APRIL 12- ~ 6, ~ 997
`SAN DIEGO, CA
`
`~997
`
`TX 1495
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`DPEM2 0002317
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`Sandoz Inc. IPR2016-00318
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`
`PROCEIED~NGS OF =[’HE
`AMIER~CAN ASSOCIATION FOR CANCER RESEARCH
`
`AMERICAN ASSOCIATION FOR CANCER RESEARCH, iNC.
`
`Executive Director and
`Director of Publications
`Margaret Foti
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`Publications Staff
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`of Publications
`Mary Anne Mennite
`
`~lanager, Editorial Services
`Heide M. Pusztay
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`Staff Editors
`Michael J. Beveridge
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`Louise C. Strong, President
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`Board of Directors
`
`Term Expiring 1997
`Webster K. Cavenee
`Philip C. Hanawalt
`Stanley J. Korsmeyer
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`Clara Derber Bloomfield
`Michael M. Gottesman
`Ann R. Kennedy
`Frederick P. Li
`
`Term Expiring 1999
`Susan P.C. Cole
`Eric R. Fearon
`Stephen H. Friend
`Waun Ki Hong
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`Administrative Staff
`Director of Administration
`Adam D. Blistein
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`The Proceedings of the American Association for Cancer Research is printed for the AACR by Cadmus Journal Services, Linthicum, MD 21090-2908 and included in
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`

`
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`Author index of Proffered Papers
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`X
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`
`y
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`
`Proceedings of the American Association for Cancer Research e Volume 38 o March 1997
`
`DPEM2 0002~J
`
`Sandoz hie. IPR2016-00318
`Sandoz v. Eli Lilly, Exhibit 1101-0003
`
`

`
`PHARMACOLOGY/THERAPEUTICS (PR~SCLINICAL AND CLINICAL) 9
`
`A55 does express a membrane protein which has a high affinity for folic acid, but
`a low affinity for folinic acid. Studies are underway to isolate and further charac-
`terize this protein.
`
`#654 Characterization of a human CEM leukemia cell line with altered
`RFC-mediated transport of relic acid, reduced folates and tetrahydrofolate-
`based antifolates. Mauritz, R.M., Kathmann, I., Peters, G.J., Pinedo, H.M.,
`Schomagei, J.H., Priest, D.G., Bunni, M., Drori, S., Assaraf, Y.G., and Jansen, G.
`Dept. of Oncology, Hosp. Vrije Universiteit, Neth. Cancer Inst., Amsterdam, The
`Netherlands, Med. Univ. S. Carolina, Charleston, SC, U.S,A, Dept. of BioL, The
`Technlon, Halfa, Israel
`CEM/MTX, a subline of human CEM leukemia cells is 200-fold resistant to
`methotrexate (MTX) due to defective transport via the reduced relate carrier
`(RFC). When adapted to grow in low folate (LF) medium containing only 2 nM relic
`acid, CEM/MTX-’LF’ cells overexpress (25-fold) an altered RFC protein that is
`characterized by strikingly altered properties (10-35 fold lowered Kin) for uptake
`of folic acid, folinic acid and tetrahydrofolate-based antifolates such as DDATHF,
`AG2032 and AG2034, but not for antifolates such as MTX, BW1843 or ZD1694.
`An 8-fold amplification of the RFC1 gene was observed in CEM/MTX-’LF’ cells,
`along with >!0-fold increased RFC1 mRNA expression. Wher~ CEM/MTX-’LF’
`were transferred to medium containing 2/~M relic acid, the lowered K~, for uptake
`of folic acid resulted in a 7-fold elevated pool of intracellular folates as compared
`to CEM cells. Under, these conditions, poiyglutamylati0n of ant!relates was abol-
`ished, resulting in resistance to antifolate drugs that are dependent on polyglu-
`tamylation for their biological activity (e.g. ZD1694 and DDATHF). (Supported by
`the Dutch Cancer Society, grant VU 96-1260.)
`
`#655 Augmented folic acid accumulation in pyrimethamine-resistant CliO
`cells. Assaraf, Y.G., Barr, H.M., Jansen, G., and Goldman, I.D. Depts. of Biology,
`Technion, Haifa, Israel Medical Oncology, Free University, Amsterdam, the Neth-
`erlands, Medicine and Molecular Pharmacology, Albert Einstein College of Med-
`icine, Bronx, NY, 10461
`We previously described a 1000-fold pyrimethamine-resistant CHO cell line
`(PyrR100) which retains parental sensitivity to MTX. This paper explores the basis
`for the 14-fold decreased folic acid growth requirement of PyrR100 cells. MTX
`influx via the reduced folate carrier (RFC) was identical in the PyrR100 and parent
`lines. The efflux rate constant was decreased 3-fold, and the steady-state MTX
`concentration was increased 3-fold in Pyr! 00 cells. After 4 h incubation with MTX,
`accumulation of polyglutamates was -5-fold greater in PyrR100 cells. Under
`conditions in which RFC activity and folic acid reduction were blocked (i.e. 20/~M
`MTX and 20 ~M trimetrexate) the initial uptake rate for 1 /~M folic acid was
`increased 2.5-fold in PyrR100 cells and there was a -14-fold increase in net
`[3H]folic acid uptake largely due to the accumulation of essentially nonexchange-
`able label. Folylpolyglutamate synthetase activity in PyrR!00 ceils was 2.5-fold
`greater than in parent CH0 cells. The data indicate that the markedly decreased
`relate growth dependence for PyrR100 cells is associated with at least two
`factors: enhanced transport of relic acid into cells via a RFC-independent route
`and enhanced polyglutamylation. Studies are in progress to clarify the basis for
`the nonexchangeable folic acid component in PyrR100 ceils which accumulates
`under conditions in which folic acid reduction is blocked.
`
`#656 Comparisons of ;the synergistic growth inhibition by trimetrexate
`(TMQ) + AG2034 and TIVlQ + Tomudex (ZD1694) of human ileocecal HCT-8
`cells and DW2, a subline deficient in folylpolyglutamate synthetase {FPGS).
`Faessel, H., Lu, K., SIocum, H.K., Rustum, Y.M., and Greco, W.R. Roswell Park
`Cancer Institute, Buffalo, NY 14263
`Folic acid (FA)-enhanced super synergy between nonpolyglutamylatable dihy-
`drofolate reductase (DHFR) inhibitors and polyglutamylatable inhibitors of other
`relate-requiring enzymes, such as glycinamide ribonucleotide formyltransferase
`(GARFT) and thymidylate synthase iTS), is a general phenomenon (Gaumont et al,
`Cancer Res. 52:2228-2235, 1992). We report here on the combined-action (96 hr
`drug exposure) of the DHFR inhibitor, TMQ, with the GARFT inhibitor, AG2034,
`and with the TS inhibitor, ZD1694. At 2.3/~.M FA, DW2 cells were 2.7 fold more
`sensitive than parent HCT-8 cells to TMQ, but 8.8-fold End 17-fold more resistant
`to AG2034 and ZD1694. The ability of 40 ~M FA to increase the Loewe synergy
`for the TMQ + AG2034 combination was 6-fold less for the FPGS-deficient DW2
`subline than for HCT-8 cells. The FA-enhancement of Loewe synergy for TMQ +
`ZD1694 in HCT-8 was absent in DW2. In both cell lines, at 2.3 and 40/~M FA, 10
`~M thymidine enhanced about 2-fold the intensity of Loewe synergy for TMQ +
`AG2034. Also, in both cell lines, at 2.3 and 40 ~.M FA, the combined-cytotoxicity
`of TMQ + AG2034 was only partially prevented by 100 /~M hypoxanthine.Thus,
`the effects of FA, antifolates, and combinations of antifolates are dependent on
`intracellular FPGS activity. [Supported by RR10742 & 0A16056.]
`
`detected using probes derived from the human FPGS cDNA cloned by Shane and
`co-workers (Garrow et al. Prec. Natl. Acad. Sci. USA 89: 9151, 1992). Protein was
`detected by Western blot analysis using an immunoaffinity-purified rabbit anti-
`body elicited to an FPGS-peptide deduced from the cDNA sequence. FPGS
`activity was assayed in extracts using the method developed in this laboratory.
`One subline resistant to MTX as a result of amplified dihydrofolate reductase
`shows no change in FPGS DNA, RNA, activity or protein relative to CCRF-CEM.
`An MTX transport-defective line, however, displays higher FPGS protein and
`activity levels. Several sublines, in which the only apparent meclianism of MTX
`resistance is decreased FPGS activity, have the same gene copy number and
`restriction map, and mRNA size and level as the parent; however, the FPGS
`protein level in these sublines is decreased similarly to their decreased activity.
`Apparently, in these sublines the mRNA is either poorly translated, the protein is
`tess catalytically active, and/or the protein [urns over more rapidly; these possi-
`bilities are now being studied. Supported by 0A43500 & The Buffalo Foundation.
`
`#658 Analysis of the ATP and Folate binding sites of recombinant human
`folylpoly-3,-glutamate synthetase (rhFPGS}. Sanghani, P., Sanghani, S., and
`Moran, R. Medical College of Virginia, Richmond, VA 23298
`An active site labeling approach using carbodiimide-activated methotrexate
`and iodoacetamide was taken to identify aminoacids interacting with the ~- and
`~/- carboxyt groups of the glutamate side chain of the folate molecule and reactive
`cysteines responsible for thiol requirement of the enzyme. Both carbodiimide-
`aetivated methotrexate and iodoacetamide inactivated rhFPGS irreversibly. The
`bond between activated methotrexate and enzyme was not stable to the condi-
`tions of peptide analysis, but enzyme activity was protected by excess metho-
`trexate. However, reaction of enzyme with iodoacetamide and subsequent tryptic
`digestion and microsequencing identified three cysteines distributed over the
`primary sequence. Modification of all three reactive cysteines was prevented by
`ATP alone, indicating that the peptides containing these residues form the ATP-
`binding pocket. The conserved aminoacids flanking the reactive cysteines are
`being studied by site-directed mutagenesis.
`
`#659 A novel posttranscriptional mechanism of antifolate resistance in
`L1210 cells decreasing synthesis of folylpolyglutamate synthetase. Roy, K.,
`and Sirotnak, F.M. Molecular Pharmacology and Therapeutics Program, Memorial
`Sloan-Kettering Cancer Center, New York, NY 10021
`The anabolism of folate analogues by foiylpolyglutmate synthetase (FPGS) is an
`important determinant of their cytotoxicit~/and acquired resistance in tumors. A
`decrease of up to 23 fold in the amount of FPGS in some edatrexate resistant
`variants was recently shown to be posttranscriptionally determined, resulting
`from an alteration of the cognate mRNA, itself, which impairs its translation. We
`now provide evidence to show that this impairment occurs from an alteration of
`progression rather than initiation of translation. No differences were seen in the
`variant versus wild-type mRNA nucleotide sequence in the 5’ UTR that is relevant
`to ribosome binding. However, base-pair differences were identified in the ORF of
`the FPGS mRNA from these variants. These base-pair differences represent
`either single purine to purine or pyrimidine to pyrimidine transitions. The most
`impaired variant exhibited two such base-pair transitions in its FPGS mRNA.
`Since some codons resulting from base-pair transitions did not evoke an amino
`acid substitution, the decrease in amount of FPGS cannot reflect merely in-
`creased degradation. This is in agreement with data provided earlier showing no
`deference in FPGS turnover in these variants. These data appear to document
`examples of altered translation mediated by mutationaliy determined differences
`in secondary structure of FPGS mRNA. Support: Grant CA56517 from the Na-
`tional Cancer Institute.
`
`#6{}0 The synthesis and biological activity of a series of 2,4-diaminopy-
`rido[2,3-d]pyrimidine based antifolates as dihydrofolate reductase !rib!bi-
`ters. Gossett, L., Habeck, L., Shackelford, K., Mendelsohn, L, Gates, S., Wor-
`zalla, J., Self, T., Theobald, K., And!s, S., Schultz, R, and Shih, C. Lilly Research
`Laboratories, Lilly Corporate Center, Indianapolis, IN 46285
`Antifolates, such as methotrexate, that contain the 2,4-diaminopyrimidine con-
`figuration have long been known to possess potent enzyme inhibition against
`dihydrofolate reductase (DHFR). As part of our continuing exploration into the
`SAR of 5,10-dideazatetrahydrofolic acid (DDATHF), we have synthesized a series
`of ant!relates which contain the 2,4-diaminopyrido[2,3-d]pyrimidine structure.
`Thede compounds displayed potent cytotoxicity against human leukemia CCRF-
`CEM cells in culture with IC50 values as low as 5 nM. Enzyme assay studies using
`human DHFR show a span of inhibition from 6.3 nM to 10 nM. In vivo ant!tumor
`results, enzyme inhibition against other folate-dependent enzymes, and the ability
`of these compounds to act as substrates for folylpolyglutamate synthetase will
`also be discussed.
`
`#657 Folylpolyglutamate synthetase (FPGS) expression in antifolate-sen-
`sitive and -resistant human leukemia cell lines. McGuire, J.J., and Russell,
`C.A. Grace Cancer Drug Center, Roswetl Park Cancer lnstitute, Buffalo, NY 14263
`USA
`FPGS expression in the methotrexate (MTX)-sensitive human Tqymphoblastic
`leukemia cell line CCRF-CEM and a number of MTX-resistant sublines has been
`investigated at the DNA, RNA, protein, and activity levels. DNA and RNA were
`
`~
`
`H2N
`
`~J.~N."
`
`H
`
`C__OOH
`
`~,~ H
`
`CH2CH2COOH
`
`~
`1,4-phenyl LY335518
`2,5-thienyl LY335580
`2,5-furanyl LY335738
`
`98
`
`Proceedings of the American Association for Cancer Research e Vobr~2e ~i~31j~7
`
`Sandoz Inc. IPR2016-00318
`Sandoz v. Eli Lilly, Exhibit 1101-0004
`
`

`
`doses. The increases of lifespan achieved with F 12158, as assessed by T/C
`ratios, ranged from 200 to 457%, and proved markedly superior to those obtained
`with any other Vieca (129 to 157%). Against sc implanted B16 melanoma, multiple
`ip administrations of F 12158 proved active both in terms of survival prolongation
`(LogRank test probability < 0.001) and tumour growth inhibition (optimal T/C of
`25%). Tumour growth inhibition of human LX-1 lung and MX-1 breast xenografts
`was also observed with intermittent ip F 12158 treatments (optimal T!C of 23 and
`26% respectively), whereas the human DLD-1 colon xenograft proved generally
`non-responsive. Overall F 12158 demonstrated antitumour activity superior to
`that of vinorelbine in each tumour model evaluated.
`
`#1512 Novel water soluble paclitaxel derivatives: Evaluation of PEG-
`paclitaxel’s in vitro and in vivo effects. Gilbert, C., Conover, C., Pendri, A., Shum;
`K., and Greenwald, R. Enzon Inc., 20 Kingsbridge Road, Piscataway, NJ 08854
`This study was designed to assess the toxicity and efficacy of water soluble
`polyethylene glycol (PEG) conjugated paclitaxel prodrug derivatives. Initial candi-
`dates were screened for in vitro cell toxicities and rates of hydrolysis in water, buffer
`and human plasma. Lead candidates were then tested in vivo within a toxicity battery
`which included hematology, blood chemistry and histopathological profiles and for
`efficacy in both murine leukemia and human xenograft models. Lead compounds
`displayed in vitro cell toxicities equivalent with paclitaxel and delayed hydrolysis in rat
`plasma. These same derivatives caused reduced in vivo toxicity with significant
`survival over paclitaxel controls in our murine leukemia model and encouraging tumor
`reduction in pilot xenograff models. Thus, PEG conjugation may offer an effective
`methodology for paclitaxel delivery.
`
`#1513 Interaction of cryptophycin with isotypically purified tubulin.
`Prasad, V., Chaudhuri, A.R., and Luduena, R.F. Dept. of Biochemistry, University
`of Texas Health Science Center., San Antonio, TX 78284
`Cryptophycin, isolated from the cyanobacterium Nostoc, is a dioxadiazacyclo-
`hexadecenetetrone with strong antimitotic activity. We have examined the inter-
`action of cryptophycin with unfractionated tubulin and its isotypes in vitro. Cryp-
`tophycin irhibits microtubule assembly at concentrations of 1-5 /zM. When the
`tubulin-cryptophycin complex was treated with different concentrations of urea,
`c~/3~ appeared more stable than unfractionated tubulin as determined by trypto-
`phan fluorescence quenching. After removing the C-termini of the tubulin, cryp-
`tophycin still inhibits alkylation of the sulfhydryl groups which implies that C-
`termini may not be involved in the binding site of the drug. Sedimentation
`experiments indicate that cryptophycin appears to make aggregates of 13r-16s
`which differs from that of tubutin, which is 6s. [Supported by grants CA26376 from
`the NIH and AQ-0726 from the Welch Foundation.]
`
`#1514 Preclinical antitumor activity of cryptophycin-52/55 (C-52;C-55)
`against human tumors in SCID mice. Polin, L., Valeriote, F., Moore, R., Tius, M.,
`Barrow, R., Hemscheidt; T., Liang, J., Paik, S., White, K., Harrison, S., Shih, J.,
`Martinelli, M., and Corbett, T. Karmanos Cancer Inst./Harper Hosp., Detroit, MI
`48202 [L.P., F.V., K.W., T.C.], University of Hawaii, Honolulu, HI 96822 [R.M., M.T.,
`R.B., T.H., J.L., S.P.], Eli Lilly Corp., Indianapolis, IN 46285 [S.H., J.S, M.M.]
`C-52 and C-55 are gem-dimethyl (Carbon-2, C-unit position) analogs of Crypto-
`phycin-1 and -8, respectively; antitumo[.agents with broad antitumor activity against
`both human tumor xenografts and routine tumors and having no cross-resistance
`with Taxol or Adriamycin (J. Exp. Then Onc. 1:95, 1996). C-55 is the chlorohydrin of
`C-52 (an .epoxide). The in vivo efficacy at the MTD for a variety of human tumors in
`SCID mice is shown below and demonstrates that both compounds have broad
`anticancer activity although C-55 has greater therapeutic efficacy.
`
`C~ypto-52
`
`Crypto-55
`
`Human
`Tumor
`
`T!C
`in %
`
`Log10
`Cell Kill
`
`Cures
`
`T/C
`in %
`
`Log10
`Ceil Kill
`
`Cures
`
`TSU-Prost. #
`LNCaP-Prost.#
`PC3-Prost. #
`H116-Colon #
`H125-Lung
`Gib-Panc
`
`15
`7
`32
`4
`5
`16
`
`1.8
`3.2
`0.4
`2.4
`1.8
`1.0
`
`0/5
`0/5
`0/5
`0/5
`0/5
`0/5
`
`0
`0
`0
`8
`0
`4
`
`# = Unstaged Tumors at 1st Rx. (100 to 500 mg).
`
`4.0
`5.9
`2.9
`3.6
`2:4
`2.4
`
`2/5
`3/5
`0/5
`0/5
`0/5
`0/5
`
`Supported by Eli Lilly Corp. and NCI-CA53001.
`
`#1515 Preclinical antitumor activity of Cq/ptophycin-52/55 (C-52;C-55) against
`mouse tumors. Corbett, T., Valeriote, F., Simpson, C., Moore, R, Tius, M., Barrow, R.,
`Hemscheidt, T., Liang, J., Paik, S., Polin, L., Pugh, S., Kushner, J., Harrison, S., Shih, J.,
`and Martinelli, M. Karmanos Cancer lnst./Harper Hosp., Detroit MI, 48202 [T.C., F.V.,
`C.S., L.P., S.P., J.K.], University of Hawaii, Honolulu, HI 96822 [R.M., M. T., R.B., T.H., J.L_,
`
`S.P.], Eli IuTty Corp., Indianapolis, IN 46285 [S.H., J.S., M.M.]
`C-52 and C-55 are gem-dimethyl (Carbon-2, C-unit-position) analogs of Cryp-
`tophycin-1 and -8, respectively; antitumor agents with broad antitumor activity
`against both human tumor xenografts and murine tumors and having no cross-
`
`PHARMACOLOGY/THERAPEUTICS (PRECLINICAL AND CLINICAL) 15
`
`resistance with Taxol or Adriamycin (J. Exp. Ther. Onc. 1:95, 1996). C-55 is the
`chlorohydrin of C-52 (an epoxide). The in vivo efficacy at the MTD for a variety of
`routine tumors is shown below and demonstrates that both compounds have
`broad anticancer activity although C-55 has greater therapeutic efficacy.
`
`Crypto-52
`
`Crypto-55
`
`Mouse
`Tumor
`
`T/C
`in %
`
`Log10
`Cell Kill
`
`Cures
`
`T/C
`in %
`
`Log10
`Cell Kill
`
`Cures
`
`Colon-38#
`Mare-16/C
`Mam-16/ADR
`Mam-17/ADR
`Panc-02
`Panc-03
`Colon-26
`
`0
`5
`2
`10
`" 9
`--
`14
`
`1.6
`1.2
`2.0
`1.6
`2.3
`--
`1.1
`
`0/5
`1/5
`0/5
`0/5
`0/5
`--
`0/5
`
`0
`0
`0
`0
`9
`0
`0
`
`4.7
`3.9
`2.4
`2.1
`2.9
`>4.5
`1.2
`
`0/5
`0/5
`0/5
`0/5
`0/5
`5/5
`0/5
`
`# = Unstaged 150-380 mg at 1st Rx.; C-52 = 1/5 CR’s; C-55 = 4/5 CR’s.
`
`Supported by Eli Lilly Corp. and NCI-CA53001,
`
`#1516 LY355702 and LY355703~ new cryptophycin analogues with antitu-
`mot activity against human tumor xenografts. Worzalla, J.F., Cao, J., Ehlhardt,
`W.J., Harrison, S.D., Law, K.L., Martinelli, M.J., Self, T.D., Starling, J.J., Shih, C.,
`Theobald, K.S., Toth, J.E., Zimmermann, J.h, and Corbett, T.H. Lilly Research
`Labs, Indianapolis, IN 46285, Wayne State U., Detroit, MI 48201 ~T.H.C.]
`LY355702 and LY355703 are new synthetic analogs of the marine natural
`product cryptophycin. Cryptophycins bind to tubulin and show potent in vitro
`cytotoxicity and in vivo antitumor activity against solid murine tumor models (G.
`Trimurtulu et at., J. Am. Chem. Soc., 116:4729, 1994). LY355702, a chlorohydrin
`cryptophycin analog, is converted in vivo to the epoxide, LY355703. The maxi-
`mum tolerated dose (MTD) of LY355702 (i.vl bolus q2dxS) was 200 to 240 mg/kg
`total and 40 to 50 mg/kg total for LY355703. Both showed 90 to 100% inhibition
`of tumor growth against LX-1 lung, MX-1 mammary and GC3 colon xenografls. A
`single course of either at MTD regressed established (300 to 400 mg size) GC3
`and H116 colon, MX-1 mammary and LNCaP prostate tumors, with some total
`regressions noted in mammary and prostate tumors. Against these established
`tumors, groups treated with the analogs took from 2.4 to 9.8 times as many days
`to reach 1 gram tumor size as compared to controls; log cell kill values of 0.7 to
`4.8 were observed in these studies. Against wild-type UCLA-P3 non-small cell
`lung cancer xenografl, LY355702 and LY355703 were not as effective as taxol,
`but the cryptophycin analogs were more effective than taxol against a P-glyco-
`protein expressing variant of this tumor.
`
`#1517 Inhibition of microtubule polymerization and dynamics by two
`novel cryptophycins, cryptophycins 52 and 55. Randa, D., Williams, D.C.,
`Wagner, M.M., Paul, D.C., Habeck, L.L., Mendelsohn, L.G., Shih, C., Moore, R.E.,
`and Wilson, L. Dept MCD BioL, Univ of California, Santa Barbara, CA, Lilly
`Research Labs, Indianapolis, IN, Dept Chemistry, Univ of Hawaii, Honolulu, HI
`Cryptophycins 52 (C-52; LY355703) and 55 (C-55; LY355702) are new syn-
`thetic analogs of cryptophycin 1. Both exe