Metabolite dFdU Research Articles

A phase I dose escalation, dose expansion and pharmacokinetic trial of gemcitabine and alisertib in advanced solid tumors and pancreatic cancer

PurposeAurora Kinase A (AKA) inhibition with gemcitabine represents a potentially synergistic cancer treatment strategy via mitotic catastrophe. The feasibility, safety, and preliminary efficacy of alisertib (MLN8237), an oral AKA inhibitor, with gemcitabine was evaluated in this open-label phase I trial with dose escalation and expansion.MethodsKey inclusion criteria included advanced solid tumor with any number of prior chemotherapy regimens in the dose escalation phase, and advanced pancreatic adenocarcinoma with up to two prior chemotherapy regimens. Four dose levels (DLs 1–4) of alisertib (20, 30, 40, or 50 mg) were evaluated in 3 + 3 design with gemcitabine 1000 mg/m2 on days 1, 8, and 15 in 28-day cycles.ResultsIn total, 21 subjects were treated in dose escalation and 5 subjects were treated in dose expansion at DL4. Dose-limiting toxicities were observed in 1 of 6 subjects each in DL3 and DL4. All subjects experienced treatment-related adverse events. Grade ≥ 3 treatment-related adverse events were observed in 73% of subjects, with neutropenia observed in 54%. Out of 22 subjects evaluable for response, 2 subjects (9%) had partial response and 14 subjects (64%) had stable disease. Median PFS was 4.1 months (95% CI 2.1–4.5). No significant changes in pharmacokinetic parameters for gemcitabine or its metabolite dFdU were observed with alisertib co-administration.ConclusionsThis trial established the recommended phase 2 dose of alisertib 50 mg to be combined with gemcitabine. Gemcitabine and alisertib are a feasible strategy with potential for disease control in multiple heavily pre-treated tumors, though gastrointestinal and hematologic toxicity was apparent.

MP03-03 TARGETING V1/CHASE TO OVERCOME GEMCITABINE RESISTANCE

You have accessJournal of UrologyCME1 May 2022MP03-03 TARGETING V1/CHASE TO OVERCOME GEMCITABINE RESISTANCE Semih Sarcan, Daley S. Morera, Marie C. Hupe, Karina Aguilar, Anuj Sharma, Sung Alexander, Joseph McDaniels, Huabin Zhu, Martin Hennig, Martha Terris, Axel S. Merseburger, and Vinata B. Lokeshwar Semih SarcanSemih Sarcan More articles by this author , Daley S. MoreraDaley S. Morera More articles by this author , Marie C. HupeMarie C. Hupe More articles by this author , Karina AguilarKarina Aguilar More articles by this author , Anuj SharmaAnuj Sharma More articles by this author , Sung AlexanderSung Alexander More articles by this author , Joseph McDanielsJoseph McDaniels More articles by this author , Huabin ZhuHuabin Zhu More articles by this author , Martin HennigMartin Hennig More articles by this author , Martha TerrisMartha Terris More articles by this author , Axel S. MerseburgerAxel S. Merseburger More articles by this author , and Vinata B. LokeshwarVinata B. Lokeshwar More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002515.03AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Precision-based treatment approaches could improve the prognosis of patients with bladder cancer (BC). We recently discovered a first-in-class human Chondroitinase (Chase) that degrades chondroitin sulfate. This Chase is a splice variant of HYAL4 (V1) and promotes a malignant phenotype and chemoresistance in BC cells. We evaluated V1/Chase expression and functions in BC and inhibitors to overcome V1/Chase-mediated Gemcitabine (Gem) resistance. METHODS: Cohort 1: 86 bladder tissues (normal (NBL)=34; tumor (TBL)=52); cohort 2: 40 cystectomy specimens from MIBC patients who received adjuvant Gemcitabine plus cisplatin (G+C) treatment. Cohort 3: 144 urine specimens (BCA=42, non-BCa=102). Gene expression was measured by q-PCR in all cohorts and urine was assayed for Chase activity. V1/Chase was stably expressed or silenced in normal urothelial and three BC cell lines. Transfectants were analyzed for sensitivity to Gemcitabine and Cisplatin. The mechanism of Gem resistance was evaluated in preclinical in vitro and in vivo models. RESULTS: In cohort 1, V1 mRNA levels were an independent predictor of metastasis and death due to BC (P=0.0008). In cohort 2, high V1 levels significantly predicted with G+C treatment failure (P <0.0001). In cohort 3, Chase levels were 13 to 75-fold elevated in low and high-grade BC patients’ urine compared to non-BC cases (92.7% sensitivity; 88.2% specificity). V1-expressing urothelial and BC cells were resistant to Gem but not to Cisplatin. V1 expression increased Gem metabolism and subsequent efflux of an inactive metabolite dFdU. V1 increased CD44 shedding the conditioned media. This triggered intracellular activation of the JAK2/STAT3 pathway, resulting in the upregulation of cytidine deaminase (CDA). CDA induces Gem metabolism and dFdU efflux. JAK2, STAT3 and CDA inhibitors, (some FDA-approved), synergistically re- sensitized V1-expressing cells to Gem. V1-induced muscle invasive tumors that were metastatic. While Gem inhibited BC xenograft growth, V1-expressing tumors were resistant. Combination of Gem with a CDA inhibitor (tetrahydrouridine) abrogated V1 tumor growth with minimal toxicity. CONCLUSIONS: V1 is a functional biomarker that drives muscle-invasion, metastasis and Gem resistance in BC. Inhibitors of the JAK2/STAT3/CDA pathway overcome V1- mediated Gem resistance, suggesting a precision-based treatment approach to improve treatment response. Source of Funding: 1R01CA227277-01A1 (VBL); Department of Defence-PRCRP (W81XWH1810277 (CA170470; PRCRP) © 2022 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 207Issue Supplement 5May 2022Page: e19 Advertisement Copyright & Permissions© 2022 by American Urological Association Education and Research, Inc.MetricsAuthor Information Semih Sarcan More articles by this author Daley S. Morera More articles by this author Marie C. Hupe More articles by this author Karina Aguilar More articles by this author Anuj Sharma More articles by this author Sung Alexander More articles by this author Joseph McDaniels More articles by this author Huabin Zhu More articles by this author Martin Hennig More articles by this author Martha Terris More articles by this author Axel S. Merseburger More articles by this author Vinata B. Lokeshwar More articles by this author Expand All Advertisement PDF DownloadLoading ...

Method To Visualize the Intratumor Distribution and Impact of Gemcitabine in Pancreatic Ductal Adenocarcinoma by Multimodal Imaging.

Gemcitabine (dFdC) is a common treatment for pancreatic cancer; however, it is thought that treatment may fail because tumor stroma prevents drug distribution to tumor cells. Gemcitabine is a pro-drug with active metabolites generated intracellularly; therefore, visualizing the distribution of parent drug as well as its metabolites is important. A multimodal imaging approach was developed using spatially coregistered mass spectrometry imaging (MSI), imaging mass cytometry (IMC), multiplex immunofluorescence microscopy (mIF), and hematoxylin and eosin (H&E) staining to assess the local distribution and metabolism of gemcitabine in tumors from a genetically engineered mouse model of pancreatic cancer (KPC) allowing for comparisons between effects in the tumor tissue and its microenvironment. Mass spectrometry imaging (MSI) enabled the visualization of the distribution of gemcitabine (100 mg/kg), its phosphorylated metabolites dFdCMP, dFdCDP and dFdCTP, and the inactive metabolite dFdU. Distribution was compared to small-molecule ATR inhibitor AZD6738 (25 mg/kg), which was codosed. Gemcitabine metabolites showed heterogeneous distribution within the tumor, which was different from the parent compound. The highest abundance of dFdCMP, dFdCDP, and dFdCTP correlated with distribution of endogenous AMP, ADP, and ATP in viable tumor cell regions, showing that gemcitabine active metabolites are reaching the tumor cell compartment, while AZD6738 was located to nonviable tumor regions. The method revealed that the generation of active, phosphorylated dFdC metabolites as well as treatment-induced DNA damage primarily correlated with sites of high proliferation in KPC PDAC tumor tissue, rather than sites of high parent drug abundance.

Comparative pharmacokinetic study of PEGylated gemcitabine and gemcitabine in rats by LC-MS/MS coupled with pre-column derivatization and MSALL technique

Gemcitabine is a small molecular antitumor compound used to treat many types of solid tumors. The clinical application of gemcitabine is limited by its short biological half-life, rapid metabolism and poor tumor tissue targeting. The covalent attachment of polyethylene glycol to gemcitabine is a promising technique to overcome these limitations. After PEGylation, PEGylated gemcitabine could be metabolized into gemcitabine and its metabolites in vivo. Due to the scale effect of PEGylated gemcitabine, the DMPK process of the original drug is greatly changed. Therefore, understanding the pharmacokinetic behavior of PEGylated gemcitabine, gemcitabine and the metabolite dFdU in vivo is really important to clarify the antitumoral activity of these compounds. It would also guide the development of other PEGylated drugs. Due to the complex structure and diverse physiochemical property of PEG, direct quantification analysis of PEGylated gemcitabine presented many challenges in terms of assay sensitivity, selectivity, and robustness. In this article, a data-independent acquisition method, MSALL-based approach using electrospray ionization (ESI) coupled quadrupole time of flight mass spectrometry (MS), was utilized for the determination of PEGylated gemcitabine in rat plasma. The technique consists of a Q1 mass window through all the precursor ions, fragmenting and recording all product ions. PEGylated gemcitabine underwent dissociation in collision cell to generate a series of PEG related ions at m/z 89.0604, 133.0868, 177.1129 of 2, 3, 4 repeating ethylene oxide subunits and PEGylated gemcitabine related ions at m/z 112.0514. PEGylated gemcitabine was detected by the high resolution extracted ions based on the specific compound. For gemcitabine and dFdU, the study used derivatization of these high polarity compounds with dansyl chloride to improve their chromatographic retention. This paper describes comparative pharmacokinetic study of PEGylated gemcitabine and gemcitabine in rats by LC-MS/MS coupled with pre-column derivatization and MSALL technique. The results show that PEGylation could reduce the drug clearance of the conjugated compounds and increase the drug plasma half-life. After administration of PEGylated gemcitabine, the exposure of the free gemcitabine in vivo is lower than administration of gemcitabine, which means that PEGylated gemcitabine possesses lower toxicity compared with gemcitabine.

Cytosolic 5'-nucleotidase 1A is overexpressed in pancreatic cancer and mediates gemcitabine resistance by reducing intracellular gemcitabine metabolites.

BackgroundCytosolic 5′-nucleotidase 1A (NT5C1A) dephosphorylates non-cyclic nucleoside monophosphates to produce nucleosides and inorganic phosphates. Here, we investigate NT5C1A expression in pancreatic ductal adenocarcinoma (PDAC) and its impact on gemcitabine metabolism and therapeutic efficacy.MethodsNT5C1A expression was determined by semiquantitative immunohistochemistry using tissue microarrays. Gemcitabine metabolites and response were assessed in several human and murine PDAC cell lines using crystal violet assays, Western blot, viability assays, and liquid chromatography tandem mass-spectrometry (LC-MS/MS).FindingsNT5C1A was strongly expressed in tumor cells of a large subgroup of resected PDAC patients in two independent patient cohorts (44–56% score 2 and 8–26% score 3, n = 414). In contrast, NT5C1A was expressed at very low levels in the tumor stroma, and neither stromal nor tumoral expression was a prognostic marker for postoperative survival. In vitro, NT5C1A overexpression increased gemcitabine resistance by reducing apoptosis levels and significantly decreased intracellular amounts of cytotoxic dFdCTP in +NT5C1A tumor cells. Co-culture experiments with conditioned media from +NT5C1A PSCs improved gemcitabine efficacy in tumor cells. In vivo, therapeutic efficacy of gemcitabine was significantly decreased and serum levels of the inactive gemcitabine metabolite dFdU significantly increased in mice bearing NT5C1A overexpressing tumors.InterpretationNT5C1A is robustly expressed in tumor cells of resected PDAC patients. Moreover, NT5C1A mediates gemcitabine resistance by decreasing the amount of intracellular dFdCTP, leading to reduced tumor cell apoptosis and larger pancreatic tumors in mice. Further studies should clarify the role of NT5C1A as novel predictor for gemcitabine treatment response in patients with PDAC.

Simultaneous determination of gemcitabine prodrug, gemcitabine and its major metabolite 2′, 2′-difluorodeoxyuridine in rat plasma by UFLC-MS/MS

To improve bioavailability and provide resistance to deamination, an array of gemcitabine (dFdC) prodrugs carrying the acyl modifications has been successful in the optimization of pharmacokinetic properties of dFdC, but the reports about 4-N-carbobenzoxy-dFdC (Cbz-dFdC), a dFdC prodrug bearing alkyloxycarbonyl modification, are relatively rare. Notably, in vivo enzymatic hydrolysis was an absolutely essential factor for the activation of these prodrugs, which is correlated with the anti-tumor activity. Therefore, detailed metabolism studies of Cbz-dFdC should be carried out for a more authentic pharmacodynamic evaluation. In order to detect the pharmacokinetic characteristics of Cbz-dFdC, a selective, sensitive and accurate method for the simultaneous determination of Cbz-dFdC, along with dFdC and its major metabolite dFdU in rat plasma was developed and validated using UFLC–MS/MS techniques. Column was at 40 °C for separation using an eluent with acetonitrile and 0.1% formic acid, 1 mM ammonium formate at a flow rate of 0.2 mL/min. Detection was performed using ESI source in positive ion selected reaction monitoring mode by monitoring the following ion transitions m/z 398.1 → 202.2 (Cbz-dFdC), m/z 264.1 → 112.0 (dFdC), m/z 265.3 → 113.2 (dFdU) and m/z 246.1 → 112.0 (IS). Analytes were extracted by simple precipitation with acetonitrile containing internal standards followed by liquid-liquid extraction with ethyl acetate. The calibration curves of Cbz-dFdC, dFdC and dFdU were linear in the concentration range of 2 to 500 ng/mL, 2 to 500 ng/mL and 40 to 10,000 ng/mL, respectively. The assay ranges selected for the three analytes were appropriate and minimized the need for reanalysis. All the validation data, such as intra- and inter-day precision, accuracy, selectivity and stability, were within the required limits. In conclusion, the sensitive analytical assay was selective and accurate for the determination of rat plasma concentrations of Cbz-dFdC, dFdC and dFdU from a single LC–MS/MS analysis and well-suited to support pharmacokinetic studies.

Population Pharmacokinetics of Gemcitabine and dFdU in Pancreatic Cancer Patients Using an Optimal Design, Sparse Sampling Approach.

Gemcitabine remains a pillar in pancreatic cancer treatment. However, toxicities are frequently observed. Dose adjustment based on therapeutic drug monitoring might help decrease the occurrence of toxicities. In this context, this work aims at describing the pharmacokinetics (PK) of gemcitabine and its metabolite dFdU in pancreatic cancer patients and at identifying the main sources of their PK variability using a population PK approach, despite a sparse sampled-population and heterogeneous administration and sampling protocols. Data from 38 patients were included in the analysis. The 3 optimal sampling times were determined using KineticPro and the population PK analysis was performed on Monolix. Available patient characteristics, including cytidine deaminase (CDA) status, were tested as covariates. Correlation between PK parameters and occurrence of severe hematological toxicities was also investigated. A two-compartment model best fitted the gemcitabine and dFdU PK data (volume of distribution and clearance for gemcitabine: V1 = 45 L and CL1 = 4.03 L/min; for dFdU: V2 = 36 L and CL2 = 0.226 L/min). Renal function was found to influence gemcitabine clearance, and body surface area to impact the volume of distribution of dFdU. However, neither CDA status nor the occurrence of toxicities was correlated to PK parameters. Despite sparse sampling and heterogeneous administration and sampling protocols, population and individual PK parameters of gemcitabine and dFdU were successfully estimated using Monolix population PK software. The estimated parameters were consistent with previously published results. Surprisingly, CDA activity did not influence gemcitabine PK, which was explained by the absence of CDA-deficient patients enrolled in the study. This work suggests that even sparse data are valuable to estimate population and individual PK parameters in patients, which will be usable to individualize the dose for an optimized benefit to risk ratio.

The radiosensitising effect of gemcitabine and its main metabolite dFdU under low oxygen conditions is in vitro not dependent on functional HIF-1 protein

BackgroundRegions within solid tumours often experience oxygen deprivation, which is associated with resistance to chemotherapy and irradiation. The aim of this study was to evaluate the radiosensitising effect of gemcitabine and its main metabolite dFdU under normoxia versus hypoxia and to determine whether hypoxia-inducible factor 1 (HIF-1) is involved in the radiosensitising mechanism.MethodsStable expression of dominant negative HIF-1α (dnHIF) in MDA-MB-231 breast cancer cells, that ablated endogenous HIF-1 transcriptional activity, was validated by western blot and functionality was assessed by HIF-1α activity assay. Cells were exposed to varying oxygen environments and treated with gemcitabine or dFdU for 24 h, followed by irradiation. Clonogenicity was then assessed. Using radiosensitising conditions, cells were collected for cell cycle analysis.ResultsHIF-1 activity was significantly inhibited in cells stably expressing dnHIF. A clear radiosensitising effect under normoxia and hypoxia was observed for both gemcitabine and dFdU. No significant difference in radiobiological parameters between HIF-1 proficient and HIF-1 deficient MDA-MB-231 cells was demonstrated.ConclusionsFor the first time, radiosensitisation by dFdU, the main metabolite of gemcitabine, was demonstrated under low oxygen conditions. No major role for functional HIF-1 protein in radiosensitisation by gemcitabine or dFdU could be shown.

Self-Assembling, Amphiphilic Polymer–Gemcitabine Conjugate Shows Enhanced Antitumor Efficacy Against Human Pancreatic Adenocarcinoma

The therapeutic efficacy of gemcitabine is severely compromised due to its rapid plasma metabolism. Moreover, its hydrophilicity poses a challenge for its efficient entrapment in nanosized delivery systems and to provide a sustained release profile. In this study, gemcitabine was covalently conjugated to poly(ethylene glycol)-block-poly(2-methyl-2-carboxyl-propylene carbonate) (PEG-PCC) which could self-assemble into micelles of 23.6 nm. These micelles afforded protection to gemcitabine from plasma metabolism as evident by negligible amount of gemcitabine and its metabolite dFdU detected in the plasma after 24 h. A controlled release of gemcitabine from the micelles was observed with 53.89% drug release in 10 days in the presence of protease enzyme Cathepsin B. Gemcitabine conjugated micelles were cytotoxic, showed internalization, and induced cell apoptosis in MIA PaCa-2 and L3.6pl pancreatic cancer cell lines. These micelles efficiently inhibited tumor growth when injected intravenously into MIA PaCa-2 cell derived xenograft tumor bearing NSG mice at a dose of 40 mg/kg in terms of reduced tumor volume and tumor weight (0.38 g vs 0.58 g). TUNEL assay revealed that gemcitabine conjugated micelles induced a much higher extent of apoptosis in the tumor tissues compared to free gemcitabine. In conclusion, gemcitabine conjugated micelles were able to enhance the drug payload, protect it from rapid plasma metabolism, and provide a sustained release and showed enhanced antitumor activity, and thus have the potential to provide a better therapeutic alternative for treating pancreatic cancer.

High sensitive assay employing column switching chromatography to enable simultaneous quantification of an amide prodrug of gemcitabine (LY2334737), gemcitabine, and its metabolite dFdU in human plasma by LC–MS/MS

In this study we report a high sensitive method for the simultaneous analysis of LY2334737 (2'-deoxy-2',2'-difluoro-N-(1-oxo-2-propylpentyl)-cytidine), an amide prodrug of gemcitabine (2', 2'-difluoro-deoxycytidine), along with its active drug gemcitabine and its major metabolite dFdU (2',2'-difluoro-deoxyuridine) by LC-MS/MS. Quantification of all three analytes within a single analysis was challenging because the physio-chemical properties of LY2334737 were significantly different from gemcitabine and dFdU and was accomplished by incorporating column-switching. The assay was fully validated to quantify LY2334737 from 0.1 to 100ng/mL, gemcitabine from 0.25 to 100ng/mL and dFdU from 1 to 1000ng/mL in order to cover the diverse concentration ranges expected in clinical samples. A 25-fold dilution was also validated to accommodate any samples outside this range. Overall, the assay had good accuracy (ranging from -7.0 to 1.2% relative error) and precision (ranging from 2.1 to 8.4% relative standard deviation). Extraction efficiency was greater than 80% for all three analytes and there were no matrix effects. Plasma samples were stable for 24h at room temperature, 660 days in frozen storage, and at least 4 freeze-thaw cycles, at both -20 and -70°C. Data from clinical trials showed that plasma concentrations for LY2334737, gemcitabine, and dFdU were successfully quantified from a single LC-MS/MS analysis and that the assay ranges selected for the three analytes were appropriate and minimized the need for reanalysis.

Phase I dose-escalation study and population pharmacokinetic analysis of fixed dose rate gemcitabine plus carboplatin as second-line therapy in patients with ovarian cancer

ObjectiveThis phase I study of fixed dose rate (FDR) gemcitabine and carboplatin assessed the maximum tolerated dose (MTD), dose-limiting toxicities (DLTs), safety, pharmacokinetic (PK)/pharmacodynamic (PD) profile and preliminary anti-tumor activity in patients with recurrent ovarian cancer (OC). MethodsPatients with recurrent OC after first line treatment were treated with carboplatin and FDR gemcitabine (infusion speed 10mg/m2/min) on days 1, 8 and 15, every 28days. Pharmacokinetics included measurement of platinum concentrations in plasma ultrafiltrate (pUF) and plasma concentrations of gemcitabine (dFdC) and metabolite dFdU. Intracellular levels of dFdC triphosphate (dFdC-TP), the most active metabolite of gemcitabine, were determined in peripheral blood mononuclear cells (PBMCs). Population pharmacokinetic modeling and simulation were performed to further investigate the optimal schedule. ResultsTwenty three patients were enrolled. Initial dose escalation was performed using FDR gemcitabine 300mg/m2 (administered at infusion speed of 10mg/m2/min) combined with carboplatin AUC 2.5 and 3. Excessive bone marrow toxicity led to a modified dose escalation schedule: carboplatin AUC 2 and dose escalation of FDR gemcitabine (300mg/m2, 450mg/m2, 600mg/m2 and 800mg/m2). DLT criteria as defined per protocol prior to the study were not met with carboplatin AUC 2 in combination with FDR gemcitabine 300–800mg/m2 because of myelosuppressive dose-holds (especially thrombocytopenia and neutropenia). ConclusionsFDR gemcitabine in combination with carboplatin administered in this 28days schedule resulted in increased grade 3/4 toxicity compared to conventional 30-minute infused gemcitabine. A two weekly schedule (chemotherapy on days 1 and 8) would be more appropriate.

Preclinical Absorption, Distribution, Metabolism, and Excretion of an Oral Amide Prodrug of Gemcitabine Designed to Deliver Prolonged Systemic Exposure

Gemcitabine is an intravenously administered nucleoside analog chemotherapeutic agent. The ability to deliver this agent as an oral drug would allow greater flexibility of administration and patient convenience; however, attempts have been fraught with high first-pass metabolism and potential intestinal toxicity. Alternatively, an amide prodrug of gemcitabine (LY2334737) was discovered, which is able to avoid the extensive first-pass metabolism that occurs following administration of gemcitabine. Preclinical in vitro and in vivo experiments were conducted to evaluate the hydrolysis and pharmacokinetics of LY2334737 and its downstream metabolites. In mice, rats, and dogs, the prodrug is absorbed largely intact across the intestinal epithelium and delivers LY2334737 to systemic circulation. The hydrolysis of LY2334737 is relatively slow, resulting in sustained release of gemcitabine in vivo. In vitro experiments identified carboxylesterase 2 (CES2) as a major enzyme involved in the hydrolysis of LY2334737, but with relatively low intrinsic clearance. Following hydrolysis of the prodrug, gemcitabine is cleared predominantly via the formation of its inactive metabolite dFdU. Both biliary and renal excretion was responsible for the elimination of LY2334737 and its metabolites in both mice and dogs.

Abstract 5722: Retention of the radiosensitizing effect of gemcitabine and its main metabolite dFdU under reduced oxygen conditions is not influenced by HIF-1 functionality

Abstract Introduction: Regions within solid tumors often experience mild to severe oxygen deprivation, associated with resistance to chemotherapy and irradiation. The aim of this study was to evaluate the radiosensitizing effect of gemcitabine and its main metabolite dFdU under normal versus reduced oxygen conditions and to determine whether hypoxia-inducible factor 1 (HIF-1) is involved in the radiosensitizing mechanism. Materials &amp; methods: The clonogenic assay was performed in three isogenic MDA-MB-231 breast cancer cell lines differing in HIF-1alpha proficiency (24h 0-8 nM gemcitabine or 0-4 microM dFdU, 0-8 Gy irradiation). Validation of the transfection with dominant negative HIF-1alpha was done by western blot and by assessment of HIF-1alpha activity. The relative expression of 84 genes related to the hypoxia signaling pathway was characterized by human hypoxia signaling pathway PCR array. Using radiosensitizing conditions, cells were collected for cell cycle analysis. Results: HIF-1 activity was significantly inhibited after transfection with a dominant negative protein. Furthermore, anoxia-induced VEGF secretion was significantly lower (p&amp;lt;0.05) in MDA-MB-231 cells stably expressing dnHIF (dominant negative HIF-1alpha) than in MDA-MB-231 EV (empty vector) cells. PCR array indicated that hypoxic conditions significantly influenced the expression of HIF-1alpha and ANGPTL4 (angiopoetin-like 4). A clear radiosensitizing effect under normoxic and anoxic conditions was observed for both gemcitabine and dFdU. The radiosensitizing effect was similar under normoxic and anoxic conditions (p=0.48 for gemcitabine, p=0.56 for dFdU) and the dose enhancement factor (DEF) was not significantly influenced by the cell line used. Under anoxia, DEFs for gemcitabine ranged from 1.26 to 1.59, from 1.11 to 1.77, and from 1.34 to 2.04 in MDA-MB-231 wt, EV and dnHIF cells, respectively. Statistical analysis revealed no significant differences in radiobiological parameters between HIF-1 proficient and HIF-1 deficient cells. Cell cycle analysis showed that, in contrast to previous findings in other cell lines, exposure of MDA-MB-231 cells to low oxygen conditions did not induce a significant increase in the percentage of G0/1 cells (p=0.21). Gemcitabine and dFdU caused a block of cells in the S phase of the cell cycle under both normoxic and anoxic conditions. Conclusion: For the first time, radiosensitization by dFdU, the main metabolite of gemcitabine, was demonstrated under low oxygen conditions. As dFdU has a prolonged half-life, the sustained presence of dFdU in the blood might induce radiosensitization despite the short half-life of the parent drug, gemcitabine. This might be highly relevant, especially considering delivery of the drug to hypoxic tumor regions. No major role for HIF-1 in radiosensitization by gemcitabine or dFdU could be shown. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5722. doi:1538-7445.AM2012-5722

Combinatorial Pharmacologic Effects of Gemcitabine and its Metabolite dFdU

Recent evidence has shown that the gemcitabine metabolite, dFdU, is pharmacologically active. Though less potent, dFdU has a longer half-life and could potentiate or antagonize the activity of gemcitabine. Hence, studies were undertaken to evaluate the combined effects. Following chemical synthesis, an improved purification procedure for dFdU was developed (80 % yield; >99 % purity). Zebrafish phenotype-based embryo screens revealed no acute toxicity after gemcitabine or dFdU treatment. Only gemcitabine affected zebrafish development in a dose-dependent manner. Synergy or antagonism for the combination was not observed. Antitumor effects for dFdU were dose dependent. Antagonism was tumor cell-line dependent and did not depend on formation of the intracellular active metabolite of gemcitabine, suggesting that the drug-metabolite interaction occurs later. These studies highlight a platform for testing the pharmacologic activity for anticancer agent and metabolite combinations. Such analyses are expected to provide insight into the beneficial or harmful effect(s) of metabolites towards parent drug activity.

Paclitaxel alters the expression and specific activity of deoxycytidine kinase and cytidine deaminase in non-small cell lung cancer cell lines

BackgroundWe observed that paclitaxel altered the pharmacokinetic properties of gemcitabine in patients with non-small cell lung cancer (NSCLC) and limited the accumulation of gemcitabine and its metabolites in various primary and immortalized human cells. Therefore, we classified the drug-drug interaction and the effects of paclitaxel on deoxycytidine kinase (dCK) and cytidine deaminase (CDA) in three NSCLC cell lines. These enzymes are responsible for the metabolism of gemcitabine to its deaminated metabolite dFdU (80% of the parent drug) and the phosphorylated metabolites dFdCMP, dFdCDP and dFdCTP. These metabolites appear to relate to sensitivity and tolerability of gemcitabine based on previous animal and laboratory studies.MethodsThree immortalized human cells representative of the most common histological subtypes identified in patients with advanced NSCLC were exposed to the individual drugs or combinations to complete a multiple drug effect analysis. These same cell lines were exposed to vehicle-control or paclitaxel and the mRNA levels, protein expression and specific activity of dCK and CDA were compared. Comparisons were made using a two-tailed paired t-test or analysis of variance with a P value of < 0.05 considered significant.ResultsThe multiple drug effect analysis indicated synergy for H460, H520 and H838 cells independent of sequence. As anticipated, paclitaxel-gemcitabine increased the number of G2/M cells, whereas gemcitabine-paclitaxel increased the number of G0/G1 or S cells. Paclitaxel significantly decreased dCK and CDA mRNA levels in H460 and H520 cells (40% to 60%, P < 0.05) and lowered dCK protein (24% to 56%, P < 0.05) without affecting CDA protein. However, paclitaxel increased both dCK (10% to 50%) and CDA (75% to 153%) activity (P < 0.05). Paclitaxel caused substantial declines in the accumulation of the deaminated and phosphorylated metabolites in H520 cells (P < 0.05); the metabolites were not measurable in the remaining two cell lines. The ratio of dCK to CDA mRNA levels corresponded to the combination index (CI) estimated for sequential paclitaxel-gemcitabine.ConclusionIn summary, paclitaxel altered the mRNA levels and specific activity of dCK and CDA and these effects could be dependent on histological subtype. More cell and animal studies are needed to further characterize the relationship between mRNA levels and the overall drug-drug interaction and the potential to use histological subtype as a predictive factor in the selection of an appropriate anticancer drug regimen.

First-in-human study of a novel nucleoside analogue, CP-4126, in patients with advanced solid tumors

2577 Background: CP-4126 (gemcitabine 5'-elaidic acid ester) is a novel nucleoside analogue with proven preclinical antitumor activity. Unlike gemcitabine, the intracellular uptake of CP-4126 is independent of nucleoside transporters. The aims of this study were to determine the safety, toxicity, MTD and the RD of CP-4126, to describe its pharmacokinetic (PK) characteristics, and to assess its preliminary antitumor activity. Methods: Patients (pts) with refractory solid tumors, performance status ECOG &lt; 2, with adequate haematologic, renal and hepatic function were enrolled in this dose escalation study (1–6 pts per dose level (DL)). CP-4126 was administered on days (d) 1, 8 and 15 every 4 week by a 30 min IV infusion. Start dose was 30 mg/m2/d and the dose was increased by 100% until toxicity &gt; CTCAE grade 2 occurred. Standard DLT definitions were used. Activity was assessed at the end of every 2nd cycle (cy). Plasma and urine PK were determined during d1 (24 hrs) of cy1; plasma at all DLs and urine at DL= 1400 mg/m2/d. Results: 39 pts have been included, (m =24; f =15 ), median age 60 (range 19–78), receiving 96 cycles (range 1–9) of treatment, with 1 pt/DL from 30 to 240 mg/m2/d. The first grade 2 AE (neutropenia) was reported at 480 mg/m2/d. Most frequent toxicities include nausea, vomiting, fatigue and anorexia, the majority of mild severity (grade 1–2). 5 DLTs have been reported; 800 mg/m2/d (1 pt - d8 dose delay &gt;2 weeks due to grade 3 thrombocytopenia and anaemia); 1000 mg/m2/d (1 pt died 48 hrs after treatment start due to acute lung damage); 1400 mg/m2/d (1 pt - fatigue grade 3); 2 pts at 1600 mg/m2/d (grade 3 ALT/AST elevation [1 pt]; 1 pt grade 4 neutropenia and grade 3 fatigue). Stabilisation of disease (≥ 3 months) reported in 5 pts (pancreas, colon and ovarian cancer) lasting between 3.5 to 8 months. One ovarian pt had a 28.3% reduction in tumor mass. CP-4126 was detected in plasma up to 24 hrs post-dosing. AUC for dFdC exposure was significantly higher than reported with gemcitabine at comparable dose levels. Urinary excretion of the main metabolite dFdU during the first 24 hrs was approximately 60% of dose. Conclusions: CP-4126 is well tolerated and accrual is ongoing at 1400 mg/m2/d to establish RD for phase II studies. Updated results including plasma and urine PK will be presented. [Table: see text]

Phase I study of oral gemcitabine prodrug (LY2334737) alone and in combination with erlotinib in patients (pts) with advanced solid tumors

2576 Background: LY2334737 (LY) is an orally available valproic acid prodrug of gemcitabine that was developed to overcome the extensive first-pass metabolism of gemcitabine to 2',2'-difluorodeoxyuridine (dFdU). The objectives of this study were to determine the maximum tolerated dose (MTD), dose limiting toxicity (DLT) and pharmacokinetics (PK) of LY as monotherapy and in combination with erlotinib. Methods: Eligible pts had ECOG PS &lt; 2 and adequate hematologic, renal and hepatic function. In Arm A, LY was given daily for 14 days in a 3-week cycle. Pts assigned to Arm B also received erlotinib daily 100 mg continuously. Dose escalation was based on observed toxicity and the modified continual reassessment method (mCRM). The dose was maximally increased by 100% depending on the toxicity observed in the previous cohort. PK of LY, gemcitabine, dFdU and intracellular metabolites were determined. Results: 33 pts (21 m, 12 f, median age 60 yrs (range 24–81)) were treated at 5 different dose-levels (range 5–50 mg/day). Pts received a median of 3 cycles (range 2–17). Three out of 7 pt treated with 50 mg experienced 5 dose limiting toxicities (DLT). DLTs observed at 40 and 50 mg include fatigue (4 pt), thrombocytopenia (1 pt), GGT elevation (1 pt), AST/ALT elevation (1 pt), fever (1 pt), and pulmonary embolism (1 pt). One death was possibly related to LY intake. This pt, treated with 40 mg LY, developed on day 15 dyspnea, hypovolemic shock, and suddenly died. No grade 3 or 4 toxicities were reported at dose-levels &lt; 40 mg. The most common adverse events were fatigue, vomiting, nausea, pyrexia, anorexia, and diarrhea. Two pts with mesothelioma were stable for &gt; 9 months. One pt with refractory prostate cancer presented a PSA CR as assessed by investigator. The PK show dose-proportional increase in exposure of both LY and gemcitabine. Both LY and gemcitabine are rapidly cleared, thus no accumulation occurs. The metabolite dFdU accumulates due to its long half life. Conclusions: LY displays linear PK. The dose level of 50-mg is non-tolerable and 40-mg is being confirmed as the MTD as single agent and in combination with 100 mg erlotinib. Antitumor activity warrants further development. Pt accrual is ongoing. [Table: see text]

Extensive Metabolism and Hepatic Accumulation of Gemcitabine After Multiple Oral and Intravenous Administration in Mice

In a clinical study with oral gemcitabine (2',2'-difluorodeoxycytidine, dFdC), we found that gemcitabine was hepatotoxic and extensively metabolized to 2',2'-difluorodeoxyuridine (dFdU) after continuous oral dosing. The main metabolite dFdU had a long terminal half-life after oral administration. Our hypothesis was that dFdU and/or phosphorylated metabolites of gemcitabine accumulated in the liver after multiple oral dosing. In this study, mice were treated with oral or i.v. dFdC at a single dose (1qdx1d) or at multiple doses once daily for 7 days (1qdx7d) or seven times daily (7qdx1d). Blood, liver, kidneys, and lungs were collected at several time points. Urine samples were collected after i.v. dFdC, and peripheral blood mononuclear cells were collected 7qdx1d dosing of dFdC. The nucleosides dFdC and dFdU as well as the nucleotides gemcitabine monophosphate (dFdC-MP), diphosphate, and triphosphate (dFdC-TP) and dFdU monophosphate, diphosphate (dFdU-DP), and triphosphate (dFdU-TP) were simultaneously quantified by high-performance liquid chromatography with ultraviolet and radioisotope detection. We demonstrate that phosphorylated metabolites of both dFdC and dFdU are formed in mice, primarily consisting of dFdC-MP, dFdC-TP, and dFdU-TP. Multiple dosing of dFdC leads to substantial hepatic and renal accumulation of dFdC-TP and dFdU-TP, which have a more pronounced liver accumulation after oral than after i.v. dosing. The presence of dFdC-MP, dFdC-TP, and dFdU-TP in plasma and urine suggests efflux of these potentially toxic metabolites. Our results show that dFdU, dFdC-TP, and dFdU-TP accumulate in the liver after multiple dosing of dFdC in mice and might be associated with hepatotoxicity of oral dFdC in patients.

Measurement of Plasma Concentration of Gemcitabine and Its Metabolite dFdU in Hemodialysis Patients with Advanced Urothelial Cancer

We investigated the pharmacokinetics of gemcitabine and its metabolite in two male patients (52 and 56-year-old) with advanced urothelial cancer receiving hemodialysis three times a week. Gemcitabine, 1000 mg/m(2) in 100 ml of saline, was intravenously administered for 30 min. The concentration of gemcitabine and its metabolite 2',2'-difluorodeoxyuridine (dFdU) was measured at several given time points using a high-pressure liquid chromatography assay. Pharmacokinetic parameters were determined using the two-compartment modeling program. Gemcitabine was rapidly eliminated from plasma even in patients with renal dysfunction. No obvious differences in pharmacokinetic parameters such as the t(1/2), AUC and C(max) of gemcitabine were observed between the patients on hemodialysis and those with normal renal function in previous reports. On the other hand, dFdU showed a sustained level until hemodialysis was initiated. Hemodialysis could reduce the plasma dFdU level by approximately 50%. According to the previous information, no dose modification of gemcitabine may be required for patients with renal impairment or hemodialysis. However, gemcitabine should be given with caution because only limited information is available, and the clinical effect of sustained and/or accumulated dFdU is unknown.

Gemcitabine (G) plamsa and intracellular pharmacokinetics in E6201: Greater metabolite levels using fixed dosing rate (FDR) delivery

2024 Background: Gemcitabine (G) is an antimetabolite which requires activation by the rate-limiting enzyme dCK to its active metabolite, dFdCTP. FDR delivery of G at 10 mg/m2/minute may allow greater formation of active metabolite and improved activity. E6201 randomly assigned first line pancreatic cancer patients to three arms: A (G 1000 mg/m2 over 30 minutes), B FDR G (1500 mg/m2 over 150 minutes), and C GemOx (1000 mg/m2 over 100 minutes with oxaliplatin 100 mg/m2). Methods: Investigators from 18 centers contributed 23 sample sets on the first dose of G. Five time points over 4 hrs were sampled (baseline, mid infusion, end infusion, 1–2 post and 4 hrs). Plasma fractionation along with the PBMC purification and subsequent perchloric acid extraction were conducted locally with THU 1 mg (cytidine deaminase inhibitor). Samples were shipped overnight to the central lab where G and its metabolite dFdU were quantified from deproteinized plasma by reverse phase HPLC. dFdCTP was quantified by ion-exchange HPLC in neutralized PBMC extracts after removal of ribonucleotide triphosphates. Data were fit to nonlinear models (WinNonLin, v4.1) and comparisons amongst dosing groups employed the non-parametric, 2-sided Mann-Whitney test. Results: For Arms A, B and C the plasma AUCs were respectively (median ± SD): 4542 ± 2472 (N=9), 8603 ± 2608 (N=8) and 9130 ± 8788 (N=6) ng/ml x hr. The difference between group A and B (p=0.003) and A and C (p=0.05) were statistically different. Intracellular dFdCTP AUCs for groups A, B, and C were: 1862 ± 794 (N=8); 3725 ± 7763 (N=8) and 5024 ± 2113 (N=5) and significantly different for both A vs. B (p=0.05) and borderline significant for A vs C (p=0.065). The latter comparison is limited by a small number of samples. Conclusions: When studied in a multi-centered, randomized cooperative group setting, FDR delivery of G results in higher plasma AUC, and increased intracellular levels of active metabolite dFdCTP. These data support the concept that fixed dose rate delivery of G results in greater intracellular metabolite production at similar and equitoxic doses. Supported by ECOG NYU620HH04–00, NCI CA16087, CA2115, and from Eli Lilly. No significant financial relationships to disclose.