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Background Cisplatin/gemcitabine is standard first-line treatment for patients with advanced biliary tract cancer (ABC). NUC-1031 (phosphoramidate transformation of gemcitabine) is designed to enhance efficacy by maximizing intratumoral active metabolites. Methods Patients with untreated ABC, Eastern Cooperative Oncology Group performance status 0–1 received NUC-1031 (625 or 725 mg/m2) and cisplatin (25 mg/m2) on days 1 and 8, every 21 days. Primary objectives were safety and maximum tolerated dose; secondary objectives were objective response rate (ORR), pharmacokinetics, progression-free survival (PFS), and overall survival (OS). Results Twenty-one patients (median age 61 years, n = 13 male; 17 cholangiocarcinoma, 2 ampullary, and 2 gallbladder cancer) received NUC-1031 625 mg/m2 (n = 8 and expansion n = 7; median six cycles) or 725 mg/m2 (n = 6; median 7.5 cycles). Treatment was well tolerated; most common treatment-emergent grade 3–4 adverse events occurring in more than one patient with 625 mg/m2 NUC-1031 were increased gamma-glutamyl transferase (GGT), 40%; alanine aminotransferase, 20%; bilirubin, 13%; neutropenia, 27%; decreased white cell count, 20%; thrombocytopenia, 13%; nausea, 13%; diarrhea, 13%; fatigue, 13%; and thrombus, 20% and with 725 mg/m2, increased GGT, 67%, and fatigue, 33%. NUC-1031 725 mg/m2 was selected as the recommended dose with cisplatin in ABC. ORR was 33% (one complete response, six partial responses), DCR was 76%, median PFS was 7.2 months (95% confidence interval [CI], 4.3–10.1), and median OS was 9.6 months (95% CI, 6.7–13.1). The median estimates of area under the plasma concentration–time curve from time 0 to last measurable time and maximum concentration were highest for NUC-1031 (218–324 μg•h/mL and 309–889 μg/mL, respectively) and lowest for di-fluoro-deoxycytidine (0.47–1.56 μg•h/mL and 0.284–0.522 μg/mL, respectively). Conclusion This is the first study reporting on the combination of NUC-1031 with cisplatin in ABC and demonstrated a favorable safety profile; 725 mg/m2 NUC-1031 in combination with cisplatin is undergoing phase III trial evaluation in ABC. (ClinicalTrials.gov ID: NCT02351765; EudraCT ID: 2015-000100-26). Implications for Practice The prognosis for patients with advanced biliary tract cancer (ABC) is approximately 1 year, and new treatment options are required. The cisplatin/gemcitabine combination is standard first-line treatment for patients with ABC. NUC-1031 is a phosphoramidate transformation of gemcitabine and is designed to enhance efficacy by maximizing intratumoral active metabolites. This phase Ib study (ABC-08) demonstrated a favorable safety profile of NUC-1031 in combination with cisplatin for the first-line treatment of patients with ABC, and 725 mg/m2 NUC-1031 was recommended in combination with cisplatin for phase III trial evaluation; the NuTide:121 global randomized study is currently enrolling.

Previous studies have shown that sphingosine kinase interacting protein (SKIP) inhibits sphingosine kinase (SK) function in fibroblasts. SK phosphorylates sphingosine producing the potent signaling molecule sphingosine-1-phosphate (S1P). SKIP gene (SPHKAP) expression is silenced by hypermethylation of its promoter in acute myeloid leukemia (AML). However, why SKIP activity is silenced in primary AML cells is unclear. Here, we investigated the consequences of SKIP down-regulation in AML primary cells and the effects of SKIP re-expression in leukemic cell lines. Using targeted ultra-HPLC-tandem MS (UPLC-MS/MS), we measured sphingolipids (including S1P and ceramides) in AML and control cells. Primary AML cells had significantly lower SK activity and intracellular S1P concentrations than control cells, and SKIP-transfected leukemia cell lines exhibited increased SK activity. These findings show that SKIP re-expression enhances SK activity in leukemia cells. Furthermore, other bioactive sphingolipids such as ceramide were also down-regulated in primary AML cells. Of note, SKIP re-expression in leukemia cells increased ceramide levels 2-fold, inactivated the key signaling protein extracellular signal-regulated kinase, and increased apoptosis following serum deprivation or chemotherapy. These results indicate that SKIP down-regulation in AML reduces SK activity and ceramide levels, an effect that ultimately inhibits apoptosis in leukemia cells. The findings of our study contrast with previous results indicating that SKIP inhibits SK function in fibroblasts and therefore challenge the notion that SKIP always inhibits SK activity.

A 3'-protected route toward the synthesis of the diastereomers of clinically active ProTides, NUC-1031 and NUC-3373, is described. The in vitro cytotoxic activities of the individual diastereomers were found to be similar to their diastereomeric mixtures. In the KG1a cell line, NUC-1031 and NUC-3373 have preferential cytotoxic effects on leukemic stem cells (LSCs). These effects were not diastereomer-specific and were not observed with the parental nucleoside analogues gemcitabine and FUDR, respectively. In addition, NUC-1031 preferentially targeted LSCs in primary AML samples and cancer stem cells in the prostate cancer cell line, LNCaP. Although the mechanism for this remains incompletely resolved, NUC-1031-treated cells showed increased levels of triphosphate in both LSC and bulk tumor fractions. As ProTides are not dependent on nucleoside transporters, it seems possible that the LSC targeting observed with ProTides may be caused, at least in part, by preferential accumulation of metabolized nucleos(t)ide analogues.

Purpose: NUC-1031 is a first-in-class ProTide modification of gemcitabine. In PRO-002, NUC-1031 was combined with carboplatin in recurrent ovarian cancer. Patients and Methods: NUC-1031 was administered on days 1 and 8 with carboplatin on day 1 every 3 weeks for up to six cycles. Four dose cohorts of NUC-1031 (500, 625, and 750 mg/m2) with carboplatin (AUC4 or 5) were investigated. Primary endpoint was recommended phase II combination dose (RP2CD). Secondary endpoints included safety, investigator-assessed objective response rate (ORR), clinical benefit rate (CBR), progression-free survival (PFS), and pharmacokinetics. Results: A total of 25 women with recurrent ovarian cancer, a mean of 3.8 prior lines of chemotherapy, and a median platinum-free interval of 5 months (range: 7–451 days) were enrolled; 15 of 25 (60%) were platinum resistant, 9 (36%) were partially platinum sensitive, and 1 (4%) was platinum sensitive. Of the 23 who were response evaluable, there was 1 confirmed complete response (4%), 5 partial responses (17%), and 8 (35%) stable disease. The ORR was 26% and CBR was 74% across all doses and 100% in the RP2CD cohort. Median PFS was 27.1 weeks. NUC-1031 was stable in the plasma and rapidly generated high intracellular dFdCTP levels that were unaffected by carboplatin. Conclusions: NUC-1031 combined with carboplatin is well tolerated in recurrent ovarian cancer. Highest efficacy was observed at the RP2CD of 500 mg/m2 NUC-1031 on days 1 and 8 with AUC5 carboplatin day 1, every 3 weeks for six cycles. The ability to deliver carboplatin at AUC5 and the efficacy of this schedule even in patients with platinum-resistant disease makes this an attractive therapeutic combination.

Background Gemcitabine is used to treat a wide range of tumours but its efficacy is limited by cancer cell resistance mechanisms. NUC-1031, a phosphoramidate modification of gemcitabine, is the first anti-cancer ProTide to enter the clinic and is designed to overcome these key resistance mechanisms. Methods Sixty-eight patients with advanced solid tumours who had relapsed after treatment with standard therapy were recruited to a dose escalation study to determine the recommended Phase II dose (RP2D) and assess safety of NUC-1031. Pharmacokinetics and anti-tumour activity was also assessed. Results Sixty-eight patients received treatment, 50% of whom had prior exposure to gemcitabine. NUC-1031 was well-tolerated with the most common Grade 3/4 adverse events of neutropaenia, lymphopaenia and fatigue occurring in 13 patients each (19%). In 49 response-evaluable patients, 5 (10%) achieved a partial response and 33 (67%) had stable disease, resulting in a 78% disease control rate. Cmax levels of the active intracellular metabolite, dFdCTP, were 217-times greater than those reported for equimolar doses of gemcitabine, with minimal toxic metabolite accumulation. The RP2D was determined as 825 mg/m2 on Days 1, 8 and 15 of a 28-day cycle. Conclusion NUC-1031 was well-tolerated and demonstrated clinically significant anti-tumour activity, even in patients with prior gemcitabine exposure and in cancers not traditionally perceived as gemcitabine-responsive. Trial registration: clinicaltrials.gov identifier: NCT01621854

Background: NUC-1031 is a ProTide transformation of the nucleoside analog gemcitabine, designed to overcome key cancer resistance mechanisms and generate significantly higher levels of the active metabolite, dFdCTP. Its incorporation into DNA interrupts cell replication and induces DNA damage, leading to cell death. NUC-1031 has shown broad clinical activity across a range of solid tumors as both a single agent and in combination with platinum agents. Increasing evidence in the clinical setting in both ovarian and biliary tract cancer populations suggests potential synergy between NUC-1031 and platinum agents, which may lead to increased anti-tumor activity. The aim of this study was to investigate the dose and time relationships between the incorporation of dFdCTP into DNA, the effect on the cell cycle and DNA damage response in biliary tract cancer cells treated with NUC-1031. Methods: Human intrahepatic cholangiocarcinoma HuCCT1 cells were treated with 500 nM (half-IC50 dose) or 1 µM (IC50 dose) NUC-1031 for 24h and samples were collected every 24h up to 96h. The intracellular conversion of NUC-1031 to its active metabolite dFdCTP was assessed by liquid chromatography mass spectrometry (LC-Q-TOF). This technique was also used to determine the incorporation of dFdCTP into DNA, using the dFdC signature as a surrogate and deoxyguanosine (2dG) for normalization. Flow cytometry was used to measure cell cycle populations and to analyse histone variant H2AX phosphorylation (γH2AX) as a marker for response to DNA damage, where increased signal is related to increase in response. Results: NUC-1031 generated dFdCTP in HuCCT1 cells. The incorporation of this fluorinated deoxycytidine into DNA was dose-dependent over time. At 48h post-treatment, the ratio of dFdC:2dG was 0.70 at half-IC50 dose, with comparable levels at IC50 dose. After 48h, the ratio continued to rise for the 1 µM dose but decreased with 500 nM. The incorporation of the active metabolite was accompanied by an increase in cells in S phase, up to 46% for 500 nM and 64% for 1 µM. This dose-dependent increase coincides with H2AX phosphorylation over time up to 48h. Active metabolite incorporation, S phase population and yH2AX signals reduced towards the 96h time point. Conclusion: NUC-1031 is a potent cytotoxic agent that causes DNA damage through the incorporation of its active metabolite into DNA, in a cell cycle dependant manner. This incorporation induces S phase arrest and evokes the DNA damage response via generation of double-strand breaks. The cytotoxic effect of NUC-1031 is prolonged post-treatment. Ongoing studies are investigating NUC-1031 in combination with cisplatin, where relative levels of dFdCTP incorporated in tumor DNA may act as a pharmacodynamic biomarker to determine synergy in patients who receive the combination treatment. Citation Format: Dillum Patel, Alison L. Dickson, Oliver J. Read, Clarissa M. Czekster, Essam A. Ghazaly, David J. Harrison, Jennifer Bré. NUC-1031 causes incorporation of fluorinated deoxycytidine into DNA, inducing persistent damage in biliary tract cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1003.

3030 Background: The inhibition of cellular nucleotide metabolism to promote apoptosis is a key principle of cancer therapy. This, in combination with platinum-induced DNA-damage, is key to promoting anti-cancer activity in a variety of tumors, including ovarian, biliary tract, lung, breast and bladder. NUC-1031, a phosphoramidate transformation of gemcitabine is designed to overcome resistance mechanisms that limit the efficacy of this nucleoside analog. NUC-1031 has shown broad clinical activity across multiple solid tumors as both a single agent and in combination with platinum agents. We show potential synergism between NUC-1031 and a platinum agent in advanced ovarian (OC) and biliary tract (BTC) cancers. Methods: PRO-002 was a phase Ib study; 25 patients (pts) with recurrent OC who had exhausted all other therapy options received NUC-1031 + carboplatin. 17 pts were considered platinum resistant (10) or platinum refractory (7). ABC-08 is a phase Ib study, 14 pts with advanced BTC treated in the first-line setting with NUC-1031 + cisplatin. Results: In PRO-002, strong efficacy signals were observed in non-platinum-responsive patients. Of the 17 response-evaluable platinum-resistant or refractory pts, 5 partial responses (PRs) and 11 stable diseases (SDs) were achieved, resulting in an ORR of 29% and a DCR of 94%. NUC-1031 + carboplatin was well-tolerated with no unexpected AEs; DLTs were myelosuppression and fatigue. Encouraging response rates were also observed in ABC-08 compared to historical standard of care (ABC-02). One CR (7%), 6 PRs (43%) and 1 SD (7%) were observed, resulting in an ORR of 50%. NUC-1031 + cisplatin was well-tolerated, with no unexpected AEs or DLTs. Complementary in vitro evidence suggests that the beneficial interaction occurs whereby platinum treatment sensitizes cells to NUC-1031. Conclusions: Increasing evidence suggests that NUC-1031 in combination with a platinum agent may have synergistic properties, leading to enhanced anti-cancer activity. In both OC and BTC, durable tumor shrinkage was observed. This was particularly encouraging in a platinum resistant/refractory OC population. Future studies utilizing both NUC-1031 plus a platinum agent will further elucidate the potential of this therapeutic combination.

TPS3617Background: 5-FU and its other forms, floxuridine and capecitabine, exert their anti-cancer activity mainly due to the active metabolite, fluorodeoxyuridine-monophosphate (FUDR-MP), which in...

TPS544 Background: The UK ABC-02 study established cisplatin and gemcitabine as the reference regimen for first-line treatment of patients (pts) with advanced biliary tract cancers (BTCs) (median overall survival (OS): 11.7 months). No clinical studies since ABC-02 have reported an extension in OS, and therefore effective new agents/combinations are required. NUC-1031 was designed to improve on gemcitabine’s relatively poor efficacy by overcoming its associated key cancer resistance mechanisms, through cellular uptake independent of nucleoside transporters, activation independent of deoxycytidine kinase and protection from cytidine deaminase inactivation, resulting in over 200x the intracellular levels of the anti-cancer metabolite, dFdCTP, greater stability and reduction in the generation of toxic metabolites. NUC-1031 showed activity as monotherapy in a phase I/II study in 7 pts with BTC, refractory to all standard treatments (Blagden et al ASCO 2015; abstract 2514). Methods: ABC-08 is a multi-centre phase Ib study of NUC-1031 combined with cisplatin in pts with non-resectable or recurrent/metastatic cholangiocarcinoma, gallbladder or ampullary carcinoma, aged ≥18 years with an ECOG performance status of 0-1, who have received no prior systemic therapy. The starting dose for NUC-1031 is 625 mg/m2 administered IV on days 1 and 8 in combination with cisplatin (standard dose of 25 mg/m2) (21 day schedule). The dose will be escalated sequentially in cohorts of 3-6 pts using an accelerated titration procedure (725mg/m2, 825mg/m2, 925mg/m2). Treatment will continue until intolerable toxicity/progressive disease. The primary endpoints are safety and RP2D. Secondary endpoints are progression-free survival, OS, response rate and pharmacokinetic endpoints, including assessments of multiple plasma and intracellular analytes: NUC-1031, cisplatin, dFdC, dFdCMP, dFdCDP, dFdCTP and dFdU, and will be correlated with safety profile and clinical activity. Planned accrual is 15-24 pts over 2 years. Cohort 1 has been completed without dose-limiting toxicities. Enrolment to cohort 2 is on-going. Clinical trial information: NCT02351765.

Ovarian cancer is the most lethal of gynecological cancers killing 60% of women diagnosed with the disease within 5 years. The major contributor to this high mortality is the emergence of chemotherapy resistance; the tumor is initially sensitive to chemotherapy (especially cisplatin, the mainstay of treatment) but recurs with increasingly resistant disease. Effective methods of overcoming treatment resistance are a major unmet medical need and would prolong survival and improve quality of life for women with this disease. We have shown previously that the RNA binding protein La-related protein 1 (LARP1) binds and post-transcriptionally regulates the stability of mRNAs encoding cell survival and stress response proteins including mTOR, BCL2 and BIK. In ovarian cancer tissue, elevated levels of LARP1 protein correlate with adverse survival outcome and chemotherapy resistance. In vivo inhibition of LARP1 using therapeutic RNA interference (packaged in DOPC nanoliposomes) restores cisplatin sensitivity in resistant ovarian cancer xenograft models. In concurrent studies, using a novel ultra-high performance liquid chromatography tandem mass spectrometry method, we have quantified LARP1 in the circulation of ovarian cancer patients and found that high levels correspond with poor prognosis. Circulating LARP1 has prognostic significance and may act as a companion biomarker to a LARP1 inhibitor. We conclude that LARP1, through its regulation of multiple mRNAs within critical pathophysiological pathways, is an important cancer therapeutic target and that RNA-based drugs designed to target LARP1 restore chemotherapy sensitivity in xenograft models. Citation Format: Essam A. Ghazaly, John Le Quesne, Dahai Jiang, Selanere L. Mangala, James Chettle, Cristian Rodriguez-Aguayo, Gabriel Lopez-Berestein, Chathunissa Gnanaranjan, Manuela Mura, Chara Stavraka, Anil K. Sood, Sarah P. Blagden. The RNA-binding protein LARP1 is a cancer therapeutic target. [abstract]. In: Proceedings of the AACR Special Conference on Translational Control of Cancer: A New Frontier in Cancer Biology and Therapy; 2016 Oct 27-30; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2017;77(6 Suppl):Abstract nr B30.

Targeted therapies have yet to have significant impact on the survival of patients with bladder cancer. In this study, we focused on the urea cycle enzyme argininosuccinate synthetase 1 (ASS1) as a therapeutic target in bladder cancer, based on our discovery of the prognostic and functional import of ASS1 in this setting. ASS1 expression status in bladder tumors from 183 Caucasian and 295 Asian patients was analyzed, along with its hypothesized prognostic impact and association with clinicopathologic features, including tumor size and invasion. Furthermore, the genetics, biology, and therapeutic implications of ASS1 loss were investigated in urothelial cancer cells. We detected ASS1 negativity in 40% of bladder cancers, in which multivariate analysis indicated worse disease-specific and metastasis-free survival. ASS1 loss secondary to epigenetic silencing was accompanied by increased tumor cell proliferation and invasion, consistent with a tumor-suppressor role for ASS1. In developing a treatment approach, we identified a novel targeted antimetabolite strategy to exploit arginine deprivation with pegylated arginine deiminase (ADI-PEG20) as a therapeutic. ADI-PEG20 was synthetically lethal in ASS1-methylated bladder cells and its exposure was associated with a marked reduction in intracellular levels of thymidine, due to suppression of both uptake and de novo synthesis. We found that thymidine uptake correlated with thymidine kinase-1 protein levels and that thymidine levels were imageable with [18F]-fluoro-L-thymidine (FLT)–positron emission tomography (PET). In contrast, inhibition of de novo synthesis was linked to decreased expression of thymidylate synthase and dihydrofolate reductase. Notably, inhibition of de novo synthesis was associated with potentiation of ADI-PEG20 activity by the antifolate drug pemetrexed. Taken together, our findings argue that arginine deprivation combined with antifolates warrants clinical investigation in ASS1-negative urothelial and related cancers, using FLT-PET as an early surrogate marker of response. Cancer Res; 74(3); 896–907. ©2013 AACR.

Background: NUC-3373 is the first nucleotide analogue to bypass the key cancer resistance mechanisms associated with 5-fluorouracil (5-FU) and 5-fluorodeoxyuridine (FUDR) that significantly impede their clinical activity. In EC50 assays, NUC-3373 demonstrated up to 330x greater activity than 5-FU across multiple cancer cell lines and retained its activity in conditions mimicking drug resistance: thymidine kinase inhibition, thymidine phosphorylase over-expression and membrane transporter inhibition. In addition, whilst dihydropyrimidine dehydrogenase (DPD) significantly degraded 5-FU in cell lysates (p = 0.039), levels of NUC-3373 remained unaffected. Importantly, NUC-3373 generated 363x higher intracellular levels of the anti-cancer agent 5-fluorodeoxyuridine monophosphate, the active metabolite of 5-FU and FUDR. In colorectal cancer xenografts, NUC-3373 administered at 30 mg/kg 5-FU equimolar dose, demonstrated significantly greater tumour growth inhibition (47%) than 5-FU (25%). Finally, in formal toxicology studies NUC-3373 was significantly better tolerated than 5-FU, showing low conversion into the metabolite dihydrofluorouracil, dhFU (AUC plasma ratio dhFU:NUC-3373 = 0.03). NUC-3373 warrants clinical evaluation. Study Design: A two-part, Phase I open label, dose escalation and expansion study will be conducted to assess safety, pharmacokinetics and clinical activity of single agent NUC-3373 in participants with advanced solid tumours. A standard 3+3 dose escalation scheme will be used to identify the recommended Phase 2 dose (RP2D) of NUC-3373 comparing, in Part 1, a weekly schedule of administrationon days 1, 8, 15 and 22 of a 28-day cycle; and in Part 2 a fortnightly schedule of administration on days 1 and 15 of a 28-day cycle. Objectives: The primary objective is to establish the RP2D of NUC-3373 administered weekly and fortnightly. The secondary objectives are to assess the safety, pharmacokinetics, pharmacodynamics and anti-tumour activity of the agent. Tertiary objectives are to identify biomarkers that may help select those patients more likely to benefit from NUC-3373 than 5-FU. Major eligibility criteria: Histologically confirmed diagnosis of solid tumour, which is not amenable to, or refractory to standard chemotherapy, or for which no standard chemotherapy exists; measurable/evaluable disease; ECOG score 0-2; adequate bone marrow, liver and renal functions. Patients with a history of allergic reactions attributed to previous 5-FU or capecitabine treatment are excluded. Conclusions: NUC-3373 overcomes key cancer resistance mechanisms associated with 5-FU and a Phase I clinical study has been initiated. Citation Format: Sarah P. Blagden, Magdalena Slusarczyk, Michaela Serpi, Christopher McGuigan, Essam A. Ghazaly. First-in-human phase I study of the nucleotide analogue NUC-3373 designed to overcome fluoropyrimidine drug resistance mechanisms. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr CT028.

Argininosuccinate synthetase 1 (ASS1) is the rate-limiting enzyme for arginine biosynthesis. ASS1 expression is lost in a range of different tumour types, including 50% of malignant pleural mesotheliomas. Starving ASS1-deficient cells of arginine with arginine blockers such as ADI-PEG20 can induce selective lethality and has shown great promise in the clinic. However, recent data has shown that ASS1-deficient tumours can become resistant to this therapy although the mechanisms behind this resistance remain unclear. We have generated the first model of ADI-PEG20 resistance in mesothelioma cells whereby we observe re-expression of ASS1, via demethylation of the ASS1 promoter. Through coordinated transcriptomic and metabolomic profiling, we have shown that ASS1-deficient cells have decreased levels of acetylated polyamines, resulting in an increased activation of the polyamine synthesis pathway. Upon arginine deprivation, we observe a decrease in polyamine metabolites in the ASS1-deficient cells only, suggesting that exogenous arginine is required to maintain polyamine biosynthesis in the absence of ASS1. We identify for the first time a compensatory increase in polyamine synthesis gene expression upon ASS1 loss and highlight a synthetic lethal dependence between ASS1-deficiency and polyamine metabolism, which could potentially be exploited for the treatment of ASS1-negative cancers. Citation Format: Peter W. Szlosarek, Matthew Locke, Essam Ghazaly, Laura Lattanzio, Cristiana Lo Nigro, Sarah A. Martin. Inhibition of the polyamine synthesis pathway is synthetically lethal with loss of argininosuccinate synthase 1 in cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1050.

5565Background: Acelarin is a first-in-class nucleotide analogue utilising phosphoramidate chemistry to overcome the key gemcitabine resistance mechanisms and generate high intracellular levels of ...

Lapatinib is a specific HER1 and 2 targeted tyrosine kinase inhibitor now widely used in combination with chemotherapy in the clinical setting. In this work, we investigated the interactions between lapatinib and specific chemotherapy agents (cisplatin, SN-38, topotecan) in a panel of cell lines [breast (n = 2), lung (n = 2), testis (n = 4)]. A high-sensitivity cell proliferation/cytotoxicity ATP assay and flow cytometry were used to determine cell viability, apoptosis, and the effect of the drugs on cell-cycle distribution. CalcuSyn analysis was employed to formally identify synergistic interactions between drugs. Intracellular concentrations of SN-38 were measured using a novel high-performance liquid chromatography (HPLC) technique. Flow cytometry and HPLC techniques were used to identify the effect of lapatinib on drug influx and efflux pumps, using specific substrates and inhibitors of these pumps. Results showed significant synergy between SN-38, and lapatinib in the majority of cell lines (combination index < 0.75), associated with increased apoptosis. This synergy was not universal but, when observed (Susa S/R, H1975, H358, and MDA-MB-231 cell lines), was related to SN-38 intracellular accumulation (2.2- to 4.8-fold increase, P < 0.05 for each), attributable to the inhibition of the breast cancer–related protein (BCRP) efflux pump by lapatinib. Flow cytometry analysis showed that lapatinib (10 μmol/L) inhibited the efflux of mitoxantrone, a specific substrate of the BCRP pump, in a manner similar to fumitremorgin C, a known BCRP inhibitor, confirming lapatinib as a BCRP inhibitor. This work shows that lapatinib has a direct inhibitory effect on BCRP accounting for the synergistic findings. The synergy is cell line dependent and related to the activity of specific efflux pumps. Mol Cancer Ther; 9(12); 3322–9. ©2010 AACR.

Background: NUC-3373 is the first Nucleotide Analogue able to bypass the key drug resistance mechanisms associated with 5-fluorouracil (5-FU) and 5-fluorodeoxyuridine (FUDR) that severely hinder their clinical activity. The key cellular mechanisms causing drug resistance in cancer cells are the limited expression of activating kinases and nucleoside transporters, and overexpression of catabolic enzymes. The anti-neoplastic activity of 5-FU is largely attributed to its active metabolite, 5-fluorodeoxyuridine monophosphate (FdUMP), which inhibits the enzyme thymidylate synthase. As it already bears the monophosphate moiety, NUC-3373 is a pre-activated form of the active anti-cancer agent FdUMP. Here, we report potent in vitro and in vivobiological activity of NUC-3373. Methods: The cytotoxicity of 5-FU and the ProTide NUC-3373 was monitored with EC50 in vitro viability assays using 4 colorectal (Colo-205-luc; HT-29; HCT-116 and SW620); 3 lung (H1975; H1703 and SK-MES-1); 2 ovarian (OVCAR3 and A2780); 1 acute lymphoblastic leukemia (CCRF-CEM) and 1 cervix (HeLa) human tumour cell lines. These assays were also performed in conditions mimicking cancer resistance, where the activating enzyme thymidine kinase (TK) and the nucleoside transporter, hENT1, were mutated. Sensitivity of NUC-3373 and 5-FU to dihydropyrimidine dehydrogenase (DPD) degradation was assessed by absorption spectroscopy and UPLC-MS/MS. NUC-3373 anti-cancer activity was further evaluated in HT-29 nude mouse xenograft models. Results: NUC-3373 was more cytotoxic than 5-FU, achieving 2 to 333 fold lower EC50 values in the majority of cancer cell lines tested. TK inhibition reduced the cytotoxicity of NUC-3373 and FUDR by 4- and 136-fold respectively, suggesting NUC-3373 is more independent of TK. Inhibition of nucleoside transport decreased cytotoxic activity of NUC-3373 only mildly compared to a 63-fold reduction in FUDR cytotoxicity. NUC-3373 and 5-FU sensitivity to DPD degradation was assessed in cell lysates with or without the DPD inhibitor, gimeracil. NUC-3373 concentrations remained unaffected in the cell lysate irrespective of gimeracil treatment. In contrast, when the experiment was conducted with 5-FU, gimeracil treatment significantly increased the 5-FU concentration (p = 0.039) suggesting that NUC-3373, unlike 5-FU, is not a substrate for DPD metabolism. In colorectal cancer xenografts, NUC-3373 demonstrated greater tumour growth inhibition (47%) than 5-FU (25%). A toxicology study in which NUC-3373 was administered daily for 5 days/week for 4 consecutive weeks, at doses greater than 4 mg/kg/day in beagle dogs, compared favourably with that described following a single bolus intravenous administration of 5-FU. Conclusions: The novel ProTide, NUC-3373, overcomes the key cancer resistance mechanisms associated with 5-FU. NUC-3373 has efficacy in vitro and in vivo, and is resistant to DPD-mediated degradation. Results from formal toxicology assessments support the initiation of human clinical studies. A Phase I/II clinical study at Oxford University has been initiated to explore the safety, pharmacokinetic and clinical activity of NUC-3373 in participants with advanced solid tumours. Citation Format: Essam A. Ghazaly, Magdalena Slusarczyk, Christopher McGuigan, David Harrison, Sarah P. Blagden. NUC-3373: A novel pyrimidine nucleotide analogue that overcomes key cancer drug resistance limiting patient survival. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B46.

Introduction: Acelarin® is the first anti-cancer ProTide to enter the clinic and has achieved a very high rate of disease control in a Phase I study (ProGem1) of patients with advanced progressive solid tumours. It is a first-in-class nucleotide analogue specifically designed to bypass key cancer resistance pathways associated with gemcitabine, including altered expression of human equilibrative nucleoside transporter 1 (hENT1), deoxycytidine kinase (dCK) and cytidine deaminase (CDA). We have used a cytological RNA-based assay, RNAscope® and quantitative Reverse Transcriptase Polymerase Chain Reaction (qPCR) analysis to quantify these three biomarkers in seven cancer cell lines, and reviewed and tested a panel of commercially available antibodies against each protein. The aim was to select and validate a method suitable for stratifying tumours according to their molecular signature and allow the identification of patients unlikely to derive clinical benefit from gemcitabine. Methods: Cells from seven cancer lines (BxPC-3, MiaPaCa-2, PANC-1, SK-MES-1, NCI-H1703, OVCAR-3 and OVCAR-8) were prepared in formalin-fixed paraffin-embedded blocks. In addition, transiently transfected MiaPaCa-2 cells overexpressing hENT1, dCK and CDA were prepared. Expression of mRNA was detected using RNAscope and quantified by SpotStudio as well as by qPCR. Antibodies were examined by Western-blot analysis, reverse phase protein arrays (RPPA) and immunohistochemistry. Protein expression by immunofluorescence was quantified using Aqua HistoRx technology. Results: RNAscope detected mRNA for CDA at low levels in all cell lines. dCK and hENT1 were detected at low (in BxPC-3) to high (in NCI-H1703) expression levels across the cell lines. The qPCR method quantified precisely the amount of RNA, which demonstrated a positive and significant correlation with RNAscope data (Spearman correlation rho = 0.76, p = 0.037 for CDA, rho = 0.67, p = 0.08 for dCK, rho = 0.67, p = 0.08). Most antibodies were rejected because of low specificity as observed by Westerns, RPPA and immunohistochemistry. In addition, both techniques identified the gene over-expression in the transfected cell lines where the expression was up to 100 fold higher in transfected cells compared to their wild-type pairs. Conclusions: Selection of antibodies against hENT1, CDA and DCK for clinical use remains challenging. The RNAscope technology was validated, allowing reliable detection of biomarkers thought to be associated with cancer cell resistance to gemcitabine. Citation Format: Essam A. Ghazaly, Chathunissa Gnanaranjan, Bill Greenhalf, Sarah P. Blagden, Peter Mullen, David Harrison, John G. Gribben. Validating the RNAscope for molecular profiling of key biomarkers associated with gemcitabine resistance. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3390. doi:10.1158/1538-7445.AM2015-3390

Background Approximately 50% of all malignant pleural mesotheliomas (MPM) are deficient in argininosuccinate synthetase (ASS1), the rate-limiting enzyme in arginine biosynthesis, and are therefore sensitive to arginine deprivation. This discovery in MPM has been translated into the clinic using the arginine depletor pegylated arginine deiminase (ADI-PEG20) which showed a halving in the risk of disease progression in a randomized phase II study (Szlosarek et al, ASCO 2014). However, unstudied to date, stromal resistance to ADI-PEG20 may reduce its efficacy. Here, we studied the effect of macrophages, which are abundant in mesothelioma, on the tumor cytotoxicity of ADI-PEG20. Methods ASS1 negative MPM cell lines treated with ADI-PEG20 were analysed using the Affymetrix Human Genome U133 Plus 2.0 array, with validation of genes involved in stromal-tumor cell communication. Additional studies involved using tumor-macrophage co-culture experiments, mass spectrometry and an MPM xenograft model. Results A distinct pro-inflammatory cytokine gene expression signature involved in macrophage recruitment and activation was identified in the ADI-PEG20-treated ASS1 negative MPM cell lines. Notably, a significant increase in ASS1 negative MPM cell viability was seen upon co-culture with macrophages in the presence of ADI-PEG20. This was accompanied by a significant increase in ASS1 expression in co-cultured macrophages, with a corresponding increase in argininosuccinate lyase (ASL) expression in co-cultured tumor cells and a doubling in levels of the arginine precursor, argininosuccinate, in cell supernatant. The addition of argininosuccinate to tumor cell media rescued ASS1 negative MPM cells from ADI-PEG20 cytotoxicity, while the macrophage-mediated resistance to ADI-PEG20 was abrogated following ASL knockdown in MPM cells. Finally, xenograft studies demonstrated a significant reduction in tumor volume in mice treated with ADI-PEG20 in combination with macrophage depletion, compared with ADI-PEG20 monotherapy. Conclusion Collectively, our data indicate that as a result of metabolic ‘cross-talk’ between macrophages and ASS1 negative MPM cells, macrophages mediate MPM resistance to ADI-PEG20 via the provision of argininosuccinate. Our studies provide a rationale for combining ADI-PEG20 with an inhibitor of macrophage recruitment in the treatment of ASS1-deficient mesothelioma. Citation Format: Melissa M. Phillips, Ramsay Khadeir, Laura Tookman, Fiona McCarthy, Jeremy Steele, John Bomalaski, Essam Ghazaly, Peter W. Szlosarek. Macrophages promote resistance to pegylated arginine deiminase in malignant pleural mesothelioma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3360. doi:10.1158/1538-7445.AM2015-3360

Blagden, Sarah Patricia, et al. "A first in human Phase I/II study of NUC-1031 in patients with advanced gynecological cancers." ASCO Annual Meeting Proceedings. Vol. 33. No. 15_suppl. 2015.

Introduction: Chemotherapeutic resistance is a common issue that severely limits the clinical utility of all nucleoside analogues. The key cellular mechanisms causing resistance are limited expression of activating kinases and nucleoside transporters, and overexpression of catabolising enzymes. Phosphoramidate chemistry has been applied to nucleoside analogues to generate a new class of anti-cancer agents, ProTides, which overcome all these resistance mechanisms. Here, we report potent in vitro and in vivo biological activity of NUC-1031, a gemcitabine-derived ProTide. Methods: The cytotoxicity of the ProTide NUC-1031 and gemcitabine was monitored through in vitro viability assays. Both compounds were tested against a broad range of cancer cell lines including the pancreatic lines MiaPaCa-2 and BxPC-3 (partially or fully resistant to gemcitabine). Mimicking cancer resistance conditions, the activating enzyme, deoxycytidine kinase (dCK) was inhibited with deoxycytidine, and the nucleoside transporter, hENT1, was blocked with dipyridamole. The resulting intracellular concentrations of the active moiety, dFdCTP, were then measured. The ability of gemcitabine and NUC-1031 to resist degradation by cytidine deaminase (CDA) was assessed using absorption spectroscopy. Activity of NUC-1031 and gemcitabine were further evaluated in MiaPaCa-2 and BxPC-3 nude mouse xenograft models. Results: NUC-1031 was more cytotoxic than gemcitabine in vitro, with a lower EC50. NUC-1031 activity was not significantly affected by dCK inhibition (EC50= 0.2nM to 0.7nM in the presence of deoxycytidine) whereas gemcitabine cytotoxicity was reduced by 75 fold (EC50= 1.4nM to 104.9nM). Inhibition of nucleoside transport with dipyridamole significantly decreased gemcitabine cytotoxicity (EC50= 611μM rose to >2000μM). In contrast, NUC-1031 cytotoxic activity remained unchanged and significantly lower than gemcitabine (EC50= 190μM to 162μM). Measured intracellular concentrations of dFdCTP were consistent with these results. Additionally, gemcitabine was catabolized within one minute of exposure to CDA, while NUC-1031 was impervious to deamination. Finally, in pancreatic cancer xenografts, at 11, 14, 21 & 25 days, NUC-1031 showed greater reduction in tumor volumes compared to gemcitabine and control. Conclusions: The novel ProTide, NUC-1031, overcomes the key cancer resistance mechanisms associated with gemcitabine. Notably, NUC-1031 reaches up to 20 fold higher intracellular levels of dFdCTP, even in resistant cells. A Phase I clinical study of NUC-1031 is nearing completion and has provided significant disease control in both patients refractory to, or deemed unsuitable for, gemcitabine therapy. Citation Format: Essam A. Ghazaly, Magdalena Slusarczyk, Malcolm Mason, John Gribben, Christopher McGuigan, Sarah Blagden. NUC-1031: A novel ProTide that overcomes the key cancer resistance mechanisms associated with poor survival. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr CT401. doi:10.1158/1538-7445.AM2014-CT401

Background Arginine is an important amino acid for tumor cell growth and development. Argininosuccinate synthetase 1 (ASS1) is a key enzyme required for biosynthesis of arginine. Preclinically, ASS1-deficient tumor cells are particularly sensitive to arginine depletion, and randomised trials exploring this strategy are in progress in hepatocellular carcinoma and mesothelioma using the drug pegylated arginine deiminase (ADI-PEG20). In this study, we determined the metabolic changes induced by ADI-PEG20 treatment in a panel of bladder cancer and mesothelioma cell lines with promoter methylation-dependent silencing of ASS1. We used two cancer cell lines expressing ASS1 as a control. Methods Malignant mesothelioma, ASS1-negative (H2591, MSTO and JU77) and ASS1- positive (H28) cell lines were used. For bladder cancer, ASS1-negative (T24, 253J, UMUC-3) and ASS1-positive (RT112) cell lines were also used. All cells were treated with 750 ng/ml of ADI-PEG20 for 24 hours which induces up to 90% killing of arginine-dependent cell lines. Then, ultra performance liquid chromatography-mass spectrometry (UPLC-MS) technique was employed for untargeted quantitation of the metabolomic changes induced by ADI-PEG20. Results: All cell lines treated with ADI-PEG20 could be clearly discriminated from their untreated control pair when using PCA multivariate analysis. Arginine depletion was noted in all treated cell lines irrespective of ASS1 expression, however the reduction was at least one-log-fold greater in the ASS1-negative tumor cells. Citrulline, n-a-acetylcitrulline, and glutamine were upregulated specifically in ASS1-negative tumor cell lines. The main impact of ADI-PEG20 treatment was on pyrimidine metabolism in the ASS1-deficient tumor cells with upregulation of thymine and downregulation of thymidine, ureidosuccinic acid, uridine monophosphate and 5-hydroxymethyluracil. Notably, we identified that the reduction of the thymidine nucleotide pool was linked to suppression of thymidylate synthetase and dihydrofolate reductase, and paradoxically, reduced uptake of 3H-FLT. Finally, ADI-PEG20 caused variable effects on cytidine, uridine and ornithine levels in different cancer cell lines. Conclusion This study provides an insight into possible metabolic pathways affected by ADI-PEG20. The impact of ADI-PEG20 on thymidine metabolism, in particular, may be employed as a potential biomarker for optimizing the efficacy of ADI-PEG20 in the treatment of arginine auxotophic cancers. Citation Format: Essam A. Ghazaly, Phuong Luong, Malgorzata Chmielewska-Kassasir, Chantelle Hudson, John S. Bomalaski, L Wozniak, Norbert E. Avril, Simon P. Joel, Peter W. Szlosarek. A comprehensive untargeted UPLC-MS based metabolomic analysis of ASS1-deficient solid tumor cell lines treated with arginine deiminase. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1885. doi:10.1158/1538-7445.AM2013-1885

Background: Sphingolipids are important in cancer cell signalling. Sphingosine 1 phosphate (S1P) promotes cell survival and resistance to apoptosis, while S1P precursors ceramide (CER) and sphingosine (SPH), mediate antiproliferative and apoptotic responses. S1P is generated from SPH by sphingosine kinase (SK) enzymes (SK1 and SK2), with SK activity and localisation regulated by other proteins, including PKC, PKA and a SK anchoring protein (SKAP) that has been reported to negatively regulate SK1 activity in fibroblasts. S1P localisation is thought to play an important role in its function. Based on our preliminary observation in primary AML cells that SKAP expression resulted in an increase in S1P, we have investigated the effect of SKAP transfection on S1P production and localisation. Methods: K562, (and for some confirmatory experiments MCF-7), cells were transfected with the SKAP gene using standard techniques. SKAP is normally silenced in both cell lines. Transfection was confirmed by RNA expression. Intracellular and extracellular S1P and SPH, and intracellular SK activity (based on the production of C17 S1P from C17 SPH, an unnatural SPH that is a SK substrate) in intact cells were measured by LC-MS/MS. Phorbol 12-myristate 13-acetate (PMA) was used to induce membrane associated SK function, and MK-571 and fumitremorgen C (FTC) were used to block S1P efflux through ABCC1 and ABCG2 efflux pumps, respectively. Chemosensitivity to doxorubicin and imatinib in transfected cells was also studied. Results: K562 cells transfected with the SKAP gene showed a 2.5 fold increase in intracellular and extracellular levels of basal S1P compared to vector alone control. (In MCF-7 cells SKAP transfection resulted in an almost 10-fold increase in S1P). Further studies in K562 cells confirmed a significant increase in intracellular SK activity in SKAP transfected compared to vector alone cells, based on C17 S1P production (8.8 ± 2.6 vs 1.4 ± 0.4 ng/106 cells respectively after 24 hrs, p< 0.05). This increase was also observed, though to a lesser extent, in extracellular C17 S1P (678 ± 50 in SKAP vs 462 ± 47 pg/ml in vector alone, p< 0.05). In a proliferation assay this increase in SK activity was associated with a 25% increase in viable cell number (p 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 3256. doi:1538-7445.AM2012-3256

Tumors deficient in argininosuccinate synthetase-1 (ASS1), a key enzyme involved in arginine synthesis, are auxotrophic for arginine and sensitive to amino acid deprivation. Currently, several trials are testing the arginine-depleting agent, pegylated arginine deiminase (ADI-PEG20) in patients with cancer, including a randomised phase II multicenter UK study in patients with ASS1-deficient mesothelioma (NCT01279967). Here, we sought to identify key pathways involved in the mechanism of action of ADI-PEG20 using a panel of ASS1-deficient malignant mesothelioma (MPM) cell lines as our model. Epithelioid (2591), biphasic (MSTO) and sarcomatoid (JU77) mesothelioma cells were exposed to ADI-PEG20 and analysed using Affymetrix gene expression profiling with validation of candidate genes by qPCR and western blotting, and metabolic studies by LC-MS and 1H-NMR. ADI-PEG20 modulated several metabolic genes involved in nucleotide synthesis in the MPM cell lines. We identified suppression of ribonucleotide reductase (M1 and M2 isoforms), thymidylate synthase and dihydrofolate reductase by 24hrs, the latter enzymes being known targets of pemetrexed, a multitargeted antifolate used in the first-line therapy of mesothelioma. Moreover, metabolic profiling revealed upregulation of several modified nucleosides, including ribothymidine and downregulation of the nucleotide, guanosine monophosphate. Lastly, in vitro studies confirmed potentiation between ADI-PEG20 and pemetrexed and sequential therapy led to reduced tumor growth in vivo. In conclusion, arginine depletion modulates several key enzymes involved in nucleotide synthesis, providing a rationale for combining ADI-PEG20 with pemetrexed-containing regimens in the clinic. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B139.

SummaryIncreasing chemotherapy delivery to tumors, while enhancing drug uptake and reducing side effects, is a primary goal of cancer research. In mouse and human cancer models in vivo, we show that coadministration of low-dose Cilengitide and Verapamil increases tumor angiogenesis, leakiness, blood flow, and Gemcitabine delivery. This approach reduces tumor growth, metastasis, and minimizes side effects while extending survival. At a molecular level, this strategy alters Gemcitabine transporter and metabolizing enzyme expression levels, enhancing the potency of Gemcitabine within tumor cells in vivo and in vitro. Thus, the dual action of low-dose Cilengitide, in vessels and tumor cells, improves chemotherapy efficacy. Overall, our data demonstrate that vascular promotion therapy is a means to improve cancer treatment.

7587 Background: Arginine is a key amino acid for tumorigenesis modulating a diverse array of metabolic pathways, from synthesis of proteins, NO and polyamines to nucleotide turnover and mTOR signa...

2531 Background: Acelarin (NUC-1031) is a first-in-class nucleotide designed to overcome key cancer cell resistance by having nucleoside transporter-independent cellular uptake, activity independen...

2514 Background: NUC-1031 is a first-in-class nucleotide analogue utilising phosphoramidate chemistry to enhance anti-cancer efficacy and safety. In preclinical studies NUC-1031 demonstrated potent...

2576 Background: NUC-1031 is a novel nucleotide (ProTide) that evades all three key cellular resistance mechanisms associated with gemcitabine (dFdC). NUC-1031 bypasses nucleoside transporters, is activated independent of deoxycytidine kinase and is resistant to cytidine deaminase-mediated degradation. NUC-1031 has demonstrated broad antiproliferative activity in vitro and in vivo. Methods: Patients with relapsed/refractory advanced solid tumors entered in sequential cohorts of up to 6 patients, with escalating doses of NUC-1031 administered as a 5-10 minute IV injection weekly or twice-weekly. Ongoing objectives are to determine recommended phase II dose, safety profile, pharmacokinetics (PK) and preliminary anti-tumor activity. Results: 8 patients (5 female, 3 male) with pancreatic (2), colorectal (2), breast (1), and ovarian (1) cancers; cholangiocarcinoma (1) and unknown primary (1) have been enrolled. Two dose levels - 500mg/m2 (4) and 1000mg/m2 (1) weekly and one dose level - 375 mg/m2(3) twice-weekly. No DLTs have been observed. Mean AUC (0 - 24 h) for NUC-1031 was 150.3 ± 84.8 µM/h (n=5). dFdC and dFdU were detected in plasma up to 24 h (range of 0 - 5.8 µM for dFdC and 0 - 14.9 µM for dFdU). NUC-1031 excreted in urine mainly as dFdU. The Table shows rapid elimination of NUC-1031 from plasma and high intracellular levels of the active gemcitabine triphosphate at 2 and 24 h. Stable disease achieved in 1 patient with rapidly progressing breast cancer. Two further patients had symptomatic relief and improved QOL, including a dramatic reduction in ascites and pain. Conclusions: PK data show NUC-1031 has ≥ 10x higher intracellular levels of the active compound, dFdCTP, and significantly lower plasma Cmax levels of the toxic metabolite, dFdU, compared to equivalent levels of gemcitabine. NUC-1031 has shown better intracellular delivery and toxicity profile than gemcitabine with some promising early indicators of clinical efficacy. Clinical trial information: NCT01621854. [Table: see text]