Kathleen M. Heinz-Taheny

Human Carboxylesterase-2 Hydrolyzes the Prodrug of Gemcitabine (LY2334737) and Confers Prodrug Sensitivity to Cancer Cells

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. ©2012 AACR.

Qualification of Cardiac Troponin I Concentration in Mouse Serum Using Isoproterenol and Implementation in Pharmacology Studies to Accelerate Drug Development

Cardiac troponin I is a useful biomarker of myocardial injury, but its use in mice and application to early drug discovery are not well described. The authors investigated the relationship between cTnI concentration in serum and histologic lesions in heart tissue from mice treated with isoproterenol (ISO). Cardiac TnI concentrations in serum increased in a dose-dependant manner and remained increased twenty-four to forty-eight hours after a single administration of isoproterenol. Increased cTnI concentration was of greater magnitude and longer duration than increased fatty acid binding protein 3 concentration, aspartate aminotransferase activity, and creatine kinase activity in serum. Isoproterenol-induced increases in cTnI concentrations were both greater and more sustained in BALB/c than in CD1 mice and correlated with incidence and severity of lesions observed in heart sections from both strains. In drug development studies in BALB/c mice with novel kinase inhibitors, cTnI concentration was a reliable stand-alone biomarker of cardiac injury and was used in combination with measurements of in vivo target inhibition to demonstrate an off-target contribution to cardiotoxicity. Additional attributes, including low cost and rapid turnaround time, made cTnI concentration in serum invaluable for detecting cardiotoxicity, exploring structure–activity relationships, and prioritizing development of compounds with improved safety profiles early in drug discovery.

Cardiovascular physiology and diseases of amphibians.

The class Amphibia includes three orders of amphibians: the anurans (frogs and toads), urodeles (salamanders, axolotls, and newts), and caecilians. The diversity of lifestyles across these three orders has accompanying differences in the cardiovascular anatomy and physiology allowing for adaptations to aquatic or terrestrial habitats, pulmonic or gill respiration, hibernation, and body elongation (in the caecilian). This article provides a review of amphibian cardiovascular anatomy and physiology with discussion of unique species adaptations. In addition, amphibians as cardiovascular animal models and commonly encountered natural diseases are covered.

Progressive Renal Disease Established by Renin-Coding Adeno-Associated Virus–Driven Hypertension in Diverse Diabetic Models

Progress in research and developing therapeutics to prevent diabetic kidney disease (DKD) is limited by a lack of animal models exhibiting progressive kidney disease. Chronic hypertension, a driving factor of disease progression in human patients, is lacking in most available models of diabetes. We hypothesized that superimposition of hypertension on diabetic mouse models would accelerate DKD. To test this possibility, we induced persistent hypertension in three mouse models of type 1 diabetes and two models of type 2 diabetes by adeno-associated virus delivery of renin (ReninAAV). Compared with LacZAAV-treated counterparts, ReninAAV-treated type 1 diabetic Akita/129 mice exhibited a substantial increase in albumin-to-creatinine ratio (ACR) and serum creatinine level and more severe renal lesions. In type 2 models of diabetes (C57BKLS db/db and BTBR ob/ob mice), compared with LacZAAV, ReninAAV induced significant elevations in ACR and increased the incidence and severity of histopathologic findings, with increased serum creatinine detected only in the ReninAAV-treated db/db mice. The uninephrectomized ReninAAV db/db model was the most progressive model examined and further characterized. In this model, separate treatment of hyperglycemia with rosiglitazone or hypertension with lisinopril partially reduced ACR, consistent with independent contributions of these disorders to renal disease. Microarray analysis and comparison with human DKD showed common pathways affected in human disease and this model. These results identify novel models of progressive DKD that provide researchers with a facile and reliable method to study disease pathogenesis and support the development of therapeutics.

Abstract B31: Potential for patient tailoring for prodrug of gemcitabine (LY2334737) by assessment carboxylesterase II expression in solid tumor biopsies

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.

Role of TGF-alpha in the Progression of Diabetic Kidney Disease

Transforming growth factor-alpha (TGFA) has been shown to play a role in experimental chronic kidney disease associated with nephron reduction, while its role in diabetic kidney disease (DKD) is unknown. We show here that intrarenal TGFA mRNA expression, as well as urine and serum TGFA, are increased in human DKD. We used a TGFA neutralizing antibody to determine the role of TGFA in two models of renal disease, the remnant surgical reduction model and the uninephrectomized (uniNx) db/db DKD model. In addition, the contribution of TGFA to DKD progression was examined using an adeno-associated virus approach to increase circulating TGFA in experimental DKD. In vivo blockade of TGFA attenuated kidney disease progression in both nondiabetic 129S6 nephron reduction and Type 2 diabetic uniNx db/db models, whereas overexpression of TGFA in uniNx db/db model accelerated renal disease. Therapeutic activity of the TGFA antibody was enhanced with renin angiotensin system inhibition with further improvement in renal parameters. These findings suggest a pathologic contribution of TGFA in DKD and support the possibility that therapeutic administration of neutralizing antibodies could provide a novel treatment for the disease.

Abstract A49: Circulating tumor cell (CTC) assay development for detection of H2AX as a clinical pharmacodynamic (PD) marker for Chk1 kinase inhibitors.

Circulating tumor cells are prognostic of survival in metastatic breast, colon, and prostate cancers. Additionally, CTCs are of interest because they may be representative of the phenotype/genotype of the primary and metastatic tumors, and a useful tool (e.g. patient stratification) for drug development. CTCs, as “liquid biopsies” are potentially useful as a pharmacodynamic marker allowing easy repeat sampling before and after drug treatment. We describe the development of a CTC assay measuring H2AX induction, a marker of DNA damage. The CTC assay was developed using the FDA cleared Veridex/CellSearch™ instrument and reagents for enumeration of CTCs from blood. CellSearch defines CTCs as EpCam+/DAPI+/CK 8,18,19+/CD45−. For assay development, cells from tumor cell lines representing major solid tumor types were chosen, cultured, treated with inhibitors of checkpoint kinase 1 (Chk1, LY2603618, LY2606368) or with various standard-of-care (SOC) chemotherapeutics. These treated cells were then spiked into whole blood drawn into CellSave™ (preservative+EDTA). LY2603618 and LY2606368 are inhibitors of Chk1 kinase currently in clinical development. LY2603618 is meant to be used in combination with a DNA damaging agent, while LY2606368 has potent single agent activity. Inhibition of Chk1 in cells with DNA damage allows the cells to progress into mitosis without DNA repair, leading to cell death. In tumor xenograft mouse models, treatment reduced tumor volume with elevation of γH2AX in tumors and anagen hair follicles at drug exposure levels similar to concentrations used for the CTC γH2AX development assay. For initial assay development, mouse whole blood was used and results reproduced subsequently with human whole blood from healthy subjects. The spiked tumor cells in blood samples were recovered using the CellSearch Mouse/Rat CTC kit (for mouse blood) or human CXC kit (for human blood), supplemented with the R-PE conjugated anti-γH2AX antibody. γH2AX in the recovered tumor cells was detected in the open/fourth channel on the CellSearch instrument. Results were confirmed by flow analysis. Without drug treatment, only about 0.2–2% of all cultured tumor cells were γH2AX positive. The magnitude of the induction of γH2AX in the cells after treatment was dose and cell-line dependent. Significant induction of H2AX (>20% cells) was observed after 24 hour treatment with only a few SOC agents (cisplatin, etoposide) in sensitive tumor cell lines; the majority induced moderate to low levels (usually 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 A49.

Abstract B36: Identification of carboxylesterase 2 as a hydrolase that cleaves the prodrug of gemcitabine LY2334737

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 29,29-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.

Synopsis of Sweet! Mouse Models of Diabetic Kidney Disease

Diabetes mellitus (types 1 and 2) is the leading cause of glomerular disease and end-stage renal disease in most developed countries, with estimates that one-third of people living with diabetes will develop diabetic kidney disease (DKD). The current standard of care medications slow but do not arrest progression of kidney disease, and therefore, therapy for DKD is a highly unmet medical need for patients. To discover and test novel and durable new therapies, it is necessary to develop animal models of human DKD, which authentically recapitulate the human disease state and provide translatable efficacy to human patients. Here, we review selected mouse models of human DKD, which demonstrate many of the features of type 2 human DKD.

Pathology and GLPs, Quality Control and Quality Assurance

Good laboratory practice (GLP) standards were authored by the United States Food and Drug Administration (FDA) to ensure sound and repeatable non-clinical research. They set the standard by which studies are designed, conducted, and reported to assure reproducibility, accuracy, and consistency. Similar guidelines were developed by the Environmental Protection Agency (EPA) and internationally, the Organisation for Economic Cooperation and Development (OECD) and regulatory organizations in other countries. The main goal of GLP is to assure that studies produce data that are reliable, repeatable, auditable, and globally accepted.