This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Project Summary Grant Number: 5R33EB000705-05 Project Start: 01-SEP-2004 Project End: 31-AUG-2009 The delivery of large molecular agents into the central nervous system (CNS) via the blood supply is often impossible as the blood brain barrier (BBB) protects the brain tissue from foreign molecules. The factors that determine penetration of substances from the blood to the CNS are lipid solubility, molecular size, and charge. The BBB prevents penetration of ionized water-soluble materials with molecular weight greater than 180 daltons. Thus most of the potential molecular imaging agents cannot reach the brain tissue via the blood supply. A technique that allows these agents to reach the brain tissue will open the door to new possibilities for the diagnosis and monitoring of brain disorders that currently cannot be performed. Laboratory experiments have shown that focused ultrasound beams can be used to noninvasively open the BBB in a highly localized tissue volume deep in the brain. While extensive research on the interaction with ultrasound and the CNS has been performed in animals, the clinical utilization of ultrasound in the brain has been seriously limited by the commonly accepted view that a piece of the skull bone must be removed for the ultra-sound beam to propagate into the brain. This additional procedure makes ultrasound treatments of the brain more complex, hazardous, and expensive. As a result, the effects of ultrasound in the brain have not been widely explored in clinical trials. We have demonstrated that highly focused ultrasound beams can be accurately delivered through an intact human skull noninvasively with a phased array of multiple transducers, thus eliminating the most significant barrier for using focused ultrasound in brain. The overall objective of this research is to combine our ability to deliver focused ultrasound through the intact skull and the method that allows us to use the ultrasound to open the BBB and to develop a device that can be used to open the BBB for molecular imaging agents. The device will be made MRI compatible so that it can be used to target specified anatomic locations in the brain. After the BBB is open, the molecular imaging agent can be injected, and the imaging can be performed using any imaging method. The system required to accurately focus and ultrasound beam is very complicated. While this complexity, as well as the need to shave the head, is acceptable for the targeted treatments, it makes the current system unrealistic for routine diagnostic imaging. The applicants'hypothesis is that the BBB opening can be performed with a much simpler system. Similarly, we estimate that the effect of human hair will most likely be reduced with our proposed approach. There is currently no date using the proposed ultrasound exposures for BBB opening. We propose to use the combined R21/R33 mechanisms to first establish the feasibility and then to develop a method for a relatively simple device to selectively open the BBB for molecular imaging. A successful implementation of this method will make many new molecular imaging approaches possible in brain. The development of even one such imaging method for routine clinical use would have a major impact on patient care. Benefit to NCIGT As BBB disruption using focused ultrasound is a major component of the Focused Ultrasound Core, these experiments will allow us to optimize the procedure and to understand its underlying mechanisms. Benefit to the Project This work benefited from collaboration with faculty and staff that are funded through the U41. Overview The delivery of large molecular agents into the central nervous system (CMS) via the blood supply is often impossible because the blood brain barrier (BBB) protects the brain tissue from foreign molecules. The factors that determine penetration of substances from the blood to the CNS are lipid solubility, molecular size, and charge. The BBB prevents penetration of ionized water-soluble materials with molecular weight greater than 180. Thus most of the potential molecular imaging agents cannot reach the brain tissue via the blood supply. A technique that allows these agents to reach the brain tissue will open the door to new possibilities for the diagnosis and monitoring of brain disorders that currently cannot be performed. Such a technique would also result in a new research tool to investigate brain function and disorders in animal models using molecular imaging. Optimization of ultrasound-induced BBB disruption To optimize the procedure, we will first investigate different acoustic parameters to determine the best values for BBB disruption. In experiments in rabbits, we will vary the ultrasound frequency, burst length, repetition frequency, and sonication duration, and gauge the BBB disruption using MRI contrast agents, or as needed, other tracers. Further, we will investigate different commercially-available ultrasound contrast agents. The goal will be to determine which parameters result in the largest BBB disruption without causing unwanted damage to the brain. At the end of this work, we anticipate that we will have the parameters that will be used clinically. We will also characterize what sized agents we can deliver to the brain using fluorescent microspheres (from a vendor such as Invitrogen, Carlsbad, CA). These spheres can be purchased at different diameters (and excitation and emission wavelengths) and imaged and quantified with fluorescent microscopy. Spheres that are excited or fluoresce at different wavelengths can be injected at the same time, thereby providing quantitative images of the extent of the distribution of different size agents. We will inject the microspheres immediately after sonication and at later times to investigate the time course of the resealing of the BBB. These times will be determined from the experiments in the first aim: we will inject the spheres at the times when the BBB is roughly 25% and 50% closed. From those measurements we can evaluate whether the molecular size of the agent that can be delivered depends on the time after sonication. We will also be able to determine whether the closing of the BBB depends on the size of the tracer or if it closes to all agents at the same time. Finally, we will continue our work investigating methods to monitor the procedure using acoustic emission signals. In our preliminary work, we found that a sharp increase in harmonics of the ultrasound frequency occurred during sonications that resulted in BBB disruption. Based on this work, we will develop an automated system that uses the emission signals in real time to control the ultrasound bursts and we hope to be able to determine online the correct ultrasound intensities to use to maximize the BBB disruption without inducing inertial cavitation. Having such a method to guide the procedure will be important because it is difficult to determine the acoustic intensity when focusing deep into living tissue ?especially when the sonications are delivered through the intact skull. Delivery of therapeutics in animal models In this work, we will continue our efforts in delivering therapeutics to the animal brain through the ultrasound-induced BBB disruption. In our preliminary work, we have demonstrated that we can deliver clinically relevant dosages of a chemotherapy agent (liposomal doxorubicin ?Doxil?) to the normal rat brain, and we have demonstrated that we can deliver antibodies (dopamine D4 receptor-targeting antibodies) into the brains of mice. We will test the delivery of Doxil? into gliomas inoculated in rat brain. We will compare the growth of these tumors for cases with and without BBB disruption of the tissue surrounding the tumors through serial MRI studies. We will also quantify the amount of doxorubicin delivered to the brain using fluorometry (excitation: 480 nm;emission: 590 nm). In addition, we will investigate the delivery of Herceptin?, a humanized anti-human epidermal growth-factor receptor 2 (HER2 / c-erbB2) monoclonal antibody that is used clinically used to treat breast cancer patients and has shown great success in controlling local and distal breast cancer lesions. When these cancers metastasize to the brain, however, this effectiveness of this agent has been limited because of the BBB. First, we will demonstrate that we can deliver this antibody into the normal brain in experiments in mice. Next, we will inoculate breast cancer tumors in the brains of nude rats to test the effectiveness of this agent when its delivery is combined with BBB disruption. Methods For the experiments, the transducer will be attached to our MRI-compatible positioning device and submerged in a tank of degassed, deionized water. We currently have systems available for our clinical 1.5 and 3T clinical scanners. In the upcoming year, we will also construct a system for our 4.7T animal magnet as well. The anesthetized animal will be placed on its back on a plastic a tray. Acoustic coupling between the water tank and the animal's head will be achieved with plastic bag filled with water. The hair in the beam path will be removed prior to the experiments. Before the animal is placed on the system, the focal position will be located in the MRI coordinate space by imaging the heating produced by sonications in a tissue-mimicking phantom. Based on this registration, we can accurately target the focus in the brain with an accuracy of ~0.5 mm using anatomical MR images as a guide. BBB opening can be confirmed immediately after sonication using standard MRI contrast agents (such as Magnevist?, Berlex Inc., Wayne NJ).
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