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.

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 cisplatin> Pd-Spm>Pt-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.

OBJECTIVETo determine whether a carboplatin dose calculation that is based on a targeted area under the concentration-versus-time curve (AUC(Target)) and individual glomerular filtration rate (GFR) accurately predicts carboplatin-associated myelotoxicoses in tumor-bearing cats, and to determine the maximum tolerated AUC(Target).ANIMALS32 cats with tumors.PROCEDURESIn each cat, plasma clearance of technetium Tc 99m-labeled diethylenetriaminepentaacetic acid was measured to assess GFR. Carboplatin was administered IV. The dose was calculated by use of an equation as follows: Dose = AUC(Target) x 2.6 x GFR x body weight. Initial AUC(Target) was 2.0 min.mg.mL(-1) and was increased in increments of 0.50 min.mg.mL(-1) in cohorts of 3 cats. To assess myelotoxic effects, CBCs were performed weekly for > or = 4 weeks. Following identification of the maximum tolerated AUC(Target), additional cats were treated at that AUC(Target) and plasma platinum concentrations were measured in 6 cats.RESULTSThe AUC(Target) values ranged from 2.0 to 3.0 min.mg.mL(-1). Neutropenia was the dose-limiting toxicosis, and the maximum tolerated AUC(Target) was 2.75 min.mg.mL(-1). Nineteen cats received this dose of carboplatin; 13 became neutropenic, but only 1 developed severe neutropenia (< 500 neutrophils/microL), and none had neutropenia-associated clinical signs. In the cats that had plasma platinum concentration determined, the difference between AUC(Target) and the measured value ranged from -0.23 to 0.31 min.mg.mL(-1) (median, 0.20 min.mg.mL(-1)).CONCLUSIONS AND CLINICAL RELEVANCEIn cats, carboplatin-associated myelotoxicoses were accurately and uniformly predicted by use of the proposed dosing strategy. The maximum tolerated AUC(Target) for a single dose of carboplatin was 2.75 min.mg.mL(-1).

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.

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].

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.

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 <24 hrs) in 1 pt at the 1700 mg DL. Grade 2 nausea, fatigue, and anorexia were common; especially at DL ≥ 1700 mg. Antitumor activity was noted in pts with CRC despite prior refractoriness to 5-FU and failure to oxaliplatin, irinotecan, and cetuximab in all pts. 12/21 CRC pts had a confirmed SD (11) or PR (1). CRC pts had a median PFS of 4 months, a ≥ 6 months PFS rate of 43%, and a ≥ 8 months PFS rate of 33%. Conclusions: The maximum tolerated dose (MTD) of vorinostat in combination with sFULV2 is 1700 mg PO QD x 3 every 2 weeks. This combination is associated with considerable activity in pts with 5-FU-refractory CRC and warrants further investigation. An expanded MTD cohort is accruing to investigate 5-FU-vorinostat PK interaction and intra-tumor TS down-regulation. (This work was supported by a grant from CTEP and the ACS.) No significant financial relationships to disclose.

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.

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.

BACKGROUND:Time-course and concentration-effect experiments with multiple time-points and drug concentrations provide far more valuable information than experiments with just two design-points (treated vs. control), as commonly performed in most microarray studies. Analysis of the data from such complex experiments, however, remains a challenge.MATERIALS AND METHODS:Here we present a semi-automated method for fitting time profiles and concentration-effect patterns, simultaneously, to gene expression data. The submodels for time-course included exponential increase and decrease models with parameters, such as initial expression level, maximum effect, and rate-constant (or half-time). The submodel for concentration-effect was a 4-parameter Hill model.RESULTS:The method was applied to an Affymetrix HG-U95Av2 dataset consisting of 51 arrays. The specific study focused on the effects of two platinum drugs, cisplatin and oxaliplatin, on A2780 human ovarian carcinoma cells. Replicates were available at most time points and concentrations. Eighteen genes were selected, and after selection, time-course and concentration-effect were modeled simultaneously.CONCLUSION:Comparisons of model parameters helped to distinguish genes with different expression patterns between the two drug treatments. This overall paradigm can help in understanding the molecular mechanisms of the agents, and the timing of their actions.

PurposeTo evaluate the efficacy of a combination of capecitabine, oxaliplatin, and radiotherapy (RT) in the neoadjuvant treatment of Stage II and III rectal cancers.MethodsCapecitabine was given at 725 mg/m2 orally twice daily Monday through Friday concurrently with RT. Oxaliplatin was given intravenously at 50 mg/m2 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.ResultsA 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.ConclusionCapecitabine at 725 mg/m2 orally twice daily, oxaliplatin 50 mg/m2/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.

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.

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.

3292 We previously characterized an oxaliplatin resistant clonal cell line (A2780/C10B) that exhibited changed morphology and molecular characteristics akin to epithelial to mesenchymal transition (EMT) with reduced platinum uptake and increased glutathione (Hector et al., Cancer Letters 2007). We hypothesized that the resistance is at least partially related to EMT and its reversal may restore some sensitivity to oxaliplatin. Na,K-ATPase an oligomeric protein consisting of α- and β-subunits is well studied as an ion transporter in epithelial cells. It has been shown that reduced Na,K-ATPase β-subunit (Na,K-β) level is associated with cancer progression and that its repletion induces epithelial phenotype and suppresses invasiveness and tumorigenicity of carcinoma cells (Rajasekaran et al., Mol Biol Cell 2001; Barwe et al., Mol Biol Cell 2005; Inge et al., Histol Histopathol, In Press). In this study, we evaluated whether Na,K-β expression sensitizes A2780/C10B cells to oxaliplatin to gain insight into whether Na,K-β has any diagnostic or therapeutic potential in cancer. In oxaliplatin resistant A2780/C10B ovarian cancer cells Na,K-β subunit expression was undetectable while the oxaliplatin sensitive A2780 parental cells express abundant β-subunit protein. A pooled clone of A2780/C10B (C10B-Na,K-β) expressing Na,K-β was established and levels and membrane localization of this protein were confirmed by immunoblot and immunofluorescence analyses, respectively. We found that C10B-Na,K-β cells were significantly sensitive to oxaliplatin compared to A2780/C10B cells (P N 1 -acetyl transferase mRNA associated with oxaliplatin sensitivity (A2780, ~250-fold) and resistance (C10B, ~7-fold) (Hector et al., Mol Cancer Therap 2004, Hector et al., Proc AACR 2004) did not change between C10B and C10B-Na,K-β cells. These data are consistent with a novel role for Na,K-β subunit in oxaliplatin sensitivity and that reduced expression of this protein might be associated with increased oxaliplatin resistance in cancer cells. This work is supported by NIHCA109619 and NIHDK56216

Purpose 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. Methods 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. Results 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 at >IC 50 concentrations and antagonism at <IC 50 . SMO and SSAT induction occurred in 60 and 30% of the patient samples examined. Conclusions 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.

A single cell clonal sub-line A2780/C10B that is 18-fold resistant to oxaliplatin and ∼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 γ-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: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.METHODS: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.RESULTS: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.CONCLUSION: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.

4088 Background: At 5μM, vorinostat decreases thymidilate synthase (TS) expression by ∼ 40 fold, which translates into synergistic antitumor activity when added to 5-FU. We conducted a phase I study of vorinostat plus FOLFOX in patients with CRC to determine the recommended dose of this combination. Methods: Vorinostat was escalated in a standard 3+3 design with a planned expansion of the maximum tolerated dose (MTD) cohort to 10 patients (pts). Vorinostat (100mg, 200mg, 300mg, 400 mg dose levels) was given twice daily for 1 week followed by 1 week break. FOLFOX was administered at a fixed standard dose every 2 weeks on the 4th day of vorinostat. Tumor biopsies were obtained from liver metastases before and on the 4th day of vorinostat (prior to FOLFOX) to assess TS expression. Results: 19 pts were treated on study (M/F: 12/7; median age: 58; ECOG 0/1: 6/13). All pts had failed prior FOLFOX therapy. Dose-limiting toxicities (DLT) were noted in 3 pts: 2/4 pts at dose level (DL)4 (vorinostat 400mg BID) consisting of grade (G) 3 fatigue, & diarrhea in 1 pt and G3 fatigue in the other; 1/8 pts at DL3 (MTD, vorinostat 300mg BID) consisting of G3 fatigue, anorexia, nausea, and dehydration. 8 pts have been treated at the MTD for a total of 38 cycles. “All Cycles” G3–4 toxicities at the MTD consisted of 2 pts with G3 neutropenia and 2 pts with G3 thrombocytopenia along with the above described DLT. Responses were evaluable in 17 pts: 0 Objective Response, 8 Stable Disease (4 confirmed). TS expression by IHC and by RT-PCR showed modest decreases in 2/6 patients after vorinostat treatment. Cmax of SAHA was < 2μM at all investigated DL, which could explain the lack of adequate TS down-regulation. Conclusions: vorinostat 300mg PO BID × 1 week every 2 weeks in combination with FOLFOX is the established recommended dose. The lack of significant TS down-regulation may be due to the suboptimal serum vorinostat concentrations. Alternate shorter vorinostat schedules may allow for further daily dose escalations and hence for better likelihood of TS down-regulation. This study was partly supported by CTEP, NCI. No significant financial relationships to disclose.

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 x 3 or weekly x 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.