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LY2334737, an oral prodrug of gemcitabine, is cleaved in vivo, releasing gemcitabine and valproic acid. Oral dosing of mice results in absorption of intact prodrug with slow systemic hydrolysis yielding higher plasma levels of LY2334737 than gemcitabine and prolonged gemcitabine exposure. Antitumor activitywas evaluated in human colon and lung tumor xenograft models. The dose response for efficacy was examined using 3 metronomic schedules, once-a-day dosing for 14 doses, every other day for 7 doses, and once a day for 7 doses, 7 days rest, followed by an additional 7 days of once-a-day dosing. These schedules gave significant antitumor activity andwerewell tolerated.Oral gavageof 6mg/kgLY2334737daily for 21daysgave equivalent activity to i.v. 240mg/kg gemcitabine.HCl administered once aweek for 3weeks tomice bearing a patientmesothelioma tumor PXF 1118 or a non–small cell lung cancer tumor LXFE 937. The LXFE 397 tumor possessed elevated expression of the equilibrative nucleoside transporter-1 (ENT1) important for gemcitabine uptake but not prodrug uptake and responded significantly better to treatment with LY2334737 than gemcitabine (P 0.001). In 3 colon xenografts, antitumor activity of LY2334737 plus amaximally tolerated dose of capecitabine, an oral prodrug of 5-fluorouracil, was significantly greater than eithermonotherapy.During treatment, the expression of carboxylesterase 2 (CES2) and concentrative nucleoside transporter-3was induced inHCT-116 tumors; both are needed for the activity of the prodrugs. Thus, metronomic oral low-dose LY2334737 is efficacious, well tolerated, and easily combined with capecitabine for improved efficacy. Elevated CES2 or ENT1 expression may enhance LY2334737 tumor response. Mol Cancer Ther; 12(4); 1–10. 2013 AACR.

Purpose: The oral prodrug of gemcitabine LY2334737 is cleaved systemically to gemcitabine; the mechanism responsible for hydrolysis is unknown. LY2334737 cytotoxicity was tested in the NCI-60 panel; mining of microarray expression data identified carboxylesterase (CES) as a top hydrolase candidate. Studies examined whether CES is responsible for hydrolysis and whether cellular CES expression confers prodrug sensitivity.Experimental Design: Human recombinant CES isozymes were assayed for LY2334737 hydrolysis. Stable CES-overexpressing HCT-116 transfectants and a SK-OV-3 knockdown were prepared. Cell lines were tested for drug sensitivity and CES expression by quantitative real time-PCR (qRT-PCR), Western blotting, and immunohistochemical staining. Bystander cytotoxicity studies were conducted with GFP-tagged PC-3 cells as the reporter cell line. Therapeutic response of the HCT-116 transfectants was evaluated in xenografts.Results: Of 3 human CES isozymes tested, only CES2 hydrolyzed LY2334737. Five cell lines that express CES2 responded to LY2334737 treatment. LY2334737 was less cytotoxic to a SK-OV-3 CES2 knockdown than parental cells. The drug response of CES2-transfected HCT-116 cells correlated with CES2 expression level. Bystander studies showed statistically greater PC-3-GFP growth inhibition by LY2334737 when cells were cocultured with CES2 and not mock transfectants. Oral treatment of xenograft models with 3.2 mg/kg LY2334737 once a day for 21 days showed greater tumor growth inhibition of CES2 transfectant than the mock transfectant (P <= 0.001).Conclusions: CES2 is responsible for the slow hydrolysis of LY2334737. Because intact prodrug circulates at high plasma levels after oral LY2334737 administration, improved response rates may be observed by tailoring LY2334737 treatment to patients with CES2 tumor expression. Clin Cancer Res; 19(5); 1159-68. (C) 2012 AACR.

Abstract LY2334737, an oral prodrug of gemcitabine, is cleaved in vivo, releasing gemcitabine and valproic acid. Oral dosing of mice results in absorption of intact prodrug with slow systemic hydrolysis yielding higher plasma levels of LY2334737 than gemcitabine and prolonged gemcitabine exposure. Antitumor activity was evaluated in human colon and lung tumor xenograft models. The dose response for efficacy was examined using 3 metronomic schedules, once-a-day dosing for 14 doses, every other day for 7 doses, and once a day for 7 doses, 7 days rest, followed by an additional 7 days of once-a-day dosing. These schedules gave significant antitumor activity and were well tolerated. Oral gavage of 6 mg/kg LY2334737 daily for 21 days gave equivalent activity to i.v. 240 mg/kg gemcitabine. HCl administered once a week for 3 weeks to mice bearing a patient mesothelioma tumor PXF 1118 or a non–small cell lung cancer tumor LXFE 937. The LXFE 397 tumor possessed elevated expression of the equilibrative nucleoside transporter-1 (ENT1) important for gemcitabine uptake but not prodrug uptake and responded significantly better to treatment with LY2334737 than gemcitabine (P ≤ 0.001). In 3 colon xenografts, antitumor activity of LY2334737 plus a maximally tolerated dose of capecitabine, an oral prodrug of 5-fluorouracil, was significantly greater than either monotherapy. During treatment, the expression of carboxylesterase 2 (CES2) and concentrative nucleoside transporter-3 was induced in HCT-116 tumors; both are needed for the activity of the prodrugs. Thus, metronomic oral low-dose LY2334737 is efficacious, well tolerated, and easily combined with capecitabine for improved efficacy. Elevated CES2 or ENT1 expression may enhance LY2334737 tumor response. Mol Cancer Ther; 12(4); 481–90. ©2013 AACR.

Gemcitabine (dFdC, 2',2'-difluorodeoxycytidine) is metabolized by cytidine deaminase (CDA) and deoxycytidine kinase (DCK), but the contribution of genetic variation in these enzymes to the variability in systemic exposure and response observed in cancer patients is unclear. Wild-type enzymes and variants of CDA (Lys27Gln and Ala70Thr) and DCK (Ile24Val, Ala119Gly, and Pro122Ser) were expressed in and purified from Escherichia coli, and enzyme kinetic parameters were estimated for cytarabine (Ara-C), dFdC, and its metabolite 2',2'-difluorodeoxyuridine (dFdU) as substrates. All three CDA proteins showed similar K(m) and V(max) for Ara-C and dFdC deamination, except for CDA70Thr, which had a 2.5-fold lower K(m) and 6-fold lower V(max) for Ara-C deamination. All four DCK proteins yielded comparable metabolic activity for Ara-C and dFdC monophosphorylation, except for DCK24Val, which demonstrated an approximately 2-fold increase (P < 0.05) in the intrinsic clearance of dFdC monophosphorylation due to a 40% decrease in K(m) (P < 0.05). DCK did not significantly contribute to dFdU monophosphorylation. In conclusion, the Lys27Gln substitution does not significantly modulate CDA activity toward dFdC, and therefore would not contribute to interindividual variability in response to gemcitabine. The higher in vitro catalytic efficiency of DCK24Val toward dFdC monophosphorylation may be relevant to dFdC clinical response. The substrate-dependent alterations in activities of CDA70Thr and DCK24Val in vitro were observed for the first time, and demonstrate that the in vivo consequences of these genetic variations should not be extrapolated from one substrate of these enzymes to another.

Abstract Metronomic chemotherapy is the regular administration of chemotherapy drugs at relatively low minimally toxic doses without prolonged break periods; it is currently undergoing phase III trial evaluation. Metronomic chemotherapy is thought to cause anti-tumor effects primarily by antiangiogenic mechanisms, both locally by targeting endothelial cells of the tumor neovasculature and systemically by effects on bone marrow derived cells, including circulating endothelial progenitor cells (CEPs). Previous studies have shown reduction of CEPs by metronomic administration of a number of different chemotherapeutic drugs, including cyclophosphamide, paclitaxel, topotecan, and tegafur plus uracil. Here we report results evaluating the properties of metronomic administration of an oral prodrug of gemcitabine LY2334737 (LY) in non tumor-bearing mice, and in preclinical models of human ovarian (SKOV3-13) and breast cancer (LM2-4) xenografts. Through daily gavage (at 6mg/kg/day) the schedules tested were devoid of toxicity and caused anti-tumor effects; however, a suppressive effect on CEPs was not detected. Human SKOV3-13 ovarian cancer cells were grown as ascites in SCID mice; LY monotherapy (6mg/kg/day), as well as LY in combination with metronomic cyclophosphamide (CTX, 20mg/kg/day), caused a significant increase in survival (n=5 mice per group, p<0.05) compared to controls. Human LM2-4 breast cancer cells, implanted orthotopically into SCID mice, were growth inhibited by LY monotherapy (at 6mg/kg/day as well as at 8mg/kg/day; n=5 mice per group, p<0.05). LM2-4 tumors were also growth inhibited by the combination of LY (6mg/kg/day) plus a bolus of CTX (100mg/kg, single dose) followed immediately by a maintenance regimen of metronomic CTX (20mg/kg/day); this combination did not produce any overt toxicity. Unexpectedly metronomic LY administration caused increased intratumoral blood flow, as measured by ultrasound after 1 week and after 4 weeks of continuous treatment, in luciferase-tagged LM2-4 tumor xenografts; and the increase in blood flow coincided with a relative increase in tumor bioluminescence. Paraffin embedded LM2-4 tumor sections from the therapy experiments were further analyzed by Angiogenesis and by Tumor Health Panel analyses; LY monotherapy did not caused any significant changes in tumor hypoxia, or in intratumoral vessel density, compared to controls. These results highlight the possibility of significant anti-tumor effects mediated by metronomic administration of some chemotherapy drugs without a concomitant inhibition of systemic angiogenesis. 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 4377. doi:1538-7445.AM2012-4377

Abstract Metronomic chemotherapy refers to the close, regular administration of conventional chemotherapy drugs at relatively low, minimally toxic doses, with no prolonged break periods; it is now showing encouraging results in various phase II clinical trials and is currently undergoing phase III trial evaluation. It is thought to cause antitumor effects primarily by antiangiogenic mechanisms, both locally by targeting endothelial cells of the tumor neovasculature and systemically by effects on bone marrow–derived cells, including circulating endothelial progenitor cells (CEP). Previous studies have shown reduction of CEPs by metronomic administration of a number of different chemotherapeutic drugs, including vinblastine, cyclophosphamide, paclitaxel, topotecan, and tegafur plus uracil (UFT). However in addition to, or even instead of, antiangiogenic effects, metronomic chemotherapy may cause suppression of tumor growth by other mechanisms such as stimulating cytotoxic T-cell responses or by direct antitumor effects. Here we report results evaluating the properties of metronomic administration of an oral prodrug of gemcitabine LY2334737 in nontumor–bearing mice and in preclinical models of human ovarian (SKOV3-13) and breast cancer (LM2-4) xenografts. Through daily gavage (at 6 mg/kg/d), the schedules tested were devoid of toxicity and caused antitumor effects; however, a suppressive effect on CEPs was not detected. Unexpectedly, metronomic LY2334737 administration caused increased blood flow in luciferase-tagged LM2-4 tumor xenografts, and this effect, readily measured using contrast micro-ultrasound, coincided with a relative increase in tumor bioluminescence. These results highlight the possibility of significant antitumor effects mediated by metronomic administration of some chemotherapy drugs without a concomitant inhibition of systemic angiogenesis. Mol Cancer Ther; 11(3); 680–9. ©2011 AACR.

Abstract LY2334737 (LY) is a prodrug analog of gemcitabine with an amide linked valproate. LY is orally bioavailable, non-toxic, and cleaved in the liver to release gemcitabine systemically. The studies presented identify carboxylesterase 2 (CES2) as the enzyme responsible for the hydrolysis of LY, and further outlines how tumor expression of CES2 would enhance LY efficacy. Employing HPLC and enzyme inhibitors, CES2, and not CES1 or CES3, was identified as the liver enzyme responsible for cleaving LY to gemcitabine. Carboxylesterases rapidly hydrolyze ester bonds; however the hydrolysis of the prodrug amide bond occurs at a much slower rate. This observation supports the findings that only a fraction of administered LY is converted to gemcitabine, the two agents circulating at a ratio of 10:1 respectively in humans. In view of the persistent levels of LY in the blood, it was thought that CES2 expression in tumors might provide an additional site of prodrug activation. A number of cancer cell lines, including the NCI60 panel, were screened in vitro for sensitivity to LY, which was defined as the ratio of the prodrug EC50 versus gemcitabine EC50 being less than 15. Cells which met this criterion were all found to express CES2. The ability of CES2 to confer cellular sensitivity to LY was confirmed through inhibition of cytotoxicity by 10uM loperamide, a CES2-specific inhibitory dose, and CES2 knockdown with shRNA in a sensitive cell line. Transfection of CES2 into the LY insensitive cell line HCT116 resulted in increased responsiveness to LY that directly correlated to the level of CES2 expression. In vivo studies comparing LY efficacy with xenografts of mock- and CES2-transfected HCT116 cell lines also demonstrated that CES2 expressing tumors are more sensitive to LY treatment than non-expressors. As such, employing CES2 as a tailoring biomarker would identify those tumors that would have enhanced sensitivity through their ability to intracellularly cleave circulating LY and result in increased levels of gemcitabine directly at the tumor site. Immunohistochemical characterization of human tumor types revealed that head and neck, colorectal, and squamous NSCLC tumors have a high incidence of CES2 expression. Gemcitabine administered IV is first or second line therapy in many cancers. LY, as an oral agent, is ideally suited to be dosed, and deliver gemcitabine, metronomically. The advantages of a metronomic dosing strategy include the ability to deliver sustained dosing of chemotherapeutic agents with lower toxicity and potential antiangiogenic activity. Additionally, tailoring treatment to CES2 expressing tumors could enhance efficacy through both a systemic and a local generation of gemcitabine. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2981. doi:10.1158/1538-7445.AM2011-2981

Abstract Gemcitabine hydrochloride is a nucleoside anticancer agent registered for the treatment of pancreatic, NSCLC, breast, and ovarian cancers. A prodrug of gemcitabine (LY2334737) was prepared that is a gemcitabine analog with an amide-linked valproate. This prodrug is noncytotoxic and must be hydrolyzed to release gemcitabine. In man, the prodrug is orally absorbed intact and converted to gemcitabine systemically. This study was undertaken to identify the enzyme responsible for hydrolysis of the prodrug to gemcitabine. The NCI-60 cell line panel was screened for prodrug sensitivity and microarray data were used to evaluate the expression of hydrolase enzymes in responsive and unresponsive cell lines. Cell extracts and candidate enzymes were tested for LY hydrolysis using HPLC and cell lines were evaluated for enzyme expression using qRT-PCR and Western methods. Screening of the NCI-60 panel demonstrated that LY was not cytotoxic to the majority of cell lines, however sensitivity was observed in a few cell lines such as SK-OV-3 and COLO205. Other cell lines were also evaluated. Extracts of drug sensitive cell lines hydrolyzed LY to gemcitabine and its inactive metabolite 2',2'-difluorodeoxyuridine (dFdU), indicating that LY hydrolysis was necessary for activity. Analysis of the expression of known hydrolases in the NCI-60 microarray data identified the serine ester hydrolase, carboxylesterase 1 (CES1) among the top candidates. In humans, there are 3 CESs and these are known to hydrolyze other prodrugs. Human recombinant protein of each CES was tested for LY cleavage. With long incubation periods, only CES2 hydrolyzed the drug. Loperamide is an inhibitor of CES1 and CES2 that is permeable to cells and noncytotoxic. At low micromolar concentrations this inhibitor only affects CES2, therefore cytotoxicity assays were conducted with SK-OV-3 in the presence and absence of 10 µM loperamide. The cytotoxicity of LY was decreased with loperamide CES2 inhibition without effect on gemcitabine cytotoxicity. Expression of CES2 was evaluated in drug-sensitive and -insensitive cell lines by qRT-PCR and Western analysis; LY sensitive lines expressed CES2 while insensitive lines did not. An immunohistochemical assay was developed that showed expression of CES2 to be heterogeneously expressed in these cell lines. Taken together, these data indicate that CES2 hydrolyzes the prodrug. Cancer cells that express CES2 may have an enhanced response to treatment with the prodrug LY2334737. Citation Information: Clin Cancer Res 2010;16(14 Suppl):B36.

Gemcitabine (gem) is a nucleoside anticancer agent that inhibits cell cycle in S-phase and is registered clinically for the treatment of a number of solid tumors. A prodrug of gemcitabine, LY2334737, was developed for oral administration for metronomic dosing with the aim of growth inhibition of more tumor cells as they go through the cell cycle. LY is well absorbed and systemically cleaved in humans. The rate of hydrolysis is slow resulting in circulating levels of LY that are 10-fold higher than gemcitabine in plasma. Studies with cell lines and recombinant protein indicate that LY is cleaved to gemcitabine by carboxylesterase 2 (CES2). Because LY circulates in the plasma for an extended period of time, we wondered if expression of CES2 by the tumor could alter its response to the prodrug. The present study was undertaken to determine if the expression of CES2 alters cellular response to LY in vitro and in vivo. The constitutive expression of CES2 in SKOV-3 was knocked down by shRNA. A stable subclone was obtained that had CES2 expression reduced by 80%. Stable HCT-116 transfectants were prepared that overexpressed CES2. Cell lines, transfectants and knock down cells were evaluated for drug sensitivity in a cytotoxicity assay. They were also evaluated for CES2 expression by biochemical cellular assay, Western analysis, and qRT-PCR. A xenograft study was conducted employing transfected HCT-116 cells. Immunohistochemical (IHC) staining was used to evaluate cell lines and tumors for CES2 expression. LY was less cytotoxic to SKOV-3 cells when CES2 was knocked down while sensitivity to gem was unaltered. Transfection of CES2 into low CES2 endogenously expressing HCT-116 cells enhanced LY drug sensitivity with no change in gemcitabine sensitivity. Stably transfected CES2 and mock HCT-116 transfectants were grown as a xenograft. The transfectants had identical growth rates in vivo and were equally sensitive to gem treatment. IHC staining of the tumors indicated that CES2 expression was maintained throughout the growth period. CES2 expressing tumors demonstrated a statistically significant greater response to LY than mock transfectant tumors. Taken together, these studies indicate that CES2 cleaves the prodrug and tumor expression of CES2 can enhance response to treatment with LY2334737. Citation Information: Clin Cancer Res 2010;16(14 Suppl):B30.

Abstract Carboxylesterase II (CES2), a serine ester hydrolase, is the major carboxylesterase responsible for the conversion of the prodrug of gemcitabine into its active form, gemcitabine (LY2334737), an anticancer chemotherapeutic. In humans, the ratio of plasma levels of prodrug to active form is 10:1. Therefore, increased availability of LY2334737 can result in cleavage of the prodrug into its active form at the tumor site and has potential for greater tumor cytotoxicity. Relatively high levels of expression of CES2 within non-neoplastic human tissue occur in the liver, kidney and gastrointestinal tract as visualized by immunohistochemistry (IHC). CES2 is also reported to be expressed in human cancers such as non-small cell lung carcinoma and colon adenocarcinomas. In this study, we analyzed CES2- transfected in vitro cell lines by real-time PCR, chromogenic IHC, and automated quantitative analysis (AQUA) of immunofluorescent in situ protein expression and showed that high levels of CES2 correlate with higher cytotoxicity. qRT-PCR analysis revealed that the mRNA levels of CES2 measured in formalin-fixed paraffin-embedded tumors correlate with protein levels. CES2 transcriptional profiling was performed to identify additional tumor types with high levels of expression of CES2. CES2 overexpression was detectable by IHC and/or AQUA analysis in human colon carcinoma, mesothelioma, non-small cell lung cancer, and tumors of the breast and ovary. CES2 IHC labeling in colonic adenocarcinomas was diffuse. However, labeling in non-neoplastic mucosa showed low levels in proliferating cells at the crypt base and increasingly higher expression toward the terminally differentiated cells at the tips—cells that would not progress through the cell cycle and should not be affected by antiproliferative agents. In conclusion, we have developed robust IHC and AQUA-based assays for differential expression of CES protein levels in a variety of archival human tumor types and have shown high correlation with CES2 transcript levels by qRT-PCR. These methodologies can identify tumors with high levels of CES2, a biomarker that may further be investigated in biomarker-driven clinical trials to identify patients who will more likely respond to LY2334737. Citation Information: Clin Cancer Res 2010;16(14 Suppl):B31.

The design, synthesis, and biological characterization of an orally active prodrug (3) of gemcitabine are described. Additionally, the identification of a novel co-crystal solid form of the compound is presented. Valproate amide 3 is orally bioavailable and releases gemcitabine into the systemic circulation after passing through the intestinal mucosa. The compound has entered clinical trials and is being evaluated as a potential new anticancer agent.

Multidrug resistance protein-5 (MRP5, ABCC5) is a member of the ATP-binding cassette transporter superfamily that effluxes a broad range of natural and xenobiotic compounds such as cyclic GMP, antiviral compounds, and cancer chemotherapeutic agents including nucleoside-based drugs, antifolate agents and platinum compounds. In cellular assays, MRP5 transfectants are less fluorescent after incubation with 5-chloromethylfluorescein diacetate (CMFDA). The present study examines the uptake of a close fluorescent analog, carboxydichlorofluorescein (CDCF), and drug substrates into inside-out membrane vesicles prepared from MRP transfected cells. MRP5-mediated uptake of CDCF was ATP-dependent and GSH-independent and possessed a Km of 12 microM and a Vmax of 56 pmol/min/mg prot. Comparison of kinetic parameters with drug substrates such as methotrexate (MTX), pemetrexed (Alimta), and the metabolite of 5-fluorouracil, 5-fluorodeoxyuridine monophosphate (5-FdUMP) (Km values of 0.3-1.3 mM) indicated that MRP5 has a 25-100-fold higher affinity for CDCF than for these drugs and that they share a common transport binding site. In addition, the potency of MRP5 inhibitors such as probenecid, MK571, and the phosphodiesterase 5 inhibitors correlated well between the uptake of CDCF and MTX. A survey of CDCF uptake by other MRPs revealed that MRP2 (ABCC2) also demonstrated ATP-dependent uptake with a Km of 19 microM and Vmax of 95.5 pmol/min/mg prot, while MRP1 (ABCC1) and MRP4 (ABCC4) had little to no uptake. Taken together, these data indicate that CDCF is a useful fluorescent drug surrogate with which to measure ATP-dependent MRP5-mediated transport.

Novel cyclohexyl drug resistance modulators were synthesized and evaluated for in vitro inhibition of the drug resistance transporter, MRP1. This series resulted in the potent and selective MRP1 modulator 21b, which demonstrated reversal of MRP1-mediated multidrug resistance in vivo.

The splicing factor SPF45 (RBM17) is frequently overexpressed in many solid tumors, and stable expression in HeLa cells confers resistance to doxorubicin and vincristine. In this study, we characterized stable transfectants of A2780 ovarian carcinoma cells. In a 3-day cytotoxicity assay, human SPF45 overexpression conferred 3- to 21-fold resistance to carboplatin, vinorelbine, doxorubicin, etoposide, mitoxantrone, and vincristine. In addition, resistance to gemcitabine and pemetrexed was observed at the highest drug concentrations tested. Knockdown of SPF45 in parental A2780 cells using a hammerhead ribozyme sensitized A2780 cells to etoposide by approximately 5-fold relative to a catalytically inactive ribozyme control and untransfected cells, suggesting a role for SPF45 in intrinsic resistance to some drugs. A2780-SPF45 cells accumulated similar levels of doxorubicin as vector-transfected and parental A2780 cells, indicating that drug resistance is not due to differences in drug accumulation. Efforts to identify small molecules that could block SPF45-mediated drug resistance revealed that the selective estrogen receptor (ER) modulators tamoxifen and LY117018 (a raloxifene analogue) partially reversed SPF45-mediated drug resistance to mitoxantrone in A2780-SPF45 cells from 21-fold to 8- and 5-fold, respectively, but did not significantly affect the mitoxantrone sensitivity of vector control cells. Quantitative PCR showed that ERbeta but not ERalpha was expressed in A2780 transfectants. Coimmunoprecipitation experiments suggest that SPF45 and ERbeta physically interact in vivo. Thus, SPF45-mediated drug resistance in A2780 cells may result in part from effects of SPF45 on the transcription or alternate splicing of ERbeta-regulated genes.