An Official Journal
`of the
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`Cancer Research
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`.. ti ~ovem er 2000 o Volume 6 0 Supplement
`-"? 1P. 4467s-4597s o ISSN 1078-0432
`c,i
`II
`~
`
`West-Ward Exhibit 1082
`Hidalgo NCI 2000 Abs #413
`Page 001
`
`

`

`4548s
`
`Poster Session 17
`
`Clinical Cancer Research o Volume 6 o November 2000 (Supplement)
`
`408 Inhibition of Ras expression in lymphoma by isoform specific anti(cid:173)
`sense oligonucleotides: effects on proliferation, survival and gene expres(cid:173)
`sion profiled by cDNA microarrays. Clarke, Paul, A.1. Sumpter, K.1
`·2 • Te
`• Mania. B. 4
`Poele, R. 1 , Di Stefano, F. 1 , Wooster, R. 3 • Cunningham, D.2
`, and
`
`Workman, P1 • CRC Centre for Cancer Therapeutics 1, Dept. Molecular Carcino(cid:173)
`genesis3, Lymphoma Unit2, Institute of Cancer Research and Royal Marsden
`Hospital, UK, and /SIS Pharmaceuticals, USA4
`•
`Ras proteins play a central role in signal transduction. The specific function of
`individual Ras-isofonns is unclear. but evidence suggests that they may have
`distinct functions. Different isoforms are mutated in specific tumours for example
`Kirsten (K~ - ras mutations in colorectal cancer and N-ras mutations in haemato(cid:173)
`logical malignancies. The use of recombinant knock-out mice has demonstrated
`that Ki-ras expression is essential for embryogenesis, while N-ras and Harvey (Ha)
`- ras knockout mice show few defects. Therapeutic strategies targeting the ex(cid:173)
`pression or post-translational modification of Ras are currently under development.
`However it is not entirely clear which particular isoform. if any, iS critical to tumour
`cell proliferation or survival. In this study our aims were to characterise the contri(cid:173)
`bution of each specific isofonn to lymphoma oell proliferation, survival and gene
`expression by using an antisense strategy to specifically inhibit the expression of
`eacn isofonn in non-Hodgkin's lymphoma (NHL) cell lines. Six different lymphoid
`cell lines were treated with antisense oligonucieotides specific for each individual
`Ras isofonn. Western blotting confinned that these antisense oligonucleotides
`specifically inhibited the expression of their target genes. Growth in media supple(cid:173)
`mented with 10% serum was not effected by any of the Ras antisense oligonu(cid:173)
`cleotides. However, under restrictive growth conditions (0.1 % serum) proliferation
`was significantly reduced following treatment with N-ras antisense. FACS analysis
`demonstrated there was no significant increase in cell death following treatment
`with any of the antisense molecules, nor was there a marked change in cell cycle
`distribution. Gene expression profiling using an in-house microarray with 5800
`cDNAs detected changes specific for the inhibition of each Ras-isofonn. In con(cid:173)
`clusion, N-ras appears to be required for lymphoma cell proliferation and would be
`a suitable therapeutic target in NHL
`
`409 Cerivastatin triggers apoptosis of AML cells with higher potency
`than lovastatin. Tan. M., Xia. Z .• Wong, W. W.-L, Dimitroulakos. J .. Minden,
`M., and Penn. LZ. Ontario Cancer lnstitvte, University Health Network, Toronto,
`Canada.
`The vastatin family of drugs inhibits the 3-hydroxy-3-methylglutaryl coen·
`zyme A (HMG-CoA) reductase enzyme that catalyzes the rate-limiting step in
`the mevalonate pathway. We have previously reported that lovastatin, a mem·
`ber of the vastatin family, induces apoptosis in human acute myeloid leukemic
`(AML) cells. We have recently shown that inhibiting protein geranylgeranylation
`downstream of mevalonate is critical for lovastatin-induced apoptosis. In the
`present study we evaluate the potency and mechanism of cerivastatin. a third
`generation member of the vastatin family. Unlike lovastatin, which requires
`activation from pro-drug to active form. cerivastatin is administered in active
`configuration. Our results show that cerivastatin is at least ten fold more potent
`than lovastatin at inducing apoptosis in AML cell-lines as well as in primary AML
`patient samples in vitro. Importantly, like lovastatin, cerivastatin is not cytotoxic
`to normal human bone marrow cells. Moreover, we show that inhibition of
`protein geranylgeranylation is essential for cerivastatin to trigger apoptosis.
`These results strongly suggest that the active form of the vastatin family
`induces apoptosis in AML cells by inhibiting HMG-CoA reductase. In addition,
`the apoptotic activity of the vastatin family occurs through a common mecha(cid:173)
`nism that requires the abrogation of protein geranylgeranylation. Taken to·
`gether, cerivastatin may be the drug of choice for inhibiting HMG-CoA reduc(cid:173)
`tase and triggering tumor cell apoptosis in the clinical management of AML.
`
`410 Blocking protein geranylgeranylation is essential for apoptosis of
`AML cells triggered by inhibitors of HMG-CoA reductase. Zhenlei Xia 1 •
`Melissa M. Tan 1, Jim Dimitroulakos', Mark D. Minden 1·2 , W. Wei-Lynn Wong 1·2 ,
`Linda z. Penn"2 • Department of Cellular and Molecular Biology, the Ontario
`Cancer Institute, University Health Network, Toronto 1 Department of Medical
`Biophysics, University of Toronto, Toronto2•
`We have previously reported that lovastatin, a 3-hydroxy-3-methylglutaryl
`coenzyme A (HMG-CoA) reductase inhibitor, induces apoptosis in human acute
`myeloid leukemia (AML) cells. To understand the critical biochemical mecha(cid:173)
`nism linking inhibition of the mevalonate pathway and apoptosis induced by
`lovastatin in AML cells, add-back experiments were employed in the present
`study. Apoptosis induced by lovastatin was completely prevented by meval·
`onate and geranylgeranyl pyrophosphate (GGPP), while only partially prevented
`by farnesy1 pyrophosphate (FPP). Other products of the mevalonate pathway
`Including cholesterol, squalene, lanosterol, desmosterol, dolichol. dolichol
`phosphate, ubiquinone, and isopentenyladenine had no effect. On the induction
`of apoptosis by lovastatin, our results suggest that the inhibition of geranylgera(cid:173)
`nylation of target proteins is the predominant mechanism of lovastatin-induced
`apoptosis in AML cells. Supporting evidence showed that the geranylgeranyl
`transferase inhibitor (GGTI-298) mimicked the effect of lovastatin, whereas the
`famesyl transferase inhibitor (FTl-277) was much less effective at inducing
`apoptosis in AML cells. In addition, inhibition of Rap1A and Rab5 prenylation
`was observed with lovastatin and GGTl-298 treatment, but not with FTl-277.
`GGPP completely reverted the effect of lovastatin on protein prenylation, while
`FPP had no significant effect on these processes. We conclude that blockage
`of the mevalonate pathway, especially inhibition of protein geranylgeranylation
`is a critical mechanism in lovastatin·induced apoptosis in AML cells.
`
`411 The use of expression profiling to Identify genes regulated by the
`Pl3K inhibitor L Y294002. Robert te Peele, Karine Maillard, Richard Wooster
`and Paul Workman. The Institute of Cancer Research, 15 Cotswold Road,
`Sutton, SM2 5NG, UK.
`The Pl3K pathway plays an important role in regulating cellular proliferation
`and survival, and mutations in components of this pathway have been impli(cid:173)
`cated in cancer. To determine which genes are regulated by the P13K pathway,
`we have used DNA microarrays to establish expression profiles in tumour cells
`treated with the P13K inhibitor L Y294002 versus non-treated colon adenocar(cid:173)
`cinoma cells.
`Our DNA expression arrays are based on cDNAs from the Unigene set of ESTs.
`PCR products from an initial set of 5808 cDNAs were gridded on a single double
`poly-L-Lysine coated glass microsoope slide. Hybridisations were performed using
`Cy3- and CyS-labelled first strand cDNA from treated and untreated cell line
`mRNA. After washing. the slides were scanned using an Axon Genepix 4000
`scanner. Data were analysed with the Genepix software. Ratios from this process
`were normalised and used for interpretation and cluster analysis.
`Concentrations of 30 and 100 "'M of LY294002 were used in time course
`experiments, both concentrations inhibited phosphorylation of the Akt/PKB
`protein. The lower concentration mainly caused cell cycle arrest whereas at the
`higher concentration cell death was induced. Expression changes in genes
`involved in cell cycle regulation and apoptosis were observed. Cluster analysis
`grouped the cells treated with 30 "'M and those treated with 100 "'M on
`separate branches. A subset of differentially expressed genes in the cells
`treated with 30 or 1 00 µ.M of L Y294002 could be identified. One of the genes
`was an apoptotic cysteine protease.
`Expression profiling using DNA microarrays can be used to identify P13K
`inhibitor regulated genes and may help understand signal transduction path·
`ways for proliferation and survival in tumour cells. This work was supported by
`the /CR and the CRC.
`
`412 p53 cooperates in enforcing rapamycin-induced G1 arrest and pro(cid:173)
`tects from apoptosis. S. Huang, LN.Liu, H. Hosoi, M.B.Dilling, T. Shikata, P.J.
`Houghton. Dept. Molecular Pharmacology, St. Jvde Children's Research Hos(cid:173)
`pital, Memphis, TN 38105, U.S.A
`The macrolide antibiotic rapamycin specifically inhibits the Ser!Thr kinase
`activity of mTOR/FRAP, a signaling molecule that links extracellular growth
`factor signals to protein translation. Rapamycin is a potent inhibitor of tumor cell
`growth. and an analog, CCl-779 is currently under Phase I investigation for
`treatment of cancer. In this work, the relationship between G1 checkpoint
`function and rapamycin-induced apoptosis was examined using human rhab·
`domyosarcoma cells, and mouse embryo fibroblasts (MEFs). Serum-starved
`tumor cells expressing mutated p53 alleles became apoptotic when exposed to
`rapamycin, and were protected by expression of a rapamycin resistant mutant
`mTOR. Replication defective adenovirus expressing either p53, or p21"'P'. but
`not control virus. induced Gl accumulation, up-regulation of p21c1P1 , and
`complete protection of cells from raparnycin-induced apoptosis, but not against
`apoptosis induced by cycloheximide. Under serum-free conditions rapamycin
`induced apoptosis of MEFs with p53·1• or p21c•P•-1- genotype, but not wild type
`or p19ARF-t- cells. Cycloheximide induced apoptosis independent of the geno(cid:173)
`type. Ad-p21 and Ad-p53 rescued cells from rapamycin-induced death,
`whereas expression of dominant negative p53 in wild type MEFs restored the
`ability of rapamycin to induce apoptosis. Under serum-containing conditions
`
`rapamycin suppressed levels of cyclin A, only in wild type, and p1gARF·1• MEFs.
`Rapamycin also inhibited DNA replication or S-phase progression significantly
`more in wild type, p19ARF-1-. and p21c•p1-1- MEFs compared to p53-1· cells.
`Suppression of G1 progression by p53 was therefore partly independent of
`p21c•P1
`• However, p21ciP1-mediated G1 block ultimately protected cells from
`apoptosis. The data suggest p53 cooperates in enforcing Gt cell cycle arrest
`leading to a cytostatic response to rapamycin. In contrast, in tumor cells having
`deficient p53 function the response to this agent may be apoptosis. These data
`suggest a mechanistic basis for selective tumor cytotoxicity for rapamycin and
`CCl-779. Supported by USPHS grant CA77776 and by ALSAC.
`
`413 Phase I and pharmacological study of CCI-n9, a cell cycle Inhibi(cid:173)
`tor. M Hidalgo, E Rowinsky, C Erlichman, R Drengler, B Marshall, A Adjei, L
`Hammond, E Galanis, T Edwards, J Burton, J Boni, A Teicher, G Dukart, and J
`Buckner. CTRC, San Antonio, TX; Mayo Clinic, Rochester, MN; Wyeth-Ayerst
`Research, Radnor, PA.
`CCl-779, an ester of rapamycin, binds to FKBP-12 intracellularly, forming a
`complex that inhibits the kinase activity of mammalian target of rapamycin
`(mTOR). This interferes with key signal transduction pathways, including those
`regulated by p70s6 kinase and PHAS-1 protein, resulting in inefficient translation
`of proteins involved in cell cycle progression. CCI· 779 inhibited tumor growth in
`preclinical models and is being developed for treatment of cancer. This study is
`evaluating the safety and pharmacokinetics (PK) of escalating doses of CCl-779
`administered as a 30·minute IV infusion daily x 5 every 2 weeks in patients (pts)
`with solid neoplasms. As of May 2000, 63 patients had been entered. This
`abstract is based on 45 patients in the data base as of April 2000, treated with
`<;loses ranging from 0.75-19.1 mg/m2/d. A total of 214 courses were adminis(cid:173)
`tered. Two episodes of DLT in the first cycle were observed: asymptomatic,
`grade 3 hypocalcemia (2.16 mg/m2/d) and grade 3 elevation in transaminases
`(19.1 mg/m2/d). In addition. grade 3 thrombocytopenia requiring dose reduction
`was observed in 5 pts, including 3 heavily pretreated (HP) pts at 19.1 mg/m2/d.
`Idiosyncratic reactions to the infusion (including flushing, SOB, chest pain) were
`
`Proceedings of the 2000 NCloEORTCoAACR Symposium
`
`West-Ward Exhibit 1082
`Hidalgo NCI 2000 Abs #413
`Page 002
`
`

`

`Clinical Cancer Research o Volume 6 o November 2000 (Supplement)
`
`Poster Session 17
`
`4549s
`
`observed also. Other adverse events, generally mild-moderate in severity, oc(cid:173)
`curred over a broad range of doses. Toxicities include asthenia, cutaneous
`toxicity, mucositis, and hypertriglyceridemia. In 17 pis receiving 0.75 to 3.12
`mg/m2/d, CCI-779 exhibited little accumulation from cycle to cycle, preferenlial
`binding to RBCs. dose-related increase in AUC, and mean to,., of 32.6 h.
`Preliminary evidence of antitumor activity has been· observad, with 1 PR (non(cid:173)
`small cell lung cancer) and minor responses in other tumor types. The safety
`profile and antitumor activity observed to date, associated with plasma con(cid:173)
`centrations at which biological activity was observed in vitro, are encouraging.
`
`414 CCl-779, an ester analogue of rapamycin that interacts with PTEN/
`Pl3 kinase pathways: A phase I study utilizing a weekly intravenous sched(cid:173)
`ule. E. Raymond, J. Alexandre, H. Depenbrock, N. Ady Vago, S. Faivre, A.
`Lahr-Aandak, E. Matennan, J. Boni, S. Abbas, E. Angevin, B. Escudier, J.P.
`Armand. /nstitut Gustave Roussy, Villejuif, France; Onkologische Tagesklinik &
`Wyeth Ayerst Research I Genetics Institute, Munich, Germany.
`Background. CCl-779 inhibits mTOR. thus the phosphorylation of elF4E(cid:173)
`BP1 and p7056 kinases, prevents e1F4E to initiate protein synthesis and the
`phosphorylation of the ribosomal protein S6 required for the translation of
`mRNAs. Patients and Methods. CCl-779 was given as a weekly 30-min
`infusion in patients (pts} with advanced tumors using the modified CAM. Re(cid:173)
`sults. 1 Bpts (MiF: 12/6) received: 7.5 (1 pt). 15 (2pts}, 22.5 (1 pt}, 34 (3pts}, 45
`(4pts), 60 (1pt), 80 (1pt}, 110 (1pt}, 165 (1pt} and 220 mg/m2/week (3~ts}. OLT
`was observed in only 1 pt; MTO has not been reached. No prolonged 1mmuno(cid:173)
`suppression has been induced. Grade (Gr) 1-2 skin toxicity was observed:
`dryness with mild itching (6pts), eczema-like lesions (2pts}, ?~b-acute .u.rticaria
`(2pts}, and aseptic folliculitis (11 pis}. Gr1-2 and Gr-3 m~cos1t1s/stomatit1s were
`observed in 10pts and 1pt. respectively. All pts rece1v1ng ;;,, 8 doses experi(cid:173)
`enced Gr-1 nail changes. Thrombocytopenia was observed in 9pts; 2pts with
`G·3 at 34 and 45 mg/m2/week. Leukopenia was reported in 4pts and anemia in
`7pts. Asymptomatic increases of triglyceride and cholesterol levels were ob(cid:173)
`served in 9pts and 5pts, respectively. A reversible decrease in testosterone
`concentrations with increased levels of LH/FSH were observed in 5/9 men
`receiving :;,4 doses at dosages 2:15mg/m2/week. Pharmacokinetic analysis
`from 12pts (doses: 7.5-60 mg/m2/week) indicates that CCl-779 Cmax increased
`linearly but AUC increased sub-proportionately. Clearance and volume of dis(cid:173)
`tribution at steady state increased with increasing dose. Mean half-life was
`about 20hrs. Of the 16pts evaluable for anti-tumor activity, 3pts had a partial
`response (renal cell carcinoma with lung metastases; neuro-endocrine tumor
`with hepatic metastases and breast cancer with liver, lymph node and peri(cid:173)
`orbital metastases). Conclusion. Current data show that CCl-779 has promis(cid:173)
`ing activity and mild-moderate toxicity over a broad range of doses.
`
`415 Effect of the proteasome inhibitor PS-341 on cell cycle progression
`and bcl-2: A potentially unique mechanism of action. A. Perez-Solert. YH
`L!ngt. B Ngt. J Adams', P Elliott', L Liebest. Kaplan Cancer Center, tNew York
`University School of Medicine, New York, NY, and "Millennium Pharmaceuticals,
`Cambridge, MA.
`PS-341 is a proteasome inhibitor currently in Phase I clinical evaluation. We
`studied the effects of PS-341 on cell cycle progression and related events in
`human NSCLC H460 (p53 wild type} and H358 (p53 null} cells. Exposure to 0.1
`tJ.M PS-341 for 6 h resulted in a marked accumulation of cells at G2/M. This
`blockade was associated with a 6-10 fold time-dependent accumulation of
`cyclins A and Band a 10-fold elevation of cyclins A and B kinase activities as
`assessed by 32 P-,-ATP incorporation into histone-H1. In addition, bcl-2 phos(cid:173)
`phorylation, a marker of mitotic arrest, was detected as early as 3 h after
`exposure to PS341. More importantly, a 25 kDa bcl-2 degradation product was
`detected as early as 12 h after exposure to PS341. This degradation product
`appeared specific for proteasome inhibition since it was observed with the
`proteasome inhibitors MG132 and PSI but not with the chemotherapeutic
`agents paclitaxel. vinblastine, camptothecin, etoposide, and cisplatin, ?r the
`PKC inhibitor staurosporine. In addition, it was not caspase-dependent since 1t
`was observed in the presence of caspase inhibitors and appeared to localize in
`the triton X-100 insoluble cellular fraction. In view of the ability of PS341 to
`induce arrest at G2/M we then studied in vitro cytotoxicity of the combination of
`PS341 and the antitubulin agent docetaxel against H460 and H358 cells. Cells
`were treated concomitantly with PS341 (0.1 tJ.M or 0.5tJ.M) and docetaxel (0.1 to
`4tJ.M) for 48 h. An additive cytotoxic effect was observed with the combination
`0.5tJ.M PS341 and 0.5 and 1 tJ.M docetaxel. In conclusion, our results indicate
`that PS341 induces unique changes in bcl-2 that appear to be specific for
`proteasome inhibition. The functional consequences of these bcl-2 changes
`and their potential relationship with the demonstrated ability of this agent to
`retain its cytotoxicity against bc!-2 transfected cells is being investigated.
`
`416 A phase I pharmacodynamic study of the proteasome inhibitor
`PS-341. J.P. Thomas. A. Adjei, C. Ehrlichman, P. Geiger. A. Haas, R. Arzooma(cid:173)
`nian. D. Alberti, R. Mamocha. K. Binger. J. Volkman, C. Feierabend, K. Tutsch,
`J. Adams, P. Eliot and G. Wilding. University of Wisconsin Comprehensive
`Cancer Center, Madison, WI, Mayo Clinic, Rochester, MN and Millenium Phar(cid:173)
`maceuticals, Cambridge, MA.
`The ubiquitin-proteasome pathway is the principal enzymatic degradation path(cid:173)
`way for most intracellular proteins incli.;ding those involved in cell cycle regulatio~.
`apoptosis and angiogenesis. PS-341 is a dipeptide boronic acid compound that JS
`a potent inhibitor of the 20S/26S proteasome. Proteasome inhibition affects such
`
`diverse metabolic processes including stabilization of cell cycle regulatory proteins
`and inhibition of NF-KB activation. PS·341 has broad activity including MOR and
`Bcl-2 overcxpresslng cancer cell lines. In vivo PS-341 inhibits the growth of a
`number of tumors including the HT-29, NCl-H23 and PC-3 models. Toxicity was
`seen in preclinical models when the proteasome was inhibited by greater than
`60%. We are conducting a phase I trial of PS-341 in patients with advanced
`refractory cancers. PS-341 is administered intravenously twice weekly for 4 weeks
`followed by a two week break. Dose levels of 0.5, 0.9, 1.25 and 1.50 mg/m2 have
`been explored. A total of 9 patients have been treated at the UW. Toxicities seen
`have included rash, fatigue and thrombocytopenia. A MTO has not yet been
`reached. Proteasome inhibition by PS-341 has been monitored by measuring 20$
`proteasome activity in whole blood samples using a ftourogenic peptide substrate.
`20S proteasome inhibition in this study measured 1 hour after PS-341 administra(cid:173)
`tion correlates highly with PS-341 dose. We are achieving levels of proteasome
`inhibition (> 60%) associated with anti-tumor activity in the preclinical models. We
`have also examined patient peripheral blood mononuclear cells to determine
`whether levels of proteasome inhibition achieved in this study may be associated
`with accumulation of ubiquitinated proteins. Cell lysates were analyzed by Western
`blot for ubiquitin protein conjugates. Up to a 3 fold increase in ubiquitinated
`proteins were seen in some patients, peaking at 5 hours after PS-341 admini(cid:173)
`stration.
`
`417 Proteasome inhibition by PS-341: A phase I study. A Hamilton'. JP
`Eder, A Pavlick 1
`, JW Clark", A Chachoua 1
`, DP Ryan3, K Farrell', H Wasserstrom 1,
`L Liebes 1
`• J Wright'. P Elliott5. J Adams5 and F Muggia'. 1 NYU Sch. of Med .. 2Dana
`Farber Can. Inst., 3Mass. General Hosp., 4CTEP NCl,5Millennium Pharm.
`The proteasome is a multimeric protease complex that regulates cellular
`proteins by degrading ubiquinated proteins. Proteasome inhibition results in
`increased levels of a variety of key cellular proteins that may contribute to
`anti-tumor activity; IKB inhibits nuclear factor KB (NF-KB) mediated transcrip·
`tion, p53 inhibits apoptosis, and p21 inhibits cyclin-dependent kinase (CDK)
`activity. PS-341 is a dipeptide boronic acid derivative that inhibits the protea(cid:173)
`some by stabilization of its active site. Animal models predicted dose limiting
`gastrointestinal toxicity at 2:60% proteasome inhibition (Pl). PS-341 was ad(cid:173)
`ministered as an IV bolus on 01 &4 of a 2-week cycle. Five dose levels have
`, 0.6mg/m2 • 1mg/m2
`been studied to date: 0.25mg/m2
`, 1.2mg/m2 and 1.45mg/
`m2 • 19 pts have been treated: 11M /BF. Age: median 57, range 25-76. Primary
`tumors: colorectal (3), renal (3), NSCLC (3), melanoma (2), ST sarcoma (2),
`osteosarcoma (1), lymphoma (1), prostate (1), endometrial (1), esophagus (1),
`hepatoma (1). Prior therapies: chemotherapy (17), radiotherapy (13). To~icities
`have been mild and non-specific. 1/6 pts treated at 1.2mg/m2 expe11enced
`self-limiting G3 diarrhea. No objective respenses have been documented. One
`pt with melanoma treated at 1 mg/m2 maintained a PR in lung with SO in skin for
`6 months. Pl was measured at 1, 4 and 24hrs after dosing. At all dose levels,
`peak Pl was seen at 1 hr, and recovery to approximately 50% of peak Pl was
`seen at 24h. Peak mean Pl were 21 %, 54%, 46% and 59% at dose levels 1, 2,
`3 and 4 respectively. Tumor Pl at 24h in one pt was 67% and averaged 54% in
`2 biopsies at 2-3 h in another pt. Accrual is ongoing at 1.9mg/m2 , and phase 11
`studies are planned. Supported by U01 CA76642, M01 RR00096 and the Lynne
`Cohen Foundation (NY), and U01 62490 (Boston}.
`
`418 Pharmacodynamlc evaluation of the protein kinase C (PKC) Inhib(cid:173)
`itor CGP41251 (PKC412) in patients with metastatic melanoma. M. Mill(cid:173)
`ward'. C. House'. L Webster'. B. Linahan'. I. Olver, G. Toner'. J. Zalcberg'.
`o. Bowtell'. 'Peter MacCal/um Cancer Institute, Melbourne, 2Royal Adelaide
`Hospital, Australia.
`PKC412 selectively inhibits PKC (IC50 <1 µ.M) and has preclinical activity as a
`cytostatic and modulator of MOR. The recommended Phase II dose is 75mg tds
`which produces potentially active trough plasma levels (1 Oµ.molll), and suppresses
`cytokine release and lymphocyte ERK2 levels (Thavasu 1999). Patients (pts) with
`measurable metastatic melanoma and 2 2 superficial lesions received 75mg tds;
`tumor biopsies and plasma were collected prior to and after 28 days treatment.
`lntra-tumoral total PKC activity was measured in cytosotic and particulate fractions
`using protamine sulphate as the substrate. Initial experiments showed addition of
`10tJ.M PKC412 to melanoma biopsies inhibited phosphorylation. Ability of plasma
`to modulate ex vivo intracellular daunorubicin accumulation in MOR cells was
`measured with activity of 20µ.g/ml vaJspodar (PSC833} defining 100% reversal.
`Compared to the pretreatment biopsy, cytosoiic PKC activity was reduced by 7%
`to 91 % in 7i9 pts. Particulate PKC activity was reduced by 11 % to 79% 1n 4/9 pts.
`Only 1 pt had >50% inhibition in both fractions. Tumor PKG iS?fonn profile .in 1 pt
`resistant to PKC412 (unchanged cytosolic activity and >200% increase ~~ulate
`activity) showed an abundance of PKC~. an isoform refractory to 1nh1bit1on by
`PKC412 (IC50 >1000tJ.M}. Addition of 20µ.g/ml PKC412 to pretreatment plasma
`produced 14%-64% (mean 40%) reversal of MOR. Plasma taken following 28
`days PKC412 showed <10% reversal in 818 patients. All patients had .P"?9ressive
`disease. This Phase llA trial did not demonstrate consistent target inh1b1tton or
`phannacodynamic efficacy of PKC412 in melanoma patients.
`
`Proceedings of the 2000 NC!oEORTCoAACR Symposium
`
`West-Ward Exhibit 1082
`Hidalgo NCI 2000 Abs #413
`Page 003
`
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