U.S. patent application number 13/086477 was filed with the patent office on 2011-10-20 for perturbed membrane-binding compounds and methods of using the same.
Invention is credited to Anat Shirvan, Ilan ZIV.
Application Number | 20110257434 13/086477 |
Document ID | / |
Family ID | 39969720 |
Filed Date | 2011-10-20 |
United States Patent
Application |
20110257434 |
Kind Code |
A1 |
ZIV; Ilan ; et al. |
October 20, 2011 |
PERTURBED MEMBRANE-BINDING COMPOUNDS AND METHODS OF USING THE
SAME
Abstract
The invention relates to compounds that selectively bind to
cells undergoing perturbations and alterations of their normal
plasma membrane organization, i.e., cells undergoing cell death,
apoptotic cells or activated platelets. The invention further
provides methods for utilizing the compounds in medical practice,
for diagnostic and therapeutic purposes.
Inventors: |
ZIV; Ilan; (Kfar Saba,
IL) ; Shirvan; Anat; (Herzliya, IL) |
Family ID: |
39969720 |
Appl. No.: |
13/086477 |
Filed: |
April 14, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10585928 |
Jul 13, 2006 |
7947253 |
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PCT/IL2005/000055 |
Jan 16, 2005 |
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13086477 |
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10799586 |
Mar 15, 2004 |
7270799 |
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10585928 |
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60536493 |
Jan 15, 2004 |
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60537289 |
Jan 20, 2004 |
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Current U.S.
Class: |
562/596 |
Current CPC
Class: |
A61K 49/0052 20130101;
A61P 35/00 20180101; A61K 51/0402 20130101; C07F 13/005 20130101;
A61K 49/0021 20130101; A61K 49/0002 20130101; A61K 47/542 20170801;
A61K 51/0497 20130101 |
Class at
Publication: |
562/596 |
International
Class: |
C07C 55/02 20060101
C07C055/02 |
Claims
1. A compound represented by the structure as set forth in formula
(I): ##STR00034## including pharmaceutically acceptable salts,
hydrates, solvates and metal chelates of the compound represented
by the structure as set forth in formula (I) and solvates and
hydrates of the salts; wherein, one of R or R' groups is hydrogen,
and the other of R or R' group represents C.sub.1, C.sub.2,
C.sub.3, C.sub.4, C.sub.5, C.sub.6, C.sub.7, C.sub.8, C.sub.g,
C.sub.10, C.sub.11, C.sub.12, C.sub.13, C.sub.14, C.sub.15,
C.sub.16, linear or branched alkyl, linear or branched
hydroxy-alkyl, linear or branched fluoro-alkyl, aryl or hetroaryl
composed of one or two rings, or combinations thereof; n and m each
stands for an integer of 0, 1, 2, 3 or 4; n and m may be same or
different; M is selected from null, hydrogen, --O--, --S--, and
--N(U), wherein U stands for hydrogen, or C.sub.1, C.sub.2,
C.sub.3, or C.sub.4 alkyl; x, and z each stands independently and
is an integer of 0, 1 or 2, where x and z can be the same or
different; y is an integer of 0, 1 or 2, where when y=2 the
substituent R' may be the same or different at each occurrence; and
D is hydroxyl.
2. The compound according to claim 1 represented by the structure
as set forth in formula (V): ##STR00035## wherein J represents OH
and r stands for an integer of 3, 4, 5, 6, 7 or 8.
3. The compound according to claim 2, wherein r is 4.
4. The compound according to claim 2, wherein r is 5.
5. The compound according to claim 2, wherein the functional groups
of the compound are protected by protecting groups.
6. The compound according to claim 5, wherein the protecting groups
comprise tert-butyl or ethyl.
7. A method for radio-labeling the compound represented by a
structure as set forth in formula (V), ##STR00036## wherein J
represents OH and r stands for an integer of 3, 4, 5, 6, 7 or 8,
the method comprising replacing J with a radio-isotope, thereby
radio-labeling the compound represented by a structure as set forth
in formula (V).
8. The method according to claim 7, wherein r is 4.
9. The method according to claim 7, wherein r is 5.
10. The method according to claim 7 comprising protecting the
functional groups of the compound with protecting groups.
11. The method according to claim 9, wherein the protecting groups
comprise tert-butyl or ethyl.
12. The method according to claim 9 comprising removing the
protecting groups after the step of replacing J with a
radio-isotope.
13. The method according to claim 7, wherein the radio-isotope is
.sup.18F.
Description
PRIOR APPLICATION DATA
[0001] This application is a divisional application of U.S. patent
application Ser. No. 10/585,928, entitled "PERTURBED
MEMBRANE-BINDING COMPOUNDS AND METHODS OF USING THE SAME" filed
Mar. 15, 2004, which is a National Phase application of PCT
International Application No. PCT/IL2005/000055, International
Filing Date: Jan. 16, 2005, claiming priority from U.S. Provisional
Patent Application Ser. No. 60/536,493, entitled "PERTURBED
MEMBRANE-BINDING COMPOUNDS" filed Jan. 15, 2004, U.S. Provisional
Patent Application Ser. No. 60/537,289, entitled "PERTURBED
MEMBRANE-BINDING COMPOUNDS" filed Jan. 20, 2004 and is a
Continuation in part of U.S. patent application Ser. No.
10/799,586, entitled "PERTURBED MEMBRANE-BINDING COMPOUNDS AND
METHODS OF USING THE SAME" filed Mar. 15, 2004, all of which are
incorporated by reference in their entirety.
FIELD OF THE INVENTION
[0002] The invention relates to compounds that selectively bind to
cells undergoing perturbations and alterations of their normal
plasma membrane organization, i.e., cells undergoing cell death,
apoptotic cells or activated platelets. The invention further
provides methods for utilizing the compounds in medical practice,
for diagnostic and therapeutic purposes.
BACKGROUND OF THE INVENTION
[0003] The plasma membrane (outer membrane) of intact eukaryotic
cells is characterized by a highly organized structure. This high
level of membrane organization is determined, among others, by the
molecular structure of the specific lipids constituting the
membrane; the ratio between the various lipid species from which
the membrane is composed; the distribution of the phospholipids
between the outer and inner leaflets of the membrane; and by the
membrane protein constituents.
[0004] While maintenance of the high level of plasma membrane
organization is fundamental to normal cell physiology, substantial
perturbations and alterations of the normal organization of the
cell plasma membrane (PNOM) occur in numerous physiological and
pathological conditions, and are characterizing a plurality of
diseases. Such alterations and perturbations may be evident both at
the morphological level (membrane blebbing observed in cells
undergoing apoptosis) and at the molecular level. PNOM includes,
among others, scrambling and redistribution of the membrane
phospholipids, with movement to the cell surface of
aminophsopholipids, mainly phosphatidylserine (PS) and
phosphatidylethanolamine (PE), which are normally restricted almost
entirely to the inner leaflet of the membrane bilayer, and
reciprocal movement of sphingomyelin (SM) and phosphatidylcholine
(PC) from the outer leaflet to the inner leaflet of the membrane.
This redistribution is referred herein as loss of cell membrane
lipid asymmetry (CMLA). In addition to CMLA loss, PNOM is also
often associated with reduction in the level of packing of membrane
phospholipids and an increase in membrane fluidity.
[0005] These alterations play an important role in rendering the
cell surface a catalytic platform for the assembly of several
clotting factor complexes, such as the tenase and prothrombinase
protein complexes. Accordingly, platelet activation is associated
with the platelet membrane undergoing PNOM, and these alterations
constitute an important factor in normal blood coagulation, as well
as in the initiation and/or propagation of abnormal, excessive
blood clotting in numerous disorders. These disorders include,
among others, arterial or venous thrombosis or thrombo-embolism
[e.g., cerebral stroke, myocardial infarction, deep vein thrombosis
(DVT), disseminated intravascular coagulation (DIC), thrombotic
thrombocytopenic purpura, etc.], unstable atherosclerotic plaques,
sickle cell disease, beta-thalassemia, anti-phospholipid antibody
syndrome [among others in systemic lupus erythematosus (SLE)], and
disorders associated with shedding of membrane microparticles,
e.g., neurological dysfunction in association with cardiopulmonary
bypass.
[0006] Apoptosis is another major situation in which
alterations/perturbations of cell membrane take place. Apoptosis is
an intrinsic program of cell self-destruction or "suicide", which
is inherent in every eukaryotic cell. In response to a triggering
stimulus, cells undergo a highly characteristic cascade of events
of cell shrinkage, blebbing of cell membranes, chromatin
condensation and fragmentation, culminating in cell conversion to
clusters of membrane-bound particles (apoptotic bodies), which are
thereafter engulfed by macrophages. PNOM is a universal phenomenon
of apoptosis, it occurs early in the apoptotic cascade, probably at
the point of cell commitment to the death process, and has also
been shown to be an important factor in the recognition and removal
of apoptotic cells by macrophages.
[0007] A strong correlation has been recently drawn between PNOM
and the potent procoagulant activity of apoptotic cells. PNOM in
apoptotic endothelial cells, such as those occurring in
atherosclerotic plaques, probably plays an important role in the
pathogenesis of thrombotic vascular disorders.
[0008] Since apoptosis or thrombosis each has an important role in
the majority of medical disorders, it is desirable to have tools
for detection of these biological processes and targeting of
associated cells. Compounds for selective binding to
PNOM-membranes, potentially also performing subsequent entry into
these cells having such PNOM-membranes (PNOM-cells), may therefore
serve as an important tool for detecting and targeting of imaging
agents or drugs to cells undergoing damage or death process,
especially by apoptosis, or to platelets undergoing activation.
SUMMARY OF THE INVENTION
[0009] In an embodiment of the invention, there are provided
compounds that can selectively bind to cells undergoing
perturbation of their normal organization of the plasma membrane
(PNOM-cells), while binding to a lesser degree to cells, which
maintain the normal organization of their plasma membrane, and
which are defined hereto as "normal cells". The PNOM-cells are, in
an embodiment of the invention, cells undergoing a death process.
In an embodiment of the invention the cells are apoptotic cells,
and in another embodiment, the cells may be activated platelets.
The invention further relates to methods of detecting PNOM-cells by
using these compounds, which selectively bind to the PNOM-cells. In
another embodiment of the invention, compounds are provided,
represented by structures set forth in formulae I-XIV.
[0010] The term "perturbed membrane-binding compound" (PMBC) refers
to a compound that selectively targets PNOM-cells, while binding to
a lesser degree to normal cells. According to the invention,
binding of the PMBC to the PNOM-cell should be a least 30% greater
than its binding to the normal cell.
[0011] The term "selective targeting" refers in the invention to
the selective binding of a compound to PNOM-cells, i.e., binding to
the PNOM-cell in an extent being at least 30% greater than the
binding to normal cells.
[0012] The term "diagnostic perturbed membrane-binding compound"
(diagnostic PMBC) refers to a compound capable of selective
targeting PNOM-cells, wherein the compound comprises or is linked
to a marker, whereas the marker is detectable by means known to
those skilled in the art.
[0013] The term "therapeutic perturbed membrane-binding compound"
(therapeutic PMBC) refers to a PMBC as defined above, comprising a
drug, useful in the treatment of disease.
[0014] The term "solid support" refers in the contents of the
present invention to a solid matrix, an insoluble matrix, or an
insoluble support. The solid support in accordance with the present
invention may be formed in a variety of structures such as a stack
of micro-particulates, micro-filters, or micro-capillara.
[0015] The PMBC is used in an embodiment of the invention for the
preparation of an agent for selective targeting PNOM-cells.
In one aspect, the present invention provides a compound which
selectively targets a PNOM-cell (i.e., a PMBC) wherein the compound
is represented by the structure set forth in formula
(I):
##STR00001##
[0016] or pharmaceutically acceptable salts, metal chelates,
solvates and hydrates of the compound represented by the structure
as set forth in formula (I), and solvates and hydrates of the
salts; wherein, one of R or R' groups is hydrogen, and the other of
R or R' group represents C.sub.1, C.sub.2, C.sub.3, C.sub.4,
C.sub.5, C.sub.6, C.sub.7, C.sub.8, C.sub.9, C.sub.10, C.sub.11,
C.sub.12, C.sub.13, C.sub.14, C.sub.15, C.sub.16, linear or
branched alkyl, linear or branched hydroxy-alkyl, linear or
branched fluoro-alkyl, aryl or hetroaryl composed of one or two
rings, or combinations thereof; n and m each stands for an integer
of 0, 1, 2, 3 or 4; n and m may be same or different; M is selected
from null, hydrogen, --O--, --S--, and --N(U), wherein U stands for
hydrogen, or C.sub.1, C.sub.2, C.sub.3, or C.sub.4 alkyl; x, and z
each stands independently and is an integer of 0, 1 or 2, where x
and z can be the same or different; y is an integer of 0, 1 or 2,
where when y=2 the substituent R' may be the same or different at
each occurrence; and D is a marker for diagnostics, hydrogen,
hydroxyl, F or a drug; wherein the marker for diagnostics, selected
from a marker for imaging such as F, wherein the F atom may be
either .sup.18F or .sup.19F, or a radio-labeled metal chelate; the
marker for imaging may be detected by color, fluorescence, x-ray,
CT scan, magnetic resonance imaging (MRI) or radio-isotope scan
such as single photon emission tomography (SPECT) or positron
emission tomography (PET). Alternatively, D is a drug to be
targeted to the PNOM cells.
[0017] The drug may be a medicinally-useful agent for the
prevention, amelioration, or treatment of a specific disease and
may be, for example, without being limited: an inhibitor of
apoptosis, (e.g., a caspase inhibitor, antioxidant, modulator of
the Bcl-2 system); an activator of cell death (e.g. an anticancer
drug); or a modulator of blood coagulation, which may be an
anticoagulant, an antithrombotic, or a thrombolytic agent. In such
case, the drug is preferably selected among an antiplatelet agent,
heparin, low molecular weight heparin, antagonists of glycoprotein
IIb/IIIa, tissue plasminogen activator (tPA), or an inhibitor of a
clotting factor, such as an inhibitor of thrombin or an inhibitor
of factor Xa; or an anti-inflammatory drug or an immuno-modulatory
drug. In an embodiment of the invention, there is provided a method
for improvement of anti-cancer therapy, by targeting anti-cancer
drugs to tumors, via targeting the drug to foci of apoptosis, which
occur within tumors either spontaneously, or in response to
therapy. In another embodiment of the invention, there is provided
a method of treating a thrombosis by targeting anticoagulants to
the thrombus, so as to prevent, reduce or cease coagulation.
[0018] In another embodiment of the invention, D may be a solid
support.
[0019] In another embodiment of the invention there is provided a
compound which selectively targets a PNOM cell represented by the
structure as set forth in formula (II):
##STR00002##
including pharmaceutically acceptable salts hydrates, solvates and
metal chelates of the compound represented by the structure as set
forth in formula (II) and solvates and hydrates of the salts;
wherein R.sup.1 represents hydrogen or C.sub.1, C.sub.2, C.sub.3,
C.sub.4, C.sub.5, C.sub.6, C.sub.7, C.sub.8, C.sub.9, C.sub.10,
C.sub.11, C.sub.12, C.sub.13, C.sub.14, C.sub.15, C.sub.16, linear
or branched alkyl, linear or branched hydroxy-alkyl, linear or
branched fluoro-alkyl, aryl or hetroaryl composed of one or two
rings, or combinations thereof; n and m each stands for an integer
of 0, 1, 2, 3 or 4; n and m may be same or different; M is selected
from null, hydrogen, --O--, --S--, and --N(U), wherein U stands for
a null, hydrogen, C.sub.1, C.sub.2, C.sub.3, or C.sub.4 alkyl; D is
hydrogen or a marker for diagnostics. The marker for diagnostics
may being an embodiment of the invention a marker for imaging such
as F, wherein the F may be .sup.18F or .sup.19F or a labeled metal
chelate; the marker for imaging may be detected by color,
fluorescence, x-ray, CT scan, magnetic resonance imaging (MRI) or
radio-isotope scan such as single photon emission tomography
(SPECT) or positron emission tomography (PET). Alternatively, D is
a drug to be targeted to the PNOM-cells, as define above.
[0020] In another embodiment of the invention there is provided a
compound represented by the structure as set forth in formula
(III):
##STR00003##
including pharmaceutically acceptable salts hydrates, solvates and
metal chelates of the compound represented by the structure as set
forth in formula (III) and solvates and hydrates of the salts;
wherein R.sup.3 is hydroxyl or F; R.sup.4 is C.sub.4, C.sub.5,
C.sub.6, C.sub.7, C.sub.8, C.sub.9 or C.sub.10 linear or branched
alkyl, and k is an integer selected from 0, 1, 2, 3, 4 and 5.
[0021] In another embodiment of the invention there is provided a
compound represented by the structure as set forth in formula
(IV):
##STR00004##
[0022] including pharmaceutically acceptable salts hydrates,
solvates and metal chelates of the compound represented by the
structure as set forth in formula (IV) and solvates and hydrates of
the salts; the compound is designated NST200.
[0023] In another embodiment of the invention there is provided a
compound represented by the structure as set forth in formula
(V):
##STR00005##
including pharmaceutically acceptable salts hydrates, solvates and
metal chelates of the compound represented by the structure as set
forth in formula (V) and solvates and hydrates of the salts;
wherein J is --F or --OH, and r stands for an integer of 4, 5, 6,
7, 8, 9, 10. In the case that r is 4 and J is --F, the compound is
designated NST201. In the case that r is 5 and J is --F, the
compound is designated NST-ML-10.
[0024] In yet another embodiment of the invention there is provided
a compound represented by the structure as set forth in formula
(VI):
##STR00006##
including pharmaceutically acceptable salts hydrates, solvates and
metal chelates of the compound represented by the structure as set
forth in formula (VI) and solvates and hydrates of the salts;
wherein J is selected from hydrogen, --F and --OH. In the case that
J is --F, the compound is designated NST205.
[0025] In another embodiment of the invention there is provided a
compound represented by the structure set forth in formula VII:
##STR00007##
including pharmaceutically acceptable salts hydrates, solvates and
metal chelates of the compound represented by the structure as set
forth in formula (VII) and solvates and hydrates of the salts,
wherein Q is selected from Technetium, oxo-Technetium, Rhenium and
oxo-Rhenium, R.sup.4 is selected from hydrogen, C.sub.1, C.sub.2,
C.sub.3, C.sub.4, C.sub.5, and C.sub.6 linear or branched alkyl,
and p stands for an integer, selected from 1, 2, 3, 4 and 5.
[0026] In another embodiment of the invention there is provided a
compound represented by the structure set forth in formula
VIII:
##STR00008##
including pharmaceutically acceptable salts hydrates, solvates and
metal chelates of the compound represented by the structure as set
forth in formula (VIII) and solvates and hydrates of the salts,
wherein Q is selected from technetium, oxo-technetium, rhenium and
oxo-rhenium. In another embodiment of the invention there is
provided a compound represented by the structure as set forth in
formula (IX):
##STR00009##
including pharmaceutically acceptable salts hydrates, solvates and
metal chelates of the compound represented by the structure as set
forth in formula (IX) and solvates and hydrates of the salts;
wherein R.sup.5 is selected from hydrogen, C.sub.1, C.sub.2,
C.sub.3, C.sub.4, C.sub.5, and C.sub.6 linear or branched alkyl,
C.sub.1, C.sub.2, C.sub.3, C.sub.4, C.sub.5, and C.sub.6 linear or
branched fluoro-alkyl, and C.sub.1, C.sub.2, C.sub.3, C.sub.4,
C.sub.5, and C.sub.6 linear or branched hydroxy-alkyl; q stands for
an integer, selected from 1, 2, 3, 4 and 5; and Y is a marker for
fluorescence. In an embodiment of the invention, Y is selected from
a dansyl-amide group and fluorescein.
[0027] In another embodiment of the invention there is provided a
compound represented by the structure as set forth in formula
(X):
##STR00010##
including pharmaceutically acceptable salts hydrates, solvates and
metal chelates of the compound represented by the structure as set
forth in formula (X) and solvates and hydrates of the salts; The
compound is designated NST203.
[0028] In another embodiment of the invention there is provided a
compound represented by the structure as set forth in formula
(XI):
##STR00011##
including pharmaceutically acceptable salts hydrates, solvates and
metal chelates of the compound represented by the structure as set
forth in formula (XI) and solvates and hydrates of the salts;
wherein R.sup.1 represents hydrogen or C.sub.1, C.sub.2, C.sub.3,
C.sub.4, C.sub.5, C.sub.6, C.sub.7, C.sub.8, C.sub.9, C.sub.10,
C.sub.11, C.sub.12, C.sub.13, C.sub.14, C.sub.15, C.sub.16, linear
or branched alkyl, linear or branched hydroxy-alkyl, linear or
branched fluoro-alkyl, aryl or hetroaryl composed of one or two
rings, or combinations thereof.
[0029] In another embodiment of the invention, there is provided a
compound represented by the structure set forth in formula
(XII):
##STR00012##
including pharmaceutically acceptable salts hydrates, solvates and
metal chelates of the compound represented by the structure as set
forth in formula (XII) and solvates and hydrates of the salts;
wherein F may be .sup.18F or .sup.19F.
[0030] In another embodiment of the invention there is provided a
compound represented by the structure as set forth in formula
(XIII):
##STR00013##
including pharmaceutically acceptable salts hydrates, solvates and
metal chelates of the compound represented by the structure as set
forth in formula (XIII) and solvates and hydrates of the salts;
R.sup.1 represents hydrogen or C.sub.1, C.sub.2, C.sub.3, C.sub.4,
C.sub.5, C.sub.6, C.sub.7, C.sub.8, C.sub.9, C.sub.10, C.sub.11,
C.sub.12, C.sub.13, C.sub.14, C.sub.15, C.sub.16, linear or
branched alkyl, linear or branched hydroxy-alkyl, linear or
branched fluoro-alkyl, aryl or hetroaryl composed of one or two
rings, or combinations thereof; m stands for an integer of 0, 1, 2,
3 or 4; D is a marker for diagnostics which may be in an embodiment
of the invention a marker for imaging such as F, wherein the F may
be .sup.18F or .sup.19F or a labeled metal chelate; the marker for
imaging may be detected by color, fluorescence, x-ray, CT scan,
magnetic resonance imaging (MRI) or radio-isotope scan such as
single photon emission tomography (SPECT) or positron emission
tomography (PET). Alternatively, D is a drug to be targeted to the
PNOM-cells, as define above. In another embodiment of the
invention, there is provided a compound represented by the
structure set forth in formula (XIV):
##STR00014##
including pharmaceutically acceptable salts hydrates, solvates and
metal chelates of the compound represented by the structure as set
forth in formula (XIV) and solvates and hydrates of the salts;
wherein F may be .sup.18F or .sup.19F.
[0031] In another aspect of the invention, there is provided a
pharmaceutical composition for targeting of drugs to foci of
apoptosis or blood clotting in a patient, wherein the patient may
be a human or non-human mammal, wherein the pharmaceutical
composition comprising a compound according to the structure set
forth in formulae I, II, III, IV, V, VI, VII, VIII, IX, X, XI XII,
XIII, or XIV wherein the compound comprises or is being linked to a
drug.
[0032] In an aspect of the invention, there is provided a method of
selectively targeting a medicinally-useful compound to PNOM-cells
being within a population of cells, the method comprising:
contacting the cell population with a compound represented by the
structure set forth in any one of formulae I, II, III, IV, V, VI,
VII, VIII, IX, X, XI, XII, XIII, or XIV, thereby selectively
targeting the medicinally-useful compound to the PNOM-cells within
the cell population.
[0033] In another aspect of the invention, there is provided a
method of detecting a PNOM-cell within a cell population, the
method comprising: (i). contacting the cell population with
compound represented by the structure set forth in any one of
formulae I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII, XIII, or
XIV, or pharmaceutically acceptable salts, metal chelates, solvates
and hydrates of the compound represented by the structure as set
forth in any one formulae I, II, III, IV, V, VI, VII, VIII, IX, X,
XI, XII, XIII, or XIV, and solvates and hydrates of the salts; and
(ii). determining the amount of the compound bound to the cells,
wherein a significant amount of the compound bound to a cell
indicates that the cell is being a PNOM-cell.
[0034] In another aspect of the invention, there is provided a
method for detecting of PNOM-cells in a patient or an animal, the
method comprising: (i). administering to the patient or animal a
compound represented by the structure set forth in formulae I, II,
III, IV, V, VI, VII, VIII, IX, X, XI, XII, XIII, or XIV, or
pharmaceutically acceptable salts, metal chelates, solvates and
hydrates of the compound represented by the structure as set forth
in formulae I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII, XIII,
or XIV, and solvates and hydrates of the salts; and (ii) imaging
the examined patient or animal, so as determine the amount of the
compound bound to cells, wherein a significant amount of compound
bound to a cell indicates that the cell is a PNOM-cell.
[0035] In another aspect of the invention, there is provided a
pharmaceutical composition for targeting of drugs to foci of
apoptosis or foci or activated platelets in a blood clot in a
patient or an animal, the pharmaceutical composition comprising a
compound according to the structure set forth in formulae I, II,
III, IV, V, VI, VII, VIII, IX, X, XI, XII, XIII, or XIV, wherein
the compound comprises or is being linked to a drug.
In another embodiment, the invention provides a method of detecting
cells undergoing a death process within a tumor in an examined
subject, the method comprising: (i) administering to the examined
subject a compound or a conjugate comprising the compound wherein
said compound is represented by the structure set forth in
formula
(I):
##STR00015##
[0036] or pharmaceutically acceptable salts, metal chelates,
solvates and hydrates of the compound represented by the structure
as set forth in formula (I), and solvates and hydrates of the
salts; wherein, one of R or R' groups is hydrogen, and the other of
R or R' group represents C.sub.1, C.sub.2, C.sub.3, C.sub.4,
C.sub.5, C.sub.6, C.sub.7, C.sub.8, C.sub.9, C.sub.10, C.sub.11,
C.sub.12, C.sub.13, C.sub.14, C.sub.15, C.sub.16, linear or
branched alkyl, linear or branched hydroxy-alkyl, linear or
branched fluoro-alkyl, aryl or hetroaryl composed of one or two
rings, or combinations thereof; n and m each stands for an integer
of 0, 1, 2, 3 or 4; n and m may be same or different; M is selected
from null, hydrogen, --O--, --S--, and --N(U), wherein U stands for
hydrogen, or C.sub.1, C.sub.2, C.sub.3, or C.sub.4 alkyl; x, and z
each stands independently and is an integer of 0, 1 or 2, where x
and z can be the same or different; y is an integer of 0, 1 or 2,
where when y=2 the substituent R' may be the same or different at
each occurrence; and D is a marker for diagnostics. The marker for
diagnostics may be in an embodiment of the invention a marker for
imaging such as F, wherein the F may be .sup.18F or .sup.19F or a
labeled metal chelate; the marker for imaging being selected from
the group comprising a fluorescent label, a radio-label, a marker
for X-ray, a marker for MRI, a marker for PET scan and a label
capable of undergoing an enzymatic reaction that produces a
detectable color; and (ii) determining the amount of the compound
bound to the examined tumor of the patient, wherein detection of a
significant amount of the compound bound to cells in the tumor
indicates that these tumor cells are undergoing a death
process.
[0037] In another embodiment, the invention provides a method of
detecting cells undergoing a death process within a tumor in an
examined subject, the method comprising:(i) administering to the
examined subject a compound according to the structure set forth in
formulae I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII, XIII, or
XIV, wherein the compound comprises or is linked to a marker for
imaging or a labeled metal chelate; and (ii) determining the amount
of the compound bound to cells within the tumor, wherein detection
of a significant amount of the compound bound to cells in the tumor
indicates that these tumor cells are undergoing a death
process.
[0038] In another embodiment, there is provided a method of
targeting anticancer drugs to a tumor which has foci of apoptotic
cells, the method comprising the step of administering a compound
as set forth in any of the formulae I, II, III, IV, V, VI, VII,
VIII, IX, X, XI, XII, XIII, or XIV, which either comprises a
cytotoxic drug or is being linked to a cytotoxic drug, thereby
achieving targeting of the drug to the foci of cell death within
the tumor.
[0039] In another embodiment, there is provided a method of
targeting an anticoagulant or a fibrinolytic agent to a blood clot,
comprising the step of administering a compound as set forth in any
of the formulae I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII,
XIII, or XIV, which either comprises the anticoagulant or
fibrinolytic agent, thereby achieving targeting of the drugs to a
blood clot.
BRIEF DESCRIPTION OF THE DRAWINGS
[0040] FIG. 1 demonstrates a scheme of the mechanism of action of
the compounds of the invention: the NST-ML-Action Motif.
[0041] FIG. 2 (A and B) demonstrates selective binding of
tritium-labeled NST200 to cultured Jurkat cells, undergoing
apoptosis induced by CD95 (A) and selective binding of
tritium-labeled NST205 to cultured Jurkat cells, undergoing
apoptosis induced by CD95 (B).
[0042] FIG. 3 (A and B) shows fluorescent microscopy, demonstrating
the selective binding of NST203 to cultured HeLa cells undergoing
apoptosis (FIG. 3A). The control compound, n-butyl-dansylamide
(BDA), having the same fluorophore but devoid of the NST-ML-Action
Motif, did not manifest this selectivity (FIG. 3B).
[0043] FIG. 4 (A and B) shows fluorescent microscopy of the
selective binding of NST203 to cells undergoing cell death induced
by chemotherapy in mice in vivo. (A.). Apoptosis of melanoma cells;
(B). Apoptosis of epithelial cells of the gastrointestinal
tract.
[0044] FIG. 5 A-C shows effect of chemotherapy on carcinoma cell
death as detected by tritium-labeled NST200.
[0045] FIG. 6 shows autoradiography by NST 200 in chemotherapy
treated mice with colon carcinoma.
[0046] FIG. 7 demonstrates a table showing the ratio between the
uptake in carcinoma tumor and other body organ before and after
chemotherapy.
[0047] FIG. 8 (A and B) demonstrates uptake of tritium-labeled
NST200 into colon carcinoma and the effect of chemotherapy on the
same (A). FIG. 8B demonstrates changes in the colon weight.
[0048] FIG. 9 (A and B) demonstrates autoradiographic image
analysis showing targeting of tritium-labeled NST200 to region of
apoptotic death in the brain (A) and H&E staining (B).
[0049] FIG. 10 (A and B) demonstrates autoradiography by
tritium-labeled NST200 of rat renal ischemia reperfusion; (A)
damaged kidney; (B) intact kidney.
[0050] FIG. 11 (A and B) demonstrates autoradiography by
tritium-labeled NST205 in a rat model of radiocontrast-induced
acute distal tubular necrosis; (A) damaged kidney; (B) intact
kidney.
[0051] FIG. 12 shows .sup.3H-200 imaging by autoradiography in
brain and spinal cord of Experimental Autoimmune
Encephalomyelitis.
DETAILED EMBODIMENTS OF THE INVENTION
[0052] The present invention is related to compounds, capable of
performing selective binding to cells undergoing perturbation of
their normal organization of their plasma membrane (PNOM-cells),
while binding to a lesser degree to cells maintaining the normal
organization of their plasma membrane. The PNOM-cells are selected
from cells undergoing a death process, apoptotic cells and
activated platelets. The invention further relates to methods of
detecting PNOM-cells by using compounds, which selectively bind to
the PNOM-cells.
[0053] The compounds of the invention have the advantage of being
active in performing selective targeting of PNOM-cells, while also
featuring a relatively low molecular weight, and a potentially
favorable pharmacokinetic profile.
In one embodiment of the invention, there is provided a compound
which selectively targets to a PNOM cell (i.e., a PMBC) wherein the
compound is represented by the structure set forth in formula
(I):
##STR00016##
[0055] or pharmaceutically acceptable salts, metal chelates,
solvates and hydrates of the compound represented by the structure
as set forth in formula (I), and solvates and hydrates of the
salts; wherein, one of R or R' groups is hydrogen, and the other of
R or R' group represents C.sub.1, C.sub.2, C.sub.3, C.sub.4,
C.sub.5, C.sub.6, C.sub.7, C.sub.8, C.sub.9, C.sub.10, C.sub.11,
C.sub.12, C.sub.13, C.sub.14, C.sub.15, C.sub.16, linear or
branched alkyl, linear or branched hydroxy-alkyl, linear or
branched fluoro-alkyl, aryl or hetroaryl composed of one or two
rings, or combinations thereof; n and m each stands for an integer
of 0, 1, 2, 3 or 4; n and m may be same or different; M is selected
from null, hydrogen, --O--, --S--, and --N(U), wherein U stands for
hydrogen, or C.sub.1, C.sub.2, C.sub.3, or C.sub.4 alkyl; x, and z
each stands independently and is an integer of 0, 1 or 2, where x
and z can be the same or different; y is an integer of 0, 1 or 2,
where when y=2 the substituent R' may be the same or different at
each occurrence; and D is a marker for diagnostics, which in one
embodiment of the invention may be a marker for imaging such as F,
wherein the F may be .sup.18F or .sup.19F or a labeled metal
chelate; the marker for imaging may be detected by color,
fluorescence, x-ray, CT scan, magnetic resonance imaging (MRI) or
radio-isotope scan such as single photon emission tomography
(SPECT) or positron emission tomography (PET). In another
embodiment, D is a drug to be targeted to the PNOM cells.
[0056] The drug may be a medicinally-useful agent for the
prevention, amelioration, or treatment of a specific disease and
may be, for example, without being limited: an inhibitor of
apoptosis, (e.g., a caspase inhibitor, antioxidant, modulator of
the Bcl-2 system); an activator of cell death (e.g. an anticancer
drug); or a modulator of blood coagulation, which may be an
anticoagulant, an antithrombotic, or a thrombolytic agent. In such
case, the drug is preferably selected among an antiplatelet agent,
heparin, low molecular weight heparin, antagonists of glycoprotein
IIb/IIIa, tissue plasminogen activator (tPA), or an inhibitor of a
clotting factor, such as an inhibitor of thrombin or an inhibitor
of factor Xa; or an anti-inflammatory drug or an immuno-modulatory
drug. In an embodiment of the invention, there is provided a method
of targeting the drug to the area of interest, such as a focus of
apoptosis in tumor, in order to achieve killing of the tumor cells.
In another embodiment of the invention, there is provided a method
of treating thrombosis by targeting an anticoagulant or a
fibrinolytic agent to the thrombotic area, so as to prevent, reduce
or cease coagulation.
[0057] In another embodiment of the invention, D may be a solid
support.
[0058] In another embodiment of the invention, there is provided a
compound which selectively targets PNOM cells, the compound being
represented by the structure as set forth in formula (II):
##STR00017##
[0059] including pharmaceutically acceptable salts hydrates,
solvates and metal chelates of the compound represented by the
structure as set forth in formula (II) and solvates and hydrates of
the salts; wherein R.sup.1 represents hydrogen or C.sub.1, C.sub.2,
C.sub.3, C.sub.4, C.sub.5, C.sub.6, C.sub.7, C.sub.8, C.sub.9,
C.sub.10, C.sub.11, C.sub.12, C.sub.13, C.sub.14, C.sub.15,
C.sub.16, linear or branched alkyl, linear or branched
hydroxy-alkyl, linear or branched fluoro-alkyl, aryl or hetroaryl
composed of one or two rings, or combinations thereof; n and m each
stands for an integer of 0, 1, 2, 3 or 4; n and m may be same or
different; M is selected from null, hydrogen, --O--, --S--, and
--N(U), wherein U stands for a null, hydrogen, C.sub.1, C.sub.2,
C.sub.3, or C.sub.4 alkyl; D is hydrogen or a marker for
diagnostics, selected from a marker for imaging such .sup.18F, or a
labeled metal chelate; the marker for imaging may be detected by
color, fluorescence, x-ray, CT scan, magnetic resonance imaging
(MRI) or radio-isotope scan such as single photon emission
tomography (SPECT) or positron emission tomography (PET).
Alternatively, D is a drug to be targeted to the PNOM cells, as
defined above.
[0060] In another embodiment of the invention there is provided a
compound represented by the structure as set forth in formula
(III):
##STR00018##
including pharmaceutically acceptable salts hydrates, solvates and
metal chelates of the compound represented by the structure as set
forth in formula (III) and solvates and hydrates of the salts;
wherein R.sup.3 is hydroxyl or F; R.sup.4 is C.sub.4, C.sub.5,
C.sub.6, C.sub.7, C.sub.8, C.sub.9 or C.sub.10 linear or branched
alkyl, and k is an integer selected from 0, 1, 2, 3, 4 and 5.
[0061] In another embodiment of the invention there is provided a
compound represented by the structure as set forth in formula
(IV):
##STR00019##
[0062] including pharmaceutically acceptable salts hydrates,
solvates and metal chelates of the compound represented by the
structure as set forth in formula (IV) and solvates and hydrates of
the salts; the compound is designated NST200.
[0063] In another embodiment of the invention there is provided a
compound represented by the structure as set forth in formula
(V):
##STR00020##
including pharmaceutically acceptable salts hydrates, solvates and
metal chelates of the compound represented by the structure as set
forth in formula (V) and solvates and hydrates of the salts;
wherein J is --F and --OH, and r stands for an integer of 4, 5, 6,
7, 8, 9, 10. In the case that r is 4 and J is --F, the compound is
designated NST201. In the case that r is 5 and J is --F, the
compound is designated NST-ML-10.
[0064] In yet another embodiment of the invention there is provided
a compound represented by the structure as set forth in formula
(VI):
##STR00021##
including pharmaceutically acceptable salts hydrates, solvates and
metal chelates of the compound represented by the structure as set
forth in formula (VI) and solvates and hydrates of the salts;
wherein J is selected from hydrogen, --F and --OH. In the case that
J is --F, the compound is designated NST205.
[0065] In another embodiment of the invention there is provided a
compound represented by the structure set forth in formula VII:
##STR00022##
including pharmaceutically acceptable salts hydrates, solvates and
metal chelates of the compound represented by the structure as set
forth in formula (VII) and solvates and hydrates of the salts,
wherein Q is selected from Technetium, oxo-Technetium, Rhenium and
oxo-Rhenium, R.sup.4 is selected from hydrogen, C.sub.1, C.sub.2,
C.sub.3, C.sub.4, C.sub.5, and C.sub.6 linear or branched alkyl,
and p stands for an integer, selected from 1, 2, 3, 4 and 5.
[0066] In another embodiment of the invention there is provided a
compound represented by the structure set forth in formula
VIII:
##STR00023##
including pharmaceutically acceptable salts hydrates, solvates and
metal chelates of the compound represented by the structure as set
forth in formula (VIII) and solvates and hydrates of the salts,
wherein Q is selected from technetium, oxo-technetium, rhenium and
oxo-rhenium. In another embodiment of the invention there is
provided a compound represented by the structure as set forth in
formula (IX):
##STR00024##
including pharmaceutically acceptable salts hydrates, solvates and
metal chelates of the compound represented by the structure as set
forth in formula (IX) and solvates and hydrates of the salts;
wherein R.sup.5 is selected from hydrogen, C.sub.1, C.sub.2,
C.sub.3, C.sub.4, C.sub.5, and C.sub.6 linear or branched alkyl,
C.sub.1, C.sub.2, C.sub.3, C.sub.4, C.sub.5, and C.sub.6 linear or
branched fluoro-alkyl, and C.sub.1, C.sub.2, C.sub.3, C.sub.4,
C.sub.5, and C.sub.6 linear or branched hydroxy-alkyl; q stands for
an integer, selected from 1, 2, 3, 4 and 5; and Y is a marker for
fluorescence. In an embodiment of the invention, Y is selected from
a dansyl-amide group and fluorescein.
[0067] In another embodiment of the invention there is provided a
compound represented by the structure as set forth in formula
(X):
##STR00025##
including pharmaceutically acceptable salts hydrates, solvates and
metal chelates of the compound represented by the structure as set
forth in formula (X) and solvates and hydrates of the salts; The
compound is designated NST203.
[0068] In another embodiment of the invention there is provided a
compound represented by the structure as set forth in formula
(XI):
##STR00026##
including pharmaceutically acceptable salts hydrates, solvates and
metal chelates of the compound represented by the structure as set
forth in formula (X) and solvates and hydrates of the salts;
wherein R.sup.1 represents hydrogen or C.sub.1, C.sub.2, C.sub.3,
C.sub.4, C.sub.5, C.sub.6, C.sub.7, C.sub.8, C.sub.9, C.sub.10,
C.sub.11, C.sub.12, C.sub.13, C.sub.14, C.sub.15, C.sub.16, linear
or branched alkyl, linear or branched hydroxy-alkyl, linear or
branched fluoro-alkyl, aryl or hetroaryl composed of one or two
rings, or combinations thereof.
[0069] In another embodiment of the invention, there is provided a
compound represented by the structure set forth in formula
(XII):
##STR00027##
including pharmaceutically acceptable salts hydrates, solvates and
metal chelates of the compound represented by the structure as set
forth in formula (XII) and solvates and hydrates of the salts;
wherein F may be .sup.18F or .sup.19F.
[0070] In another embodiment of the invention there is provided a
compound represented by the structure as set forth in formula
(XIII):
##STR00028##
including pharmaceutically acceptable salts hydrates, solvates and
metal chelates of the compound represented by the structure as set
forth in formula (XIII) and solvates and hydrates of the salts;
R.sup.1 represents hydrogen or C.sub.1, C.sub.2, C.sub.3, C.sub.4,
C.sub.5, C.sub.6, C.sub.7, C.sub.8, C.sub.9, C.sub.10, C.sub.11,
C.sub.12, C.sub.13, C.sub.14, C.sub.15, C.sub.16, linear or
branched alkyl, linear or branched hydroxy-alkyl, linear or
branched fluoro-alkyl, aryl or hetroaryl composed of one or two
rings, or combinations thereof; m stands for an integer of 0, 1, 2,
3 or 4; D is hydrogen or a marker for diagnostics, which may be in
an embodiment of the invention a marker for imaging such as F,
wherein the F may be .sup.18F or .sup.19F or a labeled metal
chelate; the marker for imaging may be detected by color,
fluorescence, x-ray, CT scan, magnetic resonance imaging (MRI) or
radio-isotope scan such as single photon emission tomography
(SPECT) or positron emission tomography (PET). Alternatively, D is
a drug to be targeted to the PNOM-cells, as define above. In
another embodiment of the invention, there is provided a compound
represented by the structure set forth in formula (XIV):
##STR00029##
including pharmaceutically acceptable salts hydrates, solvates and
metal chelates of the compound represented by the structure as set
forth in formula (XIV) and solvates and hydrates of the salts;
wherein F may be .sup.18F or .sup.19F.
[0071] In another embodiment of the invention each of the compounds
represented by formulae I, II, III, IV, V, VI, VII, VIII, IX, X,
XI, XII, XIII, or XIV, may comprise or may be linked to a marker
for diagnostics such as for example without being limited Tc,
Tc.dbd.O, In, Cu, Ga, Xe, Tl, Re and Re.dbd.O, .sup.123I,
.sup.131I, Gd(III), Fe(III), Fe.sub.2O.sub.3, Fe.sub.3O.sub.4,
Mn(II) .sup.18F, .sup.15O, .sup.18O, .sup.11C, .sup.13C, .sup.124I,
.sup.13N, .sup.75Br, Tc-99m or In-111.
[0072] In another aspect of the invention, there is provided a
method of detecting a PNOM-cell within a cell population, the
method comprising: (i). contacting the cell population with a
compound represented by any one of the structure set forth in
formulae I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII, XIII, or
XIV, or pharmaceutically acceptable salts, metal chelates, solvates
and hydrates of the compound represented by the structure as set
forth in formulae I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII,
XIII, or XIV, and solvates and hydrates of the salts; and (ii).
determining the amount of the compound bound to the cells, wherein
a significant amount of compound bound to a cell indicates its
being a PNOM-cell.
[0073] The term "significant amount of the compound bound to a
cell" refers according to the invention to the amount of the
compound of the invention, comprising or is being attached to a
marker for diagnostics, which binds to a PNOM-cell in an amount
which is at least 30% greater than the amount bound to a normal
cell. In another embodiment, the amount may be higher by 50%. In
another embodiment of the invention, the amount may be higher by
75%. In another embodiment, the amount may be higher by 150%. In
another embodiment the amount may be higher by about two fold. In
another embodiment the amount may be higher than at least two fold.
In another embodiment, the amount may be higher than at least five
fold. In another embodiment, the amount may be higher by at least
ten fold.
[0074] In an embodiment of the invention, relating to use of the
compounds of the invention for obtaining images of cells undergoing
a death process in a patient via radio-nuclide imaging by PET or
SPECT, the calculation of the ratio between the amount of the
compound bound to the PNOM-cells vs. the amount bound to normal
cells may be conducted by comparing the amplitude or intensity of
the signal obtained from the tissue inflicted by the death process,
with the amplitude/intensity obtained from an organ not-inflicted
by the process.
[0075] According to another aspect of the invention, there is
provided a method for detecting of PNOM-cells in a patient or an
animal, the method comprising: (i) administering to the patient or
animal a compound represented by the structure set forth in
formulae I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII, XIII, or
XIV, wherein the compound comprises a marker for imaging, such as
.sup.18F or pharmaceutically acceptable salts, metal chelates,
solvates and hydrates of the compound represented by the structure
as set forth in formulae I, II, III, IV, V, VI, VII, VIII, IX, X,
XI, XII, XIII, or XIV, and solvates and hydrates of the salts; and
(ii) imaging the examined patient or animal, so as to determine the
amount of compound bound to cells, wherein detection of a
significant amount of compound bound to cells indicates that these
cells are PNOM-cells.
[0076] The mechanism of action of the compounds of the invention
comprises, at least in part, the activity of a module shared by all
the compounds, having the general formula XV, and designated
NST-ML-Action Motif:
##STR00030##
wherein R stands for an alkyl. In an embodiment of the invention, R
is butyl.
[0077] The NST-ML-Action Motif is designed to correspond to the
structural alterations encountered in the plasma membranes of
apoptotic cells, which distinguish these membranes from the
membranes of healthy cells. This complex of membrane alterations
comprises: [0078] (i). Scrambling of membrane phospholipids, with
exposure on the cell surface of phosphatidylethanolamine (PE) and
the negatively-charged phosphatidylserine (PS). Exposure of PS on
the cell surface leads to a negative surface electric potential,
and attraction of protons form the bulk to the membrane interface.
[0079] (ii). The increase in the fraction of aminophospholipids (PE
and PS) within the outer leaflet of the membrane results in an
enhancement of the proton currents in the interface of the outer
leaflet of the membrane (interfacial proton currents, IPC). This
enhancement is due to the substantial increase in the number of
functionalities amenable to participation in proton transfer
reactions, as PE and PS replace phosphatidylcholine (PC) and
sphingomeylin (SM) in the outer membrane leaflet. PE comprises a
primary amine, while PS comprises a primary amine and a carboxyl
group. By contrast, PC and SM comprise each a quaternary ammonium,
that bears a permanent positive charge, and thus cannot participate
in proton transfer reactions. [0080] (iii). In addition, apoptotic
membranes are characterized by reduced level of packing of the
membrane constituents and increased membrane fluidity.
[0081] In a non limiting hypothesis of the mode of action of the
NST-ML-Action Motif, it comprises a switch moeity, activated
selectively upon its approaching a membrane which features the
above characteristics, i.e., the plasma membrane of an apoptotic
cell (FIG. 1). The Action Motif is soluble in physiological pH, due
to its having two negatively-charged carboxylate groups (pKa of
alkylmalonate is about 5.6 and 2.8), thus having mostly a formal
charge of -2 in physiological conditions. However, upon approaching
the apoptotic membrane, due to the more acidic surface, and due to
the reduction in the dielectric constant of the interfacial
environment, which acts to elevate pKa values of the carboxyl
groups, a proton is being captured by the malonate moiety.
[0082] The capture of the proton by the malonate group neutralizes
one of the negative charges, thus rendering the molecule more
hydrophobic, with an overall charge of -1. Moreover, the capture of
the proton further leads to a very unique situation, which includes
the following: [0083] (i). An acid-anion pair is formed, wherein an
exceptionally strong hydrogen bond is formed between the protonated
and unprotonated carboxyl groups. This hydrogen bond is strong,
symmetrical and stabilized by resonance and tautomerization. [0084]
(ii). This leads to distribution of the negative charge over the
four carboxyl atoms, i.e., its being partially delocalized. [0085]
(iii). The strong acid-anion hydrogen bond rigidifies the molecule,
creating a bulky, rigid, flat, six-membered ring, bearing a
partially-delocalized negative charge, and comprising pi-electron
clouds over the carboxyl double bonds. Such an element can undergo
a relatively favorable penetration into the membrane interface,
according to a non-limiting hypothesis of the mechanism of action
of the compounds of the invention. However, its bulky, rigid
structure directs its binding selectively to loosely packed
membranes, i.e., apoptotic membranes, while precluding binding to
highly-packed membranes, such as the plasma membranes of healthy
cells. These steric features therefore promote selectivity in
binding to the apoptotic membranes.
[0086] Upon the selective penetration of the single-protonated
malonate into the membrane interface of the apoptotic cell, it
becomes subjected to the enhanced interfacial proton currents, and
becomes integrated within the enhanced interfacial network of
hydrogen bonds. The probability for a second proton to be acquired
by the malonate moiety under these conditions is markedly
increased. This will further lead to neutralization of charge and
formation of further acid-anion pairs with adjacent phospholipid
molecules. Taken together, these events will act to stabilize the
binding of the molecule to the interface of the apoptotic
membrane.
[0087] The penetration of the protonated malonate moiety into the
membrane interface and the stabilization of its binding in the
interface, allow the alkyl chain R to traverse the membrane
interface and to reach its optimal binding environment, i.e., the
membrane hydrocarbon core, whereupon it will further contribute
through hydrophobic interactions to the free energy gain of
compound binding.
[0088] The NST-ML-Action Motif is being utilized for useful
diagnostic or therapeutic purposes, through its binding to a marker
for imaging or a therapeutic drug (moiety D in Formula I) through a
hydrocarbon linker [(CH.sub.2).sub.m of Formulae I or 2]. The
NST-ML-Action Motif according to this approach acts as a targeting
moiety, allowing selective targeting of the marker for imaging or
the drug attached to it to cells and tissues inflicted by cell
death, particularly apoptosis, or tissues inflicted by platelet
activation and thrombosis.
[0089] FIG. 1 demonstrates NST200 (Formula IV), and describes the
three stages of its approach and binding to the PNOM membrane in
physiological conditions: [0090] A: The compound is in an aqueous
solution, thus both carboxyl groups are deprotonated, i.e.,
negatively charged, and the compound is highly soluble. [0091] B:
Upon approaching the negatively-charged apoptotic membrane, the
compound acquires a proton. An anion-acid dimer is formed, thus
creating a stable six-membered, resonance-stabilized ring, which
penetrates the membrane interface. The bulky, rigid ring structure
assists in selectivity, since its steric features favor binding to
the more loosely-packed plasma membrane of the apoptotic cell,
rather than binding to the well-packed plasma membrane of the
healthy cell. [0092] C: Upon penetration of the compound into the
membrane interface, it is subjected to the interfacial network of
hydrogen bonds, and to the augmented interfacial proton currents
encountered in the interface of the apoptotic membrane. The
resultant protonation and hydrogen bonding further acts to
stabilize the binding of the compound to the interface (arrows).
Such penetration further allows the alkyl chain to reach its
optimal position within the membrane, thus further contributing to
the binding energy, through formation of hydrophobic interactions
with the membrane hydrocarbon core.
[0093] The compounds of the invention may be used for selective
targeting of medicinally-useful agents to tissues and organs
comprising PNOM-cells, in three different approaches of the
invention: [0094] (i). According to a first approach, termed
hereinafter the "detection approach", the selective binding may be
utilized to targeting a marker for imaging to PNOM-cells. This may
be used in clinical practice, either in vivo, ex vivo or in vitro,
for the diagnosis of diseases in which such cells emerge as will be
explained herein below. [0095] (ii). According to a second
approach, termed hereinafter the "therapeutic approach", the
property of selective binding is used for selective targeting of
therapeutic agents to organs and tissues in the body wherein
PNOM-cells emerge, e.g., regions of cell death, thrombus formation
or inflammation. [0096] (iii). In accordance with a third approach
of the invention termed the "clearance approach", the selective
binding of the compounds of the invention to PNOM-cells is
utilized, via attachment of the compounds to a solid support, to
clear body fluids such as blood from PNOM-cells, which may be
potentially hazardous due to their pro-coagulant properties.
[0097] In accordance with the detection approach, the present
invention concerns a composition comprising a PMBC as an effective
ingredient, comprising or linked to a marker for imaging, for the
detection of PNOM-cells, either in vitro, ex vivo or in vivo. Such
a PMBC is hereinafter designated "diagnostic PMBC". The diagnostic
PMBC is capable of performing selective binding to PNOM-cells
present in the assayed sample. Then, the binding may be identified
by any means known in the art. The diagnostic PMBC of the invention
enables the targeting of the marker, by the PMBC, to PNOM-cells in
a selective manner. Then, the detectable label can be detected by
any manner known in the art, and in accordance with the specific
label used, for example, fluorescence, radioactive emission, or a
color production, MRI, x-ray and the like. In one embodiment, the
diagnostic PMBC is linked to the detectable label by a covalent or
a non-covalent (e.g., electrostatic) binding.
[0098] In an embodiment, the detectable label may be any of the
respective radio-isotopes of the metal ions Tc, oxo-Tc, In, Cu, Ga,
Xe, Tl and Re, oxo-Re and the covalently linked atoms: .sup.123I
and .sup.131I for radio-isotope scan such as SPECT; Gd(III),
Fe(III) or Mn(II) for MRI; and .sup.18F, .sup.15O, .sup.18O,
.sup.11C, .sup.13C, .sup.124I, .sup.13N and .sup.75Br for positron
emission tomography (PET) scan.
[0099] In an embodiment, the PMBC of the invention is aimed at
clinical imaging of apoptosis via PET scan, and the PMBC comprises
.sup.18F atom(s).
[0100] Due to the short half-life of certain radio-isotopes used as
markers for imaging, such as .sup.18F, the attachment of such
marker for the purposes of clinical PET imaging may be performed
immediately before the administration of the diagnostic compound to
the patient. Therefore, it may be useful to synthesize a PMBC-PET
precursor, comprising a moiety to be substituted by the
radio-isotope such as .sup.18F before administration to the
patient. In one embodiment, the moiety to be replaced by .sup.18F
is selected from a hydroxyl group, a nitro group, or a halogen atom
such as bromine or chlorine. Such a PMBC-precursor PMBC-PET
precursor is also included in the scope of the invention.
[0101] The method for labeling a PMBC, which can be any PMBC of the
structures described above, with .sup.18F for PET imaging,
comprises the step of attaching an .sup.18F atom to the PMBC;
thereby radio-labeling the PMBC with .sup.18F for PET imaging.
Optionally, the functional groups of the PMBC may be protected by
appropriate protecting groups prior to the step of attaching
.sup.18F atom. Said protecting groups are thereafter optionally
removed after the step of attachment of the .sup.18F atom.
[0102] In the case that the marker is a metal atom (e.g., Gd,
.sup.99mTc or oxo-.sup.99mTc for MRI or SPECT, respectively), the
PMBC comprises a metal chelator. The metal coordinating atoms of
the chelator may be nitrogen, sulfur or oxygen atoms. In an
embodiment of the invention, the chelator is diaminedithiol,
monoamine-monoamide-bisthiol (MAMA), triamide-monothiol, and
monoamine-diamide-monothiol. In such case, both a PMBC-chelate
precursor, being the PMBC attached to or comprising a chelator
prior to complexation with the metal atom, and the complex
comprising the metal atom, are included in the scope of the
invention.
[0103] For fluorescent detection, the diagnostic PMBC may comprise
a fluorescent group selected from any fluorescent probe known in
the art. Examples for such probes are 5-(dimethylamino)
naphthalene-1-sulfonylamide (dansyl-amide), and fluorescein.
[0104] The compounds of the invention may be used for the detection
and diagnosis of a wide variety of medical conditions,
characterized by formation PNOM-cells. Examples of clinical
conditions characterized by PNOM-cells are as follows:
[0105] Diseases which are characterized by occurrence of excessive
apoptosis, such as degenerative disorders, neurodegenerative
disorders (e.g., Parkinson's disease, Alzheimer's disease,
Huntington chorea), AIDS, ALS, Prion Diseases, myelodysplastic
syndromes, ischemic or toxic insults, graft cell loss during
transplant rejection; tumors, and especially highly
malignant/aggressive tumors, are also often characterized by
enhanced apoptosis in addition to the excessive tissue
proliferation.
[0106] Example 3 of the invention as well as FIG. 2, exemplify the
performance of a compound of the invention in detecting brain cells
undergoing a death process. The trigger for the cell death was
ischemia/reperfusion. The damaged brain hemisphere manifested
markedly higher levels of uptake of tritium-labeled NST200,
compared to the contralateral non-damaged hemisphere.
[0107] Diseases manifested by excessive blood clotting, wherein
PNOM occurs during platelet activation, and/or during activation of
or damage to other cellular elements (e.g., endothelial cells).
These diseases include, among others, arterial or venous
thrombosis, thrombo-embolism, e.g., myocardial infarction, cerebral
stroke, deep vein thrombosis, disseminated intravascular
coagulation (DIC), thrombotic thrombocytopenic purpura (TTP),
sickle cell diseases, thalassemia, antiphospholipid antibody
syndrome, systemic lupus erythematosus.
[0108] Inflammatory disorders, and/or diseases associated with
immune--mediated etiology or pathogenesis, auto-immune disorders
such as antiphospholipid antibody syndrome, systemic lupus
erythematosus, connective tissue disorders such as rheumatoid
arthritis, scleroderma; thyroiditis; dermatological disorders such
as pemphigus or erythema nodosum; autoimmune hematological
disorders; autoimmune neurological disorders such as myasthenia
gravis; multiple sclerosis; inflammatory bowel disorders such as
ulcerative colitis; vasculitis.
[0109] Atherosclerotic plaques, and especially plaques that are
unstable, vulnerable and prone to rupture, are also characterized
by PNOM-cells, such as apoptotic macrophages, apoptotic smooth
muscle cells, apoptotic endothelial cells, and activated platelets.
Such activated platelets are encountered in the thrombi, often
associated with the unstable atherosclerotic plaque.
[0110] The detection may also be carried out in a person already
known to have the respective disease, for the purpose of evaluating
disease severity and in order to monitor disease course and/or
response to various therapeutic modalities. A non-limited example
for such monitoring is evaluation of response to anticancer
therapy. Since most anti-tumor treatments, such as chemotherapy or
radiotherapy exert their effect by induction of apoptosis,
detection by a diagnostic PMBC of therapy-induced apoptosis of
tumor cells may teach on the extent of sensitivity of the tumor to
the anti-tumor agent. This may substantially shorten the lag period
between the time of administration of the anti-cancer treatment and
the time of proper assessment of its efficacy.
[0111] Moreover, the detection may be also used to monitor adverse
effects of anti-cancer treatments. A large part of such adverse
effects is due to untoward treatment-induced apoptosis in normal,
yet sensitive cells, such as those of the gastrointestinal
epithelium or the bone marrow hematopoietic system.
[0112] In addition, the detection may aim at characterization of
intrinsic apoptotic load within a tumor, often correlated with the
level of tumor aggressiveness; and may also assist in the detection
of metastases, via detection of the intrinsic apoptosis frequently
occurring within metastases.
[0113] Similarly, the diagnostic PMBC of the invention may be
useful in monitoring graft survival after organ transplantation,
since apoptosis plays a major role in cell loss during graft
rejection.
[0114] In addition, the detection may aim at monitoring response to
cyto-protective treatments, and thus aid in screening and
development of drugs which are capable of inhibiting cell loss in
various diseases (for example those recited above) by enabling a
measure of assessment of cell death.
[0115] The detection may also be useful for the detection of
atherosclerotic plaques, since destabilization of such plaques,
rendering them vulnerable, prone to rupture, thrombosis and
embolization, is characterized by participation of several types of
PNOM-cells, including apoptotic cells (apoptotic macrophages,
smooth muscle cells and endothelial cells), and activated
platelets.
[0116] In accordance with this approach, the present invention is
related to a method of detection of PNOM-cells in a cell
population, selected from whole body, organ, tissue, tissue culture
or any other cell population, the method comprising: (i).
contacting the cell population with a diagnostic PMBC according to
any of the embodiments of the invention; and (ii). determining the
amount of PMBC bound to the cell population, wherein detection of a
significant amount of compound bound to a cell within the
population indicates that the cell is a PNOM-cell.
[0117] The examples section show the ability of the ability of
tritium-labeled NST 200, NST 203 and NST 205 to bind to apoptotic
cells in higher amount than to control, non-apoptotic cells,
demonstrate that the property of the compounds of the invention, in
performing selective binding and detection of apoptotic cells.
[0118] In another embodiment, the present invention further relates
to a method for detecting PNOM-cells in a patient or in an animal
in vivo, the method comprising: (i). administering a diagnostic
PMBC to the examined patient or animal; the administration being
performed by any means known in the art, such as parenteral (e.g.,
intravenous) or oral administration; and (ii). imaging the examined
patient or animal, by any method known in of the art (e.g., PET
scan, SPECT, MRI), to detect and determine the amount of
diagnostic-PMBC bound to cells, wherein a significant amount of
compound bound to a cell indicates that the cell is a
PNOM-cell.
[0119] In another embodiment of the invention, the present
invention is related to a method for the detection of PNOM-cells in
a tissue or cell culture sample in vitro or ex-vivo, the method
comprising: (i). contacting the sample with a diagnostic PMBC,
which may be any of the PMBC compounds described in the invention,
under conditions enabling binding of the diagnostic PMBC to the
biological membranes of PNOM-cells; and (ii). detecting the amount
of diagnostic PMBC bound to the cells; the presence of a
significant amount of bound diagnostic compound indicating the
presence of PNOM-cells within the tissue or cell culture.
[0120] The step of detection in the in vitro or ex-vivo studies may
be, for example, in the case of fluorescent-labeled compound of the
invention, without limitation by using flow cytometric analysis,
which permits cell visualization on equipment that is widely
commercially available. In an example using fluorescence to
visualize cells, a single 15 mW argon ion laser beam (488 nm) is
used to excite the FITC fluorescence, and fluorescence data is
collected using 530 nm band pass filter to provide a histogram. The
percent of fluorescent cells can be calculated, for example using
Lysis II software or any other software. The method for detection
may be used in an embodiment of the invention for screening
therapeutic drugs such as anticancer drugs.
[0121] The term "significant amount" according to the invention,
means that the amount of PMBC bound to a PNOM-cell is at least 30%
higher than the amount bound to a non-PNOM-cell. The actual amount
may vary according to the imaging method and equipment utilized,
and according to the organs or tissues examined. In another
embodiment the amount of PMBC bound to a PNOM-cell is at least 50%
higher than the amount bound to a non-PNOM-cell. In another
embodiment the amount of PMBC bound to a PNOM-cell is at least 75%
higher than the amount bound to a non-PNOM-cell. In another
embodiment the amount of PMBC bound to a PNOM-cell is at least
twice times the amount bound to a non-PNOM-cell. In another
embodiment the amount of PMBC bound to a PNOM-cell is at least four
times the amount bound to a non-PNOM-cell. In another embodiment
the amount of PMBC bound to a PNOM-cell is at least six times the
amount bound to a non-PM-cell. In another embodiment the amount of
PMBC bound to a PNOM-cell is at least eight times the amount bound
to a non-PNOM-cell. In another embodiment the amount of PMBC bound
to a PNOM-cell is at least ten times the amount bound to a
non-PNOM-cell.
[0122] The action of the binding depends inter-alia on the method
of measuring the difference in binding. The method of the present
invention may be used for the diagnosis of a disease characterized
by the occurrence of PNOM-cells, for example, without being limited
to any of the diseases mentioned above.
[0123] The method of the present invention may also be used for
monitoring the effects of various therapeutic modalities used for
treatment of diseases or medical conditions, or alternatively for
basic science research purposes as explained above.
[0124] In accordance with a second approach of the invention,
termed "the therapeutic approach", the present invention concerns a
pharmaceutical composition comprising a PMBC, used for targeting an
active drug or a pro-drug to PNOM-cells. A therapeutic PMBC
according to the invention means a PMBC comprising a drug or a PMBC
being conjugated to a medicinally-useful agent. The term
"conjugate" means two molecules being linked together by any means
known in the art.
[0125] The association between the medicinally-useful drug and the
PMBC wherein it is comprised or linked to in the therapeutic PMBC
may be by covalent binding, by non-covalent binding (e.g.,
electrostatic forces) or by formation of carrier particles (such as
liposomes) comprising the drug and having on their surface a PMBC
which targets the complex to the PNOM-cells. Once the drug reaches
the target, it should be able to exert its physiological activity,
either when still being part of the PMBC-conjugate, after
disconnecting from the PMBC unit (for example by cleavage,
destruction, etc., activity of natural enzymes), by phagocytosis of
drug-containing liposomes having PMBC on their membrane, or by any
other known mechanism.
[0126] The drug should be chosen in accordance with the specific
disease for which the composition is intended.
[0127] The pharmaceutical composition, as well as the diagnostic
composition of the invention may be administered by any of the
known routes, inter alia, oral, intravenous, intraperitoneal,
intramuscular, subcutaneous, sublingual, intraocular, intranasal or
topical administration, or intracerebral administration. The
carrier should be selected in accordance with the desired mode of
administration, and include any known components, e.g. solvents;
emulgators, excipients, talc; flavors; colors, etc. The
pharmaceutical composition may comprise, if desired, also other
pharmaceutically-active compounds which are used to treat the
disease, eliminate side effects or augment the activity of the
active component.
[0128] In accordance with this aspect, the present invention still
further concerns a method for treating a disease manifesting
PNOM-cells, comprising administering to an individual in need of
such treatment an effective amount of a therapeutic PMBC, the
therapeutic PMBC comprising a drug being active as a treatment for
the disease or a pro-drug to be converted to an active drug in the
targeted area. The therapeutic PMBC allows for selective targeting
of the drug to the tissues comprising PNOM-cells, thus augmenting
its local concentration, and potentially enhancing its therapeutic
effect at the target site. Such medical disorders are those defined
above.
[0129] In another embodiment, there is provided a method of killing
cancer cells in a tumor, comprising the step of targeting apoptotic
cells within the tumor by administration of a therapeutic PMBC,
comprising any one of the compounds set forth in formulae I, II,
III, IV, V, VI, VII, VIII, IX, X, XI, XII, XIII, or XIV, and a
cytotoxic drug, thereby killing the cancer cells. In one
embodiment, the method of killing the tumor cells involves an
"autocatalytic mechanism", whereby the amount of cytotoxic agent
being targeted to the tumor increases with sequential doses, as
each dose, due to its cytotoxic effect, enhances the load of
apoptotic cells within the tumor, thus creating more sites for the
targeting of the next dose of the therapeutic PMBC. Such strategy
may enhance the efficacy of the anticancer treatment, and augment
the chances for tumor eradication.
[0130] The term "effective amount" of the therapeutic PMBC refers
to an amount capable of decreasing, to a measurable level, at least
one adverse manifestation of the disease, and should be chosen in
accordance with the drug used, the mode of administration, the age
and weight of the patient, the severity of the disease, etc.
[0131] In another embodiment, the therapeutic PMBC of the invention
comprises or is being linked to a radioisotope which has
therapeutic effect. An Example without limitation for such a
radio-isotope is Yittrium 90, Iodine 131, Rhenium 188, Holmium 166,
Indium 111, Leutitium 177, or any other radioisotopes emitting
radiation, which is useful for therapeutic purposes.
[0132] In another embodiment, there is provided a method for
reducing/preventing a blood clot, by administration of a
therapeutic PMBC, comprising any one of the compounds set forth in
formulae I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII, XIII, or
XIV, being linked to an anticoagulant or a fibrinolytic agent,
thereby targeting the therapeutic agent to the activated platelets
in the blood clot and reducing/preventing the thrombus
formation.
[0133] This method may be used also to treat or prevent diseases
manifested by excessive blood clotting, wherein PNOM occurs during
platelet activation, and/or during activation of or damage to other
cellular elements (e.g., endothelial cells). These diseases
include, among others, arterial or venous thrombosis,
thrombo-embolism, e.g., myocardial infarction, cerebral stroke,
deep vein thrombosis, disseminated intravascular coagulation (DIC),
thrombotic thrombocytopenic purpura (TTP), sickle cell diseases,
thalassemia, antiphospholipid antibody syndrome, systemic lupus
erythematosus.
[0134] According to a third approach of the invention, termed the
"clearance approach", the properties of the PMBCs of the invention
to bind specifically to PNOM-cells are utilized to clear body fluid
of the cells. In an embodiment of the invention, the body fluid is
blood or a blood product.
[0135] Many surgical or medical interventions requiring
extracorporeal circulation are associated with exposure of blood
elements to exogenous artificial environment. This often leads to
activation of and damage to blood cells, systemic inflammation, and
thromboembolic phenomena, potentially having serious clinical
consequences, such as neurological dysfunction upon lodging of
microemboli in the cerebral blood vessels. It is therefore
desirable to detect and remove the damaged, activated or apoptotic
cells from blood.
[0136] Thus, according to one of its aspects, the present invention
concerns a PMBC immobilized on a solid support. The immobilization
may be by direct attachment, either by covalent or non-covalent
binding, or by attachment through a spacer. The immobilized PMBC is
intended to clear a body fluid from PNOM-cells.
[0137] According to another embodiment of the present invention,
the solid support features a plurality of beads to which the PMBC
are bound. Preferably, the beads are resin-coated beads.
Alternatively, the beads may be magnetic beads.
[0138] Where the solid support includes a plurality of fibers or
micro-capillara, among and/or through which the body fluid flows,
the inner and/or outer faces thereof are covered with the PMBC.
[0139] The compounds immobilized on a solid support form part of a
filter device. Thus, in accordance with the clearance approach, the
present invention further concerns a filter device comprising a
housing containing the PMBC immobilized on the solid support, and a
fluid inlet and fluid outlet. Body fluids such as blood or blood
products enter the housing through the inlet, come into contact and
adhere to the immobilized PMBC contained in the housing. Thus, the
body fluid is cleared of circulating cells having perturbed
membranes, such as damaged or dying cells, or cleared of larger
structures such as emboli having PNOM membranes. Consequently,
fluid exiting from the outlet has a reduced content of the
PNOM-cells or is essentially devoid of same.
[0140] The filter device may form a replaceable, a permanent, or an
add-on portion of an extracorporeal circulation apparatus. Thus the
present invention also concerns an extracorporeal circulation
apparatus comprising the filter device, wherein blood circulating
through the apparatus also passes through the device.
[0141] Examples of such apparatuses are a cardiopulmonary bypass
apparatus; a hemodialysis apparatus; a plasmapheresis apparatus and
a blood transfusion apparatus, such as state of the art blood
transfusion bags.
EXAMPLES
[0142] In order to understand the invention and to see how it may
be carried-out in practice, the following examples are described:
examples directed to synthesis of the compounds of the invention;
and examples directed to the performance of the compounds of the
invention in selective binding to cells undergoing death process.
In order to allow detection of the compounds of the invention, they
were radio-labeled with tritium and detected by measuring uptake to
the damaged areas or by autoradiographic methods. In some of the
Examples, the compounds were labeled by attachment to a fluorescent
label, i.e., a dansylamide group, and detected by fluorescent
microscopy. The selectivity of binding of the compounds to the
apoptotic cells was demonstrated in vitro, in tissue cultures, and
in vivo, in a murine model of cerebral stroke, wherein cell death
was induced by occlusion of the middle cerebral artery, in murine
models of kidney ischemic and toxic insults, in a murine model of
melanoma, in a murine model of colon carcinoma, and in experimental
autoimmune encephalomyelitis (EAE), a murine model related to
multiple sclerosis.
Example I
Synthesis of NST-ML-F-4 (2-butyl-2(3-fluoropropyl)-malonic acid,
NST205); (Scheme 1)
[0143] Di-t-butyl malonate (5 mL) was deprotonated with 1 eq of NaH
in dimethyl formamide (DMF), and 1 eq of n-butyl iodide was added
after the hydrogen evolution ceased. The reaction mixture was
heated to 50.degree. C. for 14 hours. 5.8 g di-t-butyl, butyl
malonate (2) were obtained in a 95% yield by using column
chromatography. 2 (3.8 g) was treated with NaOCH.sub.3 (0.05 eq,
cat.) and acrolein (1.1 eq) in toluene to afford 1.26 g of aldehyde
(3) in a 30% yield. Compound 3 was then reacted with NaBH.sub.4
(1.05 eq) in a mixture of ether/water (8:1 v/v) for two hours.
After work-up and flash chromatography, pure alcohol 4 was obtained
(93%). The resulted product was treated with 1.1 eq of
methansulfonyl chloride (MsCl) and 2.2 eq of triethyl amine
(Et.sub.3N) so as to obtain mesylate compound 5 in 97% yield. This
product was essentially pure and directly carried over to the next
reaction with no further purification.
[0144] A mixture of KF (5 eq), kryptofix (5 eq) and K.sub.2CO.sub.3
(2.5 eq) in 2 mL of acetonitrile was stripped to dryness under a
stream of nitrogen for 4 times. The mesylate compound 5 (167 mg) in
2 mL of acetonitrile was then added. The reaction was stirred in a
120.degree. C. sand bath for 10 min. Upon work-up, .sup.1H NMR of
the crude product showed a mixture of the desired product 6 and
kryptofix. Deprotection of the di-t-butyl ester 6 was performed
with (474 mg) and trifluoroacetic acid (TFA) (17 mL) at 10.degree.
C. for 30 min, and then evaporated to dryness. The residual
material was evaporated twice from chloroform and dried on the
vacuum line to afford a white solid (312 mg, 99%) of NST-ML-F-4.
NMR data of the compound are: .sup.1H NMR (300 MHz, CDCl.sub.3)
.delta. 4.41 (dt, J.sub.t=5.9 Hz, J.sub.d=47.4 Hz, 2H), 1.97-1.91
(m, 2H), 1.89-1.83 (m, 2H), 1.69-1.60 (m, 1H), 1.58-1.52 (m, 1H),
1.33 (p, J=6.9 Hz, 2H), 1.25-1.14 (m, 2H), 0.92 (t, J=7.2 Hz, 3H);
.sup.13C NMR (75 MHz, CD.sub.3OD) .delta. 175.8, 86.2, 84.1, 58.6,
34.1, 30.1, 30.0, 27.9, 27.3, 27.0, 24.5, 14.6; .sup.19F NMR (282
MHz, CD.sub.3OD) .delta. -220.9; MS (EI) m/z 219 (M-H).
##STR00031## ##STR00032##
Example 2
Synthesis of NST-ML-F: 2-methyl-2(3-fluorobutyl)-malonic acid;
NST201 (Scheme 2)
[0145] 4-bromo-1-butanol (1), 3 g, was treated with 1.5 eq of
3,4-dihydro-2H-pyran and 0.1 eq of pyridinium para tuloenesulfonate
(PPTS) in 135 mL of CH.sub.2Cl.sub.2. After work-up and
purification, 1.45 g (33%) of product 2 was obtained. 1.0 eq of
diethylmethylmalonate was deprotonated with 1 eq of NaH and 1.0 eq
of bromide 2 was added along with catalytic amount of KI at
50.degree. C. A complete conversion was observed after 10 hours and
a 90% yield was obtained. Deprotection of tetrahydro pyran (THP)
with PPTS in ethanol at 55.degree. C. went smoothly. After work-up,
a quantitative yield of alcohol 4 was obtained and directly used
for the mesylation reaction (as above). With the mesylate 5 in
hand, the kryptofix reaction was applied as above. Compound 6 was
obtained in 68% yield. Compound 6 (233 mg) was treated with 2 N
NaOH/EtOH (30 mL/5 mL) at 50.degree. C. to provide NST-ML-F ca. in
99% yield (190 mg).
[0146] The NMR data of the compound are as follows: .sup.1H NMR
(300 MHz, CDCl.sub.3) .delta. 11.89 (bs, 2H), 4.46 (dt, J.sub.t=5.9
Hz, J.sub.d=47.2 Hz, 2H), 1.99-1.92 (m, 2H), 1.82-1.64 (m, 2H),
1.50 (s, 3H), 1.50-1.40 (m, 2H); .sup.13C NMR (75 MHz, CDCl.sub.3)
.delta. 178.6, 85.1, 82.9, 54.2, 35.5, 31.0, 30.8, 20.8, 20.7,
20.2; .sup.19F NMR (282 MHz, CDCl.sub.3) .delta. -219.0; MS
(EI).
##STR00033##
Example 3
Selective Binding of Tritium-Labeled NST 200 and NST205 to Cultured
Jurkat Cells, Undergoing Apoptosis Induced by CD95
Experimental Procedure
[0147] Cultured Jurkat cells (human adult T cell leukemia cells)
were grown in suspension in RPMI medium (Beit-Haemek, Israel),
supplemented with 10% fetal calf serum (FCS), 2 mM of L-glutamine,
1 mM of sodium pyruvate, 1 mM HEPES and antibiotics (100 units/ml
penicilin; 100 .mu.g/ml streptomycin and 12.5 units/ml of
nystatin). Prior to induction of apoptosis, medium was replaced
with HBS buffer (10 mM HEPES; 140 mM NaCl, 1 mM CaCl). Apoptosis
was then triggered by treatment with CD95 (0.1 ug/10.sup.7
cells/ml). As a result, a marked percentage of the cells became
apoptotic. Non-treated cells served as control. Both control cells
and apoptotic cells were then incubated for 40 minutes at room
temperature followed by 30 minutes on ice with (2 .mu.Ci/10.sup.7
cells/0.5 ml) tritium-labeled NST 200 and NST 205.
[0148] After washing the cells twice, 1 ml of SOLVABLE.TM. reagent
(GNE9100, Packard Biosciences) was added to the pellet. Following
one hour of incubation at 60.degree. C., the extracts were
transferred into glass scintillation vials and 10 ml of
scintillation liquid (Ultima gold 6013329, Packard Biosciences) was
added to each vial. The radioactivity was counted after 1 hour of
cooling to room temperature and dark adaptation. The radioactive
values were calculated and presented in percents of total added
radiolabeled NST 200 and NST 205
Experimental Results
[0149] As can be clearly seen from FIG. 2A, the apoptotic cells
showed markedly higher uptake of NST200 (over 8 fold) compared to
the non-apoptotic cells. The experiment show that NST200 is capable
of selective binding to apoptotic cells and can serve as a marker
for the detection thereof. Similarly, as can be seen from FIG. 2B,
the apoptotic cells showed higher uptake of NST205 in comparison to
the non apoptotic cells, and this effect was completely reversed
while adding ZVAD (fluoromethyl ketone peptid (V-valine, A-alanine,
D-aspartate) inhibitor of caspase), which is a caspase
inhibitor.
Example 4
Selective Binding of NST 203 to Cultured HeLa Cells Undergoing
Apoptosis
[0150] HeLa S3 cells (ATCC CCL-2.2) were grown in Dulbecco's
modified Eagle's medium (DMEM), supplemented with 2 mM of
L-glutamine; 100 units/ml of penicillin; 100 .mu.g/ml of
streptomycin; 12.5 units/ml of nystatin and 10% of fetal calf serum
(FCS). Cells were seeded at a density of 5.times.10.sup.6
cells/plate, on a 10 cm.sup.3 culture plates, in a volume of 10 ml,
and were allowed to age by incubating the culture for 96 hours
without exchange of the growth medium. As a result, a marked
percentage of the cells became apoptotic. Cells were harvested
using a cell scraper, separated to single cells by passage through
a syringe with a 18G needle, and re-suspended at a density of
10.sup.6 cells/ml in PBS buffer at pH=7.4. As was shown before NST
203 including a dansylamide group which enables the visualization
of the flouresence of a single cell. The selective binding of the
NST203 to apoptotic cells is shown in FIG. 3A which demonstrates
the uptake of NST 203 into the population of apoptotic cells
(green, glowing color cells, representative are marked by arrows),
while non-fluorescence is observed in the non-apoptotic cells
(blue, not glowing cells, marked by arrow heads).
[0151] By contrast, the control compound n-butyl-dansylamide (BDA),
having the same fluorophore but devoid of the NST-ML-Action Motif,
did not manifest this selectivity, thus manifesting the activity of
the NST-ML-Action Motif in selective binding to the apoptotic cells
(FIG. 3B). Therefore, NST203 can serve as a marker, which performs
selective binding to apoptotic cells.
Example 5
Selective Binding of NST 203 to Melanoma Cells Undergoing a Death
Process Induced by Chemotherapy in Mice In Vivo
Experimental Procedure
[0152] Mice (c57/black; 8 weeks old male mice) were injected
subcutaneously bilaterally, in the flank, with murine
melanoma-derived B16-F10 cells (ATCC CRL-6475; 10.sup.5 cells/mice
in a volume of 100 .mu.l). Prior to injection, the cell line was
maintained in culture in Dulbecco's modified Eagle's medium (DMEM),
supplemented with 4 mM of L-glutamine; 100 units/ml of penicillin;
100 .mu.g/ml of streptomycin; 12.5 units/ml of nystatin and 10% of
fetal calf serum (FCS). After 10 days, when tumor diameter reached
the size of 5-7 mm, mice were subjected to chemotherapy treatment
(Taxol 20 mg/Kg together with Cyclophosphomide, 300 mg/Kg, in a
volume of 200 .mu.l intra-peritoneal injection). Twenty-four hours
later, NST-203 was injected intravenously, at a dose of 2.8
mg/mouse in 10% chromophore in tris-base buffer. Two hours later,
mice were sacrificed and tumors as well as other organs were
harvested and immediately frozen in liquid nitrogen. Uptake of
NST-203 by the tumors or other organs was assessed by fluorescent
microscopy of frozen sections from each tissue.
Experimental Results
[0153] FIG. 4A shows fluorescent microscopy of the tumor. Extensive
binding of NST203 to numerous tumor cells undergoing apoptosis can
be observed. Demonstrated are also the intracellular accumulation
of the compound (right side of the picture) and the high level of
selectivity, reflected by a marked uptake into the apoptotic cells,
while the surrounding viable tumor cells remain unstained (left
upper side of the picture).
[0154] Chemotherapy often acts to induce cell death no only in
target tumor tissue, but also in non-target tissues, such as the
epithelium of the gastrointestinal tract. FIG. 4B shows the
capability of NST203 to selectively detect these cells undergoing
apoptosis (see arrows). Similar to the findings in the tumor,
viable cells in the gastrointestinal tract did not manifest uptake
of NST203 and therefore remained dark.
[0155] These results therefore manifest the capability of the
compound of the invention, NST203, to target specifically apoptotic
cells in vivo, wherein the death process is being induced by
chemotherapy. The apoptotic cells are detected in a universal
manner, irrespective of the tissue involved. By contrast, viable
cells of said tissues do not manifest such binding.
Example 6
Imaging, by Autoradiographic Methods with .sup.3H-NST200, of
Response to Chemotherapy in a Mouse Model of Colon Carcinoma
Experimental Procedure
[0156] Colon carcinoma model was established in the abdomen of
Balb/c male mice. At day 12-14 when tumor size was 6-8 mm in
diameter, the uptake of NST200 into tumors was measured following
treatment of colon carcinoma tumors with i.v. injection of two
doses of doxorubicin (20 mg/kg, 72 hours apart). Fourty-eight hours
after the chemotherapy, both the control non-treated and the
chemotherapy-treated animals were injected intravenously with and
radio-labeled NST200 (80 .mu.Ci/animal) Four hours later, the
animals were sacrificed. 10 .mu.m frozen sections were prepared,
air-dried and exposed to a tritium sensitive film. The film was
exposed for a duration of 7 weeks, developed and analyzed by
densitometry measurement. Measurement of .sup.3H-DDC densities in
optical density/mm.sup.2, in the ischemic core vs. the
contralateral hemisphere signal was assessed by TINA software.
Signals were translated according to a Microscale autoradiography
standards and expressed in nCi/mg units.
Experimental Results
[0157] Non-treated tumors exhibit no uptake of NST200, and
therefore, no image could be detected by autoradiography (FIG. 5A).
However, upon induction of cell death via chemotherapy, a dramatic
increase in NST 200 uptake occurs indicating a massive process of
irradiation-induced cell death. (FIG. 5 B and C)
[0158] NST 200 uptake can be detected at multiple foci on the
surface of the tumors in the form of dark intense patches. NST-200
labeling localized to specific foci of apoptotic areas within the
tumor and was not diffused throughout the tumor. Other areas of the
treated tumors were not labeled, indicative of vital tumor tissue.
This observation emphasized the advantage of NST 200 as targeting
molecule that is accumulating selectively in large quantities in
dying and not in live cells (FIG. 5B). The heterogeneous nature of
response to therapy even within the same tumor is manifested in
FIG. 5C: the uptake of NST200 accumulates especially in one large
area of the tumor that was exhibited increased cell death, but not
in other tumor areas, that did not response to the chemotherapy
Example 7
Uptake of 3H-NST 200 into Colon Carcinoma Model: Effect of
Chemotherapy
Experimental Procedure
Colon Carcinoma Model
[0159] Murine colon carcinoma cells (CT-26) (ATCC CRL-2638) were
maintained in RPMI (Gibco, UK), 2 mM of L-glutamine; 100 units/ml
of penicillin; 100 .mu.g/ml of streptomycine; 12.5 units/ml
nystatin; and 10% heated inactivation FCS. Studies were carried out
in adult male Balb/C mice 8-10 weeks old (weighting 20-25 gr).
Inoculation of Tumor:
[0160] Cells were trypsinized, washed twice with HBS (140 mM NaCl;
0.5M Hepes, PH 7.4) and than centrifuged (5 min, 1000 rpm,
4.degree. C.) and concentrated into 2.times.10.sup.6 cells/ml in a
mixture of (2%) methyl cellulose and saline (1:3). A volume of 0.2
ml of the above solution (containing 4.times.10.sup.5 cells/dose)
was injected subcutaneously into the mice abdomen in both sides
while mice were anaesthetized. Anaesthetic stock solution was
prepared from 0.85 ml Ketamine (100 mg/ml)+0.15 ml Xilazine (2%)
diluted 1:10. 0.1 ml of diluted solution was injected (i.p.) per 10
gr body weight.
Tumor Follow Up:
[0161] Mice were examined daily for palpable tumor formation. 7-10
days after tumor injection small tumors were visible.
Evaluation of .sup.3H-NST200 Uptake into Colon Carcinoma Tumors
[0162] Uptake of .sup.3H-NST200 into tumors was measured following
treatment of colon carcinoma tumors with i.v. injection of two
doses of doxorubicin (20 mg/kg, 72 hours apart). 48 hours after the
second dose, the mice were intravenously injected with
.sup.3H-NST200 (10 .mu.Ci/animal). Four hours later, tumors were
collected, weighed and the tissue was processed: tumor lysis was
performed using SOLVABLE.TM. reagent (GNE9100, Packard Bioscience)
in a ratio of one ml reagent per 150 mg of tumor tissue at
60.degree. C. in 20 ml scintillation glass vials. Following 2-4
hours, one ml from each tissue extract was transferred to a glass
scintillation vial. To reduce color quenching problems, samples
were treated with 0.4 ml of 30% H.sub.2O.sub.2 in the presence of
0.066M EDTA. After 15 min of incubation time at room temperature,
extracts were incubated for 1 hr at 60.degree. C., followed by
further 15 min incubation at room temperature. Ten (10) ml of
scintillation liquid (Ultima gold, 6013329, Packard Bioscience) was
added to each vial. The vials were incubated for 1 hr at RT, and
than analyzed in a .beta.-counter (TRI-CARB 2100TR, liquid
scintillation analyzer, Packard Bioscience). All samples were
measured in triplicates Values of percents of injected dose (% ID/g
tissue) were calculated for each sample.
Experimental Results
[0163] The quantitative analyses of .sup.3H-NST200 accumulation in
doxorubicin treated colon carcinoma tumors versus non treated
control tumors revealed a massive accumulation of .sup.3H-NST200 at
48 hr following treatment. While in control group all tumors
accumulated similar and low amounts of .sup.3H-NST200, in the
chemotherapy-treated tumors the accumulation values were variable
with some tumors that exhibited a 40-50 fold increase in uptake as
compared with the control group. The mean value of uptake in the
treated group was 1.28% ID/gr, which is 12.1 fold more than the
mean value of the control group (see FIG. 6).
[0164] The wide spectrum of .sup.3H-NST200 accumulated values,
reflects the individual response of different tumors to the anti
cancer treatment. The above experiment clearly shows that
.sup.3H-NST200 can serve for detecting carcinoma and for detecting
the effect of cytotoxic drugs on the carcinoma cells.
Example 8
Biodistribution of .sup.3H-NST200 in Colon Carcinoma Tumor Bearing
Mice
Experimental Procedure
[0165] Colon carcinoma model was established in the abdomen of
Balb/c male mice, as described in previous example, and
.sup.3H-NST200 (10 .mu.Ci) was injected to animals treated with
doxorubicin. Four hours later, the animals were sacrificed and
various organs/tissues were collected and processed, to determine
the accumulation of .sup.3H-NST200 within them. The uptake in each
organ was expressed as % of the injected dose (ID), and the ratio
between the uptake in the tumor and other organs was calculated, as
shown in the table attached hereto as FIG. 7.
Experimental Results
[0166] The damage induced by doxorubicin to tumor tissue indeed
exceeded the damage to other non-target organs, including the heart
and the small intestine. The ratio of uptake in the tumor vs. all
organs in the table is >1, showing increased apoptosis in the
tumor and selective accumulation of .sup.3H-NST200 in the target
vs. non-target tissues.
Example 9
.sup.3H-NST200 can Detect Cell Death Occurring within BiCNU-Treated
Tumors, Before Shrinkage of Tumor Size
Experimental Procedures
[0167] Colon carcinoma model was established in the abdomen of
Balb/c male mice, as described in Example No. 10. At day 12-14 when
tumor size was 6-8 mm in diameter, mice were injected intravenously
with doxorubicin. A total of 2 doses of doxorubicin were given,
separated by 3 days interval (each dose was 20 mg/Kg). Two days
after the second doxorubicin injection, mice were injected i.v.
with 10 .mu.Ci of .sup.3H-NST200 in a volume of 0.2 ml saline. Four
hours following .sup.3H-NST200 injection, mice were sacrificed by
pental overdosing. Tumors were collected in ependorff tubes,
weighted and frozen in -20.degree. C.
[0168] Tumor lysis was performed using SOLVABLE.TM. reagent
(GNE9100, Packard Bioscience) in a ratio of 1 ml reagent per 150 mg
of tumor tissue at 60.degree. C. in 20 ml scintillation glass
vials. Following 2-4 hours, 1 ml from each tissue extract was
transferred to a glass scintillation vial. To reduce color
quenching problems, samples were treated with 0.4 ml of 30%
H.sub.2O.sub.2 in the presence of 0.066M EDTA. After 15 min of
incubation time at room temperature, extracts were incubated for 1
hr at 60.degree. C., followed by further 15 min incubation at room
temperature. Ten (10) ml of scintillation liquid (Ultima gold,
6013329, Packard Bioscience) was added to each vial. The vials were
incubated for 1 hr at RT, and than analyzed in a .beta.-counter
(TRI-CARB 2100TR, liquid scintillation analyzer, Packard
Bioscience). Values of percents of injected dose (% ID/g tissue)
were calculated for each sample. Comparison between NST200 uptake
and tumor volume is shown. The uptake values of NST200 after each
dose were correlated with tumor volume, which were calculated by
the formula (assuming spherical tumors) V=.pi.D.sup.3/6, where D is
the average tumor diameter.
Experimental Results
[0169] During the treatment with doxorubicin (lasting 5 days), the
tumor mass was not reduced (see FIG. 8A). In contrast, the tumor
continued to grow, exhibiting a 50% increase in their volume, as
compared to the control non-treated tumors, that were collected 5
days earlier than the treated tumors. This increase was found to be
non-significant. While such non-significant change in tumor volume
occurred after 2 doses of doxorubicin treatment, a dramatic
increase, by 18.5 fold in .sup.3H-NST200 uptake was detected,
indicating that .sup.3H-NST200 is a sensitive tool, capable of
sensing cell death within the tumor even in cases where no
shrinkage of tumor mass is detected (see FIG. 8B).
Example 10
Use of Tritium-Labeled NST200 for the Detection, by Autoradiography
Methods, of Apoptotic Damage Following Middle Cerebral Artery
Occlusion in Mice
Experimental Procedure
[0170] p-MCA was induced through a subtemporal approach in Balb/C
mice with an outcome of a pronounced ischemic damage. Twenty-two
hours after p-MCA, the animal's neurological score was assessed
(from 0-no clinical signs to 3-hemiplegia, circling and catatonia)
and radio-labeled NST200 (80 .mu.Ci/animal) was intravenously
injected for successive 2 h before sacrificing the animals. 10
.mu.m frozen sections were prepared, air-dried and exposed to a
tritium sensitive film (Hyperfilm-3 h, RPN535B Amersham-Pharmacia,
Eu). The film was exposed for a duration of 7 weeks, developed (GBX
Developer & Fixer, Kodak, USA) and analyzed by densitometry
measurement. Measurement of .sup.3H-DDC densities in optical
density/mm.sup.2, in the ischemic core vs. the contralateral
hemisphere signal was assessed by TINA software. Signals were
translated according to a Microscale autoradiography standards
(RPN510 Amersham-Pharmacia, Eu) and expressed in nCi/mg units.
Experimental Results
[0171] As can be seen from FIG. 9, which shows accumulation of
.sup.3H-NST 200 (a) and H&E staining (b), autoradiographic
image analysis revealed the specificity of targeting the injury by
.sup.3H-NST200 within the regions of apoptotic/necrotic cell death.
The results were further confirmed by H& E staining.
Accordingly, .sup.3H-NST200 can be used as a marker for brain
apoptotic damage in autoradiographic image analysis.
Example 11
Autoradiography by .sup.3H-Radiolabeled NST 200 of Rat Renal
Ischemia-Reperfusion (I/R)
Experimental Procedure
[0172] Operative procedures were performed in rats under general
anesthesia induced by the combination of Ketamine (80 mg/kg) and
Xylazine, (10 mg/kg), administrated intraperitoneally. Renal
ischemia was induced by unilateral left renal artery clamping,
using a small nontraumatic vascular clamp, for 45 minutes. The
contralateral, untreated kidney from the same animal was designed
as kidney from sham-operated control. Reperfusion was initiated by
removal of the clamp. Period of renal reperfusion was 24 hours.
During the course of reperfusion, animals were injected
intravenously with 100 .mu.Ci of .sup.3H-NST205 and one hour later,
both kidneys were excised, frozen in liquid nitrogen, and stored at
-70.degree. C. until use. 10 .mu.m frozen sections were prepared,
air-dried and exposed to a tritium sensitive film (Hyperfilm-3 h,
RPN535B Amersham-Pharmacia, Eu). The film was exposed for a
duration of 7 weeks, developed (GBX Developer & Fixer, Kodak,
USA) and analyzed by densitometry measurement. Measurement of
.sup.3H-DDC densities in optical density/mm.sup.2, in the ischemic
kidney vs. the contralateral kidney signal was assessed by TINA
software. Signals were translated than according to Microscale
autoradiography standards (RPN510 Amersham-Pharmacia, Eu) and
expressed in nCi/mg units.
Experimental Results
[0173] As shown in FIG. 10, during ischemia-reperfusion injury in
the rat kidney, apoptosis was observed in the distal tubules of the
cortico-medullary region and outer medulla (OM) while severe
necrosis was seen in the proximal straight tubules of the (OM).
Less damage was observed in the cortex. No damaged tubules in the
contralateral, sham kidney were observed. Autoradiographic image
analysis revealed the specificity of targeting the injury by
.sup.3H-NST205 within the regions of apoptotic/necrotic cell death,
confirmed by morphological analysis. By contrast, no .sup.3H-NST205
uptake into contralateral kidney was demonstrated, emphasizing the
specificity of .sup.3H-NST205 uptake only into the injured
kidney.
Example 12
Autoradiography by 3H NST205 in a Rat Model of
Radiocontrast-Induced Acute Distal Tubular Necrosis (ATN)
Experimental Procedures
[0174] Nephropathy with selective medullary hypoxic tubular damage
was induced by the combined administration of indomethacin (Sigma
Chemical Co.), 10 mg/kg, i.v., N.sup..omega.nitro-L-arginine methyl
ester (L-NAME, Sigma Chemical Co.), 10 mg/kg, i.v., and
radiocontrast agent sodium-iothalamate 80% (Angio-Conray,
Mallinckrodt Inc), 6 mL/kg, i.a. Additional rats injected with
vehicles served as control. Twenty-four hours after insult,
animals, both control and experimental were intravenously injected
with 100 .mu.Ci of .sup.3H-NST205 and one hour later kidneys were
excised and frozen in liquid nitrogen.
Experimental Results
[0175] As can be seen from FIG. 11, renal morphology analysis
disclosed wide-ranging extent of medullary (namely, outer and inner
strip of outer medulla) damage. Homing of .sup.3H-NST205 was
primarily restricted to injured regions within the outer medulla.
There was no observation of .sup.3H-NST205 uptake at a specified
region in the absence of morphologic damage.
Example 13
Imaging of Experimental Autoimmune Encephalomyelitis (EAE) by
.sup.3H-200; Autoradiography
Experimental Procedure
[0176] EAE was induced by immunization of C3H.SW/C57/b1 female
mice, 6-8 week-old. The animals were immunized with the peptide
encompassing amino acids 35-55 of rat myelin oligodendrocyte
glycoprotein (MOG). The peptide was synthesized using a solid-phase
technique on a peptide synthesizer. Mice were injected
subcutaneously at one site in a flank with a 200 .mu.l emulsion
containing 75 .mu.l MOG peptide in complete Freund's adjuvant (CFA)
and 200 .mu.g mycobacterium tuberculosis. An identical buster was
injected at one site in the other flank 1 week later. Following the
encephalitogenic challenge mice were observed daily and clinical
manifestation of EAE were scored (from 0=no clinical signs to
5=total paralysis of four limbs). At a selected stage of the
disease (Pre-symptoms or end-stage) animals were intravenously
injected with radio-labeled NST200 (100 .mu.Ci/animal) for one hour
of incubation before sacrificing the animals. 10 .mu.m frozen
sections were prepared, air-dried and exposed to a tritium
sensitive film. The film was exposed for duration of 7-9 weeks,
developed and analyzed by densitometry measurement. Measurement of
.sup.3H-DDC densities in optical density/mm.sup.2, in the ischemic
core vs. the contralateral hemisphere signal was assessed by TINA
software.
Experimental Results
[0177] The EAE animal model of multiple sclerosis disease mimicked
the chronic disabling autoimmune neurological disorder targeting
the white matter of the central nerve system. The severe damage of
the white matter in the experimental animals was observed in the
autoradiography demonstrated in FIG. 12. At the brain level, the
coronal sections expressed a dark staining of the pyramidal tracts
at the bottom of the brain and an excess of radio-ligand
accumulation in the whole brain in comparison to the bright
staining of the control, non-immunized mouse. An impressive
accumulation of the radio labeled .sup.3H-200 was observed also at
the spinal cord level. A longitudinal section of the spinal cords
presented a high level of labeling of the lateral tracts of the
white matter in the immunized mouse, compared to the spinal cord of
the control untreated animal.
* * * * *