Vibhudutta Awasthi - US grants

[unreadable] DESCRIPTION (provided by applicant): In the USA, trauma kills approximately 150,000 people each year. Despite such an impact on health care, fluid resuscitation in trauma continues to be a debatable issue. Crystalloids or colloids are commonly used and these fluids augment O2 delivery only by increasing preload. The only fluids that can increase both O2- carrying capacity and preload are hemoglobin based products. Thus, the long-term goal of this research is to develop encapsulated hemoglobin as a complete and universal resuscitative fluid to address multifaceted pathophysiology of hemorrhagic shock. Liposome encapsulated hemoglobin (LEH), as well as other hemoglobin-based O2 carriers (HBOCs), have undergone extensive evaluation. But, an efficient O2 carrier is not necessarily an efficient O2 delivery vehicle. PET has the capability to visually, quantitatively and non- invasively assess O2 delivery and metabolism in response to resuscitation. HYPOTHESES 1) LEH delivers O2 to the brain in the same fashion as RBCs, 2) cerebral O2 delivery from LEH is sustained for a longer period of time than the unencapsulated HBOCs, and 3) LEH encapsulating high-affinity hemoglobin delivers more O2 to the brain than LEH containing low-affinity hemoglobin in severe blood loss. SPECIFIC AIMS are: 1). Develop and validate image-derived cardiac time-activity curve as an arterial input function in MicroPET investigation of rat cerebral oxygen metabolism (CMRO2). 2). Investigate the effect of oxygen affinity of hemoglobin in LEH on cerebral oxygen metabolism after resuscitation in a rat model of hemorrhagic shock. 3).Evaluate the improvement in CMRO2 and energy metabolism after LEH resuscitation against standard resuscitative fluids in a rat model of hemorrhagic shock. We will perform microPET imaging with O-15 radiotracers (O2 gas, CO gas and H2O) to determine CMRO2 and CBF. A rat model of hemorrhagic shock will be resuscitated with LEH, whole blood, Ringer's lactate solution and 5% albumin. Cerebral energy metabolism will be established by tissue markers of aerobic metabolism. This is an innovative proposal that utilizes state-of-the-art imaging technology in a small animal model. The study will not only address important issues pertaining to oxygen metabolism in shock and resuscitation, it may also be useful in evaluation of other artificial oxygen carriers undergoing preclinical and clinical trials. [unreadable] [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): In the USA, trauma kills approximately 150,000 people each year. Despite such an impact on health care, fluid resuscitation in trauma continues to be a debatable issue. Crystalloids or colloids are commonly used and these fluids augment O2 delivery only by increasing preload. The only fluids that can increase both O2- carrying capacity and preload are hemoglobin based products. Thus, the long-term goal of this research is to develop encapsulated hemoglobin as a complete and universal resuscitative fluid to address multifaceted pathophysiology of hemorrhagic shock. Liposome encapsulated hemoglobin (LEH), as well as other hemoglobin-based O2 carriers (HBOCs), have undergone extensive evaluation. But, an efficient O2 carrier is not necessarily an efficient O2 delivery vehicle. PET has the capability to visually, quantitatively and non- invasively assess O2 delivery and metabolism in response to resuscitation. HYPOTHESES 1) LEH delivers O2 to the brain in the same fashion as RBCs, 2) cerebral O2 delivery from LEH is sustained for a longer period of time than the unencapsulated HBOCs, and 3) LEH encapsulating high-affinity hemoglobin delivers more O2 to the brain than LEH containing low-affinity hemoglobin in severe blood loss. SPECIFIC AIMS are: 1). Develop and validate image-derived cardiac time-activity curve as an arterial input function in MicroPET investigation of rat cerebral oxygen metabolism (CMRO2). 2). Investigate the effect of oxygen affinity of hemoglobin in LEH on cerebral oxygen metabolism after resuscitation in a rat model of hemorrhagic shock. 3).Evaluate the improvement in CMRO2 and energy metabolism after LEH resuscitation against standard resuscitative fluids in a rat model of hemorrhagic shock. We will perform microPET imaging with O-15 radiotracers (O2 gas, CO gas and H2O) to determine CMRO2 and CBF. A rat model of hemorrhagic shock will be resuscitated with LEH, whole blood, Ringer's lactate solution and 5% albumin. Cerebral energy metabolism will be established by tissue markers of aerobic metabolism. This is an innovative proposal that utilizes state-of-the-art imaging technology in a small animal model. The study will not only address important issues pertaining to oxygen metabolism in shock and resuscitation, it may also be useful in evaluation of other artificial oxygen carriers undergoing preclinical and clinical trials. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Pancreatic cancer is the fourth leading cause of cancer-related deaths in the USA. While early diagnosis is still a challenge, the standard of care after diagnosis is palliative in nature, and mostly ineffective in improving the prognosis in pancreatic cancer patients. Anticancer drugs are the cornerstone in managing pancreatic cancer, but they can cause severe adverse-effects in therapeutic doses. A more selective delivery of the cytotoxic agents to the primary and metastatic tumors would allow a dose escalation without a parallel increase in peripheral toxicity. It is therefore important not only to develop new chemotherapeutic molecules, but also ensure preferential drug delivery in cancerous tissue. The strategy should also include an in vivo assay to monitor adequacy and selectivity of drug accumulation in cancer. Our goal is to target a recently developed antiproliferative agent in pancreatic cancer, and enable molecular imaging of the targeted agent's distribution in live animals by nuclear techniques. We are developing a series of curcumin-based antiproliferative drugs including a diphenyl difluoroketone (DPDK) compound. DPDK is a potently cytotoxic in several cancer forms;we found it effective against cultured pancreatic cancer cells. We propose to target DPDK through the gastrin-releasing peptide receptors (GRPrs) which are over-expressed in pancreatic cancer tissue. We will modify the DPDK molecule to carry bombesin7-14 peptide which is a ligand for GRPr. The peptide will also harbor a chelator for Tc-99m radionuclide. The Tc-99m radiolabel will allow noninvasive imaging of bombesin-DPDK's distribution after administration. Our null hypothesis is that the targeted DPDK is not more antiproliferative than the non-targeted DPDK. For the rejection of this null hypothesis, the specific aims are: 1. To synthesize bombesin-DPDK conjugate amenable to radiolabeling with 99mTc radionuclide, and 2. To evaluate anticancer activity of bombesin-DPDK in a mouse model of pancreatic cancer. The experiments to accomplish these specific aims are designed to proceed from lab-based synthetic work to in vitro testing of the synthesized compounds in cell culture conditions, and finally to in vivo investigations in a mouse model of xenograft pancreatic tumor. We anticipate that the targeted delivery will help in localized higher concentration of DPDK in pancreatic cancer, allowing efficacy comparable to that achieved by larger doses of non-targeted drug. The strategy to develop a bifunctional molecule that can be monitored by non-invasive imaging is novel application of bombesin-GRPr interaction. The imaging will be useful to assess availability of the drug in the primary/metastatic pancreatic cancer tissue. Recent emphasis by the NCI and the FDA on imaging biomarkers provides support to the utility of imaging in drug development. PUBLIC HEALTH RELEVANCE: Effective non-surgical therapies are needed to alter the outcome and bleak prognosis in pancreatic cancer patients. The proposed research pertains to the development of a novel anticancer drug that can be delivered to the pancreatic cancer tissue in targeted manner, and that such preferential drug delivery can be monitored by non-invasive imaging.

DESCRIPTION (provided by applicant): Our long-term objective is to establish a small animal imaging facility (SAIF) at the College of Pharmacy in the University of Oklahoma Health Sciences Center (OUHSC). The purpose of this proposal is to acquire a single photon emission tomography (SPECT) system for small animals. The system will complement our existing capabilities of providing positron emission tomography (PET) and computed tomography (CT) for imaging animal models. We envision creating the SAIF infrastructure in order to distribute the expertise of nuclear imaging in growing drug development and pre-clinical efforts within the ranks of OUHSC, and other Oklahoma-based research centers and pharmaceutical laboratories. The acquisition of a SPECT system will facilitate the over all goal of the College of Pharmacy to expand nuclear pharmacy education, research and service. As such, the specific aims of this proposal are: 1. to acquire SPECT module for our existing small animal PET-CT system, and 2. to establish a mechanism for provision of imaging capabilities to the biomedical researchers. The College of Pharmacy has already acquired a PET-CT system for small animals. The system is modular in nature and has the capability of being upgraded to a tri-modality system by addition of a SPECT module. We propose to obtain a module based on advanced Cadmium Zinc Telluride detector with high energy, spatial, and contrast resolution. The proposed detector has high sensitivity of detection. The SPECT will be acquired with the imaging and reconstruction package which will enable us to simultaneously image multiple SPECT radionuclides within the same session. Significance: Recently, the FDA and NCI have published a Critical Path Initiative to address dropping submission rate of new molecular entities. The development of imaging biomarkers is an essential part of the strategy proposed to overcome this barrier. The proposed SAIF will be the first in Oklahoma region to provide multi-modality nuclear imaging. It will be leveraged by biomedical researchers in OUHSC as well as other research facilities in Oklahoma to hasten pre-clinical phase of drug development. With the ever increasing number of researchers seeking a variety of imaging support, acquisition of SPECT in OUHSC has become a necessity. The proposed SPECT system will also add essential value in the overall educational program of the College of Pharmacy which offers graduate program in nuclear pharmacy. In service area, the OUHSC Nuclear Pharmacy often produces `orphan'SPECT radiopharmaceuticals for clinical use. The existence of SPECT imaging within the College of Pharmacy will be of immense value in testing these products for their imaging potential and biodistribution before their clinical usage. Therefore, SPECT imaging in SAIF will serve all three components of the OUHSC mission- Research, Education and Service, and help OUHSC fulfill its ambition to improve regional health care. PUBLIC HEALTH RELEVANCE: We propose to acquire a single photon emission tomography (SPECT) system to image small animal models of human diseases. The SPECT will help speed up pre-clinical drug development research in Oklahoma region, supplement educational program in nuclear pharmacy, and provide a resource to the OUHSC Nuclear Pharmacy for the development of new radiopharmaceuticals.

DESCRIPTION (provided by applicant): Severe hemorrhage is the leading cause of death among trauma victims, and is responsible for approximately 3 million deaths worldwide. Much of the mortality may be prevented by the timely availability of a safe and uni- versal resuscitation fluid. While whole blood or red blood cells (RBCs) remain the preferred transfusion fluids, inadequate availability of safe blood remains a problem. Despite the optimism in combating transfusion-related infections and ensuring an educated donor population, the limited shelf-life of blood and the need to cross- match frequently cause blood inventory shortages. These issues have stimulated a search for safe, immune- tolerant, shelf-stable and efficacious oxygen carrier for both civilian emergency and military applications. He- moglobin based oxygen carriers increase both oxygen-carriage and cardiac preload. We are developing liposome-encapsulated hemoglobin or LEH as a substitute oxygen carrier closely mimick- ing the RBCs. Encapsulated hemoglobin is believed to have better control over hemodynamics as compared to other chemically-modified, but free hemoglobin products. Using a novel formulation and advanced processing technologies, we have overcome the barriers of low encapsulation, oxidative instability, expense and scale-up. The product (called NeoLEH) circulates in blood for a prolonged time, and improves cerebral metabolism. Pre- liminary data demonstrate that the smaller size of LEH (275 nm) as compared to the RBCs (7.5 5m) results in more efficient oxygen delivery to the brain, which in turn improves cerebral oxygen and energy metabolism. In the proposed work, we will test our overall hypothesis that NeoLEH improves survival and cerebral metabol- ism, and serves as a long-circulating and non-toxic oxygen carrier in hemorrhagic shock. The specific aims are to assess: 1) Survival and cerebral metabolism in a rat model of 40% hemorrhage, 2) Pharmacokinetics and biodistribution of NeoLEH, and the effect of reinjection, 3) Acute toxic effects of NeoLEH in the animal model of 40% hemorrhage, and 4) NeoLEH resuscitation and ischemia-reperfusion (I/R) injury. We propose a 4-y project for testing efficacy and safety of NeoLEH. We will study 24 h survival of rats receiv- ing NeoLEH resuscitation after 40% blood loss. Ringer's lactate, shed blood and Hextend will be used as con- trol fluids. Improvement in cerebral oxygen, glucose and energy metabolism will be evaluated by positron emission tomography and magnetic resonance imaging. Circulation persistence and biodistribution of NeoLEH will be assessed by monitoring the blood and organ levels of radiolabeled 99mTc-NeoLEH. In order to test the safety of NeoLEH, acute effects of NeoLEH infusion on phagocytic capacity of liver and complement-mediated platelet activation will be studied. Lastly, we will assess if NeoLEH infusion has any salutary effects in ische- mia-reperfusion phenomenon by monitoring intestinal integrity, neutrophil sequestration, etc. The results of the proposed investigations will be critical for the clinical development of NeoLEH. Targeted towards resuscitation in acute blood loss due to surgery or trauma, NeoLEH will serve civilian as well as military needs alike. PUBLIC HEALTH RELEVANCE: Transfusion practices in hemorrhagic shock would benefit from adequate and safe availability of a universal substitute for red blood cells. This research pertains to the development of a novel liposome-encapsulated hemoglobin preparation as an oxygen carrier. The product, called NeoLEH, will be evaluated in a rat model of hemorrhagic shock.