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Systems and methods for high-resolution in vivo imaging of biochemical activity in a living organism

This application is a continuation-in-part of U.S. application Ser. No. 10/252,311 filed Sep. 23, 2002, the entire contents of which are incorporated herein by reference.

The present invention relates generally to the field of medical diagnostic imaging. More particularly, the present invention relates to the design and synthesis of multifunctional contrast agents that are operable for both magnetic resonance imaging (“MRI”) and optical imaging, and methods of using such agents to obtain high-resolution in vivo images of biochemical activity in a living organism.

Magnetic resonance imaging (“MRI”) was established over two decades ago as a medical diagnostic technique that offers high-resolution anatomical information about the human body, and has since been used for the detection of a multitude of diseases. MRI creates images of a body using the principles of nuclear magnetic resonance. MRI can generate thin-section images of any part of the body from any angle and/or direction, in a relatively short period of time, and without surgical invasion. MRI can also create “maps” of biochemical compounds within any cross section of the body.

MRI is possible in the human body because the body is filled with small biological magnets—the most important, for MRI purposes, being the nucleus of the hydrogen atom, also know as a proton. Once a patient is placed into a MRI unit, their body is placed in a steady magnetic field that is more than 30,000 times stronger than the Earth's magnetic field. The MRI stimulates the body with radio waves to change the steady-state orientation of the protons, causing them to align with the magnetic field in one direction or the other. Then, the MRI stops the radio waves and “listens” to the body's electromagnetic transmissions at a selected frequency. The transmitted signal is used to construct images of the internal body using principles similar to those developed for computerized axial tomography scanners (CAT scanners). Since the nuclear magnetic relaxation times of tissues and tumors differ, abnormalities can be visualized on the MRI-constructed image.

Optical imaging continues to gain more acceptance as a diagnostic modality since it does not expose patients to ionizing radiation. Optical imaging is based on the detection of differences in the absorption, scattering and/or fluorescence of normal and tumor tissues. One type of optical imaging comprises near-infrared fluorescent (“NIRF”) imaging. Generally, in NIRF imaging, filtered light or a laser with a defined bandwidth is used as a source of excitation light. The excitation light travels through the body and when it encounters a NIRF molecule or optical imaging agent, the excitation light is absorbed. The fluorescent molecule (i.e., the optical imaging agent) then emits detectable light that is spectrally distinguishable from the excitation light (i.e, they are lights of different wavelengths). Generally, light that is detectable via NIRF imaging has a wavelength of approximately 600-1200 nm. The optical imaging agent increases the target:background ratio by several orders of magnitude, thereby enabling better visibility and distinguishability of the target area. Optical imaging agents can be designed so that they only emit detectable light upon the presence of a particular event (i.e., in the presence of a predetermined enzyme). Optical imaging, such as NIRF imaging, shows significant promise for detecting functional or metabolic changes, such as the overproduction of certain proteins or enzymes, in a body. This is useful because the majority of diseases induce early functional or metabolic changes in the body before anatomical changes occur. The ability to detect these metabolic changes allows for early detection, diagnosis and treatment of a disease, thereby improving the patient's chance of recovery and/or of being cured.

A contrast agent is often used in conjunction with MRI and/or optical imaging to improve and/or enhance the images obtained of a person's body. A contrast agent is a chemical substance that is introduced into the body to change the contrast between two tissues. Generally, MRI contrast agents comprise magnetic probes that are designed to enhance a given image by affecting the proton relaxation rate of the water molecules in proximity to the MRI contrast agent. This selective change of the T1 (Spin-Laffice Relaxation Time) and T2 (Spin-Spin Relaxation Time) of the tissues in the vicinity of the MRI contrast agents changes the contrast of the tissues visible via MRI. Generally, optical contrast agents comprise dyes designed to emit light when excited with outside radiation. This emitted light is then detected by an optical imaging device.

Contrast agents are administered to a person, typically via intravenous injection into their circulatory system, so that abnormalities in a person's vasculature, extracellular space and/or intracellular space can be visualized. Some contrast agents may stay in the person's vasculature and highlight the vasculature. Other contrast agents may penetrate the vessel walls and highlight abnormalities in the extracellular space or intracellular space through different mechanisms, like, for example, binding to receptors. After a contrast agent is injected into a tissue, the concentration of the contrast agent first increases, and then starts to decrease as the contrast agent is eliminated from the tissue. In general, a contrast enhancement is obtained in this manner because one tissue has a higher affinity or vascularity than another tissue. For example, most tumors have a greater MRI contrast agent uptake than the surrounding tissues, due to the increased vascularity and/or vessel wall permeability of the tumor, causing a shorter T1 and a larger signal change via MRI.

Typical MRI contrast agents belong to one of two classes: (1) complexes of a paramagnetic metal ion, such as gadolinium (Gd), or (2) coated iron nanoparticles. As free metal ions are toxic to the body, they are typically complexed with other molecules or ions to prevent them from complexing with molecules in the body, thereby lessening their toxicity. Some typical MRI contrast agents include, but are not limited to: Gd-EDTA, Gd-DTPA, Gd-DOTA, Gd-BOPTA, Gd-DOPTA, Gd-DTPA-BMA (gadodiamide), ferumoxsil, ferumoxide and ferumoxtran.

Another class of MRI contrast agents—called “smart” contrast agents—includes contrast agents that are activated by the physiology of the body or a property of a tumor, i.e, agents that are activated by pH, temperature and/or the presence of certain enzymes or ions. Some examples of MRI smart contrast agents include, but are not limited to, contrast agents that are sensitive to the calcium concentration in a body, or those that are sensitive to pH.

“Smart” optical contrast agents have recently been used in vivo to monitor enzyme activity in the human body. These smart contrast agents only produce contrast in the presence of specific proteases. Since proteases are key factors involved in multiple disease processes, the ability to tailor contrast agents or probes to specific enzymes should ultimately allow one to detect the expression levels of marker enzymes for various pathologic conditions. This approach is capable of providing all the necessary information for studying pathologies near the surface of the skin via optical imaging. However, since low localization information is characteristic of optical imaging, one or more additional modalities may be required for diagnosing pathologies deeper within the body.

Contrast agents are not only useful, but are often times required in order to make the presence of certain diseases detectable. For example, the mechanisms of contrast in MRI (such as T1, T2 and/or proton density) are somewhat limited, allowing certain diseases to remain undetectable by MRI in the absence of exogenous contrast agents. This is because none of the parameters that influence contrast are affected in some diseases without the addition of a contrast agent. Therefore, using contrast agents in conjunction with MRI offers excellent sensitivity for detecting some additional pathologic conditions, thereby allowing some diseases to be detected that would otherwise be undetectable via MRI alone. For example, MRI in the presence of contrast agents has very high sensitivity for detecting breast tumors, but very low specificity for the detection of cancerous tissue. The specificity for identifying cancerous tissue is so low via MRI because multiple pathologies, such as the recruitment and production of new blood vessels, are characterized by markers similar to those of cancerous tissue.

While both MRI and optical imaging provide useful information, neither independently provides all the information desired to help make early diagnoses of all diseases. As previously discussed, the majority of diseases induce early functional or metabolic changes in the body before anatomical changes occur. While these metabolic changes are almost impossible to detect via current MRI techniques, optical imaging shows significant promise in being able to detect such changes. However, when applications such as breast imaging are envisioned, optical imaging by itself is very limited by the spatial resolution that can be achieved. Roughly speaking, the spatial resolution of an optical image is about one-third of the distance between the source and the detector, which translates to about a 3 cm precision for localizing a small lesion in a 9 cm breast. This imprecision in localizing pathology via optical imaging might have proven to be an insurmountable drawback, leading an otherwise promising diagnostic technique to go unused. However, it is known to be advantageous to utilize MRI and optical imaging together to obtain more complete anatomical and functional information, thereby aiding in the early detection of disease. In fact, optical imaging and MRI are inherently compatible with one another, and concurrent MRI and optical images of breasts have already been acquired. However, no single multifunctional contrast agent comprising an always-activated magnetic resonance component for enhancing anatomical information and an activatable optical component for enhancing functional information currently exists.

Many contrast agents and/or detection agents are known. However, many are only unifunctional, not multifunctional. The prior art regarding unifunctional contrast agents does not suggest that it is possible to use a single detection agent to obtain images from two different modalities concurrently. While some multifunctional detection agents are known, none of them comprise an always-activated first component and an activatable second component that only emits detectable signals in the presence of a predetermined event (i.e., emits detectable light only in the presence of a particular enzyme). Furthermore, none of the prior art regarding multifunctional contrast agents discloses or suggests using an activatable optical imaging component, nor of combining a magnetic resonance imaging agent with an activatable optical imaging component.

Therefore, there is a need for systems and methods that can be used to further aid in the early detection of disease. There is also a need for systems and methods that allow for high-resolution localization of biochemical activity in a living organism. There is also a need for multifunctional contrast agents that can be utilized in two different modalities concurrently. There is yet a further need for multifunctional contrast agents that can be utilized in both MRI and optical imaging concurrently. There is still a further need for multifunctional contrast agents comprising an always-activated first component for obtaining enhanced anatomical information and an activatable second component for obtaining enhanced functional information. Finally, there is a need for multifunctional contrast agents wherein one component is an activatable component that is activatable only in the presence of a predetermined event.

Accordingly, the above-identified shortcomings of existing contrast agents and methods of using them are overcome by embodiments of the present invention. Embodiments of the present invention provide systems and methods that aid in the early detection of disease. These systems and methods allow for high-resolution in vivo imaging of the localization of biochemical activity in a living organism. Embodiments of the present invention may comprise multifunctional contrast agents that can be utilized in two different modalities concurrently. One embodiment comprises a multifunctional contrast agent that may be utilized in both MRI and optical imaging concurrently. Embodiments of these multifunctional contrast agents may comprise an always-activated first component for obtaining enhanced anatomical information and an activatable second component for obtaining enhanced functional information. In embodiments, these multifunctional contrast agents may comprise one activatable component that is activatable only in the presence of a predetermined event.

The present invention relates to multifunctional contrast agents or probes, and methods of using the same. One embodiment of the present invention comprises a multifunctional contrast agent or probe useful for both MRI and optical imaging simultaneously. In embodiments, these multifunctional contrast agents comprise a magnetic resonance component and an optical imaging component. In embodiments, the optical imaging component comprises one dye molecule and a quencher. In other embodiments, the optical imaging component comprises two dye molecules. In yet further embodiments, the optical imaging component comprises a plurality of dye molecules and a plurality of quenchers. In one embodiment, the dye molecule and the quencher are different from one another. In another embodiment, when the optical imaging component comprises two or more dye molecules, at least two of them are different from one another. In yet another embodiment, when the optical imaging component comprises a plurality of dye molecules, at least two of them are different from one another.

In accordance with the needs outlined above, embodiments of the present invention provide multifunctional detection agents, each comprising at least one activatable optical contrast agent or dye covalently linked to at least one magnetic resonance contrast agent. In one embodiment, the multifunctional detection agent comprises a magnetic resonance contrast agent covalently linked to both an optical dye and a quencher molecule, wherein the dye and quencher are linked such that when the multifunctional detection agent is excited with light of a certain wavelength, the quencher efficiently absorbs the emitted light so as to reduce the amount of light being detected by the optical detector. The magnetic resonance contrast agent may comprise a chelated gadolinium complex (i.e., Gd-DTPA or Gd-DOTA), a coated iron oxide nanoparticle, or the like. The linker can be designed so that close proximity of the dye and quencher molecule is achieved either through chemical bonds or through space. Furthermore, the linker may be designed such that, as the result of some biological or signaling process (i.e., enzyme cleavage), the proximity of the dye and the quencher is compromised (i.e., the distance between the dye and the quencher is increased), thereby allowing light to be emitted and detected by an optical imaging device.