U.S. patent application number 13/755506 was filed with the patent office on 2013-06-06 for in vivo mitochondrial labeling using positively-charged nitroxide enhanced and gadolinum chelate enhanced magnetic resonance imaging.
The applicant listed for this patent is Micael Joel Hardy, Joy Joseph, Balaraman Kalyanaraman, Marcos Lopez, Douglas Edward Prah, Kathleen Marie Schmainda. Invention is credited to Micael Joel Hardy, Joy Joseph, Balaraman Kalyanaraman, Marcos Lopez, Douglas Edward Prah, Kathleen Marie Schmainda.
Application Number | 20130142735 13/755506 |
Document ID | / |
Family ID | 40998510 |
Filed Date | 2013-06-06 |
United States Patent
Application |
20130142735 |
Kind Code |
A1 |
Kalyanaraman; Balaraman ; et
al. |
June 6, 2013 |
In Vivo Mitochondrial Labeling Using Positively-CHarged Nitroxide
Enhanced and Gadolinum Chelate Enhanced Magnetic Resonance
Imaging
Abstract
A system and method for acquiring MR imaging data from a subject
includes administering positively-charged nitroxides or gadolinium
chelates for in vivo mitochondrial labeling, acquiring MR imaging
data from the subject, and reconstructing an image of the subject
having enhanced contrast in areas including metabolic and/or
mitotic activity.
Inventors: |
Kalyanaraman; Balaraman;
(Wauwatosa, WI) ; Joseph; Joy; (New Berlin,
WI) ; Schmainda; Kathleen Marie; (Elm Grove, WI)
; Prah; Douglas Edward; (Muskego, WI) ; Lopez;
Marcos; (Wauwatosa, WI) ; Hardy; Micael Joel;
(Wauwatosa, WI) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Kalyanaraman; Balaraman
Joseph; Joy
Schmainda; Kathleen Marie
Prah; Douglas Edward
Lopez; Marcos
Hardy; Micael Joel |
Wauwatosa
New Berlin
Elm Grove
Muskego
Wauwatosa
Wauwatosa |
WI
WI
WI
WI
WI
WI |
US
US
US
US
US
US |
|
|
Family ID: |
40998510 |
Appl. No.: |
13/755506 |
Filed: |
January 31, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12390929 |
Feb 23, 2009 |
8388936 |
|
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13755506 |
|
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61030627 |
Feb 22, 2008 |
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Current U.S.
Class: |
424/9.33 ;
424/9.3; 424/9.36; 424/9.363; 540/465; 556/21 |
Current CPC
Class: |
C07F 9/572 20130101;
A61K 49/103 20130101; A61K 49/105 20130101; C07F 9/59 20130101;
A61K 49/106 20130101; A61K 49/108 20130101; A61K 49/20 20130101;
C07F 9/6561 20130101; C07F 9/6524 20130101 |
Class at
Publication: |
424/9.33 ;
424/9.3; 424/9.36; 424/9.363; 556/21; 540/465 |
International
Class: |
A61K 49/10 20060101
A61K049/10; C07F 15/00 20060101 C07F015/00 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0003] This invention was made with government support under
NS39958-07, RO1 CA082500, RO1 HL073056-06, and 1R21CA109280 awarded
by the National Institutes of Health. The government has certain
rights in this invention.
Claims
1. A method of in vivo MR imaging a target area of a subject
comprising the steps of: a) administering a mitochondria-targeted
contrast agent to the subject; b) applying a pulse sequence
selected to acquire MR imaging data from the target area of the
subject; and c) reconstructing an image of the target area of the
subject having enhanced contrast in areas of at least one of
metabolic and mitotic activity.
2. The method according to claim 1, wherein the
mitochondria-targeted contrast agent is administered
intravenously.
3. The method according to claim 1, wherein the
mitochondria-targeted agent is a mitochondria-targeted
nitroxide.
4. The method according to claim 3, wherein the
mitochondria-targeted nitroxide comprises a triphenylphosphonium
group or a benzyl ammonium group.
5. The method according to claim 4, wherein the
mitochondria-targeted nitroxide comprises a triphenylphosphonium
group linked to the nitroxide by a linkage selected from the group
consisting of an amide linkage, an ester linkage, and an ether
linkage.
6. The method according to claim 5, wherein the
mitochondria-targeted nitroxide is selected from the group
consisting of Mito-Tempol ether, Mito-Tempol ester, Mito-Tempol
amide, Mito-CP, Mito-Tempamide, Mito-Proxyl ether, Mito-Proxyl
ester, Mito-Proxyl amide, Tributylalkylammonium Tempol ether, and
Tribenzyalkylammonium Tempol ether.
7. The method according to claim 3, wherein the
mitochondria-targeted nitroxide is Mito-CP.
8. The method according to claim 1, wherein the
mitochondria-targeted contrast agent is a mitochondria-targeted
gadolinium chelate.
9. The method according to claim 8, wherein the
mitochondria-targeted gadolinium chelate comprises a
triphenylphosphonium group and a coordination complex consisting of
a gadolinium (III) ion bound to a hexadentate, heptadentate,
octadentate, or nonadentate organic chelating agent.
10. The method according to claim 9, wherein the organic chelating
agent is selected from the group consisting of:
1,4,7,10-tetrazacyclododecane-N,N',N'',N''' tetracetate (DOTA);
diethylenetriaminepentaacetate (DTPA);
1,4,7-triazacyclononane-N,N',N''-triacetate (NOTA);
1,5,9-triazacyclododecane-N,N',N''-triacetate (DOTRA);
1,4,8,9,11-tetraazacyclotetradecane-N,N',N'',N'''-tetraacetate
(TETA); and salts thereof.
11. The method according to claim 10, wherein the
mitochondria-targeted gadolinium chelate further comprises a
phenylthiocarbamide linking group.
12. The method according to claim 8, wherein the
mitochondria-targeted gadolinium chelate is Mito-Gd-DOTA or
Mito-Gd-DTPA.
13. A mitochondria-targeted contrast agent molecule, comprising: a)
a gadolinium (III) ion bound to a hexadentate, heptadentate,
octadentate, or nonadentate organic chelating agent; b) a
triphenylphosphonium group; and c) a phenylthiocarbamide linking
group coupling the triphenylphosphonium group to the organic
chelating agent.
14. The mitochondria-targeted contrast agent molecule according to
claim 13, wherein the organic chelating agent is selected from the
group consisting of: 1,4,7,10-tetrazacyclododecane-N,N',N'',N'''
tetracetate (DOTA); diethylenetriaminepentaacetate (DTPA);
1,4,7-triazacyclononane-N,N',N''-triacetate (NOTA);
1,5,9-triazacyclododecane-N,N',N''-triacetate (DOTRA);
1,4,8,11-tetraazacyclotetradecane-N,N',N'',N'''-tetraacetate
(TETA); and salts thereof.
15. The mitochondria-targeted contrast agent molecule according to
claim 13, wherein the phenylthiocarbamide linking group
additionally comprises a ten carbon alkyl chain.
16. A compound having the formula: ##STR00005## wherein L is a
paramagnetic metal selected from Cr, Mn, Fe, Co, Ni, Cu, Pr, Nd,
Sm, Y, Gd, Tb, Dy, Nd, Pm, Ho, Sm, Tm, Eu, Yb, Lu or Er; wherein
R.sub.1 is S, N or P, wherein R.sub.2 is a branched or straight
chain, saturated or unsaturated, substituted or unsubstituted
C.sub.1-25 group, wherein R.sub.3 is a branched or straight chain,
saturated or unsaturated, substituted or unsubstituted organic
chelating moiety comprising one or more of carboxyl, amine, amide,
ester, alcohol or thiol, wherein R.sub.4, R.sub.5 or R.sub.6 are
the same or independently a straight or branched, saturated or
unsaturated, substituted or unsubstituted C.sub.1-10 alkyl or
cycloalkyl, substituted or unsubstituted phenyl, or substituted or
unsubstituted benzyl, and wherein X.sup.- is Cl.sup.-, I.sup.-,
Fl.sup.- or another salt-forming counterion, or a solvate or
hydrate thereof.
17. The compound according to claim 16, wherein R.sub.2 is a
branched or straight chain, saturated or unsaturated, substituted
or unsubstituted C.sub.4-10 alkyl.
18. The compound according to claim 16, wherein R.sub.3 the organic
chelating moiety is a hexadentate, heptadentate, octadentate, or
nonadentate organic chelating moiety.
19. The compound according to claim 16, wherein R.sub.3 the organic
chelating moiety is selected from the group consisting of:
1,4,7,10-tetrazacyclododecane-N,N',N'',N'''tetracetate (DOTA);
diethylenetriaminepentaacetate (DTPA);
1,4,7-triazacyclononane-N,N',N''-triacetate (NOTA);
1,5,9-triazacyclododecane-N,N',N''-triacetate (DOTRA);
1,4,8,11-tetraazacyclotetradecane-N,N',N'',N'''-tetraacetate
(TETA); and salts thereof.
20. The compound according to claim 16, wherein L is Gd.
21. An injectable dosage form, comprising a compound according to
claim 16 and a pharmaceutically suitable injectable carrier
system.
22. A compound made by the steps of: (a) providing a compound
having the formula: ##STR00006## wherein R.sub.1 is S, N or P,
wherein R.sub.2 is branched or straight chain, saturated or
unsaturated, substituted or unsubstituted C.sub.1-25 group, wherein
R.sub.3 is a branched or straight chain, saturated or unsaturated,
substituted or unsubstituted organic chelating moiety comprising
one or more of carboxyl, amine, amide, ester, alcohol or thiol,
wherein R.sub.4, R.sub.5 or R.sub.6 are the same or independently a
straight or branched, saturated or unsaturated, substituted or
unsubstituted C.sub.1-10 alkyl or cycloalkyl, substituted or
unsubstituted phenyl, or substituted or unsubstituted benzyl, and,
wherein X.sup.- is Cl.sup.-, Fl.sup.- or another salt-forming
counterion, or a solvate or hydrate thereof, and (b) chelating the
compound by contacting the compound with a paramagnetic metal
selected from Cr, Mn, Fe, Co, Ni, Cu, Pr, Nd, Sm, Y, Gd, Tb, Dy,
Nd, Pm, Ho, Sm, Tm, Eu, Yb, Lu or Er.
23. A method of in vivo MR imaging a target area of a subject
comprising the steps of: a) administering a compound according to
claim 16 to the subject; b) applying a pulse sequence selected to
acquire MR imaging data from the target area of the subject; and c)
reconstructing an image of the target area of the subject having
enhanced contrast in areas of at least one of metabolic and mitotic
activity.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. NonProvisional
application Ser. No. 12/390,929, filed Feb. 23, 2009, which claims
the benefit of U.S. Provisional Application No. 61/030,627, filed
on Feb. 22, 2008, both of which are incorporated by reference
herein in its entirety for all purposes.
[0002] The present application describes technology similar to that
disclosed in commonly owned U.S. application Ser. No. 12/394,581,
titled ".sup.99mTc-Labeled Triphenylphosphonium Derivative
Contrasting Agents and Molecular Probes for Early Detection and
Imaging of Breast Tumors."
FIELD OF THE INVENTION
[0004] The present invention relates generally to the field of
magnetic resonance imaging. More particularly, the invention is
directed to in vivo mitochondrial labeling using positively-charged
nitroxide enhanced or gadolinium chelate enhanced magnetic
resonance imaging.
BACKGROUND OF THE INVENTION
[0005] When a substance such as human tissue is subjected to a
uniform magnetic field (polarizing field B.sub.0), the individual
magnetic moments of the nuclear spins in the tissue attempt to
align with this polarizing field, but process about it in random
order at their characteristic Larmor frequency. Usually the nuclear
spins are comprised of hydrogen atoms, but other NMR active nuclei
are occasionally used. A net magnetic moment Mz is produced in the
direction of the polarizing field, but the randomly oriented
magnetic components in the perpendicular, or transverse, plane (x-y
plane) cancel one another. If, however, the substance, or tissue,
is subjected to a magnetic field (excitation field B.sub.1) that is
in the x-y plane and is near the Larmor frequency, the net aligned
moment, Mz, may be rotated, or "tipped" into the x-y plane to
produce a net transverse magnetic moment Mt, which is rotating, or
spinning, in the x-y plane at the Larmor frequency. The practical
value of this phenomenon resides in the signal that is emitted by
the excited spins after the excitation signal B.sub.1 is
terminated.
[0006] When utilizing these signals to produce images, magnetic
field gradients (G.sub.x, G.sub.y, and G.sub.z) are employed.
Typically, the region to be imaged is scanned by a sequence of
measurement cycles in which these gradients vary according to the
particular localization method being used. The resulting set of
received MRI signals is received using a receiver coil. The MRI
signals are then digitized and processed to reconstruct the image
using one of many well-known reconstruction techniques.
[0007] These signals can be decomposed into directional components.
The relaxation time T1 is the time required for the z component of
M to return to 63 percent of its original value following
excitation. It is also referred to as spin-lattice relaxation or
longitudinal relaxation. The relaxation time T2 is the time
required for the transverse component of M to decay to 37 percent
of the initial value. It is also know as the spin-spin relaxation
time or transverse relaxation time.
[0008] The ability to depict anatomy and pathology using MRI is
dependent on the contrast, or difference in signal intensity
between the target and background tissue. In order to maximize
contrast, it is necessary to suppress the signal intensities of the
background tissues. Substances can be contrasted in an MR image by
the differences in either their T1 or T2 characteristics.
[0009] Contrast agents can be used to modify the T1 or T2
characteristics in vivo. The specific modification in contrast
caused by a given contrast agent is due to an effect of shortening
the relaxation time T1 and/or T2 of the hydrogen nuclei. If the
contrast agent reduces T1, a T1 hypersignal is observed in the
reconstructed image. On the other hand, if the contrast agent
shortens T2, a reduction in the T2 and T2* signal will be observed
in the reconstructed image.
[0010] One very common application of MR imaging is in vivo
screening for tumors. However, distinguishing a tumor from
surrounding tissue can, at times, be difficult. That is, it is
often difficult to maximize the signal intensity received from the
tumor while suppressing the signal intensities received from the
surrounding tissue.
[0011] Also, generally speaking, MR imaging is advantageous in
performing anatomical analysis. That is, unlike other imaging
modalities, such as positron emission tomography, MR imaging is not
as readily suited for functional imaging. To perform functional
analysis using MR imaging, a contrast agent is typically employed.
For example, when performing functional MRI (fMRI) of the brain,
oxygen is typically employed as a contrast agent using the BOLD
method. On the other hand, when attempting to image arterial or
venous flow, a contrast agent such as gadolinium is utilized. In
either case, the functional imaging is achieved by monitoring the
presence or absence of the contrast agent using MR imaging.
However, there are many functional processes within the body that
cannot be imaged using traditional contrast agents.
[0012] Therefore, it would be desirable to have a system and method
for enhancing the contrast of specific structures, such as tumors.
Furthermore, it would be desirable to have a system and method for
imaging functional processes in vivo that cannot otherwise be
imaged using traditional functional MR imaging.
SUMMARY OF THE INVENTION
[0013] The present invention overcomes the aforementioned drawbacks
by providing a system and method for enhancing the contrast of
processes or specific structures, such as tumors, during an MR
imaging process. Specifically, a contrast agent is provided that is
designed to target mitochondria as a marker for metabolic and/or
mitotic activity. Accordingly, the contrast of pathologies, such as
tumors, where metabolic activity is significantly increased, is
enhanced during MR imaging. Furthermore, functional processes
earmarked by metabolic and/or mitotic activity can be readily
identified during an MR imaging process using the contrast
agent.
[0014] Contrast agents particularly useful in methods according to
the present invention are mitochondria-targeted nitroxides and
mitochondria-targeted gadolinium (III) chelate complexes. Due to
their paramagnetic properties, the nitroxide or gadolinium chelate
moieties exhibit T.sub.1-contrast enhancement, making the molecules
useful as MR contrast agents. Mitochondria-targeted contrast agents
molecules according to the invention further include a mitochondria
targeting moiety, preferably a triphenylphosphonium group or a
benzyl ammonium group.
[0015] Preferred mitochondria-targeted nitroxides for use in the
present methods additionally contain ether, amide, or ester
linkages to couple the mitochondria targeting moiety and the
nitroxide. Thus, the preferred mitochondria-targeted nitroxides are
positively charged nitroxide ethers, positively charged nitroxide
esters or positively charged nitroxide amides. Suitable positively
charged nitroxides include, but are not limited to, Mito-Tempol
ether, Mito-Tempol ester, Mito-Tempol amide, Mito-CP,
Mito-Tempamide, Mito-Proxyl ether, Mito-Proxyl ester, Mito-Proxyl
amide, Tributylalkylammonium Tempol ether and Tribenzyalkylammonium
Tempol ether. A particularly preferred molecule for use in methods
according to the invention is Mito-CP.
[0016] Preferred mitochondria-targeted gadolinium chelates for use
in the present methods contain a coordination complex consisting of
a gadolinium (III) ion bound to a hexadentate, heptadentate,
octadentate, or nonadentate organic chelating agent. Examples of
such chelating agents include
1,4,7,10-tetrazacyclododecane-N,N',N'',N''' tetracetate (DOTA);
diethylenetriaminepentaacetate (DTPA);
1,4,7-triazacyclononane-N,N',N''-triacetate (NOTA);
1,5,9-triazacyclododecane-N,N',N''-triacetate (DOTRA);
1,4,8,11-tetraazacyclotetradecane-N,N',N'',N'''-tetraacetate
(TETA); and salts thereof.
[0017] The mitochondria-targeted gadolinium chelate contrast agent
molecules of the present invention further contain a mitochondria
targeting moiety such as a triphenylphosphonium group or a benzyl
ammonium group. The preferred mitochondria targeting moiety is a
triphenylphosphonium group.
[0018] More preferred mitochondria-targeted gadolinium chelates
additionally contain phenylthiocarbamide linkages to couple the
mitochondria targeting moiety and the gadolinium chelates. In some
preferred embodiments, the linkage may additionally contain an
alkyl chain, most preferably a ten carbon unbranched alkyl chain.
Suitable mitochondria-targeted gadolinium chelates for use in the
present methods include, but are not limited to, Mito-Gd-DOTA and
Mito-Gd-DTPA. A particularly preferred molecule for use in methods
according to the invention is Mito-Gd-DOTA.
[0019] Furthermore, it can be appreciated that the present
invention also provides novel mitochondria-targeted contrast agents
including certain compounds having the formula:
##STR00001##
wherein L is a paramagnetic metal selected from Cr, Mn, Fe, Co, Ni,
Cu, Pr, Nd, Sm, V. Gd, Tb, Dy, Nd, Pm, Ho, Sm, Tm, Eu, Yb, Lu or
Er, wherein R.sub.1 is S, N or P, wherein R.sub.2 is a branched or
straight chain, saturated or unsaturated, substituted or
unsubstituted C.sub.1-25 group, wherein R.sub.3 is a branched or
straight chain, saturated or unsaturated, substituted or
unsubstituted organic chelating moiety comprising one or more of
carboxyl, amine, amide, ester, alcohol or thiol, wherein R.sub.4,
R.sub.5 or R.sub.6 are the same or independently a straight or
branched, saturated or unsaturated, substituted or unsubstituted
C.sub.1-10 alkyl or cycloalkyl, substituted or unsubstituted
phenyl, or substituted or unsubstituted benzyl, and, wherein
X.sup.- is Cl.sup.-, I.sup.-, Fl.sup.- or another salt-forming
counterion, or a solvate or hydrate thereof. In preferred
compounds, R.sub.2 is a branched or straight chain, saturated or
unsaturated, substituted or unsubstituted C.sub.4-10 alkyl. The
organic chelating moiety is preferably a hexadentate, heptadentate,
octadentate, or nonadentate organic chelating moiety, in certain
embodiments selected from the group consisting of:
1,4,7,10-tetrazacyclododecane-N,N',N'',N''' tetracetate (DOTA);
diethylenetriaminepentaacetate (DTPA);
1,4,7-triazacyclononane-N,N',N''-triacetate (NOTA);
1,5,9-triazacyclododecane-N,N',N''-triacetate (DOTRA);
1,4,8,11-tetraazacyclotetradecane-N,N',N'',NT'''-tetraacetate
(TETA); and salts thereof. The preferred paramagnetic metal L is
the metal Gd.
[0020] One preferred mitochondria-targeted contrast agent is the
compound Mito-Gd-DOTA having the structure:
##STR00002##
[0021] An alternative mitochondria-targeted contrast agent, also
preferred, is the compound Mito-Gd-DTPA having the structure:
##STR00003##
[0022] In certain embodiments, the compounds according to the
invention are provided in the form of an injectable dosage. Such
compositions include one or more of the compounds described and
claimed herein along with a pharmaceutically suitable injectable
carrier system.
[0023] The invention further encompasses compounds made by the
steps of providing a compound having the formula:
##STR00004##
wherein R.sub.1 is S, N or P, wherein R.sub.2 is branched or
straight chain, saturated or unsaturated, substituted or
unsubstituted C.sub.1-25 group, wherein R.sub.3 is a branched or
straight chain, saturated or unsaturated, substituted or
unsubstituted organic chelating moiety comprising one or more of
carboxyl, amine, amide, ester, alcohol or thiol, wherein R.sub.4,
R.sub.5 or R.sub.6 are the same or independently a straight or
branched, saturated or unsaturated, substituted or unsubstituted
C.sub.1-10 alkyl or cycloalkyl, substituted or unsubstituted
phenyl, or substituted or unsubstituted benzyl, and, wherein
X.sup.- is Cl.sup.-, I.sup.-, Fl.sup.- or another salt-forming
counterion, or a solvate or hydrate thereof, and chelating the
compound by contacting the compound with a paramagnetic metal
selected from Cr, Mn, Fe, Co, Ni, Cu, Pr, Nd, Sm, Y, Gd, Tb, Dy,
Nd, Pm, Ho, Sm, Tm, Eu, Yb, Lu or Er.
[0024] Of course, the invention contemplates methods of in vivo MR
imaging a target area of a subject comprising the steps of: (a)
administering a compound having one of the structures described and
claimed herein to the subject; (b) applying a pulse sequence
selected to acquire MR imaging data from the target area of the
subject; and (c) reconstructing an image of the target area of the
subject having enhanced contrast in areas of at least one of
metabolic and mitotic activity.
[0025] While the invention defined by the appended claims is
susceptible to various modifications and alternative forms,
specific embodiments thereof have been shown by way of example in
the drawings and are herein described in detail. It should be
understood, however, that the description of specific embodiments
is not intended to limit the invention to the particular forms
disclosed, but on the contrary, the intention is to cover all
modifications, equivalents and alternatives falling within the
spirit and scope of the invention as defined by the appended
claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0026] The invention will be better understood when consideration
is given to the following detailed description thereof. Such
detailed description makes reference to the following drawings,
wherein:
[0027] FIG. 1 is a block diagram of an MRI system for use with the
present invention.
[0028] FIG. 2 shows the chemical structures of Carboxy-Proxyl (CP)
and Mito-Carboxy-Proxyl (Mito-CP).
[0029] FIG. 3 shows cell viability data obtained from MTT assay
results.
[0030] FIG. 4 shows the longitudinal relaxivity of buffered saline
solutions of Mito-CP at various magnetic field strengths.
[0031] FIG. 5 shows spin echo inversion recovery images (TI=1900
msec) of the eppendorf tubes containing mixtures described in Table
1.
[0032] FIG. 6 shows transient in vivo Mito-CP blood concentration
curves obtained from a single rat. Data were obtained at X-band EPR
using a femoral arterial-venous shunt catheter (PE50 tubing) that
allowed for repeated sampling (every 86 s) of the EPR spectrum.
Subsequent injections were collected in order of increasing
concentration and were spaced at least 1 hour apart.
[0033] FIG. 7 shows transient in vivo Mito-CP blood concentration
curves obtained from separate individual rats. Data were obtained
at X-band EPR using femoral arterial-venous shunt catheter that
allowed for repeated sampling (every 86 s) of the EPR spectrum.
Note that the Mito-CP was eliminated from the blood within 30
minutes.
[0034] FIG. 8 shows transient in vivo CP blood concentration curves
obtained from separate individual rats. Data were obtained at
X-band EPR using femoral arterial-venous shunt catheter that
allowed for repeated sampling (every 86 s) of the EPR spectrum.
Note that the CP concentration had significantly slower elimination
rates than the Mito-CP counterpart suggesting that the Mito-CP was
not only being reduced but also was being taken up by tissue.
[0035] FIG. 9 shows transient in vivo Mito-CP blood X-Band EPR
spectra obtained from a single rat using the AV-shunt at various
time points. Following the acquisition of the dynamic EPR spectra,
the blood within the catheter was treated with
(Fe(CN.sub.6)).sup.3- to reverse the reduction of Mito-CP. The
Mito-CP signal was recovered following treatment with
(Fe(CN.sub.6)).sup.3-, labeled as FCN.
[0036] FIG. 10 shows transient in vivo CP blood X-Band EPR spectra
obtained from a single rat using the AV-shunt at various time
points. Following the acquisition of the dynamic EPR spectra, the
blood within the catheter was treated with (Fe(CN.sub.6)).sup.3-,
labeled as FCN, to reverse the reduction of CP. Like Mito-CP, the
CP signal was recovered following treatment with
(Fe(CN.sub.6)).sup.3- but not back to its initial level. Both
Mito-CP and CP had similar increases in signal following treatment
with (Fe(CN.sub.6)).sup.3-.
[0037] FIG. 11 shows the dynamic time series of a representative
voxel from T1-weighted SPGR images obtained in regions of
aggressive breast tumor.
[0038] FIG. 12 is a Mito-CP decay map (fit .alpha.=0.05).
[0039] FIG. 13 shows the chemical structures of the positively
charged nitroxides Mito-Tempol ether, Mito-Tempol ester,
Mito-Tempol amide, Mito-Proxyl ether, Mito-Proxyl ester,
Mito-Proxyl amide, Tributylalkylammonium Tempol ether and
Tribenzyalkylammonium Tempol ether.
[0040] FIG. 14 shows the chemical structures of Mito-Gadolinium
(III)-1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid
(Mito-Gd-DOTA) and Mito-Gadolinium
(III)-diethylenetriaminepentaacetic add (Mito-Gd-DTPA).
[0041] FIG. 15A shows a dynamic time series of Mito-Gd-DOTA uptake
from within a C6 glioma (brain tumor). FIG. 15B shows post contrast
MRI images taken 40 minutes after injection with Mito-Gd-DOTA.
[0042] FIG. 16 depicts the scheme for the chemical synthesis of the
contrast agent Mito-Gd-DOTA.
[0043] FIG. 17 depicts a relaxitivity study of Mito-Gd-DOTA at 9.4
T.
DETAILED DESCRIPTION OF THE INVENTION
[0044] The present invention relates to the inventors' observation
that certain mitochondria targeted nitroxides and
mitochondria-targeted gadolinium (III) chelate complexes are useful
contrast agents in in vivo imaging applications.
[0045] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which the invention belongs. Although
any methods and materials similar to or equivalent to those
described herein can be used in the practice or testing of the
present invention, the preferred methods and materials are now
described.
[0046] Referring particularly to FIG. 1, the invention is employed
with an MRI system. The MRI system includes a workstation 10 having
a display 12 and a keyboard 14. The workstation 10 includes a
processor 16 that is a commercially available programmable machine
running a commercially available operating system. The workstation
10 provides the operator interface that enables scan prescriptions
to be entered into the MRI system.
[0047] The workstation 10 is coupled to four servers: a pulse
sequence server 18; a data acquisition server 20; a data processing
server 22, and a data store server 23. In the one embodiment, the
data store server 23 is performed by the workstation processor 16
and associated disc drive interface circuitry. The remaining three
servers 18, 20 and 22 are performed by separate processors mounted
in a single enclosure and interconnected using a backplane bus. The
pulse sequence server 18 employs a commercially available
microprocessor and a commercially available communication
controller. The data acquisition server 20 and data processing
server 22 both employ commercially available microprocessors and
the data processing server 22 further includes one or more array
processors based on commercially available parallel vector
processors.
[0048] The workstation 10 and each processor for the servers 18, 20
and 22 are connected to a serial communications network. This
serial network conveys data that is downloaded to the servers 18,
20 and 22 from the workstation 10 and it conveys tag data that is
communicated between the servers and between the workstation and
the servers. In addition, a high speed data link is provided
between the data processing server 22 and the workstation 10 in
order to convey image data to the data store server 23.
[0049] The pulse sequence server 18 functions in response to
program elements downloaded from the workstation 10 to operate a
gradient system 24 and an RF system 26. Gradient waveforms
necessary to perform the prescribed scan are produced and applied
to the gradient system 24, which excites gradient coils in an
assembly 28 to produce the magnetic field gradients Gx, Gy, and Gz
used for position encoding NMR signals. The gradient coil assembly
28 forms part of a magnet assembly 30, which includes a polarizing
magnet 32 and a whole-body RF coil 34.
[0050] The RF excitation waveforms are applied to the RF coil 34 by
the RF system 26 to perform the prescribed magnetic resonance pulse
sequence. Responsive NMR signals detected by the RF coil 34 are
received by the RF system 26, amplified, demodulated, filtered, and
digitized under direction of commands produced by the pulse
sequence server 18. The RF system 26 includes an RF transmitter for
producing a wide variety of RF pulses used in MR pulse sequences.
The RF transmitter is responsive to the scan prescription and
direction from the pulse sequence server 18 to produce RF pulses of
the desired frequency, phase, and pulse amplitude waveform. The
generated RF pulses may be applied to the whole body RF coil 34 or
to one or more local coils or coil arrays.
[0051] The RF system 26 also includes one or more RF receiver
channels. Each RF receiver channel includes an RF amplifier that
amplifies the NMR signal received by the RF coil to which it is
connected and a quadrature detector which detects and digitizes the
I and Q quadrature components of the received NMR signal. The
magnitude of the received NMR signal may thus be determined at any
sampled point by the square root of the sum of the squares of the I
and Q components: M= {square root over (I.sup.2+Q.sup.2)}, and the
phase of the received NMR signal may also be determined:
.phi.=tan.sup.-1 Q/I.
[0052] The pulse sequence server 18 also optionally receives
patient data from a physiological acquisition controller 36. The
controller 36 receives signals from a number of different sensors
connected to the patient, such as ECG signals from electrodes or
respiratory signals from a bellows. Such signals are typically used
by the pulse sequence server 18 to synchronize, or "gate", the
performance of the scan with the subject's respiration or heart
beat.
[0053] The pulse sequence server 18 also connects to a scan room
interface circuit 38 that receives signals from various sensors
associated with the condition of the patient and the magnet system.
It is also through the scan room interface circuit 38 that a
patient positioning system 40 receives commands to move the patient
to desired positions during the scan.
[0054] It should be apparent that the pulse sequence server 18
performs real-time control of MRI system elements during a scan. As
a result, it is the pulse sequence server 18 includes hardware
elements that are operated with program instructions that are
executed in a timely manner by run-time programs. The description
components for a scan prescription are downloaded from the
workstation 10 in the form of objects. The pulse sequence server 18
contains programs that receive these objects and converts them to
objects that are employed by the run-time programs.
[0055] The digitized NMR signal samples produced by the RF system
26 are received by the data acquisition server 20. The data
acquisition server 20 operates in response to description
components downloaded from the workstation 10 to receive the
real-time NMR data and provide buffer storage such that no data is
lost by data overrun. In some scans, the data acquisition server 20
does little more than pass the acquired NMR data to the data
processor server 22. However, in scans that require information
derived from acquired NMR data to control the further performance
of the scan, the data acquisition server 20 is programmed to
produce such information and convey it to the pulse sequence server
18. For example, during prescans, NMR data is acquired and used to
calibrate the pulse sequence performed by the pulse sequence server
18. Also, navigator signals may be acquired during a scan and used
to adjust RF or gradient system operating parameters or to control
the view order in which k-space is sampled. And, the data
acquisition server 20 may be employed to process NMR signals used
to detect the arrival of contrast agents. In all these examples the
data acquisition server 20 acquires NMR data and processes it in
real-time to produce information which is used to control the
scan.
[0056] The data processing server 22 receives NMR data from the
data acquisition server 20 and processes it in accordance with
description components downloaded from the workstation 10. Such
processing may include, for example: Fourier transformation of raw
k-space NMR data to produce two or three-dimensional images; the
application of filters to a reconstructed image; the performance of
a backprojection image reconstruction of acquired NMR data; the
calculation of functional MR images; the calculation of motion or
flow images, and the like.
[0057] Images reconstructed by the data processing server 22 are
conveyed back to the workstation 10 where they are stored.
Real-time images are stored in a data base memory cache (not shown)
from which they may be output to operator display 12 or a display
42 which is located near the magnet assembly 30 for use by
attending physicians. Batch mode images or selected real-time
images are stored in a host database on disc storage 44. When such
images have been reconstructed and transferred to storage, the data
processing server 22 notifies the data store server 23 on the
workstation 10. The workstation 10 may be used by an operator to
archive the images, produce films, or send the images via a network
to other facilities.
[0058] Using the above-described MRI system, a method of medical
imaging can be performed using a T.sub.1-targeted contrast agent. A
wide variety of processes are contemplated for imaging tumors or
other areas of high metabolic and/or mitotic activity. Furthermore,
a variety of processes are contemplated for performing functional
imaging processes using the above-described MRI system including,
but not limited to, assay of tissue redox status by dynamic
T.sub.1-weighted imaging.
[0059] One group of contrast agents useful in the present invention
are mitochondria-targeted nitroxides. Nitroxide radicals, due to a
single unpaired electron, exhibit T.sub.1-contrast enhancement.
Nitroxides have also been shown to exhibit T.sub.1 contrast
enhancement in vivo. As used herein, the term "nitroxide" refers to
any five-membered or six-membered ring having a stabilized
nitroxide moiety. The term "mitochondria-targeted nitroxide" refers
to any nitroxide conjugated to a molecule that increases its lipid
bilayer permeability. Such molecules include, but are not limited
to, a triphenylphosphonium group or a benzyl ammonium group.
[0060] The nitroxide moiety is generally coupled to the molecule
that increases lipid bilayer permeability via an ether, amide or
ester linkage. This linkage may be of varying chain length with
longer chain lengths increasing the mitochondria-targeted
nitroxide's hydrophobic character. Accordingly, the
mitochondria-targeted nitroxide is generally a positively charged
nitroxide ether, a positively charged nitroxide ester or a
positively charged nitroxide amide.
[0061] In specific embodiments, the mitochondria-targeted nitroxide
is Mito-Tempol ether, Mito-Tempol ester, Mito-Tempol amide,
Mito-CP, Mito-Tempamide, Mito-Proxyl ether, Mito-Proxyl ester,
Mito-Proxyl amide, Tributylalkylammonium Tempol ether, or
Tribenzyalkylammonium Tempol ether (see FIG. 13). The syntheses of
the specific molecules described herein were previously described
in U.S. patent application Ser. No. 11/520,191, filed Sep. 13,
2006, and Dhanasekaran, et al., Free Radic Biol Med, 2005. 39(5):
p. 567-83, both of which are incorporate herein by reference in
their entireties. Accordingly, mitochondria-targeted nitroxides
useful in the invention may be based on, e.g.,
2-ethyl-2,5,5-trimethyl-3-oxazolidine-1-oxyl (OXANO),
2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO),
4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPOL),
4-amino-2,2,6,6tetramethyl-1-piperidinyloxy (Tempamine),
3-Aminomethyl-PROXYL, 3-CyanoPROXYL, 3-Carbamoyl-PROXYL,
3-Carboxy-PROXYL, and 4-Oxo-TEMPO. Molecules such as TEMPO, for
example, can also be substituted, typically in the 4 position,
e.g., 4-amino, 4-(2 bromoacetamido), 4-(ethoxyfluorophosphonyloxy),
4-hydroxy, 4-(2-iodoacetamido), 4 isothiocyanato, 4-maleimido,
4-(4-nitrobenzoyloxyl), 4-phosphonooxy, and derivatives
thereof.
[0062] The structure of one preferred mitochondria-targeted
nitroxide, Mito-CP, is depicted in FIG. 2, along with the structure
of the untargeted nitroxide, CP. Mito-CP has been shown to
preferentially target mitochondria. Both CP and Mito-CP molecules
are paramagnetic, due the single unpaired electron delocalized over
the nitrogen oxygen bond, which allows detection by both MR and
EPR. Alone, the 5-membered nitroxide ring, CP, lacks the ability to
accumulate selectively within functional mitochondria. However,
when the CP is covalently bound to triphenylphosphonium with
11-carbon linkage, Mito-CP, mitochondrial accumulation occurs.
While no one theory of operation is adopted herein, Mito-CP is most
likely taken up by mitochondria because: (1) lipophilic cations,
such as the triphenylphosphonium cation, distribute their charge
over a large surface area allowing them to penetrate the lipid
bilayers; and (2) uptake of lipophilic ions through the lipid
bilayers is increased 10-fold for every 61.5 mV difference in the
membrane potential. This would apparently explain the uptake of
Mito-CP across the plasma membrane (30-60 mV) and across the
mitochondria membrane (150-180 mV).
[0063] A second group of contrast agents useful in the present
invention are mitochondria-targeted gadolinium (III) chelate
complexes. Gadolinium, due to its seven unpaired electrons, has
strong paramagnetic properties. Its large magnetic moment, which
efficiently relaxes magnetic nuclei, causes it to exhibit
T.sub.1-contrast enhancement. Because gadolinium is toxic to
animals, its use in animals for contrast enhancement is limited to
compounds such as gadolinium complexes or chelates that are
excreted without releasing the toxic gadolinium ions. See U.S. Pat.
No. 4,639,365, which is incorporated by reference herein. A number
of gadolinium chelates have been shown to exhibit T.sub.1 contrast
enhancement in vivo.
[0064] As used herein, the term "gadolinium chelate" refers to a
coordination complex consisting of a gadolinium (III) ion bound to
a hexadentate, heptadentate, octadentate, or nonadentate organic
chelating agent. Examples of such organic chelating agents include,
without limitation, 1,4,7,10-tetrazacyclododecane-N,N',N'',N'''
tetracetate (DOTA); diethylenetriaminepentaacetate (DTPA);
1,4,7-triazacyclononane-N,N',N''-triacetate (NOTA);
1,5,9-triazacyclododecane-N,N',N''-triacetate (DOTRA);
1,4,8,11-tetraazacyclotetradecane-N,N',N'',N'''-tetraacetate
(TETA); and salts thereof. The term "mitochondria-targeted
gadolinium chelate" refers to any gadolinium chelate conjugated to
a molecule that increases its lipid bilayer permeability. Such
molecules include, but are not limited to, a triphenylphosphonium
group or a benzyl ammonium group.
[0065] The molecule that increases lipid bilayer permeability is
generally coupled to the gadolinium chelate moiety via a carbon
chain attached to a phenylthiocarbamide (phenylthiourea),
thiocarbamide (thiourea), thioamide, ether, amide or ester linkage.
The linkage is attached to the gadolinium chelate moiety, either
directly or through an additional short carbon chain. The first
carbon chain, extending from the molecule that increases lipid
bilayer permeability to the linkage, may be of varying chain
lengths (preferably from two to eleven carbons, more preferably ten
carbons), with longer chain lengths increasing the
mitochondria-targeted gadolinium chelate's hydrophobic
character.
[0066] A preferred mitochondria-targeted gadolinium chelate is
generally a positively charged triphenylphosphonium
phenylthiocarbamide gadolinium chelate, wherein the
triphenylphosphonium group is attached to a ten carbon chain
coupled to a phenylthiocarbamide linking group, which is attached
through an additional one-carbon chain to a gadolinium chelate
moeity. In specific embodiments, the mitochondria-targeted
gadolinium chelate is Mito-Gd-DOTA or Mito-Gd-DOTA (see FIG. 14).
The syntheses of these specific molecules is described herein at
Example 8.
[0067] For in vivo imaging applications, mitochondria-targeted
nitroxides or mitochondria-targeted gadolinium chelates are
administered intravenously or intraperitoneally, alone or in
combination with a pharmaceutically-acceptable carrier.
Alternatively, the compounds may be administered orally where the
target area to be imaged is associated with the upper digestive
tract, particularly the mouth, pharynx or esophagus.
Mitochondria-targeted nitroxides and mitochondria-targeted
gadolinium chelates are generally stable in aqueous solutions at a
pH of about 7.0.
[0068] Dosages of mitochondria-targeted nitroxide suitable for in
vivo imaging applications are in a range of about 0.1 mg/kg to
about 100 mg/kg body weight. It is noted that the number of doses a
subject receives, the time allowed between doses and the length of
time a subject receives mitochondria-targeted nitroxides will
depend upon, for example, the subject's body weight and duration of
imaging procedure.
[0069] In another embodiment, the invention includes the disclosed
mitochondria-targeted gadolinium (III) chelate complex molecules.
Although a number of gadolinium chelates are known, Applicants for
the first time have synthesized the disclosed mitochondria-targeted
gadolinium chelates. The preferred molecules of this embodiment of
the invention are triphenylphosphonium phenylthiocarbamide
gadolinium chelates, wherein the triphenylphosphonium group is
attached to a ten carbon chain coupled to a phenylthiocarbamide
linking group, which is attached through an additional one-carbon
chain to a gadolinium chelate moeity. In specific embodiments, the
mitochondria-targeted gadolinium chelate is Mito-Gd-DOTA or
Mito-Gd-DPTA (see FIG. 14). The syntheses of these specific
molecules is described herein at Example 8.
[0070] As used herein, a "salt-forming counterion" may be a
pharmaceutically suitable (i.e., pharmaceutically acceptable) salt
including, but not limited to, acid addition salts formed by mixing
a solution of the instant compound with a solution of a
pharmaceutically acceptable acid. The pharmaceutically acceptable
acid may be hydrochloric acid, methanesulphonic acid, fumaric acid,
maleic acid, succinic acid, acetic acid, benzoic acid, oxalic acid,
citric acid, tartaric acid, carbonic acid or phosphoric acid.
Various pharmaceutically acceptable salts are well known in the art
and may be used with the instant compound such as those disclosed
in Berge S M et al., "Pharmaceutical Salts." J. Pharm. Sci. 66:1-19
(1977) and Haynes D A et al., "Occurrence of pharmaceutically
acceptable anions and cations in the Cambridge Structural
Database," J. Pharm. Sci. 94:2111-2120 (2005), which are hereby
incorporated herein by reference.
[0071] For example, the list of FDA-approved commercially marketed
salts includes acetate, benzenesulfonate, benzoate, bicarbonate,
bitartrate, bromide, calcium edetate, camsylate, carbonate,
chloride, citrate, dihydrochloride, edetate, edisylate, estolate,
esylate, fumarate, gluceptate, gluconate, glutamate,
glycollylarsanilate, hexylresorcinate, hydrabamine, hydrobromide,
hydrochloride, hydroxynaphthoate, iodide, isethionate, lactate,
lactobionate, malate, maleate, mandelate, mesylate, methylbromide,
methylnitrate, methylsulfate, mucate, napsylate, mitrate, pamoate,
pantothenate, phosphate, diphosphate, polygalacturonate,
salicylate, stearate, subacetate, succinate, sulfate, tannate,
tartrate, teoclate, and triethiodide.
[0072] As used herein, "hydrates" of the instant compound may be a
pharmaceutically suitable (i.e., pharmaceutically acceptable)
hydrate that is a compound formed by the addition of water or its
elements to a host molecule (e.g., the free form version of the
compound) including, but not limited to, monohydrates, dihydrates,
etc.
[0073] As used herein, the terms "injectable" and "infusion dosage
forms" (i.e., parenteral dosage forms) include, but are not limited
to, liposomal injectables or a lipid bilayer vesicle having
phospholipids that encapsulate an active drug substance. Injection
includes a sterile preparation intended for parenteral use.
[0074] Five distinct classes of injections exist as defined by the
USP. Emulsion injection includes an emulsion comprising a sterile,
pyrogen-free preparation intended to be administered parenterally.
Lipid complex and powder for solution injection are sterile
preparations intended for reconstitution to form a solution for
parenteral use. Powder for suspension injection is a sterile
preparation intended for reconstitution to form a suspension for
parenteral use. Powder lyophilized for liposomal suspension
injection is a sterile freeze dried preparation intended for
reconstitution for parenteral use that is formulated in a manner
allowing incorporation of liposomes, such as a lipid bilayer
vesicle having phospholipids used to encapsulate an active drug
substance within a lipid bilayer or in an aqueous space, whereby
the formulation may be formed upon reconstitution. Powder
lyophilized for solution injection is a dosage form intended for
the solution prepared by lyophilization ("freeze drying"), whereby
the process involves removing water from products in a frozen state
at extremely low pressures, and whereby subsequent addition of
liquid creates a solution that conforms in all respects to the
requirements for injections. Powder lyophilized for suspension
injection is a liquid preparation intended for parenteral use that
contains solids suspended in a suitable fluid medium, and it
conforms in all respects to the requirements for Sterile
Suspensions, whereby the medicinal agents intended for the
suspension are prepared by lyophilization.
[0075] Solution injection involves a liquid preparation containing
one or more drug substances dissolved in a suitable solvent or
mixture of mutually miscible solvents that is suitable for
injection. Solution concentrate injection involves a sterile
preparation for parenteral use that, upon addition of suitable
solvents, yields a solution conforming in all respects to the
requirements for injections. Suspension injection involves a liquid
preparation (suitable for injection) containing solid particles
dispersed throughout a liquid phase, whereby the particles are
insoluble, and whereby an oil phase is dispersed throughout an
aqueous phase or vice-versa. Suspension liposomal injection is a
liquid preparation (suitable for injection) having an oil phase
dispersed throughout an aqueous phase in such a manner that
liposomes (a lipid bilayer vesicle usually containing phospholipids
used to encapsulate an active drug substance either within a lipid
bilayer or in an aqueous space) are formed. Suspension sonicated
injection is a liquid preparation (suitable for injection)
containing solid particles dispersed throughout a liquid phase,
whereby the particles are insoluble. In addition, the product may
be sonicated as a gas is bubbled through the suspension resulting
in the formation of microspheres by the solid particles.
[0076] A parenteral or injectable carrier system according to the
invention includes one or more pharmaceutically suitable
excipients, such as solvents and co-solvents, solubilizing agents,
wetting agents, suspending agents, thickening agents, emulsifying
agents, chelating agents, buffers, pH adjusters, antioxidants,
reducing agents, antimicrobial preservatives, bulking agents,
protectants, tonicity adjusters, and special additives.
[0077] The invention will be more fully understood upon
consideration of the following non-limiting Examples.
EXAMPLES
Example 1
Materials and Methods
[0078] All chemical and reagents were obtained from Sigma-Aldrich
(Milwaukee, Wis.) unless otherwise noticed. All reagents were used
as received without further purification. All cell culture
materials and buffers were obtained from Invitrogen (Grand Island,
N.Y.).
[0079] Synthesis of Mito-CP
[0080] Mito-CP,
(11-(3-carboxy-2,2,5,5-tetramethyl-1-pyrrolidinyloxy)-undecyl)-triphenylp-
hosphonium bromide was synthesized as described previously
(Dhanasekaran, et al. at Free Radic Biol Med, 2005. 39(5): p.
567-8).
[0081] Cell Culture.
[0082] MCF7, MDA-MB-231 and MCF10-A cells were obtained from the
American Type Culture Collection (ATCC) and cultured at 37.degree.
C. in humidified 95% air/5% CO2. MCF.sub.7 cells were maintained in
MEM-alpha medium containing 10% FBS, L-glutamine (4 mM), penicillin
(100 .mu.g/ml), streptomycin (100 .mu.g/ml), non-essential amino
acids and sodium pyruvate. MDA-MB-23 were grown in Dulbecco's
Modified Eagle's medium (DMEM) containing 10% FBS, L-glutamine (4
mM), penicillin (100 .mu.g/ml), and streptomycin (100 .mu.g/ml).
MCF10-A cells were maintained in F-12 media supplemented with 20%
FHS, hydrocortisone, cholera toxin, penicillin, streptomycin, and
endothelial cell growth factor (ECGF). Typically, the day of the
treatment, various concentrations of the nitroxides were added to
the cells directly in the media.
[0083] T1 Relaxivity at 1.5 Tesla
[0084] The longitudinal relaxivities of CP and Mito-CP were
determined using a LX 1.5 T GE Scanner (GE, Waukesha, Wis.) using
an 8-channel head rf coil. Solutions of CP and Mito-CP were diluted
with Dulbecco's Phosphate Buffered Saline (DPBS) to the following
concentrations: 5.0 .mu.M, 10.0 .mu.M, 20.0 .mu.M, 50.0 .mu.M,
100.0 .mu.M, 200.0 .mu.M, 300.0 .mu.M, 500.0 .mu.M, 700.0 .mu.M,
1.0 mM, 2.0 mM, 5.0 mM, 10.0 mM, and 20.0 mM. Longitudinal
relaxation times (T.sub.1) for each solution and relaxivities
(R.sub.1) for CP and Mito-CP were calculated using images from a
standard spin echo sequence (TE=14 msec, TR=10 sec) at various
inversion times (TI=50, 125 msec incrementing by 125 msec to 3500
msec and 4000 msec).
[0085] T1 Relaxivity at 3.0 Tesla.
[0086] The longitudinal relaxivities of CP and Mito-CP were
determined using a Signa 3.0 T GE Scanner (GE, Waukesha, Wis.)
using a custom rf quadrature coil. Solutions of CP and Mito-CP were
diluted with Dulbecco's Phosphate Buffered Saline (DPBS) to the
following concentrations: 1.0 .mu.M, 10.0 .mu.M, 100.0 .mu.M, 1.0
mM, 10.0 mM, and 20.0 mM. Longitudinal relaxation times (T.sub.1)
for each solution and relaxivities (R.sub.1) for CP and Mito-CP
were calculated using images from a standard spin echo sequence
(TE=9 msec, TR=15 sec) at various inversion times (TI=100 msec
incrementing by 100 msec to 4000 msec and 50, 150, 1050, 1150
msec).
[0087] T1 Relaxivity at 9.4 Tesla.
[0088] Both solutions were diluted with Dulbecco's Phosphate
Buffered Saline (DPBS) to the following concentrations: 1.0 mM,
10.0 mM, 100.0 mM, 1.0 mM, 10.0 mM, and 20.0 mM. Longitudinal
relaxation times (T.sub.1) for each solution and relaxivities
(R.sub.1) for CP and Mito-CP were calculated using a standard spin
echo sequence (TE=9 msec, TR=15,000 msec) at various inversion
times (TI=100 msec incrementing by 100 msec to 4000 msec and 50,
150, 1050, 1150 msec).
[0089] Experimental Setup for In Vivo Rat Nitroxides Concentration
Measurements in Blood with X-Band EPR.
[0090] Each anesthetized rat was given heparin, placed on a warming
pad, and positioned above the EPR cavity. A femoral arterial line
(PE50 tubing) was fed down through the EPR cavity and connected to
the femoral venous line with a PVC couple. After the unit was
tuned, a nitroxide bolus was given via the tail vein catheter and
repeated acquisitions (every 86 s) of the EPR spectrum were
simultaneously initiated. Identical EPR parameters were maintained
within and among all rats throughout the entire study.
Example 2
Cell Viability Assay
[0091] The MTT assay (Sigma-Aldrich, Milwaukee, Wis.) was used to
measure cell survival using quantitative colorimetry (.lamda.=570
nm). This assay is based on the capacity of mitochondrial
dehydrogenases to reduce MTT to form the insoluble formazan
product. Cells were plated in 12-well plates and treated at
pre-confluency with Mito-CP and CP (0-30 .mu.M) in their respective
media for 48 h. The culture media was removed after treatment,
washed gently with DPBS and MTT (5 mg/ml in media) was added to
each well, following incubation of the plates for 1 h at 37.degree.
C. The MTT solution was removed and the formazan product in each
well was dissolved with dimethyl sulfoxide (DMSO). The optical
density of each well was measured using an Agilent 8453 UV-Vis
spectrometer at 570 nm.
[0092] MCF7, MDA-MB-231 and non-tumorigenic MCF10-A were grown to
pre-confluency and treated with either Mito-CP or parent compound
CP (1-30 uM) for 48 h and cell viability was measured using the MTT
assay. FIG. 3 shows that Mito-CP induced cancer cell apoptosis
while the parent compound CP did not. Mito-CP or CP had no
influence inducing apoptosis in normal MCF10-A breast epithelial
cells. These results suggest the differential signaling and action
mechanisms of Mito-CP and CP in breast cancer cells and normal
epithelial cells.
Example 3
Relaxivities of CP and Mito-CP
[0093] The longitudinal relaxivities of other nitroxides have been
approximate 0.2 mmol.sup.-1s.sup.-1. In order to assess the
longitudinal relaxivity of CP and Mito-CP, concentrations were
prepared with the expectation of this relaxivity. Two separate
initial solutions of CP and Mito-CP were prepared. Longitudinal
relaxivities were determined for both CP and Mito-CP at three field
strengths. Relaxivities were obtained from non-linear fits of
standard spin echo inversion recovery images calculated using
Matlab.
[0094] The longitudinal relaxivity (R1) for buffered saline
solutions of Mito-CP at various field strengths matched previous
published values for other nitroxides, R1.apprxeq.0.2
mM.sup.-1sec.sup.-1. FIG. 4 depicts slight field dependence such
that the relaxivity increased with field strength. At 1.5 T, 3.0 T,
and 9.4 T Mito-CP exhibited an R1=0.1643.+-.0.01314,
0.2322.+-.0.0151, and 0.2083.+-.0.0431 mM.sup.-1sec.sup.-1,
respectively. In addition to these measurements, CP was found to
exhibit an R1=0.1546.+-.0.00567 mIVI.sup.-1sec.sup.-1 at 3.0 T,
slightly less than its counterpart.
Example 4
In Vitro Localization of Mito-CP in Mitochondria: MRI Analysis
[0095] The experiment was carried out on a Signa 3.0 T GE scanner
(GE, Waukesha, Wis.) using a custom quadrature rf coil with a G10
fiberglass vial rack. Rabbit heart mitochondria were isolated. For
verification of MR detectable mitochondrial accumulation of
Mito-CP, isolated rabbit mitochondria were separated into three
eppendorf tubes each containing 40 .mu.L of 15 mg/ml mitochondria.
Six eppendorf tubes were prepared as indicated in Table 1 below
(1-6), and three additional eppendorf tubes were prepared from the
supernatant of tubes 4-6 (4b-6b):
TABLE-US-00001 TABLE 1 Eppendorf Tube Label Contents 1 Buffer 20 uL
0.1M succinate 180 uL DPBS 2 Buffer + 10 uM Mito-CP 20 uL 0.1M
succinate 180 uL DPBS 1.0 uL 2 mM Mito-CP/EtOH 3 Buffer + 10 uM CP
20 uL 0.1M succinate 180 uL DPBS 1.0 uL 2 mM CP/EtOH 4 Mitochondria
40 uL 15 mg/mL mitochondria 20 uL 0.1M succinate 140 uL DPBS 5
Mitochondria + 40 uL 15 mg/mL mitochondria 10 uM Mito-CP 20 uL 0.1M
succinate 139 uL DPBS 1.0 uL 2.0 mM Mito-CP/EtOH 6 Mitchondria + 10
uM CP 40 uL 15 mg/mL mitochondria 20 uL 0.1M succinate 139 uL DPBS
1.0 uL 2.0 mM CP/EtOH 4b Supernatant #4 5b Supernatant #5 6b
Supernatant #6
[0096] Note that succinate is needed to activate the mitochondrial
respiratory chain, necessary for normal function. All tubes were
incubated at 37.degree. C. for 10 minutes. Immediately following
incubation, all tubes were centrifuged at 1,000.times.g for 4
minutes at 4.degree. C. The supernatant was then transferred into
another eppendorf tube. The mitochondria in tubes 4-6 were
re-suspended with the addition of 50 mL of DPBS and allowed to
settle naturally. All tubes were simultaneously imaged using a fast
spin echo inversion recovery sequence (T.sub.E=24.25 msec,
T.sub.R=15,000 msec, and an echo train of 16) at various inversion
times for initial estimates of T.sub.1 and a standard spin echo
sequence (T.sub.E=21 msec, T.sub.R=15,000 msec) at various
inversion times (T.sub.I=63, 250, 500, 750, 1000, 1200, 1250, 1275,
1300, 1325, 1350, 1375, 1400, 1450, 1600, 1700, 1750, 1800, 1850,
1900, 2500, 3000, 3500, 4000 msec). T.sub.1 maps were then
generated from the spin echo inversion recovery data.
[0097] To determine the specificity that Mito-CP has toward
mitochondria, the compound carboxy-proxyl (CP) was used for
comparison. CP is a 5-member nitroxide ring, depicted in FIG. 1,
without the ten chain linking carbons and the triphenylphosphonium.
CP is known to be impermeable to lipid membranes but remains
detectable by both MR and EPR. These properties make CP an ideal
control for specificity of Mito-CP toward mitochondria.
[0098] FIG. 5 depicts T1 weighted images of cross-sections through
the nine eppendorf tubes at the inversion time of 1900 msec. The
overlaid numbers correspond to the labels from Table 1. Within
several minutes after the mitochondria were re-suspended in
solution (tube 4-6), settling was observed that also confirmed with
imaging as a differences in contrast at the bottom of the eppendorf
tubes containing mitochondria. The mitochondria treated with the
Mito-CP (tube 5) were found to have an shortened T.sub.1,
indicating uptake. The mitochondria treated with CP (tube 6) did
not enhance considerably compared to mitochondria alone (tube 4)
demonstrating the selectivity of Mito-CP for uptake into
mitochondria. No observed differences were found in the
supernatant. These results suggest the presence of an increased
concentration within the mitochondria compared to the incubating
solution demonstrating the ability of Mito-CP to indirectly
visualize the presences of mitochondria.
[0099] Results from the isolated rabbit mitochondria demonstrated a
clear uptake of Mito-CP. Ignoring partial voluming and assuming a
similar hydration sphere as in free water, an estimated
mitochondrial associated Mito-CP tissue concentration of 0.143 mM
can be calculated using the estimated longitudinal tissue
relaxivity and the longitudinal relaxation time from the untreated
mitochondria. This would suggest at least a 140-fold increase in
concentration within the mitochondria compared to the incubating
solution.
Example 5
In Vivo Systemic Concentration--EPR
[0100] In order to determine nitroxide accumulation and elimination
rates, in vivo systemic blood concentration was determined using
EPR and in vivo tumor concentration was determined using MRI. One
male Fisher rat (345 g) and six male Sprague-Dawley rats (240-370
g) were anesthetized with 1.2 mg/kg urethane. Using polyethylene
tubing (PE50), femoral vein and arterial catheters were placed. To
reduce clotting 400 units/kg heparin was administer intravenously.
For high arterial blood pressure, the PE50 tubing was advanced into
the descending aorta. The PE50 tubing was then run through the
X-band electron paramagnetic resonance (EPR) resonator and
connected to the venous catheter using a poly vinyl chloride (PVC)
couple creating an arterial-venous (AV) shunt. This allowed for the
continuous sampling of the in vivo Mito-CP blood concentration.
Various doses of Mito-CP and CP were administered via a tail-vein
catheter. Following each injection, EPR spectra were collected
every 86 seconds for at least 30 minutes. In order to determine if
Mito-CP or CP was reduced or eliminated, the blood remaining within
the PE50 tubing from the rats treated with 9 mmol/kg was removed
and treated with 10 uL ferricyanide following the last acquisition
of EPR spectra and an additional spectrum was obtained.
Ferricyanide, (Fe(CN).sub.6).sup.3-, will oxidize the reduced
nitroxide (i.e. hydroxylamine) back to the original nitroxide.
Rectal temperature was monitored and maintained at 37.+-.1.degree.
C.
[0101] Depicted in FIG. 6, are the transient in vivo Mito-CP blood
concentration curves obtained from a single rat using a femoral
arterial-venous shunt catheter that allowed for repeated sampling
(every 86 s) of the X-band EPR spectrum. Fitting these curves to a
monoexponential decay following the peak of the curve suggests that
Mito-CP has an in vivo half-life of approximately 2 minutes.
[0102] The transient in vivo Mito-CP and CP blood concentration
curves obtained from independent rats using femoral arterial-venous
shunt are displayed in FIGS. 7 and 8, respectively. Nitroxides are
rapidly reduced in vivo by the reduction capacity within blood.
Mito-CP was almost completely eliminated/reduced from the blood
within 30 minutes following the bolus injection. However, the CP
concentration had significantly slower elimination/reduction rates
than at the respective Mito-CP concentration suggesting that the
Mito-CP was not only being reduced but also was being taken up by
tissue. At the dose of 9 mmol/kg, CP was still detectible within
the systemic circulation while Mito-CP was not.
[0103] The transient in vivo Mito-CP and CP blood EPR spectra
obtained from one rat using the AV-shunt at various time points are
present in FIGS. 9 and 10, respectively. Following the acquisition
of the dynamic EPR spectra, the blood within the catheter was
treated with ferricyanide to reverse the reduction of CP. Both
Mito-CP and CP had similar increases in signal following treatment
with ferricyanide. Since, Mito-CP did not have super-reduction
compared to CP this further suggest that Mito-CP undergoes tissue
absorption in addition to in vivo reduction.
Example 6
In Vivo MRI
[0104] A female Fisher rat weighing 162 g was anesthetized with 75
mg/kg ketamine, 10 mg/kg xylazine and 2.5 mg/kg acepromazine and
inoculated with the Mat B III cell line (ATCC #CRL-1666). The Mat B
III cell line is a rapidly growing, well vascularized, rat mammary
adenocarcinoma. Fourteen days post inoculation the rat was
anesthetized with 1.2 mg/kg urethane. Two minutes into a dynamic
time series of T1-weighted SPGR images (TE=3 ms, TR=78.125,
Effective TR=10 seconds, Repetitions=150, Slice thickness=1 mm,
flip angle=30, slices=8) 4 mg/kg Mito-CP with 1% DMSO vehicle in
0.25 mL of PBS was injected via the tail-vein. Both pre- and
post-contrast T1-weighted images were obtained.
[0105] A representative dynamic T1-weighted time course obtained
from breast tumor shown in FIG. 11. The longer duration of the
tissue time curve compared to the blood concentration time curve
suggests uptake of the contrast agent by the aggressive breast
tumor. Results from a monoexponential fit of the dynamic
T1-weighted time course revealed tissue decay constants ranging
from 100 to 500 s.
[0106] A decay map showing exponential MR signal decay rates
overlaid on a post-contrast T1-weighted image of a highly necrotic
breast tumor is depicted in FIG. 12. The aggressive rim of the
tumor matches regions of MR signal decay due to nitroxide
reduction. Necrotic regions of the tumor lack nitroxide decay.
Necrotic regions were confirmed by gross dissection follow
euthanasia.
Example 7
In Vivo Mitochondrial Labeling Using Mito-Gd-DOTA Enhanced Magnetic
Resonance Imaging
[0107] This example demonstrates the feasibility of using an
exemplary mitochondria target gadolinium chelate complex,
Mito-Gd-DOTA (see FIG. 14), as an in vivo contrast agent.
[0108] Preliminary Data from a SD Rat with a C6 Glioma (Brain
Tumor) at 21 Days Post Inoculation.
[0109] FIG. 15A shows the uptake of Mito-Gd-DOTA within the tumor
as a time series. Each time point represents ten seconds. The
Mito-Gd-DOTA agent is taken up by the tumor slowly over time.
Although similar general patterns are seen with standard Gd-DOTA,
the uptake is significantly different than with standard
Gd-DOTA.
[0110] FIG. 15B shows post contrast images taken 40 minutes after
injection with the Mito-Gd-DOTA agent. Agent uptake can be seen in
the right hemisphere corresponding to the area of tumor
inoculation.
Example 8
Chemical Synthesis of Two Novel Mitochondria Targeting Contrast
Agents: Mito-Gd-DOTA and Mito-Gd-DTPA
[0111] Materials and Methods.
[0112] All the materials and solvents were obtained from
Sigma-Aldrich (Milwaukee, Wis.) and used without further
purification unless otherwise noted. S-2-(4-lsothiocya
natobenzyl)-1,4,7,10-tetraazacyclododeca ne-tetraacetic acid
(.rho.-SCN-Bn-DOTA, 1) and
2-(4-lsothiocyanatobenzyl)-diethylenetriaminepentaacetic acid
(.rho.-SCN-Bn-DTPA) were purchased from Macrocyclics (Dallas,
Tex.). Characterization was performed on HPLC and HRMS for all
products. HPLC experiments were performed using an Agilent 1100
system equipped with UV-Vis absorption and fluorescence detectors
using a C18 column (Alltech, Kromasil, 250.times.4.6 mm, 5 mm) that
was equilibrated with 10% CH3CN (containing 0.1% (v/v)
trifluoroacetic acid (TFA)) in 0.1% TFA aqueous solution. Mass
spectra were obtained using a 7.0 Tesla Fourier Transform Ion
Cyclotron Resonance (FTICR) Mass Spectrometer.
[0113] Synthesis of Mito-Gd-DOTA and Mito-Gd-DTPA.
[0114] Both compounds, Mito-Gd-DOTA (FIG. 16-4) and Mito-Gd-DTPA,
were synthesized according the synthetic route shown in FIG. 16.
(10-aminodecyl) triphenylphosphonium bromide (FIG. 16-3) was
synthesized and prepared as previously described. Briefly, a
mixture containing (10-bromodecyl) phthalimide (7 g, 0.019 mol) and
triphenylphosphine (5 g, 0.019 mol) in acetonitrile (60 mL) was
refluxed for 15 hours. The solvent distilled under reduced
pressure. Purification of the crude product by flash chromatography
on a silica gel (CH.sub.2Cl.sub.2/EtOH 80:20) afforded a white
solid 10-decylphthalimidyl triphenylphosphonium bromide (9 g, 73%).
The calculated MS value for [C.sub.36H.sub.39NO.sub.2P].sup.+,
Br.sup.-; [C.sub.36H.sub.39NO.sub.2P].sup.+ is 548.3. the product's
MS value was 548.3. To prepare the (10-aminodecyl)
triphenylphosphonium bromide (FIG. 16-3), hydrazine was added (0.54
mL, 0.0108 mol) to a solution of (10-decylphthalimidyl)
triphenylphosphonium bromide (7 g, 0.0108 mol) in EtOH (70 mL). The
mixture was refluxed for 15 hours. The solvent was removed under
reduced pressure and the impurity was crystallized using a mixture
Et.sub.2O/EtOH (100 mL+45 mL). The product was purified by flash
chromatography on a silica gel (CH.sub.2Cl.sub.2/EtOH 80:20)
afforded a yellow solid (4 g, 73%). The calculated HRMS for
[C.sub.28H.sub.37NP].sup.+, Br.sup.-; [C.sub.28H.sub.37NP].sup.+ is
418.2. The product's measured HRMS value was 418.2.
[0115] Synthesis of .rho.-SCN-Bn-Gd-DOTA (FIG. 16-2).
[0116] To a stirred solution of the free ligand (FIG. 16-1) (50
mg), in ddH.sub.2O (3 ml), GdCl.sub.3 (100 mg) was added. The pH of
the solution was monitored and kept between 6.0 and 7.0 using 0.1 M
NaOH. The reaction was stirred overnight and monitored by HPLC
until the total disappearance of the free ligand (FIG. 16-1) peak
was observed. Purification of the crude product by preparative HPLC
using a C18 column afforded a white powder (0.0606 g, 95%),
corresponding to .rho.-SCN-Bn-Gd-DOTA (FIG. 16-2). The calculated
HRMS for C.sub.24H.sub.29GdN.sub.5O.sub.8S,
[C.sub.24H.sub.29GdN.sub.5O.sub.8S]+ is 704.8308. The product's
measured HRMS value was 705.1130. .rho.-SCN-Bn-Gd-DTPA was
synthesized using the same procedure (data not shown).
[0117] Synthesis of Mito-Gd-DOTA.
[0118] To an argon purged solution of .rho.-SCN-Bn-Gd-DOTA (FIG.
16-2) (0.03 g, 0.0425 mmol) and (10-aminodecyl)
triphenylphosphonium bromide (FIG. 16-3) (0.025 g, 0.05 mmol) in
anhydrous DMSO (25 mL), (0.014 g, 0.143 mmol) of TEA were added.
The mixture was allowed to react overnight and monitored by HPLC.
Purification of the crude product by preparative HPLC using a C18
column afforded a white powder (0.0455 g, 93%), corresponding to
(FIG. 16-4). The calculated HRMS value for
C.sub.52H.sub.66GdN.sub.6O.sub.8PS+,
[C.sub.52H.sub.66GdN.sub.6O.sub.8PS]+ is 1123.3636. The product's
measured HRMS value was 1124.5301. FIG. 17 illustrates a
relaxitivity study of Mito-Gd-DOTA at 9.4 T. The DTPA derivative
was synthesized using substantially the same procedure (data not
shown).
[0119] The present invention has been described in connection with
what are presently considered to be the most practical and
preferred embodiments. However, the present invention has been
presented by way of illustration and is not intended to be limited
to the disclosed embodiments. Accordingly, those skilled in the art
will realize that the invention is intended to encompass all
modifications and alternative arrangements within the spirit and
scope of the invention as set forth in the appended claims.
* * * * *