Search

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.

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.

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.

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.

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.

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.

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.

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: 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.

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.

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.

Iproplatin is structurally unique among the platinum (Pt) agents in the clinic because it is a quadrivalent complex. On the basis of the redox parameters for the Pt(IV) and Pt(II) oxidation states in a chloride system, it has been suggested that Pt(IV) complexes will be reduced to Pt(II) complexes in a biological environment. To test this hypothesis, uptake and metabolism studies of [14C]-iproplatin were carried out in L1210 cells. The L1210 cells raised in DBA2/J mice were incubated in vitro with 50 and 100 microM [14C]-iproplatin at 37 degrees C in Hanks' balanced salt solution, and total uptake and radioactivity associated with acid-insoluble fractions were measured for up to 3 h. Under these conditions, the uptake of iproplatin was linear with time and increased with increasing concentrations of iproplatin in the medium. At all times measured, greater than 35% of radioactivity was associated with the acid-insoluble fraction, suggesting binding to macromolecules. The [14C]-labelled compounds in neutralized acid extracts of cells were separated by reverse-phase high-performance liquid chromatography (HPLC). Three labelled compounds were detected; based on chromatographic elution times, they appeared to be iproplatin, cis-dichloro-bis-isopropylamine platinum(II) (CIP), the reduction product of iproplatin, and a third compound more polar than iproplatin and CIP. The finding of free CIP and the macromolecular binding of radioactivity in the cells suggests that iproplatin is reduced intracellularly.

We have previously shown that a significant portion of the total platinum in the plasma of patients receiving iproplatin is protein-bound [17]. We have also identifiedcis-dichloro-bis-isopropylamine platinum(II) (CIP) as a major metabolite of iproplatin [19]. To understand the nature of the bound platinum, we carried out in vitro comparative protein-binding studies for iproplatin and CIP. These studies indicate that when CIP is incubated in plasma, protein binding occurs, with a 2.7-h half-life for the disappearance of CIP; the parent complex does not bind and is stable in plasma for at least 48 h. The time dependence of protein binding with CIP suggests the formation of other chemical species from CIP that may be responsible for the observed protein binding. The results indicate that in patients receiving the drug, the reduction of iproplatin to CIP must take place intracellularly and that CIP or its protein-binding derivatives must efflux from the cells into the plasma. Efflux studies carried out to explore this possibility with cells in the whole blood showed that iproplatin was taken up into cells, but the efflux of protein-binding iproplatin metabolites did not occur. To understand further the nature of the metabolites of iproplatin, we carried out195Pt-NMR (nuclear magnetic resonance) studies with urine from two patients who received a high dose of iproplatin (500 mg/m2). The predominant signals from the195Pt-NMR corresponded to the divalent platinum complexes and not to quadrivalent complexes, indicating that the iproplatin metabolites in urine are divalent in nature.

The ovarian carcinoma subline A2780/C10B (C10B) is an oxaliplatin resistant clone derived from the human ovarian carcinoma cell line A2780. The C10B cells are characterized by mesenchymal phenotype, decreased platinum uptake and increased glutathione levels (Hector et al. in Cancer Lett 245:195-204, 2007; Varma et al. in Oncol Rep 14:925-932, 2005). Na,K-ATPase-beta subunit (Na,K-beta(1)) functions as a cell-cell adhesion molecule in epithelial cells and is reduced in a variety of carcinoma cells that show mesenchymal phenotype. The purpose of this study is to evaluate the relationship between Na,K-beta expression and sensitivity to oxaliplatin.Cell lines used include A2780, C10B, C10B transfected with Na,K-beta(1) (C10B-Na,K-beta) and a canine kidney carcinoma cell line MSV-MDCK also transfected with Na,K-beta(1) (MSV-MDCK-beta subunit). Cytotoxicity studies were performed by sulforhodamine-blue assay. The Na,K-alpha(1) and Na,K-beta(1) subunit localization and expression were by immunofluorescence microscopy and Western blot analysis. Platinum accumulation measurements were by atomic absorption spectrophotometry.C10B cells express highly reduced levels of Na,K-beta(1) subunit. Exogenous expression of Na,K-beta(1) increased platinum accumulation and sensitized C10B cells to oxaliplatin. The pharmacological inhibitor of Na,K-ATPase ouabain did not alter the oxaliplatin accumulation indicating that Na,K-beta(1) sensitizes cells in a Na,K-ATPase enzyme activity independent manner. These findings were also confirmed in MSV-MDCK-beta subunit cells.This study for the first time reveals that reduced expression of the Na,K-beta(1) protein is associated with oxaliplatin resistance in cancer cells and demonstrates a novel role for this protein in sensitizing the cells to oxaliplatin. This study suggests a potentially important role for Na,K-beta(1) in both prognosis and therapy of oxaliplatin resistant malignancies.

Purpose To understand the mechanisms behind platinum drug/DENSPM-induced inhibition of cancer cell growth, we compared the effects of oxaliplatin and cisplatin when combined with DENSPM on the induction of SSAT mRNA, activity, polyamines and cell growth in A2780 human ovarian carcinoma cells and their oxaliplatin- and cisplatin-resistant variants A2780/C10B and A2780/CP, respectively. Methods Parental and Pt-resistant cells were treated with platinum agent alone, DENSPM alone or combination (10 μM each, 20 h). QRT–PCR, radioactive product measurement and HPLC were used for mRNA, activity and polyamine pools, respectively; drug interaction on cell growth was by SRB and isobologram analysis. Results Both platinum agents induced SSAT mRNA in parental A2780 cells, but not in resistant cells. Platinum drug/DENSPM combinations produced high levels of SSAT activity in parental cells with significant depletion of spermine and spermidine, but not in resistant cells. Co-treatment with platinum agents increased the levels of DENSPM in all cell lines. Oxaliplatin/DENSPM combination was superior to cisplatin/DENSPM in the inhibition of cell growth in parental cells. No synergy was observed in the resistant cells. Conclusions Increased DENSPM levels following co-treatment with Pt agents enhances the translation and stability of SSAT protein leading to polyamine pool depletion, facilitating more Pt–DNA adduct formation in parental cells. Oxaliplatin/DENSPM combination is superior to cisplatin/DENSPM in cell growth inhibition as DACH-Pt DNA adducts are cytotoxic even at relatively fewer numbers. Reduced platinum uptake in Pt-resistant cells contributes to reduced SSAT mRNA induction and absence of synergy when combined with DENSPM.