U.S. patent application number 15/001752 was filed with the patent office on 2016-07-21 for agents for the molecular imaging of serine-protease in human pathologies.
This patent application is currently assigned to Institut National de la Sante et de la Recherche Medicale. The applicant listed for this patent is Marie-Claude Fournie-Zaluski, Dominique Leguludec, Jean-Baptiste Michel, Herve Poras, Bernard Roques, Francois Rouzet. Invention is credited to Marie-Claude Fournie-Zaluski, Dominique Leguludec, Jean-Baptiste Michel, Herve Poras, Bernard Roques, Francois Rouzet.
Application Number | 20160206762 15/001752 |
Document ID | / |
Family ID | 45319090 |
Filed Date | 2016-07-21 |
United States Patent
Application |
20160206762 |
Kind Code |
A1 |
Michel; Jean-Baptiste ; et
al. |
July 21, 2016 |
Agents for the Molecular Imaging of Serine-Protease in Human
Pathologies
Abstract
The present invention is directed to the use of an irreversible
ligand of a serine protease selected from the group consisting of
leukocyte elastase, thrombin, tissue plasminogen activator (t-PA)
and plasmin for the molecular imaging of said serine protease and
the diagnosis of pathophysiological conditions associated with said
serine protease activity.
Inventors: |
Michel; Jean-Baptiste;
(Paris, FR) ; Rouzet; Francois; (Paris, FR)
; Leguludec; Dominique; (Paris, FR) ; Poras;
Herve; (Paris, FR) ; Fournie-Zaluski;
Marie-Claude; (Paris, FR) ; Roques; Bernard;
(Paris, FR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Michel; Jean-Baptiste
Rouzet; Francois
Leguludec; Dominique
Poras; Herve
Fournie-Zaluski; Marie-Claude
Roques; Bernard |
Paris
Paris
Paris
Paris
Paris
Paris |
|
FR
FR
FR
FR
FR
FR |
|
|
Assignee: |
Institut National de la Sante et de
la Recherche Medicale
Paris
FR
Pharmaleads
Paris
FR
Assistance Publique Hopitaux de Paris
Paris
FR
Universite Paris Diderot - Paris 7
Paris
FR
|
Family ID: |
45319090 |
Appl. No.: |
15/001752 |
Filed: |
January 20, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13990845 |
May 31, 2013 |
9272054 |
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PCT/EP2011/071774 |
Dec 5, 2011 |
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15001752 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 49/14 20130101;
C07K 5/1024 20130101; A61K 51/088 20130101; A61K 49/0004 20130101;
A61K 51/0482 20130101; A61K 51/08 20130101; A61K 51/048
20130101 |
International
Class: |
A61K 51/08 20060101
A61K051/08; A61K 51/04 20060101 A61K051/04; C07K 5/117 20060101
C07K005/117 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 3, 2010 |
EP |
10306343.4 |
May 15, 2013 |
EP |
11305405.0 |
Claims
1-11. (canceled)
12. A leukocyte elastase-targeted molecular imaging agent
comprising at least one irreversible peptide ligand of leukocyte
elastase associated with at least one detectable moiety wherein
said irreversible peptide ligand has the general formula (I):
P-X-Y-Z-Pro-Val-cmk (I) wherein: P is a protective group X is Gly,
Ala or beta-Ala Y is Gly, Ala or Leu Z is Ala, Val, Ile, or
Leu.
13. The leukocyte elastase-targeted molecular imaging agent of
claim 12, wherein said protective group is Acetyl (Ac) or
tButyloxycarbonyl (Boc).
14. The leukocyte elastase-targeted molecular imaging agent of
claim 12, wherein the at least one irreversible peptide ligand of a
leukocyte elastase is covalently bound through a Gly residue with a
metal-chelating moiety.
15. The leukocyte elastase-targeted molecular imaging agent of
claim 14, wherein the metal-chelating moiety is 1, 4, 7,
10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid (DOTA),
diethylene triaminepentaacetic acid (DTPA),
1,4,7-triaza-cyclononane N,N,N''-triacetic acid (NOTA) or
6-hydrazinopyridine-3-carboxylic acid (HYNIC).
16. The leukocyte elastase-targeted molecular imaging agent of
claim 14, wherein the metal-chelating moiety is complexed with a
radionuclide metal or a paramagnetic metal ion.
17. The leukocyte elastase-targeted molecular imaging agent of
claim 16, wherein the radionuclide metal is gallium-68 (68Ga),
technetium-99m (99mTc), gallium-67 (67Ga), yttrium-91 (91Y),
indium-111 (111In), rhenium-186 (186Re), or thallium-201
(201Tl).
18. The leukocyte elastase-targeted molecular imaging agent of
claim 16, wherein the paramagnetic metal ion is gadolinium III
(Gd3+).
19. The leukocyte elastase-targeted molecular imaging agent of
claim 12, wherein the at least one irreversible peptide ligand of a
leukocyte elastase is covalently bound through a benzylthiourea
with a metal-chelating moiety.
20. The leukocyte elastase-targeted molecular imaging agent of
claim 19, wherein the metal-chelating moiety is DOTA, DTPA or
NOTA.
21. A pharmaceutical composition comprising at least one leukocyte
elastase-targeted molecular imaging agent according to claim 12 and
at least one pharmaceutically acceptable carrier.
22. A method of molecular imaging leukocyte elastase in a patient
in need thereof, comprising: a) administering to the patient an
effective amount of a leukocyte elastase-targeted molecular imaging
agent comprising at least one irreversible peptide ligand of a
leukocyte elastase bound to at least one detectable moiety via a
spacer, whereby said leukocyte elastase-targeted molecular imaging
agent binds to leukocyte elastase present in the patient, wherein
said irreversible peptide ligand has formula (I):
P-X-Y-Z-Pro-Val-cmk (1) wherein: P is a protective group X is Gly,
Ala or beta-Ala Y is Gly, Ala or Leu Z is Ala, Val, Ile, or Leu.
wherein said detectable moiety is detectable by a molecular imaging
technique selected from the group consisting of Magnetic Resonance
Imaging (MRI), planar scintigraphy (PS), Positron Emission
Tomography (PET), Single Photon Emission Computed Tomography
(SPECT), or any combination of these techniques and b) detecting
the leukocyte elastase-targeted imaging agent bound to leukocyte
elastase present in the patient using said molecular imaging
technique.
Description
FIELD OF THE INVENTION
[0001] The present invention is directed to the use of irreversible
ligands of the serine proteases, leukocyte elastase, thrombin,
tissue plasminogen activator (t-PA) and plasmin, for the molecular
imaging of said serine proteases in pathophysiological conditions
associated with said serine protease activities.
BACKGROUND OF THE INVENTION
[0002] Serine proteases have been shown to play a role in diverse
physiological functions, many of which can play important roles in
disease processes such as cardiovascular disease (Kohler, H. T., et
al., (2000) N. Engl. J. Med. 342(24):1792-801; Hamsten, A., et al.,
(2000) Thromb. Haemost. 83(3):397403; Califf, R. M., et al., (2000)
Circulation, 101(19):2231-8; Krendel, S., et al., (2000) Ann.
Emerg. Med. 35(5):502-5), cancer (Schmidt, M., et al., (1999) Acta
Otolaryngol. 119(8):949-53; Raigoso, P., et al., (2000) Int. J.
Biol. Markers, 15(1):44-50; Haese, A., et al., (2000) J. Urol.
163(5):1491-7; Hoopera, J. D., et al., (2000) Biochim. Biophys.
Acta, 2000, 1492(l):63-71; Wallrapp, C., et al., (2000) Cancer Res.
2000, 60(10):2602-6; Cao, Y., et al., (2000) Int. J. Mol. Med.
2000, 5(5):547-51), asthma, chronic obstructive pulmonary disease
(COPD), inflammatory diseases (Rice, K. D., et al., (1998) Curr.
Pharm. Des., (5):381-96; Nadel, J. A., et al., (1998) Eur. Respir.
J., (6):1250-1; Wright, C. D., et al., (1999) Biochem. Pharmacol.,
58(12):1989-96; Burgess, L. E., et al., (1999) Proc. Natl. Acad.
Sci. U.S.A., 96(15):8348-52; Barnes, P. J., et al., (2000) Chest,
117(2 Suppl):1OS-4S) and bacterial infections (Al-Hasani, K., et
al., (2000) Infect. Immun., 68(5):2457-63; Gaillot, O., et al.,
(2000) Mol Microbiol. 35(6):1286-94; Lejal, N., et al., (2000) J.
Gen. Virol., 81(Pt 4):983-92).
[0003] Accordingly, serine protease activities of leukocyte
elastase, thrombin, tissue plasminogen activator (t-PA) and plasmin
are considered as potential useful markers for the diagnosis of
some of these pathologies. For example, the tissue pathology of
serine proteases is linked to their ability to be retained within
diseased tissue, therefore providing molecular targets for in situ
molecular imaging. However there remains a need in the art for new
approaches of the molecular imaging of serine proteases in tissues.
Serine protease molecular imaging agents that are easy and
relatively cheap to produce are therefore particularly
desirable.
[0004] Leukocyte elastase, thrombin, tissue plasminogen activator
(t-PA) and plasm in belong to the S1 family of serine proteases,
whose the mechanism of action is well known (Barrett, Handbook of
Proteolytic Enzymes, 2.sup.nd Ed). The catalytic activity of the S1
family of proteases is provided by a charge relay system involving
an aspartic acid residue that is hydrogen-bonded to a histidine,
which itself is hydrogen-bonded to a serine. The sequences in the
vicinity of the active site serine and histidine residues are well
conserved in this family of proteases. The specificity of these
proteases is essentially ensured by the "nonprime" domain of their
active site i.e. the S5 or S4 to S1 binding subsites (following the
nomenclature of Schechter and Berger (Schechter I., and Berger A.
(1967) Biochem Biophys Res Commun 27, 157-162)) In these serine
proteases, the amino acid interacting with the S1 subsite is either
an arginine residue (t-Pa, Thrombin, Plasmin) or an hydrophobic
residue (Leukocyte elastase).
[0005] Various irreversible ligands of the selected serine
proteases are reported in the literature (Teger-Nilsson, 1977),
(Kettner, 1978) (see table 1), They are constituted by small
peptides interacting with the S5 or S4-S2 domain of the peptidase,
and the amino acid binding the S1 subsite is replaced by an
aminoacyl chloromethylketone (cmk) which interacts covalently with
the serine of the catalytic site. Consequently, due to the
restricted selectivity of the P1 residue, some of these
irreversible inhibitors do not possess the required parameters of
affinity and selectivity needed for a specific labelling of each
protease. For example: in table 1, it was observed that the same
peptide is described for Thrombin and t-PA.
TABLE-US-00001 TABLE 1 Examples of irreversible ligands of the
selected serine proteases Serine protease Peptide Reference
Leukocyte MeOSucAla-Ala-Pro-Ala-cmk Navia, PNAS, (1989), 86
elastase MeOSucAla-Ala-Pro-Val-cmk Powers, Biochim Biophys Acta
(1977), 485, 156 tPA D-Phe-Pro-Arg-cmk Boatman, J Med Chem (1999),
42, 1367 Plasmin D-Val-Phe-Lys-cmk Woessner, Steroids (1989), 54,
491 Thrombin D-Phe-Pro-Arg-cmk Kettner, Thromb Res (1979), 14,
969
[0006] However, as shown in table 1, the ligand
D-Val-Phe-Lys-COCH.sub.2Cl (dVFK-cmk, M.W. 500) was developed as a
very selective irreversible peptide ligand of plasmin active site
with high affinity (Collen, Biochimica and Biophysica Acta, 165
(1980), 158-166).
[0007] Moreover, no investigations have been carried out to use
these irreversible ligands for the molecular imaging of serine
proteases.
SUMMARY OF THE INVENTION
[0008] The present invention relates to the development of
molecular imaging agents that comprise one irreversible
chloromethylketone (cmk) peptide ligand of a serine protease
selected from the group consisting of leukocyte elastase, thrombin,
tissue plasminogen activator (t-PA) and plasmin associated with one
detectable moiety.
DETAILED DESCRIPTION OF THE INVENTION
[0009] The present invention is directed to the use of an
irreversible ligand of a serine protease selected from the group
consisting of leukocyte elastase, thrombin, tissue plasminogen
activator (t-PA) and plasmin for the molecular imaging of said
serine protease and the diagnosis of pathophysiological conditions
associated with said serine protease activity. In particular, the
invention encompasses molecular imaging agents, kits and strategies
for specifically detecting the presence of said serine proteases
using molecular imaging techniques.
[0010] The inventors have indeed demonstrated that peptide
irreversible ligands of the selected serine proteases can be used
for the molecular imaging of these serine proteases in
physiological and physiopathological conditions by introducing at
the N-terminal position of the irreversible cmk peptide ligand a
detectable moiety (DOTA or DTPA) via a small spacer, in order to
minimize a possible steric hindrance between these two
moieties.
[0011] More particularly, they designed a derivative of
D-Val-Phe-Lys-COCH.sub.2Cl (dVFK-cmk), substituted by a detectable
moiety via a spacer, in order to study tissue plasm in in
pathological conditions, by in vivo molecular imaging. They tested
the ability of this new peptide to bind and inhibit plasmin in
vitro, and demonstrated the ability of this peptide to target in
vivo plasmin-rich tissues, by (.sup.99mTc) scintigraphy in several
fibrinolytic-rich experimental models of cardiovascular pathologies
in rats.
[0012] More particularly, they designed a derivative of an
irreversible cmk peptide ligand of thrombin substituted by a
detectable moiety via a spacer, in order to study tissue thrombin
in pathological conditions. They tested the ability of this new
peptide to bind and inhibit thrombin in vitro and in vivo.
[0013] They also designed a derivative of an irreversible cmk
peptide ligand of leukocyte elastase substituted by a detectable
moiety via a spacer, in order to study tissue leukocyte elastase in
pathological conditions. They tested the ability of this new
peptide to bind and inhibit leukocyte elastase in vitro and in
vivo.
[0014] They also designed a derivative of an irreversible cmk
peptide ligand of tissue plasminogen activator (tPA) substituted by
a detectable moiety via a spacer, in order to study tPA in
pathological conditions. They tested the ability of this new
peptide to bind and inhibit tPA in vitro and in vivo.
[0015] Accordingly, the present invention relates to "serine
protease-targeted molecular imaging agents" that comprise at least
one irreversible peptide ligand of a serine protease associated
with at least one detectable moiety, wherein said serine protease
is selected from the group consisting of leukocyte elastase,
thrombin, tissue plasminogen activator (t-PA) and plasmin.
[0016] As used herein, the term "molecular imaging agent" refers to
a compound that can be used to detect specific biological elements
(e.g., biomolecules) using molecular imaging techniques. Molecular
imaging agents of the present invention can be used to detect the
serine protease of interest in tissues of a subject.
[0017] The term "irreversible peptide ligand of a serine protease"
or "irreversible chloromethylketone (cmk) peptide ligand of a
serine protease" refers to any modified peptide that is capable to
form a covalent complex with the essential amino acid serine of the
catalytic triad of the serine protease. According to the invention
the irreversible ligands of the selected serine proteases have to
exhibit high affinity, specificity and/or selectivity for said
serine proteases.
[0018] In one embodiment, the present invention relates to a
leukocyte elastase-targeted molecular imaging agent that comprise
at least one irreversible peptide ligand of leukocyte elastase
associated with at least one detectable moiety wherein said
irreversible peptide ligand has the general formula (I):
P-X-Y-Z-Pro-Val-cmk (I)
[0019] wherein: [0020] P represents a protective group such as
Acetyl (Ac), tButyloxycarbonyl (Boc) [0021] X represents Gly, Ala
or .beta.-Ala [0022] Y represents Gly, Ala or Leu [0023] Z
represents Ala, Val, Ile, or Leu.
[0024] In formula (I), when Y and Z are chiral amino acids then
could be under the L or D configuration
[0025] Accordingly the present invention relates to a tPA-targeted
molecular imaging agent that comprise at least one irreversible
peptide ligand of tPA associated with at least one detectable
moiety wherein said irreversible peptide ligand has the general
formula II:
P-X1-Pro-Y1-Gly-Arg-cmk (II)
[0026] wherein: [0027] P represents a protective group such as
Acetyl (Ac), tButyloxycarbonyl (Boc) [0028] X1 represents Gly or
Ala [0029] Y1 represents Phe, Tyr, Ile or Leu
[0030] In formula (II), when X1 and Y1 are chiral amino acids, they
could be under the L or D configuration.
[0031] Accordingly the present invention relates to a
thrombin-targeted molecular imaging agent that comprise at least
one irreversible peptide ligand of thrombinassociated with at least
one detectable moiety wherein said irreversible peptide ligand has
the general formula II:
P-X2-Y2-Z2-Arg-cmk (III)
[0032] wherein: [0033] P represents a protective group such as
Acetyl (Ac), tButyloxycarbonyl (Boc) [0034] X2 represents Gly or
.beta.Ala [0035] Y2 represents Phe, Val, Ile or Leu [0036] Z2
represents Pro or Gly
[0037] In formula (III), when X2, Y2 and Z2 are chiral amino acids;
they could be under the L or D configuration.
[0038] Accordingly the present invention relates to a
plasmin-targeted molecular imaging agent that comprise at least one
irreversible peptide ligand of plasmin associated with at least one
detectable moiety wherein said irreversible peptide ligand is
D-Val-Phe-Lys-cmk.
[0039] The term "detectable moiety", as used herein refers to any
entity which, when part of a molecule, allows visualization of the
molecule by using molecular imaging techniques. In the context of
the present invention, detectable moieties are entities that are
detectable by molecular imaging techniques such as Magnetic
Resonance Imaging (MRI), planar scintigraphy (PS), Positron
Emission Tomography (PET), Single Photon Emission Computed
Tomography (SPECT), or any combination of these techniques.
Preferably, detectable moieties are stable, non-toxic entities
which, when part of a serine protease-targeted molecular imaging
agent, retain their properties under in vitro and in vivo
conditions.
[0040] In certain embodiments, the serine protease-targeted
molecular imaging agent is designed to be detectable by a nuclear
medicine molecular imaging techniques such as planar scintigraphy
(PS), Positron Emission Tomography (PET) and Single Photon Emission
Computed Tomography (SPECT). In such embodiments, the molecular
imaging agent of the invention comprises at least one irreversible
inhibitor of the selected serine protease associated with at least
one radionuclide (i.e., a radioactive isotope).
[0041] SPECT and PET acquire information on the concentration of
radionuclides introduced into a subject's body. PET generates
images by detecting pairs of gamma rays emitted indirectly by a
positron-emitting radionuclide. A PET analysis results in a series
of thin slice images of the body over the region of interest (e.g.,
brain, breast, liver). These thin slice images can be assembled
into a three dimensional representation of the examined area. SPECT
is similar to PET, but the radioactive substances used in SPECT
have longer decay times than those used in PET and emit single
instead of double gamma rays. Although SPECT images exhibit less
sensitivity and are less detailed than PET images, the SPECT
technique is much less expensive than PET and offers the advantage
of not requiring the proximity of a particle accelerator. Planar
scintigraphy (PS) is similar to SPECT in that it uses the same
radionuclides. However, PS only generates 2D-information.
[0042] Thus, in certain embodiments, the detectable moiety in a
molecular imaging agent of the invention is a radionuclide
detectable by PET such as Gallium-68 (68Ga).
[0043] In other embodiments, the detectable moiety is a
radionuclide detectable by planar scintigraphy or SPECT. Examples
of such radionuclides include technetium-99m (99mTc), gallium-67
(67Ga), yttrium-91 (91Y), indium-111 (111In), rhenium-186 (186Re),
and thallium-201 (201Tl). Preferably, the radionuclide is
technetium-99m (99mTc). Over 85% of the routine nuclear medicine
procedures that are currently performed use radiopharmaceutical
methodologies based on 99mTc.
[0044] In certain embodiments, the serine protease-targeted
molecular imaging agent is designed to be detectable by Magnetic
Resonance Imaging (MRI). MRI has the advantage (over other
high-quality molecular imaging methods) of not relying on
potentially harmful ionizing radiation. Thus, in certain
embodiments, the molecular imaging agent of the invention comprises
at least one irreversible inhibitor of the serine protease
associated with at least one paramagnetic metal ion. Example of
paramagnetic metal ions detectable by MRI is gadolinium III
(Gd.sup.3+), which is an FDA-approved contrast agent for MRI, or
iron oxide, which gives a sensitive negative signal in MRI.
[0045] The inventive molecular imaging agents may be prepared by
any synthetic method known in the art, the only requirement being
that, after reaction, the irreversible inhibitor of the serine
protease and detectable moiety retain their affinity and
detectability property, respectively. The irreversible ligands of
the serine protease and detectable moieties may be associated in
any of a large variety of ways. However, the detectable moiety
being a metal entity, the irreversible inhibitor of the serine
protease is associated to the detectable metal entity via a
metal-chelating moiety. The irreversible inhibitor of the serine
protease is associated to the metal-chelating moiety by a covalent
bond through a small spacer. Accordingly the N-terminal protective
group (P) of the "irreversible inhibitor" of thrombin, tPA or
Leukocyte Elastase as described in formula (I), (II) or (III), is
replaced by the selected spacer before the association with the
metal chelating moiety.
[0046] The small spacer is either a Gly residue or the
benzylthiourea generated by the introduction of the commercially
available metal-chelating moiety (p-SCN-Bn-DOTA, p-SCN-Bn-DTPA or
p-SCN-Bn-NOTA).
[0047] Suitable metal-chelating moieties for use in the present
invention may be any of a large number of metal ligands and metal
complexing molecules known to bind detectable metal moieties.
Preferably, metal-chelating moieties are stable, non-toxic entities
that bind radionuclides or paramagnetic metal ions with high
affinity.
[0048] Examples of metal-chelating moieties that have been used for
the complexation of paramagnetic metal ions, such as gadolinium III
(Gd.sup.3+), include DTPA (diethylene triaminepentaacetic acid);
DOTA (1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic
acid); and derivatives thereof (see, for example, U.S. Pat. Nos.
4,885,363; 5,087,440; 5,155,215; 5,188,816; 5,219,553; 5,262,532;
and 5,358,704; and D. Meyer et al., Invest. Radiol. 1990, 25:
S53-55), in particular, DTPA-bis(amide) derivatives (U.S. Pat. No.
4,687,659). Other ligands also include NOTA
(1,4,7-triaza-cyclononane N,N',N''-triacetic acid), and HYNIC
(6-Hydrazinopyridine-3-carboxylic acid).
[0049] The invention provides reagents and strategies to image the
presence of the serine proteases of interest in tissues. More
specifically, the invention provides targeted reagents that are
detectable by molecular imaging techniques and methods that allow
the detection, localization and/or quantification of serine
proteases in living subjects, including human patients.
[0050] As used herein, the term "subject" refers to a human or
another mammal (e.g., mouse, rat, rabbit, hamster, dog, cat,
cattle, swine, sheep, horse or primate). In many embodiments, the
subject is a human being. In such embodiments, the subject is often
referred to as an "individual" or a "patient if the subject is
afflicted with a disease or clinical condition. The terms
"subject", "individual" and "patient" do not denote a particular
age, and thus encompass adults, children and newborns.
[0051] More specifically, the present invention provides methods
for detecting the presence of the selected serine protease (i.e.
leukocyte elastase, thrombin, tissue plasminogen activator (t-PA)
and plasmin) in a tissue's patient. The methods comprise
administering to the patient an effective amount of a serine
protease-targeted molecular imaging agent of the invention, or a
pharmaceutical composition thereof. The administration is
preferably carried out under conditions that allow the molecular
imaging agent (1) to reach the tissue(s)'s patient that may contain
abnormal serine proteases (i.e., serine proteases associated with a
clinical condition) and (2) to interact with such serine proteases
so that the interaction results in the binding of the molecular
imaging agent to the serine proteases. After administration of the
serine protease-targeted molecular imaging agent and after
sufficient time has elapsed for the interaction to take place, the
molecular imaging agent bound to serine proteases present in the
patient is detected by a molecular imaging technique. One or more
images of at least part of the body of the patient may be
generated.
[0052] Administration of the serine protease-targeted molecular
imaging agent, or pharmaceutical composition thereof, can be
carried out by any suitable method known in the art such as
administration by oral and parenteral methods, including
intravenous, intraarterial, intrathecal, intradermal and
intracavitory administrations, and enteral methods.
[0053] Accordingly, the molecular imaging agents of the invention
represent powerful tools for the diagnosis of pathological
conditions that are associated with serine proteases selected from
the group consisting of leukocyte elastase, thrombin, tissue
plasminogen activator (t-PA) and plasmin.
[0054] The terms "pathological condition associated with serine
proteases", "disease associated with serine proteases" and
"disorder associated with serine proteases" are used herein
interchangeably. They refer to any disease condition characterized
by undesirable or abnormal tissue serine protease activity. Such
conditions may result from a tissue degeneration mediated by the
serine proteases selected from the group consisting of leukocyte
elastase, thrombin, tissue plasminogen activator (t-PA) and
plasmin. The term include for example, disease conditions
associated with or resulting from the homing of leukocytes to sites
of pathologies the interaction of platelets with activated
endothelium, platelet-platelet and platelet-leukocyte interactions
in the blood vascular compartment, the formation of intraluminal or
intraparietal thrombi, and the like. The term also includes all
tissue degenerative pathologies involve less or more serine
protease activation, including plasmin formation, t-PA, thrombin
and/or leukocyte protease release and retention. For example these
human pathologies include all forms of atherothrombotic diseases
whatever the localisation (coronary artery diseases,
cerebrovascular disease including stroke, aneurysms of the aorta,
leg ulcers, etc.), acute and chronic pulmonary pathologies,
including Acute Respiratory Distress Syndrome, emphysema and
Chronic Obstructive Pulmonary Disease (COPD), arthritis,
auto-immune diseases, certain forms of localized infectious
diseases. The term also includes cancers.
[0055] The diagnosis is thus achieved by examining and molecular
imaging parts of or the whole body of the patient. Comparison of
the results obtained from the patient with data from studies of
clinically healthy individuals will allow determination and
confirmation of the diagnosis.
[0056] These methods can also be used to follow the progression of
a pathological condition associated with serine proteases selected
from the group consisting of leukocyte elastase, tissue plasminogen
activator (t-PA) and plasmin. For example, this can be achieved by
repeating the method over a period of time in order to establish a
time course for the presence, localization, distribution, and
quantification of "abnormal" serine proteases in a patient's
tissue.
[0057] These methods can also be used to monitor the response of a
patient to a treatment for a pathological condition associated with
serine proteases selected from the group consisting of leukocyte
elastase, thrombin, tissue plasminogen activator (t-PA) and
plasmin. For example, an image of part of the patient's body that
contains tissue "abnormal" serine proteases is generated before and
after submitting the patient to a treatment. Comparison of the
"before" and "after" images allows the response of the patient to
that particular treatment to be monitored.
[0058] In the methods of molecular imaging of serine proteases and
of diagnosis of pathological conditions associated with serine
proteases described herein, the molecular imaging agents of the
present invention may be used per se or as a pharmaceutical
composition.
[0059] Accordingly, in one aspect, the present invention provides
for the use of irreversible ligands of serine protease for the
manufacture of a composition for the diagnosis of clinical
conditions associated with serine proteases selected from the group
consisting of leukocyte elastase, thrombin, tissue plasminogen
activator (t-PA) and plasmin.
[0060] In another aspect, the present invention provides
pharmaceutical compositions comprising at least one serine
protease-targeted molecular imaging agent and at least one
pharmaceutically acceptable carrier.
[0061] As used herein, the term "pharmaceutically acceptable
carrier" refers to a carrier medium which does not interfere with
the effectiveness of the biological activity of the active
ingredients and which is not excessively toxic to the hosts at the
concentrations at which it is administered. The term includes
solvents, dispersion media, coatings, antibacterial and antifungal
agents, isotonic agents, adsorption delaying agents, and the like.
The use of such media and agents for pharmaceutically active
substances is well known in the art (see for example, Remington's
Pharmaceutical Sciences, E. W. Martin, 18th Ed., 1990, Mack
Publishing Co., Easton, Pa.).
[0062] Pharmaceutical compositions will be administered by
injection. For administration by injection, pharmaceutical
compositions of molecular imaging agents may be formulated as
sterile aqueous or non-aqueous solutions or alternatively as
sterile powders for the extemporaneous preparation of sterile
injectable solutions. Such pharmaceutical compositions should be
stable under the conditions of manufacture and storage, and must be
preserved against the contaminating action of microorganisms such
as bacteria and fungi.
[0063] Pharmaceutically acceptable carriers for administration by
injection are solvents or dispersion media such as aqueous
solutions (e.g., Hank's solution, alcoholic/aqueous solutions, or
saline solutions), and non-aqueous carriers (e.g., propylene
glycol, polyethylene glycol, vegetable oil and injectable organic
esters such as ethyl oleate). Injectable pharmaceutical
compositions may also contain parenteral vehicles (such as sodium
chloride and Ringer's dextrose), and/or intravenous vehicles (such
as fluid and nutrient replenishers); as well as other conventional,
pharmaceutically acceptable, non-toxic excipients and additives
including salts, buffers, and preservatives such as antibacterial
and antifungal agents (e.g., parabens, chlorobutanol, phenol,
sorbic acid, thirmerosal, and the like). Prolonged absorption of
the injectable compositions can be brought about by adding agents
that can delay absorption (e.g., aluminum monostearate and
gelatin). The pH and concentration of the various components can
readily be determined by those skilled in the art.
[0064] Sterile injectable solutions are prepared by incorporating
the active compound(s) and other ingredients in the required amount
of an appropriate solvent, and then by sterilizing the resulting
mixture, for example, by filtration or irradiation.
[0065] In general, the dosage of a serine protease-targeted
molecular imaging agent (or pharmaceutical composition thereof)
will vary depending on considerations such as age, sex and weight
of the patient, as well as the particular pathological condition
suspected to affect the patient, the extent of the disease, the
tissue(s) of the body to be examined, and the sensitivity of the
detectable moiety. Factors such as contraindications, therapies,
and other variables are also to be taken into account to adjust the
dosage of molecular imaging agent to be administered. This,
however, can be readily achieved by a trained physician.
[0066] In general, a suitable daily dose of a serine
protease-targeted molecular imaging agent (or pharmaceutical
composition thereof) corresponds to the lowest amount of molecular
imaging agent (or pharmaceutical composition) that is sufficient to
allow molecular imaging of any relevant (i.e., generally
overexpressed) serine protease present in the patient. To minimize
this dose, it is preferred that administration be intravenous,
intramuscular, intraperitoneal or subcutaneous, and preferably
proximal to the site to be examined. For example, intravenous
administration is appropriate for molecular imaging the
cardio/neurovascular system; while intraspinal administration is
better suited for molecular imaging of the brain and central
nervous system.
[0067] In another aspect, the present invention provides kits
comprising materials useful for carrying out the diagnostic methods
of the invention. The diagnostic procedures described herein may be
performed by clinical laboratories, experimental laboratories, or
practitioners.
[0068] In certain embodiments, an inventive kit comprises at least
one irreversible ligands of serine protease as above described and
at least one detectable entity, and, optionally, instructions for
associating the irreversible ligands of serine protease and
detectable entity to form a serine protease-targeted molecular
imaging agent according to the invention. The detectable entity is
preferably a short-lived radionuclide such as technetium-99m
(99mTc), gallium-67 (67Ga), yttrium-91 (91Y), indium-111 (111In),
rhenium-186 (186Re), and thallium-201 (201Tl). Preferably, the
irreversible ligands of serine protease and detectable entity are
present, in the kit, in amounts that are sufficient to prepare a
quantity of molecular imaging agent that is suitable for the
detection of serine proteases and diagnosis of a particular
clinical condition in a subject.
[0069] In addition, the kit may further comprise one or more of:
labelling buffer and/or reagent; purification buffer, reagent
and/or means; injection medium and/or reagents. Protocols for using
these buffers, reagents and means for performing different steps of
the preparation procedure and/or administration may be included in
the kit.
[0070] The different components included in an inventive kit may be
supplied in a solid (e.g., lyophilized) or liquid form. The kits of
the present invention may optionally comprise different containers
(e.g., vial, ampoule, test tube, flask or bottle) for each
individual component. Each component will generally be suitable as
aliquoted in its respective container or provided in a concentrated
form. Other containers suitable for conducting certain steps of the
preparation methods may also be provided. The individual containers
of the kit are preferably maintained in close confinement for
commercial sale.
[0071] In certain embodiments, a kit further comprises instructions
for using its components for the diagnosis of clinical conditions
associated with serine proteases according to a method of the
present invention. Instructions for using the kit according to a
method of the invention may comprise instructions for preparing a
molecular imaging agent from the irreversible ligands of serine
protease and detectable entity, instructions concerning dosage and
mode of administration of the molecular imaging agent obtained,
instructions for performing the detection of serine proteases,
and/or instructions for interpreting the results obtained. A kit
may also contain a notice in the form prescribed by a governmental
agency regulating the manufacture, use or sale of pharmaceuticals
or biological products.
[0072] The invention will be further illustrated by the following
figures and examples. However, these examples and figures should
not be interpreted in any way as limiting the scope of the present
invention.
FIGURES
[0073] FIG. 1 shows the main player in the catalytic mechanism of
serine protease is the "catalytic triad".
[0074] FIG. 2 shows the schematic representation of a proposed
mechanism of Plasmin by DVal-Phe-Lys-cmk: a) Michaelis complex; b)
final alkylated product.
[0075] FIG. 3 shows the plasmin inhibition of VFK, PL702, PL700,
PL703, PL701 and PL704 : the dose-dependant inhibitory capacity of
each molecule (n=4) was evaluated in the presence of 10 nM of
plasmin. Plasmin with or without inhibitors was incubated at room
temperature for 15 min. The fluorescent substrate (40 .mu.M) was
added just before reading (2 excitation 390 nM, .lamda. emission
460 nM)
[0076] FIG. 4 shows ex vivo imaging of experimental aneurysm in
rat.
[0077] FIG. 5 shows ex vivo imaging of experimental left-sided
endocarditis in rat
[0078] FIG. 6 shows intraluminal thrombus in human aneurysm of the
abdominal aorta
[0079] FIG. 7 shows the thrombin, uPA, and tPA inhibition of PL714,
PL716, PL719, PL720: the dose-dependant inhibitory capacity of each
molecule was evaluated in the presence of 20 ng/mL; 50 ng/mL, 1
.mu.g/mL of thrombin, uPA and tPA respectively. Enzyme with or
without inhibitors was pre-incubated at room temperature for 30
min, following by 60 min incubation. The fluorescent substrate (20
.mu.M) was added just before reading (.lamda. excitation 390 nM,
.lamda. emission 460 nM).
[0080] FIG. 8 shows the leukocyte elastase, thrombin and tPA
inhibition of PL715, PL718: The dose-dependant inhibitory capacity
of each molecule was evaluated in the presence of 30 ng/mL, 20
ng/mL and 1 .mu.g/mL of leukocyte elastase, thrombin and tPA
respectively. Enzyme with or without inhibitors was pre-incubated
at room temperature for 30 min, following by 60 min incubation. The
fluorescent substrate (20 .mu.M) was added just before reading
(.lamda. excitation 390 nM, .lamda. emission 460 nM).
EXAMPLE 1
Design of a Peptide Ligand for Plasmin Molecular Imaging
[0081] Plasmin catalyses the cleavage of Lys-X or Arg-X bonds with
specificity similar to that of trypsin, but with a lesser
efficiency for hydrolysis of these bonds in proteins. In this
family of proteases the selectivity of the enzyme is essentially
ensured by the amino acid interacting with the S1 subsite and,
consequently plasmin is able to cleave esters or amides of Lys and
Arg as well as small peptide substrate recognizing the S3 to S1
subsites of the enzyme and containing a C-terminal lysine or
arginine {Lottenberg, 1981}. Likewise, small three amino acids
peptide analogues, such as leupeptin (Ac-Leu-Leu-Arg-H), are
efficient competitive inhibitors with Ki values in the 10.sup.-7
molar range {Chi, 1989}.
[0082] However, to obtain an efficient labeling of the peptidase
for molecular imaging, an irreversible inhibitor has been chosen as
starting material for the design of such a marker. Indeed, the
replacement of the C-terminal carboxylate of Lys, by a chloro
methyl ketone CO--CH.sub.2--Cl in the three amino acids peptide
D-Val-Phe-Lys, allowed the irreversible inhibition of plasmin by
formation of a covalent complex between the inhibitor and essential
amino acids (His and Ser) of the catalytic site, as shown in FIG.
2a, b.
[0083] Using this peptide as model, we decided to introduce the
metal chelating agent, DOTA or DTPA in N-terminal position via a
small spacer (Gly) in order to minimize the steric hindrance due to
the large size of the ligand.
[0084] Moreover, due to the covalent nature of these inhibitors,
the possible presence of an important non specifique binding has to
be considered. For this purpose, an analogue of dV-F-K-cmk, without
affinity for plasmin, dV-F-A-cmk or DOTA dV-F-A-cmk, has been
prepared and used to determine the "background noise".
[0085] DOTA is more specific for the complexation of Ga or Gd
cation whereas DTPA is use for the complexation of Tc.
[0086] Synthetic Pathway:
[0087] Synthesis of the aminoacyl chloromethylketone
##STR00001##
[0088] The carboxylate group of the N-protected amino acid,
Boc-Lys(.epsilon.N--Z)--OH or Boc-Ala-OH is transformed into a
mixed anhydride by action of i-Bu-chloroformate in presence of
N-methylmorpholine, then the addition of diazomethane
CH.sub.2N.sub.2, formed the corresponding diazoketone. Treatment by
saturated HCl in Dioxanne solution gives the chloromethylketone and
the deprotection of the amine function is performed by action of
TFA in CH.sub.2Cl.sub.2.
[0089] Synthesis of the N-Protected tripeptide Boc-Gly-dVal-Phe
##STR00002##
[0090] The peptide is synthesized by liquid phase method using
EDCI, HOBT or TBTU as coupling reagents in four steps: i) synthesis
of Boc-dVal-Phe-OCH.sub.3, ii) deprotection of the amine group by
TFA/CH.sub.2Cl.sub.2, iii) Coupling of Boc-Gly to
H-dVal-Phe-OCH.sub.3, iv) saponification of the methyl ester
leading to Boc-Gly-dVal-Phe.
[0091] Synthesis of the Final Marker
##STR00003##
[0092] The tripeptide Boc-Gly-dVal-Phe is coupled with the
chloromethylketone, Lys (.epsilon.N-Z)-cmk or Ala-cmk by the mixed
anhydride method and the Boc-group eliminated and replaced by the
DOTA group, using DOTA-NHS with DIEA in DMF, or by the DTPA group,
using DTPA di-anhydride DMSO/Hepes buffer.
[0093] For peptides containing a lysine residue, the last step
corresponds to the deprotection of the .epsilon.-amino group by HBr
in AcOH.
[0094] The different inhibitors synthesized are summarized
below:
[0095] dVFK-cmk
[0096] PL700: G-dV-F-K-cmk
[0097] PL701: DOTA-G-dV-F-K-cmk
[0098] PL702: G-dV-F-A-cmk
[0099] PL703: DOTA-G-dV-F-A-cmk
[0100] PL704: DTPA-G-dV-F-K-cmk
EXAMPLE 2
In Vitro Ligation of the Plasmin Active Site by PL704
[0101] Material and Methods:
[0102] Determination of plasmin activity: 10 nM of plasmin from
human plasma (calbiochem) was diluted in Tris-HCl buffer pH 7.4 50
mM, NaCl 100 mM and Tween-20 0.01%. The samples were then incubated
or not with the different inhibitors (see below) for 15 minutes at
room temperature. Then, the fluorescent substrate
(Suc-Ala-Phe-Lys-AMC, 40 mM) was added just before reading (.lamda.
excitation 390 nm, emission 460 nm) on the Fluoroskan Ascent plate
reader (Thermo Fisher). The fluorescence intensity was evaluated
every 10 minutes for 2 hours at 37.degree. C. The integration time
for each measure was 20 ms.
[0103] Each inhibitor was tested in a range of concentrations from
5 nM up to 1000 nM. All the inhibitors were tested in duplicates
for each concentration and 4 independent experiments were
performed.
[0104] Statistical analysis: Results are presented as % of active
plasm in. The differences between the concentrations of each
inhibitor were evaluated by paired t-test using StatView software.
Statistical significance was accepted when p<0.05.
[0105] Results:
[0106] As expected, we observed a significant dose-dependent effect
of dV-F-K and PL 700 (G-dV-F-K-cmk) on plasmin activity from 5 nM
(p=0.0038 vs control). When lysine residue is replaced by alanine
(PL702), the inhibitory effect is totally abolished. However,
adding of a DOTA group on PL702, corresponding to PL703, induced an
inhibition of plasmin activity which is not significant until 1000
nM. The PL704 (DTPA-G-dV-F-K-cmk) inhibitor induced a significant
dose dependant inhibition of plasmin activity from 5 nM (p=0.0385
vs control). As compared to dV-F-K, the plasmin inhibition by PL704
at low concentrations is higher but the difference is not
significant. PL701 (DOTA-G-dV-F-K-cmk) also induced a dose
dependant inhibition of plasmin activity which is significant from
50 nM (p=0.016 vs control) (FIG. 3).
[0107] The table 4 gives the mean and the standard deviation of %
of plasmin activity for all inhibitors
TABLE-US-00002 TABLE 4 0 5 10 50 100 500 1000 nM nM nM nM nM nM nM
VFK Mean 100.0 77.1 68.3 39.1 11.9 8.0 7.8 SEM 0.0 4.1 18.4 12.5
2.8 0.3 0.4 PL 700 VFK- Mean 100.0 89.8 95.6 65.7 43.1 15.8 5.8 CmK
SEM 0.0 5.2 15.0 9.2 8.0 8.1 4.1 PL 701 -VFK- Mean 100.0 83.0 79.1
68.7 54.6 34.1 12.8 CmK-DOTA SEM 0.0 6.7 17.9 15.9 13.5 5.3 3.0 PL
702 VFA- Mean 100.0 102.3 123.5 122.9 113.3 112.1 107.3 CmK SEM 0.0
5.2 11.0 11.4 6.4 5.1 6.2 PL 703 VFA- Mean 100.0 94.9 93.6 86.1
84.1 58.4 48.4 CmK-DOTA SEM 0.0 7.6 18.3 23.9 20.6 23.7 19.1 PL 704
VFK- Mean 100.0 56.3 46.2 27.4 16.6 4.0 1.1 CmK-DTPA SEM 0.0 13.2
7.0 0.9 2.5 1.0 0.5
EXAMPLE 3
Ex Vivo and In Vivo Scintigraphy
[0108] Material and Methods:
[0109] Experimental models: we have used several experimental
models of endovascular thrombus formation in rats, which have been
already developed for molecular molecular imaging of platelet
activation and fibrin formation, including aneurysm of the
abdominal aorta {Sarda-Mantel, 2006}, endocarditic vegetations
{Rouzet, 2008}. Aneurysm of the aorta was induced by matrix
decellularized xenograft in rats {Allaire, 1996}, and endocarditis
by left or right ventricle catheterization followed by provoked
bacteraemia {Rouzet, 2008} as previously described. These thrombus
models were completed by a model of stroke, provoked by cerebral
autologous thrombus emboli in rats (reference).
[0110] These experimental models were also compared to 99m Tc
aprotinin signal ex vivo on human aneurymal thrombus.
[0111] Radiolabelling Procedures:
[0112] Aprotinin labelling with .sup.99mTechnetium: Aprotinin
labelling was performed according to a procedure modified from
Schaadt et al. (J Nucl Med 2003; 44: 177-183).
[0113] DOTA-G-dVFKcmk, DOTA-G-dVFAcmk, and DTPA-G-dVFKcmk labelling
with .sup.111Indium: One hundred and eleven MBq of .sup.111Indium
chlorure (Mallinckrodt, France) were mixed with 10 .mu.g of the
tracer diluted in 80 .mu.l of Ammonium Acetate buffer (0.1 M; pH
6.45), and allowed to incubate for 1 hour at 40.degree. C. The
quality control was performed with instant thin-layer
chromatography (ITLC-SG), using acetone as eluant. The
radiolabeling yield was around 90%.
[0114] DTPA-G-dVFKcmk labelling with .sup.99mTechnetium:
.sup.99mTc-sodium pertechnetate (740 MBq) freshly eluted was mixted
with 10 .mu.g of DTPA-G-dVFKcmk, 4 .mu.l of stannous chloride, and
2 .mu.l of potassium borohydrure, and allowed to incubate for 1
hour at 40.degree. C. The quality control was performed with
instant thin-layer chromatography (ITLC-SG), using acetone as
eluant. The radiolabelling yield was around 90%.
[0115] Single Photon Emission Computed Tomography (SPECT):
[0116] SPECT acquisitions: All acquisitions were performed using a
dedicated small animal .gamma.IMAGER-S system (Biospace Lab,
France) equipped with parallel low-energy high resolution
collimators, 256.times.256 matrix, 15% energy window centered on
140 keV. A dual-head SPECT acquisition was performed for 60 min
associated with an helicoidal computed tomography scan (.mu.CT,
Biospace Lab, France) for image co-registration. Acquisitions were
performed under intraperitoneal (rats) pentobarbital anaesthesia
(40 mg/Kg b.w., Ceva Sante Animale, France), 2 hours after
intravenous infusion of the radiotracer. Targeted activities were
111 MBq (range 62.9-150.6) for .sup.99mTc and 28 MBq (range
25.9-37) for .sup.111 In.
[0117] Processing: After completion of acquisitions, raw CT data
were converted into 256 axial slices using the dedicated Biospace
software, then reformatted into DICOM. Raw SPECT data were
reformatted into DICOM, then transferred on a Xeleris 2 workstation
(GE Medical Systems, Buc, France) and reconstructed in a
128.times.128 matrix, using OSEM (2 iterations, 8 subsets) with
Butterworth 3D post-filtering (cut-off frequency 1.33 cycle/pixel,
order 10).
[0118] Quantitative Analysis: Fused images of both SPECT and CT
were displayed for co-registration. A focal uptake of the
radiotracer in the relevant area (heart, abdominal aorta, or brain
according to the model) was assessed visually, and its intensity
was quantified using the target to background ratio. For that
purpose, a region of interest was manually drawn over the focal
uptake to quantify its activity (counts/min/mm.sup.2). The
background activity was determined by a second region of interest
drawn over the lungs (endocarditis model), the supra-renal aorta
(AAA model), or the contralateral brain hemisphere (stroke
model).
[0119] Binding in excised human AAA samples: Aneurysm thrombi (of 5
mm thickness) obtained from 2 patients who undergone surgical AAA
resection were incubated with either .sup.99mTc-aprotinin or
.sup.99mTcDTPA-G-dVFKcmk-diluted in RPMI-1640 medium (volumetric
activity: 2 MBq/ml) for 30 min at room temperature. After
incubation, the thrombus slices were rinsed 5 times with ice-cold
RPMI-1640 medium. Then a 30 min scintigraphic planar acquisition
(Biospace Lab, France) was performed to assess the global uptake of
the tracer.
[0120] After freezing, samples were cut into 20 .mu.m transverse
sections for autoradiography. Activity (counts/mm.sup.2) were
measured on autoradiograms {Petegnief, 1998} by drawing regions of
interest on each layer of the thrombus (luminal, intermediate and
abluminal), and were corrected from the background activity.
Activity ratios between .sup.99mTc-aprotinin and .sup.99mTc-albumin
were calculated for each layer.
[0121] Quantitative Autoradiography: Relevant tissues (heart,
abdominal aorta, or brain according to the model) were carefully
dissected, frozen and cut into transverse sections of 20 .mu.m
thickness, which were exposed in a digital radioimager (Instant
Imager, Packard, Meriden, USA) for 12 hours. The activity
normalized to the region of interest area (mean counts/min/mm.sup.2
corrected for background activity) was determined on
autoradiograms. Quantification was performed by calculating the
ratio between the activity of the relevant tissue and the activity
of a region of interest drawn on either normal myocardium remote
from the vegetation in the endocarditis model, supra-renal aorta
sample in the AAA model, or the contralateral brain hemisphere in
the stroke model. According to calibration studies previously
reported, with activity standards of tissue-equivalent homogenates,
50 counts/min/mm.sup.2 of .sup.99mTc-ANX approximated 210 kBq/mg in
autoradiography {Petegnief, 1998}
[0122] Histology: Some representative samples of left- and
right-sided endocarditis vegetations in rabbits and rats, including
aortic tissue, aortic valves, superior vena cava, right atria,
tricuspid and pulmonary valves, and left and right ventricles were
fixed in paraformaldehyde for 24H, embedded in paraffin for
morphological analysis, or frozen in OCT for cryostat sectioning
and immunohistochemistry. Five-micrometer thick serial sections
were routinely stained with Masson's trichrome to visualize
erythrocytes and fibrin, hematoxylin/eosin for cells and nuclei,
Alcian blue coupled with nuclear red {Scott, 1996} to reveal areas
of mucoid accumulation and their relation to cell nuclei, orcein
for elastin and Sirius Red for collagen.
[0123] Cryostat sections were used for autoradiography and then
stained by Masson's trichrome, and the full sections were
reconstituted under the microscope using Cartograph software
(Microvision, France). Superposition of both images at the same
scale was then processed (fusion images) in order to localize
.sup.99mTc-ANX uptake (autoradiography) on histological
sections.
[0124] Results:
[0125] Whatever the model used, (99mTc)DTPA-G-dVFKcmk, give a
detectable signal related to the localized lesion, by in vivo SPECT
and by autoradiographies. (99mTc)DTPA-G-dVFKcmk has a high in vivo
uptake by bone marrow for which the specificity has not yet been
explored.
[0126] Abdominal aortic aneurysm in rats: On SPECT acquisitions
performed in vivo 2 hours after radiotracers injection, the AAA was
detectable in n/N rats with 99mTc-DTPA-G-dVFKcmk and in none/N rats
with 99mTc-Aprotinin. Autoradiography gave similar results with
greater ratios with 99mTc-DTPA-G-dVFKcmk. Despite the high uptake
intensity on autoradiography, the lack of detectability of AAA
uptake in vivo is related to: (1) the small volume of the mural
thrombus which is inferior to the spatial resolution of the
detection system (#3 mm) thus generating a partial volume effect
leading to an underestimation of the intensity of the uptake, and
(2) the high background activity coming from the surrounding
structures such as kidneys and bone marrow of the rachis (Table
5).
TABLE-US-00003 TABLE 5 Radiolabelling DTPA-G-dVFKcmk Aprotinin
SPECT (n = 10) (n = 10) P value Autoradiography 7.57 .+-. 3.32 4.95
.+-. 0.90 0.027
[0127] In order to assess the specificity of the tracer uptake by
the mural thrombus, we performed a pre-injection of 20 nmoles of
non-labelled DTPA-G-dVFKcmk in 2 animals, followed 10 minutes later
by the injection of 2 nmoles of radiolabelled DTPA-G-dVFK-cmk,
which resulted in a fivefold decrease of the intensity of the
uptake compared with animals without pre-injection of the
non-labelled compound (quantified by autoradiography: 1.34.+-.0.30
vs 7.57.+-.3.32 cpm/mm.sup.2 respectively, p=0.032 using
Mann-Whitney U test) (FIG. 4).
[0128] Right-sided endocarditis in rats: On SPECT acquisitions
performed in vivo 2 hours after radiotracers injection, the
vegetation uptake was detected in n/N rats with
99mTc-DTPA-G-dVFKcmk and in none/N rats with 99mTc-Aprotinin.
[0129] Left-sided endocarditis in rats: In this model we compared
the uptake of DTPA-G-dVFK-cmk with that of a non-specific analog
(DOTA-G-dVFA-cmk) obtained by the substitution of Lysin by Alanin,
labelled with .sup.111Indium (Table 6).
TABLE-US-00004 TABLE 6 99mTc-DTPA-G- 111In-DOTA-G- dVFKcmk dVFAcmk
SPECT (n = 5) (n = 6) P value Autoradiography 10.65 .+-. 1.07 2.66
.+-. 0.47 <0.0001
[0130] Of note, the pre-injection of 1 mg of non-labelled
DOTA-G-dVFA-cmk in 3 animals did induce a decrease of the uptake
intensity of 99mTc-DTPA-G-dVFKcmk (quantified by autoradiography:
6.43.+-.1.85 vs 10.65.+-.1.07 respectively, p=0.1 using
Mann-Whitney U test). (FIG. 5)
[0131] Stroke in rats: In this model we compared the uptake of
99mTc-DTPA-G-dVFK-cmk with that of a the 99mTc chelate
diethylenetriaminepentaacetic acid (DTPA), which is a non-specific
tracer of capillary permeability.
[0132] A DOTA-G-dVFK-cmk stained by gadolinium (Gd) has been
synthesized and injected in the model of stroke in rat (15 mg) for
MRMolecular imaging. The MRI signal of the (Gd)-DOTA-G-dVFK-cmk was
detectable and colocalized with the cerebral infarct in vivo and ex
vivo.
[0133] The chelation was carried out by stoechiometric addition of
the DOTA groups coupled to the VFK in the presence of GdCl.sub.3,
6H.sub.2O. After adjusting the pH to 6.5 in water, the solution was
maintained at room temperature overnight followed by heating at
60.degree. C. for 4 h. Free gadolinium was removed by dialysis
against NaCl 0.1M then by dialysis against water through a 100 Da
cut-off membrane. The product was freeze-dried. The reaction yield
was about 88% in mass and nearly 100% in chelation. No free
gadolinium was detected.
[0134] Human samples of abdominal aortic aneurysms: using
99mTc-Aprotinin as a control (Houard et al), human samples of AAA
incubated in radiolabelled VFK showed a twofold increase of the
activity. Autoradiography allowed evidencing a greater uptake both
at the luminal and abluminal layers of the thrombus, but also
within the intermediate layer. The latter may be related, at least
in part, to the smaller size of 99mTc-DTPA-G-dVFKcmk (1 kDa)
compared to 99mTc-Aprotinin (6 kDa) (FIG. 6).
[0135] In order to assess whether the higher tracer uptake at the
luminal and abluminal layers of the thrombus was specific or merely
due to the contact with the radio-labelled solution, prior to
incubation with 99mTc-DTPA-G-dVFKcmk, a thrombus slice has been
pre-incubated with a RPMI solution containing the non-specific
analog DOTA-G-dVFA-cmk at a concentration of 0.05 mg/ml. The
overall uptake of 99mTc-DTPA-G-dVFKcmk has been decreased by about
25%, but the ratio between the (ab)luminal layers and the
intermediate layer remained unchanged.
[0136] In left ventricular endocarditis, 99mTc-DTPA-G-dVFKcmk gave
localized positive image corresponding to the sceptic thrombus
surrounding the catheter, and aortic valve and left ventricular
myocardium positivity, which were confirmed by
autoradiographies.
[0137] Similar images were obtained in right ventricular
endocarditis, associated with detectable pulmonary emboli.
[0138] Histology: Whatever the model, 99mTc-DTPA-G-dVFKcmk uptake
was co-localized with the thrombus. Of note, in the endocarditis
model, it was also co-localized with clusters of inflammatory cells
within the myocardium.
[0139] Conclusions: The new tracer radiolabelled DTPA-G-dVFK-cmk is
able to report plasmin activity in pathologic tissues in various
experimental models (AAA, left and right-sided endocarditis,
stroke) as well as in human samples of mural thrombus in AAA. The
uptake of the radiotracer is specific as evidenced by its
significant decrease by the pre-injection of the non-labelled
compound in the AAA model, by the lack of inhibition of its uptake
by the pre-injection of the non-specific analog DOTA-G-dVFA-cmk in
left-sided endocarditis and in human samples of AAA, and by the
lower uptake intensity of the non-specific radiolabelled analog
.sup.111In-DOTA-G-dVFA-cmk.
REFERENCES
[0140] Throughout this application, various references describe the
state of the art to which this invention pertains. The disclosures
of these references are hereby incorporated by reference into the
present disclosure.
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