U.S. patent application number 11/875245 was filed with the patent office on 2008-05-22 for systems and methods for high-resolution in vivo imaging of biochemical activity in a living organism.
This patent application is currently assigned to GENERAL ELECTRIC COMPANY. Invention is credited to Mohan Mark Amaratunga, Paritosh Dhawale, Ileana Hancu, Nadeem Ishaque, Bruce Fletcher Johnson, Faisal Ahmed Syud, Denyce Kramer Wicht, Amy Casey Williams.
Application Number | 20080118439 11/875245 |
Document ID | / |
Family ID | 31992929 |
Filed Date | 2008-05-22 |
United States Patent
Application |
20080118439 |
Kind Code |
A1 |
Hancu; Ileana ; et
al. |
May 22, 2008 |
SYSTEMS AND METHODS FOR HIGH-RESOLUTION IN VIVO IMAGING OF
BIOCHEMICAL ACTIVITY IN A LIVING ORGANISM
Abstract
This invention relates to multifunctional detection agents
useful for providing high-resolution, in vivo imaging of
biochemical activity in a living organism. Methods of using these
multifunctional detection agents may comprise administering them
into a living organism, and then estimating the localization of the
detection agent using one modality (i.e., MRI), while concurrently
estimating the level of biological activity using a second modality
(i.e., optical imaging). One of the multifunctional detection
agents comprises a magnetic resonance component and an optical
imaging component. The magnetic resonance component comprises a
contrast agent that is always activated or "on". The optical
imaging component comprises a plurality of activatable contrast
agents or dyes, at least two of which are different from one
another, wherein at least one of the activatable contrast agents
can be activated or turned "on" only in the presence of a
particular event.
Inventors: |
Hancu; Ileana; (Clifton
Park, NY) ; Amaratunga; Mohan Mark; (Clifton Park,
NY) ; Wicht; Denyce Kramer; (Saratoga Springs,
NY) ; Dhawale; Paritosh; (Brookfield, WI) ;
Ishaque; Nadeem; (Clifton Park, NY) ; Syud; Faisal
Ahmed; (Guilderland, NY) ; Johnson; Bruce
Fletcher; (Scotia, NY) ; Williams; Amy Casey;
(Clifton Park, NY) |
Correspondence
Address: |
GENERAL ELECTRIC COMPANY;GLOBAL RESEARCH
PATENT DOCKET RM. BLDG. K1-4A59
NISKAYUNA
NY
12309
US
|
Assignee: |
GENERAL ELECTRIC COMPANY
Schenectady
NY
|
Family ID: |
31992929 |
Appl. No.: |
11/875245 |
Filed: |
October 19, 2007 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10252311 |
Sep 23, 2002 |
7303741 |
|
|
11875245 |
|
|
|
|
Current U.S.
Class: |
424/9.3 ;
424/9.4 |
Current CPC
Class: |
A61K 49/0056 20130101;
A61K 49/0002 20130101; A61K 49/0032 20130101; A61K 49/14 20130101;
A61K 49/10 20130101; A61K 49/085 20130101 |
Class at
Publication: |
424/9.3 ;
424/9.4 |
International
Class: |
A61K 49/06 20060101
A61K049/06; A61K 49/04 20060101 A61K049/04 |
Claims
1. A multifunctional detection agent comprising: a magnetic
resonance imaging component; and a plurality of activatable optical
imaging components, at least two of which are different from one
another; wherein the magnetic resonance imaging component and the
optical imaging component are contained in a single multifunctional
detection agent, and wherein at least one activatable optical
imaging component is activated only in the presence of a
predetermined event.
2. The multifunctional detection agent of claim 1, wherein the
magnetic resonance imaging component comprises at least one of: a
paramagnetic material and a superparamagnetic material.
3. The multifunctional detection agent of claim 2, wherein the
paramagnetic material comprises at least one of: a chelated
gadolinium complex, a chelate of a paramagnetic ion, and a coated
iron nanoparticle.
4. The multifunctional detection agent of claim 3, wherein the
paramagnetic ion comprises at least one of: manganese (Mn),
praseodymium (Pr), neodymium (Nd), samarium (Sm), europium (Eu),
terbium (Tb), dysprosium (Dy), holmium (Ho), erbium (Er), thulium
(Tm), ytterbium (Yb) and lutetium (Lu).
5. The multifunctional detection agent of claim 2, wherein the
superparamagnetic material comprises a chelate of a
superparamagnetic ion.
6. The multifunctional detection agent of claim 5, wherein the
superparamagnetic ion comprises at least one of: manganese (Mn),
praseodymium (Pr), neodymium (Nd), samarium (Sm), europium (Eu),
terbium (Tb), dysprosium (Dy), holmium (Ho), erbium (Er), thulium
(Tm), ytterbium (Yb) and lutetium (Lu).
7. The multifunctional detection agent of claim 1, wherein the
plurality of activatable optical imaging components comprises at
least one optical dye.
8. The multifunctional detection agent of claim 7, wherein the
plurality of activatable optical imaging components further
comprises at least one quencher.
9. The multifunctional detection agent of claim 1, wherein the
magnetic resonance imaging component is covalently linked to the
plurality of activatable optical imaging components.
10. The multifunctional detection agent of claim 1, wherein the
predetermined event comprises the presence of light of a
predetermined wavelength and at least one of: the presence of a
predetermined enzyme, enzymatic cleavage at fluorescence activation
sites, when temperature exceeds a predetermined value, when
temperature falls below a predetermined value, when pH exceeds a
predetermined value, and when pH falls below a predetermined
value.
11. The multifunctional detection agent of claim 1, wherein the
magnetic resonance imaging component is always activated.
12. The multifunctional detection agent of claim 1, wherein the
plurality of activatable optical imaging components is a dye or
pigment selected from the group consisting of anthranones and their
derivatives; anthraquinones and their derivatives; croconines and
their derivatives; fluorescein and its derivatives; rhodamine and
its derivatives such as tetramethylrhodamine; eosin and its
derivatives; erythrosine and its derivatives; coumarin and its
derivatives such as methyl-coumarins; pyrene and its derivatives;
monoazos, disazos, trisazos and their derivatives; benzimidazolones
and their derivatives; diketo pyrrole pyrroles and their
derivatives; dioxazines and their derivatives; diarylides and their
derivatives; indanthrones and their derivatives; isoindolines and
their derivatives; stilbene and its derivatives; isoindolinones and
their derivatives; naphtols and their derivatives; perinones and
their derivatives; perylenes and their derivatives such as
perylenic acid anhydride or perylenic acid imide; ansanthrones and
their derivative; dibenzpyrenequinones and their derivatives;
pyranthrones and their derivatives; bioranthorones and their
derivatives; isobioranthorone and their derivatives;
diphenylmethane, and triphenylmethane, type pigments; cyanine and
azomethine type pigments; indigoid type pigments; bisbenzoimidazole
type pigments; azulenium salts; pyrylium salts; thiapyrylium salts;
benzopyrylium salts; phthalocyanines and their derivatives,
pryanthrones and their derivatives; quinacidones and their
derivatives; quinophthalones and their derivatives; squaraines and
their derivatives; squarilylums and their derivatives; leuco dyes
and their derivatives, deuterated leuco dyes and their derivatives;
leuco-azine dyes; acridines; di- and tri-arylmethane, dyes;
quinoneamines; o-nitro-substituted arylidene dyes, aryl nitrone
dyes, and a combination comprising at least one of the foregoing
dyes and pigments.
13. A multifunctional detection agent comprising: a first component
capable of enhancing anatomical information in a living organism;
and a plurality of activatable second components capable of
enhancing functional information in a living organism, wherein the
first component and the plurality of activatable second components
are contained in a single multifunctional detection agent, and
wherein the activatable second component is activated only in the
presence of a predetermined event.
14. The multifunctional detection agent of claim 13, wherein the
first component is always activated.
15. The multifunctional detection agent of claim 13, wherein the
functional information comprises metabolic information.
16. The multifunctional detection agent of claim 13, wherein the
predetermined event includes the presence of light of a
predetermined wavelength and at least one of: the presence of a
predetermined enzyme, enzymatic cleavage at fluorescence activation
sites, when temperature exceeds a predetermined value, when
temperature falls below a predetermined value, when pH exceeds a
predetermined value, and when pH falls below a predetermined
value.
17. A method of obtaining high-resolution, in vivo imaging of
biochemical activity in a living organism, comprising the steps of:
obtaining an image of anatomical information of the living
organism; and obtaining an image of functional information of the
living organism, wherein a multifunctional detection agent is
present within the living organism, wherein the multifunctional
detection agent comprises a first component capable of enhancing
anatomical information of the living organism and a plurality of
activatable second components capable of enhancing functional
information of the living organism, and wherein the plurality of
activatable second components is activated only upon the occurrence
of a predetermined event.
18. The method of claim 17, wherein the multifunctional detection
agent is administered to the living organism before the obtaining
steps are performed.
19. The method of claim 17, wherein the multifunctional detection
agent is administered in at least one of the following ways:
intravenously, orally and intramuscularly.
20. The method of claim 17, wherein the image of the anatomical
information of the living organism is obtained via at least one of:
computed tomography, positron emission tomography, and magnetic
resonance imaging and wherein the image of the functional
information of the living organism is obtained via optical
imaging.
21. The method of claim 17, wherein the obtaining an image of
anatomical information and the obtaining an image of functional
information are performed concurrently.
22. A system for obtaining high-resolution, in vivo images of
biochemical activity in a living organism, comprising: a first
imaging device capable of detecting a first imaging component of a
multifunctional detection agent to obtain images of anatomical
information of the living organism; and a second imaging device
capable of detecting an activatable second imaging component of the
multifunctional detection agent to obtain images of functional
information of the living organism, wherein the activatable second
imaging component of the multifunctional detection agent is
activated only in the presence of a predetermined event; the
multifunctional detection agent comprising a plurality of
activatable second imaging components at least two of which are
different from one another.
23. The system of claim 22, wherein the functional information
comprises metabolic information.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] 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.
FIELD OF THE INVENTION
[0002] 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.
BACKGROUND OF THE INVENTION
[0003] 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.
[0004] 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.
[0005] 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.
[0006] 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
T.sub.1 (Spin-Laffice Relaxation Time) and T.sub.2 (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.
[0007] 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 T.sub.1 and a larger signal change via MRI.
[0008] 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.
[0009] 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.
[0010] "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.
[0011] 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
T.sub.1, T.sub.2 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.
[0012] 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.
[0013] 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.
[0014] 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.
SUMMARY OF THE INVENTION
[0015] 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.
[0016] 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.
[0017] 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.
[0018] One embodiment of the present invention provides
multifunctional detection agents that are designed to allow
simultaneous MRI and optical imaging of a target area so as to
allow both anatomical and functional (i.e., metabolic) information
to be obtained contemporaneously. The anatomical information is
obtained via MRI and the magnetic resonance contrast agent, while
the functional/metabolic information is obtained via optical
imaging and the optical dye(s) and/or quencher. In embodiments of
the present invention, the magnetic resonance contrast agent is
designed so as to always be "on" (i.e., always be activated and/or
detectable), while the optical contrast agent is designed so as to
only be "on" when the dye is no longer in close proximity to the
quenching molecule (i.e., the optical contrast agent is activatable
so as to only be "on" or activated or detectable when light of a
specific wavelength activates or excites the optical contrast agent
and the presence of a particular enzyme causes cleavage between the
dye and the quenching molecule). If one excites the contrast agent
with light of the appropriate wavelength, the dye in the contrast
agent will absorb the excitation light and re-emit radiation. As
long as the dye and the quencher are in close proximity to one
another, this re-emitted radiation/light will be absorbed by the
quencher, so no significant fraction of the re-emitted light will
hit the optical detector. In this case, the optical contrast agent
is deemed to be "off." However, if the dye and the quencher move
apart from one another (i.e., because of cleavage, bond breakage or
conformation change), the dye will still absorb the excitation
light, and will re-emit radiation of a slightly different
wavelength. This re-emitted radiation/light will not be absorbed by
the quencher because the dye and the quencher are too far apart,
and therefore, the light will be detected by the optical detector.
In this case, the optical contrast agent is deemed to be "on."
[0019] One aspect of the present invention relates to the
co-localization of the multifunctional agent. This co-localization
is possible because the magnetic resonance contrast agent and the
optical contrast agent/dye are covalently bound together. Another
aspect of embodiments of the present invention is that the magnetic
resonance component and the optical component are also covalently
bound to a quencher molecule that, due to its close proximity to
the dye, efficiently absorbs the emitted light of the excited
optical contrast agent. Yet another aspect of the present invention
is that the magnetic resonance contrast agent and the optical dye
remain covalently bound while a biological signaling process (i.e.,
enzyme cleavage) diminishes the close proximity of the quencher
molecule, thereby activating the optical contrast agent and turning
it "on". The optical signal produced by the activatable optical
contrast agent is directly related to the biological signaling
process, thereby allowing functional information to be
detected.
[0020] The present invention has all the advantages associated with
anatomical imaging using current magnetic resonance contrast
agents, but also allows functional information to be obtained
simultaneously via optical imaging. The multifunctional MRI/optical
imaging contrast agents of the present invention make the
simultaneous gathering of high-resolution anatomical and functional
information possible. Preferably, the optical component of these
contrast agents is only activated in the presence of a particular
event (i.e., only when a particular enzyme is present in a person's
body and light of a specific wavelength excites the optical
contrast agent).
[0021] Embodiments of the present invention comprise a
multifunctional detection agent comprising a magnetic resonance
imaging component and an activatable optical imaging component,
preferably, covalently linked to one another. In an embodiment, the
magnetic resonance imaging component and the optical imaging
component are contained in a single multifunctional detection
agent. The multifunctional detection agents of the present
invention allow magnetic resonance images and optical images of a
living organism to be obtained, preferably concurrently. In an
embodiment, the activatable optical imaging component is activated
only in the presence of light of a predetermined wavelength and
only in the presence of a predetermined event, such as in the
presence of a predetermined enzyme, and/or when enzymatic cleavage
occurs at fluorescence activation sites.
[0022] As noted above, the multifunctional detection agent may
comprise a plurality of magnetic resonance imaging components. The
respective magnetic resonance imaging components may be the same or
different. In one exemplary embodiment, the magnetic resonance
imaging components are different from one another.
[0023] In one embodiment, the magnetic resonance imaging component
of the present invention may be continually activated and comprise
a paramagnetic material such as a chelated gadolinium complex; a
chelate of a paramagnetic ion such as europium (Eu), dysprosium
(Dy), terbium (Tb), holmium (Ho), erbium (Er), thulium (Tm), or
ytterbium (Yb); a coated iron nanoparticle; or the like.
[0024] The activatable optical imaging component of the present
invention comprises at least one optical dye, and may also comprise
at least one quencher (which may also be a dye). As noted above,
the optical dye and the quencher are different from one another.
The magnetic resonance imaging component of the present invention
allows enhanced anatomical information to be obtained, while the
activatable optical imaging component allows enhanced
functional/metabolic information to be obtained. Herein, "enhanced"
means that the image or information obtained by using the
multifunctional contrast agent is of improved quality over the
image or information that would be obtained by using no contrast
agent.
[0025] Embodiments of the present invention comprise a
multifunctional detection agent comprising a first component
capable of enhancing anatomical information in a living organism
and an activatable second component capable of enhancing
functional/metabolic information in a living organism. In an
embodiment of the present invention, the first component and the
second component are contained in a single multifunctional
detection agent. In embodiments, the enhanced anatomical
information and the enhanced functional information are obtained
simultaneously. In an embodiment, the first component is always
activated or "on", while the activatable second component is
activated or turned "on" only in the presence of a predetermined
event, such as in the presence of light of a predetermined
wavelength and (1) in the presence of a predetermined enzyme, (2)
when enzymatic cleavage occurs at fluorescence activation sites,
(3) when the temperature exceeds or falls below a predetermined
value, or (4) when the pH exceeds or falls below a predetermined
value. The enhanced anatomical information may be obtained via
computed tomography, positron emission tomography, or magnetic
resonance imaging. The enhanced functional information may be
obtained via near-infrared fluorescence imaging.
[0026] Embodiments of the present invention also comprise a method
of obtaining high-resolution, in vivo imaging of biochemical
activity in a body, comprising the steps of administering a
multifunctional detection agent of the present invention to a
living organism; obtaining an image of anatomical information of
the living organism; and obtaining an image of functional/metabolic
information of the living organism. These images may be obtained
concurrently. In an embodiment, the multifunctional detection agent
is administered intravenously, but it may also be administered in
any other suitable manner such as orally or intramuscularly. In
embodiments, the image of anatomical information may be obtained
via computed tomography, positron emission tomography, or magnetic
resonance imaging. In embodiments, the image of functional
information may be obtained via optical imaging.
[0027] Embodiments of the present invention also comprise systems
for obtaining high-resolution, in vivo imaging of biochemical
activity in a body. One system comprises a first imaging device
capable of detecting a first imaging component of a multifunctional
detection agent to obtain images of anatomical information of the
living organism and a second imaging device capable of detecting an
activatable second imaging component of the multifunctional
detection agent to obtain images of functional/metabolic
information of the living organism. The first imaging device and
the second imaging device may be capable of being utilized
simultaneously, and the activatable second imaging component of the
multifunctional detection agent may be activated only in the
presence of a predetermined event. The first imaging device may
comprise a magnetic resonance imaging device, a computed tomography
device, or a positron emission tomography device. The second
imaging device may comprise an optical imaging device. In an
embodiment, the first imaging device comprises a magnetic resonance
imaging device and the second imaging device comprises an optical
imaging device. In embodiments, the first imaging device and the
second imaging device may be utilized simultaneously.
[0028] Further features, aspects and advantages of the present
invention will be more readily apparent to those skilled in the art
during the course of the following description, wherein references
are made to the accompanying figures which illustrate some
preferred forms of the present invention, and wherein like
characters of reference designate like parts throughout the
drawings.
DESCRIPTION OF THE DRAWINGS
[0029] FIGS. 1A-1E show the synthesis of a multifunctional
detection agent in accordance with one exemplary embodiment of the
present invention;
[0030] FIGS. 2A-2D show the synthesis of a multifunctional
detection agent in accordance with another exemplary embodiment of
the present invention; and
[0031] FIGS. 3A-3D show the synthesis of a multifunctional
detection agent in accordance with a third exemplary embodiment of
the present invention.
DETAILED DESCRIPTION OF THE INVENTION
[0032] For the purposes of promoting an understanding of the
invention, reference will now be made to some preferred embodiments
of the present invention as illustrated in FIGS. 1A-3D, and
specific language used to describe the same. The terminology used
herein is for the purpose of description, not limitation. Specific
structural and functional details disclosed herein are not to be
interpreted as limiting, but merely as a basis for the claims as a
representative basis for teaching one skilled in the art to
variously employ the present invention. Any modifications or
variations in the depicted detection agents and methods of using
the same, and such further applications of the principles of the
invention as illustrated herein, as would normally occur to one
skilled in the art, are considered to be within the spirit of this
invention.
[0033] Embodiments of the present invention comprise
multifunctional detection agents that function as both MRI contrast
agents and optically detectable agents or dyes. These
multifunctional detection agents aid in the detection of
physiological changes associated with biochemical changes in the
tissue, which may indicate tissue abnormality, cardiovascular
disease, thrombosis, cancer, etc. As used herein, the term "MRI
contrast agent" or "magnetic resonance contrast agent" means a
molecule that can be used to enhance an MRI image. MRI contrast
agents generally comprise a paramagnetic metal ion bound to a
chelator. As used herein, a "paramagnetic metal ion" means a metal
ion that is magnetized parallel or antiparallel to a magnetic field
to an extent proportional to the field.
[0034] As noted above, the multifunctional detection agent can
comprise one or more MRI contrast agents or MRI imaging components.
In one embodiment, the multifunctional detection agent can comprise
a plurality of MRI contrast agents or MRI imaging components at
least two of which are not identical to one another. In another
words, they are different from each other. Examples of suitable MRI
contrast agents are paramagnetic metal ions. Generally,
paramagnetic metal ions are metal ions having unpaired electrons.
Some non-limiting examples of suitable paramagnetic ions include:
manganese (Mn), praseodymium (Pr), neodymium (Nd), samarium (Sm),
europium (Eu), terbium (Tb), dysprosium (Dy), holmium (Ho), erbium
(Er), thulium (Tm), ytterbium (Yb), lutetium (Lu), gadolinium (Gd),
and combinations comprising at least one of the foregoing
paramagnetic ions.
[0035] As used herein, "optical imaging component", "optically
detectable agent", "optically detectable dye", "optical contrast
agent" and/or "optical dye" mean a photoluminescent compound (i.e.,
a compound that will emit detectable energy after excitation with
light). The optical dye may be dyes that emit fluorescent light. As
noted above, multifunctional detection agent can comprise a
plurality of optically detectable agents or optically detectable
dyes. In one embodiment, at least two of the optically detectable
agents in the multifunctional detection agent are different from
one another. In another embodiment, the optically detectable agent
and a quencher, if present, are different from one another.
[0036] In embodiments, the multifunctional detection agents of the
present invention comprise a magnetic resonance component and an
optical imaging component. The magnetic resonance component
comprises a contrast agent that is preferably always activated or
"on" so that the localization of this agent can always be detected.
Suitable magnetic resonance contrast agents include, but are not
limited to, one or more paramagnetic chelates such as Gd-DTPA,
Gd-DOTA, a coated iron nanoparticle, or the like. The optical
imaging component comprises an activatable contrast agent or dye
that is activated only in the presence of a particular event. When
the optical imaging component is activated or turned "on",
detectable light is emitted. When the optical imaging component is
not activated or is not "on", no significant fraction of the
re-emitted light falls onto the optical detector. For example, the
optical imaging component may be activated or turned "on" by the
presence a certain wavelength of light and (1) the presence of a
particular biochemical marker, (2) enzymatic cleavage at
fluorescence activation sites, (3) when there is an increase or
decrease in the temperature, or (4) when there is an increase or
decrease in the pH. Some non-limiting examples of biochemical
markers that may activate the optical imaging component of the
present invention include matrix metalloproteinases, cysteine and
serine proteases, or other biochemical markers that tend to be
preferentially over-expressed in pathological conditions.
[0037] Some non-limiting examples of suitable optically detectable
agents are anthranones and their derivatives; anthraquinones and
their derivatives; croconines and their derivatives; fluorescein
and its derivatives; rhodamine and its derivatives such as
tetramethylrhodamine; eosin and its derivatives; erythrosine and
its derivatives; coumarin and its derivatives such as
methyl-coumarins; pyrene and its derivatives; monoazos, disazos,
trisazos and their derivatives; benzimidazolones and their
derivatives; diketo pyrrole pyrroles and their derivatives;
dioxazines and their derivatives; diarylides and their derivatives;
indanthrones and their derivatives; isoindolines and their
derivatives; stilbene and its derivatives; isoindolinones and their
derivatives; naphtols and their derivatives; perinones and their
derivatives; perylenes and their derivatives such as perylenic acid
anhydride or perylenic acid imide; ansanthrones and their
derivative; dibenzpyrenequinones and their derivatives;
pyranthrones and their derivatives; bioranthorones and their
derivatives; isobioranthorone and their derivatives;
diphenylmethane, and triphenylmethane, type pigments; cyanine and
azomethine type pigments; indigoid type pigments; bisbenzoimidazole
type pigments; azulenium salts; pyrylium salts; thiapyrylium salts;
benzopyrylium salts; phthalocyanines and their derivatives,
pryanthrones and their derivatives; quinacidones and their
derivatives; quinophthalones and their derivatives; squaraines and
their derivatives; squarilylums and their derivatives; leuco dyes
and their derivatives, deuterated leuco dyes and their derivatives;
leuco-azine dyes; acridines; di- and tri-arylmethane, dyes;
quinoneamines; o-nitro-substituted arylidene dyes, aryl nitrone
dyes, or the like, or a combination comprising at least one of the
foregoing. Suitable optical dyes are described in the 1989-1991
Molecular Probes Handbook by Richard P. Haugland, hereby expressly
incorporated by reference. Suitable optical and fluorescent dyes
are described in the Invitrogen, Handbook of Fluorescent Probes and
Research at http://probes.invitrogen.com/handbook/temp.html, the
entire contents of which are hereby incorporated by reference.
[0038] Suitable optical imaging contrast agents include, but are
not limited to, compounds comprising the following dyes or modified
versions of these dyes: Cy5, Cy5.5, Cy7, IRD41, IRD700, NIR-1,
LaJolla Blue, IR780, Indocyanine Green (ICG), Alexa Fluor dyes,
Oregon Green 488 and Oregon Green 514, Texas Red, Texas Red
Sulfonyl Chloride, Texas Red-X Succinimidyl Ester, Texas Red-X STP
Ester, Texas Red C2-Dichlorotriazine Malachite green, Lucifer
Yellow, Cascade Blum, or the like, or a combination comprising at
least one of the foregoing dyes.
[0039] Examples of Alexa Fluor dyes are Alexa Fluor 350, Alexa
Fluor 405, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 500 and
Alexa Fluor 514, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 555,
Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 610, Alexa Fluor 633,
Alexa Fluor 635, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680,
Alexa Fluor 700, Alexa Fluor 750, or the like, or a combination
comprising at least one of the foregoing Alexa Fluor dyes.
[0040] In one embodiment, the multifunctional detection agents
comprise a magnetic resonance contrast agent covalently linked to
an optical dye and a quencher molecule. In another embodiment, the
multifunctional detection agents comprise a magnetic resonance
contrast agent covalently linked to at least two optical dyes. In
yet another embodiment, the multifunctional detection agents
comprise a magnetic resonance contrast agent covalently linked to
multiple optical dyes and multiple quencher molecules.
[0041] The dye(s) and/or quencher(s) of the optical imaging
component are generally located in close proximity to one another,
for example less than 100 Angstroms apart. Additionally, in some
embodiments, the dyes used in the optical imaging component emit
light slightly shifted towards the red part of the spectrum when
excited from outside the body at the proper excitation frequency.
Generally, when the optical imaging component is not activated or
"on", the dye(s) or the dye(s) and the quencher(s) are in close
proximity to one another, causing the emitted light to be
reabsorbed and therefore, be undetected by the optical detector. In
these embodiments, when the spacing between the dye(s), or the
spacing between the dye(s) and the quencher(s), is compromised or
increased, the optical imaging component is activated or turned
"on", thereby causing light to be emitted that can be detected by
the optical detector. The optical imaging component may be designed
so that the spacing between the dye(s), or the spacing between the
dye(s) and the quencher(s), is compromised or increased only as the
result of some biological or signaling process. Some non-limiting
examples of such biological or signaling processes include: the
presence of certain enzymes or biochemical changes in the body for
which the optical imaging component has been precisely designed to
detect; a change in the temperature of the tissue; or a change of
the local pH of the tissue. The presence of such biological or
signaling processes causes bond breakage or conformation change,
thereby compromising and increasing the spacing between the dye(s),
or the spacing between the dye(s) and the quencher(s), and causing
detectable light to be emitted.
[0042] The present invention also comprises methods of obtaining
high-resolution, in vivo images of biochemical activity in a living
organism. One method comprises estimating the localization of the
detection agent using one modality (i.e., MRI), while concurrently
estimating the level of biological activity using a second modality
(i.e., optical imaging). Another method comprises obtaining an
image of anatomical information of a living organism and obtaining
an image of functional information of the living organism, wherein
a multifunctional detection agent is present within the living
organism. In this embodiment, the multifunctional detection agent
comprises a first component capable of enhancing anatomical
information of the living organism and an activatable second
component capable of enhancing functional information of the living
organism, and the activatable second component is activated only in
the presence of a predetermined event. The multifunctional
detection agents of the present invention may be administered in
any suitable way, preferably via intravenous injection.
[0043] The multifunctional detection agents of the present
invention allow both anatomical and functional information to be
obtained simultaneously via two different modalities. In
embodiments of the present invention, a first modality (i.e,
magnetic resonance imaging) provides high-resolution anatomical
information from which the precise anatomical localization of the
detection agent can be determined. In embodiments of the present
invention, a second modality (i.e., optical imaging) provides
functional or metabolic information. This combination of anatomical
and functional information allows for easier and earlier diagnosis
and treatment of diseases than currently exits, thereby improving
the patient's chance of recovery and/or of being cured.
[0044] In an embodiment, the multifunctional detection agents may
comprise a polymer or co-polymer selectively grafted and/or
end-terminated with optical dye #1, optical dye #2 and the MRI
contrast agent. Some non-limiting examples of such agents
include:
##STR00001##
where can be a polymer (i.e., polypeptide) or co-polymer (i.e.,
polypeptide/pNA co-polymer) and -- represents moiety covalently
linked to the polymer or co-polymer (i.e., amino acid or pNA side
chain). The optical dyes comprise commercially available dyes such
as, for example, Cy7Q (Dye #1) and Cy5.5 (Dye #2). The MRI contrast
agent is covalently linked to the polymer or co-polymer, and is
preferably a chelated gadolinium complex such as, for example,
Gd-(DTPA). Various alternative optical dyes and magnetic contrast
agents may be used without deviating from the scope of this
invention, so long as the optical dyes are activatable only in the
presence of a predetermined event(s).
[0045] In other embodiments, the multifunctional detection agents
may comprise:
##STR00002##
[0046] One multifunctional detection agent of the present invention
may be synthesized as shown in FIGS. 1A-1E, and as more fully
described below. First, a polymer, such as, for example, the
polypeptide
Fmoc-N-Gly-Pro-Leu-Gly-Val-Arg(Pmc)-Gly-Lys(Aloc)-Gly-Asp(OBut)-C-linker--
resin (SEQ. ID. NO. 1), can be synthesized via solid phase
synthesis with standard Fmoc chemistry using various linkers and
solid-state supports or resins. Any suitable amino acid sequence
may be used in the polymer. Fmoc (9-fluorenylmethyl carbamate)
protected amino acids and solid-state supports can be purchased
from commercial suppliers, and the peptide synthesis can be
automated with a peptide synthesizer, if desired. The synthesis of
the peptide is started at the C terminal end with commercially
available polystyrene resin. The carboxylic acid group on the
aspartic acid is protected with the acid-sensitive protecting group
tertiary butyl ester (OBut). The amine group on the lysine
side-chain is protected with the orthogonal protecting group
allyloxycarbonyl (Aloc). The guanidinium group on the arginine
amino acid is protected with an acid-sensitive protecting group
such as 2,2,4,6,7-pentamethyl-dihydrobenzofuran-5-sulfonyl (Pmc) to
yield:
TABLE-US-00001 (SEQ. ID. NO. 1) Pmc Aloc OBut | | |
Fmoc-N-Gly-Pro-Leu-Gly-Val-Arg-Gly-Lys-Gly-Asp-C-linker-resin
Treatment of the protected 10 mer peptide with a palladium complex
such as palladium tetrakis triphenylphosphine
(Pd(PPh.sub.3).sub.4), acetic acid, and tributyltin hydride removes
the Aloc protecting group and leaves a free amine to yield:
TABLE-US-00002 (SEQ. ID. NO. 2) Pmc NH.sub.2 OBut | | |
Fmoc-N-Gly-Pro-Leu-Gly-Val-Arg-Gly-Lys-Gly-Asp-C-linker-resin
The compound is then treated with amine-reactive Cy5.5 NHS ester to
yield:
TABLE-US-00003 (SEQ. ID. NO. 3) Pmc Cy5.5 OBut | | |
Fmoc-N-Gly-Pro-Leu-Gly-Val-Arg-Gly-Lys-Gly-Asp-C-linker-resin
De-protection of the N-terminus with 20% piperidine in DMF removes
the Fmoc group and gives a free amine:
TABLE-US-00004 (SEQ. ID. NO. 4) Pmc Cy5.5 OBut | | |
H.sub.2N-Gly-Pro-Leu-Gly-Val-Arg-Gly-Lys-Gly-Asp-C-linker-resin
Treating the compound with amine-reactive Cy7Q NHS ester
yields:
TABLE-US-00005 (SEQ. ID. NO. 5) Cy7Q Pmc Cy5.5 OBut | | |
NH-Gly-Pro-Leu-Gly-Val-Arg-Gly-Lys-Gly-Asp-C-linker-resin
Acids, such as trifluoroacetic acid, in the presence of an
appropriate scavenger may then be used to cleave the dye-peptide
conjugate from the solid-phase support, while the arginine amino
acid and the aspartic acid amino acid are de-protected at the same
time to yield:
TABLE-US-00006 (SEQ. ID. NO. 6) Cy7Q Cy5.5 CO.sub.2H | | |
HN-Gly-Pro-Leu-Gly-Val-Arg-Gly-Lys-Gly-Asp-C-NH.sub.2 || O
The compound is then treated with the appropriate activating agent,
such as disuccinimidyl carbonate, to activate the carboxylic acid
and yield:
TABLE-US-00007 (SEQ. ID. NO. 7) Cy7Q Cy5.5 OSuc | | |
HN-Gly-Pro-Leu-Gly-Val-Arg-Gly-Lys-Gly-Asp-C-NH.sub.2 || O
The compound is then treated with
p-aminobenzyl-DTPA-penta(t-Bu)ester to yield:
TABLE-US-00008 (SEQ. ID. NO. 8) Cy7Q Cy5.5
NH-benzyl-DTPA-penta(t-Bu)ester | | |
HN-Gly-Pro-Leu-Gly-Val-Arg-Gly-Lys-Gly-Asp-C-NH.sub.2 || O
The carboxylic acid groups of the multifunctional ligand are
deprotected by treatment with trifluoroacetic acid to remove the
t-butyl groups to yield:
TABLE-US-00009 (SEQ. D. NO. 9) Cy7Q Cy5.5 NH-benzyl-DTPA | | |
HN-Gly-Pro-Leu-Gly-Val-Arg-Gly-Lys-Gly-Asp-C-NH.sub.2 || O
Finally, gadolinium is chelated to the carboxylic acid groups of
DTPA by treatment with GdCl.sub.3, and the pH is adjusted to give a
multifunctional detection agent of the present invention:
TABLE-US-00010 (SEQ. ID. NO. 10) Cy7Q Cy5.5 NH-benzyl-Gd(DTPA) | |
| HN-Gly-Pro-Leu-Gly-Val-Arg-Gly-Lys-Gly-Asp-C-NH.sub.2 || O
[0047] The final chemical structure of this embodiment is shown in
FIG. 1E. In this embodiment, Cy5.5 was selected because is has the
least interference from heme, deoxy-heme, tissue, water, etc. Cy5.5
absorbs light at about 673 nm, and emits light at about 692 nm.
Cy7Q was selected as the "dark" dye because it does not emit light,
it only absorbs the light emitted from Cy5.5, as long as the Cy5.5
and Cy7Q are less than about 100 Angstroms apart (i.e., if the
string of amino acids remains intact). When the Cy7Q and the Cy5.5
are in close proximity to one another (i.e., within about 100
Angstroms of one another) the Cy7Q acts as a quencher. However,
when a predetermined event exists (i.e., a certain enzyme is
present), the amino acid chain is cleaved between the amino acid
residues, thereby activating the optical imaging component of this
compound, and allowing optically detectable light to be emitted.
The Gd-(DTPA) was selected as the magnetic resonance contrast agent
that is always "on".
[0048] FIGS. 2A-2D show the synthesis of another multifunctional
detection agent of the present invention, and the final chemical
structure of this particular embodiment is shown in FIG. 2D. This
embodiment may be synthesized by methods similar to the methods
described for FIGS. 1A-1E.
[0049] FIGS. 3A-3D show the synthesis of yet another
multifunctional detection agent of the present invention, and the
final chemical structure of this particular embodiment is shown in
FIG. 3D. This embodiment may also be synthesized by methods similar
to the methods described for FIGS. 1A-1E.
[0050] While multifunctional detection agents for concurrent use in
combination MRI/optical imaging systems have been described above,
it is understood that multifunctional detection agents may be
designed for concurrent use in alternative combination imaging
systems without deviating from the scope of the present invention.
For example, a multifunctional detection agent for concurrent use
in computed tomography (CT) and optical imaging, or for concurrent
use in positron emission tomography (PET) and optical imaging, also
falls within the scope of this invention so long as the CT/PET
contrast agent is always "on" or activated and the optical imaging
component is activatable only in the presence of a specific
predetermined event. Other diagnostic imaging techniques that may
be combined with optical imaging include: X-ray based techniques,
ultrasound, diagnostic techniques based on radioactive materials
(e.g. scintigraphy and SPECT), and the like.
[0051] Various embodiments of the invention have been described
above. However, it should be recognized that these embodiments are
merely illustrative of the principles of various embodiments of the
present invention. Numerous modifications and adaptations thereof
will be apparent to those skilled in the art without departing from
the spirit and scope of the present invention. Thus, it is intended
that the present invention cover all suitable modifications and
variations as come within the scope of the appended claims and
their equivalents.
Sequence CWU 1
1
10110PRTArtificialSynthetic Peptide 1Xaa Pro Leu Gly Val Xaa Gly
Xaa Gly Xaa1 5 10210PRTArtificialSynthetic Peptide 2Xaa Pro Leu Gly
Val Xaa Gly Xaa Gly Xaa1 5 10310PRTArtificialSynthetic Peptide 3Xaa
Pro Leu Gly Val Xaa Gly Xaa Gly Xaa1 5 10410PRTArtificialSynthetic
Peptide 4Xaa Pro Leu Gly Val Xaa Gly Xaa Gly Xaa1 5
10510PRTArtificialSynthetic Peptide 5Xaa Pro Leu Gly Val Xaa Gly
Xaa Gly Xaa1 5 10610PRTArtificialSynthetic Peptide 6Xaa Pro Leu Gly
Val Arg Gly Xaa Gly Xaa1 5 10710PRTArtificialSynthetic Peptide 7Xaa
Pro Leu Gly Val Arg Gly Xaa Gly Xaa1 5 10810PRTArtificialSynthetic
Peptide 8Xaa Pro Leu Gly Val Arg Gly Xaa Gly Xaa1 5
10910PRTArtificialSynthetic Peptide 9Xaa Pro Leu Gly Val Arg Gly
Xaa Gly Xaa1 5 101010PRTArtificialSynthetic Peptide 10Xaa Pro Leu
Gly Val Arg Gly Xaa Gly Xaa1 5 10
* * * * *
References