Detecting in vivo urokinase plasminogen activator activity with a catalyCEST MRI contrast agent.

Urokinase plasminogen activator (uPA) promotes tumor invasion and metastasis. The monitoring of uPA activity using molecular imaging may have prognostic value and be predictive for response to anti-cancer therapies. However, the detection of in vivo enzyme activity with molecular imaging remains a challenge. To address this problem, we designed a nonmetallic contrast agent, GR-4Am-SA, that can be detected with chemical exchange saturation transfer (CEST) MRI. This agent has a peptide that is cleaved by uPA, which causes a CEST signal at 5.0 ppm to decrease, and also has a salicylic acid moiety that can produce a CEST signal at 9.5 ppm, which is largely unresponsive to enzyme activity. The two CEST signals were used to determine a reaction coordinate, representing the extent of enzyme-catalyzed cleavage of the GR-4Am-SA agent during an experimental study. Initial biochemical studies showed that GR-4Am-SA could detect uPA activity in reducing conditions. Subsequently, we used our catalyCEST MRI protocol with the agent to detect the uPA catalysis of GR-4Am-SA in a flank xenograft model of Capan-2 pancreatic cancer. The results showed an average reaction coordinate of 80% ± 8%, which was strongly dependent on the CEST signal at 5.0 ppm. The relative independence of the reaction coordinate on the CEST signal at 9.5 ppm showed that the detection of enzyme activity was largely independent of the concentration of GR-4Am-SA within the tumor tissue. These results demonstrated the advantages of a single CEST agent with biomarker-responsive and unresponsive signals for reliably assessing enzyme activity during in vivo cancer studies.

Assessing Metabolic Changes in Response to mTOR Inhibition in a Mantle Cell Lymphoma Xenograft Model Using AcidoCEST MRI.

AcidoCEST magnetic resonance imaging (MRI) has previously been shown to measure tumor extracellular pH (pHe) with excellent accuracy and precision. This study investigated the ability of acidoCEST MRI to monitor changes in tumor pHe in response to therapy. To perform this study, we used the Granta 519 human mantle cell lymphoma cell line, which is an aggressive B-cell malignancy that demonstrates activation of the phosphatidylinositol-3-kinase/Akt/mammalian target of rapamycin (mTOR) pathway. We performed in vitro and in vivo studies using the Granta 519 cell line to investigate the efficacy and associated changes induced by the mTOR inhibitor, everolimus (RAD001). AcidoCEST MRI studies showed a statistically significant increase in tumor pHe of 0.10 pH unit within 1 day of initiating treatment, which foreshadowed a decrease in tumor growth of the Granta 519 xenograft model. AcidoCEST MRI then measured a decrease in tumor pHe 7 days after initiating treatment, which foreshadowed a return to normal tumor growth rate. Therefore, this study is a strong example that acidoCEST MRI can be used to measure tumor pHe that may serve as a marker for therapeutic efficacy of anticancer therapies.

Detection of in vivo enzyme activity with CatalyCEST MRI.

PURPOSE: CatalyCEST MRI compares the detection of an enzyme-responsive chemical exchange saturation transfer (CEST) agent with the detection of an unresponsive "control" CEST agent that accounts for other conditions that influence CEST. The purpose of this study was to investigate the feasibility of in vivo catalyCEST MRI. METHODS: CEST agents that were responsive and unresponsive to the activity of urokinase plasminogen activator were shown to have negligible interaction with each other. A CEST-fast imaging with steady state precession (FISP) MRI protocol was used to acquire MR CEST spectroscopic images with a Capan-2 pancreatic tumor model after intravenous injection of the CEST agents. A function of (super)-Lorentzian line shapes was fit to CEST spectra of a region-of-interest that represented the tumor. RESULTS: The CEST effects from each agent showed the same initial uptake into tumor tissues, indicating that both agents had the same pharmacokinetic transport rates. Starting 5 min after injection, CEST from the enzyme-responsive agent disappeared more quickly than CEST from the unresponsive agent, indicating that the enzyme responsive agent was being catalyzed by urokinase plasminogen activator, while both agents also experienced net pharmacokinetic washout from the tumor. CONCLUSION: CatalyCEST MRI demonstrates that dynamic tracking of enzyme-responsive and unresponsive CEST agents during the same in vivo MRI study is feasible.

Investigation of bendamustine HCL in a phase 2 study in women with resistant ovarian cancer.

We investigated the safety and efficacy of 90 mg/m(2) bendamustine HCL, administered intravenously on days 1 and 2 every 28 days in 10 women with platinum and taxane resistant epithelial ovarian cancer. There were no objective tumor responses observed; 2 patients had stable disease. Plasma samples collected at pre-treatment and end of cycle one were analyzed for changes in circulating total cytokeratin 18 and caspase cleaved cytokeratin 18 as exploratory early biomarkers of bendamustine-induced tumor cell death. All patients had measureable levels of both total and cleaved caspase 3 cytokeratin 18, but no relationship with response was possible due to the lack of clinical benefit in treated patients. Due to the high incidence of adverse events and absence of objective responses, only ten patients were treated as predefined by the Simon Two-Stage Design in the protocol. Overall, the regimen was not well tolerated and was associated with fatigue and a greater number of gastrointestinal side effects as compared to previously reported experiences in different patient populations. However, our study subjects did experience less bone marrow suppression. The lack of tolerability could reflect the degree of tumor burden within the peritoneal cavity as well as the high number of prior regimens (median of 5) received by the patients participating in this study.

The antitumor agent imexon activates antioxidant gene expression: evidence for an oxidative stress response.

PURPOSE: The aim of this study was to identify biomarkers that may be predictive for the clinical activity of the redox-active antitumor agent imexon. EXPERIMENTAL DESIGN: cDNA microarray and quantitative real-time PCR were used to identify global changes in gene expression in peripheral blood mononuclear cells (PBMC) collected from patients treated with imexon during a phase I trial. Electrophoretic mobility shift assays and Western blot analysis were done using the RPMI8226 myeloma cell line grown in vitro and PBMCs treated ex vivo to investigate the molecular mechanism responsible for these gene changes. RESULTS: Both cDNA microarray and quantitative real-time PCR showed the up-regulation of many antioxidant genes, including thioredoxin reductase-1, glutaredoxin-2, and peroxiredoxin-3 in PBMCs collected from patients treated with imexon. Studies in PBMCs treated ex vivo and RPMI8226 myeloma cells showed that imexon increased binding to the activator protein-1 consensus sequence measured by electrophoretic mobility shift assay. Supershift analysis showed that the majority of the activator protein-1 DNA binding activity was c-Jun, with minor contribution of Jun-D. Nuclear translocation of the nuclear factor (erythroid-derived 1)-like 2 transcription factor and its binding to the antioxidant response element was also increased after imexon treatment, which correlated with an increase in the message levels for nuclear factor (erythroid-derived 1)-like 2/antioxidant response element-regulated antioxidant genes. CONCLUSIONS: Together, these results show that a predominant biological effect of imexon is a change in redox state that can be detected in surrogate normal tissues as increased redox-sensitive transcription factor binding and increased antioxidant gene expression.

Characterization of carfilzomib-resistant non-small cell lung cancer cell lines.

PURPOSE: We previously showed that carfilzomib (CFZ) has potent anti-proliferative and cytotoxic activity in a broad range of lung cancer cell lines. Here we investigate possible mechanisms of CFZ acquired resistance in lung cancer cell lines. METHODS: CFZ-resistant non-small cell lung cancer (NSCLC) cell lines were developed by exposing A549 and H520 cells to stepwise increasing concentrations of CFZ. Resistance to CFZ and cross-resistance to bortezomib and other chemotherapy drugs was measured using the MTT assay. Cytotoxicity to CFZ was determined using a CytoTox assay. Western blot was used to measure apoptosis, autophagy, and drug efflux transporter-related proteins. Quantitative targeted whole transcriptome sequencing and quantitative RT-PCR was used to measure gene expression. Flow cytometry was used to analyze intracellular accumulation of doxorubicin. RESULTS: The CFZ IC50 value of the resistant cells increased versus parental lines (2.5-fold for A549, 122-fold for H520). Resistant lines showed reduced expression of apoptosis and autophagy markers and reduced death versus parental lines following CFZ treatment. Both resistant lines exhibited higher P-glycoprotein (Pgp) gene (TempO-Seq® analysis, increased 1.2-fold in A549, > 9000-fold in H520) and protein expression levels versus parental lines. TempO-Seq® analysis indicated other drug resistance pathways were upregulated. The resistant cell lines demonstrated less accumulation of intracellular doxorubicin, and were cross-resistant to other Pgp client drugs: bortezomib, doxorubicin, and paclitaxel, but not cisplatin. CONCLUSIONS: Upregulation of Pgp appears to be an important, but not the only, mechanism of CFZ resistance in NSCLC cell lines.

The skin and hair as surrogate tissues for measuring the target effect of inhibitors of phosphoinositide-3-kinase signaling.

BACKGROUND: The purpose of the study was to evaluate the use of phospho-Akt in mouse and human skin as a surrogate target for tumor phospho-Akt to measure the effect of antitumor inhibitors of phosphatidylinositol-3-kinase (PI-3-K)/Akt (protein kinase B) signaling. METHOD: The expression of phosphoSer473-Akt was quantitatively assessed by Western blotting in human HT-29 colon, MCF-7 breast, A-549 non small cell lung tumor xenografts in mice, and by immunohistochemistry in mouse skin and human hair. RESULTS: The pattern of PI-3-K isoforms in human hair keratinocytes was similar to that in tumor but mouse hair keratinocytes showed a different pattern. A high level of phospho-Akt staining was present in keratinocytes of the external root sheath of the hair and was inhibited by the PI-3-K inhibitor PX-866 administered to mice, and in human hair exposed to PX-866 in culture. The inhibition of phospho-Akt by PX-866 in mouse hair keratinocytes was greater than inhibition of phospho-Akt in HT-29 and A-549 xenografts in the same mice. Phospho-Akt in mouse hair keratinocytes was inhibited by the Akt inhibitor PX-316 to a lesser degree than in MCF-7 tumor xenografts. CONCLUSIONS: Hair offers a way of measuring the effects of PI-3-K signaling inhibitors and, in cancer patients, may provide a readily obtainable surrogate tissue for assessing PI-3-K and phospho-Akt inhibition in tumor.

Stability of phosphoprotein as a biological marker of tumor signaling.

PURPOSE: The purpose of the study was to evaluate the stability of phosphoprotein as a marker of signaling activity in human tumors using clinical samples and xenografts. EXPERIMENTAL DESIGN: The expression of phospho-Ser473-Akt (p-Akt) was assessed by immunohistochemistry in paraffin-embedded samples from patients enrolled in a Southwest Oncology Group clinical trial of gastroesophageal junction tumors and by immunohistochemistry and Western blotting in human colon tumor xenografts at various times after removal from the animal. RESULTS: Clinical samples had evaluable p-Akt staining only when obtained as biopsies (9 of 13) and no staining was observed in tumors obtained as surgically resected samples (0 of 15). In HT-29 colon cancer xenografts, p-Akt staining was present in fresh sample but not in tissue that had been allowed to stand for 30 minutes at room temperature. Western blotting of HT-29 tumor xenografts at room temperature showed a slow decrease in total Akt with a half-life of 180 minutes and a rapid decrease in p-Akt with a half-life of 20 minutes. CONCLUSIONS: Caution should be used when using phosphoprotein levels in human tumor specimens to measure intrinsic signaling activity or drug effects because of the potential for rapid dephosphorylation. Rapid processing of biopsies is essential and postoperative surgical samples may be of limited value because of the time to fixation.

The thioredoxin-1 inhibitor 1-methylpropyl 2-imidazolyl disulfide (PX-12) decreases vascular permeability in tumor xenografts monitored by dynamic contrast enhanced magnetic resonance imaging.

PURPOSE: The purpose of this study was to use dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) to measure changes in tumor xenograft permeability produced by the antitumor thioredoxin-1 (Trx-1) inhibitor 1-methylpropyl 2-imidazolyl disulfide (PX-12) and to assess the relationship to Trx-1 and vascular endothelial growth factor (VEGF) levels. EXPERIMENTAL DESIGN: DCE-MRI was used to monitor the dynamics of gadolinium-diethylenetriaminepentaacetic acid coupled bovine serum albumin as a macromolecular contrast reagent to measure hemodynamic changes in HT-29 human colon xenografts in immunodeficient mice treated with PX-12. Blood vessel permeability was estimated from the slope of the enhancement curves, and tumor vascular volume fraction from the ordinate. Tumor Trx-1 and VEGF was also measured. RESULTS: PX-12 caused a rapid 63% decrease in the average tumor blood vessel permeability within 2 hours of administration. The decrease lasted 24 hours and had returned to pretreatment values by 48 hours. The changes in vascular permeability were not accompanied by alterations in average tumor vascular volume fraction. There was a decrease in tumor and tumor-derived VEGF in plasma at 24 hours after treatment with PX-12, but not at earlier time points. However, tumor redox active Trx-1 showed a rapid decline within 2 hours following PX-12 administration that was maintained for 24 hours. CONCLUSION: The rapid decrease in tumor vascular permeability caused by PX-12 administration coincided with a decrease in tumor redox active Trx-1 and preceded a decrease in VEGF. DCE-MRI responses to PX-12 in patients of Trx-1 inhibition at early time points and decreased VEGF at later times, may be useful to follow tumor response and even therapeutic benefit.

Promoting Adoption of the 3Rs through Regulatory Qualification.

One mechanism to advance the application of novel safety assessment methodologies in drug development, including in silico or in vitro approaches that reduce the use of animals in toxicology studies, is regulatory qualification. Regulatory qualification, a formal process defined at the the U. S. Food and Drug Administration and the European Medicines Agency, hinges on a central concept of stating an appropriate "context of use" for a novel drug development tool (DDT) that precisely defines how that DDT can be used to support decision making in a regulated drug development setting. When accumulating the data to support a particular "context-of-use," the concept of "fit-for-purpose" often guides assay validation, as well as the type and amount of data or evidence required to evaluate the tool. This paper will review pathways for regulatory acceptance of novel DDTs and discuss examples of safety projects considered for regulatory qualification. Key concepts to be considered when defining the evidence required to formally adopt and potentially replace animal-intensive traditional safety assessment methods using qualified DDTs are proposed. Presently, the use of qualified translational kidney safety biomarkers can refine and reduce the total numbers of animals used in drug development. We propose that the same conceptual regulatory framework will be appropriate to assess readiness of new technologies that may eventually replace whole animal models.

Carfilzomib combined with suberanilohydroxamic acid (SAHA) synergistically promotes endoplasmic reticulum stress in non-small cell lung cancer cell lines.

PURPOSE: The endoplasmic reticulum (ER) stress response is a therapeutic target for pharmacologic intervention in cancer cells. We hypothesized that combining carfilzomib (CFZ), a proteasome inhibitor, and vorinostat (SAHA), a histone deacetylase (HDAC) inhibitor, would synergistically activate ER stress in non-small cell lung cancer (NSCLC) cell lines, resulting in enhanced anti-tumor activity. METHODS: Five NSCLC cell lines were treated with CFZ, SAHA, or the combination and cell proliferation measured using the MTT assay. Calcusyn software was utilized to determine the combination index as a measure of synergy. Cell viability and cytotoxicity were measured using trypan blue exclusion, CellTiter, and CytoTox assays. Western blot was used to measure markers of apoptosis, ER stress, and oxidative stress-related proteins. Reactive oxygen species (ROS) was measured using the fluorophore CM-H2DCFDA. RESULTS: Synergistic activity was observed for all cell lines following 48 and 72 h of combined treatment. H520 and A549 cell lines were used to assess viability and apoptosis. In both cell lines, increased death and cleaved caspase-3 were observed following combination treatment as compared with single-agent treatments. Combination therapy was associated with upregulation of ER stress-regulated proteins including activating transcription factor 4, GRP78/BiP, and C/EBP homologous protein. Both cell lines also showed increased ROS and the oxidative stress-related protein, heat shock protein 70. CONCLUSION: Combining proteasome inhibition with HDAC inhibition enhances ER stress, which may contribute to the synergistic anticancer activity observed in NSCLC cell lines. Further preclinical and clinical studies of CFZ + SAHA in NSCLC are warranted.

MR Imaging Biomarkers to Monitor Early Response to Hypoxia-Activated Prodrug TH-302 in Pancreatic Cancer Xenografts.

TH-302 is a hypoxia-activated prodrug known to activate selectively under the hypoxic conditions commonly found in solid tumors. It is currently being evaluated in clinical trials, including two trials in Pancreatic Ductal Adenocarcinomas (PDAC). The current study was undertaken to evaluate imaging biomarkers for prediction and response monitoring of TH-302 efficacy in xenograft models of PDAC. Dynamic contrast-enhanced (DCE) and diffusion weighted (DW) magnetic resonance imaging (MRI) were used to monitor acute effects on tumor vasculature and cellularity, respectively. Three human PDAC xenografts with known differential responses to TH-302 were imaged prior to, and at 24 h and 48 hours following a single dose of TH-302 or vehicle to determine if imaging changes presaged changes in tumor volumes. DW-MRI was performed at five b-values to generate apparent diffusion coefficient of water (ADC) maps. For DCE-MRI, a standard clinically available contrast reagent, Gd-DTPA, was used to determine blood flow into the tumor region of interest. TH-302 induced a dramatic decrease in the DCE transfer constant (Ktrans) within 48 hours after treatment in the sensitive tumors, Hs766t and Mia PaCa-2, whereas TH-302 had no effect on the perfusion behavior of resistant SU.86.86 tumors. Tumor cellularity, estimated from ADC, was significantly increased 24 and 48 hours after treatment in Hs766t, but was not observed in the Mia PaCa-2 and SU.86.86 groups. Notably, growth inhibition of Hs766t was observed immediately (day 3) following initiation of treatment, but was not observed in MiaPaCa-2 tumors until 8 days after initiation of treatment. Based on these preclinical findings, DCE-MRI measures of vascular perfusion dynamics and ADC measures of cell density are suggested as potential TH-302 response biomarkers in clinical trials.

Radiosensitization of Hs-766T Pancreatic Tumor Xenografts in Mice Dosed with Dodecafluoropentane Nano-Emulsion-Preliminary Findings.

Tumor hypoxia is an important mediator of radiation therapy resistance. We conducted a study to investigate whether an oxygen therapeutic based upon dodecafluoropentane (DDFP) nano-emulsion (NVX-108) could increase tumor PO2 in hypoxic tumors and improve radiation response. Pancreatic (Hs-766T) tumor xenografts were grown in the flanks of 29 SCID mice. Direct tumor PO2 measurements were performed in 9 mice treated with 0.3, 0.45 and 0.6 cc/kg NVX-108 (2% w/vol DDFP) in order to assess the dose dependent increase in tumor PO2. Twenty mice were randomized into 3 groups including control (no treatment), carbogen breathing treated with 12 Gy radiation, and carbogen breathing treated with 12 Gy radiation and NVX-108 (0.6 cc/kg NVX-108 administered as 30 minute IV infusion at time of radiation). Tumor volume was monitored to assess treatment efficacy. Results showed that tumor PO2 increased in NVX-108 treated mice up to 400% with the greatest effect seen at the highest dose of 0.6 cc/kg. Tumor growth was significantly reduced in both treatment groups relative to controls (p < 0.0001). The combination of carbogen, radiation, and NVX-108 demonstrated a 2-fold reduction in average tumor volume compared to carbogen plus radiation treatment (p = 0.01). Further study of NVX-108 as a radiation sensitizer is warranted.

The anti-tumor efficacy of 2-deoxyglucose and D-allose are enhanced with p38 inhibition in pancreatic and ovarian cell lines.

PURPOSE: The anti-tumor activity of glucose analogs 2-deoxy-glucose (2-DG) and D-allose was investigated alone or in combination with p38 mitogen-activated protein kinase (MAPK) inhibitor SB202190 or platinum analogs as a strategy to pharmacologically target glycolytic tumor phenotypes. METHODS: Hypoxia inducible factor-1 alpha (HIF-1α) protein accumulation in pancreatic cell lines treated with SB202190 alone and in combination with glucose analogs was analyzed by Western blot. HIF-1α transcriptional activity was measured in MIA PaCa-2 cells stably transfected with a hypoxia response element luciferase reporter following treatment with glucose analogs alone, and in combination with SB202190. Induction of cleaved poly(ADP-ribose) polymerase (PARP) was measured by Western blot in the MIA PaCa-2 cells. In vitro anti-proliferative activity of 2-DG and D-allose alone, or in combination with oxaliplatin (pancreatic cell lines), cisplatin (ovarian cell lines), or with SB202190 were investigated using the MTT assay. RESULTS: SB202190 decreased HIF-1α protein accumulation and transcriptional activity. 2-DG demonstrated greater anti-proliferative activity than D-allose. Pre-treatment with SB202190 enhanced activity of both 2-DG and D-allose in MIA PaCa-2, BxPC-3, ASPC-1, and SK-OV-3 cells. The combination of D-allose and platinum agents was additive to moderately synergistic in all but the OVCAR-3 and HEY cells. SB202190 pre-treatment further enhanced activity of D-allose and 2-DG with platinum agents in most cell lines investigated. CONCLUSIONS: SB202190 induced sensitization of tumor cells to 2-DG and D-allose may be partially mediated by inhibition of HIF-1α activity. Combining glucose analogs and p38 MAPK inhibitors with chemotherapy may be an effective approach to target glycolytic tumor phenotypes.

Evaluation of a hypoxia regulated gene panel in ovarian cancer.

A panel of nine hypoxia regulated genes, selected from a previously published fifty gene panel, was investigated for its ability to predict hypoxic ovarian cancer phenotypes. All nine genes including vascular endothelial growth factor A, glucose transporter 1, phosphoglycerate mutase 1, lactate dehydrogenase A, prolyl 4-hydroxylase, alpha-polypeptide 1, adrenomedullin, N-myc downstream regulated 1, aldolase A, and carbonic anhydrase 9 were upregulated in the HEY and OVCAR-3 human ovarian cell lines cultured in vitro under hypoxic compared to normoxic conditions as measured by quantitative reverse transcription polymerase chain reaction (qRT-PCR). The gene panel was also elevated in HEY xenograft tumor tissue compared to HEY cells cultured in normoxia. The HEY xenograft tissue demonstrated heterogeneous positive immunohistochemical staining for the exogenous hypoxia biomarker pimonidazole, and the hypoxia regulated protein carbonic anhydrase IX. A quantitative nuclease protection assay (qNPA) was developed which included the nine hypoxia regulated genes. The qNPA assay provided similar results to those obtained using qRT-PCR for cultured cell lines. The qNPA assay was also evaluated using paraffin embedded fixed tissues including a set of five patient matched primary and metastatic serous cancers and four normal ovaries. In this small sample set the average gene expression was higher in primary and metastatic cancer tissue compared to normal ovaries for the majority of genes investigated. This study supports further evaluation by qNPA of this gene panel as an alternative or complimentary method to existing protein biomarkers to identify ovarian cancers with a hypoxic phenotype.

Assessment of carbonic anhydrase IX expression and extracellular pH in B-cell lymphoma cell line models.

The expression of carbonic anhydrase IX (CA IX) and its relationship to acidosis in lymphomas has not been widely studied. We investigated the protein expression of CA IX in a human B-cell lymphoma tissue microarray, and in Raji, Ramos and Granta 519 lymphoma cell lines and tumor models, while also investigating the relationship with hypoxia. An imaging method, acidoCEST magnetic resonance imaging (MRI), was used to estimate lymphoma xenograft extracellular pH (pHe). Our results showed that clinical lymphoma tissues and cell line models in vitro and in vivo had moderate CA IX expression. Although in vitro studies showed that CA IX expression was induced by hypoxia, in vivo studies did not show this correlation. Untreated lymphoma xenograft tumor pHe had acidic fractions, and an acidity score was qualitatively correlated with CA IX expression. Therefore, CA IX is expressed in B-cell lymphomas and is qualitatively correlated with extracellular acidosis in xenograft tumor models.

Pyruvate sensitizes pancreatic tumors to hypoxia-activated prodrug TH-302.

BACKGROUND: Hypoxic niches in solid tumors harbor therapy-resistant cells. Hypoxia-activated prodrugs (HAPs) have been designed to overcome this resistance and, to date, have begun to show clinical efficacy. However, clinical HAPs activity could be improved. In this study, we sought to identify non-pharmacological methods to acutely exacerbate tumor hypoxia to increase TH-302 activity in pancreatic ductal adenocarcinoma (PDAC) tumor models. RESULTS: Three human PDAC cell lines with varying sensitivity to TH-302 (Hs766t > MiaPaCa-2 > SU.86.86) were used to establish PDAC xenograft models. PDAC cells were metabolically profiled in vitro and in vivo using the Seahorse XF system and hyperpolarized (13)C pyruvate MRI, respectively, in addition to quantitative immunohistochemistry. The effect of exogenous pyruvate on tumor oxygenation was determined using electroparamagnetic resonance (EPR) oxygen imaging. Hs766t and MiaPaCa-2 cells exhibited a glycolytic phenotype in comparison to TH-302 resistant line SU.86.86. Supporting this observation is a higher lactate/pyruvate ratio in Hs766t and MiaPaCa xenografts as observed during hyperpolarized pyruvate MRI studies in vivo. Coincidentally, response to exogenous pyruvate both in vitro (Seahorse oxygen consumption) and in vivo (EPR oxygen imaging) was greatest in Hs766t and MiaPaCa models, possibly due to a higher mitochondrial reserve capacity. Changes in oxygen consumption and in vivo hypoxic status to pyruvate were limited in the SU.86.86 model. Combination therapy of pyruvate plus TH-302 in vivo significantly decreased tumor growth and increased survival in the MiaPaCa model and improved survival in Hs766t tumors. CONCLUSIONS: Using metabolic profiling, functional imaging, and computational modeling, we show improved TH-302 activity by transiently increasing tumor hypoxia metabolically with exogenous pyruvate. Additionally, this work identified a set of biomarkers that may be used clinically to predict which tumors will be most responsive to pyruvate + TH-302 combination therapy. The results of this study support the concept that acute increases in tumor hypoxia can be beneficial for improving the clinical efficacy of HAPs and can positively impact the future treatment of PDAC and other cancers.

In vivo molecular pharmacology and antitumor activity of the targeted Akt inhibitor PX-316.

Akt, a serine/threonine kinase that promotes cell survival, is activated by binding of its pleckstrin homology (PH) domain to membrane phosphatidylinositol (PtdIns)-3-phosphates formed by PtdIns-3-kinase. D-3-Deoxy-phosphatidyl-myo-inositols that cannot be phosphorylated on the 3-position of the myo-inositol group are inhibitors of the Akt PH domain. The most active compound is D-3-deoxy-phosphatidyl-myo-inositol 1-[(R)-2-methoxy-3-octadecyloxypropyl hydrogen phosphate] (PX-316). PX-316 administered intraperitoneally to mice at 150 mg/kg inhibits Akt activation in HT-29 human tumor xenografts up to 78% at 10 h with recovery to 34% at 48 h. Phosphorylation of GSK-3beta, a downstream target of Akt, is also inhibited. There is no decrease in PtdIns(3,4,5)-trisphosphate levels by PX-316, showing it is not an inhibitor of PtdIns-3-K in vivo. Gene expression profiling of HT-29 tumor xenografts shows many similarities between the effects of PX-316 and the PtdIns-3-K inhibitor wortmannin, with downregulation of several ribosomal-related genes, while PX-316 uniquely increases the expression of a group of mitochondrial-related genes. PX-316 has antitumor activity against early human MCF-7 breast cancer and HT-29 colon cancer xenografts in mice. PX-316 formulated in 20% hydroxypropyl-beta-cyclodextrin for intravenous administration is well tolerated in mice and rats with no hemolysis and no hematological toxicity. Thus, PX-316 is the lead compound of a new class of potential agents that inhibit Akt survival signaling.

Reproducibility of magnetic resonance perfusion imaging.

Dynamic MR biomarkers (T2*-weighted or susceptibility-based and T1-weighted or relaxivity-enhanced) have been applied to assess tumor perfusion and its response to therapies. A significant challenge in the development of reliable biomarkers is a rigorous assessment and optimization of reproducibility. The purpose of this study was to determine the measurement reproducibility of T1-weighted dynamic contrast-enhanced (DCE)-MRI and T2*-weighted dynamic susceptibility contrast (DSC)-MRI with two contrast agents (CA) of different molecular weight (MW): gadopentetate (Gd-DTPA, 0.5 kDa) and Gadomelitol (P792, 6.5 kDa). Each contrast agent was tested with eight mice that had subcutaneous MDA-MB-231 breast xenograft tumors. Each mouse was imaged with a combined DSC-DCE protocol three times within one week to achieve measures of reproducibility. DSC-MRI results were evaluated with a contrast to noise ratio (CNR) efficiency threshold. There was a clear signal drop (>95% probability threshold) in the DSC of normal tissue, while signal changes were minimal or non-existent (<95% probability threshold) in tumors. Mean within-subject coefficient of variation (wCV) of relative blood volume (rBV) in normal tissue was 11.78% for Gd-DTPA and 6.64% for P792. The intra-class correlation coefficient (ICC) of rBV in normal tissue was 0.940 for Gd-DTPA and 0.978 for P792. The inter-subject correlation coefficient was 0.092. Calculated K(trans) from DCE-MRI showed comparable reproducibility (mean wCV, 5.13% for Gd-DTPA, 8.06% for P792). ICC of K(trans) showed high intra-subject reproducibility (ICC = 0.999/0.995) and inter-subject heterogeneity (ICC = 0.774). Histograms of K(trans) distributions for three measurements had high degrees of overlap (sum of difference of the normalized histograms <0.01). These results represent homogeneous intra-subject measurement and heterogeneous inter-subject character of biological population, suggesting that perfusion MRI could be an imaging biomarker to monitor or predict response of disease.

Clinical and economic burden of Richter syndrome in inpatient cases of chronic lymphocytic leukemia within the United States, 2001-2010.

Richter syndrome (RS) is an aggressive transformation of chronic lymphocytic leukemia (CLL) characterized by poor prognoses. The purpose of this study was to assess clinical and economic characteristics of RS within inpatient hospital settings in the United States from 2001 to 2010. This retrospective cohort study employed data from the Agency for Healthcare Research and Quality's Healthcare Cost and Utilization Project. Overall, 46 613 cases of RS were observed across 695 080 inpatient cases of CLL, representing a national bill of $2.74 billion and involving a 9.3% inpatient mortality rate. Multivariate analyses found decreased national inpatient mortality from 2001 to 2010 of - 61.1% (p < 0.001), shorter length of stay of - 15.5% (p < 0.001) and higher charges of +20.9% (p = 0.003). Numerous characteristics were also associated with increased likelihoods of death, lengths of stay and charges. Clinically, the findings allow for an increased understanding of population-based RS case-mixes, outcome prediction and clinical risk assessments. The continued burden of illness of either RS or CLL ultimately remains contingent upon the comparative- and cost-effectiveness of both existing interventions and those in development.