U.S. patent application number 17/693011 was filed with the patent office on 2022-06-23 for matrix metalloproteinase inhibitors and imaging agents, and methods using same.
The applicant listed for this patent is The United States of America as represented by the Department of Veterans Affairs, The United States of America as represented by the Department of Veterans Affairs, Yale University. Invention is credited to Henry(Yiyun) HUANG, Hye-Yeong KIM, Mehran SADEGHI, Jakub TOCZEK, Yunpeng YE.
Application Number | 20220194930 17/693011 |
Document ID | / |
Family ID | 1000006185769 |
Filed Date | 2022-06-23 |
United States Patent
Application |
20220194930 |
Kind Code |
A1 |
SADEGHI; Mehran ; et
al. |
June 23, 2022 |
Matrix Metalloproteinase Inhibitors and Imaging Agents, And Methods
Using Same
Abstract
The present invention provides certain compounds, or salts or
solvates thereof, which can be used as matrix
metalloproteinase-targeted inhibitors or imaging agents.
##STR00001##
Inventors: |
SADEGHI; Mehran; (Easton,
CT) ; YE; Yunpeng; (Staten Island, NY) ; KIM;
Hye-Yeong; (Hamden, CT) ; HUANG; Henry(Yiyun);
(Madison, CT) ; TOCZEK; Jakub; (New Haven,
CT) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Yale University
The United States of America as represented by the Department of
Veterans Affairs |
New Haven
Washington |
CT
DC |
US
US |
|
|
Family ID: |
1000006185769 |
Appl. No.: |
17/693011 |
Filed: |
March 11, 2022 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16088868 |
Sep 27, 2018 |
11286251 |
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PCT/US2017/026610 |
Apr 7, 2017 |
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17693011 |
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62320039 |
Apr 8, 2016 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07D 273/01 20130101;
C07D 413/12 20130101; A61K 51/044 20130101 |
International
Class: |
C07D 413/12 20060101
C07D413/12; A61K 51/04 20060101 A61K051/04; C07D 273/01 20060101
C07D273/01 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] This invention was made with government support under grants
HL112992 and HL114703 awarded by National Institutes of Health, and
grant I0-BX001750 awarded by the Department of Veterans Affairs.
The government has certain rights in the invention.
Claims
1. A compound of formula I, or a salt, solvate, stereoisomer, or
tautomer thereof: ##STR00048## wherein: R is selected from the
group consisting of OH, --NH.sub.2, --NHR', --NR'R', --NH(aryl),
--NH(heteroaryl) and --NHR.sup.1; R.sup.1 is selected from the
group consisting of: ##STR00049## R.sup.2 is selected from the
group consisting of: ##STR00050## R.sup.3 is selected from the
group consisting of H, OH, OCH.sub.3, F, .sup.18F, ##STR00051##
##STR00052## each occurrence of n is independently an integer
ranging from 0 to 30; each occurrence of R' is independently
selected from the group consisting of C.sub.1-C.sub.6 alkyl and
C.sub.3-C.sub.7 cycloalkyl; and LG is a group capable of undergoing
nucleophilic displacement.
2. A pharmaceutical composition comprising at least one compound of
claim 1 and further comprising at least one pharmaceutically
acceptable carrier.
3. A method of evaluating a subject's risk of developing a
cardiovascular disease or disorder, the method comprising:
administering to a subject at least one compound of claim 1,
wherein the at least one compound comprises a radioisotope or
fluorophore, acquiring an image of at least a portion of the
subject's body, and measuring the amount of compound bound to the
imaged portion of the subject's body, wherein, if the measured
amount of bound compound is above a determined control amount, the
subject is diagnosed as having an increased risk of developing the
cardiovascular disease or disorder.
4. The method of claim 3, wherein the subject is a mammal.
5. The method of claim 3, wherein the subject is a human.
6. A method of treating a matrix metalloproteinase-related disease
or disorder in a subject, the method comprising administering to
the subject in need thereof a therapeutically effective amount of
at least one compound of claim 1.
7. The method of claim 6, wherein the disease or disorder is at
least one selected from the group consisting of cancers,
inflammatory diseases, cardiovascular diseases, stroke, aneurysm,
periodontitis, hepatitis, cirrhosis, portal hypertension,
glomerulonephritis, atherosclerosis, emphysema, asthma, pulmonary
fibrosis, autoimmune disorders of skin and dermal photoaging,
rheumatoid arthritis, osteoarthritis, multiple sclerosis,
Alzheimer's disease, chronic ulcerations, uterine involution and
bone resorption.
8. The method of claim 6, wherein the at least one compound is
administered to the subject through a route selected from the group
consisting of oral, nasal, inhalational, topical, buccal, rectal,
pleural, peritoneal, intra-peritoneal, vaginal, intramuscular,
subcutaneous, transdermal, epidural, intratracheal, otic,
intraocular, intrathecal, and intravenous.
9. The method of claim 6, wherein the subject is a mammal.
10. The method of claim 6, wherein the subject is a human.
11. A kit comprising at least one compound of claim 1, an
applicator, and instructions to use the at least one compound to
evaluate a subject's risk of developing a cardiovascular disease or
disorder or treat a matrix metalloproteinase-related disease or
disorder in a subject.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of, and claims priority
to, U.S. patent application Ser. No. 16/088,868, filed Sep. 27,
2018, now allowed, which is a national phase application under 35
U.S.C. .sctn. 371 from, and claims priority to, International
Application No. PCT/US2017/026610, filed Apr. 7, 2017, and
published under PCT Article 21 (2) in English, which claims
priority under 35 U.S.C. .sctn. 119(e) to U.S. Provisional Patent
Application No. 62/320,039, filed Apr. 8, 2016, which are
incorporated herein by reference in their entireties.
BACKGROUND OF THE INVENTION
[0003] Matrix metalloproteinases (MMPs) are a family of
structurally related zinc-containing enzymes that have the ability
to breakdown matrix and other proteins. Upregulation of MMPs is
associated with a wide range of diseases, including cancer, as well
as pulmonary, musculoskeletal and cardiovascular diseases. MMP
inhibitors have thus been proposed as potential therapeutic agents
against these diseases. For example, hydroxamate-based MMP
inhibitors act by binding to the active site Zn(II) ion in
activated MMPs. However, MMP inhibitors as a class of drugs are
generally toxic, have debilitating side effects at effective doses
(such as musculoskeletal pain or inflammation) and/or exhibit
mutagenic properties.
[0004] Further, due to the involvement of MMPs in diseases and
disorders, there is a high demand for imaging agents that bind to
MMPs, helping characterize their expression and/or activation. Such
imaging agents would allow physicians to accurately diagnose and
treat MMP-associated diseases, such as cardiovascular inflammation.
Unfortunately, currently available MMP imaging agents (e.g.,
.sup.99mTc-RP805; FIG. 12, D) exhibit poor target specificity,
prolonged blood circulation time, poor stability and/or poor
aqueous solubility of their precursors. Such undesired properties
limit their utility in clinical applications.
[0005] Abdominal aortic aneurysm (AAA) accounts for 10,000-15,000
recorded deaths per year, mainly due to rupture, in the U.S.
Current clinical guidelines for surgical repair of AAA are based on
aneurysm size, expansion rate and clinical symptoms. However, a
significant portion of AAA ruptures occurs in patients who do not
meet the criteria for AAA repair, while some large AAA may remain
stable for many years. As such, new risk stratification tools are
needed to overcome limitations of the current approach to patient
selection for AAA repair. Molecular imaging targeted at the
determinants of AAA expansion and rupture appears particularly
promising in this regard. MMP activation is a main
pathophysiological feature of AAA, and is believed to be closely
related to aneurysm progression and rupture risk. Thus, molecular
imaging of MMP activation can be a useful tool for AAA risk
stratification.
[0006] There is a need in the art for clinically useful imaging
agents that can be used to image MMP activity and/or activation in
a patient. There is also a need in the art for compounds that can
bind and inhibit MMP activity and/or activation. Such compounds can
be used to treat MMP-related diseases and/or evaluate aneurysm
(e.g., AAA) progression and rupture risk. The present invention
addresses these needs.
BRIEF SUMMARY OF THE INVENTION
[0007] The invention provides a compound of formula I, or a salt,
solvate, stereoisomer, or tautomer thereof:
##STR00002##
R.sup.2 is selected from the group consisting of:
##STR00003##
R.sup.3 is selected from the group consisting of H, OH, OCH.sub.3,
F, .sup.18F,
##STR00004## ##STR00005##
each occurrence of n is independently an integer ranging from 0 to
30; each occurrence of R' is independently selected from the group
consisting of C.sub.1-C.sub.6 alkyl and C.sub.3-C.sub.7 cycloalkyl;
and LG is a group capable of undergoing nucleophilic
displacement.
[0008] The invention further provides a compound of formula II or a
salt, solvate, stereoisomer, or tautomer thereof:
##STR00006##
wherein R is selected from the group consisting of H, OH, OR',
aroxy, heteroaroxy, SH, thioalkoxy, thiocycloalkoxy, --NH.sub.2,
--NHR' [such as but not limited to --NHCH.sub.3,
--NHCH.sub.2CH.sub.3, --NH(CH.sub.2).sub.2CH.sub.3 or
--NHCH(CH.sub.3).sub.2], --NR'R' [such as but not limited to
--N(CH.sub.3).sub.2, --N(CH.sub.3)CH.sub.2CH.sub.3, or
--N(CH.sub.2CH.sub.3).sub.2], --NH(aryl) and --NH(heteroaryl),
wherein each occurrence of R' is independently selected from the
group consisting of C.sub.1-C.sub.6 alkyl and C.sub.3-C.sub.7
cycloalkyl.
[0009] In certain embodiments, the compound of formula I is at
least one selected from the group consisting of:
##STR00007## ##STR00008## ##STR00009## ##STR00010##
##STR00011##
or a salt, solvate, stereoisomer, or tautomer thereof.
[0010] In certain embodiments, R.sup.3 is present, and the compound
of formula I further comprises a radioisotope-containing group,
wherein the radioisotope is bound to and/or present in R.sup.3. In
other embodiments, the radioisotope is at least one selected from
the group consisting of .sup.99mTc, .sup.18F, .sup.111In, .sup.64Cu
and .sup.68Ga. In yet other embodiments, the
radioisotope-containing group further comprises one or more ligands
that are bound to the radioisotope. In yet other embodiments, the
one or more ligands help stabilize the radioisotope within the
compound of formula I.
[0011] In certain embodiments, R.sup.3 is present, and the compound
of formula I further comprises a fluorophore-containing group.
[0012] In certain embodiments, R.sup.3 is present, and the compound
further comprises a radioisotope- and/or fluorophore-containing
group comprising a radioisotope selected from the group consisting
of .sup.99mTc, .sup.18F, .sup.111In, .sup.64Cu and .sup.68Ga, and
optionally further comprising one or more additional ligands,
wherein the radioisotope is bound to R.sup.3.
[0013] In certain embodiments, R.sup.3 is present, and the compound
further comprises a radioisotope- and/or fluorophore-containing
group comprising a radioisotope selected from the group consisting
of .sup.99mTc, .sup.18F, .sup.111In, .sup.64Cu and .sup.68Ga, and
further comprising one or more additional ligands, wherein the
radioisotope is bound to R.sup.3.
[0014] In certain embodiments, R.sup.1 and/or R.sup.2 comprises the
group N.sub.3 or C.ident.CH, and the compound is capable of
undergoing a click reaction with a substrate comprising the group
C.ident.CH or N.sub.3, respectively.
[0015] In certain embodiments, the compound of formula I is at
least one selected from the group consisting of:
##STR00012##
or a salt, solvate, stereoisomer, or tautomer thereof, wherein M is
a metal, such as but not limited to a metal radioisotope.
[0016] In certain embodiments, the compound of formula II is at
least one selected from the group consisting of:
##STR00013## ##STR00014##
[0017] In certain embodiments, the compound is part of a
pharmaceutical composition further comprising a pharmaceutically
acceptable carrier, such as but not limited to normal saline, 5%
dextrose, sodium bicarbonate, sodium phosphate and/or citrate.
[0018] The invention further provides a method of evaluating a
subject's risk of developing a cardiovascular disease or disorder.
The invention further provides a method of treating a matrix
metalloproteinase-related disease or disorder in a subject.
[0019] In certain embodiments, the method comprises administering
to the subject in need thereof a therapeutically effective amount
of at least one compound of the invention. In other embodiments,
the method comprises administering to a subject at least one
compound of the invention, wherein the at least one compound
comprises a radioisotope or fluorophore. In yet other embodiments,
the method comprises acquiring an image of at least a portion of
the subject's body. In yet other embodiments, the method comprises
measuring the amount of compound bound to the imaged portion of the
subject's body. In yet other embodiments, if the measured amount of
bound compound is above a determined control amount, the subject is
diagnosed as having an increased risk of developing the
cardiovascular disease or disorder.
[0020] The invention further provides a kit comprising at least one
compound and/or at least one pharmaceutical composition of the
invention, an applicator, and instructions to use the at least one
compound and/or the at least one composition to evaluate a
subject's risk of developing a cardiovascular disease or disorder
and/or treat a matrix metalloproteinase-related disease or disorder
in a subject.
[0021] In certain embodiments, the subject is a mammal. In other
embodiments, the subject is a human. In yet other embodiments, the
disease or disorder is at least one selected from the group
consisting of cancers, inflammatory diseases, cardiovascular
diseases, stroke, aneurysm, periodontitis, hepatitis, cirrhosis,
portal hypertension, glomerulonephritis, atherosclerosis,
emphysema, asthma, pulmonary fibrosis, autoimmune disorders of skin
and dermal photoaging, rheumatoid arthritis, osteoarthritis,
multiple sclerosis, Alzheimer's disease, chronic ulcerations,
uterine involution and bone resorption.
[0022] In certain embodiments, the therapeutically effective amount
of the at least one compound ranges from about 10 ng to 1000 mg. In
other embodiments, the at least one compound is administered to the
subject through a route selected from the group consisting of oral,
nasal, inhalational, topical, buccal, rectal, pleural, peritoneal,
intra-peritoneal, vaginal, intramuscular, subcutaneous,
transdermal, epidural, intratracheal, otic, intraocular,
intrathecal, and intravenous.
BRIEF DESCRIPTION OF THE DRAWINGS
[0023] For the purpose of illustrating the invention, there are
depicted in the drawings certain embodiments of the invention.
However, the invention is not limited to the precise arrangements
and instrumentalities of the embodiments depicted in the
drawings.
[0024] FIG. 1 illustrates a non-limiting radio-HPLC analysis of
.sup.99mTc-2.
[0025] FIG. 2 illustrates a non-limiting effect of
.sup.99mTc-labeling on recombinant human MMP-12 (rhMMP-12) activity
inhibition by 1.
[0026] FIG. 3 illustrates a non-limiting radio-HPLC analysis of
.sup.99mTc-1 and its stability in solution.
[0027] FIGS. 4A-4F illustrate .sup.99mTc-1 imaging of carotid
aneurysm. FIGS. 4A-4D illustrate non-limiting examples of
morphology (FIGS. 4A-4B) and autoradiography (FIGS. 4C-4D) of
aortae and carotid arteries from apoE.sup.-/- mice with
CaCl.sub.2-induced carotid aneurysm injected with .sup.99mTc-1 with
(FIGS. 4A, 4C) and without (FIGS. 4B, 4D) excess of unlabeled
analogue (19). FIGS. 4E-4F illustrate autoradiographic
quantification of .sup.99mTc-1 uptake in carotid aneurysm and aorta
(FIG. 4E) and aneurysm-to-aorta uptake ratio (FIG. 4F) for control
(.circle-solid.) and blocking () groups. ID: injected dose. *
P<0.05, ** P<0.01.
[0028] FIG. 5 illustrates non-limiting MMP activity of the lung
tissue of hypoxia-exposed mice with pulmonary arterial hypertension
(PAH) and control, normoxia mice, quantified using a panMMP
substrate (AU: arbitrary units).
[0029] FIG. 6 illustrates ex vivo planar images of the lungs
harvested about 60 minutes after intravenous administration of
.sup.99mTc-1, demonstrating considerably higher signal in the lung
of hypoxia-exposed mouse with pulmonary arterial hypertension
(PAH).
[0030] FIGS. 7A-7B illustrate .sup.99mTc-1 stability.
Representative radiochromatograms of .sup.99mTc-1 after
radiolabeling (FIG. 7A) and in urine collected from a C57BL/6J
mouse at 2 hours post-injection (FIG. 7B). t.sub.R: retention
time.
[0031] FIGS. 8A-8B illustrate tracer biodistribution and clearance.
Blood kinetics (FIG. 8A) and biodistribution at 2 hours (FIG. 8B)
of .sup.99mTc-1 (.circle-solid.) and .sup.99mTc-RP805 () in
C57BL/6J mice. SG: Salivary Glands, WAT: White Adipose Tissue; pAT:
Periaortic Adipose Tissue; ID: injected dose. n=5 in each group.
*P<0.05, **P<0.01.
[0032] FIGS. 9A-9D illustrate .sup.99mTc-1 imaging of AAA. FIGS.
9A-9B: Examples of fused .sup.99mTc-1 SPECT/CT images of animals
from the low remodeling (FIG. 9A) and aneurysm (FIG. 9B) groups,
classified based on visual in situ analysis of the abdominal aorta.
Transversal (left), coronal (middle) and sagittal (right) views are
shown. Arrows point to the areas of maximal tracer uptake in the
abdominal aorta. FIG. 9C: Quantification of .sup.99mTc-1 signal in
area of maximal tracer uptake in the suprarenal abdominal aorta in
low remodeling (LR) and aneurysm (AAA) groups. *P<0.05. FIG. 9D:
Correlation between .sup.99mTc-1 signal in vivo and MMP activity
quantified by zymography ex vivo. cpv: counts per voxel, AU:
arbitrary units.
[0033] FIGS. 10A-10B illustrate ex vivo characterization of
suprarenal abdominal aorta. FIG. 10A: Maximal external diameter of
the abdominal aorta in low remodeling (LR) and aneurysm (AAA)
groups. FIG. 10B: Aortic MMP activity quantified by zymography in
LR and AAA groups. ** P<0.01. AU: arbitrary units.
[0034] FIGS. 11A-11B illustrate correlates of aortic .sup.99mTc-1
signal on in vivo microSPECT/CT images. Correlation between
suprarenal abdominal aorta .sup.99mTc-1 uptake and
.beta.-actin-normalized CD68 (FIG. 11A) and MMP-12 (FIG. 11B) gene
expression. cpv: counts per voxel.
[0035] FIG. 12 illustrates chemical structures of formula II,
R.dbd.NHCH.sub.3 (19), 1 (also described as RYM1 herein; B),
.sup.99mTc-1 (C) and .sup.99mTc-RP805 (D).
[0036] FIG. 13 illustrates .sup.99mTc-1 stability in blood.
Radiochromatograms of .sup.99mTc-1 obtained after incubation in
mouse blood at 37.degree. C. for 0, 2 and 5 hours (h) demonstrate a
single peak.
[0037] FIGS. 14A-14B illustrate .sup.99mTc-1 biodistribution and
clearance. Blood kinetics (FIG. 14A) and biodistribution at two
hours (FIG. 14B) of .sup.99mTc-1 in apoE.sup.-/- with
CaCl.sub.2-induced carotid aneurysm without (.circle-solid.) and
with the pre-injection of an excess of 19 () SG: Salivary Glands,
WAT: White Adipose Tissue. n=6 and 5, respectively for .sup.99mTc-1
and .sup.99mTc-1+19 (RYM). **P<0.01
[0038] FIG. 15 illustrates .sup.99mTc-1 imaging of AAA. Examples of
CT (A) and fused .sup.99mTc-1 SPECT/CT (B) images of an animal from
the aneurysm group. Transversal (left), coronal (middle) and
sagittal (right) views are shown. Arrows points to the area of
maximal tracer uptake and arrowheads to the center of aneurysm on
contrast-enhanced CT images. Scale: 0 to 1.5 counts per voxel per
MBq.
[0039] FIGS. 16A-16B illustrate representative examples of
hematoxylin and eosin staining of suprarenal abdominal aortic
sections in angiotensin II-infused apoE.sup.-/- mice with low
aortic remodeling (FIG. 16A) or AAA (FIG. 16B). Scale bar: 500
.mu.m.
[0040] FIGS. 17A-17B illustrate correlations between suprarenal
abdominal aorta .sup.99mTc-1 signal on in vivo microSPECT/CT images
and .beta.-actin-normalized MMP-2 (FIG. 17A) and MMP-9 (FIG. 17B)
gene expression. cpv: counts per voxel.
[0041] FIGS. 18A-18D illustrate gene expression in suprarenal
abdominal aorta. Aortic .beta.-actin actin-normalized CD68 (FIG.
18A), MMP-2 (FIG. 18B), MMP-9 (FIG. 18C) and MMP-12 (FIG. 18D) mRNA
expression in angiotensin II-infused apoE.sup.-/- mice with low
aortic remodeling (LR) or aneurysm (AAA). *P<0.05,
**P<0.01.
DETAILED DESCRIPTION OF THE INVENTION
[0042] The invention relates, in certain aspects, to the unexpected
discovery that compounds of formula I strongly and selectively bind
to MMPs. The compounds of the invention can further be labelled
with one or more radioisotope-containing and/or
fluorophore-containing labels. In certain embodiments, labeled
compounds of formula I are used as MMP-targeted imaging agents. In
other embodiments, labeled compounds of formula I are used to
diagnose MMP-related diseases.
[0043] In certain embodiments, labeled compounds of formula I
exhibit improved solubility when compared to currently used
MMP-targeted imaging agents (such as, but not limited to,
.sup.99mTc-RP805). In other embodiments, labeled compounds of
formula I have faster excretion (shorter retention times in blood)
than currently used MMP-targeted imaging agents (such as
.sup.99mTc-RP805).
[0044] The invention relates in other aspects to the unexpected
discovery that compounds of formula II strongly and selectively
bind to MMPs. In certain embodiments, compounds of formula I and/or
II may be used to inhibit MMP activity and/or treat diseases
characterized by MMP upregulation and activation.
[0045] As demonstrated herein, an illustrative water-soluble
zwitterionic MMP inhibitor 1 was designed and evaluated, using
RP805 as a comparator. 1 was labeled with .sup.99mTc- to yield
(.sup.99mTc-1), which radiochemical stability was evaluated by
radio-high-performance liquid chromatography analysis. Tracer blood
kinetics and biodistribution for that compound were compared with
.sup.99mTc-RP805 in C57BL/6J mice (n=10). .sup.99mTc-1 binding to
aneurysm and specificity were evaluated by quantitative
autoradiography in apolipoprotein E-deficient (apoE.sup.-/-) mice
with CaCl.sub.2-induced carotid aneurysm (n=11). Angiotensin II
(Ang 11)-infused apoE.sup.-/- (n=16) were used for
micro-single-photon emission computed tomography (SPECT)/computed
tomography (CT) imaging. Aortic tissue MMP activity and macrophage
marker, CD68 expression were assessed by zymography and reverse
transcription-polymerase chain reaction.
[0046] 1 showed nanomolar range inhibition constants for several
MMPs. .sup.99mTc-1 was radiochemically stable in mouse blood for 5
hours, and demonstrated rapid renal clearance and lower blood
levels in vivo compared to .sup.99mTc-RP805.
[0047] .sup.99mTc-1 binding to aneurysm and its specificity were
shown by autoradiography in carotid aneurysm. Ang II infusion in
apoE.sup.-/- mice for 4 weeks resulted in AAA formation in 36%
(4/11) of surviving animals. In vivo .sup.99mTc-1 microSPECT/CT
images showed higher uptake of the tracer in AAA compared to
non-dilated aortae. Specific aortic uptake of .sup.99mTc-1 in vivo
correlated with aortic MMP activity, CD68 expression and
inflammation.
[0048] 1 showed good water solubility, while retaining MMP binding
potential. In comparison with .sup.99mTc-RP805, .sup.99mTc-1 has a
faster blood clearance, which (without wishing to be limited by any
theory) is favorable for early time point imaging.
Definitions
[0049] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
any methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present
invention, exemplary methods and materials are described. As used
herein, each of the following terms has the meaning associated with
it in this section.
[0050] Generally, the nomenclature used herein and the laboratory
procedures in cell culture, oncology, cardiology, molecular
genetics, pharmacology and organic chemistry are those well-known
and commonly employed in the art.
[0051] Standard techniques are used for biochemical and/or
biological manipulations. The techniques and procedures are
generally performed according to conventional methods in the art
and various general references (e.g., Sambrook and Russell, 2012,
Molecular Cloning, A Laboratory Approach, Cold Spring Harbor Press,
Cold Spring Harbor, N.Y., and Ausubel et al., 2002, Current
Protocols in Molecular Biology, John Wiley & Sons, N.Y.), which
are provided throughout this document.
[0052] The articles "a" and "an" are used herein to refer to one or
to more than one (i.e., to at least one) of the grammatical object
of the article. By way of example, "an element" means one element
or more than one element.
[0053] "About" as used herein when referring to a measurable value
such as an amount, a temporal duration, and the like, is meant to
encompass variations of .+-.20% or .+-.10%, more preferably .+-.5%,
even more preferably .+-.1%, and still more preferably .+-.0.1%
from the specified value, as such variations are appropriate to
perform the disclosed methods.
[0054] The term "cancer" as used herein is defined as disease
characterized by the rapid and uncontrolled growth of aberrant
cells. Cancer cells can spread locally or through the bloodstream
and lymphatic system to other parts of the body. Examples of
various cancers include but are not limited to, breast cancer,
prostate cancer, ovarian cancer, cervical cancer, skin cancer,
pancreatic cancer, colorectal cancer, renal cancer, liver cancer,
brain cancer, lymphoma, leukemia, lung cancer and the like. In
certain instances, hyperproliferative disorders are referred to as
a type of cancer including but not limited to primary or metastatic
melanoma, thymoma, lymphoma, sarcoma, lung cancer, liver cancer,
non-Hodgkin's lymphoma, Hodgkin's lymphoma, leukemias, uterine
cancer, cervical cancer, bladder cancer, kidney cancer and
adenocarcinomas such as breast cancer, prostate cancer, ovarian
cancer, pancreatic cancer, and the like.
[0055] The terms "cardiovascular disease" and "cardiovascular
disorder" as used herein refer to diseases or disorders affecting
the heart and/or peripheral vascular system. Examples of
cardiovascular diseases or disorders include but are not limited to
coronary artery diseases, angina, myocardial infarction ("heart
attack"), stroke, hypertensive heart disease, rheumatic heart
disease, cardiomyopathy, myocardial remodeling, heart arrhythmia,
congenital heart disease, valvular heart disease such as calcific
aortic valve disease, carditis, vascular remodeling, restenosis,
aortic aneurysms, brain aneurysms, peripheral artery disease such
as carotid stenosis, pulmonary arterial hypertension, device (for
example pacemaker, defibrillator, left ventricular assist device)
infection or clot formation, and arterial and venous thrombosis.
Cardiovascular diseases and disorders may be related to, or caused
by, for example, atherosclerosis, high blood pressure, smoking,
diabetes, lack of exercise, obesity, high blood cholesterol, poor
diet, focal or systemic inflammation, and excessive alcohol
consumption.
[0056] A "disease" is a state of health of an animal wherein the
animal cannot maintain homeostasis, and wherein if the disease is
not ameliorated then the animal's health continues to deteriorate.
In contrast, a "disorder" in an animal is a state of health in
which the animal is able to maintain homeostasis, but in which the
animal's state of health is less favorable than it would be in the
absence of the disorder. Left untreated, a disorder does not
necessarily cause a further decrease in the animal's state of
health.
[0057] The phrase "inhibit," as used herein, means to reduce a
molecule, a reaction, an interaction, a gene, an mRNA, and/or a
protein's expression, stability, function or activity by a
measurable amount or to prevent entirely. Inhibitors are compounds
that, e.g., bind to, partially or totally block stimulation,
decrease, prevent, delay activation, inactivate, desensitize, or
down regulate a protein, a gene, and an mRNA stability, expression,
function and activity, e.g., antagonists.
[0058] The term "musculoskeletal diseases" as used herein are
defined as diseases or disorders affecting the joints, muscles and
bones. Examples of musculoskeletal diseases include arthritis,
gout, joint infection, and abnormalities of bones, joints and
muscles associated with systemic diseases. Musculoskeletal diseases
may be caused by trauma, infection, inflammation, genetics, or
idiopathic.
[0059] As used herein, the term "pharmaceutical composition" or
"composition" refers to a mixture of at least one compound useful
within the invention with a pharmaceutically acceptable carrier.
The pharmaceutical composition facilitates administration of the
compound to a subject.
[0060] As used herein, the term "pharmaceutically acceptable
carrier" means a pharmaceutically acceptable material, composition
or carrier, such as a liquid or solid filler, stabilizer,
dispersing agent, suspending agent, diluent, excipient, thickening
agent, solvent or encapsulating material, involved in carrying or
transporting a compound useful within the invention within or to
the patient such that it may perform its intended function.
Typically, such constructs are carried or transported from one
organ, or portion of the body, to another organ, or portion of the
body. Each carrier must be "acceptable" in the sense of being
compatible with the other ingredients of the formulation, including
the compound useful within the invention, and not injurious to the
patient. Some examples of materials that may serve as
pharmaceutically acceptable carriers include: sugars, such as
lactose, glucose and sucrose; starches, such as corn starch and
potato starch; cellulose, and its derivatives, such as sodium
carboxymethyl cellulose, ethyl cellulose and cellulose acetate;
powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa
butter and suppository waxes; oils, such as peanut oil, cottonseed
oil, safflower oil, sesame oil, olive oil, corn oil and soybean
oil; glycols, such as propylene glycol; polyols, such as glycerin,
sorbitol, mannitol and polyethylene glycol; esters, such as ethyl
oleate and ethyl laurate; agar; buffering agents, such as magnesium
hydroxide and aluminum hydroxide; surface active agents; alginic
acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl
alcohol; phosphate buffer solutions; and other non-toxic compatible
substances employed in pharmaceutical formulations. As used herein,
"pharmaceutically acceptable carrier" also includes any and all
coatings, antibacterial and antifungal agents, and absorption
delaying agents, and the like that are compatible with the activity
of the compound useful within the invention, and are
physiologically acceptable to the patient. Supplementary active
compounds may also be incorporated into the compositions. The
"pharmaceutically acceptable carrier" may further include a
pharmaceutically acceptable salt of the compound useful within the
invention. Other additional ingredients that may be included in the
pharmaceutical compositions used in the practice of the invention
are known in the art and described, for example in Remington's
Pharmaceutical Sciences (Genaro, Ed., Mack Publishing Co., 1985,
Easton, Pa.), which is incorporated herein by reference.
[0061] As used herein, the language "pharmaceutically acceptable
salt" refers to a salt of the administered compound prepared from
pharmaceutically acceptable non-toxic acids and bases, including
inorganic acids, inorganic bases, organic acids, inorganic bases,
solvates, hydrates, and clathrates thereof. Suitable
pharmaceutically acceptable acid addition salts may be prepared
from an inorganic acid or from an organic acid. Examples of
inorganic acids include sulfate, hydrogen sulfate, hydrochloric,
hydrobromic, hydriodic, nitric, carbonic, sulfuric, and phosphoric
acids (including hydrogen phosphate and dihydrogen phosphate).
Appropriate organic acids may be selected from aliphatic,
cycloaliphatic, aromatic, araliphatic, heterocyclic, carboxylic and
sulfonic classes of organic acids, examples of which include
formic, acetic, propionic, succinic, glycolic, gluconic, lactic,
malic, tartaric, citric, ascorbic, glucuronic, maleic, fumaric,
pyruvic, aspartic, glutamic, benzoic, anthranilic,
4-hydroxybenzoic, phenylacetic, mandelic, embonic (pamoic),
methanesulfonic, ethane sulfonic, benzenesulfonic, pantothenic,
trifluoromethanesulfonic, 2-hydroxyethanesulfonic,
p-toluenesulfonic, sulfanilic, cyclohexylaminosulfonic, stearic,
alginic, .beta.-hydroxybutyric, salicylic, galactaric and
galacturonic acid. Suitable pharmaceutically acceptable base
addition salts of compounds of the invention include, for example,
ammonium salts, metallic salts including alkali metal, alkaline
earth metal and transition metal salts such as, for example,
calcium, magnesium, potassium, sodium and zinc salts.
Pharmaceutically acceptable base addition salts also include
organic salts made from basic amines such as, for example,
N,N'-dibenzylethylene-diamine, chloroprocaine, choline,
diethanolamine, ethylenediamine, meglumine (N-methylglucamine) and
procaine. All of these salts may be prepared from the corresponding
compound by reacting, for example, the appropriate acid or base
with the compound.
[0062] The terms "pharmaceutically effective amount" and "effective
amount" refer to a nontoxic but sufficient amount of an agent to
provide the desired biological result. That result can be reduction
and/or alleviation of the signs, symptoms, or causes of a disease
or disorder, or any other desired alteration of a biological
system. An appropriate effective amount in any individual case may
be determined by one of ordinary skill in the art using routine
experimentation. By "pharmaceutical formulation" it is further
meant that the carrier, solvent, excipient(s) and/or salt must be
compatible with the active ingredient of the formulation (e.g. a
compound of the invention). It is understood by those of ordinary
skill in this art that the terms "pharmaceutical formulation" and
"pharmaceutical composition" are generally interchangeable, and
they are so used for the purposes of this application.
[0063] As used herein, the term "prevent," "prevention," or
"preventing" refers to any method to partially or completely
prevent or delay the onset of one or more symptoms or features of a
disease, disorder, and/or condition. Prevention is causing the
clinical symptoms of the disease state not to develop, i.e.,
inhibiting the onset of disease, in a subject that may be exposed
to or predisposed to the disease state, but does not yet experience
or display symptoms of the disease state. Prevention may be
administered to a subject who does not exhibit signs of a disease,
disorder, and/or condition.
[0064] The term "pulmonary disease" as used herein are defined as
diseases or disorders affecting the lungs and respiratory system.
Examples of pulmonary diseases include but are not limited to
chronic obstructive pulmonary disease, pulmonary fibrosis, asthma,
pulmonary hypertension, lung inflammation, and lung infection.
Pulmonary diseases may be caused by smoking, exposure to irritants,
allergy, genetics, or unknown causes (idiopathic).
[0065] As used herein, the term "subject," "patient" or
"individual" to which administration is contemplated includes, but
is not limited to, humans (i.e., a male or female of any age group,
e.g., a pediatric subject (e.g., infant, child, adolescent) or
adult subject (e.g., young adult, middle-aged adult or senior
adult)) and/or other primates (e.g., cynomolgus monkeys, rhesus
monkeys); mammals, including commercially relevant mammals such as
cattle, pigs, horses, sheep, goats, cats, and/or dogs; and/or
birds, including commercially relevant birds such as chickens,
ducks, geese, quail, and/or turkeys.
[0066] As used herein, the term "therapeutically effective amount"
is an amount of a compound of the invention, that when administered
to a patient, treats, minimizes and/or ameliorates a symptom of the
disease or disorder. The amount of a compound of the invention that
constitutes a "therapeutically effective amount" will vary
depending on the compound, the disease state and its severity, the
age of the patient to be treated, and the like. The therapeutically
effective amount can be determined routinely by one of ordinary
skill in the art having regard to his own knowledge and to this
disclosure.
[0067] The terms "treat," "treating," and "treatment," refer to
therapeutic or preventative measures described herein. The methods
of "treatment" employ administration to a subject, in need of such
treatment, a composition of the present invention, for example, a
subject afflicted a disease or disorder, or a subject who
ultimately may acquire such a disease or disorder, in order to
prevent, cure, delay, reduce the severity of, or ameliorate one or
more symptoms of the disorder or recurring disorder, or in order to
prolong the survival of a subject beyond that expected in the
absence of such treatment.
[0068] Ranges: throughout this disclosure, various aspects of the
invention can be presented in a range format. It should be
understood that the description in range format is merely for
convenience and brevity and should not be construed as an
inflexible limitation on the scope of the invention. Accordingly,
the description of a range should be considered to have
specifically disclosed all the possible subranges as well as
individual numerical values within that range. For example,
description of a range such as from 1 to 6 should be considered to
have specifically disclosed subranges such as from 1 to 3, from 1
to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as
well as individual numbers within that range, for example, 1, 2,
2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of
the range.
[0069] The following abbreviations are used herein: AAA, abdominal
aortic aneurysm; AU, arbitrary units; cpv, counts per voxel; CT,
computed tomography; DCM, dichloromethane; DTPA, diethylene
triamine pentaacetic acid, or a salt thereof; ID, injected dose;
LR, low remodeling; MAG, S-acetylmercaptoacetyl triglycine, or a
salt thereof; MAS, S-acetylmercaptoacetyltriserine, or a salt
thereof; MMP, matrix metalloproteinase; NOTA,
1,4,7-triazacyclononane-1,4,7-trisacetic acid, or a salt thereof;
PAH, pulmonary arterial hypertension; pAT, periaortic adipose
tissue; PEG, polyethylene glycol; PET, positron emission
tomography; rhMMP, recombinant human matrix metalloproteinase; SG,
salivary glands; SPECT, single-photon emission computed tomography;
.sup.99mTc-RP805, compound D in FIG. 12, or a salt or solvate
thereof; t.sub.R, retention time; WAT, white adipose tissue.
Compounds and Compositions
[0070] The present invention relates to a compound of formula I, or
a compound of formula II, or a salt, solvate, stereoisomer, or
tautomer thereof, as recited elsewhere herein.
[0071] In certain embodiments, the compound of formula I binds to
at least one MMP. In other embodiments, the compound of formula I
binds to at least one MMP and allows for MMP imaging. In yet other
embodiments, the compound of formula I is useful as imaging agents
for positron emission tomography (PET) and/or single-photon
emission computed tomography (SPECT) and/or optical imaging. In yet
other embodiments, the compound of formula I is used to diagnose
cardiovascular diseases and/or disorders, pulmonary diseases and/or
disorders, musculoskeletal diseases and/or disorders, cancer and
other diseases and disorders.
[0072] The invention provides a compound of formula I, or a salt,
solvate, stereoisomer, or tautomer thereof:
##STR00015##
R.sup.2 is selected from the group consisting of:
##STR00016##
R.sup.3 is selected from the group consisting of H, OH, OCH.sub.3,
F, .sup.18F,
##STR00017## ##STR00018##
each occurrence of n is independently an integer ranging from 0 to
30; each occurrence of R' is independently selected from the group
consisting of C.sub.1-C.sub.6 alkyl and C.sub.3-C.sub.7 cycloalkyl;
and LG is a group capable of undergoing nucleophilic displacement
(such as, but not limited to, fluoride, chloride, bromide, iodide,
mesylate, tosylate, triflate, and the like).
[0073] The invention further provides a compound of formula II or a
salt, solvate, stereoisomer, or tautomer thereof:
##STR00019##
wherein R is selected from the group consisting of H, OH, OR',
aroxy, heteroaroxy, SH, thioalkoxy, thiocycloalkoxy, --NH.sub.2,
--NHR' (such as but not limited to --NHCH.sub.3,
--NHCH.sub.2CH.sub.3, --NH(CH.sub.2).sub.2CH.sub.3 or
--NHCH(CH.sub.3).sub.2), --NR'R', --NH(aryl) and --NH(heteroaryl),
wherein each occurrence of R' is independently selected from the
group consisting of C.sub.1-C.sub.6 alkyl and C.sub.3-C.sub.7
cycloalkyl.
[0074] In certain embodiments, the compound of formula I is a
macrocyclic hydroxamate, or a salt, solvate, stereoisomer, or
tautomer thereof, selected from the group consisting of 1-18.
[0075] In certain embodiments, the labeled compound of formula I is
one selected from the group consisting of .sup.99mTc-1, M-2, and
M-3 (wherein M is a metal).
[0076] In certain embodiments, the compound of formula II is at
least one selected from the
##STR00020## ##STR00021##
[0077] In certain embodiments, each occurrence of n is
independently an integer ranging from 0 to 2, 0 to 4, 0 to 6, 0 to
8, 0 to 10, 0 to 12, 0 to 14, 0 to 16, 0 to 18, 0 to 20, 0 to 22, 0
to 24, 0 to 26, 0 to 28, 2 to 4, 4 to 6, 6 to 8, 8 to 10, 10 to 12,
12 to 14, 14 to 16, 16 to 18, 18 to 20, 20 to 22, 22 to 24, 24 to
26, 26 to 28, 28 to 30, or any interval therein.
[0078] The compounds of the invention may possess one or more
stereocenters, and each stereocenter may exist independently in
either the (R) or (S) configuration. In certain embodiments,
compounds described herein are present in optically active or
racemic forms. The compounds described herein encompass racemic,
optically active, regioisomeric and stereoisomeric forms, or
combinations thereof that possess the therapeutically useful
properties described herein. Preparation of optically active forms
is achieved in any suitable manner, including by way of
non-limiting example, by resolution of the racemic form with
recrystallization techniques, synthesis from optically active
starting materials, chiral synthesis, or chromatographic separation
using a chiral stationary phase. A compound illustrated herein by
the racemic formula further represents either of the two
enantiomers or mixtures thereof, or in the case where two or more
chiral center are present, all diastereomers or mixtures
thereof.
[0079] In certain embodiments, the compounds of the invention exist
as tautomers. All tautomers are included within the scope of the
compounds recited herein.
[0080] Compounds described herein also include isotopically labeled
compounds wherein one or more atoms is replaced by an atom having
the same atomic number, but an atomic mass or mass number different
from the atomic mass or mass number usually found in nature.
Examples of isotopes suitable for inclusion in the compounds
described herein include and are not limited to .sup.2H, .sup.3H,
.sup.11C, .sup.13C, .sup.14C, .sup.36Cl, .sup.18F, .sup.123I,
.sup.125I, .sup.13N, .sup.15N, .sup.15O, .sup.17O, .sup.18O,
.sup.32P, and .sup.35S. In certain embodiments, substitution with
heavier isotopes such as deuterium affords greater chemical
stability. Isotopically labeled compounds are prepared by any
suitable method or by processes using an appropriate isotopically
labeled reagent in place of the non-labeled reagent otherwise
employed.
[0081] In certain embodiments, the compounds described herein are
labeled by other means, including, but not limited to, the use of
chromophores or fluorescent moieties, bioluminescent labels, or
chemiluminescent labels.
[0082] In all of the embodiments provided herein, examples of
suitable optional substituents are not intended to limit the scope
of the claimed invention. The compounds of the invention may
contain any of the substituents, or combinations of substituents,
provided herein.
Methods
[0083] In one aspect, the present invention includes methods of
using compounds of formula I as MMP-targeted imaging agents. In
certain embodiments, the method comprises contacting a compound of
the invention with a cell or tissue that expresses at least one
MMP. In other embodiments, the method further comprises detecting
the compound bound to the MMP-expressing cell or tissue.
[0084] In certain embodiments, the compounds of formula I can be
used to diagnose matrix metalloproteinase related diseases and
disorders. In other embodiments, the diseases or disorders are at
least one selected from the group consisting of cancers,
inflammatory diseases, cardiovascular diseases (including valvular
diseases), periodontitis, hepatitis, cirrhosis, portal
hypertension, glomerulonephritis, atherosclerosis, emphysema,
asthma, pulmonary fibrosis, autoimmune disorders of skin and dermal
photoaging, rheumatoid arthritis, osteoarthritis, multiple
sclerosis, Alzheimer's disease, chronic ulcerations, uterine
involution and bone resorption.
[0085] In another aspect, the invention includes methods of
evaluating a subject's risk of developing a cardiovascular disease
or disorder, the method comprising administering to the subject a
compound of formula I and acquiring an image of at least a portion
of the subject's body, wherein the level of compound of formula I
bound to the imaged portion of the subject's body is measured. If
the measured level of bound compound of formula I is above a
determined control level, the subject is diagnosed as having an
increased risk of developing a cardiovascular disease or disorder.
In other embodiments, the methods of the invention allow for
tracking the effect of therapeutic interventions, adjusting the
dose or level of therapeutic intervention, and/or assessing its
effectiveness. In yet other embodiments, the methods of the
invention are used during surgery and other invasive procedures to
assess the extent of focal disease, or to characterize the disease,
e.g., to assess atherosclerotic plaque vulnerability. In yet other
embodiments, the methods of the invention identify a subject that
has yet-undiagnosed and/or early-onset disease. In yet other
embodiments, the methods of the invention stratify the subject's
disease risk.
[0086] In yet another aspect, the invention includes methods of
using a compound of formula I or II to treat MMP-related diseases.
These diseases and disorders can be at least one selected from the
group consisting of cancers, inflammatory diseases, cardiovascular
diseases, including valvular diseases, periodontitis, hepatitis,
cirrhosis, portal hypertension, glomerulonephritis,
atherosclerosis, emphysema, asthma, pulmonary fibrosis, autoimmune
disorders of skin and dermal photoaging, rheumatoid arthritis,
osteoarthritis, multiple sclerosis, Alzheimer's disease, chronic
ulcerations, uterine involution and bone resorption.
[0087] In certain embodiments, the method comprises administering
to a patient in need thereof a therapeutically effective amount of
a compound of formula I or formula II, or a salt, solvate,
stereoisomer, or tautomer thereof, wherein the compound is
optionally formulated in a pharmaceutical composition. In other
embodiments, the compound of the invention is administered by a
route comprising parenteral (such as for example intravenous),
oral, subcutaneous, and/or intradermal administration. After a
delay to allow for tracer biodistribution (which can range for 10
seconds to 3 days), uptake of the compound in the subject's tissues
can be detected and quantified by PET, SPECT, nuclear planar, or
optical imaging, or any other applicable method. The dosage range
of the compound of the invention may be, for example, from about 10
ng to 1,000 mg. In certain embodiments, the subject is a mammal. In
other embodiments, the subject is human.
Administration/Dosage/Formulations
[0088] The regimen of administration may affect what constitutes an
effective amount. The therapeutic formulations may be administered
to the subject either prior to or after the onset of a disease or
disorder contemplated in the invention. Further, several divided
dosages, as well as staggered dosages may be administered daily or
sequentially, or the dose may be continuously infused, or may be a
bolus injection. Further, the dosages of the therapeutic
formulations may be proportionally increased or decreased as
indicated by the exigencies of the therapeutic or prophylactic
situation.
[0089] Administration of the compositions of the present invention
to a patient, preferably a mammal, more preferably a human, may be
carried out using known procedures, at dosages and for periods of
time effective to treat a disease or disorder contemplated in the
invention. An effective amount of the therapeutic compound
necessary to achieve a therapeutic effect may vary according to
factors such as the state of the disease or disorder in the
patient; the age, sex, and weight of the patient; and the ability
of the therapeutic compound to treat a disease or disorder
contemplated in the invention. Dosage regimens may be adjusted to
provide the optimum therapeutic response. For example, several
divided doses may be administered daily or the dose may be
proportionally reduced as indicated by the exigencies of the
therapeutic situation. A non-limiting example of an effective dose
range for a therapeutic compound of the invention is from about 1
and 5,000 mg/kg of body weight/per day. The pharmaceutical
compositions useful for practicing the invention may be
administered to deliver a dose of from 1 ng/kg/day and 100
mg/kg/day. One of ordinary skill in the art would be able to study
the relevant factors and make the determination regarding the
effective amount of the therapeutic compound without undue
experimentation.
[0090] Actual dosage levels of the active ingredients in the
pharmaceutical compositions of this invention may be varied so as
to obtain an amount of the active ingredient that is effective to
achieve the desired therapeutic response for a particular patient,
composition, and mode of administration, without being toxic to the
patient. In particular, the selected dosage level depends upon a
variety of factors including the activity of the particular
compound employed, the time of administration, the rate of
excretion of the compound, the duration of the treatment, other
drugs, compounds or materials used in combination with the
compound, the age, sex, weight, condition, general health and prior
medical history of the patient being treated, and like factors well
known in the medical arts.
[0091] A medical doctor, e.g., physician or veterinarian, having
ordinary skill in the art may readily determine and prescribe the
effective amount of the pharmaceutical composition required. For
example, the physician or veterinarian could start doses of the
compounds of the invention employed in the pharmaceutical
composition at levels lower than that required in order to achieve
the desired therapeutic effect and gradually increase the dosage
until the desired effect is achieved.
[0092] In certain embodiments, it is advantageous to formulate the
compound in dosage unit form for ease of administration and
uniformity of dosage. Dosage unit form as used herein refers to
physically discrete units suited as unitary dosages for the
patients to be treated; each unit containing a predetermined
quantity of therapeutic compound calculated to produce the desired
therapeutic effect in association with the required pharmaceutical
vehicle.
[0093] In certain embodiments, the compositions of the invention
are formulated using one or more pharmaceutically acceptable
excipients or carriers. In other embodiments, the pharmaceutical
compositions of the invention comprise a therapeutically effective
amount of a compound of the invention and a pharmaceutically
acceptable carrier. In yet other embodiments, the compound of the
invention is the only biologically active agent (i.e., capable of
treating a disease or disorder contemplated herein) in the
composition. In yet other embodiments, the compound of the
invention is the only biologically active agent (i.e., capable of
treating a disease or disorder contemplated herein) in
therapeutically effective amounts in the composition. The carrier
may be a solvent or dispersion medium containing, for example,
water, ethanol, polyol (for example, glycerol, propylene glycol,
and liquid polyethylene glycol, and the like), suitable mixtures
thereof, and vegetable oils.
[0094] In certain embodiments, the compositions of the invention
are administered to the patient in dosages that range from one to
five times per day or more. In other embodiments, the compositions
of the invention are administered to the patient in range of
dosages that include, but are not limited to, once every day, every
two days, every three days to once a week, and once every two
weeks. It is readily apparent to one skilled in the art that the
frequency of administration of the various combination compositions
of the invention varies from individual to individual depending on
many factors including, but not limited to, age, disease or
disorder to be treated, gender, overall health, and other factors.
Thus, the invention should not be construed to be limited to any
particular dosage regime and the precise dosage and composition to
be administered to any patient is determined by the attending
physical taking all other factors about the patient into
account.
[0095] Compounds of the invention for administration may be in the
range of from about 1 .mu.g to about 10,000 mg, about 20 .mu.g to
about 9,500 mg, about 40 .mu.g to about 9,000 mg, about 75 .mu.g to
about 8,500 mg, about 150 .mu.g to about 7,500 mg, about 200 .mu.g
to about 7,000 mg, about 3050 .mu.g to about 6,000 mg, about 500
.mu.g to about 5,000 mg, about 750 .mu.g to about 4,000 mg, about 1
mg to about 3,000 mg, about 10 mg to about 2,500 mg, about 20 mg to
about 2,000 mg, about 25 mg to about 1,500 mg, about 30 mg to about
1,000 mg, about 40 mg to about 900 mg, about 50 mg to about 800 mg,
about 60 mg to about 750 mg, about 70 mg to about 600 mg, about 80
mg to about 500 mg, and any and all whole or partial increments
therebetween.
[0096] In some embodiments, the dose of a compound of the invention
is from about 1 mg and about 2,500 mg, or less than about 10,000
mg, or less than about 8,000 mg, or less than about 6,000 mg, or
less than about 5,000 mg, or less than about 3,000 mg, or less than
about 2,000 mg, or less than about 1,000 mg, or less than about 500
mg, or less than about 200 mg, or less than about 50 mg. Similarly,
in some embodiments, a dose of a second compound as described
herein is less than about 1,000 mg, or less than about 800 mg, or
less than about 600 mg, or less than about 500 mg, or less than
about 400 mg, or less than about 300 mg, or less than about 200 mg,
or less than about 100 mg, or less than about 50 mg, or less than
about 40 mg, or less than about 30 mg, or less than about 25 mg, or
less than about 20 mg, or less than about 15 mg, or less than about
10 mg, or less than about 5 mg, or less than about 2 mg, or less
than about 1 mg, or less than about 0.5 mg, and any and all whole
or partial increments thereof.
[0097] In certain embodiments, the present invention is directed to
a packaged pharmaceutical composition comprising a container
holding a therapeutically effective amount of a compound of the
invention, alone or in combination with a second pharmaceutical
agent; and instructions for using the compound to treat, prevent,
or reduce one or more symptoms of a disease or disorder
contemplated in the invention.
[0098] Formulations may be employed in admixtures with conventional
excipients, i.e., pharmaceutically acceptable organic or inorganic
carrier substances suitable for oral, parenteral, nasal,
intravenous, subcutaneous, enteral, or any other suitable mode of
administration, known to the art. The pharmaceutical preparations
may be sterilized and if desired mixed with auxiliary agents, e.g.,
lubricants, preservatives, stabilizers, wetting agents,
emulsifiers, salts for influencing osmotic pressure buffers,
coloring, flavoring and/or aromatic substances and the like. They
may also be combined where desired with other active agents.
[0099] Routes of administration of any of the compositions of the
invention include oral, nasal, rectal, intravaginal, parenteral,
buccal, sublingual or topical. The compounds for use in the
invention may be formulated for administration by any suitable
route, such as for oral or parenteral, for example, transdermal,
transmucosal (e.g., sublingual, lingual, (trans)buccal,
(trans)urethral, vaginal (e.g., trans- and perivaginally),
(intra)nasal and (trans)rectal), intravesical, intrapulmonary,
intraduodenal, intragastrical, intrathecal, subcutaneous,
intramuscular, intradermal, intraperitoneal, intra-arterial,
intravenous, intrabronchial, inhalation, and topical
administration.
[0100] Suitable compositions and dosage forms include, for example,
tablets, capsules, caplets, pills, gel caps, troches, dispersions,
suspensions, solutions, syrups, granules, beads, transdermal
patches, gels, powders, pellets, magmas, lozenges, creams, pastes,
plasters, lotions, discs, suppositories, liquid sprays for nasal or
oral administration, dry powder or aerosolized formulations for
inhalation, compositions and formulations for intravesical
administration and the like. It should be understood that the
formulations and compositions that would be useful in the present
invention are not limited to the particular formulations and
compositions that are described herein.
Oral Administration
[0101] For oral application, particularly suitable are tablets,
dragees, liquids, drops, suppositories, or capsules, caplets and
gelcaps. The compositions intended for oral use may be prepared
according to any method known in the art and such compositions may
contain one or more agents selected from the group consisting of
inert, non-toxic pharmaceutically excipients that are suitable for
the manufacture of tablets. Such excipients include, for example an
inert diluent such as lactose; granulating and disintegrating
agents such as cornstarch; binding agents such as starch; and
lubricating agents such as magnesium stearate. The tablets may be
uncoated or they may be coated by known techniques for elegance or
to delay the release of the active ingredients. Formulations for
oral use may also be presented as hard gelatin capsules wherein the
active ingredient is mixed with an inert diluent.
Parenteral Administration
[0102] As used herein, "parenteral administration" of a
pharmaceutical composition includes any route of administration
characterized by physical breaching of a tissue of a subject and
administration of the pharmaceutical composition through the breach
in the tissue. Parenteral administration thus includes, but is not
limited to, administration of a pharmaceutical composition by
injection of the composition, by application of the composition
through a surgical incision, by application of the composition
through a tissue-penetrating non-surgical wound, and the like. In
particular, parenteral administration is contemplated to include,
but is not limited to, subcutaneous, intravenous, intraperitoneal,
intramuscular, intrasternal injection, and kidney dialytic infusion
techniques.
[0103] Formulations of a pharmaceutical composition suitable for
parenteral administration comprise the active ingredient combined
with a pharmaceutically acceptable carrier, such as sterile water
or sterile isotonic saline. Formulations for parenteral
administration include, but are not limited to, suspensions,
solutions, emulsions in oily or aqueous vehicles, pastes, and
implantable sustained-release or biodegradable formulations. Such
formulations may further comprise one or more additional
ingredients including, but not limited to, suspending, stabilizing,
or dispersing agents. In one embodiment of a formulation for
parenteral administration, the active ingredient is provided in dry
(i.e., powder or granular) form for reconstitution with a suitable
vehicle (e.g., sterile pyrogen-free water) prior to parenteral
administration of the reconstituted composition.
Controlled Release Formulations and Drug Delivery Systems
[0104] In certain embodiments, the formulations of the present
invention may be, but are not limited to, short-term, rapid-offset,
as well as controlled, for example, sustained release, delayed
release and pulsatile release formulations.
[0105] The term sustained release is used in its conventional sense
to refer to a drug formulation that provides for gradual release of
a drug over an extended period of time, and that may, although not
necessarily, result in substantially constant blood levels of a
drug over an extended time period. The period of time may be as
long as a month or more and should be a release that is longer that
the same amount of agent administered in bolus form. For sustained
release, the compounds may be formulated with a suitable polymer or
hydrophobic material that provides sustained release properties to
the compounds. The compounds useful within the methods of the
invention may be administered in the form of microparticles, for
example by injection, or in the form of wafers or discs by
implantation. In one embodiment of the invention, the compounds of
the invention are administered to a patient, alone or in
combination with another pharmaceutical agent, using a sustained
release formulation.
[0106] The term delayed release is used herein in its conventional
sense to refer to a drug formulation that provides for an initial
release of the drug after some delay following drug administration
and that may, although not necessarily, includes a delay of from
about 10 minutes up to about 12 hours.
[0107] The term pulsatile release is used herein in its
conventional sense to refer to a drug formulation that provides
release of the drug in such a way as to produce pulsed plasma
profiles of the drug after drug administration.
[0108] The term immediate release is used in its conventional sense
to refer to a drug formulation that provides for release of the
drug immediately after drug administration.
[0109] As used herein, short-term refers to any period of time up
to and including about 8 hours, about 7 hours, about 6 hours, about
5 hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour,
about 40 minutes, about 20 minutes, about 10 minutes, or about 1
minute and any or all whole or partial increments thereof after
drug administration after drug administration.
[0110] As used herein, rapid-offset refers to any period of time up
to and including about 8 hours, about 7 hours, about 6 hours, about
5 hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour,
about 40 minutes, about 20 minutes, about 10 minutes, or about 1
minute and any and all whole or partial increments thereof after
drug administration.
Dosing
[0111] A suitable dose of a compound of the present invention may
be in the range of from about 0.01 mg to about 5,000 mg per day,
such as from about 0.1 mg to about 1,000 mg, for example, from
about 1 mg to about 500 mg, such as about 5 mg to about 250 mg per
day. The dose may be administered in a single dosage or in multiple
dosages, for example from 1 to 5 or more times per day. When
multiple dosages are used, the amount of each dosage may be the
same or different. For example, a dose of 1 mg per day may be
administered as two 0.5 mg doses, with about a 12-hour interval
between doses. The amount of compound dosed per day may be
administered, in non-limiting examples, every day, every other day,
every 2 days, every 3 days, every 4 days, or every 5 days. For
example, with every other day administration, a 5 mg per day dose
may be initiated on Monday with a first subsequent 5 mg per day
dose administered on Wednesday, a second subsequent 5 mg per day
dose administered on Friday, and so on.
[0112] In the case wherein the patient's status does improve, upon
the doctor's discretion the administration of the inhibitor of the
invention is optionally given continuously; alternatively, the dose
of drug being administered is temporarily reduced or temporarily
suspended for a certain length of time (i.e., a "drug holiday").
The length of the drug holiday optionally varies between 2 days and
1 year, including by way of example only, 2 days, 3 days, 4 days, 5
days, 6 days, 7 days, 10 days, 12 days, 15 days, 20 days, 28 days,
35 days, 50 days, 70 days, 100 days, 120 days, 150 days, 180 days,
200 days, 250 days, 280 days, 300 days, 320 days, 350 days, or 365
days. The dose reduction during a drug holiday includes from
10%-100%, including, by way of example only, 10%, 15%, 20%, 25%,
30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,
95%, or 100%.
[0113] Once improvement of the patient's conditions has occurred, a
maintenance dose is administered if necessary. Subsequently, the
dosage or the frequency of administration, or both, is reduced, as
a function of the disease or disorder, to a level at which the
improved disease is retained. In certain embodiments, patients
require intermittent treatment on a long-term basis upon any
recurrence of symptoms and/or infection.
[0114] Toxicity and therapeutic efficacy of such therapeutic
regimens are optionally determined in cell cultures or experimental
animals, including, but not limited to, the determination of the
LD.sub.50 (the dose lethal to 50% of the population) and the
ED.sub.50 (the dose therapeutically effective in 50% of the
population). The dose ratio between the toxic and therapeutic
effects is the therapeutic index, which is expressed as the ratio
between LD.sub.50 and ED.sub.50. The data obtained from cell
culture assays and animal studies are optionally used in
formulating a range of dosage for use in human. The dosage of such
compounds lies preferably within a range of circulating
concentrations that include the ED.sub.50 with minimal toxicity.
The dosage optionally varies within this range depending upon the
dosage form employed and the route of administration utilized.
[0115] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, numerous
equivalents to the specific procedures, embodiments, claims, and
examples described herein. Such equivalents were considered to be
within the scope of this invention and covered by the claims
appended hereto. For example, it should be understood, that
modifications in reaction conditions, including but not limited to
reaction times, reaction size/volume, and experimental reagents,
such as solvents, catalysts, pressures, atmospheric conditions,
e.g., nitrogen atmosphere, and reducing/oxidizing agents, with
art-recognized alternatives and using no more than routine
experimentation, are within the scope of the present
application.
[0116] The following examples further illustrate aspects of the
present invention. However, they are in no way a limitation of the
teachings or disclosure of the present invention as set forth
herein.
EXAMPLES
[0117] The invention is now described with reference to the
following Examples. These Examples are provided for the purpose of
illustration only, and the invention is not limited to these
Examples, but rather encompasses all variations that are evident as
a result of the teachings provided herein.
Materials and Methods
Mouse Model of Carotid Aneurysm
[0118] Arterial aneurysm was induced by exposing the left common
carotid artery of apolipoprotein E-deficient (apoE.sup.-/-) mice to
calcium chloride. Briefly, 4- to 6-wk-old female apoE.sup.-/- mice
(n=96; Jackson Laboratory) were fed high-cholesterol chow ad
libitum. After 1 wk, the carotid arteries were exposed by blunt-end
dissection under anesthesia. The left common carotid artery just
below the carotid bifurcation was adventitially exposed to a 10%
solution of CaCl.sub.2 for 20 min. The opposite carotid artery was
exposed to normal saline and served as a control for imaging
studies (Razavian, et al., 2010, J Nucl Med. 51 (7):1107-15).
[0119] In addition, sixteen weeks-old male apoE.sup.-/- (n=16) were
infused with human angiotensin II (Ang II, 1000 ng/kg/min,
Calbiochem), delivered by a subcutaneous osmotic minipump (Model
2004, Alzet) implanted under anesthesia (isoflurane 2%).
Mouse Model of PAH
[0120] Six to eight week old C57BL/6J mice of either sex were
exposed to chronic hypoxia (10%) in a hypoxia chamber for up to 4
weeks. Age and sex-matched normoxic mice were used as control.
Affinity and Selectivity Profile Assessment
[0121] MMPs were purchased from R&D Systems (Minneapolis,
Minn.). MMP inhibition assays were carried out in 50 mM Tris/HCl
buffer, pH=6.8, 10 mM CaCl.sub.2 at 25.degree. C. Pro-MMPs were
pre-activated by p-aminophenylmercuric acetate (described in Devel,
et al., 2006, J. Biol. Chem. 281:11152-11160). Titration
experiments were carried out to determine active enzyme
concentration for each MMP prior to the assay. For each probe, the
% of inhibition was determined from five different concentrations
in triplicates, chosen to reach a range of 20-80% inhibition.
K.sub.i values were determined using the method described in
Horovitz & Levitzki, 1987, Proc. Natl. Acad. Sci. USA
84:6654-6658.
Probe Stability Assessment
[0122] Tracers were incubated in blood (200 .mu.L) with gentle
agitation (Thermo mixer, 1000 rpm) at 37.degree. C. for 4 h. At 0,
2, and 4 h time points, 40 .mu.L of blood sample were collected and
centrifuged at 4.degree. C. (3000 rpm, 20 min). 10 .mu.L of the
supernatant (plasma) were collected and diluted in methanol (90
.mu.L). The sample was then vortexed for 30 s at room temperature
and centrifuged at 4.degree. C. (3000 rpm, 15 min). 50 .mu.L of the
supernatant were concentrated, filtered and analysed by radioHPLC
or LC-MS.
Biodistribution
[0123] For comparison of .sup.99mTc-1 and .sup.99mTc-RP805
biodistribution, C57BL/6J mice were injected intravenously with
16.+-.5 MBq of either .sup.99mTc-1 (n=5) or .sup.99mTc-RP805 (n=5).
To investigate biodistribution in apoE.sup.-/- mice, animals at
seven weeks after peri-adventitial application of CaCl.sub.2 or
NaCl to carotid arteries were injected with 31.+-.14 MBq (14.+-.2
pg/kg) of .sup.99mTc-1, with (n=5) or without (n=6) pre-injection
of an excess of the parent inhibitor 19 (23.+-.8 ng/kg). Animals
were kept under anesthesia for 60 min and blood samples were
collected at various times post-injection. Tissue samples and body
fluids were collected and weighed at 2 hours post-injection, and
measured for their radioactivity by gamma-well counting
(WIZARD2.RTM., PerkinElmer). Data were expressed as percentage of
injected dose (ID) per gram of tissue or mL of blood.
Autoradiography
[0124] For autoradiography, the specimens were mounted on a board
and placed in contact with to a reusable phosphor screen
(MultiSensitive Phosphor Screen, PerkinElmer) along with standards
of known activity. The screen was scanned in a phosphor imager
system (Typhoon Trio, GE Healthcare Life Sciences) at a pixel size
of 25.times.25 .mu.m and images were analyzed using the Fiji
software. Tracer uptake was quantified using a standard curve and
expressed a % injected dose (ID)/pixel.
Quantitative Autoradiography
[0125] In apoE.sup.-/- mice at seven weeks after peri-adventitial
application of CaCl.sub.2 or NaCl to carotid arteries, the aorta
and carotid arteries were dissected from surrounding adherent
tissues under a stereoscopic microscope (MZ9.5, Leica) at 2 hours
post-tracer injection. The tissues were placed on a phosphor screen
(MultiSensitive Phosphor Screen, PerkinElmer) along with standards
of known activity. The phosphor screen was scanned with a phosphor
imager (Typhoon Trio, GE Healthcare Life Sciences), and the
digitalized images were quantified by drawing regions of interest
around various tissues to determine tissue activity (Fiji/ImageJ
software, NIH).
MicroSPECT/CT
[0126] Animals were imaged using a dedicated high resolution small
animal imaging system (X-SPECT, GammaMedica), using one mm low
energy (for .sup.99mTc) pinhole collimators. The spatial resolution
of this system for .sup.99mTc in tomographic images with a 1-mm
pinhole collimator and 4 cm radius of rotation was 1.1 mm
full-width half-maximum. Radiotracer (1 mCi for mice) was injected
through an intravenous catheter. Anesthetized mice (with
isoflurane) were placed in a fixed position under the camera. Three
point sources of known activity (.about.1 .mu.Ci) were placed in
the field of view, but outside the body, to quantify tracer uptake
and to verify the accuracy of image fusions. One hour after tracer
administration microSPECT imaging was performed in a step and shoot
manner, using 1 mm pinhole collimators and the following
acquisition parameters empirically optimized for similar imaging
studies: 180.degree., 64 projections, 30 sec/projection (.about.40
minute image acquisition), matrix 82.times.82, 140 keV
photopeak.+-.10% window.
[0127] After completion of microSPECT images CT imaging was
performed (energy: 75 kV/280 .mu.A, matrix: 512.times.512) to
identify anatomical structure. Compared with the microSPECT system,
the CT system had a larger field of view, with a spatial resolution
of .about.60 .mu.m. After in vivo imaging, the tissues were
harvested and placed on a holder, and planar imaging was performed
using 1 mm pinhole low energy high resolution collimators. Images
were analyzed using Xeleris Functional Imaging Workstation (General
Electric, Waukesha, Wis.). Several regions of interest (ROIs) were
placed over selected organs to calculate average activity/pixel.
Activities of each organ was expressed as % ID using the point
sources.
[0128] CT projection images were reconstructed using commercial
software (Cobra, Exxim Computing Corp., Pleasanton, Calif.), that
implement a cone-beam reconstruction algorithm. MicroSPECT images
were reconstructed through iterative reconstruction (5 iterations,
4 subsets) using system software and filtered post-reconstruction
using Butterworth filter (cut-off: 0.5 Nyquist frequency, order:
6). SPECT images were smoothed by a low-pass Butterworth filter to
reduce random noise. Reconstructed microSPECT images were
reoriented according to the CT anatomical images, fused, and
exported in "Analyze" format (Analyze, Mayo Clinic, Rochester, USA)
for further processing using Amide (a Medical Imaging Data
Examiner, amide.sf.net). ROIs were drawn around the aorta and other
organs and the uptake was measured and expressed as counts per
voxel and converted to %ID using point sources of known activity
placed in the field of view.
Tissue Analysis
[0129] After microSPECT/CT imaging, the supra-renal abdominal aorta
was rapidly cleaned from adherent tissues under stereoscopic
microscope, and was frozen in OCT. In order to determine the
maximal external diameter, 5 .mu.m-thick serial sections of the
abdominal aorta (typically 10 sections, 200-300 .mu.m apart) were
used for morphometric analysis (Fiji/ImageJ software, NIH) after
hematoxylin and eosin staining. Adjacent tissues were processed to
extract protein and RNA at the sites of maximum uptake observed on
SPECT images, identified based on anatomical landmarks.
Zymography
[0130] Aortic tissue was lysed in a lysis buffer (NaCl 0.3 M, Tris
50 mM, Triton X-100 1%, cOmplete.TM. Protease Inhibitor Cocktail,
Sigma-Aldrich), and protein concentration was measured using a
colorimetric assay (Protein Assay Dye Reagent Concentrate, Bio-Rad;
BioMate 3, Thermo Scientific). MMP activity was assessed in 1 .mu.g
of protein lysate using a fluorometric zymography assay
(SENSOLYTE.RTM. 520 Generic MMP Activity Kit, AnaSpec), according
to the manufacturer's instructions and presented in relative
arbitrary units.
Quantitative Reverse Transcription Polymerase Chain Reaction
[0131] RNA was isolated from aortic tissue using GenElute Mammalian
Total RNA Miniprep Kit (Sigma-Aldrich) and reverse-transcribed
using QuantiTect Reverse Transcription Kit (Qiagen). Quantitative
reverse transcription polymerase chain reaction analysis was
performed with a 7500 Real-Time PCR System (Applied Biosystems)
using the following primers and probe sets (MMP-2: Mm00439498_m1;
MMP-9: Mm00442991_m1; MMP-12: Mm0050054_m1; CD68: Mm03047343_m1;
.beta.-actin: Mn00607939_s1, TaqMan Gene Expression Assays, Thermo
Fisher Scientific), according to the manufacturers' instructions.
CD68 and MMP gene expression were normalized to .beta.-actin.
Mass Spectroscopy and NMR
[0132] Mass spectra were recorded using electrospray ionization
(ESI+/-) or Q-TOF high-resolution mass analyzers (Agilent). .sup.1H
and .sup.13C NMR data were obtained using a 400 MHz spectrometer
(Agilent), and TMS was used as an internal reference; chemical
shifts were reported in parts per million (.delta.).
Radiochemistry and Stability Analysis
[0133] Radiolabeling quality control was performed by reverse phase
radio-HPLC analysis (HPLC system 2489, Waters) with a flow rate of
1 mL/min using an analytical column (JUPITER.RTM. 4 .mu.m Proteo 90
.ANG., Phenomenex) with gradients of solvent A (0.16% ammonium
formate in aqueous solution) and solvent B (0.16% ammonium formate
in 90% acetonitrile). The HPLC gradients were programmed as
follows: 10% B for 2 min, 10-70% B in 5 min, 5 min 70% B and 70-90%
B in 5 min. In vitro and in vivo stability of the tracer were
analyzed by radio-HPLC following in vitro incubation in mouse blood
at 37.degree. C. for up to 5 hours, and from urine collected at 2
hours after tracer injection, respectively.
Statistical Analysis
[0134] All data are presented as mean.+-.standard deviation.
Mann-Whitney U test was used to compare data sets from two
experimental groups. Two-way analysis of variance with post-hoc
Bonferroni correction was used to compare blood activity between
groups. Spearman's rank correlation was used to assess the
significance of correlations (Prism 7, GraphPad). A P-value below
0.05 was considered statistically significant.
Example 1: Synthesis of 1, and Corresponding .sup.99mTc Complex
(.sup.99mTc-1)
##STR00022##
[0136] Synthesis of 1 used an anti-2,3-disubstituted succinic acid
derivative, i.e.
(2R,3S)-3-(tert-butoxycarbonyl)-2-iso-butylhex-5-enoic acid, with a
protected macrocyclic acid 1-7 as a key intermediate (Scheme 1).
1-7 was reacted with an Arg fragment to give I-14 (Scheme 2). I-14
was further conjugated with Boc-HYNIC to afford 1 (Scheme 4).
##STR00023## ##STR00024##
Intermediate 1 (I-1)
[0137] To a stirred solution of
(2R,3S)-3-(tert-butoxycarbonyl)-2-iso-butylhex-5-enoic acid (10.0
g, 95%, 35.1 mmol) and benzyl bromide (15 g, 85.9 mmol) in toluene
(40 mL), were added dropwise 13.0 g (86 mmol)
1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) in 20 mL toluene. The
resulting mixture was stirred at room temperature for 2 h and then
at 60.degree. C. for 1 h. The toluene solution was separated from
the precipitated solid residue. The residue was dissolved in water
(20 mL), and extracted with ethyl acetate (20 mL.times.3). The
combined toluene and ethyl acetate solution (100 mL) was washed
with 1 N HCl (20 mL.times.2), water (20 mL.times.2) and brine (20
mL.times.2). It was dried over anhydrous MgSO.sub.4 overnight,
filtered and concentrated under vacuum. The resulting residue was
purified by chromatography (silica gel, hexanes/ethyl acetate) to
afford 9.0 g (71%) of the title compound as oil. ES-MS: Observed
[MH].sup.+ 360.1, [MNa].sup.+ 383.2.
Intermediate 2 (I-2)
[0138] To a stirred solution of I-1 (9.0 g, 24.9 mmol) in 30 mL
anhydrous THF cooled in an ice bath, was added dropwise 9-BBN in
THF (200 mL, 100 mmol) over a period of 30 min. The mixture was
further stirred at room temperature overnight. 10 mL water were
added dropwise after the solution was cooled in an ice bath. A
solution of 9.9 g NaOAc in water (30 mL) was added, followed by
adding 30% H.sub.2O.sub.2 (30 mL) dropwise. The mixture was stirred
at room temperature for 60 min and concentrated under vacuum. The
resulting aqueous solution was extracted with ethyl acetate (20
mL.times.4). The combined toluene and ethyl acetate solution (100
mL) was washed with 1 N HCl (20 mL.times.2), water (20 mL.times.2),
and brine (20 mL.times.2). After it was dried over anhydrous
MgSO.sub.4 overnight, the solution was filtered and concentrated
under vacuum. The resulting residue was purified by silica gel
chromatography (silica gel, ethyl acetate/hexanes) to give the
title compound (7.2 g, 70%). ES-MS: observed [MH-tert-butyl].sup.+
323.0, [MNa].sup.+ 401.1, [MH-tert-butyl-H.sub.2O].sup.+ 305.2.
Intermediate 3 (I-3)
[0139] To a stirred solution of I-2 (7.0 g, 19.0 mmol) and
CBr.sub.4 (12.6 g, 38 mmol) in anhydrous DCM (30 mL), was added
Ph.sub.3P (10.4 g, 40 mmol) in small portions for 30 min. The
resulting mixture was stirred at room temperature for another 2 h,
followed by adding 30 mL hexanes. The resulting mixture was
transferred to a short column of silica gel for quick elution using
DCM and hexanes (1:1). The desired fractions were combined and
concentrated to get a crude product which was further purified by
column chromatography (silica gel, hexanes/ethyl acetate). 5.1 g
(63%) of the title compound was obtained. ES-MS: Observed double
peak [MNa].sup.+ 463.0/465.2, [M-tert-butyl].sup.+ 385.0/387.0.
Intermediate 4 (I-4)
[0140] A mixture of I-3 (5.0 g, 11.3 mmol), Pd/C (2.0 g, 10%, wet)
and HCOONH.sub.4 (5 g) in 30 mL methanol was stirred at room
temperature until the hydrogen gas evolved was observed. The
mixture was stirred for another 10 min, filtered, followed by
washing the Pd/C with methanol (5 mL.times.4). The combined
methanol filtrate was concentrated and the residue was acidified
with 1 N HCl solution. The product was extracted with ethyl acetate
(20 mL.times.3). The solution was washed with water (10 mL.times.2)
and brine (10 mL.times.2), dried over anhydrous MgSO.sub.4,
filtered, and concentrated. The product (3.1 g, 80%) obtained was
used for the next step without further purification. ES-MS:
Observed double peak [MNa].sup.+ 373.0/375.0,
[M-tert-butyl-H.sub.2O].sup.+ 277.0/279.0 (100%),
[MNa-tert-butyl].sup.+ 318.0/320.0.
Intermediate 5 (I-5)
[0141] To a stirred solution of I-4 (3.0 g, 8.5 mmol), HOBT (1.7 g,
12.7 mmol), and Tyr-OBz (3.4 g, 12.7 mmol) in 20 mL anhydrous DMF
cooled in ice bath, was added EDCI (2.4 g, 12.7 mmol). The
resulting mixture was stirred at room temperature for another 2.5
h. The resulting mixture was concentrated under high vacuum and
re-dissolved with ethyl acetate. The ethyl acetate solution was
washed with water, 1 N HCl, water, 1 N Na.sub.2CO.sub.3 solution,
water, and brine. The solution was dried over anhydrous MgSO.sub.4,
filtered, and concentrated under vacuum. The resulting residue was
purified by chromatography (silica gel, ethyl acetate/hexanes). 3.8
g (74%) of the title compound was obtained. ES-MS: Observed
[MNa].sup.+ double peak 626.0/628.0 and[MH].sup.+ 604.2/606.0.
.sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 0.8 (6H, d, J=8 Hz), 1.01
(1H, m), 1.24 (2H, m), 1.45 (9H, s), 1.60-1.77 (4H, m), 2.36 (2H,
m), 2.97 (1H, m), 3.09 (1H, m), 3.21 (1H, m), 3.35 (1H, m), 4.97
(1H, m), 5.16 (2H, m), 5.50 (1H, s), 6.01 (1H, m), 6.50-7.37 (9H,
aromatic H); .sup.13C NMR (100 MHz, CDCl.sub.3) .delta. 21.52,
23.96, 25.85, 28.21, 29.77, 30.60, 33.28, 37.64, 40.69, 48.42,
49.17, 53.15, 67.54, 81.41, 115.72, 127.70, 128.73, 128.75, 128.79,
130.51, 135.14, 155.09, 171.58, 173.42, 173.68.
Intermediate 6 (I-6)
[0142] To a stirred solution of Cs.sub.2CO.sub.3 (3.2 g, 23.4 mmol)
in 500 mL anhydrous acetonitrile at 60.degree. C., was added
dropwise a solution of I-5 (3.0 g, 5.3 mmol) in 50 mL over a period
of 1 h. The resulting mixture was stirred at 60.degree. C. for
another 3 h and concentrated under vacuum. The product was
redissolved with ethyl acetate and filtered, followed by washing
the solid with ethyl acetate for 5 times (10 mL.times.5). The
combined ethyl acetate filtrate was washed with 1 N HCl solution,
water, and brine. The solution was dried over anhydrous MgSO.sub.4,
filtered, and concentrated. The residue was purified by silica gel
chromatography using (silica gel, DCM-CH.sub.3OH) to yield the
product (1.6 g, 60%). ES-MS: observed [MH].sup.+ 524.2, [MNa].sup.+
546.2, [MH-tert-butyl].sup.+ 468, [MCs].sup.+ 656.2; .sup.1H NMR
(400 MHz, CDCl.sub.3) .delta. -0.47 (1H, m), 0.61 (1H, m), 0.75
(6H, m), 0.81 (1H, m), 1.21-1.55 (4H, m), 1.37 (9H, s), 1.86 (1H,
m), 2.02 (1H, m), 2.52 (1H, m), 3.57 (1H, m), 4.04 (1H, m), 4.2
(1H, m), 5.11-5.24 (3H, m), 5.51 (1H, m), 6.94-7.40 (9H, aromatic
H); .sup.13C NMR (100 MHz, CDCl.sub.3) .delta. 21.29, 24.05, 25.61,
28.20, 29.93, 31.20, 37.85, 40.55, 49.45, 50.01, 51.61, 67.56,
73.91, 80.81, 120.52, 123.59, 128,67, 128.77, 128.83, 129,87,
131.44, 132.01, 135.21, 159.38, 171.73, 173.11, 174.01.
Intermediate 7 (I-7)
[0143] A mixture of I-6 (5.0 g, 9.5 mmol), 10% Pd/C (2.0 g), and
HCOONH.sub.4 (5 g) in 15 mL CH.sub.3OH was stirred at room
temperature for 2-3 h until the hydrogen gas evolved was observed.
The mixture was further stirred for another 20 min and filtered,
followed by washing the Pd/C with CH.sub.3OH (5 mL.times.4). The
methanol filtrate was concentrated and acidified with 1N HCl
solution to get a residue, which was extracted with ethyl acetate.
The solution was washed with water (10 mL.times.2) and brine (10
mL.times.2). The solution was dried over anhydrous MgSO.sub.4,
filtered, and concentrated. The product (3.7 g, 90%) was used for
the next step without further purification. LC-MS: Observed
[MH].sup.+ 434.2, [MNa].sup.+ 456.2.
##STR00025## ##STR00026##
Intermediate 8 (I-8)
[0144] A mixture of 1.2 g Fmoc-Arg(Mtr)-OH (1.97 mmol), 0.32 g
(2.36 mmol) HOBT, and 0.38 g (2.36 mmol)
NH.sub.2CH.sub.2CH.sub.2NHBoc was dissolved in 5 mL anhydrous DMF
and cooled at 0.about.5.degree. C. in ice bath, followed by adding
543.0 mg EDCI (2.83 mmol). After stirred at room temperature
overnight, the mixture was concentrated under high vacuum. The
residue was triturated with 1 N HCl, filtered, and washed with 1 N
HCl, 1 N Na.sub.2CO.sub.3, and H.sub.2O. The solid product was
dried to give 1.3 g (90%) of the title compound. ES-MS: Observed
[MH].sup.+ 529.6.
Intermediate 9 (I-9)
[0145] 1.0 g I-8 (1.33 mmol) was dissolved in 7 mL DCM and 3 ml
piperidine. After stirred at room temperature for 30 min, the
mixture was concentrated under high vacuum. The residue was
purified by flash column chromatography using DCM and methanol to
give 0.52 g (75%) of the title compound. ES-MS: Observed
[MH-Boc].sup.+ 429.2.
Intermediate 10 (I-10)
[0146] A mixture of 200.0 mg (0.46 mmol) I-9, 291.0 mg (0.55 mmol)
H-Arg(Mtr)-NHCH.sub.2CH.sub.2NH-Boc, and 74.8 mg (0.55 mmol) HOBT
was dissolved in 5 mL DMF and cooled at 0.about.5.degree. C. in an
ice bath, followed by adding 126.5 mg (0.66 mmol) EDCI. The mixture
was stirred at room temperature overnight and concentrated. The
residue was dissolved in 10 mL CH.sub.2Cl.sub.2, washed with 1N
HCl, H.sub.2O, and brine. The DCM solution was dried over
MgSO.sub.4, filtered, and concentrated. The product was purified by
flash column chromatography using DCM and methanol to give 260 mg
(60%) of the title compound. ES-MS: Observed [MH-Boc].sup.+ 845.8,
[MH.sub.2-tert-butyl].sup.2+ 394.7.
Intermediate 11 (I-11)
[0147] 0.2 g (0.21 mmol) I-10 were dissolved in 8 mL TFA and 2 mL
DCM. After stirred at room temperature for 2 h, the solution was
concentrated and dried under vacuum to give 0.18 g (98%) of the
title compound. ES-MS: Observed [MH].sup.+ 788.3, [MH.sub.2].sup.2+
394.8.
Intermediate 12 (I-12)
[0148] 0.15 g (0.16 mmol) I-11 were dissolved in 5 mL dioxane and 5
mL 1 N Na.sub.2CO.sub.3 solution. The mixture was stirred in ice
bath, followed by adding 70.0 mg (0.32 mmol) (Boc).sub.2O. After
stirred at room temperature for 2 h, the solution was concentrated
and the residue was acidified with 1 N HCl to pH 3. The product was
extracted with DCM, washed with water, and dried over MgSO.sub.4.
The filtrate was concentrated to give the title compound (135.0 mg,
95%). ES-MS: Observed [MH].sup.+ 888.3.
Intermediate 13 (I-13)
[0149] A mixture of 100.0 mg (0.11 mmol) I-12, 30.4 mg (0.22 mmol)
HOAT, and 83.6 mg (0.22 mmol) HATU, and 57.0 mg (0.44 mmol) DIEA
was dissolved in 5 mL anhydrous DMF. The mixture was stirred at
room temperature for 20 min, followed by adding 147.2 mg (1.0 mmol)
TBDMS-ONH.sub.2. The mixture was stirred at room temperature
overnight and concentrated under vacuum. The residue was triturated
with 1 N HCl, filtered, and washed with water, 1 N
Na.sub.2CO.sub.3, and brine. The product was further purified by
column chromatography to give 30.0 mg (30%) of the title compound.
ES-MS: Observed [MH].sup.+ 903.2, [MNa].sup.+ 925.4.
Intermediate 14 (I-14)
[0150] 20 mg (0.022 mmol) I-13 was dissolved in a mixture of 9.0 mL
TFA, 0.5 mL H.sub.2O, 0.25 mL TIS, and 0.25 g phenol. The mixture
was stirred at room temperature overnight and concentrated under
vacuum. The residue was added into 10 mL cooled diethyl ether. The
precipitated product was collected by centrifugation and purified
by semi-preparative HPLC using an eluent of aqueous acetonitrile.
The desired fractions were collected and lyophilized to give the
title compound (5.4 mg, 30%). ES-MS: Observed [MH].sup.+ 591.4,
[MH.sub.2].sup.2+ 296.3, [M.sub.2H.sub.3].sup.3+ 394.6.
##STR00027##
Intermediate 15 (I-15)
[0151] A mixture of 6-chloronicotinic acid (10.0 g, 63.4 mmol),
hydrazine (20 mL), and water (20 mL) was refluxed at
95.about.100.degree. C. for 4 h. The mixture was concentrated under
vacuum and the residue was dissolved in 40 mL water. The resulting
solution was acidified with 1 N HCl to reach a pH 5.about.5.5. The
solution was kept in a refrigerator overnight. The precipitated
solid was collected by filtration, washed with cold water (10
mL.times.2) and ether (10 mL.times.2). The solid was dried to give
8.5 g (87%) of the title compound. ES-MS: Observed [MH].sup.+
154.1.
Intermediate 16 (I-16)
[0152] A mixture of 4.0 g I-15 and 8.0 g Na.sub.2CO.sub.3 was
stirred in 60 mL 1,4-dioxane and 60 mL water and cooled in ice
bath. 8.2 g Boc.sub.2O were added, and the mixture was further
stirred at room temperature for 5 h. The mixture was concentrated
and the solid residue was acidified with 1 N HCl solution to get
some precipitated solid which was collected by filtration and dried
to give the title compound.
Intermediate 17 (I-17)
[0153] A mixture of 10.0 mg (0.039 mmol) 1-16, 5.5 mg HOAT (0.039
mmol), and 15.0 mg HATU (0.039 mmol) was dissolved in 1 mL
anhydrous DMF, followed by adding 10.0 mg (0.078 mmol) DIEA. The
mixture was stirred at room temperature for 30 min. To the
resulting solution was added a solution of 16.0 mg (0.0195 mmol)
I-14 and 5 .mu.L (3.71 mg, 0.028 mmol) DIEA in 0.5 mL anhydrous
DMF. The resulting solution was stirred at room temperature for 1 h
and concentrated under vacuum. The residue was triturated with 1 N
HCl, filtered, and washed with 1 N Na.sub.2CO.sub.3 and water. The
solid product was used in the next reaction without further
purification. ES-MS: Observed [MH].sup.+ 826.2, [MH.sub.2].sup.2+
413.6.
##STR00028##
Compound 1
((6S,7R,10S)-N.sup.10-((S)-5-guanidino-1-((2-(6-hydrazinylnicotinamido)et-
hyl)
amino)-1-oxopentan-2-yl)-N.sup.6-hydroxy-7-isobutyl-8-oxo-2-oxa-9-aza-
-1(1,4)-benzenacyclo undecaphane-6,10-dicarboxamide)
[0154] I-17 was dissolved in a mixture of 4.5 mL TFA, 0.25 mL
H.sub.2O, 0.125 mL TIS, and 0.125 g phenol. The mixture was stirred
at room temperature for 30 min and concentrated under vacuum. The
residue was added into 10 mL cooled diethyl ether. The precipitated
product was collected by centrifugation and purified by
semi-preparative HPLC using an eluent of aqueous acetonitrile. The
desired fractions were collected and lyophilized to give the title
compound (3.0 mg, 16%) as confirmed by both LC-MS and analytical
HPLC. ES-MS: Observed [MH].sup.+ 726.3, [MH.sub.2].sup.2+
363.7.
Compound .sup.99mTc-1
##STR00029##
[0156] 1 was labeled with .sup.99mTc by heating .sup.99mTc in a
vehicle solution containing tricine and
3,3',3''-phosphanetriyltris(benzenesulfonic acid) trisodium salt
(TPPTS) in high purity and yield. Typically, 2 .mu.g of 1 was mixed
5.about.10 mCi/100 .mu.L .sup.99mTcO.sub.4, followed by addition of
with 200 .mu.L of the vehicle solution in a vial. The mixture was
heated at 95.degree. C. for 10 min, and cooled to room temperature
to yield the .sup.99mTc-labeled product as analyzed by radio-HPLC.
Radio-HPLC analysis was performed using Waters RP-HPLC (Milford,
Mass.) on a reverse-phase analytical column (Phenomenex, Jupiter
4.mu. Proteo 90A, 25033 4.6 mm, 4 micron) with a gradient from 10%
to 70% aqueous acetonitrile containing 25 mM ammonium formate at a
flow rate of 1 mL/min over 40 min.
Example 2: Synthesis of 2, and Corresponding Cu Complex (Cu-2)
##STR00030##
[0157]
(S)-1-amino-14-(2-(bis(carboxymethyl)amino)ethyl)-17-(carboxymethyl-
)-6-((6S,7R,10S)-6-(hydroxycarbamoyl)-7-isobutyl-8-oxo-2-oxa-9-aza-1(1,4)--
benzenacycloundecaphane-10-carboxamido)-1-imino-7,12-dioxo-2,8,11,14,17-pe-
ntaazanonadecan-19-oic acid
##STR00031##
[0158] Intermediate 18 (I-18)
[0159] DTPA-tetra (t-Bu ester) (20 mg, 0.032 mmol), HOAT (5.2 mg,
0.038 mmol), HATU (14.4 mg, 0.038 mmol), and DIEA (10.0 mg, 0.076
mmol) was mixed in 2 mL anhydrous DMF. The mixture was stirred at
room temperature for 30 min, followed by addition of a solution of
I-14 (20 mg, 0.02 mmol) and DIEA (5 .mu.L) in 0.5 mL DMF. The
mixture was stirred at room temperature for 3 h and concentrated
under vacuum. The residue was triturated with water, filtered, and
washed sequentially with 1 N HCl, water, 1 N Na.sub.2CO.sub.3, and
brine. The solid collected was used in the next reaction without
further purification. ES-MS: Observed [MH].sup.+ 1190.6,
[MNa].sup.+ 1212.6, [MH.sub.2].sup.2+ 595.8,
[MH.sub.2-tert-butyl].sup.2+ 567.8.
Compound 2
[0160] I-18 was dissolved in a mixture of 9.0 mL TFA, 0.5 mL
H.sub.2O, 0.25 mL TIS, and 0.25 g phenol. The mixture was stirred
at room temperature overnight and concentrated under vacuum. The
residue was added to 10 mL diethyl ether and cooled. The
precipitate was collected by centrifugation and purified by
semi-preparative HPLC using gradient elution with aqueous
acetonitrile. The desired fractions were identified by LC-MS,
combined, and lyophilized to give the title compound 2 (6.0 mg,
21%) as confirmed by both LC-MS and analytical HPLC. ES-MS:
Observed [MH].sup.+ 966.4, [MH.sub.2].sup.2+ 483.8,
[MH.sub.3].sup.3+ 322.8.
Preparation of Cu-2 Complex
##STR00032##
[0162] DTPA conjugate 2 (30 .mu.g) was dissolved in 50%
acetonitrile (30 .mu.L). To this solution were added ammonia (1
.mu.L) and CuCl.sub.2 (5 .mu.g) in 50% acetonitrile (30 .mu.L). The
mixture was swirled at room temperature for 30 min. LC-MS analysis
confirmed the Cu-complexation with 2: [MCuH.sub.2].sup.2+ 514.2,
[MCuH.sub.3].sup.3+ 343.2.
Example 3: Synthesis of 3, and its Corresponding Cu-3
##STR00033##
[0163]
2,2'-(7-(2-((2-((S)-5-guanidino-2-((6S,7R,10S)-6-(hydroxycarbamoyl)-
-7-isobutyl-8-oxo-2-oxa-9-aza-1(1,4)-benzenacycloundecaphane-10-carboxamid-
o)pentanamido)
ethyl)amino)-2-oxoethyl)-1,4,7-triazonane-1,4-diyl)diacetic
acid
##STR00034## ##STR00035##
[0164] Intermediate 19 (I-19)
[0165] 50.0 mg (0.055 mmol) I-13 were dissolved in 5.0 mL TFA, 5 mL
DCM. The mixture was stirred at room temperature for 3 h and
concentrated under vacuum, and dried to give 50.0 mg (100%) of the
title compound. ES-MS: Observed [MH].sup.+ 803.3, [MH.sub.2].sup.2+
402.2.
Intermediate 20 (I-20)
[0166] A mixture of 13.3 mg (0.032 mmol) NOTA-bis(t-Bu ester), 5.2
mg (0.038 mmol) HOBT, and 8.8 mg (0.046 mmol) EDCI was dissolved in
1 mL anhydrous DMF. The mixture was stirred at room temperature for
30 min, followed by adding a solution of 10 mg (0.01 mmol) I-19 and
3 .mu.L DIEA in 0.5 mL DMF. The mixture was stirred at room
temperature overnight and concentrated under vacuum. The residue
was triturated with water, filtered, and washed with 1 N HCl,
water, 1 N Na.sub.2CO.sub.3, and brine. The solid product collected
was used in the next reaction without further purification. ES-MS:
Observed [MH.sub.2].sup.2+ 600.8, [MH.sub.3].sup.3+ 401.0.
Compound 3
[0167] 10 mg I-20 were dissolved in a mixture of 4.5 mL TFA, 0.25
mL H.sub.2O, 0.125 mL TIS, and 0.125 g phenol. The mixture was
stirred at room temperature overnight and concentrated under
vacuum. The residue was added into 10 mL cooled diethyl ether. The
precipitated product was collected by centrifugation and purified
by semi-preparative HPLC using gradient elution with aqueous
acetonitrile from 5% to 50% over 20 min. The desired fractions were
identified by LC-MS, combined and lyophilized to give the title
compound (2.0 mg, 15%) as confirmed by both LC-MS and analytical
HPLC. ES-MS: Observed [MH].sup.+ 876.4, [MH.sub.2].sup.2+
438.7.
Preparation of Cu-3 Complex
##STR00036##
[0169] A solution of 30 .mu.g (30 .mu.L) the NOTA conjugate, 1
.mu.L ammonia, and 30 .mu.L (5 .mu.g) CuCl.sub.2 solution in 30
.mu.L 50% acetonitrile was swirled at room temperature for 30 min.
LC-MS analysis confirmed the Cu-complexation with 3: [MCuH].sup.+
937.2, [MCuH.sub.2].sup.2+ 469.2.
Example 4: Synthesis of Compounds of Formula II
##STR00037## ##STR00038## ##STR00039##
[0170] Intermediate 21 (I-21)
[0171] A mixture of 1.2 g Fmoc-Arg(Mtr)-OH (1.97 mmol), 0.27 g HOBT
(2.0 mmol), 2 mL (2 M) CH.sub.3NH.sub.2 in THF(4.00 mmol) was
dissolved in 5 mL anhydrous DMF and cooled at 0.about.5.degree. C.
in ice bath, followed by adding 600 mg EDCI (3.1mmol). The mixture
was stirred at room temperature overnight and concentrated under
high vacuum. The residue was triturated with 1 N HCl, filtered, and
further washed with 1 N HCl, 5 N Na.sub.2CO.sub.3, and H.sub.2O.
The solid was dried to give 1.03 g (84%) of the title compound.
ES-MS: Observed [MH].sup.+ 622.2.
Intermediate 22 (I-22)
[0172] 1.0 g of I-21 obtained above was dissolved in 10 mL DCM and
3 mL piperidine. The mixture was stirred at room temperature for 30
min and concentrated under vacuum. The product was purified by
flash column chromatography using DCM and its mixture of 2%
methanol as eluents to give the title compound (0.54 g, 85%).
ES-MS: Observed [MH].sup.+ 400.2, [M-H].sup.- 398.1.
Intermediate 23 (I-23)
[0173] A mixture of 200.0 mg (0.46 mmol) I-7, 219.7 mg (0.55 mmol)
I-22, and 115.0 mg (0.55 mmol) HOBT were dissolved in 5 mL DMF and
cooled at 0.about.5.degree. C. in an ice bath, followed by adding
100 mg (0.6 mmol) EDCI. The mixture was stirred at room temperature
overnight and concentrated. The residue was dissolved in 10 mL
CH.sub.2Cl.sub.2, washed with 1N HCl, H.sub.2O, and brine. The DCM
solution was dried over MgSO.sub.4, filtered, and concentrated. The
product was purified by flash column chromatography using DCM and
its mixture of 2% methanol to give the title compound (300.0 mg,
80%). ES-MS: Observed [MH].sup.+ 815.4.
Intermediate 24 (I-24)
[0174] 250 mg of I-23 were dissolved in 4 mL TFA and 1 mL DCM.
After stirred at room temperature for 2 h, the solution was
concentrated and dried under vacuum to get the title compound in a
quantitative yield. ES-MS: Observed [MH].sup.+ 759.3,
[MH.sub.2].sup.2+ 392.1.
Intermediate 25 (I-25)
[0175] A mixture of 200 mg (0.26 mmol) I-24, 86.0 mg (0.6 mmol)
HOAT, and 236.0 mg (0.6 mmol) HATU, and 16.0 mg (0.9 mmol) DIEA was
dissolved in 4 mL anhydrous DMF. The mixture was stirred at room
temperature for 20 min, followed by adding 137.0 mg (0.93 mmol)
TBDMS-ONH.sub.2. The mixture was stirred at room temperature
overnight and concentrated. The residue was triturated with 1 N
HCl, filtered, and washed with 1 N Na.sub.2CO.sub.3, water, and
brine. The product was further purified by flash column
chromatography to give 100.0 mg (50%) of the title compound.
Compound 19
[0176] 50 mg (0.065 mmol) I-25 was dissolved in a mixture of 9.0 mL
TFA, 0.5 mL H.sub.2O, 0.25 mL TIS, and 0.25 g phenol. The mixture
was stirred at room temperature overnight and concentrated under
vacuum. The residue was added into 10 mL cooled diethyl ether. The
precipitated product was collected by centrifugation and purified
by semi-preparative HPLC using an eluent of aqueous acetonitrile.
The desired fractions were collected and lyophilized to give the
title compound (10.0 mg, 22%;
(6S,7R,10S)-N10-((S)-5-guanidino-1-(methylamino)-1-oxopentan-2-yl)-N6-hyd-
roxy-7-isobutyl-8-oxo-2-oxa-9-aza-1(1,4)-benzenacycloundecaphane-6,10-dica-
rboxamide) as confirmed by both LC-MS and analytical HPLC. ES-MS:
Observed [MH].sup.+ 562.3, [MH.sub.2].sup.2+ 281.7, [M-H].sup.-
560.2.
##STR00040##
Intermediate 26 (I-26)
[0177] The tert-butyl ester (I-6, 67 mg) was dissolved in DCM (1.6
mL) followed by addition of TFA (1.6 mL). The reaction mixture was
stirred at room temperature for 1 h. The solvent was removed and
dried under vacuum. The resulting acid, O-benzylhydroxylamine
hydrochloride (67 mg), and HBTU (58.3 mg) was dissolved in DMF (0.8
mL) followed by addition of DIEA (112 .mu.L). The reaction was
stirred at room temperature overnight and quenched with 10% citric
acid. Organic phase was washed with 1 N HCl, water, saturated
NaHCO.sub.3, and brine. After drying (MgSO.sub.4) and
concentration, I-26 was purified by silica gel column
chromatography (EtOAc:Hexanes=1:2) to afford 67% product; Q-TOF-MS
(ESI) m/z 392.2075 (calcd. 572.29 for
C.sub.34H.sub.40N.sub.2O.sub.6 [M]+); .sup.1H NMR (400 MHz,
CD.sub.3OD) .delta. -0.135 (1H, m), 0.82 (6H, m), 0.96 (2H, m),
1.12 (2H, m), 1.46 (6H, m), 1.94 (1H, m), 2.12 (1H, m), 2.63 (1H,
m), 3.33 (2H, m), 3.48 (1H, m), 4.12 (1H, m), 4.89 (2H, m), 1.72
(2H, m), 1.81 (1H, m), 2.15 (1H, m), 2.57 (1H, m), 2.74 (1H, m),
3.63 (1H, dd, J=4 and 12 Hz), 4.14 (1H, m), 4.35 (1H, m), 5.16-5.30
(3H, m), 5.72 (1H, d, J=6 Hz), 6.98-8.07 (14H, aromatic H);
.sup.13C NMR (100 MHz, CD.sub.3OD) .delta. 21.18 24.03 25.68,
29.85, 30.01, 31.41,37.89, 41.06, 47.27, 49.01, 51.84, 67.76,
107.96, 120.44, 120.80, 123.65, 125.02, 128.47, 128.73, 128.89,
129.02, 130.08, 131.48, 132.19, 135.10, 143.53, 159.32, 171.45,
171.51, 171.63.
Compound 20
((6S,7R,10S)-6-(hydroxycarbamoyl)-7-isobutyl-8-oxo-2-oxa-9-aza-1(1,4)-ben-
zenacycloundecaphane-10-carboxylic acid)
[0178] The benzylhydroxamate (I-26, 41 mg) was dissolved in MeOH (3
mL), and 10% Pd/C (.about.7 mg) was added. The reaction was stirred
under H.sub.2 (balloon) for 3 h. The Pd/C was removed by passing
through celite pad, and the solvent was removed by a rotary
evaporator. Compound 20 was further purified by recrystallization
with acetonitrile (.about.50% yield); Q-TOF-MS (ESI) m/z 392.2075
(calcd. 392.19 for C.sub.20H.sub.28N.sub.2O.sub.6 [M]+); .sup.1H
NMR (400 MHz, CD.sub.3OD) .delta. -0.43 (1H, m), 0.715 (1H, m),
0.805 (6H, m), 0.89 (1H, m), 1.25-1.49 (6H, m), 1.94 (1H, m), 2.12
(1H, m), 2.63 (1H, m), 3.33 (2H, m), 3.48 (1H, m), 4.12 (1H, m),
4.89 (1H, m), 6.86-7.21 (4H, aromatic H); .sup.13C NMR (100 MHz,
CD.sub.3OD) .delta. 14.43, 21.75, 23.70, 24.54, 26.62, 31.37,
32.27, 32.75, 38.29, 42.02, 49.47, 49.57, 49.85, 74.58, 111.42,
121.39, 123.85, 130.39, 133.60, 134.41, 160.24, 175.68.
Example 5: Synthesis of 17 and 18
##STR00041## ##STR00042## ##STR00043##
[0179] Intermediate 27 (I-27)
[0180] To a solution of I-7 and HATU (147 mg) in DMF (1 mL) was
added dropwise DIEA (135 .mu.L) and tetraethylene glycol monoamine
(45 .mu.L). After stirring at room temperature for 5 h, the
reaction was quenched by 10% aqueous citric acid, and the reaction
mixture was extracted with EtOAc. The organic phase was washed with
1N HCl, brine, and saturated NaHCO.sub.3. After drying over
MgSO.sub.4 and filtration, solvent was removed with a rotary
evaporator, and the mixture was purified by silica gel
chromatography (DCM:MeOH=30:1) to afford I-27 in 70% yield. Q-TOF
LC/MS m/z 609.3797 [M+H]+ (calcd. 608.37 for
C.sub.32H.sub.52N.sub.2O.sub.9 [M]+); .sup.1H NMR (400 MHz,
CDCl.sub.3) .delta. -0.45 (1H, m), 0.685 (1H, m), 0.825 (6H, m),
1.13 (9H, s), 1.27-1.64 (6H, m), 1.94 (1H, m), 2.05 (1H, m), 2.64
(2H, m), 3.38-3.91 (16H, m), 4.07 (1H, t, J=8 and 12 Hz), 4.25 (1H,
m), 4.99 (1H, m), 6.35-7.06 (4H, aromatic H); .sup.13C NMR (100
MHz, CDCl.sub.3) .delta. 21.45, 24.12, 25.92, 28.20, 30.00, 31.10,
38.40, 39.51, 40.65, 49.28, 50.18, 52.77, 61.65, 69.98, 70.22,
70.60, 70.90, 72.60, 73.92, 80.30, 120.40, 123.35, 129.95, 132.23,
132.46, 159.12, 171.62, 173.23, 174.34.
Intermediate 28 (I-28)
[0181] To the solution of alcohol (I-27, 250 mg), DMAP (catalytic
amount) and TsCl (86.2 mg) in DCM (4.1 mL) was added pyridine (83.1
.mu.L). The reaction mixture was stirred at room temperature
overnight. The mixture was washed with 1N HCl, water, sat.
NaHCO.sub.3, and organic phase was collected. After drying
(MgSO.sub.4) and concentration, the residue was purified by silica
gel column chromatography (DCM:MeOH=30:1) to afford 43% product.
Q-TOF LC/MS m/z 763.3845 [M+H]+ (calcd. 762.38 for
C.sub.39H.sub.58N.sub.2O.sub.11S [M]+); .sup.1H NMR (400 MHz,
CDCl.sub.3) .delta. -0.46 (1H, m), 0.64 (1H, m), 0.79 (6H, m), 0.86
(2H, m), 1.40 (9H, s), 1.23-1.58 (6H, m), 1.88-2.05 (2H, m), 2.45
(3H, s), 2.66 (1H, m), 3.38-3.74 (16H, m), 4.0/5 (1H, m), 4.19 (2H,
m), 4.90 (1H, m), 6.94-7.83 (8H, aromatic H); .sup.13C NMR (100
MHz, CDCl.sub.3) .delta. 21.46, 21.80, 24.07, 26.00, 28.21, 29.90,
31.18, 37.62, 39.56, 40.53, 49.36, 50.08, 53.13, 68.84, 69.49,
69.76, 70.46, 70.56, 70.74, 70.97, 73.94, 77.36, 80.79, 120.63,
123.41, 128.13, 129.74, 130.03, 132.14, 132.25, 145.06, 159.16,
171.41, 173.44, 173.99.
Intermediate 29 (I-29)
[0182] To the solution of I-27 (60 mg) in THF (0.5 mL) was added
TBAF (1.0 M in THF, 200 .mu.L), and the reaction mixture was
stirred at 50.degree. C. for 3 h. After removal of solvent, the
crude mixture was dissolved in DCM and washed with water and brine.
After drying (MgSO.sub.4) and concentration, the mixture was
purified by silica gel column chromatography (DCM:MeOH=30:1) to
afford 58% product. LC/MS m/z 611.3 [M+H]+ (calcd. 610.36 for
C.sub.32H.sub.51FN.sub.2O.sub.8 [M]+).
Intermediate 30 (I-30)
[0183] The tert-butyl ester (I-29, 28 mg) was dissolved in DCM (0.7
mL) followed by addition of TFA (0.7 mL). The reaction mixture was
stirred at room temperature for 1 h. The solvent was removed and
dried under vacuum. The resulting acid, O-benzylhydroxyl amine
hydrochloride (29.4 mg), and HBTU (38.5 mg) were dissolved in DMF
(0.6 mL) followed by addition of DIEA (32 .mu.L). The reaction was
stirred at room temperature overnight and quenched with 10% citric
acid. Organic phase was washed with 1 N HCl, water, saturated
NaHCO.sub.3, and brine. After drying (MgSO.sub.4) and
concentration, I-30 was purified by silica gel column
chromatography (DCM:MeOH=20:1) to afford 63% product. LC/MS m/z
660.3 [M+H]+ (calcd. 659.36 for C.sub.35H.sub.50FN.sub.3O.sub.8
[M]+); .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. -0.53 (1H, m),
0.74 (6H, d, J=8 and 16 Hz), 0.90 (1H, m), 0.97 (1H, m), 1.19-1.43
(2H, m), 1.74 (1H, m), 2.13 (1H, m), 2.69 (1H, m), 3.37-3.81 (16H,
m), 4.12 (2H, m), 4.53 (1H, m), 4.63 (1H, m), 4.86-4.90 (3H, m),
6.86-7.42 (9H, aromatic H); .sup.13C NMR (100 MHz, CDCl.sub.3)
.delta. 21.43, 23.97, 25.98, 29.58, 29.82, 30.38, 39.62, 37.68,
40.01, 46.89, 48.42, 53.53, 69.63, 70.42, 70.58, 70.62, 70.66,
70.88, 73.61, 84.14, 120.67, 123.13, 128.66, 129.21, 129.74,
132.06, 132.37, 135.43, 150.23, 159.11, 160.69, 171.4, 174.3.
17
[(6S,7R,10S)-N10-(2-(2-(2-(2-fluoroethoxy)ethoxy)ethoxy)ethyl)-N.sup.6--
hydroxy-7-isobutyl-8-oxo-2-oxa-9-aza-1(1,4)-benzenacycloundecaphane-6,10-d-
icarboxamide]
[0184] The benzylhydroxamate (I-30, 19 mg) was dissolved in MeOH
(0.5 mL), and 10% Pd/C (.about.10 mg) was added. The reaction was
stirred under H.sub.2 (balloon) for 3 h. The Pd/C was removed by
passing through celite pad, and solvent was removed by a rotary
evaporator. 17 was further purified by a reverse phased
semi-preparative C-18 HPLC (solvent A: water, 25 mM NH.sub.4OAc;
solvent B; acetonitrile; 20% to 90% of B for 17 min; flow rate=5
mL/min). LC/MS m/z 570.2 [M+H]+ (calcd. 569.31 for
C.sub.28H.sub.44FN.sub.3O.sub.8 [M]+); .sup.1H NMR (400 MHz,
CD.sub.3OD) .delta. -0.53 (1H, m), 0.82 (6H, m), 0.90 (1H, m),
1.24-1.39 (4H, m), 1.68 (1H, m), 2.15 (1H, m), 2.67 (1H, m),
3.30-3.78 (16H, m), 4.08-4.19 (2H, m), 4.46 (1H, m), 4.58 (1H, m),
6.87-7.25 (4H, aromatic H); .sup.13C NMR (100 MHz, CD.sub.3OD)
.delta. 21.62, 24.49, 26.92, 30.98, 31.32, 38.30, 40.49, 41.49,
54.74, 70.48, 71.34, 71.50, 71.59, 71.63, 71.68, 74.53, 83.26,
84.93, 121.51, 123.81, 130.34, 133.76, 133.82, 160.037, 172.79,
173.61, 175.90.
##STR00044##
Intermediate 31 (I-31)
[0185] The tert-butyl ester (I-28, 112.4 mg) was dissolved in DCM
(1 mL) followed by addition of TFA (1 mL). The reaction mixture was
stirred at room temperature for 1 h. The solvent was removed and
dried under vacuum. The resulting acid, O-benzylhydroxylamine
hydrochloride (28.1 mg), and HBTU (67 mg) were dissolved in DMF (1
mL) followed by addition of NMM (26 .mu.L). The reaction was
stirred at room temperature overnight and quenched with 10% citric
acid at 0.degree. C. Organic phase was washed with 1 N HCl, water,
saturated NaHCO.sub.3, and brine. After drying (MgSO.sub.4) and
concentration, I-31 was purified by silica gel column
chromatography (DCM:MeOH=20:1) to afford 61% product. Q-TOF LC/MS
m/z 812.3740 [M+H]+ (calcd. 811.37 for
C.sub.42H.sub.57N.sub.3O.sub.11S [M]+); .sup.1H NMR (400 MHz,
CDCl.sub.3) .delta. -0.17 (1H, m), 0.841 (1H, m), 0.89 (7H, m),
0.96(1H, m), 1.19 (1H, m), 1.45 (1H, m), 1.65 (1H, m), 1.82 (1H,
m), 2.27 (1H, m), 2.45 (3H, s), 2.65 (2H, m), 3.43-3.73 (17H, m),
4.13 (2H, m), 4.17 (1H, m), 4.30 (1H, m), 5.00 (1H, m), 7.26-7.82
(13H, aromatic H); .sup.13C NMR (100 MHz, CDCl.sub.3) .delta.
21.29, 21.84, 24.13, 26.02, 30.00, 31.28, 37.98, 39.62, 41.03,
47.34, 48.76, 53.34, 68.78, 69.54, 69.79, 70.46, 70.52, 70.77,
70.96, 73.35, 108.01, 120.48, 120.77, 123.42, 125.02, 128.11,
129.01, 129.91, 130.09, 132.41, 132.73, 143.51, 145.26, 158.99,
171.20, 171.57, 171.80.
Intermediate 32 (I-32)
[0186] The benzylhydroxamate (I-31, 67 mg) was dissolved in MeOH (2
mL), and 10% Pd/C (20 mg) was added. The reaction was stirred under
H.sub.2 (balloon) for 3 h. The Pd/C was removed by passing through
a celite pad, and the solvent was removed by a rotary evaporator.
I-32 was obtained with 87% yield after HPLC purification and
lyophilization (a reverse phased semi-preparative C-18 HPLC;
solvent A: water with 25 mM NH.sub.4OAc; solvent B; acetonitrile;
45% to 90% of B for 20 min; flow rate=5 mL/min). Q-TOF LC/MS m/z
721.3353 [M]+ (calcd. 721.32 for C.sub.35H.sub.51N.sub.3O.sub.11S
[M]+); .sup.1H NMR (400 MHz, CD.sub.3OD) .delta. -0.23 (1H, m),
0.74 (3H, m), 0.78 (1H, m), 0.84 (3H, m), 0.97 (1H, m), 1.33 (1H,
m), 1.49 (1H, m), 1.53 (1H, m), 1.69 (1H, m), 1.89 (1H, m), 2.26
(1H, m), 2.45 (3H, s), 2.78 (1H, m), 3.56-3.66 (16H, M), 3.81 (1H,
m), 4.14 (2H, m), 4.73 (1H, m), 6.95-7.80 (8H, aromatic H);
.sup.13C NMR (100 MHz, CD.sub.3OD) .delta. 21.44, 21.61, 24.16,
26.94, 29.02, 29.98, 37.96, 40.35, 40.50, 49.57, 50.70, 51.3,
52.05, 69.77, 70.43, 70.93, 71.33, 71.52, 71.57, 71.65, 73.89,
123.65, 123.81, 129.10, 130.93, 131.08, 133.15, 134.98, 146.48,
159.0, 173.63, 176.01
Example 6: Preparation of .sup.99mTc-2
##STR00045##
[0188] 10 .mu.l (30 .mu.g) the DTPA conjugate 2, and 50 .mu.L (6.6
mCi) .sup.99mTc pertechnetate solution were mixed in a vial,
followed by adding 4 .mu.L (300 .mu.g) freshly prepared SnCl.sub.2
solution and 500 .mu.l succinic acid buffer. The mixture was
incubated at room temperature for 20 min. Radio-HPLC analysis
showed the desired labeling product with 99% yield and specific
activity of 0.22 mCi/.mu.g. The product is reformulated with saline
for in vitro and in vivo studies (FIG. 1).
Example 7: Preparation of .sup.67Ga-3
##STR00046##
[0190] NOTA conjugate 3 is radiolabeled with .sup.67Ga in a 0.5 M
NH.sub.4OAc-buffered solution (pH 3.5) at 40.degree. C. for 30 min.
Briefly, 10 .mu.L of .sup.67GaCl.sub.3 (1 mCi in 0.05 M HCl), 10
.rho.L (10 .mu.g) the NOTA conjugate 3 in water, and 100 .mu.L of
0.5 M NH.sub.4OAc (pH 3.5) are mixed in a reaction vial and
incubated at room temperature for 30 min. The labeling is analyzed
by both radio-HPLC and radio-TLC to demonstrate its radiochemical
identity, retention time, radiochemical purity, radiochemical
specificity, and radiochemical yield. It is further purified by
radio-HPLC and C.sub.18 Sep-Pak cartridge. The labeled product is
reformulated with saline for in vitro and in vivo studies.
Example 8: Preparation of .sup.64Cu-3
##STR00047##
[0192] .sup.64CuCl.sub.2 is converted to .sup.64Cu(OAc).sub.2 by
adding 0.5 mL of 0.4 M ammonium acetate (NH.sub.4OAc) solution
(pH=5.5) to 20 .mu.L .sup.64CuCl.sub.2. .sup.64Cu(OAc).sub.2
solution (1.0 mCi) is added into a solution of the NOTA conjugate
(4.0 .mu.g) in 0.4 M NH.sub.4OAc (pH=5.5). The solution is stirred
for 20 min at 40.degree. C. The .sup.64Cu-labeled product is
monitored by both radio-HPLC and radio-TLC. It is further purified
by radio-HPLC and C.sub.18 Sep-Pak cartridge. Quality control is
performed by analytical HPLC to identify retention time,
radiochemical yield, radiochemical purity, radiochemical
specificity, and radiochemical stability. The labeled product is
reformulated with saline for in vitro and in vivo studies.
Example 9: Synthesis, Stability and Solubility
[0193] Arginine-containing macrocyclic hydroxamate 19 and its
HYNIC-conjugated analog (1, or RYM1) for Tc-99m labeling were
prepared. Without wishing to be limited by any theory,
incorporation of Arg increased hydrophilicity, and improved
pharmacokinetics and MMP targeting.
[0194] All compounds were synthesized in multiple steps starting
from an anti-succinic acid analog,
(2R,3S)-3-(tert-butoxycarbonyl)-2-iso-butylhex-5-enoic acid. Both
19 and 1 had MMP binding profiles similar to the RP805 precursor. 1
showed a high affinity for recombinant human rhMMP-2, rhMMP-9 and
rhMMP-12. .sup.99mTc-1 was obtained with a high radiochemical yield
and purity (>98%).
[0195] .sup.99mTc-1 showed a high radiochemical stability in the
radiolabeling media, and in urine and blood. The radioactive
material extracted after in vitro incubation of .sup.99mTc-1 in
mouse blood for up to 5 hours (FIG. 13) or urine collected from
mice 2 hours after intravenous injection of the tracer showed
similar radio-HPLC profiles with a single major peak at a similar
retention time without significant degradation (FIGS. 7A-7B).
[0196] Based on log P values and HPLC retention time, .sup.99mTc-1
showed higher hydrophilicity than .sup.99mTc-RP805. .sup.99mTc-1
showed high water solubility (Log
P.sub.n-octanol/water=-4.0.+-.0.1), with high radiochemical
stability and favorable properties for cardiovascular imaging.
Example 10: Biodistribution
[0197] Biodistribution at 2 h post-injection (p.i.) and blood
clearance were evaluated in C57Bl/6J mice injected with 16.+-.5 MBq
of .sup.99mTc-1 (n=6) or .sup.99mTc-RP805 (n=6). In vivo targeting
to aneurysm and specificity of .sup.99mTc-1 was evaluated in
high-fat fed apolipoprotein E-deficient mice, 4 weeks after carotid
aneurysm induction through peri-adventital application of
CaCl.sub.2. The animals were injected with 31.+-.14 MBq of
.sup.99mTc-1 with (n=3) or without (n=4) pre-injection of
non-labeled 1. This was followed by quantitative autoradiography of
arterial tracer uptake at 2 h p.i. .sup.99mTc-1 showed a lower
blood pool activity compared to .sup.99mTc-RP805 at 1 and 2 h p.i.
[1.3.+-.0.4 vs 2.8.+-.1.2% injected dose (ID)/mL and 1.0.+-.0.4 vs
1.8.+-.0.7% ID/mL, respectively, P<0.05 for both] and lower
hepatobiliary excretion (bile: 1.5.+-.0.3 vs 17.8.+-.13.3% ID/g,
P<0.05) (FIG. 8B).
[0198] Tissue uptake was higher in several collected tissues, but
not in control aorta (6.0.+-.1.4 vs 8.4.+-.1.9% ID/g, P<0.05).
This difference was primarily reflected in the initial activity
values, suggesting a difference in first-pass clearance of the
tracer from blood. Despite a lower blood level, .sup.99mTc-1 tissue
uptake at 2 hours post-injection was significantly higher than
.sup.99mTc-RP805 in several organs, but not in the normal aorta.
Both tracers displayed a high activity in the kidneys and urine,
indicative of renal clearance. However, contrary to the animals
injected with .sup.99mTc-1 who had limited bile activity, animals
injected with .sup.99mTc-RP805 showed elevated activity in the bile
(FIG. 8B).
[0199] .sup.99mTc-1 binding to carotid aneurysm and specificity in
vivo were evaluated in apoE.sup.-/- mice at seven weeks after
peri-adventitial application of CaCl.sub.2 to left carotid
arteries. Similar to C57BL/6J mice. .sup.99mTc-1 (31.+-.14 MBq,
i.v.) displayed a fast blood clearance, resulting in a residual
blood pool activity at 1 and 2 hours post-injection of 1.8.+-.0.2
and 1.3.+-.0.1% ID/mL, respectively. Autoradiographic evaluation of
the carotids and aorta harvested at 2 h showed high uptake of the
tracer in the left carotid artery aneurysm (n=6, FIGS. 4A-4F).
Pre-injection of an excess of the parent pan-MMP-inhibitor 1 led to
a 4.6-fold decrease in carotid aneurysm tracer uptake (n=5,
P<0.01). Albeit to a smaller degree, tracer uptake was also
reduced in the aorta, resulting in a significant 1.8-fold decrease
in the aneurysm-to-aorta uptake ratio by autoradiography under
blocking conditions (P<0.05, FIGS. 4A-4F). Pre-injection of the
parent pan-MMP-inhibitor 1 significantly reduced .sup.99mTc-1
uptake, as assessed by gamma well counting in all tissues
evaluated, but not in the bile (FIGS. 14A-14B), indicating a
certain degree of systemic MMP activation in these mice. As such,
the novel hydroxamate-based panMMP inhibitor-derived tracer
.sup.99mTc-1 demonstrated improved pharmacokinetics for
cardiovascular imaging and specific in vivo binding to
aneurysm.
[0200] The performance of the tracers was also evaluated in a mouse
model of pulmonary arterial hypertension (PAH) following exposure
to hypoxia (10%) for 2-3 weeks. This led to significant
upregulation of MMP activity (FIG. 5). In vivo microSPECT-CT
imaging of a mouse exposed to 10% hypoxia for 2 weeks was performed
30 minutes following intravenous injection of .sup.99mTc-1 (1 mCi)
and showed considerably higher .sup.99mTc-1 signal in the lungs of
this mouse compared to a control, normoxic animal. In certain
embodiments, the background indicated a more delayed imaging,
possibly at 1 hour, could be implemented. On ex vivo planar images
acquired at 1 hour, the lung signal was considerably higher
(>2-fold) in hypoxia-exposed lungs than normoxic controls (FIG.
6).
Example 11: .sup.99mTc-1 Imaging in AAA
[0201] Ang II infusion resulted in the death of 31% (5/16) of
apoE.sup.-/- mice within 4 weeks. The surviving animals underwent
.sup.99mTc-1 microSPECT/CT imaging at 1 h post-tracer injection. On
visual and quantitative analysis of the images, a range of tracer
uptake was detectable in suprarenal abdominal aortae of the
animals. While in a subset of animals, the aortic .sup.99mTc-1
signal was readily detectable on in vivo SPECT/CT images, other
animals displayed only modest uptake of the tracer in their
suprarenal abdominal aortae (FIGS. 9A-9D and 15).
[0202] Visual examination of the aorta at the time of tissue
harvesting immediately after microSPECT/CT image acquisition
detected varying degrees of aortic remodeling. Based on this visual
analysis, 25% of animals (4/11) showed major focal dilation, and
were classified as those with suprarenal AAA. Conversely, 44% of
animals (7/11) showed no or only modest remodeling, and were
categorized as the low remodeling group. This visual categorization
was confirmed on morphometric analysis of tissue sections, which
showed a significantly higher maximal external aortic diameter in
the AAA, compared to the low remodeling group (1.74.+-.0.35 vs
0.99.+-.0.08 mm, P<0.01, FIGS. 10A-10B and 16A-16B).
[0203] When categorized based on aortic size, there was a
significant difference in suprarenal aortic .sup.99mTc-1 signal
between the AAA and low remodeling groups (0.66.+-.0.16 vs
0.40.+-.0.18 counts per voxel (cpv)/MBq, P<0.05). Similarly,
fluorometric assessment of MMP activity showed significantly higher
MMP activity in the AAA group compared to the low remodeling group
(FIGS. 10A-10B). Consistent with the MMP-specificity of the tracer,
a significant correlation existed between the aortic .sup.99mTc-1
signal in vivo and MMP activity detected by zymography ex vivo
(r.sup.2=0.65, P<0.01, FIGS. 9A-9D). There was no difference in
the left ventricle blood pool activity between the two groups of
animals (0.14.+-.0.10 vs 0.12.+-.0.08 cpv/MBq, for AAA and low
remodeling groups, respectively, P=NS).
Example 12: Gene Expression Analysis
[0204] There was no significant difference in MMP-2 and MMP-9 gene
expression between AAA and low remodeling groups, whereas
macrophage marker, CD68 and MMP-12 expression were significantly
higher in the AAA, compared to the low remodeling group (FIGS.
17A-17B). Moreover, aortic .sup.99mTc-1 signal in vivo correlated
with CD68 and MMP-12 (FIGS. 11A-11B), but not MMP-2 and MMP-9 gene
expression (FIGS. 18A-18D). Similarly, CD68 and MMP-12, but not
MMP-2 and MMP-9, expression correlated with tissue MMP activity
(Table 1).
TABLE-US-00001 TABLE 1 Spearman's rank correlation coefficient
(p-value) between .sup.99mTc-1 uptake (counts per voxel/MBq),
external diameter (mm), MMP activity (arbitrary units) and
.beta.-actin-normalized expression of CD68, MMP-2, MMP-9 and MMP-12
in the abdominal aorta of angiotensin II-infused apoE.sup.-/- mice.
.sup.99mTc-1 External MMP uptake diameter activity CD68 MMP-2 MMP-9
MMP-12 0.86 0.38 0.77 0.85 0.33 0.23 (0.001) (0.25) (0.007) (0.002)
(0.33) (0.50) MMP-9 0.22 -0.13 -0.03 0.14 0.37 (0.52) (0.71) (0.95)
(0.69) (0.26) MMP-2 0.43 0.59 0.35 0.36 (0.19) (0.06) (0.29) (0.27)
CD68 0.80 0.63 0.93 (0.005) (0.04) (0.000) MMP 0.81 0.68 activity
(0.004) (0.03) External 0.44 diameter (0.18)
Example 13: MMP Binding Screens
[0205] MMPs inhibition assays were all carried out based on the
effects of an inhibitor on MMP-mediated catalytic cleavage of a
fluorogenic substrate i.e.
Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH.sub.2. The recombinant human
MMPs including rhMMP-2, rhMMP-9, and rhMMP-12 obtained from R&D
Systems (Minneapolis, Minn., USA) were activated by 1 mM
p-aminophenylmercuric acetate (APMA, Sigma) at 37.degree. C. for
certain hours according to the manufacturer's instructions. The
activated MMPs were diluted in the assay buffer consisting of 50 mM
Tris, 10 mM CaCl.sub.2, 150 mM NaCl, 0.05% Brij-35 (w/v), pH 7.5
(TCNB). The assays were carried out in 96-well non-binding surface
black microplates (Fisher Scientific).
[0206] Typically, 10 .mu.L of an inhibitor solution at several
different concentrations such as 250 nM, 125 nM, 62.5 nM, 31.25 nM,
and 15.6 nM were added into 70 .mu.L assay buffer in each well,
followed by adding 10 .mu.L (10 ng) of the activated MMP solutions
into each well. The solutions were incubated at room temperature
for 20 min. 10 .mu.L of the fluorogenic MMP substrate i.e.
Mca-KPLGL-Dpa-AR-NH.sub.2 (R&D Systems) at 4 different
concentrations (such as 50.0 .mu.M, 40.0 .mu.M, 30.0 .mu.M, and
20.0 .mu.M) in assay buffer were added before starting the
fluorescence measurements. The kinetics (v) of fluorescence changes
were measured at excitation and emission wavelengths of 320 nm and
405 nm for 30 min (1.44 min intervals, sensitivity 100, shaking
intensity 4, duration 30 s) by a fluorescent plate reader
(BIO-TEK/Synergy HT). The inhibition constants (K.sub.i) of the
inhibitor with MMPs were calculated from the mean velocity values
using GraphPad Prism 6 by the equation:
v = V max .function. [ S ] K m .function. ( 1 + [ I ] K i ) + [ S ]
##EQU00001##
TABLE-US-00002 TABLE 2 K.sub.i values of (1), (17), (19) and (20)
Compounds K.sub.i (nM) rhMMP-2 rhMMP-7 rhMMP-9 rhMMP-12 rhMMP-13
Compound 1 10.4 .+-. 0.0 33.2 .+-. 5.0 17.4 .+-. 2.1 2.2 .+-. 0.5
16.9 .+-. 2.7 Compound 19 5.8 .+-. 0.4 17.2 .+-. 0.1 20.4 .+-. 2.0
1.0 .+-. 0.2 15.3 .+-. 5.4 Compound 20* 101.0 .+-. 12.2 60.6 .+-.
11.0 1.7 .+-. 0.2 Compound 17* 8.6 .+-. 1.3 8.8 .+-. 0.8 2.1 .+-.
0.3 GM-6001 0.5 .+-. 0.1 0.4 .+-. 0.0 1.2 .+-. 0.0 0.57 .+-. 0.0
0.3 .+-. 0.0 RP-805 precursor 19.1 .+-. 3.1 19.2 .+-. 3.8 4.6 .+-.
0.5 (*n > 3, mean .+-. SE)
[0207] A similar protocol was used to check the effect of
.sup.99mTc-labeling on MMP inhibition. A solution of .sup.99mTc-1
(250 .mu.L) was prepared from 2 .mu.g of 1 as described elsewhere
herein. After two days at -80.degree. C. for, the mixture was
lyophilized and redissolved in saline buffer for MMP inhibition
assays. The decayed .sup.99mTc-1 showed a similar inhibition
tendency as the precursor 1 (FIG. 2), indicating that
.sup.99mTc-labeling has no significant effect on MMP binding.
[0208] Accordingly, through in vitro binding screens, compounds of
formulas I and II were shown exhibited strong binding to rhMMPs.
Without wishing to be limited by any theory, changes to the length
and characteristics of the groups distal to the hydroxamate group
had very little effect on the binding of these compounds to MMP.
Compounds with arginine groups, PEG groups or both arginine and PEG
groups, bound MMP similarly well and they did not have significant
effects on the compound's binding to the rhMMP catalytic
domain.
Example 14: Hydrophilicity and Blood Clearance Comparisons
[0209] Matrix metalloproteinase-targeted imaging agents known in
the art suffer from poor solubility of their precursors and have
blood clearance times that make them ill-suited for practical
application. The partition coefficient (Log D) values of
.sup.99mTc-1 and .sup.99mTc-RP805 were determined based on their
proportional distribution between n-octanol and water or Tris
buffer at pH 7.4. As shown in Table 3, .sup.99mTc-1 had lower log D
values and was more hydrophilic than .sup.99mTc-RP805, which
characteristics were reported in WO2013070471A1 and
US20150023873A1, which are incorporated herein in their entireties
by reference. Compared with .sup.99mTc-RP805 and its precursor, the
increased hydrophilicity of .sup.99mTc-1 (and 1) can in one aspect
be ascribed to the introduction of arginine residue. The .sup.99mTc
labeling compounds showed a little higher solubility in tris buffer
at pH 7.4 than in water.
[0210] .sup.99mTc-1 was analyzed for its degradation and stability
by RP-HPLC, and showed good stability in saline at room temperature
and in murine blood samples incubated at 37.degree. C. for
different times. HPLC analysis of urine samples collected after
intravenous injection of .sup.99mTc-1 also showed its in vivo
radiochemical stability in mice (FIG. 7).
[0211] Biodistribution study of .sup.99mTc-1 in mice showed faster
blood clearance than .sup.99mTc-RP805, through a renal clearance
pathway. .sup.99mTc-1 showed a lower residual blood activity at 1
and 2 h post injection compared to .sup.99mTc-RP805 (1.3.+-.0.4 vs
2.8.+-.1.2% injected dose (ID)/mL and 1.0.+-.0.4 vs 1.8.+-.0.7%
ID/mL, respectively) and lower hepatobiliary excretion (bile:
1.5.+-.0.3 vs 17.8.+-.13.3% ID/g)]; both differences were
statistically significant (FIG. 8).
TABLE-US-00003 TABLE 3 Compound Coligands Two phases for partition
Log D .sup.99mTc-RP805 Tricine-TPPTS Octanol/Tris (pH = 7.4) -3.2
.+-. 0.1 Octanol/water -2.8 .+-. 0.0 .sup.99mTc-1 Tricine-TPPTS
Octanol/Tris (pH = 7.4) -4.4 .+-. 0.1 Octanol/water -4.0 .+-.
0.1
Example 15: Imaging Tests Using Compounds of Formula I
[0212] In vivo binding characteristics of .sup.99mTc-1 was
addressed in murine models of carotid aneurysm and pulmonary
arterial hypertension by autoradiography and in vivo SPECT/CT
imaging followed by ex vivo planar imaging. Autoradiography,
SPECT/CT and planar imaging studies indicated MMP specific binding
of .sup.99mTc-1 in in vivo murine models of CaCl.sub.2 induced
carotid aneurysm and pulmonary arterial hypertension that express
high levels of MMPs (FIGS. 4A-4E, 5-6). .sup.99mTc-1 uptake in
carotid aneurysm mouse studies was specific as pre-injection of an
excess of unlabeled 1 led to 46% signal reduction in
aneurysm-to-aorta relative uptake (FIG. 4F).
Example 16
[0213] The present studies provide preclinical evaluation of an
illustrative pan-MMP inhibitor-based radiotracer, .sup.99mTc-1,
which was designed to address the shortcomings for clinical
translation of other commonly used preclinical MMP-targeting SPECT
tracers for cardiovascular applications. The data demonstrate high
affinity of this novel tracer for a set of MMPs involved in
aneurysm development, its good radiochemical stability, favorable
properties for vascular imaging, and specific uptake in aneurysm in
vivo that correlates with tissue MMP activity and inflammation.
[0214] Increased MMP activation is a key feature of aneurysm, and
plays a central role in aortic remodeling. Accordingly, in vivo
imaging of MMP activation can help predict the evolution of the
disease and guide therapeutic decisions. The feasibility and
potential value of in vivo MMP-targeted imaging in cardiovascular
pathology has been shown in preclinical studies. Despite the wealth
of the available preclinical data, a number of shortcomings,
including a relatively slow blood clearance, can potentially limit
clinical translation of .sup.99mTc-RP805 as an effective MMP tracer
for cardiovascular applications. In addition, the limited aqueous
solubility of RP805 precursor is a barrier to establishing uptake
specificity in vivo. As such, only a few studies, mainly in
vascular and valvular disease models, have attempted to demonstrate
uptake specificity of this imaging agent in vivo.
[0215] As designed, 1 was found to have good aqueous solubility,
even at relatively high concentrations needed for blocking studies.
In addition, in comparison to .sup.99mTc-RP805, .sup.99mTc-1 showed
a faster blood clearance, which facilitates early imaging and
improves vessel wall-to-blood contrast in vivo. These favorable
characteristics were empirically demonstrated in AAA microSPECT
imaging studies, performed starting at 1 hour post-injection.
[0216] The optimal imaging time depends on many factors and for
vascular imaging the vessel wall-to-blood ratio (contrast) is
important. A longer tracer circulation time would increase tissue
uptake, provided that the tracer has not reached a plateau or is
increasingly retained, e.g., through internalization. Despite a
lower blood level, .sup.99mTc-1 uptake in many organs was
significantly higher than .sup.99mTc-RP805 (FIGS. 8A-8B) and the
final activity in urine was lower. Without wishing to be limited by
any theory, this latter point can in part be explained by a
combination of high first-pass clearance, higher global tissue
uptake, and random nature of urine samples. In vivo blocking
studies (FIGS. 14A-14B) showed that the major component of tissue
uptake is specific, reflecting a hitherto less recognized basal MMP
activation in those tissues. This higher uptake, which indicates a
higher sensitivity for the target, is not explained by differences
in MMP affinity, as both tracers have comparable affinities toward
MMPs, and in non-limiting embodiments can reflect better tissue
penetration and easier accessibility to the target of .sup.99mTc-1.
In contrast to many other tissues, there was a trend toward higher
uptake of .sup.99mTc-RP805 compared to .sup.99mTc-1 in the normal
aorta at 2 hours post-injection. This basal uptake in the normal
vessel is a disadvantage for .sup.99mTc-RP805 and highlights the
necessity of blocking studies with the non-labeled precursor to
demonstrate specificity for any imaging application, whether
vascular or not. In the absence of such blocking studies, it is
impossible to ascertain the validity of the conclusions of any
study. As alternative, other MMP inhibitors can be used in vivo to
demonstrate signal specificity.
[0217] Evaluation of .sup.99mTc-1 in two preclinical models not
only demonstrated the feasibility of in vivo imaging in aneurysm,
but also provided complementary information regarding .sup.99mTc-1
uptake in aneurysm. Pre-injection of an excess of 1 in the carotid
aneurysm model resulted in a significant decrease in .sup.99mTc-1
uptake in aneurysm, establishing uptake specificity of the tracer.
Consistent with a certain level of basal MMP activation in the
normal artery, this blocking also led to a reduction in aortic
tracer uptake. The more prominent blocking effect in aneurysm,
reflected in a reduced aneurysm-to-aorta uptake ratio under
blocking conditions, mirrored the higher level of MMP activation in
aneurysm. In Ang II-infused mice, animals that had developed
aneurysm showed higher tracer uptake in suprarenal abdominal aorta.
There was no correlation between aortic size and MMP signal in
vivo, indicating that this enhanced uptake is not primarily related
with aortic size. Of note, tracer uptake was often higher in
aneurysm shoulders, at the border of areas of arterial enlargement
(FIG. 15).
[0218] The heterogeneity of the response to Ang II infusion in
apoE.sup.-/- mice was leveraged to investigate .sup.99mTc-1 uptake
and its correlates in AAA. .sup.99mTc-1 signal was significantly
higher in the AAA group and correlated well with aortic MMP
activity detected by zymography ex vivo. Tissue MMP activity is
tightly regulated at several levels, including MMP gene expression,
MMP activation (through proteolytic cleavage of the pro-domain or
allosteric activation), as well as the presence of endogenous
inhibitors [e.g., tissue inhibitors of MMPs (TIMPs)]. A significant
correlation was observed between in vivo aortic .sup.99mTc-1 signal
with MMP-12 mRNA expression in Ang II-infused animals. Inflammatory
cells are major sources of MMPs production, and protease activity
is closely linked to tissue inflammation (e.g., link between
macrophages and MMP-12 (macrophage elastase)). Strong correlations
between aortic .sup.99mTc-1 signal, MMP activity and MMP-12
expression on one hand, and macrophage marker, CD68 expression on
the other hand, were observed.
[0219] Qualitatively, while .sup.99mTc-1 and .sup.99mTc-RP805 share
similar affinity profile to activated MMPs, higher uptake of
.sup.99mTc-1 was observed in various tissues. This is apparent when
comparing SPECT images with .sup.99mTc-1 to those obtained with
.sup.99mTc-RP805 in a similar animal model (Golestani, et al.,
2015, Circ. Cardiovasc Imaging 8:e002471). As shown by the blocking
study, a large portion of this uptake is specific, and related to
basal MMP activity.
[0220] In addition to AAA, dysregulated MMP activity is involved in
other cardiovascular disorders, such as left ventricle remodeling
post-myocardial infarction and atherosclerosis, as well as many
primarily non-cardiovascular pathologies, including
neurodegenerative diseases and cancer. Therefore, molecular imaging
using compounds of the invention, such as but not limited to
.sup.99mTc-1, can improve diagnosis and assessment of therapeutic
response, and influence patient management in a wide range of
applications. In clinical trials, several non-selective MMP
inhibitors have shown adverse effects that preclude their use as
therapeutic agents. In certain embodiments, evaluation of global
proteolytic activity of MMPs, more integrative of different
processes at play, is more informative, especially if the signal is
stronger and less prone to background noise.
[0221] As demonstrated herein, in one aspect the compounds of the
invention are MMP-targeted tracers, with favorable pharmacokinetics
for early in vivo imaging. In vivo, the compounds' signal in
aneurysm is specific and correlates with MMP activity and
inflammation in murine AAA. Molecular imaging using compounds of
the invention can improve patient management in AAA, as well as
other disorders associated with dysregulated MMP activity.
[0222] The disclosures of each and every patent, patent
application, and publication cited herein are hereby incorporated
herein by reference in their entirety. While this invention has
been disclosed with reference to specific embodiments, it is
apparent that other embodiments and variations of this invention
may be devised by others skilled in the art without departing from
the true spirit and scope of the invention. The appended claims are
intended to be construed to include all such embodiments and
equivalent variations.
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