Expression of genes related to activity of oxaliplatin and 5-fluorouracil in endoscopic biopsies of primary esophageal cancer in patients receiving oxaliplatin, 5-flourouracil and radiation: characterization and exploratory analysis with survival.

With a goal of identifying relations between gene expression and response (mucosal or pathological) or survival in esophageal cancer patients (stages II to IV) receiving oxaliplatin, 5-fluorouracil (5FU) and radiation, we measured in endoscopic primary tumor biopsies from 38 patients, the expression of seven genes (gammaGCS, gammaGT, MRP-2, ERCC-1, XPA, TS and DPD) prior to treatment, 1 week following oxaliplatin alone and at the end of the combined radio-chemotherapy cycle using real time QRT-PCR. A higher pretreatment level of XPA was related to shorter survival with a hazard ratio of 2.43 (90% confidence interval 1.09 to 5.43) using Cox regression modeling. However, multivariate analysis with a Cox model indicated low expression of XPA or TS and combined stages II and III had a higher probability of survival (for XPA: hazard ratio 3.0 and 90% C.I. of 1.3 to 6.9, with adjustment for stage included; for TS: hazard ratio is 1.98 with 90% C.I. of 0.94 to 4.20. The expression of TS, gammaGCS, ERCC-1 and MRP-2 declined from D 1 to the end of the cycle (p<0.05, sign test). A validation and further understanding of the findings need to be carried out in a larger study with a more homogeneous population of patients.

S-phase modulation by irinotecan: pilot studies in advanced solid tumors.

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.

A phase I and pharmacokinetic study of BAY59: a novel taxane.

PURPOSE: To determine the maximum tolerated dose (MTD), the dose limiting toxicities (DLT) and the pharmacokinetics of BAY59, a novel taxane given as a 1-hour intravenous infusion every 3 weeks in patients with advanced refractory solid tumors. EXPERIMENTAL DESIGN: Initially, 15 patients with previously treated (median of 4 prior chemotherapy regimens) refractory cancers, but with normal marrow, hepatic and renal function were treated with BAY59 at doses of 15, 30, 50, 75 and 100 mg/m2 using a standard dose escalation design. Subsequently, 11 patients were treated, 5 at 90 mg/m2 and 6 who had had prior oxaliplatin at 75 mg/m2. RESULTS: At 75 mg/m2, grade 4 neutropenia was noted in 2/6 patients, of whom 1 had grade 4 neutropenia lasting more than 5 days (DLT). At 100 mg/m2, 2/2 patients had febrile neutropenia, with 1 fatality. At 90 mg/m2, 2/5 patients had DLTs, including grade 3 neuropathy, severe lower extremity pain, dehydration and grade 4 neutropenia. The MTD was determined to be 75 mg/m2. A cohort of 6 patients, previously exposed to oxaliplatin, were enrolled at the MTD to evaluate the incidence of neurotoxicity. While DLTs (grade 3 arthralgia, grade 4 neutropenia) were noted in 3/6 patients, there was no increase in the incidence of neurotoxicity. There were no responses. Pharmacokinetics of BAY59 was linear over the doses studied, with a median terminal half-life of 21 h. CONCLUSIONS: The recommended phase II dose for BAY59 is 75 mg/m2.

Phase I/pharmacodynamic study of N-acetylcysteine/oltipraz in smokers: early termination due to excessive toxicity.

An N-acetylcysteine (NAC)/oltipraz (OLZ) combination was studied in healthy volunteer smokers who received daily NAC (1200 mg/day) and were randomized to weekly placebo (Arm A), OLZ 200 mg (Arm B), or 400 mg (Arm C). Treatment was for 12 weeks with follow-up at 16 weeks. The objective was to study toxicity and the modulation of pharmacodynamic end points. After treatment of 19 of a planned 60 subjects, (Arm A, six; Arm B, four; and Arm C, nine), the study was closed because of toxicity. Eight subjects failed to complete 12 weeks of drug administration, (Arm A, two, and Arm C, six). The most frequent side effects were gastrointestinal, fatigue, conjunctival irritation, and skin rash. Pharmacodynamic end points were measured pretreatment and 48 h after the dose of OLZ at weeks 1, 5, and 12 and 4 weeks after the end of treatment. Glutathione (GSH) was measured in plasma and in peripheral blood lymphocytes (PBLs). Other end points measured in PBLs were the enzyme activities of total glutathione-S-transferase (GST), GSTpi, and NAD(P)H:quinone oxidoreductase; and the mRNA expression of gamma-glutamylcysteine synthetase gammaGCS), GSTpi, and NAD(P)H:quinone oxidoreductase. GSH in PBLs, GST (total), and the mRNA of gammaGCS showed increases at some time points in some subjects. Most consistent was the mRNA of gammaGCS, which showed a > or = 30% increase at one or more time points in 11 of 19 subjects. Other end points were unchanged. We concluded that NAC/OLZ modulates some end points related to GSH but is too toxic for chemoprevention at the doses used.

Oral uracil and Ftorafur plus leucovorin: pharmacokinetics and toxicity in patients with metastatic cancer.

PURPOSE: To assess the pharmacokinetics of Ftorafur (tegafur, FT), 5-fluorouracil (5-FU), and uracil in 31 cancer patients who were enrolled in phase I studies of oral uracil and FT (UFT). The correlation between pharmacokinetic parameters and toxic effects of UFT was evaluated. METHODS: Uracil and FT were orally administered in a 4:1 molar ratio at FT doses of 200-400 mg/m2 per day. Patients also received leucovorin at 150 mg/day. Daily doses were divided into three doses and administered at 8-h intervals for 28 consecutive days. Plasma FT concentrations were measured by high-performance liquid chromatography, and plasma 5-FU and uracil concentrations were determined using gas chromatography-mass spectrometry. National Institutes of Health Common Toxicity Criteria were used for assessment of toxicity. RESULTS: The concentrations of FT, 5-FU, and uracil showed wide interpatient variations. Maximum plasma concentrations (Cp(max)) of all three compounds were achieved in 0.3 to 4.0 h. At the various study doses, the terminal half-life (t 1/2beta) of FT ranged from 3.9 to 5.9 h, the area under the concentration-versus-time curve (AUC0-6h) ranged from 16,220 to 52,446 (ng/ml)h, the total clearance (ClT) ranged from 100 to 175 ml/min, and the steady-state volume of distribution (Vd(ss)) ranged from 18.3 to 28.7 l. The 5-FU generated from FT had an apparent distribution half-life (t 1/2alpha) and an apparent elimination half-life (t 1/2beta) of 0.3-1.3 h and 4.9-7.0 h, respectively. The AUC0-6h of 5-FU ranged from 120 to 325 (ng/ml)h. Uracil had a t 1/2alpha of 0.2-0.5 h and the level quickly returned to the endogenous level. The AUC0-6h for uracil ranged from 605 to 3764 (ng/ml)h, the ClT ranged from 3225 to 7748 ml/min, and the Vd(ss) ranged from 341 to 1354 l. The Cp(max) and AUC0-6h of both FT and uracil were significantly correlated with FT doses (P-values of 0.0244 and 0.0112) and with uracil doses (P-values of 0.0346 and 0.0083), respectively. In addition to interpatient variations, intrapatient variations were also observed in six patients who had pharmacology studies done on days 1 and 26+/-2 at the same study dose. We found that the repeated treatment with UFT caused cumulative increases in the values of Cp(max), Ctrough, and AUC0-6h of FT and 5-FU. The major toxic effects observed were diarrhea and nausea and vomiting. The occurrence of these toxic effects correlated significantly with the Cp(max) and AUC0-6h of 5-FU. CONCLUSIONS: The pharmacology studies showed that FT and uracil were readily absorbed orally and that FT was rapidly converted to 5-FU. The preliminary findings suggest that determination of plasma levels of 5-FU after oral administration of UFT may help predict subsequent toxic effects.

Intravenous ifosfamide/mesna is associated with depletion of plasma thiols without depletion of leukocyte glutathione.

Depletion of cellular glutathione (GSH) enhances the efficacy of many anticancer agents in preclinical systems. Limited published data showing depletion of GSH in vitro and in patients by ifosfamide and/or mesna provided the rationale for a Phase I trial. Ifosfamide and mesna were infused over 24 and 36 h, respectively, at equal daily doses; carboplatin was given after ifosfamide to a target plasma area under the curve of 4 mg x min x ml(-1). Plasma and peripheral WBC thiols were quantitated by high-performance liquid chromatography. The dose of ifosfamide was escalated from 2 to 8 g/m2; the maximum tolerated dose was 6 g/m2. Significant depletion in plasma cysteine and homocysteine, precursors for GSH synthesis, was observed (maximum, 95% to >99% at 8 g/m2). Plasma mesna and cysteine/ homocysteine levels were inversely correlated; nadir levels of cysteine/homocysteine were maintained for several hours after ifosfamide infusion had stopped and while mesna infusion was continuing. In vitro coincubation experiments confirmed that mesna reduces these thiols from disulfides to sulfhydryls, which are readily cleared, as evidenced by the significantly increased rate of excretion of cysteine in urine. In contrast, ifosfamide/mesna treatment caused a moderate depletion of plasma GSH in only 60% of the patients, with a nadir at 24 h and recovery immediately after the end of ifosfamide infusion. The GSH depletion in these patients was not dose related. The profile of GSH recovery in plasma after ifosfamide and the fact that mesna could not reduce GSH disulfides in vitro suggest that the observed GSH depletion in plasma in 60% of the patients may be related to direct reactions of GSH with ifosfamide metabolites and/or mesna. Our results indicate that mesna is a modulator of GSH precursors and that a prolonged infusion of mesna may be required to achieve GSH precursor starvation and the consequent GSH depletion in cells.

Phase I and pharmacokinetic study of weekly 5-fluorouracil administered with granulocyte-macrophage colony-stimulating factor and high-dose leucovorin: a potential role for growth factor as mucosal protectant.

Diarrhea is dose-limiting with weekly 5-fluorouracil (5-FU) plus high-dose leucovorin (LV). Granulocyte-macrophage colony-stimulating factor (GM-CSF) has been associated with a decrease in chemotherapy-associated mucosal toxicity. This study was conducted to determine the maximum tolerated dose (MTD) of weekly 5-FU when administered with GM-CSF and high-dose LV. Patients were treated with intravenous LV 500 mg/m2 plus 5-FU weekly for six doses followed by a 2-week rest. GM-CSF 250 mg/m2 was administered subcutaneously 5 days each week. Cohorts were treated with 5-FU at 600, 700, and 800 mg/m2 weekly. Twenty-nine patients were treated. The MTD of 5-FU in this schedule was 700 mg/m2/week, with diarrhea dose-limiting. 5-FU delivered dose intensity at the MTD was 424 +/- 23.7 mg/m2/week, including rest periods. 5-FU and LV pharmacokinetics were not altered by concurrent treatment with GM-CSF. In a weekly schedule with high-dose LV and GM-CSF, the MTD of 5-FU and 5-FU delivered dose intensity were higher than previously reported with 5-FU and LV administered without GM-CSF.

Preliminary studies of a novel oral fluoropyrimidine carbamate: capecitabine.

PURPOSE: To evaluate the toxicology and pharmacology of an orally active fluoropyrimidine given as a continuous daily dose divided into two portions for 6 weeks, and to determine the maximal-tolerated daily dose (MTD) and the suggested phase II daily dose. PATIENTS AND METHODS: Solid-tumor patients with a Karnofsky performance status greater than 70 who had normal organ function and resolution of the effects of prior therapy, and who gave informed written consent, were enrolled. Oral capecitabine, as a divided morning and evening dose, was administered to cohorts of a minimum of 3 patients starting at 110 mg/m2 and escalating by means of a modified Fibonacci scheme to 1,657 mg/m2/d. Pharmacologic samples were obtained on days 1 and 15. Toxicity evaluations were performed approximately every 3 days for the first 43 days. Antitumor effect was evaluated at day 42 of therapy. RESULTS: Thirty-three patients entered the study. Few side effects occurred at or below 1,331 mg/m2/d. The MTD was 1,657 mg/m2/d with limiting toxicities of palmar-plantar erythrodysesthesia, nausea, vomiting, vertigo, abdominal pain, diarrhea, and thrombocytopenia. All toxicities were reversible. A mixed response was seen in one breast cancer patient. Pharmacologic studies showed rapid and extensive metabolism of the parent drug into cytotoxic metabolites with a maximum plasma concentration (Cmax) 1 hour after ingestion. Linear increases in the area under the concentration-time curve (AUC) and Cmax were seen with linear increases in administered dose. CONCLUSION: The suggested phase II dose on a continuous 42-day dosing schedule is 1,331 mg/m2/d. Linear pharmacologic parameters of the parent compound and metabolites are demonstrated.

Octreotide does not prevent diarrhea in patients treated with weekly 5-fluorouracil plus high-dose leucovorin.

The somatostatin analog, octreotide, is effective in treating diarrhea associated with cancer chemotherapy. This study was undertaken to determine whether octreotide could be used as prophylaxis against chemotherapy-induced diarrhea and, thereby, permit increased dose intensity. Adult cancer patients were treated with a standard regimen of intravenous 5-fluorouracil (5-FU) (600 mg/m2) plus leucovorin (LV) (500 mg/m2) weekly x 6 weeks. In addition, 150 microg of octreotide was administered subcutaneously twice daily, beginning on the first day of chemotherapy and continuing for 43 days. Escalation of 5-FU was planned for successive cohorts based upon toxicity. Eleven patients were treated at the initial 5-FU dose level. In 10 evaluable patients, dose-limiting toxicities were diarrhea (two patients), fatigue (one patient), and hyperbilirubinemia (one patient). Diarrhea was experienced by six of 10 patients, and only three patients were able to receive six weekly chemotherapy treatments without dose reduction or delay. At a dose of 150 microg twice daily, octreotide did not prevent diarrhea associated with 5-FU plus LV, and 5-FU dose escalation was not possible. While octreotide is successful in the treatment of 5-FU-induced diarrhea, we were unable to demonstrate a role in toxicity prophylaxis.

Initial clinical trial and pharmacokinetics of Thymitaq (AG337) by 10-day continuous infusion in patients with advanced solid tumors.

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 Thymitaq. 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 x 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.

Paclitaxel and carboplatin in early phase studies: Roswell Park Cancer Institute experience in the subset of patients with lung cancer.

The combination of paclitaxel (Taxol; Bristol-Myers Squibb Company, Princeton, NJ) given by 3-hour infusion followed by carboplatin infused over 30 minutes has been evaluated in a series of phase I studies and is currently being explored in a phase II study in patients with limited- and extensive-stage small cell lung cancer. Pharmacokinetic measurements were performed at all dose levels in the phase I studies, in which the use of granulocyte colony-stimulating factor in previously treated patients enabled more than twice the dose of paclitaxel to be given with low to moderate doses of carboplatin (dosed to a target area under the concentration-time curve of 4.0 mg x min x mL[-1]). Treatment-naive patients tolerated high paclitaxel doses (270 mg/m2) with carboplatin (dosed to a target area under the curve of 4.5 mg x min x mL[-1]) without granulocyte colony-stimulating factor support. Twenty-three patients (including previously treated and untreated) with non-small cell lung cancer were entered at a variety of paclitaxel doses in the phase I studies. At 100 to 205 mg/m2 paclitaxel, none of nine treated patients responded; at 230 to 290 mg/m2, four (29%) of 14 responded. In the phase II study of paclitaxel 250 mg/m2 in previously untreated patients with small cell lung cancer, two of five evaluable patients with extensive-stage disease have shown a partial response. In a preliminary analysis of the pharmacodynamics of paclitaxel in relation to neurotoxicity (dose limiting in two of three phase I studies), neurotoxicity correlated with the total dose of paclitaxel, the area under the curve, and the peak paclitaxel concentration, but not with the length of time plasma paclitaxel levels remained above 0.05 micromol/L. These correlations were not strong, however, and analysis of these data is ongoing.

Translational studies of glutathione in bladder cancer cell lines and human specimens.

Glutathione (GSH) levels were measured in 13 human tumor cell lines derived from carcinomas of the bladder, ovary, and colon and from melanoma and glioblastoma. High levels were found in four of five bladder cell lines. The average GSH concentration in the bladder cell lines was approximately 6-fold higher than in the non-bladder cell lines. Because this difference suggested the possibility of elevation of GSH in urothelial neoplasia, we measured GSH in bladder tumor tissue from patients with transitional cell carcinoma (TCC) of the bladder (Group I, n = 17). GSH was also measured in two types of control tissues: (a) nontumor bladder tissue from patients with TCC or a history of TCC of the bladder (Group II, n = 23); and (b) bladder tissue from patients without bladder cancer (Group III, n = 14). Thirteen sets of paired specimens of tumor and nontumor bladder tissue from the same patient were evaluated. The tissues were flash-frozen, and GSH was measured after histological assessment of the same samples. Free and total GSH (free + mixed disulfides) were measured by a high-performance liquid chromatography assay with fluorescence detection and expressed as nanomoles/mg protein. The mean free GSH (+/- SD) for groups I, II, and III was 32.0 +/-18.7, 17.3 +/- 11.4, and 9.3 +/- 4.0, respectively, and the mean total GSH was 45.9 +/- 32.5, 23.7 +/- 17.1, and 12.2 +/- 6.7. The respective differences between groups (I and II, I and III, and II and III) were statistically significant for both free and total GSH (Ps ranging from <0.0001 to

Phase I and pharmacokinetic study of etoposide phosphate by protracted venous infusion in patients with advanced cancer.

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.

Unusual central nervous system toxicity in a phase I study of N1N11 diethylnorspermine in patients with advanced malignancy.

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.

Effect of glutathione depletion on the cytotoxicity of cisplatin and iproplatin in a human melanoma cell line.

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 of > 7 and that of cisplatin by < 2. 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.

Early phase studies with paclitaxel/low-dose carboplatin in patients with solid tumors.

In preparation for the design of phase II studies in lung cancer, low-dose carboplatin, fixed at a target area under the concentration-time curve (AUC) of 4.0 or 4.5 mg x min/mL, has been combined with escalating doses of paclitaxel (Taxol; Bristol-Myers Squibb Company, Princeton, NJ) in a series of studies to establish the maximum tolerated dose of the combination. In patients who had received prior chemotherapy, the maximum tolerated paclitaxel dose was 135 mg/m2 (carboplatin target AUC 4.0); the dose-limiting toxicity was febrile neutropenia. Without granulocyte colony-stimulating factor support in chemotherapy-naive patients (carboplatin target AUC 4.5), and with granulocyte colony-stimulating factor in chemotherapy-pretreated patients, the current paclitaxel dose is 290 mg/m2. The maximum tolerated dose has not been defined. In a study in which paclitaxel was given by 1-hour infusion with carboplatin (target AUC 4.5), a 205 mg/m2 dose was poorly tolerated. No evidence of pharmacokinetic interactions between paclitaxel and carboplatin was found. Twenty-one evaluable patients with lung cancer have been treated to date. There have been two partial responses, one minor response, and 10 patients with stable disease at paclitaxel doses of 100 to 270 mg/m2.

Altered glutathione metabolism in oxaliplatin resistant ovarian carcinoma cells.

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.

Daily subcutaneous injection of low-dose interleukin 2 expands natural killer cells in vivo without significant toxicity.

We aimed to determine the toxicity and immunological effects of daily s.c. administered low-dose interleukin (IL) 2. Adult cancer patients received a single daily s.c. injection of IL-2 as outpatients for 90 consecutive days. Cohorts of four to nine patients were treated at escalating IL-2 dose levels until the maximum tolerated dose (MTD) was defined. Peripheral blood mononuclear cell phenotyping, IL-2 serum levels, and the presence of anti-IL-2 antibodies were investigated. Thirty-eight patients were treated at seven IL-2 dose levels ranging from 0.4 to 1.75 million International Units (mIU)/m2 daily. The MTD was 1.25 mIU/m2, with constitutional side effects, vomiting, and hyperglycemia dose limiting. Severe toxicity did not occur at or below the MTD, although mild local skin reaction and mild constitutional side effects were common. Objective tumor regressions were not observed during this Phase I trial. Low-dose IL-2 resulted in natural killer (NK) cell (CD3(-) CD56(+)) expansion at all dose levels. This effect was dose dependent (P < 0.01), ranging from a 154 to 530% increase over baseline. Peak NK levels were achieved at 6-8 weeks and sustained through 12 weeks of therapy. As predicted by in vitro studies of IL-2 receptor structure-activity relationships, the subset of NK cells that constitutively express high-affinity IL-2 receptors (CD3(-)CD56(bright+)) showed more profound dose-dependent expansion, with increases ranging from 368 to 2763% (P = 0.015). NK expansion occurred at peak IL-2 levels <10 pM (2.3 IU/ml). Three patients developed nonneutralizing anti-IL-2 antibodies. Thus, we concluded that selective expansion of NK cells may be achieved in vivo with daily s.c. injections of low-dose IL-2 with minimal toxicity.

Induction of gamma-glutamyl transpeptidase mRNA by platinum complexes in a human ovarian carcinoma cell line.

Elevation of glutathione (GSH) is widely observed in cellular resistance to platinum agents. Our previous studies have shown that sublines of human ovarian carcinoma cell line A2780, which exhibited low levels of resistance to oxaliplatin, showed elevated steady state levels of mRNA and activity of gamma-glutamyl transpeptidase (gamma-GT, EC 2.3.2.2), but not of gamma-glutamylcysteine synthetase (gamma-GCS, EC 6.3.2.2) [El-akawi et al., Cancer Lett. 105:5-14; 1966]. To understand this phenomenon better, we have studied the effect of single exposures of oxaliplatin or cisplatin on the mRNA expression of gamma-GT and gamma-GCS in A2780 cells. The mRNAs of gamma-GT and gamma-GCS were measured by reverse transcriptase 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 was measured by HPLC. The gamma-GT activity was measured by a colorimetric assay. Single exposures of cells to oxaliplatin induced a time- and concentration-dependent increase in the mRNA of gamma-GT, but not of gamma-GCS. Cisplatin also induced an elevation in gamma-GT mRNA, but to a lower degree. The gamma-GT enzyme activity increased corresponding to the elevation in mRNA expression. The gamma-GT-induced cells showed an increase in cellular GSH when incubated in medium containing GSH. The data suggest that a) single, brief exposures to pharmacologically relevant concentrations of platinum complexes induce elevation in mRNA of gamma-GT, b) elevation in gamma-GT mRNA translates into elevated gamma-GT activity and increase in GSH salvage, and c) the degree of induction of gamma-GT mRNA differs between platinum complexes.

A phase I and pharmacokinetic study of oral uracil, ftorafur, and leucovorin in patients with advanced cancer.

A phase I and pharmacokinetics study of oral uracil, ftorafur, and leucovorin was performed in patients with advanced cancer. Uracil plus ftorafur (UFT) was given in a 4:1 molar ratio in three divided doses for 28 consecutive days. Patient cohorts were treated at 200, 250, 300, and 350 mg/m2 of UFT daily. For all patients, 150 mg of leucovorin was given daily in three oral doses. A 1-week rest period followed each 28-day treatment course. Gastrointestinal toxicity, characterized by diarrhea, nausea, and vomiting, was dose-limiting at 350 mg/m2 UFT in patients who had received prior chemotherapy. Mild fatigue and transient hyperbilirubinemia were also common. In previously untreated patients, UFT at 350 mg/m2 was well-tolerated, suggesting this as an acceptable phase II dose in this schedule with leucovorin. Two of eight previously untreated patients with advanced colorectal cancer had partial responses with UFT (350 mg/m2) plus leucovorin. Pharmacokinetic parameters [ftorafur, uracil, 5-fluorouracil (5-FU), 5-methyltetrahydrofolate] showed wide interpatient variations. Plasma levels of 5-FU (Cmax 1.4 +/- 1.9 microM) were comparable to those achieved with protracted venous infusions, and folate levels (Cmax 6.1 +/- 3.6 microM) were sufficient for biochemical modulation. Ongoing study will determine if this convenient oral regimen will compare favorably in terms of efficacy, toxicity, and cost with intravenous fluoropyrimidine programs.