1
117 31 118 IIIGII-TQ- OFARA4IAND-I @GrosAcrIvE-~~~mwrInBIIslsr~ TOTOPOIITARGETRK ALKYLATINGA~. P.B Jensen, B. Helm, M. Smensm, M. Sebesied, H.H. HEmsen Dept of Oncology, Rigshospitalet and Dept of Pathology, Sundby Hospital,DK-2100 Copenhagen,Denmark. By use of a clonogenic assay we have previously hem able to demonstrate the inteneIatimship between different anticancer agents. 19 anticancer agents were compared in a panel of six “wild type” and three. VP-16 resistant SCLC cell lines and we found that the differential cytotmicity patterns can 1) give information about drug mechanism of action, 2) enable the selez~icm and combiia-tion of non-cross-resistant drugs, and 3) show where new drugs “fit in” amongestablished agents (Br J Cancer 67: 311-320, 1993). In addition to the three diffemd VP-16 (multidrugresistant (MDR)) phenotypes; H69/DAU, NYHNM, and H69NP. we have now included four new drug resistantcell lines in the panel; hvo resistant to alkylatiig agents H69lBCNU and NYHICIS, and two resistant to the tqm I agents camptothecin and tqmthecao, NYH/CAM and NYHITGPG. The patternswere compared by correlationanalysis. This showed high correlation coefficients (CC) among drug analogues (e.g. VP-16NM-26 0.94, vincristine/ vindwine 0.84). and beWe5n drugs with similar m&mism of action (e.g. BCNUICisplatin0.76, VP-16/DoxombicinO.94), whereas different drug classes demonstrated low or even negative CC (e.g. CispIatinNP-16 -0.43). Ammg 23 drugs tested, gem&&in und mpotbecan showed promising patterns 1s candidate8 for combination with alkylatingagents and top0 II agents. Correlation Coefficients VP-16 Gmptotbecin Tqmtbecm Am-C Gemitabin Cisplatin -0.43 0.45 0.53 0.05 0.23 BCNU -0.39 0.07 0.18 -0.10 -0.05 VP-16 * -0.16 -0.09 -0.08 -0.22 Doxombicin 0.94 -0.12 -0.05 -0.09 0.11 Camptothecin and its malogue topothecan lack cross-resistanceto BCNU and topo II targeting agents; Am-C and its analogue gemcitabin lack cross resistance both to cisplatinand BCNU and to top0 II targeting agents. 119 EXPRESStON OF P53 AND MM1 GENES H LUNG CARCINOMA : CGRRELAllON TO CLfNfCAL CHEYlMIEStSTANCE M. Pecc’h, E. Brambilla, D. Mom, S. Gazzeri, C. Brambilla. AirwaysCancer Research Group, Hopitaf A. Michalbn 33043 GRENOBLE Csdex 9 FRANCE Since Wild type P53 proteinplays an imporiant role in Gl armst or apoptosis of celfs aa@ing DNA tesbns after chemo or radiotherapy. loss of P53 wfkltype functiins may lead b chemoresistanrx. In an other hand, MDRl (pGP170) expmssbn is tfmught b be responsble br chemoresistance. Furthsnnorewild type p53 was shownIO repress MDRI promoter while mutant P53 enhances MDRI actMty. P53 mutatbn is one of the mostfrequent mutation inkrngcancar.The~study~done~iinvestigatethelinkofP53 mutation and MDRI expressbn in smatlcefl (SCLC) and non smallcell hmg carcinoma (NSCLC) and b examtne their ntlatiin withclinical chemoresistance. lncasesof~cMloercorrrprishp39aquamarsceHuvdnrma(SCC),25 adenocarcinomas (ADC), 3 lalpe cell carcinomas(LCC), 10 carcinoi and 35 Small ceil lungcaminonla (SCLC). 7 well differentiated NE carcinoma (WDNEC), 3 NE non SCLC, were studied.PS3 analy& was perloormed by immun0hfsbchembtry (IHC) ustng6 antibodies (pAb 421, pAb 1901, pAb 240, pAb 122. pDo7 and polycbnal CMl) a&wing recognition of mutantP53 protein abnorrnaliy acoumutated in cell nucleus. In add&n, Northernblot analysts allowedthe recogniibn of cases with a defect of P53 transcription (P53 mRNA) revealing“bss of function” mutatbn with “nufl protein” phenotype.MDRI expressbn was performedby IHC usingJSBI antiiy. P53 mutant bnrnunophenotyps was found b 52/l 22 cases (43 %) and loss of mRNA in 25/122 cases (20 %), MDRI (PgP 170) expression was found in 601122 cases (19/39 SCC, 17l25 ADC, 2i3 LCC, 8ilO carcinoids, 15/45 NE tumors). No wrrelatbn was found between P53 rmtatbn and MDRI overexpressbn in the non NE carcinomawhereas P53 mutation was correlated with MDRl expression in SCLC and other malignant NE tumors(p = 0,02). Double staining of JSBI and CM1 could not confirm the linkof P53 mutation and MDRI expressionat indiikbal cell level. There was no correlatbn between MDA1 expressionand response to chemotherapy using (JSBI hl &&em&y) in SCLC patients,hut the frequency of complete resqonw was sIgnHbanNy higherIn MDRI negativecases (p = 0,02). However the chemoresistance was s@niflcantty correlatedwith P53 mutantphenc+pe in SCLC (p - 0,015). We conclude that P53 mutation is a best pfsdMor of dfnical chsmomsiitan@ in SCLC than MDRI expression. AcknowMgements lo : INSFRM AssociatiOn ESPOIR. RESISTANCE TO THE TGPGISGMEZASE I ACTIVE DRUG TGPGTECAN IN SMALL CELL LUNG CANCER CELL LINES. M.S@rensen, M.Sehested, P.B.Jensen.Dept of Patho- logy,Sundby Hospital,DK-2300 Copenhagen S,Denmark The topoisomerase (topo) I active drug topo- tecan (TPT), a derivative of camptothecin (CPT), is in phase II clinical trials. We have investi- gated TPT resistance in two human small cell lung cancer cell (SCLC) lines. Human SCLC GC-NYH cells were selected in the presence of TPT. GC-NYH/TPT cells were 10 fold resistant to TPT as well as to CPT assessed by clonogenic assay. GC-NYH/TPT cells expressed no P-glycoprotein (Pgp) and accumulation of 14C-TPT and 3H-daunorubicin (DNR) was equal. Immunoreactive topo I level was de- creased by 40%. DNA single strand breaks (SSBs) measured by alkaline elution were reduced by the same degree as was topo I activity measured by relaxation of supercoiled DNA. CPT inhibition of top0 I activity was equal. In contrast, VP-16 induced DNA SSBs were increased and immuno- reactive topo II was.do*Lid in OC-NYH/TPT cells. Human SCLC Ii69lDAU cells selected in the presence of DNR with a high content of Pgp showed cross-resistance to TPT, but not towards CPT. Accumulation of 3H-DNR is markedly reduced in H69/DAU cells, while no major difference is seen in 3H-CPT and 14C-TPT accumulation. TPT inhibi- tion of asidopine photoaffinity labeling of Pgp was equal to that of CPT. Resistance to TPT in SCLC cells selected by TPT is associated with down regulation of topo I levels. Pgp positive SCLC cells show cross- resistance to TPT but TPT accumulation is not reduced. 120 PHASE I STUDY OF DIPYDAMOLR AS AN EFFLUX BLOCKER OF DOXORUBICIN IN LUNG AND OTIlER CANCERS. K.S. Sridhar, A. Krishan, T.S.A. Samy A. Ucar, H. Chatnor.Uniwsity of Miami School of Mediclw, Sylvester canpehensive Cmw Cmter, Mimi, FL. Doxombicln @OX) &flux cm be bknked by 10 t@ml dip- (DPL) & a. ‘II& cliical study was designed to test the hypomesis tba sustainedplasma DPL levels adqwte to block DGX &lux can be whieved in IIberapy consisted of amcUrrent IV infusionof aomg/mhn DGX and escalaing doses (10.5, 13.5, 17.5, 23.1, 30, 39, 8 50 mg/lrg) of DPL along titb 6L normal saline oyer 72b in 26 pts. wlrb &actay cmxr. Toxicltles of DOX wae as expected and lndqmdmt of DPL dose. Media Nadirs were WBC 3,800, AGC 2,1&1,Hb 10.7 and plt 242,000 and no cl&al cadiomxiclty. Toxicities of DPL (headache, mwaandvomit@)wuedwrelated AIlpatlentsmated,39mg/kgDPL needed mdge8ics (aa-& codeine, + kelomlac) and antiemetics (deurmethapone. ondammm. +loriaepam, +dqmickd). DPL ccmc(L l&q/ml) was acldlved in a majorityof pm mated? DPL ~17.5 n&g. CR (111) ommed in large cell lymphma S/I’ MACOP-B, PmhlACFQtaBOM, RT and stem cell rescue pm ICE ~y.PRoawedinnoa-smallffilllungrancerinapt S/pmi~+velban;RT,platinol+Fu,DoX+~;andinenotberp( S&'platiol+ %Fu+VP16andRT. PRwuenoledinl~melanomaand l/l @striccancer. DOXbloclringDmlevelsoftheDPL~besustainedandactivity ofDOX+DPLneedtobecmd~ed.

Phase I study of dipydamole as an efflux blocker of doxorubicin in lung and other cancers

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Page 1: Phase I study of dipydamole as an efflux blocker of doxorubicin in lung and other cancers

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IIIGII-TQ- OFARA4IAND-I @GrosAcrIvE-~~~mwrInBIIslsr~ TOTOPOIITARGETRK ALKYLATINGA~. P.B Jensen, B. Helm, M. Smensm, M. Sebesied, H.H. HEmsen Dept of Oncology, Rigshospitalet and Dept of Pathology, Sundby Hospital, DK-2100 Copenhagen, Denmark.

By use of a clonogenic assay we have previously hem able to demonstrate the inteneIatimship between different anticancer agents. 19 anticancer agents were compared in a panel of six “wild type” and three. VP-16 resistant SCLC cell lines and we found that the differential cytotmicity patterns can 1) give information about drug mechanism of action, 2) enable the selez~icm and combiia-tion of non-cross-resistant drugs, and 3) show where new drugs “fit in” among established agents (Br J Cancer 67: 311-320, 1993). In addition to the three diffemd VP-16 (multidrug resistant (MDR)) phenotypes; H69/DAU, NYHNM, and H69NP. we have now included four new drug resistant cell lines in the panel; hvo resistant to alkylatiig agents H69lBCNU and NYHICIS, and two resistant to the tqm I agents camptothecin and tqmthecao, NYH/CAM and NYHITGPG.

The patterns were compared by correlation analysis. This showed high correlation coefficients (CC) among drug analogues (e.g. VP-16NM-26 0.94, vincristine/ vindwine 0.84). and beWe5n drugs with similar m&mism of action (e.g. BCNUICisplatin0.76, VP-16/DoxombicinO.94), whereas different drug classes demonstrated low or even negative CC (e.g. CispIatinNP-16 -0.43). Ammg 23 drugs tested, gem&&in und mpotbecan showed promising patterns 1s candidate8 for combination with alkylating agents and top0 II agents.

Correlation Coefficients VP-16 Gmptotbecin Tqmtbecm Am-C Gemitabin

Cisplatin -0.43 0.45 0.53 0.05 0.23 BCNU -0.39 0.07 0.18 -0.10 -0.05 VP-16 * -0.16 -0.09 -0.08 -0.22 Doxombicin 0.94 -0.12 -0.05 -0.09 0.11 Camptothecin and its malogue topothecan lack cross-resistance to BCNU and topo II targeting agents; Am-C and its analogue gemcitabin lack cross resistance both to cisplatin and BCNU and to top0 II targeting agents.

119

EXPRESStON OF P53 AND MM1 GENES H LUNG CARCINOMA : CGRRELAllON TO CLfNfCAL CHEYlMIEStSTANCE M. Pecc’h, E. Brambilla, D. Mom, S. Gazzeri, C. Brambilla. Airways Cancer Research Group, Hopitaf A. Michalbn 33043 GRENOBLE Csdex 9 FRANCE

Since Wild type P53 protein plays an imporiant role in Gl armst or apoptosis of celfs aa@ing DNA tesbns after chemo or radiotherapy. loss of P53 wfkl type functiins may lead b chemoresistanrx. In an other hand, MDRl (pGP170) expmssbn is tfmught b be responsble br chemoresistance. Furthsnnore wild type p53 was shown IO repress MDRI promoter while mutant P53 enhances MDRI actMty. P53 mutatbn is one of the most frequent mutation inkrngcancar.The~study~done~iinvestigatethelinkofP53 mutation and MDRI expressbn in smatl cefl (SCLC) and non small cell hmg carcinoma (NSCLC) and b examtne their ntlatiin with clinical chemoresistance. lncasesof~cMloercorrrprishp39aquamarsceHuvdnrma(SCC),25 adenocarcinomas (ADC), 3 lalpe cell carcinomas (LCC), 10 carcinoi and 35 Small ceil lung caminonla (SCLC). 7 well differentiated NE carcinoma (WDNEC), 3 NE non SCLC, were studied. PS3 analy& was perloormed by immun0hfsbchembtry (IHC) ustng 6 antibodies (pAb 421, pAb 1901, pAb 240, pAb 122. pDo7 and polycbnal CMl) a&wing recognition of mutant P53 protein abnorrnaliy acoumutated in cell nucleus. In add&n, Northern blot analysts allowed the recogniibn of cases with a defect of P53 transcription (P53 mRNA) revealing “bss of function” mutatbn with “nufl protein” phenotype. MDRI expressbn was performed by IHC using JSBI antiiy. P53 mutant bnrnunophenotyps was found b 52/l 22 cases (43 %) and loss of mRNA in 25/122 cases (20 %), MDRI (PgP 170) expression was found in 601122 cases (19/39 SCC, 17l25 ADC, 2i3 LCC, 8ilO carcinoids, 15/45 NE tumors). No wrrelatbn was found between P53 rmtatbn and MDRI overexpressbn in the non NE carcinoma whereas P53 mutation was correlated with MDRl expression in SCLC and other malignant NE tumors (p = 0,02). Double staining of JSBI and CM1 could not confirm the link of P53 mutation and MDRI expression at indiikbal cell level. There was no correlatbn between MDA1 expression and response to chemotherapy using (JSBI hl &&em&y) in SCLC patients, hut the frequency of complete resqonw was sIgnHbanNy higher In MDRI negative cases (p = 0,02). However the chemoresistance was s@niflcantty correlated with P53 mutant phenc+pe in SCLC (p - 0,015). We conclude that P53 mutation is a best pfsdMor of dfnical chsmomsiitan@ in SCLC than MDRI expression. AcknowMgements lo : INSFRM AssociatiOn ESPOIR.

RESISTANCE TO THE TGPGISGMEZASE I ACTIVE DRUG TGPGTECAN IN SMALL CELL LUNG CANCER CELL LINES. M.S@rensen, M.Sehested, P.B.Jensen.Dept of Patho- logy,Sundby Hospital,DK-2300 Copenhagen S,Denmark

The topoisomerase (topo) I active drug topo- tecan (TPT), a derivative of camptothecin (CPT), is in phase II clinical trials. We have investi- gated TPT resistance in two human small cell lung cancer cell (SCLC) lines. Human SCLC GC-NYH cells were selected in the presence of TPT. GC-NYH/TPT cells were 10 fold resistant to TPT as well as to CPT assessed by clonogenic assay. GC-NYH/TPT cells expressed no P-glycoprotein (Pgp) and accumulation of 14C-TPT and 3H-daunorubicin (DNR) was equal. Immunoreactive topo I level was de- creased by 40%. DNA single strand breaks (SSBs) measured by alkaline elution were reduced by the same degree as was topo I activity measured by relaxation of supercoiled DNA. CPT inhibition of top0 I activity was equal. In contrast, VP-16 induced DNA SSBs were increased and immuno- reactive topo II was.do*Lid in OC-NYH/TPT cells.

Human SCLC Ii69lDAU cells selected in the presence of DNR with a high content of Pgp showed cross-resistance to TPT, but not towards CPT. Accumulation of 3H-DNR is markedly reduced in H69/DAU cells, while no major difference is seen in 3H-CPT and 14C-TPT accumulation. TPT inhibi- tion of asidopine photoaffinity labeling of Pgp was equal to that of CPT.

Resistance to TPT in SCLC cells selected by TPT is associated with down regulation of topo I levels. Pgp positive SCLC cells show cross- resistance to TPT but TPT accumulation is not reduced.

120

PHASE I STUDY OF DIPYDAMOLR AS AN EFFLUX BLOCKER OF DOXORUBICIN IN LUNG AND OTIlER CANCERS. K.S. Sridhar, A. Krishan, T.S.A. Samy A. Ucar, H. Chatnor. Uniwsity of Miami School of Mediclw, Sylvester canpehensive Cmw Cmter, Mimi, FL.

Doxombicln @OX) &flux cm be bknked by 10 t@ml dip- (DPL) & a. ‘II& cliical study was designed to test the hypomesis tba sustained plasma DPL levels adqwte to block DGX &lux can be whieved in IIberapy consisted of amcUrrent IV infusion of aomg/mhn DGX and escalaing doses (10.5, 13.5, 17.5, 23.1, 30, 39, 8 50 mg/lrg) of DPL along titb 6L normal saline oyer 72b in 26 pts. wlrb &actay cmxr. Toxicltles of DOX wae as expected and lndqmdmt of DPL dose. Media Nadirs were WBC 3,800, AGC 2,1&1, Hb 10.7 and plt 242,000 and no cl&al cadiomxiclty. Toxicities of DPL (headache, mwaandvomit@)wuedwrelated AIlpatlentsmated,39mg/kgDPL needed mdge8ics (aa-& codeine, + kelomlac) and antiemetics (deurmethapone. ondammm. +loriaepam, +dqmickd). DPL ccmc (L l&q/ml) was acldlved in a majority of pm mated? DPL ~17.5 n&g. CR (111) ommed in large cell lymphma S/I’ MACOP-B, PmhlACFQtaBOM, RT and stem cell rescue pm ICE ~y.PRoawedinnoa-smallffilllungrancerinapt S/pmi~+velban;RT,platinol+Fu,DoX+~;andinenotberp( S&'platiol+ %Fu+VP16andRT. PRwuenoledinl~melanomaand l/l @striccancer. DOXbloclringDmlevelsoftheDPL~besustainedandactivity ofDOX+DPLneedtobecmd~ed.