Sandra Buitrago

Conservation of IL-6 trans-signaling mechanisms controlling L-selectin adhesion by fever-range thermal stress.

Fever is associated with improved survival during infection in endothermic and ectothermic species although the protective mechanisms are largely undefined. Previous studies indicate that fever‐range thermal stress increases the binding activity of the L‐selectin homing receptor in human or mouse leukocytes, thereby promoting trafficking to lymphoid tissues across high endothelial venules (HEV). Here, we examined the evolutionary conservation of thermal regulation of L‐selectin‐like adhesion. Leukocytes from animals representing four taxa of vertebrates (mammals, avians, amphibians, teleosts) were shown to mediate L‐selectin‐like adhesion under shear to MECA‐79‐reactive ligands on mouse HEV in cross‐species in vitro adherence assays. L‐selectin‐like binding activity was markedly increased by fever‐range thermal stress in leukocytes of all species examined. Comparable increases in L‐selectin‐like adhesion were induced by thermal stress, IL‐6, or the IL‐6/soluble IL‐6 receptor fusion protein, hyper‐IL‐6. Analysis of the molecular basis of thermal regulation of L‐selectin‐like adhesion identified a common IL‐6 trans‐signaling mechanism in endotherms and ectotherms that resulted in activation of JAK/STAT signaling and was inhibited by IL‐6 neutralizing antibodies or recombinant soluble gp130. Conservation of IL‐6‐dependent mechanisms controlling L‐selectin adhesion over hundreds of millions of years of vertebrate evolution strongly suggests that this is a beneficial focal point regulating immune surveillance during febrile inflammatory responses.

In vivo toxicity evaluation of gold-dendrimer composite nanodevices with different surface charges

Abstract Composite nanodevices (CNDs) are multifunctional nanomaterials with potential uses in cancer imaging and therapy. Poly(amidoamine) dendrimer-based composite nanodevices are important members of this group and consist of an organic dendrimer component and an incorporated inorganic component, in this case, gold. This study addresses the short- (14 days) and long-term (78 days) in vivo toxicity of generation-5 (G5; 5 nm) PAMAM dendrimer-based gold-CNDs (Au-CNDs) with varying surface charges (positive, negative and neutral) in C57BL/6J male mice. Detailed toxicological analyses of (1) body weight changes, (2) serum chemistry and (3) histopathological examination of 22 organs showed no evidence of organ injury or organ function compromise. Zeta potential of Au-CNDs showed significant change from their parent dendrimers upon gold incorporation, making the normally lethal positive surface dendrimer biologically safe. Also homeostatic mechanisms in vivo may compensate/repair toxic effects, something not seen with in vitro assays.

Influence of the implantation site on the sensitivity of patient pancreatic tumor xenografts to Apo2L/TRAIL therapy.

Objectives We have previously demonstrated activity of Apo2L/TRAIL against patient pancreatic tumor xenografts. Here, we have examined the influence of the tumor implantation site on therapeutic response of orthotopic tumors and their metastases to Apo2L/TRAIL. Methods Sensitivity of 6 patient pancreatic tumor xenografts to Apo2L/TRAIL was determined in a subcutaneous model. To compare the response of orthotopic tumors, cells from subcutaneous xenografts were injected into the pancreas. Tumor growth was confirmed by histological examination of selected mice, and then treatment was started. When all control mice developed externally palpable tumors, the experiment was terminated, and pancreatic weights compared between control and treated groups. Magnetic resonance imaging was used to quantitate the response of orthotopic and metastatic tumors. Results The sensitivity to Apo2L/TRAIL observed in subcutaneous tumors was maintained in orthotopic tumors. Metastatic spread was observed with orthotopic tumor implantation. In an orthotopic model of a sensitive tumor, primary and metastatic tumor burden was significantly reduced, and median survival significantly extended by Apo2L/TRAIL therapy. Conclusions Our data provide evidence that the site of tumor engraftment does not alter the inherent sensitivity of patient xenografts to Apo2L/TRAIL, and these results highlight the potential of Apo2L/TRAIL therapy against primary and metastatic pancreatic cancer.

Abstract 4946: Developing anti-angiogenic therapies for human hepatocellular cancer (HCC)- studies of suntinib in the woodchuck model of hepatitis B related HCC

Introduction: Better animal models that recapitulate the liver milieu of human HCC are needed. The eastern woodchuck is an established model of human hepatitis B viral infection and spontaneously develops HCC in the context of chronic woodchuck hepatitis viral infection (WHV). The translational relevance of this model for developing anti-angiogenic therapies was evaluated using sunitinib (S), a potent oral, anti-angiogenic agent. Methods: Woodchucks were bred and inoculated at birth with titered infectious WHV pools obtained from chronic WHV carriers. By 12 months of age, the rate of chronic WHV infection was >60%. Carriers were followed by USG, upon developing HCC, 12 animals were randomized 1:1 to S or placebo (P) given once orally daily for 30 days. From a single treatment S PK study at 4 dose levels, n=3/group, simulations showed 12mg/kg daily was expected to be optimal for achieving steady state serum concentrations between 50- 100 ng/ml in woodchucks. Tumor size and blood flow were assessed using dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) before treatment and on day 28 using standardized protocols. At study completion or when animals were humanely euthanized, tumors and any other small nodules were fixed overnight in 10% buffered formalin. After standard processing and embedding in paraffin, 4 μm sections were prepared, deparaffinized, stained with hematoxylin-eosin (HE 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4946. doi:10.1158/1538-7445.AM2014-4946

Abstract 4257: Pharmacokinetics (PK) and tissue penetration of the novel VEGFR-3/FAK inhibitor, Chloropyramine

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Purpose: Previous reports demonstrate that chloropyramine has anti-tumor efficacy as a single agent, and in combination with cytotoxic agents like doxorubicin through modulation of the FAK-VEGFR-3 pathway. Since limited information is available about the pharmacokinetics of chloropyramine, we investigated the pharmacokinetics and tissue distribution of the drug following a single intraperitoneal (IP) injection. Methods: Female CD-1 mice received a single 50mg/kg dose of chloropyramine; plasma and tissue samples were collected at serial timepoints of 0.5, 1.0, 2.0, 4.0, and 6.0 hours. The tissues included brain, heart, spleen, liver, lung, muscle, and sternum. Each timepoint contained three mice. Chloropyramine concentrations were determined in both plasma and tissues, using a validated LC-MS/MS. Assay was validated with an LLOQ of 0.25ng/ml with the calibration curve ranging from 0.25-100ng/ml (3.42-12.4%CV). Noncompartmental PK analysis was performed using WinNonlin (Pharsight, Version 5.3). Pharmacokinetic parameters including: Maximal concentration (Cmax), terminal elimination half life (T1/2), area under the curve (AUC0-6hr), apparent clearance (Cl/F), apparent volume of distribution (Vd/F), and partition coefficients (Kp). Results: Following IP injection, chloropyramine followed either a mono- or bi-exponential decay in both plasma and tissues. Chloropyramine is cleared from plasma and all tissues within 12hr; thus chloropyramine does not exhibit a great potential for accumulation. Cmax was achieved within 1.0 hour for plasma and all tissues. Chloropyramine distributes well into various tissues such as the spleen, liver, lung, kidney, brain, and heart. The highest level of chloropyramine was achieved in lung tissue with a mean (sd) Cmax concentrations of 41.6 (11.6) μg/ml, respectively. In contrast, the mean Cmax in plasma was approximately 0.5 μg/ml; much less compared to tissues. The mean T1/2 was 0.83 (0.19) hr. The lungs exhibited the highest total tissue penetration of chloropyramine, representing a mean AUC of 51.3 μg-hr/g. The least total exposure was in muscle, with a mean AUC of 8.5 μg-hr/g. The apparent CL/F from plasma was 28.5 L/hr/kg while it was 0.9 L/hr/kg for lung tissue. Conclusions: These results illustrate the wide tissue distribution of chloropyramine, despite of the short plasma half-life. In addition, these results are of great importance because it provides supportive evidence of the tissue levels that will lead to anti-tumor efficacy through inhibition of FAK and VEGFR-3. Thus, evaluation of plasma and tissue concentrations of the drug elucidates the pharmacokinetics of this compound in an effort to identify target tissues into which it has a high degree of penetration to maximize antitumor efficacy. 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 4257. doi:10.1158/1538-7445.AM2011-4257

Abstract 3372: Pharmacokinetic analysis of CFAK-C4 in dogs and estimation of first dose in man.

Purpose: Inhibition of the FAK-VEGFR-3 interaction by the small molecule inhibitor chloropyramine hydrochloride (CFAK-C4) has been shown to reduce tumor growth both alone and synergistically with doxorubicin and gemcitabine in an animal tumor model. A multiple dose study of CFAK-C4 in dogs was investigated to gain knowledge in estimating a first in man dose. Methods: CFAK-C4 was given to two female and two male dogs as an IV infusion at 1.25 or 2.5 mg/kg for five minutes, on three consecutive days, days 1, 2 and 3. Single- and multiple-dose PK samples were collected on days 1 and 3 of dosing. Plasma samples were collected at 0.5, 1, 2, 4, 8, and 24 hours post-dose. CFAK-C4 concentrations were determined using LC-MS/MS, with a LLOQ of 2.5 pg/ml. Noncompartmental PK analysis was performed using WinNonlin (Pharsight, version 5.3). Results: The mean CFAK-C4 plasma concentration-time profiles revealed a biexponential decline of drug following IV infusion, independent of day of dosing. The median (range) half-life was 3.42 hours (2.75-4.06 hours) and 3.79 (3.5-5.0) on days 1 and 3 following administration of 1.25 mg/kg CFAK-C4. At the 2.5 mg/kg dose level, the median half-life was similar. The median (range) Cmax of 1.25 mg/kg CFAK-C4 was determined to be 75 ng/ml (41-87 ng/ml) on day 1 and 99 ng/ml (84-102 ng/ml) on day 3. After the 2.5 mg/kg dose the Cmax was 185 ng/ml (165-244 ng/ml) and 171 ng/ml (153-181 ng/ml) on days 1 and 3 respectively. The AUC0-24 at the 1.25 mg/kg dose level was similar on both days 1 and 3 with a median (range) of 167 ng*hr/ml (117-231 ng*hr/ml) and 178 ng*hr/ml (170-194 ng*hr/ml), respectively. The AUC0-24 was also similar after the 2.5 mg/kg dose on days 1 and 3 with a median (range) of 447 ng*hr/ml (434-495 ng*hr/ml) and 406 ng*hr/ml (400-460 ng*hr/ml), respectively. The median (range) clearance for dose 1.25 mg/kg was 7.46 L/hr/kg (5.36-10.59 L/hr/kg) on day 1 and 6.47 L/hr/kg (6.07-6.91L/hr/kg) on day 3. For the 2.5 mg/kg dose level, the median (range) clearance was slightly lower compared with the 1.25 mg/kg dose, with values of 5.48 L/hr/kg (5.02-5.66 L/hr/kg) and 5.85 L/hr/kg (5.04-5.93 L/hr/kg) on days 1 and 3, respectively. Utilizing allometric scaling techniques of PK parameters from mice and dogs for CFAK-C4, the estimated human PK parameters for volume of distribution, clearance and half-life were 1695 L, 126 L/hr, and 9.34 hours, respectively. Subsequently, using a no observable adverse event level (NOAEL) of 44.5 mg/kg IP for mice and a NOAEL of 2.5 mg/kg IV for dog, the human starting dose (assuming a 70 kg human) would be a daily dose of 25 mg and 10 mg based on mouse or dog, respectively. Given that dogs are the most sensitive species, 10 mg daily is the suggested first dose in man for CFAK-C4. Conclusions: These results demonstrate that CFAK-C4 has linear kinetics and that no accumulation of drug occurs after multiple dosing. Allometric scaling of mouse and dog data allowed for the estimation of the first in man dose of CFAK-C4. Citation Format: Allison Gaudy, John Wilton, Leslie Curtin, Elena Kurenova, Sandra Buitrago, William Cance, Gerald Fetterly. Pharmacokinetic analysis of CFAK-C4 in dogs and estimation of first dose in man. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3372. doi:10.1158/1538-7445.AM2013-3372

Abstract 4546: Variability analysis of dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) of hepatocellular carcinoma (HCC)

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL INTRODUCTION: HCC is a highly vascular, rapidly fatal cancer and antiangiogenic drugs have shown clinical benefit despite lack of radiographic tumor shrinkage. We standardized imaging methods to assess HCC in the woodchuck animal model (Marmota monax). This is a large unique animal model of spontaneous HCC with several similarities to human Hepatitis b(HBV) related HCC and such studies would allow utilization of this model to assess the functional changes with currently used targeted therapies. METHODS: Fourteen woodchucks infected at birth with known titers of sera from a chronic infectious pool of chronic woodchuck hepatitis virus (WHV), which is similar in structure and replicative function to HBV. Serial measurement of WHV antigen titers was conducted. Chronic WHV infection occurred in greater than 60% of woodchucks by 12 months. The WHV carrier woodchucks were screened with serial ultrasound and ten animals which developed HCC at a median age of 24- 36 months were included. Mediports were placed in the femoral vein under aseptic precautions for intravenous (IV) contrast administration. DCE-MRI methods for assessment of woodchuck HCC blood flow and size were optimized in a 1.5T magnet human head coil and IV gadolinium injected at 5cc per second using a rapid injector. Four variables - the median Ktrans, median AUCBN90, necrotic volume (NV) and total volume (TV) were analyzed by Virtualscopics using published methods. A total of 44 images were obtained, 32 were included in the study. Death due to disease progression or missing paired data resulted in exclusion of 12 images. Statistical analysis: We performed a variability analysis by calculating the concordance correlation coefficient (CCC). This coefficient was calculated for all four variables and compared at different time points. RESULTS: The CCC calculated for images obtained on day 1 and day 8 images was 0.85 for Ktrans, 0.59 for AUCBN90, 0.93 for NV and 0.99 for TV. Strong concordance was seen in 3 of the 4 DCE-MRI variables recorded a week apart. This lack of change over an 8-day period is expected with this cancer. We also compared CCC for day 8 and day 14 with Ktrans 0.76, AUCBN 0.6, NV 0.76, TV 0.89 showing less agreement than day 1 and 8. When compared to day 14 and day 30 CCC for different variables were Ktrans 0.59, AUCBN90 0.38, NV 0.82, TV 0.85, suggesting a downward trend with increasing time. Our data using this technique is able to identify clinically relevant changes in tumor size with little variability in short interval scanning. CONCLUSION: This is the first study validating the accuracy of DCE-MRI in a translational animal model for HCC and has potential to aid development of newer antiangiogenic therapies. 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 4546. doi:1538-7445.AM2012-4546