Search

The bromodomain (BRD) and extraterminal (BET) proteins including BRD2, BRD3 and BRD4 have been identified as key targets for leukemia maintenance. A novel oral inhibitor of BRD2/3/4, the thienotriazolodiazepine compound OTX015, suitable for human use, is available. Here we report its biological effects in AML and ALL cell lines and leukemic samples. Exposure to OTX015 lead to cell growth inhibition, cell cycle arrest and apoptosis at submicromolar concentrations in acute leukemia cell lines and patient-derived leukemic cells, as described with the canonical JQ1 BET inhibitor. Treatment with JQ1 and OTX15 induces similar gene expression profiles in sensitive cell lines, including a c-MYC decrease and an HEXIM1 increase. OTX015 exposure also induced a strong decrease of BRD2, BRD4 and c-MYC and increase of HEXIM1 proteins, while BRD3 expression was unchanged. c-MYC, BRD2, BRD3, BRD4 and HEXIM1 mRNA levels did not correlate however with viability following exposure to OTX015. Sequential combinations of OTX015 with other epigenetic modifying drugs, panobinostat and azacitidine have a synergic effect on growth of the KASUMI cell line. Our results indicate that OTX015 and JQ1 have similar biological effects in leukemic cells, supporting OTX015 evaluation in a Phase Ib trial in relapsed/refractory leukemia patients.

Our purpose was to determine, in patients with metastatic colorectal carcinoma treated with irinotecan single-agent after 5-FU failure, the most significant predictive parameters for tumour response, progression-free survival and toxicity. Between October 1992 and April 1995, 455 patients with 5-FU resistant metastatic colorectal carcinoma entered four consecutive phase II trials. The first two studies assessed tumour response, the other two were randomized studies which assessed the efficacy of racecadotril to prevent irinotecan-induced diarrhoea. Due to homogeneous main eligibility criterias, data from those studies could be pooled for statistical analysis. Potential clinical and biological predictive factors (PF) for toxicity, tumour growth control, e.g. response or stabilization and progression-free survival (PFS), were studied in multivariate analysis. 363 patients were evaluable for response, 432 were evaluable for PFS, 368 for neutropenia and 416 for delayed diarrhoea, respectively. Normal baseline haemoglobin level (Hb), time since diagnosis of colorectal carcinoma, grade 3 or 4 neutropenia or diarrhoea at first cycle and a low number of organs involved were the most PF for tumour growth control (P< 0.05). Significant prognostic variables for PFS were WHO Performance Status, liver and lymph-node involvement, time since diagnosis, age and CEA value (P≤ 0.02). Six groups of patients based on the number of unfavourable prognostic factors are presented. Baseline bilirubin, haemoglobin level, number of organs involved and time from diagnosis were PF for neutropenia; PS, serum creatinine, leukocyte count, time from 5-FU progression and prior abdominopelvic irradiation were PF for delayed diarrhoea (P≤ 0.05). These PF should help clinicians to anticipate for a given patient the probability to observe a response/stabilization or a toxicity. These results should also be prospectively confirmed in ongoing or future trials using irinotecan, both as a single agent and in combination with other drugs. © 2000 Cancer Research Campaign

Summary Background In phase II studies, irinotecan is active in metastatic colorectal cancer, but the overall benefit has not been assessed in a randomised clinical trial. Methods Patients with proven metastatic colorectal cancer, which had progressed within 6 months of treatment with fluorouracil, were randomly assigned either 300–350 mg/m 2 irinotecan every 3 weeks with supportive care or supportive care alone, in a 2:1 ratio. Findings 189 patients were allocated irinotecan and supportive care and 90 supportive care alone. The mean age of the participants was 58·8 years; 181 (65%) were men and 98 (35%) were women. WHO performance status was 0 in 79 (42%) patients, 1 in 77 (41%) patients, and 2 in 32 (17%) patients. Tumour-related symptoms were present in 134 (71%) patients and weight loss of more than 5% was seen in 15 (8%) patients. With a median follow-up of 13 months, the overall survival was significantly better in the irinotecan group (p=0·0001), with 36·2% 1-year survival in the irinotecan group versus 13·8% in the supportive-care group. The survival benefit, adjusted for prognostic factors in a multivariate analysis, remained significant (p=0·001). Survival without performance-status deterioration (p=0·0001), without weight loss of more than 5% (p=0·018), and pain-free survival (p=0·003) were significantly better in the patients given irinotecan. In a quality-of-life analysis, all significant differences, except on diarrhoea score, were in favour of the irinotecan group. Interpretation Our study shows that despite the sideeffects of treatment, patients who have metastatic colorectal cancer, and for whom fluorouracil has failed, have a longer survival, fewer tumour-related symptoms, and a better quality of life when treated with irinotecan than with supportive care alone.

PURPOSE Irinotecan (CPT-11), a camptothecin derivative, has shown efficacy against colorectal cancer. Delayed-onset diarrhea is its main limiting toxicity. The aim of this study was to determine the pathophysiology of CPT-11-induced delayed-onset diarrhea and assess the efficacy of combined antidiarrheal medication in a phase II, prospective, successive-cohorts, open study. PATIENTS AND METHODS Twenty-eight patients with advanced colorectal cancer refractory to fluorouracil (5-FU) therapy received CPT-11 350 mg/m2 every 3 weeks. The first cohort of 14 consecutive patients explored for the mechanism of diarrhea received acetorphan (a new enkephalinase inhibitor) 100 mg three times daily; the second 14-patient cohort received, in addition to acetorphan, loperamide 4 mg three times daily. Before treatment, and if late diarrhea occurred, patients underwent colon mucosal biopsies for CPT-11 and topoisomerase I levels; intestinal transit time; fecalogram; fat and protein excretion; alpha1-antitrypsin clearance; D-xylose test; blood levels for vasoactive intestinal polypeptide, glucagon, gastrin, somatostatin, prostaglandin E2, and carboxylesterase; CPT-11/SN-38 and SN-38 glucuronide pharmacokinetics; and stool cultures. RESULTS Delayed-onset diarrhea occurred during the first three treatment cycles in 23 patients (82%). Electrolyte fecal measurements showed a negative or small osmotic gap in nine of nine patients and an increased alpha1-antitrypsin clearance in six of six patients. There were no modifications in stool cultures or hormonal dysfunction. Four of 11 patients (36%) with delayed-onset diarrhea in the first cohort responded to acetorphan, whereas nine of 10 patients (90%) responded to the combination of acetorphan and loperamide (P < .02). CONCLUSION CPT-11-induced delayed-onset diarrhea is caused by a secretory mechanism with an exudative component. Early combined treatment with loperamide and acetorphan seems effective in controlling the diarrheal episodes.

PURPOSE A phase I study was undertaken to determine the maximum-tolerated dose (MTD), principal toxicities, and pharmacokinetics of the novel topoisomerase I inhibitor irinotecan (CPT-11). PATIENTS AND METHODS Sixty-four patients meeting standard phase I eligibility criteria were included (24 women, 40 men; median age, 51 years; primary sites: colon, head and neck, lung, pleura; 60 of 64 had been previously treated). Pharmacokinetics was determined by high-performance liquid chromatography (HPLC). RESULTS One hundred ninety CPT-11 courses were administered as a 30-minute intravenous (IV) infusion every 3 weeks (100 to 750 mg/m2). Grade 3 to 4 nonhematologic toxicities included diarrhea (16%; three hospitalizations), nausea and vomiting (9%), asthenia (14%), alopecia (53%), elevation of hepatic transaminases (8%), and one case of skin toxicity. An acute cholinergic syndrome was observed during CPT-11 administration. Diarrhea appeared dose-limiting at 350 mg/m2, but this was circumvented by using a high-dose loperamide protocol that allowed dose escalation. Dose-dependent, reversible, noncumulative granulocytopenia was the dose-limiting toxicity (nadir, days 6 to 9; median recovery time, 5 days). Grade 3 to 4 anemia was observed in 9% of patients. One patient died during the study, 8 days after CPT-11 treatment. Two complete responses (cervix, 450 mg/m2; head and neck, 750 mg/m2) and six partial responses in fluorouracil (5-FU)-refractory colon cancer were observed (260 to 600 mg/m2). Pharmacokinetics of CPT-11 and active metabolite SN-38 were performed in 60 patients (94 courses). CPT-11 plasma disposition was bi- or triphasic, with a mean terminal half-life of 14.2 +/- 0.9 hours (mean +/- SEM). The mean volume of distribution (Vdss) was 157 +/- 8 L/m2, and total-body clearance was 15 +/- 1 L/m2/h. The CPT-11 area under the plasma concentration versus time curves (AUC) and SN-38 AUC increased linearly with dose. SN-38 plasma decay had an apparent half-life of 13.8 +/- 1.4 hours. Both CPT-11 and SN-38 AUCs correlated with nadir leukopenia and granulocytopenia, with grade 2 diarrhea, and with nausea and vomiting. CONCLUSION The MTD of CPT-11 administered as a 30-minute IV infusion every 3 weeks is 600 mg/m2, with granulocytopenia being dose-limiting. At 350 mg/m2, diarrhea appeared dose-limiting, but high-dose loperamide reduced this toxicity and allowed dose escalation. For safety reasons, the recommended dose is presently 350 mg/m2 every 3 weeks; more experience must be gained to establish the feasibility of a higher dose in large multicentric phase II studies. However, when careful monitoring of gastrointestinal toxicities is possible, a higher dose of 500 mg/m2 could be recommended in good-risk patients. The activity of this agent in 5-FU-refractory colorectal carcinoma makes it unique and mandates expedited phase II testing.

Intra-arterial hepatic (i.a.h.) administration of the doxorubicin analogue pirarubicin was evaluated in a phase I trial, based on preclinical studies that showed an advantage of pirarubicin over doxorubicin after locoregional hepatic administration. Pirarubicin was given to 9 patients with metastatic liver disease with intrapatient dose escalation. Of the 58 cycles evaluable for tolerance, no hepatobiliary or vascular toxicity was observed. The doselimiting toxicity was granulocytopenia: the maximum administered doses ranged from 50 to 120 mg/m 2 , suggesting variable rates of pirarubicin hepatic extraction between patients. Pharmacokinetic data obtained in 7 patients, in which a direct comparison of intravenous (i.v.) and i.a.h. administration was possible, indicated a median i.v./i.a.h. ratio of 7.4 for the maximal plasma concentration, and a median ratio of 4 for the area under the plasma concentrations versus time curves, suggesting a high pirarubicin hepatic extraction. An unexpectedly high response rate was observed: two complete (colorectal carcinoma) and two partial responses. These data demonstrate that i.a.h. pirarubicin not only produced high locoregional concentrations and reduced systemic exposure, but can also achieve responses in metastatic liver disease of colorectal origin.

From 1986 to 1990, a multicentric phase II study was conducted with pirarubicin, a new semi-synthetic anthracyclin [4′- O -tetrahydropyranyl-adriamycin (THP)]. 87 patients with advanced gynaecological cancers were treated: epidermoid cervical carcinoma ( n = 31), adenocarcinoma of the endometrium ( n = 28) and ovarian adenocarcinoma ( n = 28). THP was administered by short intravenous infusion, for 3 consecutive days, every 3 weeks. The initial dose of THP was 25 mg/m 2 day (25% of patients) which was then reduced to 20 mg/m 2 day. The average number of courses was 3.7 (range 1–10). The cumulative THP dose was 180 mg/m 2 (range 56–594) in cervix and endometrial tumours and 121 mg/m 2 (range 58–425) in ovarian tumours. Myelosuppression was the major observed toxicity with grade 3–4 leukopenia and thrombocytopenia in 62 and 19% of the patients, respectively. Severe general complications occurred in 6% of the patients with three fatalities due to infections. Gastro-intestinal side-effects were frequent and usually mild (7% of grade 3 vomiting). 48% of the patients showed alopecia, which was complete in 9 cases (10%). 3 patients experienced cardiac events. No significant antitumoral activity was observed in patients who had failed to respod to previous chemotherapy. Promising antitumoral activity was noticed in untreated cervico-uterine carcinomas with 19% partial responses and 12% complete responses (CR). THP activity was lower in endometrial carcinomas (9.5% CR). Results were found to be negligible in ovarian cancer patients, most of them being refractory to previous chemotherapy containing an anthracyclin compound. On the basis of these results, the definite role of THP in gynaecological cancers deserves to be studied in more favourable programmes (e.g. in combined protocols as first-line chemotherapy).

Background: Single agent OTX015 has shown activity in several preclinical models of lymphoid tumors, including MCL and MM (AACR 2014), and notably in the ongoing phase I study (AACR 2014). We evaluated the synergy of OTX015 administered in combination with other anticancer compounds in several MCL and MM cell lines. Material and Methods: Two mantle cell lymphoma (REC1, MAVER1) and three multiple myeloma (RPMI8226, U266B1, KMS11) cell lines were exposed to increasing doses of OTX015 alone or in combination with increasing doses of other anticancer drugs. MTT assays were performed after 72 hours exposure. Synergy was assessed by Chou– Talalay combination index (CI) with the Synergy R package: CI < 0.3, strong synergy; 0.3−0.9, synergy; 0.9−1.1, additive effects. Changes in CCND1 protein and mRNA expression after OTX015 exposure were assessed in 4 MCL cell lines (REC1, MAVER1, Granta519, JeKo1). Results: Additive effects were seen in 2/2 MCL cell lines when OTX015 was combined with the demethylating agent 5-AZA (CI = 0.8 and 1), the HDAC-inhibitor vorinostat (CI = 0.8 and 1), and the proteasome inhibitor carfilzomib (CI = 0.8 and 0.9). Whereas, synergy was observed with the BTK-inhibitor ibrutinib in 2/2 MCL cells (CI = 0.7 and 0.4), however in ibrutinib-resistant MAVER1 cells, synergy was only seen with high ibrutinib doses (5−10mM). Synergy was observed in 1/2 MCL cell lines with OTX015 combined with the dual PI3K/mTOR inhibitor BEZ235 (CI = 0.1), the mTOR inhibitor everolimus (CI = 0.5), the glucocorticoid dexamethasone (CI = 0.1), and the immunomodulator pomalidomide (CI = 0.2). CCND1 mRNA and protein levels were not altered in 4/4 MCL cell lines after OTX015 (500 nM) treatment for 4 h and 24 h, suggesting that the antiproliferative activity of OTX015 is not CCND1mediated. Synergy was observed in 3/3 MM cell lines following OTX015 combined with carfilzomib (CI = 0.5, 0.7 and 0.9), or vorinostat (CI = 0.7, 0.6 and 0.7), in 2/3 cell lines with pomalidomide (KMS11, CI = 0.3; RPMI8226, CI = 0.4;) or 5-AZA (KMS11, CI = 0.4; RPMI8226, CI = 0.4) and in 1/3 with dexamethasone (U266B1, CI = 0.4). Conclusions: Preclinical experiments support the exploration of OTX015 combined with other anticancer agents in the clinical setting. Additional studies should be performed to clarify the mechanistic or molecular expression profile associated with sensitivity/resistance to individual combinations.

Background: Lymphomas are still incurable in many patients and novel active compounds are actively being sought. We previously reported single agent activity of the BET bromodomain OTX015 in lymphoma cell lines and in vivo (AACR 2012; ICML 2013). Here, we report a study of the mechanism of action of OTX015 and its activity in combination with other anti-cancer compounds. Methods: Cell lines: 22 diffuse large B-cell lymphoma (DLBCL), 4 mantle cell lymphomas, 3 multiple myelomas, 3 splenic marginal zone lymphoma and 1 prolymphocytic leukemia. Anti-proliferative of OTX015 (OncoEthix SA, Switzerland) was assessed by MTT and its cytotoxic activity by Annexin V staining and gene expression profiling (GEP) with Illumina HumanHT-12 Expression BeadChips. Data mining was done with LIMMA, GSEA, Metacore. Synergy was assessed in cells (2-5 cell lines) exposed for 72 h to increasing doses of OTX015 alone or in combination with increasing doses of other agents. MTT assays were performed and Chou-Talalay combination index (CI) calculated. Results: OTX015 (500 nM, 72h) showed cytostatic activity in 29/33 (88%) cell lines and apoptosis in 3/22 (14%). Mutations in genes coding for MYD88 and components of BCR (P=0.027), and ABC signaling phenotypes (P=0.008) were significantly associated with apoptosis induction. We performed GEP on 2 cell lines (SU-DHL-6, SU-DHL-2), treated with DMSO or OTX015 (500 nM) for 1, 2, 4, 8 or 12 hours. Most upregulated genes were histones. MYC target genes were highly significantly enriched among all OTX015 regulated transcripts and MYC was the most frequently downregulated gene. OTX015 also downregulated MYD88, IRAK1, TLR6, IL6, STAT3, and TNFRSF17, members of the NFKB, TLR and JAK/STAT pathways. NFKB target genes (IRF4, TNFAIP3 and BIRC3) were also downregulated (PCR). Immunoblotting and immunohistochemistry showed a reduction of transcriptionally active pSTAT3 in 2 ABC cell lines, and a reduction in nuclear localization of p50 (NFKB1), indicating an inhibitory effect of OTX015 on the canonical NFKB pathway. Finally, IL10 and IL4 production was reduced after 24 hours OTX015 treatment. Synergy was observed with everolimus (median CI=0.1; range 0.1-0.2), ibrutinib in ABC-DLBCL (CI=0.04; 0.02-0.1), idelalisib (CAL101) (CI=0.5; 0.04-2.4), vorinostat (CI=0.5; 0.3-0.6), rituximab (CI=0.5; 0.4-0.5), decitabine (CI=0.6; 0.6-0.7), lenalidomide (CI=0.7; 0.6-0.7), and all-trans retinoic acid (CI=0.4; .1-1.6). Additive effects were observed for combinations with romidepsin (CI=1.08; 1-1.22), bendamustine (CI=0.92; 0.83-1.1), and doxorubicin (CI=0.83; 0.71-0.96). Conclusions: OTX015 is a promising candidate for targeted combination therapies. A phase I study (NCT01713582) in patients with hematological neoplasias is underway and together with our preclinical data, may support further clinical investigations. Citation Format: Eugenio Gaudio, Elena Bernasconi, Ivo Kwee, Michela Boi, Paola Bonetti, Chiara Tarantelli, Andrea Rinaldi, Monica Testoni, Maurilio Ponzoni, Anastasios Stathis, Georg Stüssi, Eugenia Riveiro, Patrice Herait, Emanuele Zucca, Francesco Bertoni. The BET Bromodomain inhibitor OTX015 targets the NFKB, TLR and JAK/STAT pathways and shows pre-clinical activity as single agent and in combination in mature B-cell tumors. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5528. doi:10.1158/1538-7445.AM2014-5528

Background: The human BET family bromodomains, including BRD2-4 and BRDT proteins, has become a druggable target for the development of specific gene transcription inhibitors. Here, we report the preclinical anti-leukemic activity of OTX015 (OncoEthix SA, Switzerland), a novel pan-BET-BRD inhibitor, as single agent and in combination with different drugs. Material and Methods: Eight established human cell lines from acute and chronic myeloid leukemia (AML, i.e. HL-60 and U-937; CML, i.e. K-562 and NALM-1) and acute lymphoblastic leukemia (ALL, i.e. Jurkat, CCRF-CEM, MOLT-3 and -4) were treated by increasing doses of OTX015. The growth inhibition 50% (GI50) values of OTX015 were evaluated by MTT the assay at 72 h. Protein levels were analyzed by Western blot using commercial antibodies. RNA was extracted using the Qiagen RNAEasy and RT-PCR was performed using Fast SYBR Green on a StepOnePlus Real-Time PCR System. Combination effects were evaluated in HL60, U937, Jurkat and K562 cell lines; OTX015 was administered with daunorubicin, azacytidine, dexamethasone, cytarabine or methotrexate. The 48-h combination index (CI) was determined by median effect plot analysis using CalcuSyn software expressed as the median and range of CI values among cell lines (Chou & Talalay analysis). CI1: antagonism. Results: Firstly, the anti-proliferative effect of OTX015 was assessed. In HL60, U937 and Jurkat cells, GI50s values were between 230 and 384 nM, while ≥ 1,000 nM for the remaining cell lines. Both OTX015-sensitive and -resistant cells showed similar basal expression levels of BRD2-4, c-MYC, BCL-2, p21 and Cyclin D1 proteins. In sensitive cell lines, OTX015 caused cell cycle arrest in G1 in a time-dependent manner without induction of apoptosis. Likewise, c-MYC mRNA and protein levels were down-regulated after 6-h and up to 72-h treatments with 500 nM OTX015. In Jurkat cells, OTX015 induced up-regulation of BRD2 and 4 mRNA without modifying protein levels. On the other hand, although the OTX015-resistant cell line K562 showed decreased levels of c-MYC mRNA after 4-h and 24-h exposure, protein levels were constant even after 72 h of treatment. Synergy was observed with daunorubicin in OTX015-sensitive and -resistant cell lines (CI=0.8;0.4-0.9). The combination of OTX015 with azacytine or cytarabine was synergistic (CI=0.6;0.6-0.7 and CI=0.8;0.4-0.9, respectively) despite primary resistance of Jurkat and K562 lines to either of the individual drugs. Methotrexate, dexamethasone and panobinostat exerted synergistic effects with OTX015 in 3 out of 4 cell lines (CI=0.5;0.2-0.9; CI=0.3;0.1-0.7 and CI=0.6;0.5-0.7). Conclusion: Our findings indicate that OTX015 enhances in vitro anti-proliferative effects of standard antileukemic agents supporting combination therapy as an important aspect of the clinical development plan. Citation Format: Lucile Astorgues-Xerri, Ramiro Vázquez, Mohamed Bekradda, Esteban Cvitkovic, Patrice Herait, Eric Raymond, María E. Riveiro. In vitro evaluation of OTX015, a novel pan-BET-bromodomain (BET-BRD) inhibitor, as single agent and in combination with standard chemotherapy drugs in human leukemic cell lines. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5529. doi:10.1158/1538-7445.AM2014-5529

Summary Background In phase II trials, irinotecan is active in patients with advanced colorectal cancer, but the survival and clinical benefit of irinotecan compared with secondline fluorouracil by continuous infusion is not known. Methods 267 patients who had failed to respond to firstline fluorouracil, or whose disease had progressed after treatment with first-line fluorouracil were randomly allocated irinotecan 300–350 mg/m 2 infused once every 3 weeks or fluorouracil by continuous infusion. Treatment was given until disease progression, unacceptable toxic effects, or the patient refused to continue treatment. The primary endpoint was survival, while progression-free survival, response rate, symptom-free survival, adverse events, and quality of life (QoL) were secondary endpoints. Findings 133 patients were randomly allocated irinotecan and 134 were allocated fluorouracil by continuous infusion. Patients treated with irinotecan lived for significantly longer than patients on fluorouracil (p=0·035). Survival at 1 year was increased from 32% in the fluorouracil group to 45% in the irinotecan group. Median survival was 10·8 months in the irinotecan group and 8·5 months in the fluorouracil group. Median progression-free survival was longer with irinotecan (4·2 vs 2·9 months for irinotecan vs fluorouracil, respectively; p=0·030). The median pain-free survival was 10·3 months and 8·5 months (p=0·06) for irinotecan and fluorouracil, respectively. Both treatments were equally well tolerated. QoL was similar in both groups. Interpretation Compared with fluorouracil by continuous infusion second-line irinotecan significantly improved survival in patients with advanced colorectal cancer.

PURPOSE To assess, on a multicenter basis, the feasibility of treating advanced cancer patients with high-dose irinotecan. PATIENTS AND METHODS Thirty-five patients who met the usual phase I criteria (26 men and nine women) were included. Primary tumor sites were colon, head and neck, unknown primary, kidney, liver, and others. All had been previously treated. Irinotecan was given at the maximum-tolerated dose (MTD) (600 mg/m2) or the level below (500 mg/m2) as a 30-minute infusion once every 3 weeks. RESULTS Eighteen patients were entered in the four participating centers at the MTD of 600 mg/m2. This dose level was clearly shown not to be feasible: 14 patients (78%) had grade 3 to 4 neutropenia, with febrile episodes in 11 patients; grade 3 to 4 diarrhea was observed in nine patients; and one toxic death occurred. Subsequently, 17 not heavily pretreated patients were included at 500 mg/m2 and carefully monitored. The safety of this dose level was considered acceptable: 41% of patients had grade 3 to 4 neutropenia, 24% experienced grade 3 to 4 diarrhea, and no febrile granulocytopenia or toxic death occurred. Six partial responses were documented in metastatic colorectal cancer, all in patients who had previously received conventional chemotherapy, four in patients who had exhibited progressive disease under fluorouracil (5FU)-based chemotherapy. CONCLUSION We plan to study the higher dose-intensity 500-mg/m2 level on good-risk and carefully monitored patients. This could enlarge the spectrum of tumors sensitive to irinotecan and improve the already good results observed in colorectal cancers.

PURPOSE To assess the efficacy of irinotecan (CPT-11) in the treatment of advanced colorectal cancer in both chemotherapy-naive and pretreated patients. PATIENTS AND METHODS Two hundred thirteen patients (aged 18 to 75 years) with metastatic colorectal cancer, World Health Organization (WHO) performance status < or = 2, and life expectancy > or = 3 months were treated with CPT-11 350 mg/m2 every 3 weeks. All 178 patients eligible for efficacy analysis had not received more than one prior fluorouracil (5-FU)-based chemotherapy regimen (adjuvant or palliative) and had adequate hematologic, renal, and hepatic function. RESULTS Primary tumor sites were the colon (71%) and rectum (28%). Sixty-six percent of the patients had > or = two metastatic sites. Ninety-eight percent of the patients had undergone previous surgery, and 77.5% had received prior chemotherapy. Thirty-two of 178 eligible patients achieved on objective response (four complete responses [CRs] and 28 partial responses [PRs]; response rate, 18%; 95% confidence interval, 12.6% to 24.4%), 65 were stable, and 59 progressed. The response rate was 17.7% in the pretreated group and 18.8% in the chemotherapy-naive group. Within the former subgroup, response rates of 16.1% were reported in patients who were progressive on prior 5-FU chemotherapy and 19.1% in patients who were progressive off such treatment. The median duration of objective response (9.1 months) and median time to achievement of a response (9.3 weeks) did not differ between chemotherapy-naive and pretreated patients. The most frequent adverse events were neutropenia, which developed in 80% of the patients, delayed diarrhea (87%), alopecia (88%), fatigue (81%), and nausea/vomiting (77%). All these adverse events were manageable. Severe (WHO grade 3 or 4) neutropenia was only observed in 18% of the cycles, leukopenia in 11%, delayed diarrhea in 11%, and nausea and vomiting in 3%. Development of simultaneous grade 3 or 4 neutropenia and delayed diarrhea during 4% of the cycles was the safety issue of greatest concern. CONCLUSION CPT-11 has definite activity in the treatment of advanced metastatic colorectal cancer both in chemotherapy-naive and in pretreated patients who experienced disease progression on 5-FU, which suggests a lack of cross-resistance between CPT-11 and 5-FU. Diarrhea and neutropenia, the major toxicities of CPT-11, contribute to the risk to develop febrile neutropenic sepsis.

Colorectal cancer is one of the most common cancers in the Western World. Although 50% of patients are cured by surgery alone, the outcome is poor in high-risk patients (Dukes stages B2 and C) despite adjuvant chemotherapy with 5-fluorouracil (5-FU)-based regimens. CPT-11 (irinotecan) is a promising new agent for the treatment of colorectal cancer with a unique mechanism of action. CPT-11 is a DNA topoisomerase I inhibitor, which has not demonstrated susceptibility to the P-glycoprotein-mediated multidrug-resistant phenotype. Phase II studies with CPT-11 have demonstrated definite activity against colorectal cancer in both chemotherapy-naive and pretreated patients (response rates of 15-32% observed) even with clinical evidence of resistance to 5-FU. The response rate appears to be consistent, reproducible and equivalent to that achieved with 5-FU plus folinic acid in chemotherapy-naive patients.

Background. Galectin-1 is a multifunctional protein involved in various aspects of tumorigenesis and has been described as a promising cancer target. It has been previously shown that galectin-1 has high sensitivity to oxidative inactivation. During oxidation, galectin-1 may forms disulfide bridges resulting in profound conformational changes, which prevent galectin-1 dimerization and ligand recognition. OTX-008 is a non-peptide chemical antagonist designed to bind galectin-1. We previously showed that OTX-008 displays direct antiproliferative effects and inhibits galectin-1 expression in cancer cell lines (AACR 2011, Abstract n°685). The aim of the present study was to further the understanding on the molecular mechanism implicated on galectin-1 downregulation mediated by OTX-008 in cancer cells. Material and Methods. Proliferative SQ20B cancer cells were exposed to 3µM OTX-008 during 48h with or without i) 10µM N-acetylcysteine (an antioxidant agent), ii) 1mM Tempol (an antioxidant agent), iii) 10µM co-enzyme Q10 (a naturally occurring antioxidant agent), iv) 55µM α-mercaptoethanol (a strong reducing agent), or v) 10nM bortezomib (a proteasome inhibitor). Effects on galectin-1 protein levels were assessed by western blot analysis. Results. The head and neck SQ20B human cancer cell line is sensitive to OTX-008 (GI50 = 3µM). In this cell line, 48h-exposure to OTX-008 inhibits galectin-1 protein level (40% inhibition respect to control cells) without effect on galectin-1 mRNA levels. It has been described that galectin-1 oxidation represents a mechanism by which galectin-1 activity is regulated. SQ20B cells were exposed to OTX-008 for 48h with or without co-treatment with well-known antioxidant or reducing agents. Interestingly, these agents counteract the effects of OTX-008 on galectin-1 expression (galectin-1 protein levels by densitometry analysis: control SQ20B 100%, OTX-008 60%, N-acetylcystein+OTX-008 110%, Tempol+OTX-008 120%, co-enzyme Q10 +OTX-008 140% and α-mercaptoethanol+OTX-008 120%), suggesting that oxidation plays a key role in galectin-1 down-expression by OTX-008 in SQ20B cells. We further observed that pre-treatment with bortezomib abrogated the decrease in galectin-1 protein levels by OTX-008, pointing out that oxidized galectin-1 is recognized and degradated by the proteasome system. Conclusion. Our findings show that OTX-008 mechanism of action involves galectin-1 oxidation followed by proteosomal degradation in SQ20B cells. The mechanism by which OTX-008 can enhance the oxidation of galectin-1 will require further investigations. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2836. doi:1538-7445.AM2012-2836

Background Diarrhea is a serious side effect that may prevent the administration of high doses of the antitumor drug Irinotecan (CPT-11). Purpose Intensive, high-dose loperamide was used in an attempt to control or downstage CPT-11-induced diarrhea and thus permit the use of higher dose intensities of CPT-11. Methods Twenty-three patients with various cancers were treated with doses of CPT-11 ranging from 400 to 600 mg/m2, administered as a 30-minute intravenous infusion every 3 weeks. Starting 8 hours or more after the administration of CPT-11, any episode of diarrhea was treated with 2 mg of loperamide taken every 2 hours. Patients stopped taking loperamide only after a 12-hour diarrhea-free period. If diarrhea was not controlled after 3 consecutive days of nonstop loperamide intake, or if the patient was dehydrated, loperamide was stopped and the patient was hospitalized for intravenous fluids. If blood or mucus were found in the stools at any time during diarrhea, loperamide was stopped and the patient was hospitalized. Results Seventeen of 23 patients had diarrhea while on CPT-11 treatment. Eighty-two CPT-11 cycles were administered to these 17 patients, and diarrhea occurred in 49 of these cycles, at a median time-to-onset of 6 days after CPT-11 administration. The loperamide protocol was followed in 46 of the 49 episodes of diarrhea, with 21 capsules of loperamide the median number being taken (range, 5-72). Only one patient was hospitalized for failure to respond to loperamide, and no major toxicity was associated with loperamide use. Fourteen of the 17 patients who experienced diarrhea were rechallenged with CPT-11 three or more times, and seven patients six or more times. Conclusions High-dose loperamide controlled diarrhea in patients receiving CPT-11 and allowed administration of higher doses of CPT-11. Implications The effectiveness of CPT-11 might be increased by higher dose intensities, which can be made tolerable by control of diarrhea with loperamide.

Eighteen patients with either metastatic or locally advanced pancreatic carcinoma were treated with intravenous infusion of Pirarubicin 50 mg/m2/day every 3 weeks. Seventeen patients were evaluable for response. One had minor response, 5 had stable disease, and 11 had progression of disease. Hematological toxicity was moderate, leading to a dose reduction in only 1 patient; there was no clinical cardiac toxicity. We conclude that Pirarubicin used with this dosage and schedule has no activity in advanced pancreatic carcinoma.

Background. Sensitivity to targeted agents inhibiting VEGFR/PDGFR signaling, including sunitinib, may be dependent of the concomitant modulation of alternative survival pathways. The lectin galectin-1 (gal1) is overexpressed in several tumors, selectively binds neuropilin-1 and enhances VEGFR2 signaling thereby activating the MAPK and/or AKT pathways in cancer cells. Those effects may result in modulation of cancer cell proliferation, apoptosis, cell cycle, and tumour angiogenesis. Optimizing the inhibition of gal1/neuropilin-1/VEGFR-dependent signaling may be a useful approach to improve the activity of multitargeted drugs in cancer cells. The aim of the present study was to evaluate the effects of PTX-008, a novel drug that targets gal1, alone or in combination with sunitinib on several human cancer cell lines. Material and Methods. Antiproliferative effects were evaluated in human colon, breast, ovarian, head & neck, lung, prostate and HCC cancer cell lines by MTT assay. Effects of PTX-sunitinib combinations were determined by median effect plot analysis (Chou & Talalay). Results. Antiproliferative effects of sunitinib as single agent were evaluated on a panel of 20 human cancer cell lines including SQ20B, COLO205 and HT29 with IC50=2; 3 and 8µM, respectively. Antiproliferative effects of single agent PTX-008 were detected in several cell lines including head & neck SQ20B, colon HT29 and COLO205, lung HOP62 and ovarian cancer cells OVCAR3 and IGROV1 with GI50 of 3; 9; 9; 27; 3 and 27µM respectively. In contrast, head & neck HEP2, prostate DU145, breast MCF7 and SKBR3, renal CAKI1, hepatocarcinoma SK-HEP1 and colon HCC2998 lines appear to be resistant to PTX-008 (GI50>27µM). Effects of PTX-008 were slowing cell proliferation and G2/M accumulation, along with blockage of ERK and AKT survival pathways. In order to evaluate the effects of PTX-008 in combination with other multitargeted inhibitors, we used sequential and simultaneous exposure to PTX-008 with sunitinib. Combinations of PTX-008 with sunitinib in two colon cancer cell lines COLO205 and HT29 (which express low gal1, VEGFR and PDGFR mRNA), result in additive (CI=1) and synergistic (CI Conclusion. Combination of PTX-008 with sunitinib appears to be synergistic both in PTX-008-sensitive and -resistant cells, suggesting that modulation of gal1 signaling may be used to enhance the activity of sunitinib. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1548.

DD is the main toxicity of CPT-11 at the currently recommended dose (RD) of 350 mg/sqm IV (30’) q 3 weeks. In previously treated colorectal cancer patients (pts), we tried to determine the mechanism of DD and assess the efficacy of combined antidiarrheal medication. From Dec 93 until March 95, 24 pts having failed ≥ 1 ≤ 3 lines of 5-FU based treatment, entered a CPT-11 Phase II trial at the above RD. In the first cohort (14 pts), Acetorphan (Acet), a specific enkephalinase inhibitor, was given as 100 mg tid PO after the second loose stool, and supplemented, if DD > 48 hrs, with Loperamide (Lop) 2 mg q 2 hrs PO till 12 hrs after last loose stool. Pts had at baseline and if DD occurred endoscopy, with biopsies for Topo I and CPT-11 assays as well as transit time, stool frequency, weight, culture, electrolytes, osmotic gap, pH, fat and protein excretion, α antitrypsin ( α AT) clearance, D-xylose test; blood tests for VIP, glucagon, somatostatin, gastrin. Twelve/14 pts (first cohort) had CFT-11 DD: 5 responded to Acet alone, and the other 7 responded within 24 hrs to addition of Lop. Transit time normal in 5/7 pts, α AT increased in 4/4 pts. Stools weight >800 gr/day and fecal Na/K increased in 6/6 pts. Osmotic gap small in 3/6 pts. The second cohort (pts 15–25) received simultaneous Acet/Lop after first DD loose stool. Eight/11 pts had DD, and 7/8 had resolution of diarrhea within 12 hrs of treatment start. Available PK's of CPT-11 and SN-38 (active metabolite) show no pharmacodynamic relationship. Results suggest that CPT-11 DD is due to a secretory exudative mechanism, as attested by its response to early simultaneous antisecretory medications.