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Background After promising preclinical studies using a thoracoscopic regional lung chemotherapy technique less morbid than open perfusion methods, we initiated a Phase I clinical study. Methods Four performance status 0 to 1 patients with oligometastatic stage IV lung cancer underwent unilateral thoracoscopic lung suffusion targeting the bulk of primary disease and regional lymph nodes. We used the term suffusion (permeation of an organ) to describe the total lung distribution of chemotherapy afforded by venous distention akin to retrograde cardioplegia physiology. This was obtained by temporary thoracoscopic pulmonary vein occlusions and fluoroscopy-guided transfemoral intravascular balloon occlusion, drainage, and cisplatin distention of the main pulmonary artery. Single-lung ventilation allowed atelectasis that helped to drain the blood under pulmonary artery occlusion, then cisplatin (5% systemic dose) was instilled during venous occlusion and lung reexpansion. Chemotherapy dwelled for 30 minutes before lung reperfusion. Results All four suffusions were successful (three right, one left). Cisplatin remained concentrated in the pulmonary circulation by the end of the dwell (1,124 versus 236 ng/mL systemic). There were no changes in the postsuffusion pulmonary function tests or lung perfusion scans. All patients were discharged early (24 to 48 hours) without chest tubes, began standard chemotherapy without delay, and completed follow-up. After two systemic chemotherapeutic cycles primary tumors had volume reductions of 96%, 88%, 64%, and 14%, with the latter showing a 100% volume increase in a nonsuffused osseous metastasis. Conclusions Our initial clinical experience of thoracoscopic lung suffusion suggests that this approach is safe and merits future study with higher dose levels.

Purpose: To determine the maximal tolerated dose of capecitabine with oxaliplatin + radiotherapy in a phase I study of localized esophageal cancer. Patients and Methods: Oxaliplatin (85 mg/m2) administered on days 1, 15, and 29. Capecitabine administered twice daily 5 days weekly; dose levels (DL) were 1, 1000; 2, 1250; and 3, 1500 mg/m2 with 50.4 Gy radiation. Results: Dose-limiting toxicity was reached at DL 3. Carboxylesterase expression in day 2 tumor specimens and induction correlated with response (p ≤ 0.05). Conclusion: The maximal tolerated dose was 85 mg/m2 of oxaliplatin, 1,250 mg/m2/day of capecitabine, and 50.4 Gy of radiation.

We conducted a phase I study to determine the recommended dose of selenomethionine (SLM) in combination with irinotecan that consistently results in a protective plasma selenium (Se) concentrations15 microM after 1 week of SLM loading.A 3-3 standard escalation design was followed. SLM was given orally twice daily (BID) for one week (loading) followed by continuous once daily (QD) dosing (maintenance). Seven dose levels of selenomethionine were investigated. Irinotecan was given intravenously at a fixed standard weekly dose, starting on the first day of maintenance SLM.Thirty-one patients were treated on study. Dose limiting diarrhea complicated by sepsis was noted in one of six patients at each of the dose-levels 1 and 7. Dose-levelsor = 5 (4,800 mcg/dose loading maintenance) resulted in day 8 Se concentrations15 microM while dose-level 7 (7,200 mcg/dose loading and maintenance) resulted in day 8 Se concentrations20 muM. No significant variations in SN-38 or biliary index were noted between weeks 1 and 4 of treatment. Despite achieving target Se concentrations, gastrointestinal and bone marrow toxicities were common and irinotecan dose modification was prevalent. Objective responses were seen in two patients and nine patients had disease control for 6 months or longer.Selenomethionine can be escalated safely to 7,200 mcg BID x 1 week followed by 7,200 mcg QD in combination with a standard dose of irinotecan. No major protection against irinotecan toxicity was established; however, interesting clinical benefits were noted-supporting the investigation of this combination in future efficacy trials.

This study was designed to understand the basis for the efficacy of methylselenocysteine (MSC) in increasing the therapeutic index of irinotecan against human tumor xenografts. Nude mice bearing human FaDu and A253 tumors were treated orally with different doses of MSC and irinotecan. Plasma, tumor and normal tissue samples were collected at different times after MSC treatments and were analyzed for selenium (Se) concentration using electrothermal atomic absorption spectrophotometry. MSC is highly effective in modulating the therapeutic index of irinotecan in the treatment of human squamous cells carcinoma of the head and neck xenografts (FaDu and A253). Enhanced irinotecan efficacy was greater in FaDu tumors (100% CR) than in A253 tumors (60% CR), and depended on MSC dose with a minimum effective dose of 0.01 mg/d x 28. The highest plasma Se concentration was achieved 1 h after a single dose and 28 d after daily treatments of MSC. The ability of FaDu tumors to retain Se was significantly better than A253 tumors, and the highest Se concentration in normal tissue was achieved in the liver. Peak plasma and tissue Se concentrations were functions of the dose and duration of MSC treatment. The MSC-dependent increase in Se level in normal tissues may contribute to the protective effect against irinotecan toxicity observed in those tissues. Intratumoral total Se concentration was not found to be predictive of the combination therapy response rates. There is a critical need to develop a method to measure the active metabolite of MSC, rather than total Se.

A single cell clonal sub-line A2780/C10B that is 18-fold resistant to oxaliplatin and approximately threefold cross-resistant to cisplatin and exhibiting a metastasis associated cellular phenotype was characterized for mechanisms of resistance. The cell line exhibited a 50% reduction in the accumulation of both oxaliplatin and cisplatin relative to the parent line, while extensive decline in Pt-DNA adduct levels occurred only following oxaliplatin treatment. The basal GSH levels were fivefold higher in A2780/C10B compared to A2780 and had a fivefold elevation in gamma-GT suggesting this may be the mechanism involved in GSH elevation. The basal levels of ERCC-1, XPA and MRP-2 mRNA levels in A2780/C10B were not higher than those in A2780. The highly reduced Pt-DNA adduct formation only for oxaliplatin, but not cisplatin may be a reflection of the fact that at equimolar concentrations oxaliplatin makes fewer Pt-DNA adducts than cisplatin. The data indicate that multiple lesions occur in a single cell to produce the resistant phenotype.

Purpose: We conducted a Phase I study to determine the maximum tolerated dose (MTD) of neoadjuvant capecitabine, oxaliplatin, and radiation therapy (RT) in Stage II to III rectal adenocarcinoma. Methods and Materials: Capecitabine was given orally twice daily Monday through Friday concurrently with RT. Oxaliplatin was given i.v. once weekly × 5 (for 5 weeks) starting the first day of RT. RT was given daily except on weekends and holidays at 1.8 Gy per fraction × 28. Escalation for capecitabine or oxaliplatin was to occur in cohorts of three patients until the maximum tolerated dose (MTD) was defined. Endorectal tumor biopsy samples were obtained before and on Day 3 of treatment to explore the effects of treatment on thymidine phosphorylase, thymidylate synthase, dihydropyrimidine dehydrogenase, DNA repair, and apoptosis. Results: Twelve patients were enrolled on this study. Two of 6 patients at dose level (DL) 1 (capecitabine 825 mg/m 2 orally (p.o.) given twice daily (b.i.d.); oxaliplatin 50 mg/m 2 /week) had a dose-limiting diarrhea. One of 6 patients at DL (−)1 (capecitabine 725 mg/m 2 p.o., b.i.d.; oxaliplatin 50 mg/m 2 /week) experienced-dose-limiting diarrhea. Three of 11 patients who underwent resection had a complete pathologic response. No remarkable variations in rectal tumor biologic endpoints were noted on Day 3 of treatment in comparison to baseline. However, a higher apotosis index was observed at baseline and on Day 3 in complete pathologic responders (no statistical analysis performed). Conclusions: Capecitabine 725 mg/m 2 p.o., twice daily in combination with oxaliplatin 50 mg/m 2 /week and RT 50.4 Gy in 28 fractions is the recommended dose for future studies.

PURPOSE: We conducted a phase I study to determine the maximum tolerated dose (MTD) of irinotecan with fixed, nontoxic high dose of selenomethionine. EXPERIMENTAL DESIGN: Selenomethionine was given orally as a single daily dose containing 2,200 mug of elemental selenium (Se) starting 1 week before the first dose of irinotecan. Irinotecan was given i.v. once weekly x 4 every 6 weeks (one cycle). The starting dose of irinotecan was 125 mg/m(2)/wk. Escalation occurred in cohorts of three patients until the MTD was defined. Pharmacokinetic studies were done for selenium and irinotecan and its metabolites. RESULTS: Three of four evaluable patients at dose level 2 of irinotecan (160 mg/m(2)/wk) had a dose-limiting diarrhea. None of the six evaluable patients at dose level 1 (125 mg/m(2)/wk irinotecan) had a dose-limiting toxicity. One patient with history of irinotecan-refractory colon cancer achieved a partial response. The long half-life of selenium resulted in a prolonged accumulation towards steady-state concentrations. No significant changes in the pharmacokinetics of CPT-11, SN-38, or SN-38G were identified; however, the coadministration of selenomethionine significantly reduced the irinotecan biliary index, which has been associated with gastrointestinal toxicity. CONCLUSIONS: Selenomethionine at 2,200 mug/d did not allow the safe escalation of irinotecan beyond the previously defined MTD of 125 mg/m(2). None of the patients receiving 125 mg/m(2) of irinotecan had grade >2 diarrhea. Unexpected responses and disease stabilizations were noted in a highly refractory population. Further escalation of selenomethionine is recommended in future trials to achieve defined protective serum concentrations of selenium.

Two studies of irinotecan (CPT-11) followed 24 h later by an antimetabolite were conducted. The objectives of the studies were: (1) to determine whether the increase in S-phase in tumor cells seen 24 h after CPT-11 administration in animal studies is seen in advanced solid tumors in patients, (2) to determine the dose of CPT-11 required to produce this effect, (3) to compare two methods (immunohistochemistry, IHC, for cyclin A, and DNA flow cytometry, FC) for evaluating S-phase in tumor biopsies from patients, and (4) to establish the maximum tolerated dose (MTD) and dose-limiting toxicity (DLT) of CPT-11, given 24 h before gemcitabine (GEM, 1000 mg/m(2)). In one study CPT-11 was followed 24 h later by 5-fluorouracil (5-FU), 400 mg/m(2) per week for 4 weeks every 6 weeks. Tumor biopsies were obtained before and 24 h after CPT-11 administration before administration of 5-FU and assayed for S-phase by IHC for cyclin A and by FC. The starting dose of CPT-11 was 80 mg/m(2) per week with subsequent exploration of 40 and 60 mg/m(2) per week to establish the dose-effect relationship of the increase in tumor cells in S-phase. In the second study, CPT-11 was given 24 h before GEM 1000 mg/m(2) per week for 2 weeks every 3 weeks. Doses of 20-80 mg/m(2) were explored to establish the MTD and DLT and to study tumor cell S-phase in selected patients. CPT-11 80 mg/m(2) produced a mean increase in S-phase by IHC for cyclin A of 137%. Lesser increases were seen with 40 and 60 mg/m(2). CPT-11 followed 24 h later by 5-FU 400 mg/m(2) per week for 4 weeks was well tolerated. In the study of CPT-11 followed by GEM 1000 mg/m(2), 60 mg/m(2) of CPT-11 was the MTD.

In this report, electrospray ionization tandem mass spectrometry (ESI-MS/MS) for a pharmacokinetic study of IDN 5390, a novel C-seco taxane derivative, which is under preclinical evaluation, has been investigated. Our results showed that IDN 5390 and other taxanes including paclitaxel and IDN 5109 could ionize well in not only positive-, but also in negative-ion mode. Under collision-induced dissociation (CID) conditions, these compounds could fragment into similar M- (molecular), T- (taxane ring) and S- (side chain) series ions. In positive-ion ESI, the formation of both T- and S-series ions involved the breaking of the C-13 ester bond. In negative-ion ESI, however, while the formation mechanism of S-series ions remained the same, the breaking of the C-1' carboxylic ester bond resulted in T-series ions. At optimum collision energy (CE) values, M-, T- and S-series ions of IDN 5390 in both positive- and negative-ion ESI-MS/MS spectra had good intensity. This phenomenon makes both positive- and negative-ion ESI-MS/MS good methods for IDN 5390 metabolite structural characterization, i.e. to reveal the location of modification groups in IDN 5390 metabolites versus IDN 5390 either on the side chain or the taxane ring. A liquid chromatography (LC)/ESI-MS/MS method using the multiple-reaction monitoring (MRM) technique was thereafter developed to quantify IDN 5390 in dog plasma using paclitaxel as internal standard. The method was validated using a concentration range between 5 and 1000 ng/mL and had a limit of detection of 1 ng/mL. The inter-day %CV (%coefficient of variation) of the calibration standards ranged between 4.36 and 9.64%, the intra-day %CV of the calibration standards between 0.61 and 13.44%, and the mean % accuracy of the quality control samples at the low, middle and high end of the concentration curves were 12.5, 6.8 and 9.6%, respectively.

A great deal of experimental evidence connects induction of polyamine catabolism via spermidine/spermine N1-acetyltransferase (SSAT) to antiproliferative activity and apoptosis. Following our initial observation from gene expression profiling that platinum drugs induce SSAT, we undertook this present study to characterize platinum drug induction of SSAT and other polyamine catabolic enzymes and to examine how these responses might be enhanced with the well-known inducer of SSAT and clinically relevant polyamine analogue, N1,N11-diethylnorspermine (DENSPM). The results obtained in A2780 ovarian cancer cells by real-time quantitative RT-PCR and Northern blot analysis show that a 2-hour exposure of A2780 cells to platinum drugs induces expression of SSAT, a second SSAT (SSAT-2), spermine oxidase, and polyamine oxidase in a dose-dependent manner. At equitoxic doses, oxaliplatin is more effective than cisplatin in SSAT induction. The most affected enzyme, SSAT, increased 15-fold in mRNA expression and 2-fold in enzyme activity. When combined with DENSPM to further induce SSAT and to enhance conversion of mRNA to activity, oxaliplatin increased SSAT mRNA 50-fold and activity, 210-fold. Polyamine pools declined in rough proportion to levels of SSAT induction. At pharmacologically relevant oxaliplatin exposure times (20 hours) and drug concentrations (5 to 15 μmol/L), these responses were increased even further. Combining low-dose DENSPM with oxaliplatin produced a greater than additive inhibition of cell growth based on the sulforhodamine-B assay. Taken together, the findings confirm potent induction of polyamine catabolic enzymes, such as SSAT by platinum drugs, and demonstrate that these biochemical responses as well as growth inhibition can be potentiated by co-treatment with the polyamine analogue DENSPM. With appropriate in vitro and in vivo optimization, these findings could lead to clinically relevant therapeutic strategies.

Purpose: The pipecolinate derivative VX-710 (biricodar; Incel) is a clinically applicable modulator of P-glycoprotein (Pgp) and multidrug resistance protein (MRP-1); we studied its activity against the third multidrug resistance (MDR)-associated drug efflux protein, breast cancer resistance protein (BCRP). Experimental Design: VX-710 modulation of uptake, retention, and cytotoxicity of mitoxantrone, daunorubicin, doxorubicin, topotecan, and SN38 was studied in cell lines overexpressing Pgp, MRP-1 and wild-type (BCRPR482) and mutant (BCRPR482T) BCRP. Results: In 8226/Dox6 cells (Pgp), VX-710 increased mitoxantrone and daunorubicin uptake by 55 and 100%, respectively, increased their retention by 100 and 60%, respectively, and increased their cytotoxicity 3.1- and 6.9-fold, respectively. In HL60/Adr cells (MRP-1), VX-710 increased mitoxantrone and daunorubicin uptake by 43 and 130%, increased their retention by 90 and 60%, and increased their cytotoxicity 2.4- and 3.3-fold. In 8226/MR20 cells (BCRPR482), VX-710 increased mitoxantrone uptake and retention by 60 and 40%, respectively, and increased cytotoxicity 2.4-fold. VX-710 increased daunorubicin uptake and retention by only 10% in 8226/MR20 cells, consistent with the fact that daunorubicin is not a substrate for BCRPR482, but, nevertheless, it increased daunorubicin cytotoxicity 3.6-fold, and this increase was not associated with intracellular drug redistribution. VX-710 had little effect on uptake, retention, or cytotoxicity of mitoxantrone, daunorubicin, doxorubicin, topotecan, or SN38 in MCF7 AdVP3000 cells (BCRPR482T). Conclusions: VX-710 modulates Pgp, MRP-1, and BCRPR482, and has potential as a clinical broad-spectrum MDR modulator in malignancies such as the acute leukemias in which these proteins are expressed.

Cellular glutathione (GSH) levels are related to the resistance of tumor cells to platinum and alkylating agents, and depletion of GSH may enhance the activity of these drugs. The pharmacodynamic effects of mesna on depleting plasma cysteine, a GSH precursor, were evaluated in 22 patients as part of a Phase I study. Escalating doses of ifosfamide and mesna were administered; carboplatin was administered to achieve an AUC of 4 mg x min/mL. Plasma samples were collected and assayed by reverse-phase high-performance liquid chromatography (HPLC) for total mesna and total cysteine concentrations at 0, 1, 3, 6, 24, 25, 28, and 48 hours. A one-compartment pharmacokinetic model was fit to the mesna plasma concentrations, using M.A.P. Bayesian estimation (ADAPT II). Pharmacodynamics were evaluated by fitting an inhibitory Emax model to the cysteine concentration data. Both the pharmacokinetic (median R2 = 0.95; range = 0.85-0.98) and pharmacodynamic (median R2 = 0.96; range = 0.74-1.0) models fit the data well. Mean (coefficient of variation [CV%]) mesna pharmacokinetic parameter estimates were as follows: Vss of 15.3 (29) L/m2, CL of 4.6 (29) L/h/m2, and half-life of 2.2 (37) hours. Mean (CV%) pharmacodynamic parameter estimates were as follows: Emax of 31.7 (19) microg/mL and EC50 of 10.3 (52) microg/mL. Mesna produced a rapid, concentration-dependent reduction in plasma cysteine concentrations that could be adequately characterized by an inhibitory Emax pharmacodynamic model. The depletion of plasma cysteine was facilitated by ifosfamide, suggesting a pharmacodynamic interaction between these two agents. Further increases in mesna doses beyond those administered in this study would be unlikely to provide additional benefit.

This article gives a brief discussion of "state-of-the-art" therapeutic strategies for esophageal and gastroesophageal junction tumors, with an emphasis on combined-modality therapy. In addition, we review the results of a multimodality trial conducted using a new agent against esophageal cancer, oxaliplatin. In this trial, the emphasis was on the efficacy and toxicity of oxaliplatin in combination with protracted infusion 5-fluorouracil and radiation. A secondary endpoint of the study was the relationship between efficacy and toxicity to specific intratumoral gene expressions within the primary esophageal tumor.

Patients with stage IIIB breast carcinoma represent only a small proportion of women with breast cancer in western countries but may constitute up to 50% of cases in underdeveloped countries. The prognosis remains poor despite aggressive treatment. Nineteen patients (11 with inflammatory breast carcinoma) received at least three courses of neoadjuvant chemotherapy of methotrexate, vinblastine, adriamycin, cisplatin, and folinic acid (MVAC/FA) followed by mastectomy. Six months of cyclophosphamide, methotrexate, and 5-fluorouracil were given after surgery. Radiation therapy followed chemotherapy. Seventy percent of patients achieved complete and 14% partial response afrer MVAC/FA chemotherapy alone. Eleven patients (58%) survived 5 years, and 30% survived at least 8 years. The addition of cisplatin in combination chemotherapy used as first-line treatment for stage IIIB breast carcinoma was well tolerated, resulted in higher response rates, and appeared to have an effect on overall survival.

Paclitaxel and carboplatin have nonoverlapping toxicities with a broad range of clinical activity. The combination of escalating dose paclitaxel and carboplatin dosed to a fixed area under the curve (AUC) was explored in a series of phase I studies. 76 patients were treated with paclitaxel over three hours followed by a 30 min carboplatin infusion, dosed by the Calvert formula to a target AUC of 4.0 or 4.5 mg/min/ml −1 . The maximum tolerated dose of paclitaxel was 270 to 290 mg/m 2 , with a dose limiting toxicity of peripheral sensory neuropathy. Activity was seen in lung cancer, with a paclitaxel dose at or above 230 mg/m 2 . Neuropathy correlated with paclitaxel AUC due to nonlinear pharmacokinetics at higher doses. Ongoing studies include the use of amifostine as a neuroprotectant and phase II studies of the paclitaxel/carboplatin regimen in head and neck cancer, small cell lung cancer and sarcomas.

Purpose: To establish the maximum tolerated dose (MTD), dose-limiting and other major toxicities and the major pharmacokinetic parameters of a 10-day infusion of the nonclassical antifolate ThymitaqTM. Methods: The drug was given by 10-day infusion via a portable pump. The starting dose was 286 mg/m2 per day with escalation to 572 and 716 mg/m2 per day. Thymitaq in plasma was assayed by a validated isocratic reverse-phase HPLC assay with detection at 273 nm. Results: The dose of 716 mg/m2 per day × 10 was considered too high as none of three patients completed a 10-day infusion and two of three developed grade IV myelotoxicity. At 572 mg/m2 per day three of four patients completed a 10-day infusion. Dose-limiting myelosuppression was seen in one of four but owing to a high incidence of thrombotic phenomena, no further patients were added. Conclusion: Continuous 10-day infusions of Thymitaq should be limited to low doses until further studies can be done.

PURPOSE Etoposide has schedule-dependent cytotoxic activity, and clinical resistance may be overcome with prolonged low-dose therapy. Oral bioavailability is variable, and protracted intravenous administration is limited by water insolubility, which requires large infusion volumes. Etoposide phosphate (EP) is a water-soluble prodrug that is rapidly converted in vivo to etoposide, and may be administered in concentrated solution. A phase I study was conducted to determine the toxicity, pharmacokinetics, and pharmacodynamics of EP administered as a protracted venous infusion in the ambulatory setting. METHODS Twenty-three patients with advanced cancer were treated with a continuous infusion of EP using ambulatory pumps for 6 weeks followed by a 2-week rest. Cohorts were treated with EP at 10, 20, 25, and 30 mg/m2/d. Steady-state plasma etoposide levels (Css) and stability of EP in infusion pumps were measured using high performance liquid chromatography (HPLC). RESULTS Myelosuppression, mucositis, and fatigue were dose-limiting. The maximum-tolerated dose (MTD) of EP was 20 mg/m2/d. The mean Css (+/- SD) of etoposide were 0.67 +/- 0.25, 1.14 +/- 0.24, 1.38 +/- 0.64, and 2.19 +/- 0.52 microg/mL at daily EP doses of 10, 20, 25, and 30 mg/m2, respectively. Neutropenia correlated with Css (r = 0.65, P = .008). EP was stable in infusion pumps for at least 7 days. Partial responses were observed in patients with hepatoma and non-small-cell lung cancer (one each). CONCLUSION EP may be conveniently and safely administered as a low-volume protracted venous infusion in the ambulatory setting. Cytotoxic plasma concentrations of etoposide are obtained at the MTD. The pharmacodynamic relationships observed suggest the possibility of pharmacologically based dosing of EP.

Elevation of glutathione (GSH) is commonly observed in cellular resistance to a number of anticancer agents. Most frequently reported change in GSH metabolism that is associated with the elevated GSH levels is increased mRNA expression and activity of gamma-glutamyl cysteine synthetase (gamma GCS), the first enzyme of the GSH biosynthetic pathway. We have isolated sublines of the A2780 ovarian carcinoma cell line (C10 and C25) that are 8- and 12-fold resistant to oxaliplatin by repeatedly exposing the cells to increasing concentrations of the platinum agent. The GSH levels in C10 and C25 cell sublines are 3.1- and 3.8-fold higher than the parent A2780 cell line. The mRNA levels and activities for gamma GCS and that for gamma-glutamyl transpeptidase (gamma GT), the GSH salvage pathway enzyme, were measured in these cells. The mRNA for gamma GT and gamma GCS were measured by RT-PCR, with quantitation of the PCR product by HPLC; mRNA levels are expressed as ratios to beta-actin mRNA, used as an endogenous standard. GSH and gamma GCS activity were measured by HPLC assays and gamma GT activity by a colorimetric assay. The increase in GSH in C10 and C25 was associated with an elevation in gamma GT mRNA (2.5- and 8-fold) and gamma GT activity (2.7- and 2.8-fold). No changes were observed in gamma GCS mRNA levels or activity. The data indicate that alterations in GSH metabolism leading to elevations in cellular GSH in A2780 ovarian carcinoma cells selected for low levels of resistance to oxaliplatin are mediated by gamma GT, the "salvage' pathway, rather than an increase in GSH biosynthesis.

Oxaliplatin (trans-l-1,2-diaminocyclohexane oxalato Pt(II); 1R,2R-dach, l-OHP), its trans-d isomer (1S,2S-dach) and cis-dach (1R,2S-dach) isomers were compared in in vitro testing against human ovarian carcinoma cell lines A2780, A2780/CP (cisplatin resistant), A2780/l-OHP (oxaliplatin resistant), colon carcinoma cell line HT-29, and murine leukemia cell lines L1210, L1210/CP (cisplatin resistant), and L1210/dach (tetraplatin resistant). The relative molar potency of the three complexes in all the cell lines except A2780/l-OHP and L1210/dach are trans-l > trans-d > cis-dach; in A2780/l-OHP they are trans-l = trans-d > cis-dach; in L1210/dach trans-l > trans-d = cis-dach. The A2780/l-OHP selected for trans-l resistance is 3.6-fold resistant to oxaliplatin, showed no resistance to trans-d isomer and is 6-fold resistant to cis-dach. However, L1210/dach which is selected for carboxyphthalato 1,2-dach (trans-dl) platinum(II) is 140-fold resistant to oxaliplatin, 73-fold resistant to trans-d, and 41-fold resistant to cis-OHP. The accumulation and DNA binding of platinum following a 2-h treatment of A2780 cells with each of the isomers (60 μM) is in the order of trans-l > cis-dach > trans-d which corresponded to the cytotoxicity of trans-l, but not the others. The data suggest that other processes, such as differential formation of specific adducts and/or repair may be involved. Of the three isomers l-OHP is the superior and its accumulation and DNA binding are consistent with its cytotoxicity.

Although there have been a number of reports correlating cellular GSH levels with cytotoxicity of platinum agents, none has examined the relationship between GSH concentrations and cytotoxicity. In this study, using a highly specific HPLC method for measuring GSH and expressing GSH as concentration and also per cell number, we evaluated the correlation between GSH levels and the cytotoxicity to five agents in ten human tumor cell lines. The five platinum agents included the platinum(II) complexes cisplatin, carboplatin and oxaliplatin and platinum(IV) complexes iproplatin and tetraplatin. The correlation between intracellular GSH concentration and cytotoxicity was highly significant only for iproplatin (P = 0.002) followed by tetraplatin, which demonstrated a trend toward statistical significance (P = 0.06). Cytotoxicity of the other platinum complexes showed no relation to GSH concentration, cisplatin itself showing a P-value of 0.09. In contrast, the GSH levels normalized to cell number showed a statistically significant correlation with the cytotoxicity of four of the five platinum agents, the exception being carboplatin; the strongest correlation observed was that for iproplatin and tetraplatin. Glutathione-S-transferase (GST) activity in these cell lines showed no correlation with cytotoxicity of any of the platinum complexes. Our results, from the analyses of both GSH concentration as well as GSH per cell number, suggest a significantly higher interaction between GSH and iproplatin compared with the other platinum agents. Moreover, our data suggest that relationships between cytotoxicity and GSH levels on a per-cell basis may not persist when differences in cell volume are taken into account.

We have previously showed that platinum drugs up-regulate SSAT and SMO and down-regulate ODC and SAMDC in the polyamine pathway. Several studies including our own established that platinum drugs combined with polyamine analog DENSPM produces synergistic increase in SSAT activity with polyamine depletion. Since polyamine pathway is an important therapeutic target, we investigated whether agents containing both platinum and polyamines have similar effects on the polyamine pathway. Two complexes i) Pt-spermine with two cisplatin molecules linked to a spermine in the center and ii) Pd-spermine with similar structure i, but Pd (II) substituted for Pt (II) were analyzed with respect to their effect on the expression of genes in polyamine pathway, SSAT and SMO protein expression, SSAT activity and polyamine pools. Pt-, Pd-spermine complexes induced significant down-regulation of SMO, arginase 2 and NRF-2, with no change in SSAT, while cisplatin as a single agent or in combination with DENSPM induced significant up-regulation of SSAT and SMO. The SSAT activity was not induced by either Pt- or Pd-spermine in A2780 cells; SMO protein levels were significantly elevated compared to the no-drug control and to a similar extent as cisplatin/DENSPM. The Pd-spm treatment induced a fall in putrescine levels to 33%, spermidine to 62% and spermine to 72% while Pt-spm did not induce such a decline. Comparative cytotoxicity studies in A2780 cells indicated the potency to be cisplatinPd-SpmPt-Spm. Although both complexes exhibit a lower potency, the degree of resistance itself is much lower for Pt-spermine and Pd-spermine in that order (2.5 and 7.5, respectively) compared to cisplatin ( approximately 12) as tested in cisplatin resistant A2780/CP cells. These studies suggest that Pd (II)-polyamine complexes may constitute a promising group of inorganic compounds for further studies in the development of novel chemotherapy/adjuvant chemotherapy strategies.

Purpose: We conducted a phase I study to determine the maximum tolerated dose of vorinostat in combination with fixed doses of 5-fluorouracil (FU), leucovorin, and oxaliplatin (FOLFOX). Experimental Design: Vorinostat was given orally twice daily for 1 week every 2 weeks. FOLFOX was given on days 4 and 5 of vorinostat. The vorinostat starting dose was 100 mg twice daily. Escalation occurred in cohorts of three to six patients. Pharmacokinetics of vorinostat, FU, and oxaliplatin were studied. Results: Twenty-one patients were enrolled. Thrombocytopenia, neutropenia, gastrointestinal toxicities, and fatigue increased in frequency and severity at higher dose levels of vorinostat. Two of 4 evaluable patients at dose level 4 (vorinostat 400 mg orally twice daily) developed dose-limiting fatigue. One of 10 evaluable patients at dose level 3 (vorinostat 300 mg orally twice daily) had dose-limiting fatigue, anorexia, and dehydration. There were significant relationships between vorinostat dose and the area under the curve on days 1 and 5 (Pearson, < 0.001). The vorinostat area under the curve increased (P = 0.005) and clearance decreased (P = 0.003) on day 5 compared with day 1. The median Cmax of FU at each dose level increased significantly with increasing doses of vorinostat, suggesting a pharmacokinetic interaction between FU and vorinostat. Vorinostat-induced thymidylate synthase (TS) modulation was not consistent; only two of six patients had a decrease in intratumoral TS expression by reverse transcription-PCR. Conclusions: The maximum tolerated dose of vorinostat in combination with FOLFOX is 300 mg orally twice daily × 1 week every 2 weeks. Alternative vorinostat dosing schedules may be needed for optimal down-regulation of TS expression.

We found previously that inactivation of the FCY2 gene, encoding a purine-cytosine permease, or the HPT1 gene, encoding the hypoxanthine guanine phosphoribosyl transferase, enhances cisplatin resistance in yeast cells. Here, we report that in addition to fcy2Delta and hpt1Delta mutants in the salvage pathway of purine nucleotide biosynthesis, mutants in the de novo pathway that disable the feedback inhibition of AMP and GMP biosynthesis also enhanced cisplatin resistance. An activity-enhancing mutant of the ADE4 gene, which constitutively synthesizes AMP and excretes hypoxanthine, and a GMP kinase mutant (guk1), which accumulates GMP and feedback inhibits Hpt1 function, both enhanced resistance to cisplatin. In addition, overexpression of the ADE4 gene in wild-type cells, which increases de novo synthesis of purine nucleotides, also resulted in elevated cisplatin resistance. Cisplatin cytotoxicity in wild-type cells was abolished by low concentration of extracellular purines (adenine, hypoxanthine, and guanine) but not cytosine. Inhibition of cytotoxicity by exogenous adenine was accompanied by a reduction of DNA-bound cisplatin in wild-type cells. As a membrane permease, Fcy2 may mediate limited cisplatin transport because cisplatin accumulation in whole cells was slightly affected in the fcy2Delta mutant. However, the fcy2Delta mutant had a greater effect on the amount of DNA-bound cisplatin, which decreased to 50 to 60% of that in the wild-type cells. Taken together, our results indicate that dysregulation of the purine nucleotide biosynthesis pathways and the addition of exogenous purines can modulate cisplatin cytotoxicity in Saccharomyces cerevisiae.

Heat shock protein 60 (HSP60) participates in protein assembly, folding and transport. Increased HSP60 mRNA expression is associated with cisplatin resistance in some in vitro models and with shorter survival among ovarian cancer patients. HSP60 mRNA expression was quantitated in three independent model systems by reverse-transcription PCR (A2780 human ovarian carcinoma cell line and sublines selected for cisplatin or oxaliplatin resistance in vitro and also in the UCRU-BL13 human bladder carcinoma cell line and a cisplatin-resistant subline). Increased HSP60 mRNA expression was observed in all resistant sublines (range 2.5-15 fold), correlated with relative resistance to cisplatin and oxaliplatin. No differences in HSP60 gene copy number were apparent in resistant sublines. These data provide further evidence of a strong association between in vitro resistance to platinum compounds and increased HSP60 mRNA expression.

Purpose To evaluate the efficacy of a combination of capecitabine, oxaliplatin, and radiotherapy (RT) in the neoadjuvant treatment of Stage II and III rectal cancers. Methods Capecitabine was given at 725 mg/m 2 orally twice daily Monday through Friday concurrently with RT. Oxaliplatin was given intravenously at 50 mg/m 2 once weekly five times starting the first day of RT. The radiation dose was 50.4 Gy in 28 fractions (1.8 Gy/fraction), five fractions weekly. Endorectal tumor biopsies were obtained before treatment and on the third day of treatment to explore the effects of treatment on thymidine phosphorylase, thymidylate synthase, excision repair cross-complementing rodent repair deficiency complementation group 1 (ERCC1), and apoptosis. Results A total of 25 patients were enrolled in this study; 6 patients (24%) had a complete pathologic response. T-downstaging occurred in 52% of patients, and N-downstaging occurred in 53%. Grade 3 diarrhea was the most common Grade 3-4 toxicity, occurring in 20% of patients. Only 2 patients experienced disease recurrence, with a median of 20 months of follow-up. Thymidylate synthase, thymidine phosphorylase, ERCC1, and apoptosis did not vary significantly between the pretreatment and Day 3 tumor biopsies, nor did they predict for T-downstaging or a complete pathologic response. Conclusion Capecitabine at 725 mg/m 2 orally twice daily, oxaliplatin 50 mg/m 2 /wk, and RT at 50.4 Gy is an effective neoadjuvant combination for Stage II and III rectal cancer and results in a greater rate of complete pathologic responses than historically shown in fluoropyrimidine plus RT controls.

Our previous studies showed that combined treatment of oxaliplatin and N(1), N(11) diethyl-norspermine (DENSPM) results in massive induction of spermidine/spermine N(1)-acetyltransferase (SSAT) mRNA and activity. Since oxaliplatin and 5-fluorouracil (5FU) are used clinically in treatment of colorectal cancers, this study examines the effect of adding DENSPM to oxaliplatin/5FU combination on SSAT and spermine oxidase (SMO) in HCT-116 cells.HCT-116 cells were treated with clinically relevant concentrations of drugs for 20 h followed by 24 h in drug free medium. SSAT and SMO mRNA and protein were assayed by QRT-PCR and Westerns respectively; polyamine pools were measured by HPLC. SSAT and SMO mRNA in tumor biopsies from patients with rectal cancer receiving oxaliplatin, capecitabine and radiation were measured by QRT-PCR.Oxaliplatin + 5FU + DENSPM produced significantly higher levels of SSAT and SMO mRNA, protein and activity than those seen with oxaliplatin+5FU with a significant depletion of cellular spermine and spermidine pools. Oxaliplatin/DENSPM was superior to 5FU/DENSPM in SSAT induction but similar for SMO. Oxaliplatin + DENSPM revealed synergistic growth inhibition atIC(50) concentrations and antagonism atIC(50). SMO and SSAT induction occurred in 60 and 30% of the patient samples examined.These studies demonstrated that combining DENSPM with oxaliplatin + 5FU provides an added benefit by aiming at the clinically relevant therapeutic target, the polyamine catabolism. Further, we show for the first time, that SMO and SSAT induction could be measured in tumor biopsies in patients receiving chemo-radiation. Optimization of treatment conditions in vivo should facilitate a clinical evaluation of the three drug combination.

Human carbonyl reductase 1 (CBR1) metabolizes endogenous and xenobiotic substrates such as the fever mediator, prostaglandin E2 (PGE2), and the anticancer anthracycline drug, daunorubicin. We screened 33 CBR1 full-length cDNA samples from white and black liver donors and performed database analyses to identify genetic determinants of CBR1 activity. We pinpointed a single nucleotide polymorphism on CBR1 (CBR1 V88I) that encodes for a valine-to-isoleucine substitution for further characterization. We detected the CBR1 V88I polymorphism in DNA samples from individuals with African ancestry (p = 0.986, q = 0.014). Kinetic studies revealed that the CBR1 V88 and CBR1 I88 isoforms have different maximal velocities for daunorubicin (V(max) CBR1 V88, 181 +/- 13 versus V(max) CBR1 I88, 121 +/- 12 nmol/min . mg, p0.05) and PGE2 (V(max) CBR1 V88, 53 +/- 7 versus V(max) CBR1 I88, 35 +/- 4 nmol/min . mg, p0.01). Concomitantly, CBR1 V88 produced higher levels of the cardiotoxic metabolite daunorubicinol compared with CBR1 I88 (1.7-fold, p0.0001). Inhibition studies demonstrated that CBR1 V88 and CBR1 I88 are distinctively inhibited by the flavonoid, rutin (IC50 CBR1 V88, 54.0 +/- 0.4 microM versus IC50 CBR1 I88, 15.0 +/- 0.1 microM, p0.001). Furthermore, isothermal titration calorimetry analyses together with molecular modeling studies showed that CBR1 V88I results in CBR1 isoforms with different binding affinities for the cofactor NADPH (K(d) CBR1 V88, 6.3 +/- 0.6 microM versus K(d) CBR1 I88, 3.8 +/- 0.5 microM). These studies characterize the first functional genetic determinant of CBR1 activity toward relevant physiological and pharmacological substrates.

Purpose: Chemotherapy-induced diarrhea occurs secondary to mucosal inflammation and may be cyclooxygenase-2 mediated. Cyclooxygenase-2 inhibitors may ameliorate chemotherapy-induced mucosal toxicity and enhance its antitumor effect. We investigated this hypothesis in the Ward colorectal cancer rat model and in a phase I clinical study. Experimental Design: In the Ward rat model, irinotecan was given daily × 3 or weekly × 4 with or without celecoxib. In the phase I clinical study, we planned to escalate the dose of irinotecan in the FOLFIRI regimen (irinotecan, 5-fluorouracil, and leucovorin) with a fixed dose of celecoxib. Irinotecan was escalated in four dose levels: 180, 200, 220, and 260 mg/m2. Celecoxib was administered as 400 mg, twice daily starting on day 2 of cycle 1. Pharmacokinetics of irinotecan, SN-38, and SN-38G were obtained on days 1 and 14. A standard 3 + 3 dose escalation scheme was used. Plasma concentrations of irinotecan, SN-38, and SN-38G were measured using high-pressure liquid chromatography. Results: Celecoxib ameliorated diarrhea, weight loss, and lethality and resulted in synergistic antitumor effect in the rat model. Twelve patients with advanced cancers were enrolled and evaluable for dose-limiting toxicity (DLT). Diarrhea was the cause for discontinuation in one. Grade 2 and 3 diarrhea occurred in three and two patients, respectively. One patient had DLT at dose level 2 (grade 3 diarrhea). Two had a DLT at DL3 (G3 emesis and myocardial infarct). Celecoxib had limited influence on the pharmacokinetics of irinotecan in this data set. Conclusions: Maximum tolerated dose of irinotecan in FOLFIRI schedule with celecoxib is 200 mg/m2.

As a follow-up to our previous findings that platinum drugs induce a key enzyme in polyamine catabolism, gene expression profiling and mathematical modeling were used to define the effects of cisplatin and oxaliplatin on the expression of polyamine metabolic pathway genes in A2780 human ovarian carcinoma cells.Time-course and concentration-effect experiments were each carried out with cisplatin or oxaliplatin in two separate experiments and cells subjected to gene expression profiling using Affymetrix array technology. Time-course data were modeled using exponential increase and decrease models. Concentration-effect data were modeled using a four parameter Hill model.Gene expression profiling of human ovarian carcinoma A2780 cells after exposure to either cisplatin or oxaliplatin indicates that the expression of several genes involved in polyamine pathway is affected by the platinum drugs. Mathematical/Statistical modeling of the data from time-course and concentration-effect experiments of gene expression from nine polyamine pathway genes represented on the HGU95Av2 chip, indicates that three biosynthetic pathway genes (SAMDC, ODC1 and SRM) are down-regulated and one catabolic pathway gene (SSAT) is up-regulated. Expression changes were similar for different probesets for a given gene on the array. Studies on the induction of SSAT by platinum drugs suggested by the Affymetrix data have been previously validated from this laboratory (Hector et al. in Mol Cancer Ther 3:813-822, 2004). Here, the effects of oxaliplatin exposure on SAMDC and ODC observed by Affymetix are validated with real time QRT-PCR.The data indicate a concerted effect of platinum drugs on the polyamine metabolic pathway with down-regulation in the expression of several enzyme genes involved in biosynthesis and many-fold up-regulation in expression of SSAT, an acetylating enzyme gene that is critically involved in polyamine catabolism and export.

The efficacy of platinum drugs in the treatment of cancer is often restricted by the acquisition of tumor cell resistance subsequent to treatment. To better understand mechanisms involved in this phenomenon, a clonal subline (A2780/C10B) isolated from an oxaliplatin-resistant human ovarian carcinoma cell line (A2780/C10) was developed, as reported previously. This cell line is 18-fold resistant to oxaliplatin and shows a 3-fold cross resistance to cisplatin. Here, we report on the gene expression analysis using Affymetrix HG-U95Av2 oligonucleotide arrays of cells in log phase growth from both the parental cell line and drug-resistant variant. Probe level analysis was perfomed using the model based expression index (dChip) and robust multichip average (RMA) methods. Genes that were differentially expressed between the two groups were identified using the significance analysis of microarrays (SAM) method with a minimum false discovery rate

Objective: To study irinotecan (CPT-11)–induced changes in expression profiles of genes associated with cell cycle control and apoptosis in myeloid leukemia cells in vitro and in vivo. Methods: HL60 cells were exposed to clinically achievable concentrations of 7-ethyl-10-hydroxycamptothecin (SN-38), the active metabolite of CPT-11, and blood sampled from patients with acute myeloid leukemia and chronic myeloid leukemia in myeloid blast transformation treated with CPT-11. Gene expression changes were studied by cDNA microarray and correlated with biological responses by studying DNA distributions by flow cytometry. Results: cDNA microarray analysis showed down-regulation and up-regulation of specific cell cycle–associated genes, consistent with loss of S-phase cells and temporary delay of G1-S-phase transition seen by flow cytometry. Flow cytometry showed that cells in S phase during SN-38 exposure underwent apoptosis, whereas cells in G2-M and G1 were delayed in G1 and entered S phase only 6 to 8 hours after drug removal, consistent with the observed changes in gene expression. Proapoptotic changes in gene transcription included down-regulation of antiapoptotic genes and up-regulation of proapoptotic genes. Many gene expression changes observed following in vitro SN-38 exposure were also seen following in vivo administration of 10 or 15 mg/m2 CPT-11; notably, proapoptotic changes included reduced transcription of survivin pathway-associated genes and increased transcription of death receptor 5. Conclusion: CPT-11-induced changes in gene expression profiles in vitro and in vivo are consistent with temporary delay in G1-S transition and enhanced responsiveness to apoptosis, both of which may contribute to the synergistic interactions of this drug with antimetabolites.

The combination of methylselenocysteine and irinotecan (CPT-11) is synergistic against FaDu and A253 xenografts. Methylselenocysteine/CPT-11 increased tumor cure rate to 100% in FaDu and to 60% in A253. In this study, the effect of methylselenocysteine on pharmacokinetic and pharmacogenetic profiles of genes relevant to CPT-11 metabolic pathway was evaluated to identify possible mechanisms associated with the observed combinational synergy. Nude mice bearing tumors (FaDu and A253) were treated with methylselenocysteine, CPT-11, and a combination of methylselenocysteine/CPT-11. Samples were collected and analyzed for plasma and intratumor concentration of CPT-11 and 7-ethyl-10-hydroxyl-camptothecin (SN-38) by high-performance liquid chromatography. The intratumor relative expression of genes related to the CPT-11 metabolic pathway was measured by real-time PCR. After methylselenocysteine treatment, the intratumor area under the concentration-time curve of SN-38 increased to a significantly higher level in A253 than in FaDu and was associated with increased expression of CES1 in both tumors. Methylselenocysteine/CPT-11 treatment, compared with CPT-11 alone, resulted in a significant decrease in levels of ABCC1 and DRG1 in FaDu tumors and an increase in levels of CYP3A5 and TNFSF6 in A253 tumors. No statistically significant changes induced by methylselenocysteine/CPT-11 were observed in the levels of other investigated variables. In conclusion, the significant increase in the cure rate after methylselenocysteine/CPT-11 could be related to increased drug delivery into both tumors (CES1), reduced resistance to SN-38 (ABCC1 and DRG1) in FaDu, and induced Fas ligand apoptosis (TNFSF6) in A253. No correlation was observed between cure rate and other investigated variables (transporters, degradation enzymes, DNA repair, and cell survival/death genes) in either tumor.

Purpose: Based on reported synergy of the topoisomerase-I (topo-I) inhibitor irinotecan with antimetabolites, irinotecan and cytarabine (Ara-C) were administered sequentially to patients with acute myeloid leukemia (AML) refractory to or relapsed following high-dose Ara-C and anthracycline therapy. Pharmacokinetic and pharmacodynamic studies were performed with the first irinotecan dose. Experimental Design: In vitro synergy of irinotecan followed by Ara-C was confirmed in a human AML cell line as a basis for the clinical trial. Irinotecan was administered daily for 5 days, with Ara-C 1 g/m2 12 h after each irinotecan dose. Irinotecan was initiated at 5 mg/m2, and the dose was escalated by 5 mg/m2 increments in cohorts of three patients and in individual patients. Pre-treatment samples were studied for topo-I activity and serial samples after the first irinotecan dose were analyzed for pharmacokinetics and for pharmacodynamic effects, including DNA damage and DNA synthesis rate. Results: The irinotecan dose reached 15 mg/m2 in three-patient cohorts without reaching the maximum tolerated dose, and reached 30 mg/m2 in individual patients. The AUC and Cmax of both irinotecan and its active metabolite SN38 increased linearly in proportion to dose, and the mean half-lives of irinotecan conversion to SN38 and SN38 elimination were 6.2 h (CV 171%) and 7.2 h (CV 48%). Irinotecan rapidly induced DNA damage, and DNA synthesis inhibition varied among patients and treatment cycles. All courses resulted in rapid cytoreduction, and two patients achieved complete remission. Topo-I activity did not predict response. Conclusion: Irinotecan can be safely administered with Ara-C. This combination is active in refractory AML and warrants further study.

Cisplatin, a widely used chemotherapeutic agent, has been implicated in the induction of secondary tumors in cancer patients. This drug is presumed to be mutagenic because of error-prone translesion synthesis of cisplatin adducts in DNA. Oxaliplatin is effective in cisplatin-resistant tumors, but its mutagenicity in humans has not been reported. The polymerases involved in bypass of cisplatin and oxaliplatin adducts in vivo are not known. DNA polymerase η is the most efficient polymerase for bypassing platinum adducts in vitro. We evaluated the role of polymerase η in translesion synthesis past platinum adducts by determining cytotoxicity and induced mutation frequencies at the hypoxanthine guanine phosphoribosyltransferase (HPRT) locus in diploid human fibroblasts. Normal human fibroblasts (NHF1) were compared with xeroderma pigmentosum variant (XPV) cells (polymerase η-null) after treatment with cisplatin. In addition, XPV cells complemented for polymerase η expression were compared with the isogenic cells carrying the empty expression vector. Cytotoxicity and induced mutagenicity experiments were measured in parallel in UVC-irradiated fibroblasts. We found that equitoxic doses of cisplatin induced mutations in fibroblasts lacking polymerase η at frequencies 2- to 2.5-fold higher than in fibroblasts with either normal or high levels of polymerase η. These results indicate that polymerase η is involved in error-free translesion synthesis past some cisplatin adducts. We also found that per lethal event, cisplatin was less mutagenic than UVC. Treatment with a wide range of cytotoxic doses of oxaliplatin did not induce mutations above background levels in cells either expressing or lacking polymerase η, suggesting that oxaliplatin is nonmutagenic in human fibroblasts.

Purpose: Although the combination of irinotecan and 5-Fluorouracil is clinically active, it is associated with significant toxicity and resistance. Studies were carried out to define the optimal dosage, sequence, and timing for the combination in mice bearing xenografted human tumors. Experimental Design: The maximum tolerated dose of irinotecan and 5-Fluorouracil in combination was determined in nude mice. Therapeutic efficacy against established human colon carcinoma xenografts, HCT-8 and HT-29, and human head and neck squamous cell carcinoma xenografts, FaDu and A253, was determined using the rugs individually, simultaneously, and in sequence with various intervals in between. Treatments were i.v. weekly × 4. Immunohistochemical and reverse transcription-PCR measurements of relevant drug-metabolizing enzymes, apoptosis-related proteins, cell cycle distribution, cyclin A, and S phase fraction expression were carried out and compared with the therapeutic outcome. Results: The maximum tolerated dose of irinotecan resulted in cure rates of 30% or less in all xenografts. No cures were achieved with FUra alone. Concurrent administration of irinotecan and FUra, or of FUra 24 h before irinotecan, resulted in cure rates of Conclusions: The optimal therapeutic synergy was achieved when irinotecan was administered 24 h before 5-Flurouracil. Sensitivity to this combination was associated with poor differentiation status, higher cyclin A index, recruitment of cells into S phase, and induction of Bax expression and apoptosis.

Heat shock protein 60 (HSP60, HSPD1) is a "chaperonin" that facilitates folding of nascent proteins into proper conformations (1). It is thought to play a critical role in the assembly, folding, and transport of proteins in the mitochondria. HSP60 also interacts with nascent cellular proteins to prevent their denaturation under heat stress (2). The HSP60 gene sequence is known and is highly conserved (3). Expression of the HSP60 gene has been associated with cisplatin resistance in several preclinical model systems and in ovarian carcinoma patients (1-6); we quantitated HSP60 mRNA expression in preclinical human ovarian and bladder carcinoma models.

BNP7787 (disodium 2,2'-dithio-bis-ethane sulfonate) was evaluated in a phase I clinical trial with paclitaxel and cisplatin to assess the safety and potential efficacy for preventing or reducing cisplatin- and paclitaxel-induced toxicities. During this trial the effects of BNP7787 administration on the total concentrations (oxidized plus free) of cysteine, homocysteine and GSH in plasma, free and total GSH in WBC and rate of urinary excretion of cysteine were studied. The pharmacokinetics of ultrafilterable (free, non-protein bound) platinum were also determined after cisplatin (75 mg/m(2)) treatment which followed paclitaxel (175 mg/m(2)) and BNP7787 (8.2 to 27.6 g/m(2)).Plasma thiols were measured by HPLC with fluorescence detection and platinum was measured by atomic absorption spectrophotometry.BNP7787 administration produced a significant depletion of all plasma thiols in all the patients studied. Differences were noted in the kinetics of BNP7787-induced depletion of cysteine and other thiols. A significant depletion of cysteine occurred with a time lag of about 2 h after the end of BNP7787 infusion, while a reversible depletion of GSH and homocysteine occurred immediately following the start of BNP7787 infusion, with the plasma thiol/disulfide nadir corresponding to the end of infusion. The mean half-life of cysteine depletion following BNP7787 administration was 2.2 h, significantly longer than for homocysteine (0.23 h), or GSH (0.18 h; P0.05 for both). A several-fold increase in the urinary excretion of cysteine occurred following BNP7787 administration in all patients. The BNP7787-induced thiol/disulfide depletion in plasma was not affected by cisplatin administration ( P0.05). BNP7787 administration had no effect on the ultrafilterable platinum pharmacokinetics. The 2-h lag in the depletion of cysteine, the most abundant thiol in plasma, suggests that the process may be related to the formation of free mesna from BNP7787 and that increased levels of mesna are not in circulation until after 2 h after BNP7787 administration. No effect of BNP7787 was seen on the GSH concentration in WBC, possibly reflecting the inability of these cells to take up BNP7787.The results suggest that BNP7787 has the potential to enhance cisplatin antitumor activity by depleting the reactive thiols in plasma.

PURPOSE: To identify a dose and schedule of oxaliplatin (OXP) to be safely administered in combination with protracted-infusion (PI) fluorouracil (5-FU) and external-beam radiation therapy (XRT) for patients with primary esophageal carcinoma (EC). PATIENTS AND METHODS: Eligibility included therapeutically naïve EC patients with clinical disease stages II, III, or IV. Initial doses and schedules for cycle 1 consisted of OXP 85 mg/m2 on days 1, 15, and 29; PI 5-FU 180 mg/m2 for 24 hours for 35 days; and XRT 1.8 Gy in 28 fractions starting on day 8. At completion of cycle 1, eligible patients could undergo an operation or begin cycle 2 without XRT. Postoperative patients were eligible for cycle 2. Stage IV patients were allowed three cycles in the absence of disease progression. OXP and 5-FU increases were based on dose-limiting toxicity (DLT) encountered in cohorts of three consecutive patients. RESULTS: Thirty-eight eligible patients received therapy: 22 noninvasively staged as IV and 16 noninvasively staged as II and III. Thirty-six patients completed cycle 1, 29 patients started cycle 2, and 24 patients completed cycle 2. The combined-modality therapy was well tolerated, but DLT prevented OXP and 5-FU escalation. No grade 4 hematologic toxicity was noted. Eleven grade 3 and two grade 4 clinical toxicities were noted in eight patients. After cycle 1, 29 patients (81%) had no cancer in the esophageal mucosa. Thirteen patients underwent an operation with intent to resect the esophagus; five patients (38%) exhibited pathologic complete responses. CONCLUSION: OXP 85 mg/m2 on days 1, 15, and 29 administered with PI 5-FU and XRT is safe, tolerable, and seems effective against primary EC. The role of OXP in multimodality regimens against EC deserves further evaluation.

Purpose: We have previously reported that elevation of glutathione mediated by γ-glutamyl transpeptidase is one mechanism of oxaliplatin resistance. This study explored other potential oxaliplatin resistance mechanisms with two aims: (1) to identify the differences between cisplatin and oxaliplatin in terms of drug accumulation, DNA-Pt adduct formation and repair, and (2) to determine whether defects in drug accumulation and enhanced repair of the DNA-Pt adduct contribute to oxaliplatin resistance. Methods: The human ovarian carcinoma cell line A2780, an oxaliplatin-resistant variant A2780/C25 and a cisplatin-resistant variant A2780/CP along with an inherently cisplatin-resistant HT-29 colon carcinoma cell line were used in the study. The methods consisted of sulforhodamine-B assays, atomic absorption spectrophotometry and real-time quantitative RT-PCR. Results: Significantly higher drug accumulation and DNA-Pt adduct formation were observed after exposure to cisplatin compared to after oxaliplatin in the parent A2780 cells and the oxaliplatin-resistant A2780/C25 cells. The DNA-Pt adduct formed after treatment with either drug was repaired with equal efficiency by all cell lines except A2780/CP, which repaired the DNA-cisplatin adduct more efficiently than the DNA-oxaliplatin adduct. Relative to the parent line, the oxaliplatin-resistant A2780/C25 cells showed reduced Pt accumulation and DNA-Pt adduct levels following exposure to oxaliplatin, but only reduced accumulation after exposure to cisplatin. The cisplatin-resistant A2780/CP cells showed reduced accumulation and DNA-Pt adduct levels after exposure to cisplatin, but only reduced DNA-Pt adduct after exposure to oxaliplatin. In comparison to A2780 cells, the inherently cisplatin-resistant HT-29 cells showed lower accumulation and DNA-Pt adduct levels after exposure to cisplatin, but displayed no difference after exposure to oxaliplatin. An enhanced repair of the DNA-cisplatin adduct was observed only in A2780/CP cells relative to A2780 cells in an 8-h period. The steady-state levels of ERCC-1 mRNA, but not of XPA, were moderately elevated in the resistant cells. Exposure to either one of the drugs resulted in an induction of XPA in all the cell lines and of ERCC-1 in cisplatin-resistant cells. There was no relationship between the level of expression of the repair genes and the DNA-Pt adduct levels or repair. Conclusions: Relative to cisplatin a lower intracellular concentration and fewer DNA-Pt adducts are sufficient for oxaliplatin to exert its cytotoxicity. Resistance to oxaliplatin is mediated by similar mechanisms of reduced drug accumulation and DNA-Pt adduct formation as resistance to cisplatin. There is no clear evidence that enhanced repair is a mechanism of oxaliplatin resistance in the cell line (A2780/C25) studied here. The findings are suggestive of yet unidentified differences between the two drugs with respect to cellular uptake and/or efflux and repair of DNA-Pt adducts.

The objectives of this study were to determine the dose limiting toxicity (DLT) and other major toxicities, the maximum tolerated dose (MTD) and the human pharmacokinetics of N1N11 diethylnorspermine (DENSPM), a new polyamine analog which in experimental systems inhibits the biosynthesis of intracellular polyamines and promotes their degradation by inducing the enzyme spermine/spermidine N-acetyl transferase. These objectives were incompletely achieved because of the occurrence of an unusual syndrome of acute central nervous system toxicity which forms the basis of the present report. Fifteen patients with advanced solid tumors were entered into a phase I study of DENSPM given by a 1 h i.v. infusion every 12 h for 5 days (10 doses). The starting dose was 25 mg/m2/day (12.5 mg/m2/dose) with escalation by a modified Fibonacci search. Doses of 25 and 50 mg/m2/day were tolerated with only minor side effects of facial flushing, nausea, headache and dizziness (all grade I). At doses of 83 and 125 mg/m2/day, a symptom complex of headache, nausea and vomiting, unilateral weakness, dysphagia, dysarthria, numbness, paresthesias, and ataxia, was seen in 3 patients, one after 2 courses of 83 and 2 after 1 course of 125 mg/m2/day. This syndrome occurred after drug administration was complete and the patients had returned home. Lesser CNS toxicity was seen in 2 other patients at lower daily doses. Preliminary pharmacokinetics of DESPM measured in plasma by HPLC in 8 patients showed linearity with dose and a rapid plasma decay with a t1/2 of 0.12 h. We conclude that great caution is warranted in administering DENSPM on this schedule at doses of > or = 83 mg/m2/day.

Previous studies from our laboratory have indicated that glutathione (GSH) may affect the cytotoxicity of iproplatin to a greater extent than four other platinum agents tested including cisplatin. Therefore we studied the effect of GSH depletion by buthionine sulfoximine (BSO) on the cytotoxicity of iproplatin and cisplatin in a human melanoma cell line SK-MEL-2. Depletion of GSH was dependent on the concentration and time of incubation with BSO. BSO (100 microM) depleted GSH by 85% at 24 h and by 91% at 48 h. BSO (10 to 100 microM) by itself was not cytotoxic to SK-MEL-2 cells. At 85% depletion of GSH, cytotoxicity of iproplatin was increased by a factor of7 and that of cisplatin by2. These results confirm the previous finding that GSH interferes with the cytotoxicity of iproplatin to a significantly greater extent than that of cisplatin. Equitoxic IC65 and IC90 values of cisplatin (2 microM and 5 microM) or iproplatin (25 microM and 50 microM) had no effect on the intracellular GSH levels in SK-MEL-2 cells. Also, depletion of GSH by BSO had no effect on the accumulation of platinum from either cisplatin or iproplatin in this cell line. Our results suggest that the effect of GSH on the cytotoxicity of cisplatin and iproplatin in this cell line was not a consequence either of differences in GSH-Pt conjugate formation, or of differences in platinum accumulation induced by GSH depletion. GSH may have modulated the cytotoxicity of these platinum complexes by other means such as effects on DNA repair, apoptosis, free radical scavenging or through other yet unidentified mechanisms.

4083 Background: Thymidylate synthase (TS) over-expression is associated with 5-FU resistance. Pre-clinical studies demonstrate that vorinostat down-regulates intra-tumor TS in a dose-dependent fashion and augments 5-FU antitumor activity in xenograft models. We conducted a phase I clinical trial of an intermittent schedule of QD x 3 vorinostat in combination with a fixed dose of fluorouracil (5-FU) and leucovorin (LV) in patients (pts) with refractory solid tumors. Methods: Vorinostat was escalated in a standard 3 x 3 design in combination with a fixed dose of 5-FU and LV (simplified de Gramont regimen, sFULV2). Vorinostat was given QD x 3 on an every-2-week cycle. sFULV2 started on day 2 of vorinostat and consisted of leucovorin 400 mg/m2 i.v. over 2 hrs followed by 5-FU 400 mg/m2 bolus and 5-FU 2400 mg/m2 over 46 hrs. Results: 24 pts were enrolled: Male/Female: 11/13; ECOG 0/1: 6/18; Age: median 60 (range 42–77) yrs. 21 pts had colorectal cancer (CRC), 1 had gastric, 1 had esophageal, and 1 had anal cancer. Vorinostat dose-levels (DL) were 600 mg, 800 mg, 1000 mg, 1200 mg, 1400 mg, 1700 mg, and 2000 mg. Dose-limiting toxicities (DLT), consisting of fatigue and hand-and-foot syndrome (H&F), were seen in 2 of 3 pts at the 2000 mg DL. None of the 6 pts at the 1700 mg DL had a DLT. Cycle 1 grade 3/4 toxicities consisted of thrombocytopenia, GI bleeding, fatigue, and H&F in 2 pts at the 2000 mg DL and a non-DLT G3 diarrhea (lasted No significant financial relationships to disclose.

e15543 Background: Novel chemotherapy regimens in combination with RT aim to improve the pathologic complete response (pCR) in EC. Following our dose-finding phase I study, the present phase II neo-adjuvant (NA) EC trial was designed to examine the pCR rate using C, OXP and RT, with secondary end-points of evaluating toxicity, quality of life, and GEP of tumor tissue for correlation to therapeutic response. Methods: EC patients (PTS) with stages II-IVa, adequate organ function and performance status (ECOG 0–1) were eligible. Treatment consisted of OXP, 85mg/m2 iv on days 1, 15 and 29, C (oral or enteral tube) 625 mg/m2 bid on days of RT, and 50.4 Gy RT (3-D conformal) in 28 fractions, followed by an esophagectomy (E) 4–6 weeks later. 2 cycles of OXP + C were administered post-operatively. GEP using Agilent microarrays was conducted on primary tumor tissue pre-treatment (Rx), day (D) 17 and at E; > 50% viable tumor cells were required. Results: 20 PTS have been enrolled (17 male, 3 female); median age 59.5 yrs; 17 adenocarcinomas & 3 squamous-cell cancers. Clinical stage: II (3), III (13) and IVa (4). 18 PTS have completed NA therapy; Grade 4 toxicity includes anemia (1), lymphopenia (2); grade 3 toxicity includes esophagitis (1), pneumonia (1), wound infection (1), anastomotic leak (2), esophageal fistula (1), bowel obstruction (1), fatigue (1), hyperbilirubinemia (1), elevated ALT, AST (1 & 2, respectively), hypoalbuminemia (3), OXP hypersensitivity (2) & leucopenia (1). One PT died > 60d post- operatively secondary to infection. 15 PTS have undergone an E with 3 pCR (20%). Analysis on pre-Rx GEP on 17 PTS revealed a distinct pattern for pCR PTS with 325 over-expressed and 79 under-expressed genes. Ongoing functional analysis will characterize GEP changes in 1) pCR PTS pre-Rx & at D17, 2) pCR & non-pCR PTS pre-Rx, and 3) by histology. Validation will be performed via RT-PCR. Accrual to the trial continues. Conclusions: C, OXP & RT appears to be a tolerable and efficacious NA regimen for EC. The exploratory GEP analysis may provide insight on predicting response to NA therapy. Acknowledgement: The study was approved and funded by the National Comprehensive Cancer Network (NCCN) from general research support provided by Roche Laboratories, Inc. [Table: see text]

Data are available on the pharmacokinetics and metabolism of nilutamide in the rat, dog and human (normal volunteers and patients with advanced prostatie carcinoma). Studies using 14 Carbon-nilutamide, radioimmunoassay, and high-performance liquid chromatography (HPLC) are reviewed. In the rat, bioavailability by the oral route was complete. The majority of the plasma radioactivity was unchanged nilutamide up to six hours, t 1/2 was seven hours, and clearance was 150 mL/hour/kg body weight. Metabolism studies identified 6 urinary metabolites. The major metabolites result from reduction of the nitro group initially to an hydroxylamine (17%) and then to a primary amino (26%) group. In normal volunteers the compound was rapidly absorbed, displayed linear kinetics over a dose range of 100–300 mg, and declined slowly in plasma with a terminal phase t 1/2 of forty-three to forty-nine hours. In studies in 12 patients with advanced (Stage D) prostatic carcinoma, single-dose kinetics after 14 C-nilutamide and kinetics with repetitive twice-daily dosing of two to seven weeks were measured. Terminal phase plasma t 1/2 of unchanged nilutamide was 56 ± 19 hours and of total radioactivity 87 ± 27 hours (mean ± SD). Area under the curve of plasma radioactivity was 23 to 38 percent unchanged nilutamide. Urinary excretion of radioactivity was slow and incomplete because the collection time was not long enough in regard to t 1/2 (mean after 5 days, 62 ± 10%) and consisted almost entirely of metabolites. Steady-state plasma levels of nilutamide were reached in about two weeks. It can be concluded that in humans, unlike other species, plasma decay of nilutamide is very slow. Elimination is almost exclusively by metabolism. Single-daily dosing is appropriate. Hepatic impairment could be expected to prolong plasma decay; renal impairment is likely to have little effect.

3-Deazaguanine (3DG), an antipurine antimetabolite, has recently completed a phase I clinical trial at this Institute. The drug was given on a daily x 5 schedule by i.v. infusion over 0.25-2.16 h. The pharmacokinetics of 3DG during 16 courses were studied in 12 patients at doses of 200-800 mg/m2. 3DG in plasma was measured by an isocratic reverse-phase high-performance liquid chromatographic (HPLC) procedure carried out on IBM phenyl columns at 40 degrees C using 10 mM phosphate buffer (pH 7) as the mobile phase and detection at 300 nm. Plasma decay of 3DG was biexponential in all patients. The AUC correlated linearly with dose at 200-600 mg/m2 but deviated from linearity at doses greater than 600 mg/m2. The drug was cleared rapidly from plasma; at doses of 200-600 mg/m2, the mean plasma clearance was 61.64 +/- 9.97 l/h and the mean terminal-phase elimination half-life was 1.6 +/- 0.6 h. The steady-state volume of distribution (98.9 +/- 29.1 l) and distribution coefficient (1.24 +/- 0.39 l/kg) indicated extensive tissue distribution for the drug. No statistically significant difference was observed between the pharmacokinetics of 3DG on day 1 and that on day 4 as evaluated in three patients for whom complete plasma data were available on both days.

2574 Background: SLM reduces Iri toxicity in xenograft models at associated selenium (Se) concentrations (Con) = 15μM. We conducted a phase I clinical trial of a fixed dose Iri with escalating doses of SLM in order to identify the highest safe dose of SLM that achieves and maintains Se Conc > 15μM. Methods: A standard 3–3 escalation was conducted. Iri was given at 125mg/m2/week x 4 weeks every 6 weeks. SLM was started 1 week prior to 1st dose of Iri. A loading BID dose of SLM was given for 1 week, followed by a daily maintenance dose. Selenium trough levels were obtained on days 8 and 29 of the study (days 1 and 22 irinotecan) and once every 6 week-cycle. Seven dose-levels of SLM were investigated (loading/maintenance, in mcg): 3,200/2,800, 3,200/3,200, 4,000/3,200, 4,000/4,000, 4,800/4,800, 5,600/5,600, and 7,200/7,200. Results: 31 patients enrolled on this study: age (median 57, range 21–80), Male/Female 21/10, ECOG 0/1 (15/16), 31 with prior chemotherapy, 12 with prior radiation, 22 colorectal and 9 mixed solids. Dose escalation was successful up to dose level 7 (7,200mcg SLM PO BID × 1 week followed by 7,200mcg SLM PO QD), which was declared the recommended dose. 2 Dose limiting toxicities occurred on study: one DLT of Grade (G) 3 febrile neutropenia, G3 sepsis, G3 dehydration occurred on dose-level 1; one DLT of G3 febrile neutropenia, dehydration occurred on dose-level 7. Both DLT occurred in a setting of partial small bowel obstruction related to peritoneal carcinomatosis. Non-DLT = G3 treatment-related toxicities included: 3 G3 neutropenia (< 1 week) at DL2, 3 & 7; 2 G3 diarrhea lasting < 48 hours on DL5 & 7. A lower incidence of G2+ toxicities was noted in patients with higher Se Conc (86% for Se < 15; 67% for Se 15–20; 57% for Se> 20 μM). Toxicities related to SLM were limited to garlic-like smell to breath, sweat, and urine in few patients. Responses included 7 confirmed stable diseases and 2 confirmed partial responses. A Se Con > 15 μM was achieved at all SLM dose levels of 4,800 mcg and above. Conclusions: Doses of SLM up to 7,200 mcg BID × 7 days followed by 7,200 mcg QD can be administered safely with standard doses of Iri. Formal phase II and III studies are needed to determine if SLM reduces Iri toxicity. No significant financial relationships to disclose.