mp3 file (10 MB). In the March edition of the Cancer Discovery podcast, Executive Editor Mark Landis talks with David A. Tuveson about his paper, which provides mechanistic insight into the clinical cooperation observed between gemcitabine and nab-paclitaxel in the treatment of pancreatic cancer.

Fig. S1 (related to Fig. 1) shows in vitro effects of treatment. Fig. S2 (related to Fig. 2) shows live single-cell FastFUCCI analyses. Fig. S3 (related to Fig. 3) shows spatiotemporal effects of treatment. Fig. S4 (related to Fig. 4) shows CHK1/WEE1 expression analyses and in vitro effects of treatment. Fig. S5 (related to Fig. 5) shows in vivo effects of treatment.

Candidate drugs may exhibit higher unbound intrinsic clearances (CLint,u) in human liver microsomes (HLMs) relative to human hepatocytes (HHs), posing a challenge as to which value is more predictive of in vivo clearance (CL). This work was aimed at better understanding the mechanism(s) underlying this 'HLM:HH disconnect' via examination of previous explanations, including passive permeability limited CL or cofactor exhaustion in hepatocytes. A series of structurally related, passively permeable (Papps > 5 × 10-6 cm/s), 5-azaquinazolines were studied in different liver fractions, and metabolic rates and routes were determined. A subset of these compounds demonstrated a significant HLM:HH (CLint,u ratio 2-26) disconnect. Compounds were metabolized via combinations of liver cytosol aldehyde oxidase (AO), microsomal cytochrome P450 (CYP) and flavin monooxygenase (FMO). For this series, the lack of concordance between CLint,u determined in HLM and HH contrasted with an excellent correlation of AO dependent CLint,u determined in human liver cytosol[Formula: see text], r2 = 0.95, P < 0.0001). The HLM:HH disconnect for both 5-azaquinazolines and midazolam was as a result of significantly higher CYP activity in HLM and lysed HH fortified with exogenous NADPH relative to intact HH. Moreover, for the 5-azaquinazolines, the maintenance of cytosolic AO and NADPH-dependent FMO activity in HH, relative to CYP, supports the conclusion that neither substrate permeability nor intracellular NADPH for hepatocytes were limiting CLint,u Further studies are required to identify the underlying cause of the lower CYP activities in HH relative to HLM and lysed hepatocytes in the presence of exogenous NADPH. SIGNIFICANCE STATEMENT: Candidate drugs may exhibit higher intrinsic clearance in human liver microsomes relative to human hepatocytes, posing a challenge as to which value is predictive of in vivo clearance. This work demonstrates that the difference in activity determined in liver fractions results from divergent cytochrome P450 but not aldehyde oxidase or flavin monooxygenase activity. This is inconsistent with explanations including substrate permeability limitations or cofactor exhaustion and should inform the focus of further studies to understand this cytochrome P450 specific disconnect phenomenon.

EDITORIAL article Front. Oncol., 29 July 2022Sec. Pharmacology of Anti-Cancer Drugs Volume 12 - 2022 | https://doi.org/10.3389/fonc.2022.972718

The desmoplastic stroma of pancreatic cancers forms a physical barrier that impedes intratumoral drug delivery. Attempts to modulate the desmoplastic stroma to increase delivery of administered chemotherapy have not shown positive clinical results thus far, and preclinical reports in which chemotherapeutic drugs were coadministered with antistromal therapies did not universally demonstrate increased genotoxicity despite increased intratumoral drug levels. In this study, we tested whether TGF beta antagonism can break the stromal barrier, enhance perfusion and tumoral drug delivery, and interrogated cellular and molecular mechanisms by which the tumor prevents synergism with coadministered gemcitabine. TGF beta inhibition in genetically engineered murine models (GEMM) of pancreas cancer enhanced tumoral perfusion and increased intratumoral gemcitabine levels. However, tumors rapidly adapted to TGF beta-dependent stromal modulation, and intratumoral perfusion returned to pre-treatment levels upon extended TGF beta inhibition. Perfusion was governed by the phenotypic identity and distribution of cancer-associated fibroblasts (CAF) with the myelofibroblastic phenotype (myCAFs), and myCAFs which harbored unique genomic signatures rapidly escaped the restricting effects of TGF beta inhibition. Despite the reformation of the stromal barrier and reversal of initially increased intratumoral exposure levels, TGF beta inhibition in cooperation with gemcitabine effectively suppressed tumor growth via cooperative reprogramming of T regulatory cells and stimulation of CD8 T cell-mediated antitumor activity. The antitumor activity was further improved by the addition of anti-PD-L1 immune checkpoint blockade to offset adaptive PD-L1 upregulation induced by TGF beta inhibition. These findings support the development of combined antistroma anticancer therapies capable of impacting the tumor beyond the disruption of the desmoplastic stroma as a physical barrier to improve drug delivery.

Identification of the enzymes responsible for the metabolism of drugs entering the clinic is an important activity of a drug metabolism scientist. This activity forms the basis of important risk assessment for drug-drug interactions (DDIs) for new molecules. The majority of marketed drugs are substrates for cytochrome P450s (CYP or P450), and this pathway is an important clearance route for many drugs. If a metabolic route is inhibited or induced, a DDI will occur with the possible consequences of toxicity or lack of efficacy. As our understanding of P450s has evolved over the past 20 plus years, assays to identify the specific P450 enzymes responsible for a compound's metabolism have been developed and continue to evolve. Additionally, medicinal chemists have developed their knowledge on how to design compounds that are not metabolized by P450s, shifting toward other pathways such as aldehyde oxidase (AO) or flavin monooxygenase (FMO). With this, DMPK has continued to evolve its assays and risk assessments to better quantify the risk of metabolism-based DDIs. Finally, regulatory agencies such as the US Food and Drug Administration (FDA), the European Medicines Agency (EMA), and the Pharmaceuticals and Medical Devices Agency (PMDA) have issued guidance on what is required from a DDI assessment to support the marketing of a new drug. This includes both laboratory-based assessments as well as modeling and simulation. This chapter will provide an overview on how to identify which P450s are involved in the metabolism of a compound, discrimination of P450 vs non-P450 metabolic routes, and examples of the physiological-based pharmacokinetic (PBPK) models that were used in risk assessment.

AbstractPurpose:KRAS is mutated in the majority of pancreatic ductal adenocarcinoma. MAPK and PI3K-AKT are primary KRAS effector pathways, but combined MAPK and PI3K inhibition has not been demonstrated to be clinically effective to date. We explore the resistance mechanisms uniquely employed by malignant cells.Experimental Design:We evaluated the expression and activation of receptor tyrosine kinases in response to combined MEK and AKT inhibition in KPC mice and pancreatic ductal organoids. In addition, we sought to determine the therapeutic efficacy of targeting resistance pathways induced by MEK and AKT inhibition in order to identify malignant-specific vulnerabilities.Results:Combined MEK and AKT inhibition modestly extended the survival of KPC mice and increased Egfr and ErbB2 phosphorylation levels. Tumor organoids, but not their normal counterparts, exhibited elevated phosphorylation of ERBB2 and ERBB3 after MEK and AKT blockade. A pan-ERBB inhibitor synergized with MEK and AKT blockade in human PDA organoids, whereas this was not observed for the EGFR inhibitor erlotinib. Combined MEK and ERBB inhibitor treatment of human organoid orthotopic xenografts was sufficient to cause tumor regression in short-term intervention studies.Conclusions:Analyses of normal and tumor pancreatic organoids revealed the importance of ERBB activation during MEK and AKT blockade primarily in the malignant cultures. The lack of ERBB hyperactivation in normal organoids suggests a larger therapeutic index. In our models, pan-ERBB inhibition was synergistic with dual inhibition of MEK and AKT, and the combination of a pan-ERBB inhibitor with MEK antagonists showed the highest activity both in vitro and in vivo.

Pharmacotherapy aims to provide cost-effective relief of disease symptoms for improved patient quality of life. However, most drugs are not 100% effective due to several factors, including genetic variability in drug targets and drug-metabolizing enzymes. 1 There are efforts to address these challenges focusing on evaluating gene-drug-response associations that can be used to stratify patient responses or phenotypes for pharmacotherapy.

AZD9496 ((E)-3-(3,5-difluoro-4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylic acid) is an oral selective estrogen receptor degrader currently in clinical development for treatment of estrogen receptor–positive breast cancer. In a first-in-human phase 1 study, AZD9496 exhibited dose nonlinear pharmacokinetics, the mechanistic basis of which was investigated in this study. The metabolism kinetics of AZD9496 were studied using human liver microsomes (HLMs), recombinant cytochrome P450s (rP450s), and hepatocytes. In addition, modeling approaches were used to gain further mechanistic insights. CYP2C8 was predominantly responsible for biotransformation of AZD9496 to its two main metabolites whose rate of formation with increasing AZD9496 concentrations exhibited complete substrate inhibition in HLM, rCYP2C8, and hepatocytes. Total inhibition by AZD9496 of amodiaquine N-deethylation, a specific probe of CYP2C8 activity, confirmed the completeness of this inhibition. The commonly used substrate inhibition model analogous to uncompetitive inhibition fit poorly to the data. However, using the same model but without constraints on the number of molecules occupying the inhibitory binding site (i.e., nS1ES) provided a significantly better fit (F test, P< 0.005). With the improved model, up to three AZD9496 molecules were predicted to bind the inhibitory site of CYP2C8. In contrast to previous studies showing substrate inhibition of P450s to be partial, our results demonstrate complete substrate inhibition of CYP2C8 via binding of more than one molecule of AZD9496 to the inhibitory site. As CYP2C8 appears to be the sole isoform catalyzing formation of the main metabolites, the substrate inhibition might explain the observed dose nonlinearity in the clinic at higher doses.

Abstract Combination of cytotoxic therapy with emerging DNA damage response inhibitors (DDRi) has been limited by tolerability issues. However, the goal of most combination trials has been to administer DDRi with standard-of-care doses of chemotherapy. We hypothesized that mechanism-guided treatment scheduling could reduce the incidence of dose-limiting toxicities and enable tolerable multitherapeutic regimens. Integrative analyses of mathematical modeling and single-cell assays distinguished the synergy kinetics of WEE1 inhibitor (WEE1i) from CHEK1 inhibitor (CHK1i) by potency, spatiotemporal perturbation, and mitotic effects when combined with gemcitabine. These divergent properties collectively supported a triple-agent strategy, whereby a pulse of gemcitabine and CHK1i followed by WEE1i durably suppressed tumor cell growth. In xenografts, CHK1i exaggerated replication stress without mitotic CDK hyperactivation, enriching a geminin-positive subpopulation and intratumoral gemcitabine metabolite. Without overt toxicity, addition of WEE1i to low-dose gemcitabine and CHK1i was most effective in tumor control compared with single and double agents. Overall, our work provides quantitative insights into the mechanisms of DDRi chemosensitization, leading to the rational development of a tolerable multitherapeutic regimen. Significance: Multiple lines of mechanistic insight regarding DNA damage response inhibitors rationally guide the preclinical development of a tolerable multitherapeutic regimen. Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/11/3054/F1.large.jpg. Cancer Res; 78(11); 3054–66. ©2018 AACR.

Stromal targeting for pancreatic ductal adenocarcinoma (PDAC) is rapidly becoming an attractive option, due to the lack of efficacy of standard chemotherapy and increased knowledge about PDAC stroma. We postulated that the addition of stromal therapy may enhance the anti-tumour efficacy of chemotherapy. Gemcitabine and all-trans retinoic acid (ATRA) were combined in a clinically applicable regimen, to target cancer cells and pancreatic stellate cells (PSCs) respectively, in 3D organotypic culture models and genetically engineered mice (LSL-Kras(G12D/+); LSL-Trp53(R172H/+); Pdx-1-Cre: KPC mice) representing the spectrum of PDAC. In two distinct sets of organotypic models as well as KPC mice, we demonstrate a reduction in cancer cell proliferation and invasion together with enhanced cancer cell apoptosis when ATRA is combined with gemcitabine, compared to vehicle or either agent alone. Simultaneously, PSC activity (as measured by deposition of extracellular matrix proteins such as collagen and fibronectin) and PSC invasive ability were both diminished in response to combination therapy. These effects were mediated through a range of signalling cascades (Wnt, hedgehog, retinoid, and FGF) in cancer as well as stellate cells, affecting epithelial cellular functions such as epithelial-mesenchymal transition, cellular polarity, and lumen formation. At the tissue level, this resulted in enhanced tumour necrosis, increased vascularity, and diminished hypoxia. Consequently, there was an overall reduction in tumour size. The enhanced effect of stromal co-targeting (ATRA) alongside chemotherapy (gemcitabine) appears to be mediated by dampening multiple signalling cascades in the tumour-stroma cross-talk, rather than ablating stroma or targeting a single pathway. (C) 2016 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

A fast, sensitive and accurate method for the determination of gemcitabine (difluorodeoxycytidine; dFdC) and deoxycytidine (CdR) in human plasma/tissue was developed using LC–MS/MS techniques. Effectiveness of the method is illustrated with the analysis of plasma from a phase I trial of dFdC administered as a 24 h infusion. The method was developed using 15N3 CdR as an internal standard across the concentration range of 1–500 ng/ml, using a cold alcohol-protein precipitation followed by desorption with freeze drying. Sample clean-up for LC–MS/MS analysis was performed by an innovative liquid/liquid back extraction with ethyl acetate and water. Chromatography was performed using a Chrompak-spherisorb-phenyl-column (3.1 mm × 200 mm, 5 μm) with a 50 mM formic acid: acetonitrile (9:1) mobile phase eluted at 1 ml/min. Extracted samples were observed to be stable for a minimum of 48 h after extraction when kept at 4 °C. Detection was performed using an atmospheric pressure chemical ionization (APCI) source and mass spectrometric positive multi-reaction-monitoring-mode (+MRM) for dFdC (264 m/z; 112 m/z), CdR (228 m/z; 112 m/z), and 15N3 CdR (231 m/z; 115 m/z) at an ion voltage of +3500 V. The accuracy, precision and limit-of-quantitation (LOQ) were as follows: dFdC: 99.8%, ±7.9%, 19 nM; CdR: 100.0%, ±5.3%, 22 nM, linear range LOQ to 2 μM. During 24 h infusion dFdC levels were detected with no interference from either CdR or difluorodeoxyuridine (dFdU). CdR co-eluted with dFdC but selectivity demonstrated no "crosstalk" between the compounds. In conclusion the analytical assay was very sensitive, reliable and robust for the determination of plasma and tissue concentrations of dFdC and CdR.

Porous graphitic carbon (PGC) is an important tool in a chromatographer's armory that retains polar compounds with mass spectrometry (MS)-compatible solvents. However, its applicability is severely limited by an unpredictable loss of retention, which can be attributed to contamination. The solutions offered fail to restore the original retention and our observations of retention time shifts of gemcitabine/metabolites on PGC are not consistent with contamination. The mobile phase affects the ionization state of analytes and the polarizable PGC surface that influences the strength of dispersive forces governing retention on the stationary phase. We hypothesized that failure to maintain the same PGC surface before and after running a gradient is a cause of the observed retention loss/variability on PGC. Herein, we optimize the choice of mobile phase solvent in a gradient program with three parts: a preparatory phase, which allows binding of analytes to column; an elution phase, which gives the required separation/peak shape; and a maintenance phase, to preserve the required retention capacity. Via liquid chromatography/tandem mass spectrometry (LC-MS/MS) analysis of gemcitabine and its metabolites extracted from tumor tissue, we demonstrate reproducible chromatography on three PGC columns of different ages. This approach simplifies use of the PGC to the same level as that of a C-18 column, removes the need for column regeneration, and minimizes run times, thus allowing PGC columns to be used to their full potential.

Design Pharmacokinetic and pharmacodynamic parameters of cremophor-paclitaxel, nab-paclitaxel (human-albumin-bound paclitaxel, Abraxane) and a novel mouse-albumin-bound paclitaxel (m-nab-paclitaxel) were evaluated in genetically engineered mouse models (GEMMs) by liquid chromatography-tandem mass spectrometry (LC-MS/MS), histological and biochemical analysis. Preclinical evaluation of m-nab-paclitaxel included assessment by three-dimensional high-resolution ultrasound and molecular analysis in a novel secreted protein acidic and rich in cysteine (SPARC)deficient GEMM of pancreatic ductal adenocarcinoma (PDA).Results nab-Paclitaxel exerted its antitumoural effects in a dose-dependent manner and was associated with less toxicity compared with cremophor-paclitaxel. SPARC nullizygosity in a GEMM of PDA, Kras(G12D); p53(flox/-); p48Cre (KPfC), resulted in desmoplastic ductal pancreas tumours with impaired collagen maturation. Paclitaxel concentrations were significantly decreased in SPARC null plasma samples and tissues when administered as low-dose m-nab-paclitaxel. At the maximally tolerated dose, SPARC deficiency did not affect the intratumoural paclitaxel concentration, stromal deposition and the immediate therapeutic response.Conclusions nab-Paclitaxel accumulates and acts in a dose-dependent manner. The interaction of plasma SPARC and albumin-bound drugs is observed at low doses of nab-paclitaxel but is saturated at therapeutic doses in murine tumours. Thus, this study provides important information for future preclinical and clinical trials in PDA using nab-paclitaxel in combination with novel experimental and targeted agents.

Capecitabine (CAP) is a 5-FU pro-drug approved for the treatment of several cancers and it is used in combination with gemcitabine (GEM) in the treatment of patients with pancreatic adenocarcinoma (PDAC). However, limited pre-clinical data of the effects of CAP in PDAC are available to support the use of the GEMCAP combination in clinic. Therefore, we investigated the pharmacokinetics and the efficacy of CAP as a single agent first and then in combination with GEM to assess the utility of the GEMCAP therapy in clinic. Using a model of spontaneous PDAC occurring in Kras(G12D); p53(R172H); Pdx1-Cre (KPC) mice and subcutaneous allografts of a KPC PDAC-derived cell line (K8484), we showed that CAP achieved tumour concentrations (∼25 µM) of 5-FU in both models, as a single agent, and induced survival similar to GEM in KPC mice, suggesting similar efficacy. In vitro studies performed in K8484 cells as well as in human pancreatic cell lines showed an additive effect of the GEMCAP combination however, it increased toxicity in vivo and no benefit of a tolerable GEMCAP combination was identified in the allograft model when compared to GEM alone. Our work provides pre-clinical evidence of 5-FU delivery to tumours and anti-tumour efficacy following oral CAP administration that was similar to effects of GEM. Nevertheless, the GEMCAP combination does not improve the therapeutic index compared to GEM alone. These data suggest that CAP could be considered as an alternative to GEM in future, rationally designed, combination treatment strategies for advanced pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDA) is characterized by abundant desmoplasia and poor tissue perfusion. These features are proposed to limit the access of therapies to neoplastic cells and blunt treatment efficacy. Indeed, several agents that target the PDA tumor microenvironment promote concomitant chemotherapy delivery and increased antineoplastic response in murine models of PDA. Prior studies could not determine whether chemotherapy delivery or microenvironment modulation per se were the dominant features in treatment response, and such information could guide the optimal translation of these preclinical findings to patients. To distinguish between these possibilities, we used a chemical inhibitor of cytidine deaminase to stabilize and thereby artificially elevate gemcitabine levels in murine PDA tumors without disrupting the tumor microenvironment. Additionally, we used the FG-3019 monoclonal antibody (mAb) that is directed against the pleiotropic matricellular signaling protein connective tissue growth factor (CTGF/CCN2). Inhibition of cytidine deaminase raised the levels of activated gemcitabine within PDA tumors without stimulating neoplastic cell killing or decreasing the growth of tumors, whereas FG-3019 increased PDA cell killing and led to a dramatic tumor response without altering gemcitabine delivery. The response to FG-3019 correlated with the decreased expression of a previously described promoter of PDA chemotherapy resistance, the X-linked inhibitor of apoptosis protein. Therefore, alterations in survival cues following targeting of tumor microenvironmental factors may play an important role in treatment responses in animal models, and by extension in PDA patients.

In vitro, gemcitabine has potent growth inhibitory effects on pancreatic cancer cells. However, clinically, its effectiveness is modest, despite being used widely as the reference treatment for both advanced disease and as adjuvant therapy. Drug delivery, due to the dense stroma associated with pancreatic cancer, has been identified as a potential limiting factor in the utility of gemcitabine. Gemcitabine (difluorodeoxycytidine, dFdC) requires metabolic activation by phosphorylation, the diphosphate metabolite inhibiting ribonucleotide reductase and the triphosphate form (dFdCTP) being incorporated into DNA. It is also a substrate for the enzyme cytidine deaminase (CDA), which is an inactivating reaction. As a result of this, its pharmacokinetics (PK) would be expected to impact on outcome following treatment. However, measurement of activated gemcitabine (dFdCTP) in tumor tissue had proved elusive, but a new LC-MS/ MS approach developed recently (Bapiro et al, CCP, 2011) has allowed the routine assessment of intratumoral dFdCTP concentrations in small fragments of tissue (≥ 10mg), following gemcitabine administration in mouse models (both allograft and in situ pancreatic adenocarcinoma in a genetically engineered mouse model). The assay is also providing new insights into the pharmacology of gemcitabine and further development allows the assessment of dFdCTP incorporation into DNA. It has been applied to the preclinical assessment of gemcitabine combination strategies, demonstrating increased dFdCTP formation following nabpaclitaxel, due to destabilization of CDA protein (Frese et al, Cancer Discovery, 2012) and depletion of hyaluronan, using PEGPH20 (Jacobetz et al, Gut, 2012). In studies with the γ-secretase inhibitor (MRK003), the increased activity of the combination was not associated with increased dFdCTP formation, prompting the search for the alternative mechanism; endothelial damage leading to hypoxic necrosis (Cook et al, J. Exp Med. 2012). These studies demonstrate the ongoing importance of assessing whether the drug gets to its target and our current focus is on improving the sensitivity further, for it to be applicable to needle biopsy specimens in clinical trials. Citation Format: Duncan I. Jodrell, Tashinga Bapiro, Natalie Cook, Kristopher Frese, Mike Jacobetz, Albrecht Neesse, Frances Richards, David Tuveson. How much gemcitabine reaches the target… and does it matter? [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Progress and Challenges; Jun 18-21, 2012; Lake Tahoe, NV. Philadelphia (PA): AACR; Cancer Res 2012;72(12 Suppl):Abstract nr IA13.

Abstract Nanoparticle albumin-bound (nab)-paclitaxel, an albumin-stabilized paclitaxel formulation, demonstrates clinical activity when administered in combination with gemcitabine in patients with metastatic pancreatic ductal adenocarcinoma (PDA). The limited availability of patient tissue and exquisite sensitivity of xenografts to chemotherapeutics have limited our ability to address the mechanistic basis of this treatment regimen. Here, we used a mouse model of PDA to show that the coadministration of nab-paclitaxel and gemcitabine uniquely demonstrates evidence of tumor regression. Combination treatment increases intratumoral gemcitabine levels attributable to a marked decrease in the primary gemcitabine metabolizing enzyme, cytidine deaminase. Correspondingly, paclitaxel reduced the levels of cytidine deaminase protein in cultured cells through reactive oxygen species–mediated degradation, resulting in the increased stabilization of gemcitabine. Our findings support the concept that suboptimal intratumoral concentrations of gemcitabine represent a crucial mechanism of therapeutic resistance in PDA and highlight the advantages of genetically engineered mouse models in preclinical therapeutic trials. Significance: This study provides mechanistic insight into the clinical cooperation observed between gemcitabine and nab-paclitaxel in the treatment of pancreatic cancer. Cancer Discovery; 2(3); 260–9. ©2012 AACR. Read the Commentary on this article by Clark, p. 208 This article is highlighted in the In This Issue feature, p. 193

Abstract Capecitabine (CAP) is an oral fluoropyrimidine, converted sequentially and selectively to 5-FU at the tumour site. It is used in the treatment of a number of cancers as a single agent and in patients with pancreatic cancer, in combination with gemcitabine. However, pre-clinical data in pancreatic cancer models are limited. In this study, we investigated the pharmacokinetics (PK) and efficacy of CAP in a GEMM of spontaneous pancreatic adenocarcinoma (PDA) occurring in KrasG12D; p53R172H; Pdx1-Cre (KPC) mice, compared to an allograft model of a cell line isolated from a PDA arising in the KPC mice. In the PK study, tumour was collected 2 hours after CAP treatment (755 mg/kg by oral gavage), homogenates were analysed using an LC-MS/MS assay developed to simultaneously detect capecitabine and its 3 metabolites DFCR, DFUR and 5-FU (modified from S.M.Guichard, et al., J. Chrom. B. 2005). Data were compared to our previously reported studies in an allograft model (Proc. AACR 2011 a 5446). After a QDx7 treatment, 5-FU concentrations of 27 ± 13 μM were achieved (compared to 23.0 ± 8.1 μM and 22.7 ± 7.7 μM in allograft tumours after 1 and 5 consecutive doses respectively), confirming adequate drug delivery to the in situ tumour following oral administration of CAP. Therefore we proceeded to efficacy studies in this model. In the allograft model we had identified a significant reduction of the tumour doubling time with 755 mg/kg CAP (5 days/week, 3 weeks), compared to control (7.5 ± 3.0 vs 3.5 ± 0.5 days; P<0.001). In in situ tumours, a short term study over 7 days showed a reduction in tumour growth in CAP-treated KPC PDA tumours compared to control (199% ± 22% vs 121% ± 10%; P<0.01). In a survival study in KPC mice, CAP (755 mg/kg, 5 days/week) was compared to the standard treatment for advanced pancreatic cancer, gemcitabine (GEM, 100 mg/kg, Q3D), and there was no difference in the median survival of mice with spontaneous PDA tumours (P=0.61) suggesting a similar efficacy of CAP to GEM. There is conflicting evidence regarding the utility of the combination of GEM and CAP in this disease, so we also investigated the combination in mice bearing allograft PDA tumours. Full doses of both drugs were not tolerated, but the combination of GEM (75 mg/kg Q3D, 2 weeks) plus CAP (539 mg/kg, 5 days/week, 2 weeks) was feasible. This regimen was associated with significant growth inhibition, but this was not superior to GEM alone (75 mg/kg) at the same dose (tumour doubling time: 8.6 ± 10.8 vs 7.2 ± 2.8 days respectively). In summary, orally administered CAP achieves active concentrations of 5-FU in PDA tumours. Similar effects on survival compared to GEM are seen in PDA tumours. Growth inhibition data in allograft tumours did not show any additional benefit for the GEMCAP combination, when compared to GEM alone. CAP could be considered as an alternative to GEM in future, rationally designed, combination treatment strategies. 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 3771. doi:1538-7445.AM2012-3771