24.03 Phase I Clinical Trial of Nitroglycerin added to 5-FU & IR for Treatment of Operable Rectal Cancer

Posted on December 22, 2014

S. Huerta1,2, D. H. Wang2, J. Dowell2, W. J. Hittson2, J. R. Torrosi2, H. Illum2  1University Of Texas Soutwhestern,Surgery,Dallas, TX, USA 2North Texas VA Health Care System,Surgery/Hematology Oncology/Radiation Oncolgy,Dallas, TX, USA

Introduction:
Neoajuvant chemoradiation (CRT) is currently the standard of care for patients with rectal cancer. The ability of neoadjuvant CRT to reduce tumor load in rectal cancer is heterogeneous and unpredictable.  The nitric oxide (NO) donor DETANONOate reduced tumor load in vitro and in vivo models of rectal cancer subjected to ionizing radiation (IR).  Low doses of NO from nitroglycerine (NTG) radio-sensitized breast cancer cells and prostate cancer xenografts.  Transcutaneous NTG patches in combination to chemotherapy were safe and effective in patients with lung cancer in clinical trials.  Our hypothesis is that NO from NGT can radiosensitize tumors more effectively when added to conventional treatment in patients with rectal cancer.  

Methods:
We designed an open label, non-randomized, multi-cohort, dose escalation, Phase I Clincal Trial with primary endpoint to evaluate the safety, tolerability, feasibility and maximum tolerated dose (MTD) of topical nitroglycerin in addition to 5-flourouracil and radiation therapy for neo-adjuvant treatment of loco-regionally advanced operable rectal cancer. A secondary endpoint was rate of pathological complete response (pCR) and tumor response assessed by tumor regression (TGR).  Patients were assigned to 4 sequential cohorts (3 each) of escalating dose levels of commercially available nitro glycerin patches (0.2; 0.4; 0.6 and 0.8mg/hour). All patients received radiation therapy [45-50 Gy] in 25-28 fractions to the pelvis along with continuous infusion 5-FU [225mg/m2 / day] for the duration of the radiation therapy. The radiation therapy was planned and delivered as per institutional standard of care.

Results:
From 12/2010 to 8/2014, 15 patients were enrolled in the trial as a dose-escalation protocol. They were all male age (59.9±SD 8.5; range 42-70 years-old). The observed toxicities during the study protocol were mild to moderate and manageable. Four patients developed asymptomatic grade 3 lymphopenia during CRT that resolved promptly upon completion. This was not considered a serious side-effect and was not used for decision making in regards to therapy adjustments or dose-escalation.  Two other patients developed grade 3 toxicity (diarrhea and mucositis).  These were well managed and were not considered to be related to the NTG patches.  Seven patients developed headaches [HA] (5/15 grade I, 1/15 grade II, and 1/15 grade III).  One patient in the 0.2 mg dose developed a grade III HA requiring and additional group at this dose.   No further grade III HAs were observed following dose escalation.  HAs were managed with oral pain medication.  Two patients experience pCR (0.2 mg and 0.4 mg), six had a TGR of 1 and three of 2.  All of the patients in the 0.6 mg group were down staged from stage III to stage II.  Patients in the 8 mg cohort await surgical intervention.

Conclusion:
This data demonstrate that NTG patches can be safely utilized in conjunction with neoadjuvant CRT in patients with rectal cancer.

 

24.04 Targeting Colorectal Cancer Metastasis with Folate-Conjugated Ultrastable RNA Nanoparticles

Posted on December 22, 2014

P. Rychahou1,2, F. Haque4,5, Y. Shu4,5, Y. Zaytseva1, H. L. Weiss1, E. Y. Lee1,2,3, W. Mustain2, J. Valentino2, P. Guo1,4,5, B. M. Evers1,2  1University Of Kentucky,Markey Cancer Center,Lexington, KY, USA 2University Of Kentucky,Department Of Surgery,Lexington, KY, USA 3University Of Kentucky,Pathology And Laboratory Medicine,Lexington, KY, USA 4University Of Kentucky,Nanobiotechnology Center,Lexington, KY, USA 5University Of Kentucky,Department Of Pharmaceutical Sciences,Lexington, KY, USA

Introduction: The majority of deaths from all cancers, including colorectal cancer (CRC), is the result of systemic metastasis, usually to the liver and lung. If advances are to be made in the survival of patients with stage IV CRC, more selective, highly innovative, and better targeted therapies are required. The purpose of this study was to: (i) test primary and metastatic CRCs for expression of folate receptor α (FRα), which could be used to target nanoparticles, and (ii) construct multifunctional and chemically stable RNA nanoparticles, that take advantage of receptor targeting, to specifically target CRC liver and lung metastases.

Methods: (1) To determine FRα expression, we obtained primary CRCs (n=12), CRC liver metastases (n=22) and CRC lung metastases (n=10). All samples were tested for FRα expression by immunohistochemistry (IHC) and blindly analyzed by a pathologist. (2) Thermodynamically stable 3-way junction (3WJ) RNA nanoparticles were constructed and confirmed by atomic force microscopy (AFM) and gel electrophoresis; folate and the fluorescent dye Alexa647 were conjugated to the nanoparticles to serve as a ligand for the binding to CRC cells and to provide a fluorescent marker, respectively. Nanoparticle binding to KM20 and HT29 human CRC cells was evaluated in vitro with confocal microcopy. (3) The conjugated RNA nanoparticles were next evaluated in murine (athymic nude mice) models of human CRC liver and lung metastasis.

Results: (1) FRα expression was detected in 78% of primary CRCs, 91% of CRC liver metastases, and 80% of CRC lung metastases. (2) In vitro evaluation demonstrated that folate conjugated RNA nanoparticles are selectively taken up by CRC cells via receptor-mediated endocytosis. (3) In vivo evaluation showed, for the first time, that folate-conjugated RNA nanoparticles remained intact after systemic injection and strongly bound to CRC liver and lung metastases. Conversely, no accumulation of nanoparticles was detected in normal liver or lung parenchyma. 

Conclusion: RNA nanoparticles specifically targeted metastatic liver and lung CRC cells, demonstrating the therapeutic potential of RNA-3WJ nanoparticles as a selective delivery system for the treatment of CRC metastasis. Our enthusiasm for current project is driven not only by its inherent scientific importance, but also by its translational potential, clinical impact, and the possibility to provide a more effective and less toxic delivery system targeting CRC metastases specifically in the liver and lung.

 

24.05 Nanoparticle-targeting of breast cancer stem cells improves efficacy and durability of chemotherapy.

Posted on December 22, 2014

P. T. White1, C. Subramanian1, P. T. Grogan1,2, S. Cai3, M. L. Forrest3, M. S. Cohen1  1University Of Michigan,Department Of Surgery,Ann Arbor, MI, USA 2University Of Kansas,Department Of Pharmacology, Toxicology And Therapeutics,Kansas City, KS, USA 3University Of Kansas,Department Of Pharmaceutical Sciences,Lawrence, KS, USA

Introduction: As the second leading cause of cancer-related death in US women, breast cancer recurrence, metastatic spread, and drug resistance remain a significant challenge despite current therapeutic advances. If only a small subpopulation of tumor cells called progenitor or stem cells survive treatment, they have the ability to transform and recreate a tumor or metastasize. To date no therapeutic specifically targets this cancer stem cell (CSC) population mainly due to the heterogeneity of its surface markers, although CD44 positivity is a commonality among most CSCs. We have developed a CD44-targeted hyaluronic acid nanoparticle drug-delivery platform and hypothesize that this targeting when conjugated to standard chemotherapy drugs will selectively target breast CSCs leading to an improved, more durable therapeutic response.

Methods: A panel of validated human breast cancer cell lines (SUM159,MDA-MB-468LN,T47D,SK-BR-3) were evaluated for CD44 expression levels by flow cytometry(FC). Cy5-labeled HA-drug conjugates (cisplatin, doxorubicin, and docetaxel) were evaluated by FC and fluorescent microscopy for cellular uptake and CD44 targeting. Breast CSCs were identified in the cell lines by CD44,ALDH1,CD133 marker positivity. Drug efficacy, toxicity and survival was performed using our prior published MDA-468LN triple neg model in female Nu/Nu mice via breast fat pad injection(10^6 cells). Animals were treated with either 50% or 75% MTD cis, dox, docetaxel(ACT) combination therapy weekly x 3wks vs. same doses conjugated to 35kDa HA nanoparticles vs. control then followed for survival, relapse, and tumor CD44 expression by Western Blot (WB).

Results: Receptor-mediated active drug uptake was CD44 dependent with SUM159 and MDA468LN having 80-90% uptake and >90% expression compared to 1-5% expression and uptake with CD44-low SK-BR-3 and T47D cells (p<0.001). Uptake is time dependent with a peak at 19hrs. Drug uptake was abrogated by 70-80% via CD44 saturation with excess free HA or CD44 antibody vs. unsaturated(p<0.01). 100% of HA-ACT therapy mice(75%MTD) had a complete clinical response during treatment without observed toxicity vs. 40% std ACT group(p<0.01). 85% of the HA-ACT mice(75% MTD) had a complete durable pathologic response 8 weeks post end of treatment vs. 0% survival in the std ACT and control arms by end of study(p<0.001). Tumor lysates of the HA-triple therapy mice had almost complete inhibition of CD44 expression by WB even 8 weeks after completion of treatment compared to no inhibition with the std. ACT and control groups.

Conclusions: HA-conjugation of chemotherapeutics is a novel approach to selectively target CD44 expressing breast tumor cells which includes the vast majority of breast CSCs. This in combination with improved tumoral drug delivery lead to significant improvement in survival and durability of response compared to standard combination therapy supporting further translation toward clinical applications.

24.06 Hydrogen Sulfide Improves Resuscitation via Non-Hibernatory Mechanisms in a Porcine Shock Model

Posted on December 22, 2014

S. Satterly1, J. Stallings1, S. Salgar1, Z. Hoffer1, M. Martin1  1Madigan Army Medical Center,Surgery,Tacoma, WA, USA

Introduction: Hydrogen sulfide (H2S) has been demonstrated to induce a “suspended animation-like” state in rodent models by reversible inhibition of cellular respiration and marked metabolic suppression, and has been proposed as a potential pharmacologic adjunct to resuscitation from shock states. There is little data currently available about the mechanisms and efficacy of H2S in larger animals or humans. We examined H2S as a pharmacologic adjunct to resuscitation in a porcine model of severe traumatic shock.

Methods:  Twenty-one adult swine were assigned to 3 study arms: sham, H2S, and saline vehicle controls (SC). All pigs underwent laparotomy and instrumentation, and the 2 study arms then underwent a 35% controlled hemorrhage followed by 50 minutes of truncal ischemia via aortic cross-clamp. H2S (5mg/kg) or saline was administered immediately prior to reperfusion, followed by 6 hours of resuscitation. Resuscitation requirements, laboratory parameters, end-organ histology, and inflammatory/oxidative product gene expression (by rtPCR) were measured and compared between groups.

Results: All animals survived to the 6-hour post resuscitation time point. Both treatment arms demonstrated severe shock characterized by fluid and vasopressor requirements, metabolic acidosis, and hypotension compared to sham animals. Animals treated with H2S demonstrated significantly lower resuscitative requirements (total epinephrine 727 µg vs 3052 µg, p<0.05), decreased fluid requirements, and lower serum lactate levels (7 mmol/L vs 10 mmol/L) versus SC. Cardiac output was slightly decreased with H2S treatment but all other hemodynamic and metabolic parameters were equivalent between H2S and C groups. Serum liver and kidney biomarkers were unchanged, but administration of H2S was associated with a significant improvement in histopathologic liver and kidney injury scores compared to SC (see Figure, both p<0.05).   Both study groups demonstrated significantly increased gene expression of hypoxia inducible factor (HIF-1α), and nitric oxide synthase (eNOS, iNOS2, iNOS3) relative to sham animals. However, H2S was associated with increased expression of HIF-1α and decreased iNOS2 levels compared to SC.

Conclusion: Administration of H2S in a large animal model of severe traumatic shock resulted in a significant decrease in resuscitative requirements, decreased metabolic acidosis, and less end-organ histologic injury compared to standard resuscitation. H2S did not induce profound metabolic suppression as seen in rodents, and appears to have alternative mechanisms of action in large animals.

 

24.07 Selective Inhibition of PAD4 Improves Survival in a Rat Model of Lethal Hemorrhagic Shock

Posted on December 22, 2014

W. He1, P. Zhou1, Z. Chang1, B. Liu1, J. Maxwell1, I. Halaweish1, Y. Li1, H. B. Alam1  1University Of Michigan,General Surgery,Ann Arbor, MI, USA

Introduction: We have recently shown that inhibition of peptidylarginine deiminase (PAD) improves survival in a rodent model of lethal cecal ligation and puncture. However, the role of PAD inhibitor in hemorrhagic shock is largely unknown. The goal of this study was to investigate whether and how YW3-56, a selective PAD4 inhibitor, could improve survival following lethal hemorrhagic shock.

Methods: Anesthetized male Wistar-Kyoto rats (n=10/group) were subjected to 55% blood loss over 40 minutes (35% arterial hemorrhage in 10 minutes and 20% venous hemorrhage in 30 minutes), and randomized into three groups: (1) Dimethyl sulfoxide (DMSO) vehicle, (2) YW3-56 (10 mg/kg) in DMSO, and (3) Sham (no hemorrhage, no treatment). Survival was monitored for 24 h. In a second study, RAW 264.1 mouse macrophages were exposed to hypoxic conditions (0.5% O2, 5% CO2, and 94.5% N2) at 37°C in the presence or absence of YW3-56 (2.5 μM). The cells and cultured medium were harvested at 1, 3, and 6 hours. Sham (no hypoxia, no YW3-56) group served as a control.  Cell viability was determined by MTT assay. Enzyme-linked immunosorbent assay was performed to analyze the secreted tumor necrosis factor α (TNF-α) and interleukin (IL)-6 in the culture medium.

Results: Hemorrhage was associated with 100% mortality in the DMSO treated animals within 3 hours, whereas 57% of the YW3-56-treated animals survived over 24 hours (Fig 1A, p< 0.05). YW3-56 treatment also significantly increased the cellular viability in the hypoxic macrophages (Fig 1B, p<0.001). Moreover, hypoxic insult induced TNF-α and IL-6 secretion in the medium, which was significantly attenuated by YW3-56 treatment (p<0.05).

Conclusion: Our results demonstrate for the first time that administration of YW3-56 significantly improves survival in vivo and in vitro, and inhibits hypoxia-induced production of key pro-inflammatory cytokines.  

 

« Previous Page