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Background: This open-label, phase IB study was undertaken to determine the safety/toxicity profile and recommended dose of oral once-daily PTK787/ZK 222584 (PTK/ZK) combined with oxaliplatin/5-fluorouracil (5-FU)/leucovorin (FOLFOX4) chemotherapy in patients with advanced colorectal cancer. Secondary objectives were to assess full pharmacokinetics and gather preliminary evidence of antitumor activity. Patients and methods: Thirty-five patients received escalating doses of PTK/ZK (range 500–2000 mg daily) continuously. Concurrent FOLFOX4 chemotherapy was administered on days 1 and 2 and repeated every 14 days. Dose escalation of PTK/ZK was continued until maximum tolerated dose (MTD) was established and additional patients were then enrolled at MTD dosage. Results: Mean treatment duration of PTK/ZK was 9.5 months. The MTD was 1250 mg daily with dizziness being the most frequent dose-limiting toxicity (DLT). Hypertension (23%, grade 3) and neutropenia (37%, grades 3 + 4) were the most frequent grade 3 or 4 adverse events. Pharmacokinetic analyses found no evidence for interactions between PTK/ZK and the combination of 5-FU, leucovorin, and oxaliplatin during concomitant use. Median progression-free survival was 11.4 months. Conclusion: The MTD of PTK/ZK in combination with FOLFOX4 in this patient population is 1250 mg daily. The combination is feasible and safe and is not associated with significant pharmacokinetic interactions.

The implantation of tumour cells in normal tissues and the subsequent induction of angiogenesis by the growing xenograft were studied by means of immunohistochemistry and digital image analysis. Tumour growth was induced by injection of a human spindle cell sarcoma (ES3) into the subcutis of HsdCpb:NMRI- nu/nu mice. In vivo injection of Hoechst 33342 was used as a marker of perfusion. The vasculature was stained with specific antibodies and subsequently analysed by digital image analysis. Starting at day 3 up to day 6, angiogenesis could be detected and the relative amount of perfusion within the investigated area reached a peak at day 6. This method, which allows investigation of both functional and morphometric characteristics of human xenograft vasculature, serves as an excellent assay for evaluation of antiangiogenic therapies in translational research of experimental tumours.

Intravenous (i.v.) vinorelbine (VRL) generally given on days 1 and 8 of an every three-week cycle in combination with paclitaxel (PTX) is an effective option for the treatment of metastatic breast cancer (MBC). In an effort to improve both patient and chemotherapy unit convenience, oral VRL was used at equivalent doses of i.v. VRL.The maximal tolerated dose (MTD) was determined during the first cycle of oral VRL given on days 1 and 8 or 15 and PTX infused over 3 h on day 1 every 3 weeks, maximum of 6 cycles. The dose of oral VRL was escalated from 60 to 80 mg/m2 in 10 mg/m2 increments. Paclitaxel was administered at 110 and then 135 mg/m2. The combination regimen was given as first-line chemotherapy of MBC. Three to six patients per cohort were treated.Twenty-two patients were treated in the first four cohorts (oral VRL/PTX): 60/110, 70/110, 80/110 and 80/135. In cohort 4, seven patients were treated, one patient being non-evaluable for MTD, three of them presented a dose-limiting toxicity (DLT) consisting of febrile neutropenia and neutropenic infection. Therefore 80/135 was the MTD. Because 36% of oral VRL administrations on day 8 were delayed to day 15 at 80/110, two additional cohorts were tested: in cohort 5, oral VRL 60 mg/m2 on days 1 and 15 and PTX 135 mg/m2 on day 1 and in cohort 6, oral VRL 80 mg/m2 on days 1 and 15 and PTX 110 mg/m2 on day 1, every 3 weeks. In cohort 5, six out of eight patients had DLTs: omission of oral VRL on day 15 for five patients, grade 4 neutropenia7 days for another one. Therefore the recommended dose (RD) for further clinical testing was oral VRL 80 mg/m2 on days 1 and 15 and PTX 110 mg/m2 on day 1 of an every 3-week cycle. Two of the three evaluable patients treated at the RD had a partial response. The pharmacokinetics of VRL and PTX is being analysed and will be further presented in a separate publication.This phase I study has determined the doses of oral VRL and PTX to be used in combination for the benefit of the patient and of the chemotherapy unit in term of nurse's workload. The recommended regimen of oral VRL 80 mg/m2 on days 1 and 15 and PTX 110 mg/m2 on day 1 given every 3 weeks will be further tested in phase II.

Targeted therapies that are approved for metastatic colorectal cancer are divided into two groups: those affecting vascular endothelial growth factor (VEGF) known to interrupt tumor growth and metastasis (also called neo-angiogenesis), and agents that affect the tumor directly by interrupting the epidermal growth factor (EGF) and its receptor. Anti-angiogenic VEGF therapies are divided into two categories: one affecting the VEGF ligand, such as bevacizumab, and those that inhibit the VEGF receptor, such as PTK/ZK. Epidermal growth factor receptor (EGFR) therapies are divided into monoclonal antibodies that affect EGFR, such as cetuximab, and EGFR tyrosine kinase inhibitors, such as gefitinib. Both VEGF and EGFR areas of treatment have shown promising efficacy in first-line, combination therapy settings. Future targeted therapeutic strategies include gene profiling, combinations of capecitabine and oxaliplatin, with bevacizumab and/or cetuximab therapies.

Purpose To demonstrate that adding irinotecan to a standard weekly schedule of high-dose, infusional fluorouracil (FU) and leucovorin (folinic acid [FA]) can prolong progression-free survival (PFS). Patients and Methods Four hundred thirty patients with measurable or assessable metastatic colorectal cancer were randomly assigned to receive either FA 500 mg/m2 as a 2-hour infusion and FU 2.6 g/m2 by intravenous 24-hour infusion, both administered weekly for 6 weeks, followed by a 2-week rest (Arbeitsgemeinschaft für Internistische Onkologie [AIO] arm, n = 216), or a similar schedule but with FU 2.3 or 2.0 g/m2 preceded by irinotecan 80 mg/m2 administered over 30 minutes (experimental group, n = 214). Results The median PFS time in the experimental group was 8.5 months (95% CI, 7.6 to 9.9 months) compared with 6.4 months (95% CI, 5.3 to 7.2 months) in the AIO arm (P < .0001). The median overall survival time was increased from 16.9 to 20.1 months (P = .2779). The objective response rate was 62.2% (95% CI, 55.0% to 69.5%) in the experimental group and 34.4% (95% CI, 27.5% to 41.3%) in the AIO arm (P < .0001). Conclusion The addition of irinotecan to the standard AIO FU/FA regimen was associated with a highly significant improvement in PFS and response rate and was well tolerated. The results of this study confirm that irinotecan in combination with high-dose infusional FU/FA is a reference first-line treatment.

Purpose To define the maximum-tolerated dose (MTD) and to evaluate the dose-limiting toxicities (DLT) of the combination of capecitabine and vinorelbine in patients with metastatic breast cancer who relapse after adjuvant and/or first-line treatment. In addition, we aimed to obtain data on efficacy and safety at the recommended dose. Patients and methods Patients with measurable metastatic breast cancer after failure of prior chemotherapy (including anthracyclines and/or taxanes) were eligible. Capecitabine was administered with a fixed dose of 1000 mg/m2 orally twice daily for 2 weeks followed by 1 week rest. One treatment cycle consisted of 6 weeks of treatment containing two treatment periods of capecitabine. Vinorelbine was given intravenously at escalated doses of 25 mg/m2 (dose level 1) and 30 mg/m2 (dose level 2) on days 1 and 8, and 22 and 29. Results Thirty-three patients received a total of 91 cycles of capecitabine and vinorelbine. The median number of administered cycles per patient was three (range one to six). Thirty-one patients were evaluable for toxicity. At dose level 2 four out of seven patients experienced DLTs (nausea/vomiting, febrile neutropenia, grade 4 neutropenia, infection and diarrhea); thus, the MTD was defined. In order to confirm the safety and efficacy, dose level 1 was extended to 24 patients. Two patients [8.3%; 95% confidence interval (CI) 1% to 27%] showed DLTs (hospitalization due to febrile neutropenia and prolonged neutropenia). The main toxicity was neutropenia, which was observed at National Cancer Institute Common Toxicity Criteria grade 3 and 4 in 39% of patients. The overall response rate for capecitabine and vinorelbine was 55% (95% CI 36% to 72.7%), including three patients with a complete remission. The median time to disease progression was 8 months (95% CI 4.3–11.7) with an overall survival of 19.2 months (95% CI 11.3–27.1) based on intention-to-treat analysis. Conclusions The combination of capecitabine and vinorelbine can be administered with manageable toxicity and showed significant efficacy for patients with metastatic breast cancer even after failure of a anthracycline- and/or taxane-based therapy.

PTK787/ZK 222584 (PTK/ZK) is an oral potent and selective inhibitor of the vascular endothelial growth factor (VEGF)-mediated Flt-1 and KDR receptor tyrosine kinases. PTK/ZK has been shown to reduce growth and microvasculature in subcutaneously implanted human tumor xenografts in nude mice. A clinical difficulty in evaluating angiogenesis inhibitors has been the usefulness of conventional study endpoints. Therefore, dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) has been studied as a pharmacodynamic marker of efficacy of PTK/ZK. Phase I studies are under way evaluating the optimum dose and schedule of oral PTK/ZK administered continuously to patients with advanced cancers of types known to overexpress VEGF. To date, particularly in patients with liver metastases from colorectal cancer treated with PTK/ZK, DCE-MRI has been a useful predictor of the biological response of VEGF-receptor inhibition. Toxicities have been manageable and have included lightheadedness, ataxia, nausea, vomiting, and hypertension. Stabilization of disease for ≥ 6 months has been seen in heavily pretreated patients receiving PTK/ZK at higher doses. Preliminary data suggest that PTK/ZK can be administered safely on a continuous daily dosing schedule, efficacy data look promising, and DCE-MRI correlates with biological response. DCE-MRI will be used to guide dose optimization of PTK/ZK and perhaps of other angiogenesis inhibitors in future studies. Semin Oncol 30 (suppl 6):32-38. © 2003 Elsevier Inc. All rights reserved.

Therapeutic options for patients with metastatic colorectal cancer have clearly improved during the last years. The regular use of irinotecan and oxaliplatin in first- and second-line treatment led to a clear improvement of median overall survival time. For the first time a new therapeutic concept--the inhibition of tumor angiogenesis--has been realized for clinical use by combining the anti-VEGF monoclonal antibody bevacizumab with an irinotecan-based first-line therapy. The monoclonal antibody cetuximab, targeted against EGFR, offers another new and very effective therapeutic option to patients with advanced irinotecan-refractory colorectal cancer--even those who are already pretreated with oxaliplatin. Further clinical studies are going to evaluate the future role of these new molecular treatment options as part of those therapeutic possibilities which are already available and established for the treatment of colorectal cancer patients with advanced disease (such as the optimal sequencing, the role of orale fluoropyrimidine-based combination therapy with cetuximab or bevacizumab).

Summary Background In metastatic breast cancer patients who have had prior exposure to anthracyclines, single agents induce less than 15% and combination chemotherapy less than 20%–30% of objective responses. Therefore more active and tolerable salvage regimens are needed. Patients and methods Forty-three patients with advanced breast cancer pretreated with 1–5 (median 2) different chemotherapy regimens were entered into this phase I/II trial. Treatment consisted of folinic acid (FA) (500 mg/m2 i.v., 2-hour infusion) followed by a 24-hour infusion of 5-fluorouradil (FU) which was escalated from 1.5 g/m2 (dose level (dl 1)), to 1.8 g/m2 (dl 2) to 2.1 g/m2 (dl 3). Therapy was given as outpatient treament once weekly times 6 followed by a 2-week rest. Results HD-FU/FA was well tolerated. No dose-limiting toxicity occurred at dl 1 or 2. Only 3/32 (9%) patients had WHO grade 3/4 toxicities (gastrointestinal toxicities, hand-foot-syndrome) at dl 3. The response rate for all 32 of the patients treated at dl 3 was 41% (13/32). In the 24 patients with anthracycline-refractory disease, a response rate of 41% (10/24) was achieved. The median remission duration was 11 months and the median survival time 19 months. Conclusions This schedule of FU/FA is a safe outpatient treatment with substantial activity in intensively pretreated breast cancer patients.

Paclitaxel has demonstrated broad clinical activity in a variety of malignancies both alone and in combination with other chemotherapeutic agents. The in vitro cytotoxicity of paclitaxel and cisplatin alone, in combination and in sequence, were evaluated against established human gastric and ovarian carcinoma cell lines using 2-h drug exposure. The combination of cisplatin and paclitaxel was found to be additive or even synergistic when paclitaxel was given 24 h prior to cisplatin as demonstrated by isobologram analysis. However, when both drugs were given simultaneously or when cisplatin was given prior to paclitaxel, a strong antagonistic interaction was observed. This antagonism was evident for up to 72 h after a 2-h exposure to cisplatin. Pretreatment with cisplatin caused no alteration in [3H]paclitaxel uptake in HM2 gastric carcinoma cells, but resulted in decreased intracellular retention of paclitaxel. Since cisplatin treatment led to a reduction in cellular glutathione content in these cells and reduced levels of glutathione have been associated with protection against cytotoxicity of paclitaxel, cells were pretreated with L-buthionine sulfoximine (L-BSO). However, depletion of glutathione had no influence on the activity of paclitaxel. A significant accumulation of cells in S-phase was observed 24 h after cisplatin, which resolved after 48 h and resulted in a pronounced increase of G2M phase. These data demonstrate that the interactions of paclitaxel and cisplatin are highly schedule-dependent and applications of cisplatin simultaneously with or prior to paclitaxel may result in pronounced antagonism. These findings could have implications for the design of further clinical protocols.

Over the last ten years we have witnessed a meaningful change in the treatment options of patients with metastatic colorectal cancer. However, not only does the composition of chemotherapy regimens, integrating the three anti-neoplastic agents fluoropyrimidines, irinotecan and oxaliplatin and the antibodies directed against the epidermal growth factor-receptor (EGFR) or vascular endothelial growth factor (VEGF), have to be considered, but the concepts of chemotherapy, aiming for cure in case of potential secondary resection of metastases or aiming for palliation, also provide challenges. Therefore, it does not come as any surprise that clinicians experience difficulties in defining a standard of care for their patients.

Der Prozes der Tumorneoangiogenese beschreibt die Neubildung von Gefasen, die zur Homoostase eines neoplastischen Zellverbandes notwendig sind.

Die Zytostatikaresistenz maligner Zellen ist ein Hauptproblem der zytostatischen Chemotherapie maligner Tumoren und hamatologischer System-erkrankungen. Dabei werden die zellkinetische Resistenz, die auf dem Proliferationsverhalten beruht, und die zellulare Resistenz, die durch pharmakologische und biochemische Besonderheiten charakterisiert ist, voneinander unterschieden.

Background: The current two studies evaluate the feasibility, toxicity and efficacy of an adjuvant combined modality treatment strategy containing a three to four-drug chemotherapy regimen plus 5-fluorouracil (FU)-based radiochemotherapy. Patients and methods: Between December 2000 and October 2003, a total of 86 patients were included in both studies. Patients with completely resected gastric adenocarcinoma including a D1 or D2 lymph node dissection (LND) were eligible. Treatment consisted of two cycles of folinic acid 500 mg/m2, 5-FU 2000 mg/m2 continuous infusion over 24 h once weekly for 6 consecutive weeks, paclitaxel 175 mg/m2 in weeks 1 and 4 and cisplatin 50 mg/m2 in weeks 2 and 5 (FLPP; n=41) or two cycles of the same 5-FU/folinic acid schedule but with cisplatin 50 mg/m2 only in weeks 1, 3 and 5 (FLP; n=45). Radiation with 45 Gy plus concomitantly applied 5-FU 225 mg/m2/24 h was scheduled in between the two cycles. Results: Patients characteristics were: D1/D2 LND FLP group 53%/42%; FLPP group 27%/68%; stage distribution: UICC stages III/IV(M0) FLP group 63% and FLPP group 66%. Median follow-up was 10 months (3–25) for FLP and 18 months (2–51) for FLPP patients. CTC grade 3/4 toxicities during the first cycle/chemoradiation/second cycle of FLP: granulocytopenia 3%/0/27%, anorexia 6%/10%/8%; diarrhea 8%/0/4%, nausea 3%/0/4%; FLPP: granulocytopenia 0/0/37%, anorexia 5%/11%/6%; diarrhea 5%/0/3, nausea 3%/8%/0%; early death in one patient due to Pneumocystis carinii pneumonia. Projected 2-year progression-free survival was 64% (95% CI 56% to 68%) for the FLP and 61% (95% CI 42% to 78%) for the FLPP group. Conclusions: Both chemoradiation regimens appear feasible with an acceptable toxicity profile indicating that cisplatin can be added to 5-FU/FA and that even a four-drug regimen can be investigated further in prospective clinical trials in completely resected gastric cancer patients. Treatment should be given in experienced centres in order to avoid unnecessary toxicity.

In comparison with supportive measures alone, chemotherapy in advanced gastric carcinoma is associated with a significant increase in survival and improvement in quality of life. The following substances are considered to be effective and suitable for combination therapy: 5-FU +/- folic acid, cisplatin, irinotecan, etoposide, taxol, and taxotere. In contrast, the effect of doxorubicin and epidoxorubicin is only moderate. Although "second generation" combinations, such as FAMTX, ELF or cisplatin/5-FU, induced higher remission rates as the "first generation" combinations, they failed to improve survival times to any appreciable extent. Currently accepted standard treatment of metastatic gastric carcinoma is infusional 5-FU + cisplatin or ECF. The third generation combinations containing taxane, irinotecan, oxaliplatin appear to be at least equally effective.

Therapeutic options for patients with metastatic colorectal cancer have clearly improved during the last years. The regular use of irinotecan and oxaliplatin in first- and second-line treatment led to a clear improvement of median overall survival time. For the first time a new therapeutic concept--the inhibition of tumor angiogenesis--has been realized for clinical use by combining the anti-VEGF monoclonal antibody bevacizumab with an irinotecan-based first-line therapy. The monoclonal antibody cetuximab, targeted against EGFR, offers another new and very effective therapeutic option to patients with advanced irinotecan-refractory colorectal cancer--even those who are already pretreated with oxaliplatin. Further clinical studies are going to evaluate the future role of these new molecular treatment options as part of those therapeutic possibilities which are already available and established for the treatment of colorectal cancer patients with advanced disease (such as the optimal sequencing, the role of orale fluoropyrimidine-based combination therapy with cetuximab or bevacizumab).

Aktuelle Ansatze in der Systemtherapie gastrointestinaler Tumoren fokussieren v. a. auf molekulare Strategien, die eine hohere Tumorselektivitat der eingesetzten Medikamente bewirken sollen. Zu diesen zellularen Angriffszielen zahlen v. a. wachstumsfaktorvermittelte Proliferations- und Differenzierungsprozesse, Signal-transduktion und neoplastische Angiogenese (Tabelle 46. 1).

Background To define the maximum-tolerated dose (MTD) and to evaluate the dose-limiting toxicities (DLTs) of the combination of capecitabine and irinotecan in patients with metastatic colorectal cancer. Patients and methods Thirty-seven patients with measurable metastatic colorectal cancer with no prior chemotherapy for metastatic disease were treated at three dose levels (DLs). For the first two dose levels, irinotecan (70 mg/m2) was administered once a week for 6 weeks in combination with 2 weeks of capecitabine at 1000 mg/m2 (DL1) or 1250 mg/m2 (DL2) twice daily, starting on days 1 and 22. In the last dose escalation step, the dose of irinotecan was increased to 80 mg/m2 (DL3). One cycle lasted 7 weeks. Results In the subsequent phase I trial, 96 cycles of capecitabine and irinotecan were administered. At DL3, three out of six patients experienced DLTs (diarrhea, neutropenia, asthenia). In order to confirm the safety of the recommended dose, DL2 was extended to 15 patients. Five patients (33%) showed DLTs at this dose level, which was considered too high to embark on further clinical studies. Subsequently, the starting dose (DL1)was extended to a total of 16 patients, with diarrhea being the main toxicity. The overall response rate was 38% [95% confidence interval (CI) 21% to 58%], with a median response duration of 8.7 months (95% CI 6.4–11.5 months). Conclusions The recommended doses for further studies are irinotecan 70 mg/m2 and capecitabine 1000 mg/m2. The combination of capecitabine and irinotecan appears to have significant therapeutic efficacy with manageable toxicity.

Phase I/II studies suggest that the combination of irinotecan with capecitabine is feasible and has promising activity. Diarrhea and neutropenia are dose limiting. Overall response rates (RRs) in the 40% to 60% range are seen from preliminary data. Work in progress is assessing the combination of irinotecan with UFT/leucovorin (LV). The use of irinotecan together with raltitrexed is also being investigated, as is its combination with oxaliplatin. Two phase II studies of irinotecan plus oxaliplatin in second-line patients report median survivals of 11-12 months. It seems possible to safely escalate the dose of single-agent irinotecan to 500 mg/m(2) in patients showing good tolerance of the drug. Irinotecan can be used in combination with LV5FU2 at doses up to 260 mg/m(2), especially if only one bolus of 5-fluorouracil (5-FU) is given. Control of tumor growth is achieved in 90% of patients. Preliminary data suggest that regimens based on 5-FU/LV and irinotecan can safely be combined with the anti-epidermal growth factor receptor (EGFR) antibody cetuximab. In patients with EGFR-positive tumors, this may prove an effective means of increasing response rate or combating treatment resistance. Following evidence that COX-2 inhibition can slow progression in familial adenomatous polyposis, celecoxib is to be studied in metastatic colorectal cancer (CRC). In vitro, the cyclin-dependent kinase inhibitor flavopiridol enhances the induction of apoptosis by chemotherapy. Clinically, it can safely be administered with irinotecan, and studies in CRC are planned.

N-(pyridin-4-yl)-[1-(4-chlorbenzyl)-indol-3-yl]-glyoxyl-amid (D-24851) is a novel synthetic compound that was identified in a cell-based screening assay to discover cytotoxic drugs. D-24851 destabilizes microtubules and blocks cell cycle transition specifically at G2-M phase. The binding site of D-24851 does not overlap with the tubulin binding sites of known microtubule-destabilizing agents like vincristine or colchicine. In vitro, D-24851 has potent cytotoxic activity toward a panel of established human tumor cell lines including SKOV3 ovarian cancer, U87 glioblastoma, and ASPC-1 pancreatic cancer cells. In vivo, oral D-24851 treatment induced complete tumor regressions (cures) in rats bearing Yoshida AH13 sarcomas. Of importance is that the administration of curative doses of D-24851 to the animals revealed no systemic toxicity in terms of body weight loss and neurotoxicity in contrast to the administration of paclitaxel or vincristine. Interestingly, multidrug-resistant cell lines generated by vincristine-driven selection or transfection with the Mr 170,000 P-glycoprotein encoding cDNA were rendered resistant toward paclitaxel, vincristine, or doxorubicin but not towards D-24851 when compared with the parental cells. Because of its synthetic nature, its oral applicability, its potent in vitro and in vivo antitumoral activity, its efficacy against multidrug-resistant tumors, and the lack of neurotoxicity, D-24851 may have significant potential for the treatment of various malignancies.

Ifosfamide is an alkylating antineoplastic agent with documented activity against a variety of solid tumor types, most notably lung cancer, testicular cancer, and sarcoma. Ifosfamide has been included in various drug combination protocols, usually on an empirical basis. To gather more insight into the mechanisms that underlie these drug interactions and to develop guidelines for further improvement of clinical combination protocols, we performed a broad preclinical evaluation program of ifosfamide-based combination regimens using isobologram analysis of drug interactions. In established cisplatin-sensitive and cisplatin-refractory ovarian carcinoma cell lines, a schedule-dependent drug interaction between paclitaxel and activated hydroperoxy-ifosfamide (4-OOH-IF) could be demonstrated. When both drugs were given for 2 hours, simultaneous exposure or the sequence of paclitaxel followed by 4-OOH-IF were additive or synergistic. In contrast, application of 4-OOH-IF before paclitaxel resulted in pronounced antagonism. Based on the sequence-dependent synergistic interactions a phase I trial was initiated with paclitaxel given on day 1 and ifosfamide given on days 2 to 5 in patients with cisplatin-refractory ovarian cancer. Four dose levels were evaluated in 18 patients. The maximum tolerated dose was paclitaxel 175 mg/m2 on day 1 and ifosfamide 2,000 mg/m2 on days 2 to 5, with central nervous system toxicity and nephrotoxicity being dose-limiting. The recommended dose for further evaluation of this combination was paclitaxel 175 mg/m2 on day 1 and ifosfamide 1,500 mg/m2 on days 2 to 5. Although all patients were heavily pretreated with multiple agents, nine of 18 patients achieved an objective response. Ifosfamide also has been shown to reduce cellular glutathione content; thus, a series of experiments evaluated the ability of activated cyclophosphamide or ifosfamide to deplete cellular glutathione in vitro. It was demonstrated that glutathione depletion is dose- and time-dependent, with 4-OOH-IF leading to a more pronounced suppression of cellular glutathione compared with 4-OOH-Cy. The decrease in cellular glutathione content was maximal at 2 hours after drug treatment; however, cellular glutathione levels returned to normal within 24 hours. When 4-OOH-IF was combined in vitro with cisplatin, schedule-dependent interactions again became obvious. The highest antitumor activity was seen when both drugs were given concurrently; sequential application with 4-OOH-IF given before cisplatin resulted in antagonism. Since adequate glutathione levels are necessary for multidrug resistance protein (MRP) function, glutathione depletion might lead to reversal of MRP-mediated drug resistance. Preliminary data showed that 4-OOH-IF significantly decreases glutathione concentrations in MRP-expressing human HT1080/DR4 sarcoma cells, leading to maximum steady-state reduction after a 90-min exposure to 4-OOH-IF. Taken together the data reported here demonstrate that in vitro ifosfamide may potentiate the antitumor activity of a variety of cytotoxic agents and therefore merits further clinical evaluation in drug combinations (eg, taxanes, anthracyclines).

PURPOSE: To determine the maximum-tolerated dose (MTD) of a weekly schedule of irinotecan (CPT-11), leucovorin (LV), and a 24-hour infusion of fluorouracil (5-FU24h) as first-line chemotherapy in advanced colorectal cancer and to assess preliminary data on the antitumor activity. PATIENTS AND METHODS: Twenty-six patients with measurable metastatic colorectal cancer were entered onto this phase I study. In the first six dose levels, fixed doses of CPT-11 (80 mg/m2) and LV (500 mg/m2) in combination with escalated doses of 5-FU24h ranging from 1.8 to 2.6 g/m2 were administered on a weekly-times-four (dose levels 1 to 4) or weekly-times-six (dose levels 5 to 6) schedule. The dose of CPT-11 was then increased to 100 mg/m2 (dose level 7). RESULTS: Seventy-nine cycles of 5-FU24h/LV with CPT-11 were administered in an outpatient setting. No dose-limiting toxicities were observed during the first cycle at dose levels 1 to 6, but diarrhea of grade 4 (National Cancer Institute common toxicity criteria) was observed in three patients after multiple treatment cycles. Other nonhematologic and hematologic side effects, specifically alopecia and neutropenia, did not exceed grade 2. With the escalation of CPT-11 to 100 mg/m2 (dose level 7), diarrhea of grade 3 or higher was observed in four of six patients during the first cycle; thus, the MTD was achieved. Sixteen of 25 response-assessable patients (64%; 95% confidence interval, 45% to 83%) achieved an objective response. CONCLUSION: The recommended doses for further studies are CPT-11 80 mg/m2, LV 500 mg/m2, and 5-FU24h 2.6 g/m2 given on a weekly-times-six schedule followed by a 1-week rest period. The addition of CPT-11 to 5-FU24h/LV seems to improve the therapeutic efficacy in terms of tumor response with manageable toxicity.

The purpose of this trial was to examine the feasibility of intensive, sequential chemo- and radiochemotherapy followed by surgery in patients with locally advanced carcinomas of the lower oesophagus and the gastro-oesophageal junction (GO junction). The chemotherapy consisted of two courses of 6 weekly administrations of 5-fluorouracil (5-FU) (2.0 g/m2, 24 h infusion) and folinic acid (FA) (500 mg/m2, 2 h infusion) combined with twice weekly cisplatin (50 mg/m2, 1 h infusion). Irradiation of 30 Gy was given concurrently with one course of cisplatin and etoposide. 25 patients with locally advanced (T3-T4 NX M0) squamous cell or adenocarcinoma of the lower oesophagus and GO junction were included and evaluated. Toxicity was usually mild to moderate (WHO grade 1 and 2) with mucositis as the most important side-effect of the pre-operative treatment. Of the patients, 94 and 88% completed the chemo- and radiochemotherapy according to the protocol, respectively. A major response (=partial remission with subjective improvement) to chemotherapy was achieved in 6/10 patients with squamous cell carcinoma and 10/15 with adenocarcinoma. 19 patients had subsequent surgery and complete resection was achieved in 16 (3 patients had intra-abdominal metastases observed at laparotomy). The operative mortality rate was 16% (3/19). 10 of the 16 patients with tumour resection had a pathological complete response. 15 patients (43%) remain alive at a median follow-up of 20 months and the median survival exceeds 16+ months. Our data suggest that this intensive pre-operative chemoradiotherapy programme is feasible and remarkably effective in patients with locally advanced carcinomas of the lower oesophagus or GO junction.

Background Multidrug resistance has been associated with expression of the multidrug resistance protein (MRP). Recently, MRP-expression has been detected in human tumor samples of patients with breast cancer and non-small-cell lung cancer. Since taxoids are the most active drugs in the treatment of both tumor entities, the antitumor efficacies of paclitaxel and docetaxel were compared in nude mice bearing human tumor xenografts that express MRP. Materials and methods Athymic nude mice (nu/nu) bearing tumor xenografts of parental human sarcoma HT1080 or MRP-expressing HT1080/DR4 cells (as confirmed by Northern blot analysis) were treated with the maximum tolerated doses (MTD) of doxorubicin ([Dx] 10 mg/kg i.v. push), paclitaxel ([PC] 50 mg/kg three-hour i.v. infusion), or docetaxel ([DC] 40 mg/kg three-hour i.v. infusion). In vitro, the activity of doxorubicin, paclitaxel and docetaxel was evaluated by the sulphorhodamine B (SRB) assay using the pyridine analogue PAK-104P (5 μM), a potent inhibitor of MRP-function. Results At their MTDs both taxoids showed significant activity against MRP-negative HT1080 xenografts with response rates of 80% (40% CR) for PC and 100% (60% CR) for DC. In contrast, DC was significantly more active than PC in nude mice bearing doxorubicin resistant MRP-expressing HT1080/DR4 tumor xenografts (overall response rates: 100% (60% CR) for DC; 10% (0% CR) for PC; 0% for Dx). Since treatment of mice with the MTD of PC or DC yielded similar overall toxicity (maximum weight loss for HT1080: PC 8.6 ± 2.2%; DC 7.5 ± 2.2% and for HT1080/DR4: PC 11.6 ± 3.0%; DC 7.6 ± 1.8%, respectively), these results demonstrate the increase in the therapeutic index for docetaxel against MRP-expressing tumors. In vitro, HT1080/DR4 cells were 270-fold, 6.4-fold and 2.8-fold more resistant than parental cells to doxorubicin, PC and DC, respectively. Pyridine analogue PAK-104P completely restored drug sensitivity to PC and DC, while no effect of PAK-104P on parental HT1080 cells was observed. Conclusions Both taxoids, when given at their MTDs, showed significant efficacy against parental HT1080 tumor xenografts. However, docetaxel at its MTD was significantly more active against MRP-expressing tumor xenografts than paclitaxel. Furthermore, in vitro resistance of HT1080/DR4 cells was higher for PC (6.4-fold) than for DC (2.8-fold). Since PAK-104P completely restored sensitivity to both taxoids, the observed resistance appears to be related to MRP. These data suggest, that docetaxel is not as readily transported by MRP as paclitaxel leading to an increased therapeutic ratio in MRP-expressing tumors in vivo. Therefore, docetaxel may have therapeutic advantages in the clinical treatment of MRP expressing tumors.

The prognostic value of topoisomerase I (Topo I) catalytic activity and expression of the multidrug resistance (MDR) marker P-glycoprotein (Pgp) and multidrug resistance protein (MRP) for in vitro sensitivity to Topo I interactive agents were evaluated. The efficacy of short term (2 h) and long term (24 h) exposures of camptothecin (CPT), two CPT derivatives (SN-22, SN-38) and the indolocarbazole compound NB-506, was determined against human ovarian carcinoma (A2780 and A2780 DX5), human fibrosarcoma (HT1080 and IIT1080/DR4) and human ileocecal carcinoma (HCT-8). For each cell line the Topo I protein levels and catalytic activity were determined and correlated with drug-induced cytotoxicity. In general, the Topo I protein levels correlated with Topo I catalytic activity. Drug-induced cytotoxicity increased significantly with prolongation of the exposure time. With the 2 h exposure, the multidrug resistant A2780 DX5 cell line (Pgp+, MRP-) was moderately resistant to all four drugs compared to its parental cell line. In case of CPT and SN-22 but not for SN-38 and NB-506, this resistance was no longer detectable following 24 h drug exposure. No resistance was detectable for the multidrug resistant HT1080/DR4 (Pgp-, MRP+) cell line when compared to its parental cell line. With short term exposures a strong trend was observed toward increased cytotoxicity with increased Topo I catalytic activity, especially if this correlation was studied between derivative cell lines (A2780 vs. A2780 DX5 and HT1080 vs. HT1080/DR4). This correlation weakened when all 5 cell lines and both exposure conditions were considered. Thus, although overall the correlation between Topo I catalytic activity and sensitivity to Topo I interactive drugs between different cell lines is weak, this correlation may be stronger when comparing derivative cell lines. For CPT and SN-22 but not for SN-38 and NB-506, the moderate resistance levels observed in the Pgp-expressing cell line could be negated by prolongation of exposure duration. MRP expression did not effect drug efficacy. The data demonstrate that the importance of Topo I catalytic activity as single prognostic factor for drug response to Topo I interactive agents is weak and that additional mechanisms affecting drug response have to be taken into consideration.

Membrane protein-associated alterations in cellular drug accumulation have been recently implicated in resistance to topoisomerase I (TOP-I)-interactive drugs. The present study investigated the cellular determinants of resistance to the indolocarbazole compound NB-506 [6-N-formylamino-12,13-dihydro-1,11-dihydroxy-13(beta-D-glucopyranosyl)- 5H-indolo[2,3-alpha]pyrrolo[3,4-c]carbazole-5,7(6H)-dione], a structurally novel TOP-I-interactive drug, in parental and multidrug-resistant tumor cells expressing either the P-170 glycoprotein (Pgp170) or multidrug resistance protein (MRP). MRP-expressing 250-fold doxorubicin-resistant human fibrosarcoma HT1080/DR4 tumor cells were drug sensitive to NB-506 and camptothecin (CPT) (resistance factor: 0.7 and 0.8, respectively) with no alterations of TOP-I parameters including DNA relaxation, expression of TOP-I protein and mRNA. In contrast, doxorubicin-resistant human ovarian A2780/Dx5 tumor cells [pgp170 phenotype] were 6.2-fold resistant to NB-506, whereas resistance to CPT was 2.6-fold. HPLC analysis of cellular NB-506 accumulation showed no significant differences between A2780 and A2780/Dx5 cells (peak intracellular concentrations after 120-min exposure to 10 microM NB-506: 400+/-85.0 and 352+/-95.1 nmol NB-506/mg protein, respectively). However, resistant A2780/Dx5 cells expressed a lower amount of TOP-I mRNA and 29% protein levels of TOP-I compared to parental A2780 cells, resulting in decreased TOP-I catalytic activity (3.17+/-0.02 vs. 1.16+/-0.15 rel.U/microg nuclear protein) and reduced induction of NB-506-mediated cleavable complex formation in A2780/Dx5 cells. Furthermore, the lower induction of NB-506-induced protein-linked DNA breaks (PLDB) in A2780/Dx5 cells correlated with significantly decreased DNA 12.2-440 kb size fragmentation in these cells. The present study demonstrates that expression of MRP and Pgp170 does not confer resistance to NB-506. Resistance to indolocarbazole substance NB-506 in A2780/Dx5 cells was only related to downregulation of TOP-I associated with lower induction of cleavable complex formation and DNA fragmentation. The data reported herein may indicate that the new indolocarbazole compound NB-506 has potent antitumor efficacy in membrane-associated multidrug resistance.

Intracellular glutathione (GSH) has been implicated as a regulatory determinant of multidrug resistance protein (MRP) function. The objective of the present study was to evaluate in vivo the ability of d,l-buthionine-(S,R)-sulfoximine (d,l-BSO), a potent inhibitor of GSH biosynthesis, to reverse MRP-mediated drug resistance to doxorubicin. Athymic nude mice (nu/nu) bearing advanced parental human fibrosarcoma HT1080 and MRP-expressing HT1080/DR4 tumors were treated with the maximum tolerated dose of doxorubicin (10 mg/kg, i. v. push). This therapy produced an overall response rate of 50% (20% complete response and 30% partial response) in mice bearing parental HT1080 xenografts, whereas no significant antitumor activity against HT1080/DR4 tumors was observed. Treatment of mice bearing HT1080 and HT1080/DR4 xenografts with a continuous i.v. infusion of nontoxic doses of d,l-BSO (300 and 600 mg/kg/day) produced a 60% reduction of GSH plasma levels and greater than 95% reduction in GSH tumor levels in both parental and multidrug-resistant tumors; however, this treatment possessed no in vivo antitumor activity by itself. Under these treatment conditions, a combination of d,l-BSO with the maximum tolerated dose of doxorubicin administered at 24 h during a 48-h i.v. infusion of d,l-BSO completely restored the response of MRP-expressing HT1080/DR4 tumors to doxorubicin (overall response rate, 63%; complete response rate, 38%) with no potentiation of host toxicity. The d,l-BSO-induced in vivo reversal of MRP-mediated drug resistance correlated in vitro with the restoration of intracellular doxorubicin retention in cultured HT1080/DR4 cells. Depletion of GSH by d,l-BSO in drug-sensitive HT1080 tumors that do not express MRP did not alter the in vivo response to doxorubicin. Using the same treatment schedule, dose, and administration of doxorubicin with and without d,l-BSO in nude mice bearing P-170 glycoprotein-expressing A2780/Dx5 tumors, no potentiation of the therapeutic index of doxorubicin was found, demonstrating the in vivo selectivity of d, l-BSO-induced GSH depletion on MRP-function. The data reported herein indicate that in vivo function of MRP as a mediator of doxorubicin resistance requires the presence of sufficient GSH pools. d,l-BSO may provide an example of an effective in vivo modulator of MRP-mediated drug resistance.

Multidrug resistance (MDR) is often related to expression of P-glycoprotein (Pgp) or Multidrug Resistance Protein (MRP). Pgp-mediated MDR can be evaluated by determining cellular retention of fluorescent substrates by flow cytometry. This study determined if agents used to evaluate Pgp function also can be used to evaluate MRP function. Cellular retention of doxorubicin (Dox), Rhodamine-123 (Rh-123), and 3,3'-diethyloxacarbocyanine iodide (DiOC2(3)) were studied in MRP-expressing cell lines (HL60/Adr and HT1080/DR4), whereas a Pgp expressing cell line (A2780/Dx5) served as a positive control. Overexpression of Pgp correlated inversely with retention of Dox, Rh-123, and DiOC2(3); however, under identical experimental conditions (1 h reincubation in drug-free medium), no retention difference of the three agents was detected between parental and MRP-expressing resistant cells. Upon extending the reincubation time to 4 h, an efflux of Rh-123 and Dox in the resistant lines became apparent and even more pronounced after 24h; however, still no efflux was detectable for DiOC2(3). Incubation of the cells with a modulator of MDR, PAK-104P, negated the observed drug efflux in Pgp and MRP expressing cells, which correlated with increased sensitivity of the MDR lines to doxorubicin. Thus both Dox and Rh-123 can be used to evaluate MRP-function, but DiOC2(3) can not.

Multidrug resistance (MDR) is considered multifactorial and has been associated with overexpression of the multidrug resistance protein (MRP). However, effective compounds for reversal of MRP-related MDR are limited. In the present study, the modulatory activity of the novel pyridine analogue PAK-104P on MRP-mediated resistance to doxorubicin and paclitaxel was investigated in two doxorubicin-selected human tumor cell lines [HT1080/DR4 (sarcoma) and HL60/ADR (leukemia)] and compared with the nonimmunosuppressive cyclosporine analogue PSC-833. In cell lines HT1080/DR4 (MRP/lung resistance-related protein phenotype) and HL60/ADR (MRP phenotype), doxorubicin resistance was significantly higher (250-fold and 180-fold, respectively) than that to paclitaxel (6-fold and 9-fold, respectively). With noncytotoxic concentrations of PAK-104P (1 and 5 microM), the reversal of doxorubicin resistance was significant but partial in HT1080/DR4 and HL60/ADR cells (dose-modifying factor for 5.0 microM PAK-104P, 25.0 and 31.2, respectively), whereas complete reversal of paclitaxel resistance was achieved in HL60/ADR cells. In contrast, PSC-833 modulation of doxorubicin and paclitaxel resistance was modest. Cellular drug uptake and retention studies by flow cytometry analysis demonstrated that PAK-104P was effective in restoring cellular doxorubicin concentrations in resistant cells to levels comparable to those obtained in parental cells. In athymic nude mice, PAK-104P significantly potentiated the therapeutic efficacy of doxorubicin and paclitaxel against resistant HT1080/DR4 xenografts. Of significance is that the maximum tolerated doses of doxorubicin and paclitaxel were administered in combination with PAK-104P, documenting improvement in the therapeutic index of these agents. In addition to reversing P-glycoprotein-mediated MDR, the pyridine analogue PAK-104P provides an example of an effective in vivo modulator of MRP-mediated MDR.

Summary Background A weekly schedule of a 24-h high-dose 5-FU infusion in combination with folinic acid was investigated as salvage therapy in patients with advanced gastric cancer. Patients and methods Seventeen patients with progressive disease after chemotherapy were treated with a weekly schedule of 500 mg folinic acid/sqm by i.v. 2-h infusion followed by an i.v. 24-h infusion of 2.6 g 5-FU/sqm times six. Fourteen of the 17 patients (82%) had been pretreated in regimens which included 5-FU/folinic acid i.v. bolus. Results Toxicity was mild, with diarrhea, mucositis and nausea; WHO grade m/IV diarrhea was observed in only two patients. Three patients achieved partial remissions (18%; 95% confidence interval: 0%–38%) and 7 patients stable disease (41%; 95% confidence interval: 17%–64%). Their median survival time was five months (range 1.5–10 months) after the onset of salvage therapy. Conclusions This schedule of weekly 24-h infusions of high-dose 5-FU and folinic acid is effective, even in patients pretreated with or resistant to regimens including 5-FU/folinic acid i.v. bolus. Based on these data, it appears that this schedule of high-dose 5-FU/folinic acid in first-line combination chemotherapy merits investigation.

3558 Background: Increased angiogenesis is correlated with poor prognosis in patients with advanced metastatic colorectal cancer (CRC). PTK/ZK is a novel, oral angiogenesis inhibitor that potently inhibits all known VEGF receptor tyrosine kinases, important mediators in the formation of new blood vessels that contribute to tumor growth and metastasis. Methods: This trial determined the MTD and DLTs of once-daily oral PTK/ZK in combination with infusional 5-fluorouracil (5-FU)/leucovorin (LV) plus irinotecan (FOLFIRI) as first-line treatment in patients with metastatic CRC. PTK/ZK was administered orally once daily in escalating doses of 500, 1,000, and 1,250 mg/day to cohorts of 3–7 patients. FOLFIRI was administered q 2 weeks as irinotecan (180 mg/m2, day 1) plus LV (200 mg/m2, 2-hour infusion) and 5-FU (400 mg/m2 bolus followed by 600 mg/m2 as a 22-hour infusion) on days 1 and 2. Results: To date, 16 patients have been enrolled at 500 (n = 6), 1,000 (n = 7), and 1,250 (n = 3) mg/day. PTK/ZK was well tol...

3156 Background: EG cancer is diagnosed in more than 60% of patients (pts) in a locally advanced or metastatic stage. In this situation treatment options are limited; therefore exploration of a new therapeutic regimen is warranted. EGFR is expressed in a high proportion of gastric cancers associated with poor prognosis. Matuzumab, a humanized monoclonal anti-EGFR IgG1 antibody, inhibits ligand-mediated receptor signalling and has demonstrated antitumor activity in solid tumors. In a phase I study we evaluated the safety, tolerability, and pharmacokinetics of matuzumab in combination with standard chemotherapy in pts with advanced EG adenocarcinoma. Methods: Weekly doses of matuzumab (400 mg or 800 mg) were administered in addition to PFL (cisplatin [50 mg/m2] days 1, 15, 29, and leucovorin [500 mg/m2] and 5-fluorouracil [2000 mg/m2] days 1, 8, 15, 22, 29, 36) on a 7-week cycle up to a maximum of 3 cycles. Tumor response was assessed by endoscopy and CT/MRI in the last week of each cycle. Blood samples wer...

4036 Background: The US-Intergroup study 0116 has demonstrated a significant survival benefit for pts receiving adjuvant Rtx/CTx following complete resection of gastric cancer. The current study evaluates the feasibility, toxicity and efficacy of a novel adjuvant combined modality treatment strategy containing a 3–4-drug chemotherapy regimen (CTx) plus 5-FU-based RTx/CTx. Patients and methods: Between 12/2000 and 10/2003 96 pts were included. Treatment consisted of 2 cycles of FA 500 mg/m2 (2h) / 5-FU 2000 mg/m2 (24h) once weekly for 6 consecutive weeks, P 175 mg/m2 (3h) weeks 1 and 4 and DDP 50 mg/m2 (1h) weeks 2 and 5 or 2 cycles of FA 500 mg/m2 (2h) / 5-FU 2000 mg/m2 (24h) once weekly for 6 consecutive weeks with DDP 50 mg/m2 (1h) weeks 1,3,5. The applied regimen was individually chosen by each center. Radiation therapy with 45 Gy plus concomitantly applied 5-FU 225mg/m2/24h was scheduled in between the 2 cycles. Pts with completely resected adenocarcinoma of the stomach including a systematic D1 or D2...

3018 Background: Growth factor signaling through EGFR drives tumor proliferation, motility, angiogenic stimulation, and protects the cells from stress induced apoptosis. EMD 72000 is a humanized monoclonal anti-EGFR IgG1 antibody that blocks ligand binding, EGFR signaling, induces receptor downregulation, and potentially participates in antibody-dependent cell-mediated tumor cell killing. Methods: A phase I study was conducted to assess the safety and anti-tumor activity of EMD 72000 in 24 patients with advanced refractory gastrointestinal tumors. Antitumor activity was assessed using CT and 18FDG PET dual-modality tomography. Subjects were randomized in groups of 6 to receive absolute doses of 100, 200, 400, or 800 mg EMD 72000 weekly for 4 wks followed by treatment with 400 mg per wk until disease progression. EMD 72000 was administered by 1-hr IV infusion without routine premedication. CT and 18FDG PET images were obtained before treatment and in wks 4 and 8. Skin and tumor biopsies were obtained befor...