Telcphoncz (800) 633-4931 or (205) 9954567; Paar:
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`The Pmccrdlngr aft/M /lmm'z'czz7z /l.t.mcz'zztz'rmfr)r Cezmrr Research is copyrighted © 2002 by the AACR. All rights reserved. Redistribution or resale of
`the P7‘ocem'2'ngr or ofany materials in the /’mcrrrtd[71g.r, whether in machine readable, other electronic, or any other form, is prohibited. Reproduction
`for advertising or promotional purposes, or republication in any form, may be permitted only under license from the AACR. Any reproduction,
`whether electronic or otherwise, of abstracts beyond that permitted by copyright law must be authorized in writing in advance by the AACR.
`Requests to reproduce abstracts will be considered on an individual basis and permission may be granted contingent upon payment of an appropri-
`ate fee. Reproduction requests must include a brief description of intended use. Third parties should also obtain the approval of the authors before
`corresponding with the AACR. Failure to comply with the foregoing restrictions and unauthori7.ed duplication of any portion of these materials are
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`No responsibility is accepted by the Editors, by the American Association for Cancer Research,
`Communications for the opinions expressed by the contributors herein.
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`Ii1c., or by Cadmus Professional
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` -1
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`p
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`93rd Annual Meeting
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`Par Pharm., Inc.
`Exhibit 1030
`Page 002
`
`Par Pharm., Inc.
`Exhibit 1030
`Page 002
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`

`
`lines (NB4, HL—60 and U937). S8 was
`differentiation of myeloid leukemia cell
`identified in our recent studies as a potent inhibitor of PTPases. Herein, we
`present data demonstrating that SS (250 pg/ml, 6 days) induced 87% of NB4
`cells to reduce nitroblue tetrazolium (NB'D, in comparison to the 90% induced by
`ATRA (1 pM, 6 days). SS—induced NB4 cell differentiation was confirmed by
`increased CD11b expression and associated with growth arrest at S phase and
`increased cell death. Our results showed further that SS~incluced NB4 differen-
`tiation was irreversible and required continuous drug exposure for optimal induc-
`iion. Moreover, SS (400 pg/ml, 6 days) induced 60% and 55% of NBT—positive
`cells in HL—60 and U937 cell
`lines, which were augmented in the presence of
`GM—CSF (2 ng/ml) to levels (85% and 81%, respectively) comparable to those
`induced by ATRA. These results provide the first evidence of a differentiation
`induction activity of PTPase inhibitor SS in myeloid leukemia cell
`lines and
`Ssuggest its potential therapeutic use in myeloid leukemia. Since SS induces
`differentiation via targeting PTPases, a mechanism distinct from that of ATRA,
`it
`may be particularly useful in AML cases unresponsive or developed resistance to
`ATRA.
`
`#356 NAD(P)H:quinone oxidoreductase (NQ01)-dependent and -indepen-
`dent cytotoxicity of potent quinone cdc25 phosphatase inhibitors. Yusheng
`Han, Hongmei Shen, Brian Carr, John S. Lazo, and Su—Shu Pan. University of
`Pittsburgh Cancer Institute, Pittsburgh, PA, and University of Pittsburgh, Pitts-
`burgh, PA.
`A vitamin K analogue, compound 5 (Cpd5. a thioethanol naphthoquinone),
`inhibits oncogenic Cdc25 phosphatases, and arrests cell cycle progression at
`both G1 and G2/M. Recently, we evaluated ;>10,000 compounds in the NCI
`chemical repository for in vitro inhibition against recombinant human Cdc25B
`phosphatase and identified a quinone substructure in many of the active com-
`pounds. Bioreductive enzymes in cells, however, are known to reduce various
`quinones resulting in either detoxification or activation. Therefore, we used an
`isogenic set of human colon cancer cell lines to evaluate the effect of NQO1 on
`the cytotoxic activity of Cpd5 and the two most potent phosphatase inhibitors
`from the repository: NSC 95397 (a bis—thioethanol haphthoquinone) and NSC
`663284 (a quinolinedione). The two cancer cell lines used were HCT116, which
`has intermediate NQO1 activity. and its mitomycin C—resistant sub—line HCT116-
`Fi3OA (HSOA), which has minimum NQO1 activity. Cell survival was measured by
`colony formation after 7 days drug exposure. Cell cycle arrest was evaluated by
`flow cytometry after 6 hr drug exposure. Cpd5 had an lC50 of 22:03 nM for
`HCT116 and 0.23*0.05 j.LM for RSOA,
`i.e. a 10-fold difference.
`Inclusion of
`dicoumarol
`(10 piM), an inhibitor of NQO1, decreased the lC5,, of Cpd5 for
`HCT116 to 0.24:0.04 iiM, but had no effect on Ft30A. in contrast, HCT116 and
`R30A cells were equally inhibited by NSC 95375 with |C5Os of 1.4+0.3 p.M and
`1.3tO.2 pM, respectively. Similarly, HCT116 and RSOA cells were equally inhib-
`ited by NSC 663284 with lC50s of 24:03 p.M and 2.6:0,5 ;.i.l\/l, respectively. All
`three compounds blocked the two cell lines at the G2/M phase transition, con-
`sistent with cdc25 inhibition. Cpd5 at 2.5 ].LM arrested Fi30A cells at G2/M but 7.5
`pM Cpd5 was needed to arrest HCT116 cells to a similar degree. NSC 95375 and
`663284 arrested cell cycle progression at G2/M of HCT116 and RSOA cells
`similarly, and did so in a concentration—dependent manner between 2.5 and 7.5
`i.iM. Our data imply that NQOl in HCT116 cells protected cells from the action of
`Cpd5, probably by the reduction of Cpd5 to a less active hydroxylquinone.
`In
`contrast, both NSC 95397 and 663284 displayed cytotoxicity that was indepen-
`dent cf NQO1 levels. (Support: NCl CA61862 and CA78039)
`
`#357 Antitumor and anticarcinogenic action of Cpd 5: A new class of
`protein phosphatase inhibitor. Siddhartha Kar, Meifang Wang, Zhenggang Ren,
`Xiangbai Chen, and Brian I. Carr. University of Pittsburgh, Pittsburgh, PA.
`Background: We have chemically synthesized a new class of inhibitors of dual
`Specificity phosphatases (DSP), which play an important role in cell cycle and
`Signal transduction. Cpd 5 or 2—(2—mercaptoethanol)—3—methy|—1,4—naphthoqui—
`none is one of the most potent. It inhibits DSPS (especially the Cdc25 family) in
`tissue culture cells and induces tyrosine phosphorylation of various DSP sub~
`Strates,
`including Cdks and inhibits cell growth both in vitro and in vivo (JBC
`27028304, 1995; Proc. AACR 39:224, 1998). Purpose: In this study we evaluated
`(a) the antitumor and (b) the anticarcinogenic activity of Cpd 5 for the first time.
`Methods: (a) JM1 hepatomas were grown in 2 month old Fischer male rats by
`Subcutaneous injection on the back or intra—portally in the liver. Rats were treated
`with Cpd 5 by intratumor, subcutaneous (nearby site), intramuscular (distant site),
`Or intraperitoneal injection, either as a single high acute dose or chronically as
`Several low doses. (b) Rats were injected intraperitoneally with a single dose of the
`Carcinogen N-Nitrosodiethylamine (DEN). lmmunostained liver sections for gluta-
`i_hione—S-transferase—pi (GST—pi) detected pre—neoplastic foci after 3 weeks. Cpd
`°_ Was injected subcutaneously or intraperitoneally two weeks after DEN as a
`Single high acute dose or chronically as several low doses. Results: (a) Cpd 5 had
`Significant inhibitory effect on both intrahepatic (14% of control, p<0.0000000B)
`and subcutaneous (33% of control, p<0.00008) tumor growth and also had
`Significant inhibitory effect when injected intramuscularly at a site distant from the
`lumor (50% of control, p-110.002). There was no significant difference between the
`effects after acute or chronic injections. However, toxicity was much lower with
`Chronic treatment. (b) The number of enzyme altered foci was also significantly
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`EXPERIMENTAL/MOLECULAR THERAPEUTICS 3
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`reduced when rats were treated with acute (40% of control, p<0.00002) or
`chronic (50% of control, p<0.02) Cpd 5. Conclusions: Cpd 5 had significant
`inhibitory effect on growth of tumors and foci.
`
`#358 Bosentan, a novel endothelin-A and -B receptor antagonist inhibits
`proliferation of malignant melanoma cells. Aleksandar Sekulic, Padma Suresh,
`Mark R. Pittelkow, and Svetomir N. Markovic. Mayo Foundation, Rochester, MN.
`Here we tested a feasibility of endothelin (ET) receptor blockade with a dual
`endothelin—A and »B receptor (ETR—A and ETR-B) antagonist, Bosentan, as a
`novel therapeutic approach for malignant melanoma. ETs are 21aa peptides
`primarily produced by endothelial cells and implicated in a variety of physiological
`functions. Binding of ET to ETR—A on vascular structures potently stimulates
`angiogenesis and,
`thus,
`likely plays an important role in growth of multiple
`cancers. Activation of ETR—Bs, among other things, regulates melanocyte devel-
`opment and function. We first examined patterns of ETR subtype expression on
`six established melanoma cell lines using flow cytometry and immunocytochem—
`istry. Following this sections of primary and metastatic melanoma tissues were
`evaluated for ETRA/ETRB expression by immunohistochemistry. To test func-
`tional effects Bosentan on melanoma cell proliferation, six melanoma cell lines
`were subjected to standard 3H»thymidine incorporation assays in presence or
`absence of various concentrations of Bosentan. All examined melanoma tissues
`(2 primary, 9 metastatic) express ETRB, albeit to different levels, whereas ETRA
`was expressed to low levels in only 3 metastatic tumors.
`in functional assays
`Bosentan inhibited proliferation of all examined cell lines with the |C50 ranging
`between 7 and 40 pg/ml. Our results suggest that malignant melanocytes express
`functional ETRs, and their treatment with Bosentan leads to significant growth
`inhibition. Concurrent inhibition of ETR-A and ETFi—B in vivo by low toxicity, orally
`available inhibitor Bosentan might therefore prove useful as a novel mode of
`anti—melanoma therapy through simultaneous inhibition of cancer cell growth and
`process of angicgenesis.
`
`#359 In vivo activity of RADOO1, an orally active rapamycin derivative, in
`experimental tumor models. Terence O’Fiei|ly, Juliane Vaxelaire, Melanie Muller,
`Heinz—Herbert Fiebig, Marc Hattenberger, and Heidi A. Lane. Business Unit On-
`cology, Novartis Pharma AG, Basel, Switzerland, and Oncotest gmbH, Freiberg,
`Germany.
`RADD01 is a hydroxyethyl ether derivative of rapamycin that is orally bioavai|—
`able. RADOO1 has demonstrated in vitro anti—proli1erative activity against a panel
`of human tumor lines. For in vivo testing, tumor—bearing nude mice were admin-
`istered RAD001 in a variety of doses and schedules. Tumors were established by
`transplantation of fragments generated from injection of cells, or by transplanta-
`tion of fragments from stabilized tumors originating from surgically removed
`human tumors. When administered once daily p.o., at doses ranging from 0.5-5.0
`mg/kg/day, FlAD001 was a potent
`inhibitor of tumor growth in 10 different
`xenograft models of human tumors (including pancreatic, colon, epidermoid, lung
`and melanoma).
`In general, RAD001 was well tolerated and better tolerated in
`mouse xenograft models than standard cytotoxic agents (i.e. doxorubicin and
`5—fluorouracil), while possessing similar antitumor activity. Only one instance of in
`vivo resistance has been observed (MAXF 401 mammary xenograft model),
`otherwise the activity of RADO01 was generally inhibition of tumor growth (per-
`sistent regressions in one tumor line, T/C values of 9 to 45 % in 8 tumor lines).
`Xenograft models sensitive to RAD001 treatment
`included tumors exhibiting
`comparative resistance in vitro (KB—31 and HCT116). Persistent tumor regres-
`sions (41 %) were observed in a line displaying sensitivity to RADOO1 in v/'tro
`(A549). Pharmacokinetic analyses, following a 5 mg/kg administration, indicated
`rapid uptake into plasma (Cmax 2513 ng/ml; Tmax1 h), but the time to Cmax was
`delayed in tumors (Cmax 102 ng/g; Tmax 2 h). Elimination from the tumor (t1/2,
`16 hr) was apparently slower than for plasma (t1/2, 7.5 hr). RAD001 levels were
`above the lC5O of A549 cells for a 72 h period. interestingly, tumor RAD001 levels,
`following a single 5 mg/kg administration, never exceeded the in vitro antipro|if—
`erative lC50 for either KB—31 or HCT116 cells; despite the sensitivity of these lines
`in vivo. From these observations, and given the extreme sensitivity of endothelial
`cells to RAD001, it is plausable that RAD001 may not only act on tumor cells but
`may also affect angicgenesis. Taken together, these data support the application
`of RADO01 as an antitumor agent.
`
`#360 Discovery of anticancer agents from sponge-associated fungi. Fred-
`erick A. Valeriote, Karen Tenney, Charles Grieshaber, Halina Pietraszkiewicz,
`Akiko Amagata, Taro Amagata, Jeff Gautschi, Joseph Media, Joseph Stayanoff,
`Richard Wiegand, and Phil Crews. Henry Ford Health System, Detroit, MI, and
`University of California Santa Cruz, Santa Cruz, CA.
`We have evaluated 1,112 extracts (from 660 sponge—associated fungi) for
`assessment of potential anticancer activity. Both broth and mycelia extracts were
`assayed in most cases. Each sample was assayed in vitro against up to 8 cell
`types (murine and human) in a disk diffusionl clonogenic assay. From these
`results, the samples were assigned into one of 4 categories: Inactive (79% of the
`extracts), Equally active across cell types (16% of the extracts), Equally active and
`potent (9 extracts or 1%), and Solid tumor selective (42 extracts or 3.8%). The
`equally active and potent category is studied further since solid tumor selective
`compounds might exist in the extract but be concealed by one or more potent,
`cytotoxic compounds. Further, a novel, potent compound could form the basis
`for analog synthesis in an attempt to develop an active anticancer agent. One
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`Proceedings of the American Association for Cancer Research 0 Volume 43 0 March 2002
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`71
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`This material may be protected by Copyright law (Title 17 U.S. Code)
`
`Par Pharm., Inc.
`Exhibit 1030
`Page 003
`
`Par Pharm., Inc.
`Exhibit 1030
`Page 003