U.S. patent application number 14/218733 was filed with the patent office on 2014-09-25 for agents and devices for use in prevention of restenosis.
This patent application is currently assigned to DiscoveRx Corporation. The applicant listed for this patent is DiscoveRx Corporation. Invention is credited to Evangelos Hytopoulos, Eric J. Kunkel, Ivan Plavec.
Application Number | 20140288032 14/218733 |
Document ID | / |
Family ID | 38802134 |
Filed Date | 2014-09-25 |
United States Patent
Application |
20140288032 |
Kind Code |
A1 |
Kunkel; Eric J. ; et
al. |
September 25, 2014 |
AGENTS AND DEVICES FOR USE IN PREVENTION OF RESTENOSIS
Abstract
Agents that inhibit or prevent restenosis are identified by
assaying test agents in a battery of assays to measure the effect
of the test agent on cell proliferation, thrombosis, tissue
modeling, and inflammation. Treatment for restenosis is provided
using compositions of the invention.
Inventors: |
Kunkel; Eric J.; (San Mateo,
CA) ; Hytopoulos; Evangelos; (San Mateo, CA) ;
Plavec; Ivan; (Sunnyvale, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
DiscoveRx Corporation |
Fremont |
CA |
US |
|
|
Assignee: |
DiscoveRx Corporation
Fremont
CA
|
Family ID: |
38802134 |
Appl. No.: |
14/218733 |
Filed: |
March 18, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12302756 |
Aug 3, 2009 |
8697387 |
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PCT/US07/13230 |
Jun 4, 2007 |
|
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14218733 |
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60938031 |
May 15, 2007 |
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Current U.S.
Class: |
514/171 ;
514/182; 514/252.13; 514/252.17; 514/254.07; 514/261.1; 514/313;
514/656 |
Current CPC
Class: |
A61K 31/496 20130101;
A61L 31/16 20130101; A61K 31/519 20130101; A61K 31/4706 20130101;
G01N 33/5064 20130101; C12Q 1/02 20130101; A61K 31/517 20130101;
A61P 17/00 20180101; A61K 31/13 20130101; G01N 33/5061 20130101;
G01N 2800/323 20130101; A61K 31/565 20130101; A61K 31/135 20130101;
A61K 45/06 20130101 |
Class at
Publication: |
514/171 ;
514/656; 514/261.1; 514/313; 514/182; 514/252.13; 514/252.17;
514/254.07 |
International
Class: |
A61L 31/16 20060101
A61L031/16; A61K 45/06 20060101 A61K045/06; A61K 31/517 20060101
A61K031/517; A61K 31/4706 20060101 A61K031/4706; A61K 31/565
20060101 A61K031/565; A61K 31/496 20060101 A61K031/496; A61K 31/135
20060101 A61K031/135; A61K 31/519 20060101 A61K031/519 |
Claims
1. A device for use in a lumen of a vessel, comprising one or more
drugs selected from the group consisting of 8-Azaguanine,
Amodiaquin Dihydrochloride Dihydrate, Atovaquone, Betulin,
Chlorambucil, Ciclopirox Ethanolamine, Cis-(Z)-Flupentixol
Dihydrochloride, Clofilium Tosylate, Deferoxamine Mesylate,
Doxazosin Mesylate, Esculetin, Monobenzone, Nifedipine, Primaquine
Diphosphate, Securinine, Syrosingopine, Terconazole, a colchicine
analog as defined in Structure I ##STR00007## wherein R.sub.1 and
R.sub.2 are independently H, C.sub.1-10alkyl, C.sub.1-6cycloalkyl,
C.sub.1-10alkenyl, C.sub.1-6cycloalkyl, C.sub.1-6cycloalkenyl or
C.sub.1-10 alkynyl, or together form a ring containing from 3 to 6
carbon atoms; provided that a carbon atom comprising an alkene or
alkyne bond of R.sub.1 and R.sub.2 is not also simultaneously bound
to N; R.sub.1 and R.sub.2 optionally may be substituted with one or
more of F, OR.sub.9, or NR.sub.10R.sub.11 where R.sub.9, R.sub.10
and R.sub.11 are independently H or C.sub.1-5 alkyl; R.sub.3 is H,
C.sub.1-10alkyl, C.sub.1-6cycloalkyl, C.sub.1-10alkenyl,
C.sub.1-6cycloalkyl, C.sub.1-6cycloalkenyl or C.sub.1-10 alkynyl; X
is O or NOR.sub.9; R.sub.4 is H; OR.sub.9, provided R.sub.9 is not
CH3; SR.sub.9; NR.sub.1R.sub.2 and R.sub.5 is H, OR.sub.9,
SR.sub.9, NR.sub.1R.sub.2; and R.sub.6, R.sub.7 and R.sub.8 are
independently H or C.sub.1-5 alkyl, or R.sub.6 and R.sub.7 or
R.sub.7 and R.sub.8 independently together form a cycle containing
1 or 2 carbon atoms, and a colchicine analog as defined in
Structure II ##STR00008## wherein R.sub.1 and R.sub.2 are
independently H, C.sub.1-10 alkyl, C.sub.1-6cycloalkyl,
C.sub.1-10alkenyl, C.sub.1-6cycloalkyl, C.sub.1-6cycloalkenyl or
C.sub.1-10 alkynyl, or together form a ring containing from 3 to 6
carbon atoms; provided that a carbon atom comprising an alkene or
alkyne bond of R.sub.1 and R.sub.2 is not also simultaneously bound
to N. R.sub.1 and R.sub.2 optionally may be substituted with one or
more of F, OR.sub.9, or NR.sub.10R.sub.11 where R.sub.9, R.sub.10
and R.sub.11 are independently H or C.sub.1-5 alkyl; R.sub.3 is H,
C.sub.1-10alkyl, C.sub.1-6cycloalkyl, C.sub.1-10alkenyl,
C.sub.1-6cycloalkyl, C.sub.1-6cycloalkenyl or C.sub.1-10 alkynyl; X
is O or NOR.sub.9; R.sub.4 and R.sub.5 are H, OR.sub.9, SR.sub.9,
NR.sub.1R.sub.2; and R.sub.6, R.sub.7 and R.sub.8 are independently
H or C.sub.1-5 alkyl, or R.sub.6 and R.sub.7 or R.sub.7 and R.sub.8
independently form a cycle containing 1 or 2 carbon atoms.
2. The device of claim 1, and further comprising therein an
anti-inflammatory, anti-thrombotic or pro-healing agent.
3. The device of claim 1, wherein said drug is selected as one
defined in structure I, or a pharmaceutically acceptable salts
thereof.
4. The device of claim 3, wherein R.sub.1 and R.sub.2 are
independently H, C.sub.1-10 alkyl, or C.sub.1-6cycloalkyl R.sub.3
is H; X is O; R.sub.4 is H; OR.sub.9, provided R.sub.9 is not
CH.sub.3; SR.sub.9; or NR.sub.1R.sub.2 R.sub.5 is H; and R.sub.6,
R.sub.7 and R.sub.8 are independently H or C.sub.1-5 alkyl.
5. The device of claim 4, wherein R.sub.1 and R.sub.2 are H,
R.sub.4 is OH and R.sub.6, R.sub.7 and R.sub.8 are H or
CH.sub.3.
6. The device of claim 1, wherein the drug is selected according to
Formula I and has the structure ##STR00009##
7. The device of claim 1, wherein said device contains one or more
active agents and a compound of structure II or pharmaceutically
acceptable salts thereof of.
8. The device of claim 7, wherein R.sub.1 and R.sub.2 are
independently H, C.sub.1-10 alkyl, or C.sub.1-6cycloalkyl R.sub.3
is H; X is O; R.sub.4 is H, OR.sub.9, SR.sub.9, or NR.sub.1R.sub.2;
R.sub.5 is H; and R.sub.6, R.sub.7 and R.sub.8 are independently H
or C1-5 alkyl.
9. The device of claim 8, wherein R.sub.1 and R.sub.2 are H,
R.sub.4 is OH or OCH3, and R.sub.6, R.sub.7 and R.sub.8 are H or
CH3.
10. The device of claim 9, wherein the compound has the structure
##STR00010##
11. A method of use of a device according to claim 1, wherein said
device is a stent and comprising the step of inserting said stent
inserted into a subject in need thereof.
12. A method for reducing the incidence of restenosis, said method
comprising the steps of: (a) providing a stent or other device as
defined in claim 1, and (b) implanting said stent or other device
into the vasculature of a patient in need thereof.
13. The device of claim 1 which is a stent.
14. The stent of claim 13 combined with a biologically active agent
selected from the group consisting of an anti-inflammatory agent, a
pro-healing agent, a lipid metabolism modulating agent and an
anti-thrombotic agent.
15. The stent of claim 13 wherein said drug has as defined in
structure I or a pharmaceutically acceptable salts thereof.
16. The stent of claim 14, wherein R.sub.1 and R.sub.2 are
independently H, C.sub.1-10 alkyl, or C.sub.1-6 cycloalkyl; R.sub.3
is H; X is O; R.sub.4 is H; OR.sub.9, provided R.sub.9 is not CH3;
SR.sub.9; or NR.sub.1R.sub.2 R.sub.5 is H; and R.sub.6, R.sub.7 and
R.sub.8 are independently H or C1-5 alkyl.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional application of U.S. patent
application Ser. No. 12/302,756, filed on Aug. 3, 2009, which is a
national stage entry of PCT/US07/13230 filed Jun. 4, 2007 which
claims priority from provisional application 60/938,031 filed May
15, 2007, which claims priority from provisional application
60/810,508 filed Jun. 2, 2006, all of which are hereby incorporated
by reference.
STATEMENT OF GOVERNMENTAL SUPPORT
[0002] None
BACKGROUND OF THE INVENTION
[0003] 1. Field of the Invention
[0004] The present invention provides methods for identifying
agents that prevent restenosis, and the use of one or more agents
identified in the screen, including without limitation colchicine
analogs as defined herein, in the treatment or prevention of
restenosis and so relates to the fields of biology, molecular
biology, chemistry, medicinal chemistry, pharmacology, and
medicine.
[0005] 2. Description of Related Disclosures
[0006] The success of stents in balloon angioplasty has been
limited by thrombotic complications and restenosis of the vessel
wall, both of which occur as a result of normal injury and repair
processes. Anti-platelet drugs and procedural anticoagulation have
reduced the incidence of in-stent thrombosis, while drug-eluting
stents have significantly reduced the incidence of restenosis.
Currently approved drugs used on drug-eluting stents (paclitaxel,
rapamycin) are strongly antiproliferative, effectively inhibit
smooth muscle proliferation and block neointimal hyperplasia.
Antiproliferative drugs, however, may delay or prevent complete
healing of the injured vessel. Accordingly, the perpetuation of the
wounded vessel can result in late loss (progressive loss of vessel
diameter due to neointimal thickening) and late thrombosis,
increasing the frequency of follow-up target lesion
revascularization procedures, and thus reducing the effectiveness
of stents coated with such drugs.
[0007] The biological processes that contribute to restenosis and
late stage thrombosis include platelet and inflammatory cell
recruitment and activation, cell proliferation and migration,
vascular remodeling, and compromised re-endothelialization with
reduced endothelial cell function. Inhibition of proliferation
(e.g. smooth muscle cells, inflammatory cells) is a key process, as
the drugs that have been successfully employed in drug-eluting
stents (e.g. rapamycin, paclitaxel, everolimus) are strong
anti-proliferatives.
[0008] Drugs that have been tested on drug eluting stents include
agents from several categories: anti-proliferative (paclitaxel,
actinomycin D, 17.beta.-estradiol, imatinib mesylate),
anti-proliferative/immunosuppressive (rapamycin, FK-506,
mycophenolic acid), anti-inflammatory (methylprednisolone,
dexamethasone, tranilast), anti-thrombotic (hirudin, iloprost), and
others (rosiglitazone) (Abizaid, 2004, D'Amato, 1994, Serruys,
2004, Sousa, 2003a, 2003b). While paclitaxel, rapamycin and
everolimus have been shown to be effective and approved for
clinical use, other compounds, including some from the same classes
have failed (e.g. actinomycin D, mycophenolic acid). Blocking
inflammatory processes alone (e.g. with glucocorticoids) has not
been effective, although inflammatory mediators and growth factors
produced by recruited leukocytes and activated cell types do
regulate vascular cell functions and can affect the recovery and
repair process.
[0009] One of the major health risks of approved drug eluting
stents, which are coated with taxol or rapamycin, is late
thrombosis, which can lead to serious complications including death
months to years following stenting procedure. Current treatment to
prevent stent-related thrombosis is the use of oral anti-thrombotic
agents such as Plavix, which cannot be maintained long term due to
side effects or due to other medical procedures such as surgery
where anti-thrombotic agents are contraindicated. Since endothelial
cells play a key role in controlling thrombosis and fibrinolysis,
and provide a non-procoagulant surface, it is believed that the
main cause for the late thrombosis is incomplete
re-endothelialization on the stented site. Unfortunately, owing to
the complex biological processes that affect the function of stents
in vivo, there have been no methods developed to date for the
efficient identification and use of agents that would be most
effective in inhibiting restenosis without compromising complete
healing of the wounded vessel.
[0010] Human primary cell-based assay systems (BioMAP.RTM. Systems)
that model in vitro the complex biology of human disease, including
biology relevant to inflammation and restenosis, and which can be
used for screening and development of drugs eliciting complex
biological activities, have been developed: see U.S. Pat. Nos.
6,656,695 and 6,763,307 and PCT publication Nos. 01/67103,
03/23753, 04/22711, 04/63088, 04/94609, 05/23987, 04/94992,
05/93561, each of which is incorporated herein by reference. BioMAP
Systems are capable of detecting and distinguishing activities of a
broad range of mechanistically diverse compound classes, including
anti-proliferative drugs, immunosuppressive drugs,
anti-inflammatory drugs etc. For example, see Kunkel et al. (2004)
Assay Drug Dev Technol. 2:431-41; and Kunkel et al. (2004). FASEB
J. 18:1279-81.
[0011] Activity profiling of compounds, including experimental
compounds as well as drugs approved for human or veterinary use, in
BioMAP Systems, provides an enhanced understanding of the mechanism
of action of compounds and allows the identification of compounds
which are suitable for a particular therapeutic use, based on the
favorable combinations of biological activities which these
compounds induce in BioMAP Systems.
[0012] There remains a need for stents and other devices intended
for in vivo use with better agents and combination of agents to
prevent and treat restenosis. The present invention meets these
needs.
SUMMARY OF THE INVENTION
[0013] The present invention provides a method for identifying an
agent useful in preventing restenosis. In the method, the agent is
tested in a panel of assays employing multiple different cell types
to identify whether a candidate agent possesses the combination of
features desired of an anti-restenosis agent, which features
include (1) inhibition of smooth muscle proliferation, (2) little
or no effect on endothelial cell proliferation, (3) inhibition of
matrix remodeling with promotion of wound healing, (3) not
pro-thrombotic, and (4) selected anti-inflammatory activities.
[0014] Because few agents have all of the desired features of an
anti-restenosis agent, the present invention also provides a method
for identifying pairs of agents and combinations of two or more
agents that collectively provide the desired features of an
anti-restenosis agent more effectively than any of the agents
acting alone. In this method of the invention, combinations of
agents are tested together in the assays, and a subset of the
combinations tested is identified that collectively provide the
desired set of features.
[0015] The present invention also provides stents and other devices
intended for in vivo vascular use, e.g. vascular implantation, for
use in percutaneous coronary intervention (PCI), etc. which devices
are modified to contain one or more agents identified herein or by
the methods of the invention as having desired anti-restenotic
features. In one embodiment, the present invention provides a stent
or other device intended for in vivo use, wherein said stent or
device comprises one or more drugs selected from the group
consisting of 8-Azaguanine, Amodiaquin Dihydrochloride Dihydrate,
Atovaquone, Betulin, Chlorambucil, Ciclopirox Ethanolamine,
Cis-(Z)-Flupentixol Dihydrochloride, Clofilium Tosylate,
Deferoxamine Mesylate, Doxazosin Mesylate, Esculetin, Monobenzone,
Nifedipine, Primaquine Diphosphate, Securinine, Syrosingopine,
Terconazole, and a colchicine analog as defined herein, where the
preferred set of agents or drugs is selected from a group
consisting of, Cis-(Z)-Flupentixol Dihydrochloride, Clofilium
Tosylate, Monobenzone, Nifedipine, Primaquine Diphosphate,
Securinine, and a colchicine analog as defined herein. A stent or
device according to the invention may comprise said one or more
drugs as a coating or any other carrier formulation suitable for
elution of the drug when positioned in vivo.
[0016] In certain embodiments of the invention, a stent or other
device for vascular use, e.g. vascular implantation, for use in
percutaneous coronary intervention (PCI), etc. is provided, wherein
the stent or other device comprises a colchicine analog as defined
herein, where such analogs include, without limitation,
trimethylcolchicinic acid and its derivatives as further described
below.
[0017] In another embodiment, the present invention provides a
stent or other device intended for in vivo use, vascular use, e.g.
vascular implantation, for use in percutaneous coronary
intervention (PCI), etc. which device comprises one or more drugs
selected from the group: 8-Azaguanine, Amodiaquin Dihydrochloride
Dihydrate, Atovaquone, Betulin, Chlorambucil, Ciclopirox
Ethanolamine, Cis-(Z)-Flupentixol Dihydrochloride, Clofilium
Tosylate, Deferoxamine Mesylate, Doxazosin Mesylate, Esculetin,
Monobenzone, Nifedipine, Primaquine Diphosphate, Securinine,
Syrosingopine, Terconazole, and a colchicine analog as defined
herein in combination with a second biologically active agent. Such
agent may include, without limitation, an anti-inflammatory such as
a glucocorticoid receptor agonist (e.g. prednisolone,
methylprednisolone, budesonide), an anti-proliferative or
proliferation modifier (e.g. estradiol), a lipid metabolism
modulating drug (e.g. statins), an insulin sensitizer (e.g.
rosiglitazone), an anti-thrombotic drug, etc.
[0018] The present invention also provides methods and compositions
for the treatment or prevention of restenosis utilizing stents and
other devices intended for in vivo vascular use, e.g. vascular
implantation, for use in percutaneous coronary intervention (PCI),
etc. for vascular administration of anti-restenotic agents, where
the device is modified to contain a colchicine analog, which
analogs include, without limitation, trimethylcolchicinic acid and
analogs and derivatives thereof. A stent or device according to the
invention may comprise said one or more drugs as a coating or any
other carrier formulation suitable for elution of the drug when
positioned in vivo. The methods of the invention include the
vascular administration to a subject of a pharmaceutical
composition comprising a therapeutically effective amount of a
colchicine analog is effective to treat, delay or prevent
restenosis proximal to the site of administration.
DEFINITIONS
[0019] The practice of the present invention will employ, unless
otherwise indicated, conventional techniques of molecular biology
(including recombinant techniques), microbiology, cell biology,
biochemistry and immunology, which are within the skill of the art.
Such techniques are explained fully in the literature, such as,
"Molecular Cloning: A Laboratory Manual", second edition (Sambrook
et al., 1989); "Oligonucleotide Synthesis" (M. J. Gait, ed., 1984);
"Animal Cell Culture" (R. I. Freshney, ed., 1987); "Methods in
Enzymology" (Academic Press, Inc.); "Handbook of Experimental
Immunology" (D. M. Weir & C. C. Blackwell, eds.); "Gene
Transfer Vectors for Mammalian Cells" (J. M. Miller & M. P.
Calos, eds., 1987); "Current Protocols in Molecular Biology" (F. M.
Ausubel et al., eds., 1987); "PCR: The Polymerase Chain Reaction",
(Mullis et al., eds., 1994); and "Current Protocols in Immunology"
(J. E. Coligan et al., eds., 1991).
[0020] The compounds of the invention, or their pharmaceutically
acceptable salts may contain one or more asymmetric centers and may
thus give rise to enantiomers, diastereomers, and other
stereoisomeric forms that may be defined, in terms of absolute
stereochemistry, as (R)- or (S)- or, as (D)- or (L)- for amino
acids. The present invention is meant to include all such possible
isomers, as well as, their racemic and optically pure forms.
Optically active (+) and (-), (R)- and (S)-, or (D)- and
(L)-isomers may be prepared using chiral synthons or chiral
reagents, or resolved using conventional techniques as known in the
art, e.g. by chromatography. When the compounds described herein
contain olefinic double bonds or other centers of geometric
asymmetry, and unless specified otherwise, it is intended that the
compounds include both E and Z geometric isomers. Likewise, all
tautomeric forms are also intended to be included.
[0021] As used throughout, "modulation" is meant to refer to an
increase or a decrease in the indicated phenomenon (e.g.,
modulation of a biological activity refers to an increase in a
biological activity or a decrease in a biological activity).
[0022] As used herein, the terms "treatment," "treating," and the
like, refer to obtaining a desired pharmacologic and/or physiologic
effect. The effect may be prophylactic in terms of completely or
partially preventing a disease or condition, or symptom thereof,
and/or may be therapeutic in terms of a partial or complete cure
for a condition and/or adverse affect attributable to the
condition. "Treatment," as used herein, covers any treatment of a
disease or condition in a mammal, particularly in a human, and
includes: (a) preventing the condition from occurring in a subject
which may be predisposed to the condition but has not yet been
diagnosed as having it; (b) inhibiting the development of the
condition; and (c) relieving the condition, i.e., causing its
regression.
[0023] An "effective amount" is an amount sufficient to effect
beneficial or desired results. An effective amount can be
administered in one or more administrations. An effective amount
corresponds with the quantity required to provide a desired average
local concentration of a particular biologic agent, in accordance
with its known efficacy, within the vascular lumen, vascular wall,
or other site, for the intended period of therapy. A dose may be
determined by those skilled in the art by conducting preliminary
animal studies and generating a dose response curve, as is known in
the art. Maximum concentration in the dose response curve would be
determined by the solubility of the compound in the solution and by
toxicity to the animal model, as known in the art.
[0024] Any suitable and effective amount can be supported on one or
more implants to constitute an individual "treatment" or
"dose".
[0025] The effective amount further corresponds with the quantity
required to provide a desired average local concentration of the
particular biologic agent, in accordance with its known efficacy,
in the region of insertion of a stent or other device, for the
intended period of therapy. Due allowance can be made for losses
due to urination or circulatory fluctuation due to physical
activity, for example, from ten to ninety percent loss allowance
could be made depending upon the individual patient and their
routines.
[0026] The terms "individual," "subject," "host," and "patient,"
used interchangeably herein, refer to a mammal, including, but not
limited to, humans, murines, simians, felines, canines, equines,
bovines, mammalian farm animals, mammalian sport animals, and
mammalian pets. Human subjects are of particular interest.
[0027] As used herein, the terms "determining", "assessing",
"assaying", "measuring" and "detecting" refer to both quantitative
and qualitative determinations and as such, the term "determining"
is used interchangeably herein with "assaying," "measuring," and
the like. Where a quantitative determination is intended, the
phrase "determining an amount" and the like is used. Where either a
qualitative or quantitative determination is intended, the phrase
"determining a level of proliferation" or "detecting proliferation"
is used.
[0028] Before the present invention is further described, it is to
be understood that this invention is not limited to particular
embodiments described, as such may, of course, vary. It is also to
be understood that the terminology used herein is for the purpose
of describing particular embodiments only, and is not intended to
be limiting, since the scope of the present invention will be
limited only by the appended claims.
[0029] Where a range of values is provided, it is understood that
each intervening value, to the tenth of the unit of the lower limit
unless the context clearly dictates otherwise, between the upper
and lower limit of that range and any other stated or intervening
value in that stated range, is encompassed within the invention.
The upper and lower limits of these smaller ranges may
independently be included in the smaller ranges, and are also
encompassed within the invention, subject to any specifically
excluded limit in the stated range. Where the stated range includes
one or both of the limits, ranges excluding either or both of those
included limits are also included in the invention.
[0030] 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, the preferred methods and materials are now described.
All publications mentioned hereunder are incorporated herein by
reference. Unless mentioned otherwise, the techniques employed
herein are standard methodologies well known to one of ordinary
skill in the art.
[0031] It must be noted that as used herein and in the appended
claims, the singular forms "a," "and," and "the" include plural
referents unless the context clearly dictates otherwise. Thus, for
example, reference to "a biomarker" includes a plurality of such
biomarkers and reference to "the sample" includes reference to one
or more samples and equivalents thereof known to those skilled in
the art, and so forth. It is further noted that the claims may be
drafted to exclude any optional element. As such, this statement is
intended to serve as antecedent basis for use of such exclusive
terminology as "solely," "only" and the like in connection with the
recitation of claim elements, or use of a "negative" limitation.
Moreover any positively recited element of the disclosure provides
basis for a negative limitation to exclude that element from the
claims.
[0032] All publications and patents cited in this specification are
herein incorporated by reference as if each individual publication
or patent were specifically and individually indicated to be
incorporated by reference and are incorporated herein by reference
to disclose and describe the methods and/or materials in connection
with which the publications are cited. The citation of any
publication is for its disclosure prior to the filing date and
should not be construed as an admission that the present invention
is not entitled to antedate such publication by virtue of prior
invention. Further, the dates of publication provided may be
different from the actual publication dates which may need to be
independently confirmed.
[0033] Other objects, features and advantages of the present
invention will become apparent from the following detailed
description. It should be understood, however, that the detailed
description and specific examples, while indicating preferred
embodiments of the invention, are given by way of illustration
only, since various changes and modifications within the spirit and
scope of the invention will become apparent to those skilled in the
art from this detailed description.
DETAILED DESCRIPTION OF THE INVENTION
[0034] The present invention provides methods for the
identification of drugs for use with drug-eluting coronary stents
and other devices, e.g. for percutaneous coronary intervention,
that inhibit restenosis, promote wound healing, and prevent
thrombotic events. The long term success of a stenting procedure
generally includes (1) the prevention of the initial vascular
smooth muscle hyperplasia resulting from the wound to the vessel
induced by stent insertion, (2) promotion of re-endothelialization
to normalize the vessel morphology and function, and (3) inhibition
of local inflammation to allow wound healing to occur. Many of the
complications of the stenting procedure (e.g. need for target
lesion revascularization and thrombotic events once patients are
taken off blood thinners) are due predominantly to incomplete wound
healing and re-endothelialization.
[0035] Conventional drugs delivered by stent, for example
rapamycin, paclitaxel and everolimus, have been selected for their
ability to inhibit the proliferative events involved in rapid
neointimal hyperplasia without regard to other relevant biological
events. Patients treated with stents coated with these drugs may
have incomplete wound healing and are at risk for late restenosis
and thrombotic events. Thus, there is a need for better
anti-restenosis drugs.
[0036] The present invention provides methods to identify those
drugs and provide stents coated with new drugs and drug
combinations. The methods and stents of the invention are based on
the identification of drugs that display a favorable combination of
features (or biological activities of importance), defined as (1)
inhibition of smooth muscle cell proliferation (2) little or no
effect on endothelial cell proliferation (3) inhibition of matrix
remodeling with promotion of wound healing, (4) no promotion of
thrombosis, (5) promotion of vessel relaxation and (6) selected
anti-inflammatory activities. Description of the desired features,
and the corresponding markers (or readout parameters), which are
used to measure these features are listed in Table 1.
TABLE-US-00001 TABLE 1 Desired biological activities of an
anti-restenosis drugs. Desired Parameter and cells Change
Biological Rationale proliferation (SMC)* decrease prevent
neointimal hyperplasia proliferation (EC)* none or promote
reendothelialization increase uPAR (EC, SMC) decrease prevent
migration and remodeling PGE.sub.2 (EC, monocytes) increase
promotes relaxation and or none diagnostic of inhibition of e.g.
PGI.sub.2 expression Thrombomodulin (EC, increase promotes
anti-thrombotic SMC) environment Tissue factor (EC, decrease
promotes anti-thrombotic SMC) environment CD40 (monocytes, decrease
reduces activation of cells by EC) platelets and T cells IP-10 (EC,
HDF) decrease reduces SMC proliferation and promotes pro-angiogenic
enviroment MCP-1 (VEC, SMC) none reduces monocyte infiltration but
can promote endothelial precursor recruitment and angiogenesis
collagen I (HDF) increase indicative of wound healing or none
VEGFR2 (EC) increase promotes angiogenic state of or none
endothelium PAI-1 (HDF, SMC) decrease reduce SMC proliferation and
migration IL-8 (SMC) decrease prevent local infiltration by or none
granulocytes M-CSF (EC, SMC) decrease promotes reendothelialization
or none but can also cause monocyte activation/differentiation
TNF-.alpha. (monocytes) increase possible effects on cell
proliferation and/or apoptosis *Proliferation under both growth
factor-mediated and inflammatory conditions. EC, endothelial cell
(either coronary artery or umbilical vein); SMC, smooth muscle cell
(either coronary artery or umbilical artery), monocytes, peripheral
blood monocyte; HDF, dermal fibroblast.
[0037] The BioMAP Systems that model the relevant biological
processes (smooth muscle cell hyperplasia, tissue remodeling,
inflammation etc, including the measurements of readout parameters
listed in Table 1), and are used for screening are listed in Table
2. Each of the cells listed in Table 1 can be utilized in one or
more of the systems described below. For example, smooth muscle
cells are used in CASM3C; CASMNo; SM3C and SMNo systems. Such
BioMap systems are generally primary human cell based assays.
Compounds are screened in one or more such BioMap systems, usually
in at least about two such systems, and may be screened in at least
three, at least four, at least five, at least ten, and up to all of
the assays set forth in Table 2.
[0038] In some embodiments of the invention, compounds are screened
in at least one system utilizing smooth muscle cells, and at least
one system utilizing endothelial cells. In some embodiments, the
compounds are further screened in at least one system related to
inflammation, for example systems LPS, 3C, 4H and HSM3C, which
incorporate inflammatory cells (monocytes, T cells, endothelial
cells) and mediators (TNF-.alpha., IFN-.gamma. etc.). In other
embodiments, compounds are further screened in at least one system
related to tissue remodeling, for example systems HDF3CT, HDFT and
CASM3C, which incorporate cell types (fibroblasts, smooth muscle
cells) and factors (e.g. growth factors) involved in tissue
remodeling.
[0039] These assays are scored according to their modulation of the
selected readout parameters. Compounds receive a score of positive
(score of 1) or negative (score of 0) for each readout parameter
set forth in Table 1 that is modulated. The compounds that score
higher than a control compound, usually an approved therapeutic
agent for inhibition of restenosis, e.g. rapamycin or paclitaxel,
are considered an improvement over the approved therapeutic agent,
and may be selected for further use.
TABLE-US-00002 TABLE 2 BioMAP Systems used to screen for anti-
restenosis agents with desired features. System Cell Types
Environment Readout Parameters 3C Umbilical Vein IL-1.beta. +
MCP-1, uPAR, Endothelial Cells TNF-.alpha. + tissue factor,
IFN-.gamma. thrombomodulin, SRB*, Proliferation HNo Umbilical Vein
None Proliferation Endothelial Cells 4H Umbilical Vein IL-4 +
VEGFRII, uPAR, SRB Endothelial Cells histamine LPS Peripheral Blood
TLR4 CD40, M-CSF, PGE2, Mononuclear Cells + TNF-.alpha., SRB
Umbilical Vein Endothelial Cells CA3C Coronary Artery IL-1.beta. +
MCP-1, CD141, Endothelial Cells TNF-.alpha. + CD142, IP-10,
IFN-.gamma. IL-8, tissue factor, thrombomodulin, SRB, Proliferation
CANo Coronary Artery None Proliferation Endothelial Cells CASM3C
Coronary Artery IL-1.beta. + MCP-1, uPAR, Smooth Muscle TNF-.alpha.
+ tissue factor, Cells IFN-.gamma. thrombomodulin, SRB,
Proliferation CASMNo Coronary Artery None Proliferation Smooth
Muscle Cells SM3C Umbilical Artery IL-1.beta. + MCP-1, CD141,
Smooth Muscle TNF-.alpha. + CD142, IP-10, Cells IFN-.gamma. IL-8,
SRB, Proliferation SMNo Umbilical Artery None Proliferation Smooth
Muscle Cells HSM3C Umbilical Vein IL-1.beta. + CD40, uPAR,
Endothelial Cells + TNF-.alpha. + IP-10, tissue Umbilical Artery
IFN-.gamma. factor, Smooth Muscle cells thrombomodulin, M-CSF, SRB
HDF3CT Fibroblasts IL-1.beta. + Collagen I, IP-10 TNF-.alpha. +
IFN-.gamma. + TGF-.beta. HDFT Fibroblasts TGF-.beta. Collagen I,
SRB, PAI-1 SRB, sulforhodamine B
[0040] The relative importance of each parameter may be weighted by
the screener to select those parameters of greatest interest for a
particular application. While a preferred drug modulates all of the
readout parameters in the desired way; desirable bioactive
compounds may also modulate only one or several of the parameters
in the desired fashion. One can combine two or more drugs such that
more of the desired parameter changes are obtained than either drug
is capable of inducing alone. For restenosis, preferred compounds
are those which have high overall score and which inhibit smooth
muscle cell proliferation without affecting endothelial cell
proliferation.
[0041] A library of over 1000 physiologically active compounds was
screened in the assays listed in Table 2, for the activities listed
in Table 1. Because of the importance of the inhibition the
neointimal hyperplasia that occurs after stenting procedure,
compounds that selectively inhibit smooth muscle cell proliferation
versus endothelial cell proliferation were identified. The
resulting compounds were evaluated and scored according to the
activities listed in Table 2, receiving a 1 value for each activity
present (Table 3). Overall scores of paclitaxel and rapamycin are
shown for comparison. Thus, compounds that have overall scores
equal or higher than paclitaxel and rapamycin, but at the same time
differentially affect proliferation of smooth muscle and
endothelial cells proliferation, are considered an improvement over
the existing drugs for the treatment of restenosis. Among the
compounds with high scores in the BioMAP screen (Table 4),
esculetin has been reported to inhibit neointimal hyperplasia after
balloon vascular injury in the rat (Pan et al., 2003), thus
independently confirming the validity of the screening method
described here.
TABLE-US-00003 TABLE 3 Parameter scores for selected drugs.
##STR00001## ##STR00002## ##STR00003##
TABLE-US-00004 TABLE 4 Total score for desired parameter
modulation. Score Drug Drug Class (out of 45) Cis-(Z)-Flupentixol
Dopamine receptor antagonist 33 Dihydrochloride Betulin Natural
product, antiviral 31 Chlorambucil Alkylating agent 30
Trimethylcolchicinic Inactive colchicine analog 29 Acid
8-Azaguanine Purine antagonist, 28 antiproliferative Nifedipine
L-type calcium channel blocker 28 Securinine GABA receptor
antagonist 28 Primaquine Diphosphate Anti-malarial 27 Deferoxamine
Mesylate Iron chelator 26 Esculetin Lipoxygenase inhibitor 25
Monobenzene Bleaching agent 24 Amodiaquin Dihydro- Anti-malarial 23
chloride Dihydrate Terconazole Fungal cytochrome P-450 inhibitor 22
Doxazosin Mesylate Alpha(1)-adrenoreceptor inhibitor 22 Atovaquone
Anti-malarial 22 Syrosingopine Catecholamine depletion 22 Clofilium
Tosylate Potassium channel inhibitor 22 Ciclopirox Ethanolamine
Anti-fungal 20 Paclitaxel (average of Tubulin binder 26 .+-. 2 11
runs) Rapamycin (average of mTOR inhibitor 24 .+-. 1 13 runs)
[0042] Thus, in one embodiment, the present invention provides a
stent or other device intended for in vivo use, wherein said stent
or device comprises one or more drugs selected from the group
consisting of 8-Azaguanine, Amodiaquin Dihydrochloride Dihydrate,
Atovaquone, Betulin, Chlorambucil, Ciclopirox Ethanolamine,
Cis-(Z)-Flupentixol Dihydrochloride, Clofilium Tosylate,
Deferoxamine Mesylate, Doxazosin Mesylate, Esculetin, Monobenzone,
Nifedipine, Primaquine Diphosphate, Securinine, Syrosingopine,
Terconazole, and a colchicine analog, e.g. Trimethylcolchicinic
Acid, where the preferred set of agents or drugs is selected from a
group consisting of, Cis-(Z)-Flupentixol Dihydrochloride, Clofilium
Tosylate, Monobenzone, Nifedipine, Primaquine Diphosphate,
Securinine, and a colchicine analog, e.g. Trimethylcolchicinic
Acid, and where the most preferred set of agents includes
Trimethylcolchicinic Acid and its derivatives as further described
below.
[0043] In another embodiment, the present invention provides a
stent or other device intended for in vivo use, wherein said stent
or device comprises one of the drugs selected from the group
8-Azaguanine, Amodiaquin Dihydrochloride Dihydrate, Atovaquone,
Betulin, Chlorambucil, Ciclopirox Ethanolamine, Cis-(Z)-Flupentixol
Dihydrochloride, Clofilium Tosylate, Deferoxamine Mesylate,
Doxazosin Mesylate, Esculetin, Monobenzone, Nifedipine, Primaquine
Diphosphate, Securinine, Syrosingopine, Terconazole, and a
colchicine analog, e.g. Trimethylcolchicinic Acid in combination
with a second biologically active agent. Such biologically active
agents may include, without limitation, clinically proven
anti-inflammatory such as a glucocorticoid receptor agonist (e.g.
prednisolone, methylprednisolone, budesonide), a pro-healing drug
(e.g. estradiol), a lipid metabolism modulating drug (e.g.
statins), an anti-thrombotic drug etc.
[0044] Compounds of interest as a second agent may include
chemotherapeutic agents for neoplastic tissues, anti-inflammatory
agents for ischemic or inflamed tissues, hormones or hormone
antagonists for endocrine tissues, ion channel modifiers for
cardiovascular or other tissues, and neuroactive agents for the
central nervous system. Exemplary of pharmaceutical agents suitable
for this invention are those described in The Pharmacological Basis
of Therapeutics, Goodman and Gilman, McGraw-Hill, New York, N.Y.,
(1993) under the sections: Drugs Acting at Synaptic and
Neuroeffector Junctional Sites; Drugs Acting on the Central Nervous
System; Autacoids: Drug Therapy of Inflammation; Water, Salts and
Ions; Drugs Affecting Renal Function and Electrolyte Metabolism;
Cardiovascular Drugs; Drugs Affecting Gastrointestinal Function;
Drugs Affecting Uterine Motility; Chemotherapy of Parasitic
Infections; Chemotherapy of Microbial Diseases; Chemotherapy of
Neoplastic Diseases; Drugs Used for Immunosuppression; Drugs Acting
on Blood-Forming organs; Hormones and Hormone Antagonists;
Vitamins, Dermatology; and Toxicology, all incorporated herein by
reference. Agents may be in the form of simple drugs, peptides,
peptide fragments, DNA, RNA, ribozymes or engineered hybrids of
nucleic acids and peptides or peptide fragments, or derivatives of
each.
[0045] Specific agents of interest include therapeutic agents that
inhibit in-stent restenosis. Such agents may include rapamycin;
antiplatelet agents; GPIIb/IIIa inhibitors, e.g. RheoPro; DNA;
ribozymes; RNA; antiplatelet drugs, e.g. aspirin and dipyridamole;
anticoagulant drugs, including heparin, coumadin, protamine, and
hirudin; antimitotics (cytotoxic agents) that work directly to
prevent cell mitosis (replication) and antimetabolites that prevent
replication, e.g. methotrexate, colchicine, azathioprine,
vincristine, vinblastine, fluorouracil, adriamycin, mutamycin, etc.
Anti-inflammatory drugs such as glucocorticoids, e.g.
dexamethasone, betamethasone, etc. can also be useful to locally
suppress inflammation caused by injury to luminal tissue during
angioplasty.
[0046] Angiotensin converting enzyme inhibitors (ACE-I) are used
for antihypertensive and renoprotective actions. ACE inhibitor
include, but are not limited to, captopril, benazepril, enalapril,
fosinopril, lisinopril, quinapril, Ramipril, imidapril,
perindopril, erbumine, and trandolapril. ACE receptor blockers may
also be used in place of or as well as ACE inhibitors, and these
include losartan, irbesartan, candesartan, cilexetil, and
valsartan.
[0047] Nicotine receptor agonist, e.g. nicotine
(S-3-(1-methyl-2-pyrrolidinyl)pyridine) and other compounds that
substantially specifically bind a nicotine receptor and provide a
pharmacological effect. "Nicotine receptor agonists" encompass
naturally-occurring compounds (including, but not limited to, small
molecules, polypeptides, peptides, etc., particularly
naturally-occurring plant alkaloids, and the like), endogenous
ligands (e.g., purified from a natural source, recombinantly
produced, or synthetic, and further including derivatives and
variants of such endogenous ligands), and synthetically produced
compounds (e.g., small molecules, peptides, etc.) The term
"nicotine" further includes any pharmacologically acceptable
derivative or metabolite of nicotine which exhibits
pharmacotherapeutic properties similar to nicotine. Such
derivatives, metabolites, and derivatives of metabolites are known
in the art, and include, but are not necessarily limited to,
cotinine, norcotinine, nornicotine, nicotine N-oxide, cotinine
N-oxide, 3-hydroxycotinine and 5-hydroxycotinine or
pharmaceutically acceptable salts thereof.
[0048] Agents that increase nitric oxide are of interest as
anti-restonic agents, e.g. S-nitrosopenicillamine, sodium
nitroprusside,
N-ethyl-2-(1-ethyl-2-hydroxy-2-nitrosohydrazino)ethanamine (NOC
12), etc. The production of nitric oxide may also be modulated by
cytokines, such as .gamma.-interferon, tumor necrosis factor, IL-1,
IL-2 and endotoxin due to their effect on the enzyme, nitric oxide
synthase. The inducible form of NO synthase is increased by
cytokines and the constitutive form seems to be decreased by
cytokines. HMG-CoA reductase inhibitors have been found to
upregulate endothelial cell NOS activity, as described by U.S. Pat.
No. 6,147,109, Liao et al. Any of the forms of nitric oxide
synthase can be utilized, as the protein or an active fragment
derived therefrom, or as a DNA construct for expression.
[0049] Also of interest for the inhibition of restenosis are
compounds with an anti-angiogenic effect. These include the
anti-angiogenic polypeptides: angiostatin (O'Reilly et al. (1994)
Cell 79:315-328); endostatin (O'Reilly et al. (1997) Cell 88:
277-285); and anti-angiogenic anti-thrombin III (Bock et al. (1982)
Nucleic Acids Res. 10 (24), 8113-8125); and the like, and further
include functionally active variants and derivatives thereof. Other
anti-angiogenic agents include inhibitors of matrix
metalloproteases, e.g. amifostine, WR-1065; marimastat,
primomastat, alpha-1 antitrypsin; and the like.
[0050] Alternatively, compounds that block thrombin, and other
anti-coagulants, may be used to inhibit restenosis, such compounds
based on the tripeptide motif D-Phe-Pro-Arg; e.g. LY287045, etc.
Many compounds, such as inogatran and melagatran, are known in the
art. For non-limiting examples, see U.S. Pat. Nos. 6,326,386;
6,232,315; 6,201,006; 6,174,855; 6,060,451; and 5,985,833; among
others.
[0051] Agonists of the TGF-beta receptor are also of interest.
TGF-.beta. receptor Type I and type II mediate most activities of
TGF-beta (Ebner et al. (1993) Science 260:1344-1348; and Franzen et
al. (1993) Cell 75: 681-692). Ligands include TGF-.beta., and
mimetics and biologically active derivatives thereof.
[0052] Complex systems of drugs may be carried by the prosthesis,
i.e. stent or other device. An anticoagulant or antiplatelet may be
included in the outermost surface of the device in order to elute
off very quickly for the first several days. Antiinflammatories and
antireplicates can be formulated into the device to continue to
elute later, when in contact with non-blood cells after neointima
overgrowth has surrounded the device. The drug elution rate does
not need to be uniform, and may be tailored to fit the need of the
patient.
[0053] As used herein, the term stent is used as is known in the
art, to refer to a prosthesis which can be inserted and held, when
desired, in a lumen of a vessel or organ in the body. Uses include
the support of blood vessels, the trachea, renal and urethral
tubules, fallopian tubes, eustachian, large and small intestines,
etc. Materials commonly used in stent construction include
biologically compatible metals, e.g. stainless steel, titanium,
tantalum, gold, platinum, copper and the like, as well as alloys of
these metals; low shape memory plastic; a shape-memory plastic or
alloy, such as nitinol; and the like. Any of these materials can be
fabricated to form channels for use in the present invention, and
can form, or be derivatized to form, covalent bonds with the
matrix.
[0054] There are a multiplicity of different stents that may be
utilized. Although any number of stents may be utilized in
accordance with the present invention, for simplicity, several
representative stents will be described in exemplary embodiments of
the present invention. The skilled artisan will recognize that any
kind of stent may be utilized in connection with the present
invention. Non-limiting examples of commercially available stents
include the Gianturco-Roubin stent and the Palmaz-Schatz stent,
commonly used for tandem short segment stenotic lesions; Wallstent
(Boston Scientific, Natick, Mass.), a self expanding stainless
stent used for long lesions; Mammotherm stent, Symphony stent,
Smart stent, all of self expanding nitinol; the balloon exapandable
Perflex, AVE, Intrastent, and Herculink stents, self-expanding
Instent, Gianturco Z-stent (Wilson-Cook, Winston-Salem, N.C.),
Ultraflex nitinol mesh stent (Microinvasive, Natick, Mass.), and
Esophacoil (IntraTherapeutics, Eden Prairie, Minn.).
Tracheobronchial stents include the Gianturco Z tracheobronchial
tree stent and the Wallstent tracheobronchial endoprosthesis. The
stent may be self-expanding, or may be expandable with a balloon,
as is known in the art.
[0055] Additional platforms for the invention include polymeric
biodegradable stents, anastomotic devices, and scaffolds, including
synthetic biodegradable or bioerodible porous scaffolds produced
using solid free-form fabrication techniques which include
selective laser sintering, three-dimensional printing, fused
deposition manufacturing, and stereolithography for micro- or
nano-fabrication.
[0056] In one embodiment of the invention, the drug or drugs are
formulated as a liquid for release from a stent. For example, a
stent may include a chamber with a drug transport wall, where the
anti-restenotic agent is loaded into the chamber, then selectively
transported through the wall (see U.S. Pat. No. 5,498,238). Other
variations of this approach include the use of a hollow tubular
wire stent, or a stent comprising a reservoir. Such stents are
described in the art as having side walls facing outwardly having
holes for delivery of the liquid formulation to the targeted site,
where the stent is implanted (U.S. Pat. No. 5,891,108). The
anti-restenotic agent may be diffused from a reservoir directly to
the walls of a blood vessel, through directional delivery openings
arranged on an outer surface of the stent. Such devices may also
comprise an osmotic engine assembly for controlling the delivery of
the agent from the reservoir (U.S. Pat. No. 6,071,305).
[0057] An alternative to liquid formulation is provided by devices
that comprise a drug compounded to the device itself. In one
embodiment, the stent itself is formed of a polymeric material
comprising the anti-restenotic agent, where the stent is
biodegradable or bioabsorbable (see U.S. Pat. No. 6,004,346).
Alternatively, the prostheses may be biostable in which case the
drug is diffused out from the biostable materials in which it is
incorporated. With metal stents, the device can include a
drug-carrying coating overlying at least a portion of the
metal.
[0058] Alternatively the device may comprise a drug carrying
coating. For example a porous stent can be made from a powdered
metal or polymer, where the anti-restenotic agents are then
compressed into the pores of the stent (see U.S. Pat. Nos.
5,972,027; and 6,273,913). Stents for drug delivery can also
comprise multiple coatings, where the rate of release is different
for the two coatings (see U.S. Pat. No. 6,258,121), where one of
the anti-restenotic agents can be present in both coatings to
provide for an extended release profile; or where two or more
anti-restenotic agents are differentially released. Other composite
coatings include at least one composite layer of the
anti-restenotic agent and a polymer material, and at least a
barrier layer positioned over the composite layer and being of
thickness adequate to provide a controlled release of the bioactive
agent (see U.S. Pat. No. 6,335,029). The sheath over the coating
containing the anti-restenotic agent can also be perforated, so
that when the stent is compressed, the perforation is closed. Upon
placement in the vessel, the stent is expanded, and the perforation
is opened (see U.S. Pat. No. 6,280,411).
[0059] Drugs may be held by covalent bonds (eg, C--C bonds, sulfur
bridges) or noncovalent bonds (eg, ionic, hydrogen bonds). The
blended matrix may then be attached to the stent surface by dipping
or spraying the stent, or other coating methods known in the art.
The drug can alternatively be encapsulated in microparticles or
nanoparticles and dispersed in a stent coating. A diffusion
limiting top-coat may optionally be applied to the above coatings.
Drugs may also be released by particle dissolution or diffusion
when nonbioerodable matrices are used, or during polymer breakdown
when incorporated (absorbed) into a biodegradable substance.
[0060] In some embodiments, a drug delivery device which is
distinct from the stent may be implanted proximally to the site of
stent insertion to effect the long-term release of a drug. Without
limitation, such drug delivery devices may include biodegradable
microparticles injected into, or coated so as to adhere to, the
vascular wall; sheets of material fabricated from biodegradable
hydrogels (i.e., gelatin, alginate), ethylene vinyl acetate,
chitosan, silk fibroin, poly(D,L-lactide-co-glycolide),
poly-L-lactide-co-caprolactone, poly(hydroxyvalerate),
poly(L-lactic acid), poly(D,L-lactic acid), poly(glycolic acid),
polycaprolactone, polyanhydride, polydiaxanone, polyorthoester,
polyamino acids, poly(trimethylene carbonate), peptide nanofibers
or other or other suitable synthetic polymers as sheets, particles,
rings, or other convenient geometries. As discussed above, drugs
may be released by particle dissolution or diffusion when
nonbioerodable matrices are used, or during polymer breakdown when
incorporated (absorbed) into a biodegradable substance.
[0061] The screening methods of the invention are practiced by
assaying a test agent for its ability to affect a number of
biological activities that are positively or negatively correlated
with the ability of drugs to inhibit restenosis. These biological
activities are indicative of whether the agent can affect (1)
selected inflammatory responses (which selected activities may
include T cell responses, endothelial and smooth muscle cell
responses, production of chemokines), (2) cell proliferation, for
example of smooth muscle cells and/or endothelial cells, (3) tissue
remodeling (expression of proteins involved in matrix production
and degradation: collagen 1, uPAR, PAI-1), (4) vasorelaxation (PGE2
levels) and (5) thrombosis (expression of proteins involved in
thrombosis: tissue factor, thrombomodulin). Preferred agents for
preventing restenosis inhibit smooth muscle proliferation with
little or no effect on endothelial cell proliferation, inhibit
matrix remodeling, is vasorelaxant, is not pro-thrombotic, and
induces select anti-inflammatory activities.
[0062] While any method for assaying one of the biological
activities of interest can be used in the present method, BioMAP
Systems are the most informative and efficient to use in the
method. The BioMAP Systems included assays utilizing endothelial
cells, smooth muscle cells, monocytes, T cells, and fibroblasts,
and the assays were conducted to assess the impact of these agents
on cell proliferation (smooth muscle and endothelial cell) and on
markers of tissue remodeling, inflammation, and thrombosis.
[0063] In general, BioMAP Systems are designed to model complex
human disease biology in a practical in vitro format. This is
achieved by stimulating human primary cells (single cell type or
defined mixtures of cell types) such that multiple disease-relevant
signaling pathways are simultaneously active. The choice of cell
types and stimulations is guided by the knowledge of disease
biology and mechanisms. Incorporating appropriate cell types and
stimulating signaling pathways relevant to disease states allows
association of biological activities detected in BioMAP Systems
with disease processes. Drug effects are then recorded by measuring
biologically meaningful protein readouts that provide coverage of
biological space of interest (e.g., inflammation, tissue
remodeling) and allow discrimination between different drug classes
tested. The BioMAP systems useful for this evaluation are listed in
Table 2.
[0064] Without being limited to the theory, some of the rationale
for inclusion of certain parameters is outlined below. The
successful stent drugs paclitaxel, rapamycin and everolimus are
potent inhibitors of smooth muscle cell proliferation, suggesting
that this is a required biological effect in order to prevent
restenosis. These compounds are also potent inhibitors of
endothelial cell proliferation and therefore would prevent wound
healing, which activity is desirably avoided.
[0065] Cell migration and matrix remodeling processes are regulated
by certain proteins such as uPAR and PAI-1, and play important
roles in neointima formation (Behrendt, 2004; Stefansson, 2003).
High levels of uPA are a risk factor for restenosis (Strauss,
1999). PGE.sub.2 and other prostaglandin pathway components (e.g.
PGI.sub.2) that upregulate cAMP are known smooth muscle cell
relaxants and growth inhibitors (Wong, 2001). Inflammation plays a
key role in atherosclerosis, and inflammatory markers, including
MCP-1 and CD40, are increased in patients (Garlichs, 2001; Deo,
2004). High pre-angioplasy levels of the inflammatory protein CD40L
(the ligand for CD40) are predictive of higher restenosis rates
after angioplasty (Cipollone et al., 2003; L'Allier, 2005). Other
chemokines such as IL-8 and IP-10 will positively and negatively
regulate angiogenesis, respectively (Belperio, 2000) as well as
being involved in the recruitment of inflammatory leukocytes.
Collagen I production is associated with the resolution of wound
healing. VEGFR2 is the main VEGF receptor on angiogenic endothelium
mediating migration and proliferation, suggesting that promotion of
expression of this receptor is desired (Rahimi, 2000). M-CSF is
involved in differentiation of macrophages from monocytes as well
as being a promoter of VEGF production and potentially
reendothelialization (Eubank, 2003). The two approved stent drugs
(paclitaxel and rapamycin) increase the level of TNF-.alpha.
produced by monocytes as previously reported (Allen, 1993).
Increases in TNF-.alpha. may induce other factors like
prostaglandins to relax the smooth muscle, and inhibition of
TNF-.alpha. has shown to worsen congestive heart failure patients
(Chung, 2003), indicating that some level of TNF-.alpha. is
protective. Preventing local thrombus formation may modulate the
processes of wound healing and re-endothelialization, so that
reductions in tissue factor and increases in thrombomodulin are
important.
[0066] The method of the invention can be practiced with assays to
assess the effect of an agent on the four key biological activities
(cell proliferation, inflammation, thrombosis, and tissue
remodeling), but because BioMAP Systems utilize co-cultures of
different primary cell types as well as complex stimulation
conditions with multiple disease-relevant factors, the present
invention also provides additional embodiments that provide more
information regarding an agent's utility in the inhibition of
restenosis. Such additional assays include assays that reveal
interactions of endothelial cells and monocytes/macrophages,
effects of inflammatory factors on proliferation of smooth muscle
and endothelial cells, differential effects of various growth
factors stimuli on proliferation of smooth muscle and endothelial
cells under inflammatory conditions, and additional readouts (e.g
extracellular matrix).
[0067] The methods of the present invention are directed to the
identification of novel anti-restenosis drugs. Agents suitable for
testing in the method include, without limitation small molecular
compounds, natural products, proteins peptides, plant or other
extracts, in general any agent or substance with biological
activity. In one embodiment, the invention is practiced to identify
combinations of two or more agents that prevent restenosis.
[0068] A variety of different candidate agents may be screened by
the above methods. Candidate agents encompass numerous chemical
classes, though typically they are organic molecules, preferably
small organic compounds having a molecular weight of more than 50
and less than about 2,500 daltons. Candidate agents comprise
functional groups necessary for structural interaction with
proteins, particularly hydrogen bonding, and typically include at
least an amine, carbonyl, hydroxyl or carboxyl group, preferably at
least two of the functional chemical groups. The candidate agents
often comprise cyclical carbon or heterocyclic structures and/or
aromatic or polyaromatic structures substituted with one or more of
the above functional groups. Candidate agents are also found among
biomolecules including peptides, saccharides, nucleic acids, fatty
acids, steroids, purines, pyrimidines, derivatives, structural
analogs or combinations thereof.
[0069] Candidate agents are obtained from a wide variety of sources
including libraries of synthetic or natural compounds. For example,
numerous means are available for random and directed synthesis of a
wide variety of organic compounds and biomolecules, including
expression of randomized oligonucleotides and oligopeptides.
Alternatively, libraries of natural compounds in the form of
bacterial, fungal, plant and animal extracts are available or
readily produced. Additionally, natural or synthetically produced
libraries and compounds are readily modified through conventional
chemical, physical and biochemical means, and may be used to
produce combinatorial libraries. Known pharmacological agents may
be subjected to directed or random chemical modifications, such as
acylation, alkylation, esterification, amidification, etc. to
produce structural analogs.
[0070] In another embodiment, the invention is practiced by
additionally examining the effect on these biological activities of
known drugs (including but not limited to anti-platelet drugs,
statin drugs, and anti-hypertensive drugs) and then selecting the
agent(s) for preventing restenosis on the basis of their
complementarity of action with those drugs. Evaluation of drug
combinations is useful, as patients receiving paclitaxel-coated
stents are frequently prescribed other medications including
statins and various anti-hypertensive drugs. Identification of
particular drugs (or drug classes) that together with a known
anti-restenosis agent, such as paclitaxel, provides enhanced
anti-restenotic activities, without unexpected adverse activities,
would provide optimal patient benefit. The methods of the invention
are ideal for identifying synergistic activities, and the BioMAP
systems exemplified detect the activity of an agent on a wide
variety of biological mechanisms relevant to restenosis.
[0071] The present invention identifies drugs and provides stents
coated with such drugs and drug combinations, where the drugs
provide for a favorable combination of features (or biological
activities of importance), defined as (1) inhibition of smooth
muscle cell proliferation (2) little or no effect on endothelial
cell proliferation (3) inhibition of matrix remodeling with
promotion of wound healing, (4) no promotion of thrombosis, (5)
promotion of vessel relaxation and (6) selected anti-inflammatory
activities.
[0072] One can further refine the selection of drugs for the
treatment of restenosis by measuring their relative effects at
multiple doses on proliferation of smooth muscle cells and
endothelial cells derived from different sources (umbilical cord or
coronary artery), and under different growth conditions
(proliferation driven by growth factors only, or in the presence of
pro-inflammatory factors that are normally found at the restenotic
site). Statistical analysis of cell proliferation in BioMAP systems
3C, SM3C, CANo, CASMNo, CA3C, and CASM3C listed in Table 1 was
performed for candidate drugs 8-Azaguanine, Amodiaquin
Dihydrochloride Dihydrate, Atovaquone, Ciclopirox,
Cis-(Z)-Flupentixol Dihydrochloride, Clofilium Tosylate,
Monobenzone, Primaquine Diphosphate, Securinine, Syrosingopine,
Terconazole, and Trimethylcolchicinic Acid. Table 5 below shows the
log ratios of smooth muscle cell to endothelial cell proliferation
for the compounds. Drugs showing a ratio of >1 preferentially
inhibit smooth muscle cell proliferation relative to endothelial
cell proliferation and are especially promising candidates as
inhibitors of restenosis (cells with no ratio means that both
values of proliferation were not significantly different from DMSO
control).
[0073] This analysis singles out Trimethylcolchicinic Acid (a
colchicine analog) as promising because it shows preferential
effect on smooth muscle cells over endothelial cells under all
conditions, at all doses tested and independent of the source of
primary cells.
[0074] Thus, in one embodiment, the present invention provides a
stent or other device intended for in vivo use, wherein said stent
or device comprises one or more colchicine analogs and their
pharmaceutically acceptable salt as an active agent, where the
agent has the structure I:
##STR00004##
[0075] In another embodiment, the present invention provides a
stent or other device intended for in vivo use, wherein said stent
or device comprises one or more isocolchicine analogs and their
pharmaceutically acceptable salt as an active agent, where the
agent has the structure II:
##STR00005##
[0076] In structures I and II R.sub.1 and R.sub.2 are independently
selected from H; C.sub.1-10 alkyl, alkenyl, and alkynyl. R.sub.1
and R.sub.2 may also form a cycle containing between 1-6 carbon
atoms provided that the cycle thus formed does not contain an
alkynyl group. The alkyl, alkenyl and alkynyl groups can be
optionally substituted with one or more of OH, OR.sub.9,
NR.sub.10R.sub.11 and F where R.sub.9, R.sub.10 and R.sub.11 are
independently chosen from branched and unbranched C.sub.1-5 alkyl,
provided that the OH and F substituents are not attached to the
same carbon atom as is N in the structure above, that any F
substituents are not allylic or propargylic, and that OH and
OR.sub.9 are not attached to the same carbon nor are OH and
NR.sub.10R.sub.11 attached to the same carbon atom.
[0077] R.sub.3 is selected from H, branched and unbranched
C.sub.1-6 alkyl, alkenyl and alkynyl.
[0078] X is O or NOR.sub.9.
[0079] R.sub.4 and R.sub.5 are selected independently from H, OH,
OR.sub.9, NR.sub.1R.sub.2, provided that R.sub.9 is not CH.sub.3
when incorporated into R.sub.4 when the structure is I.
[0080] Each of R.sub.6, R.sub.7 and R.sub.8 are independently
selected from branched or unbranched C.sub.1-6 lower alkyl and
R.sub.6 and R.sub.7 or R.sub.7 and R.sub.8 may form cycles
containing 1 or 2 carbon atoms in the ring.
[0081] In one embodiment of the invention, the agent is compound
III or a pharmaceutically acceptable salt thereof.
##STR00006##
[0082] In another embodiment, the present invention provides a
stent or other device intended for in vivo use, wherein said stent
or device comprises a colchicine analog as set forth herein, in
combination with a second biologically active agent. Such
biologically active agents may include, without limitation,
clinically proven anti-inflammatory such as a glucocorticoid
receptor agonist (e.g. prednisolone, methylprednisolone,
budesonide), a pro-healing drug (e.g. estradiol), a lipid
metabolism modulating drug (e.g. statins), an anti-thrombotic drug
etc.
[0083] Such stents and devices find use as is known in the art for
the treatment or prevention of restenosis. For example, a stent may
be into the vasculature of a patient following balloon angioplasty,
where the stent or a device implanted proximally to the stent is
designed to release one or more drugs according to description set
forth above, to delay or prevent the onset of restenosis. In such
an embodiment, the colchicine analog is delivered at an effective
local concentration of about 0.01 to about 100 .mu.M. In some
embodiments, the apparatus is configured so as to release the drugs
for at least a portion of the intended duration of stent
implantation, from 1 to about 60 days.
Examples
Cell Culture
[0084] Human umbilical vein endothelial cells (HUVEC) were pooled
from multiple donors, cultured according to standard methods, and
plated into microtiter plates at passage 4. Human neonatal foreskin
fibroblasts (HDF) from 3 donors were pooled and cultured according
to standard methods. 24 hr before stimulation with cytokines,
confluent HDF in microtitre plates were serum starved. Coronary
artery endothelial cells (CAEC), coronary artery smooth muscle
cells (CASMC) and umbilical artery smooth muscle cells (SMC)
cultured according to standard methods. Peripheral blood
mononuclear cells (PBMC) were prepared from buffy coats from normal
human donors according to standard methods. Concentrations/amounts
of agents added to confluent microtitre plates to build each system
were as follows: cytokines (IL-1beta, 1 ng/ml; TNF-alpha, 5 ng/ml;
IFN-gamma, 20 ng/ml; TGF-beta, 5 ng/ml; IL-4, 5 ng/ml), activators
(histamine, 10 microM; SAg, 20 ng/ml or LPS, 0.2 ng/ml), PBMC
(7.5.times.10.sup.4 cells/well).
[0085] Compounds.
[0086] Compounds were prepared in the solvent as directed, added 1
hr before stimulation of the cells, and were present during the 24
hr stimulation period for the measurement of parameters listed in
Table 2. For proliferation, compounds added 1 hr before stimulation
of the cells with cytokines, and were present during the 96 hr. If
prepared in DMSO, the final concentration of solvent was 0.1% or
less.
[0087] Readout Parameter Measurements.
[0088] The levels of readout parameters were measured by ELISA.
Briefly, microtiter plates are treated, blocked, and then incubated
with primary antibodies or isotype control antibodies (0.01-0.5
microg/ml) for 1 hr. After washing, plates were incubated with a
peroxidase-conjugated anti-mouse IgG secondary antibody or a
biotin-conjugated anti-mouse IgG antibody for 1 hr followed by
streptavidin-HRP for 30 min. Plates were washed and developed with
TMB substrate and the absorbance (OD) was read at 450 nm
(subtracting the background absorbance at 650 nm). Quantitation of
TNF-alpha in the LPS system was done using a commercially available
kit according to the manufacturer's directions. Proliferation of
endothelial cells (HUVEC and CAEC), smooth muscle cells (SMC and
CASMC), and PBMC (T cells) was quantitated by Alamar blue or SRB
reduction.
[0089] Toxicity Assessments.
[0090] Adverse effects of compounds on cells were determined by (1)
measuring alterations in total protein (Sulforodhamine B or SRB
assay); (2) measuring the viability of peripheral blood mononuclear
cells (incorporation of propidium iodide, PI); and (3) microscopic
visualization. SRB was performed by staining cells with 0.1%
sulforhodamine B after fixation with 10% TCA, and reading wells at
560 nm. PBMC viability was assessed by adding propidium iodide (10
microgram/ml) to PBMC that had been cultured for 24 hours in the
presence of activators and measuring the percentage of cells that
incorporated dye by flow cytometry after 10 minutes. Samples were
assessed visually according to the following scheme:
2.0=cobblestone (unactivated phenotype); 1.0=activated (normal
phenotype); 0.5=lacy or sparse; 0.375=rounded; 0.25=sparse and
granular; 0.1=no cells in well.
[0091] Data Analysis.
[0092] Mean OD values for each parameter were calculated from
triplicate samples per experiment. The mean value obtained for each
parameter in a treated sample was then divided by the mean value
from an appropriate control to generate a ratio. All ratios were
then log.sub.10 transformed. 99% prediction envelopes (grey shading
in Figures) were calculated for historical controls. Mean
values+/-SD are shown.
[0093] Cellular toxicity in BioMAP Systems is indicated if the SRB
parameter (sulforhodamine B, measure of total protein loss) is
<-0.3, the PI parameter (propidium iodine, measure of
lymphotoxicity) is <-0.1, and the Visual parameter (change in
cell morphology) is <-0.6. Paclitaxel, rapamycin and
dexamethasone showed no signs of toxicity at tested doses, and were
active over the entire dose range. 17beta-estradiol and Actinomycin
D showed signs of cellular toxicity at 10 microM and 4.572 nM, and
were inactive at doses lower that 3.3 microM and 0.5 nM,
respectively.
[0094] Statistical analysis of cell proliferation in the systems
listed in Table 1 was performed for candidate drugs. Table 5 shows
the log ratios of smooth muscle cell to endothelial cell
proliferation for a subset of compounds including the highly
promising compound Trimethylcolchicinic Acid. Drugs showing a ratio
of >1 (green) preferentially inhibit smooth muscle cell
proliferation relative to endothelial cell proliferation and are
especially promising candidates as inhibitors of restenosis (cells
with no ratio means that both values of proliferation were not
significantly different from DMSO control).
TABLE-US-00005 TABLE 5 Log ratios of proliferation in endothelial
and smooth muscle cell model systems for selected compounds. 3C 3C
None None 3C 3C Drug Concentration HUVEC SMC Ratio CAEC CASMC Ratio
CAEC CASMC Ratio 8-Azaguanine 33000 nM -0.3 -0.2 0.7 -0.2 -0.1 0.9
-0.2 -0.2 0.8 8-Azaguanine 11000 nM -0.3 -0.1 0.5 -0.1 -0.1 1.1
-0.2 -0.1 0.6 8-Azaguanine 3666.667 nM -0.2 -0.1 0.4 -0.1 -0.1 1.7
0.0 0.0 8-Azaguanine 1222.222 nM 0.0 0.0 0.0 -0.1 98.0 0.0 0.0
Amodiaquin 33000 nM -0.8 -0.1 0.2 -0.3 -0.3 1.1 -0.6 -0.3 0.4
Dihydrochloride Dihydrate Amodiaquin 11000 nM -0.3 -0.1 0.3 0.0
-0.1 3.5 -0.1 -0.1 1.1 Dihydrochloride Dihydrate Amodiaquin
3666.667 nM -0.1 0.0 0.4 0.0 -0.1 4.4 -0.1 -0.1 1.1 Dihydrochloride
Dihydrate Amodiaquin 1222.222 nM 0.0 0.0 0.0 0.0 0.0 0.0
Dihydrochloride Dihydrate Atovaquone 40000 nM -0.2 -0.2 -0.2 1.1
-0.3 -0.2 0.7 Atovaquone 13333.333 nM -0.3 0.0 0.0 0.0 -0.1 1.7 0.0
-0.1 7.6 Atovaquone 4444.444 nM 0.0 0.0 0.0 -0.1 1.5 0.0 0.0
Atovaquone 1481.481 nM 0.0 0.0 0.0 0.0 0.0 0.0 Ciclopirox 55000 nM
-0.5 -0.4 0.8 -0.6 -0.5 0.8 -0.6 -0.5 0.9 Ethanolamine Ciclopirox
18333.333 nM -0.3 -0.4 1.4 -0.4 -0.4 1.0 -0.4 -0.5 1.1 Ethanolamine
Ciclopirox 6111.111 nM -0.1 -0.3 2.5 -0.2 -0.3 1.5 -0.2 -0.3 1.3
Ethanolamine Ciclopirox 2037.037 nM -0.1 -0.1 1.0 0.0 0.0 -0.1 -0.1
1.5 Ethanolamine Cis-(Z)-Flupentixol 30000 nM -0.7 -1.3 1.7 -1.2
-1.0 0.9 -1.2 -1.1 0.9 Dihydrochloride Cis-(Z)-Flupentixol 10000 nM
-1.0 -0.4 0.4 -0.3 -0.6 1.9 -0.9 -0.8 0.9 Dihydrochloride
Cis-(Z)-Flupentixol 3333.333 nM -0.2 0.0 0.2 0.0 0.0 -0.1 -0.1 0.8
Dihydrochloride Cis-(Z)-Flupentixol 1111.111 nM -0.1 0.0 0.1 0.0
0.0 0.0 0.0 Dihydrochloride Clofilium Tosylate 30000 nM -1.0 -1.3
1.3 -0.1 -0.8 6.5 -0.5 -1.1 2.3 Clofilium Tosylate 10000 nM -0.1
-0.3 1.8 0.0 -0.1 16.0 0.0 -0.5 20.9 Clofilium Tosylate 3333.333 nM
0.0 0.0 0.0 0.0 0.1 0.0 Clofilium Tosylate 1111.111 nM 0.0 0.0 0.0
0.0 0.0 0.0 Everolimus 370.37 nM -0.3 -0.2 0.6 -0.3 -0.2 0.6 -0.3
-0.2 0.6 Everolimus 123.457 nM -0.3 -0.1 0.5 -0.3 -0.2 0.6 -0.3
-0.2 0.6 Everolimus 41.152 nM -0.3 -0.1 0.5 -0.3 -0.2 0.6 -0.3 -0.2
0.7 Everolimus 13.717 nM -0.2 -0.1 0.4 -0.3 -0.1 0.4 -0.2 -0.1 0.6
Monobenzone 75000 nM -0.3 -0.2 0.6 -0.4 -0.2 0.4 -0.3 -0.2 0.6
Monobenzone 25000 nM -0.2 -0.2 1.0 -0.1 -0.1 2.2 -0.1 -0.2 1.3
Monobenzone 8333.333 nM 0.0 -0.1 8.5 0.0 0.0 0.0 -0.1 7.4
Monobenzone 2777.778 nM 0.0 0.0 0.0 0.0 0.0 0.0 Primaquine 33000 nM
-0.3 0.0 0.1 -0.1 -0.1 0.9 -0.3 -0.2 0.9 Diphosphate Primaquine
11000 nM -0.1 0.0 0.4 0.0 0.0 -0.1 -0.1 0.5 Diphosphate Primaquine
3666.667 nM 0.0 0.0 0.0 0.0 -0.1 0.0 0.4 Diphosphate Primaquine
1222.222 nM 0.0 0.0 0.0 0.0 0.0 0.0 Diphosphate Securinine 44000 nM
-0.4 -0.5 1.2 -0.5 -0.4 0.9 -0.6 -0.5 0.9 Securinine 14666.667 nM
-0.3 -0.2 0.8 -0.1 -0.2 2.2 -0.2 -0.3 1.3 Securinine 4888.889 nM
-0.1 -0.1 0.8 0.0 -0.1 2.7 -0.1 -0.1 1.3 Securinine 1629.63 nM 0.0
0.0 0.0 0.0 0.0 -0.1 1.6 Syrosingopine 22222.222 nM -0.7 -0.3 0.5
-0.3 -0.6 1.7 -0.5 -0.7 1.3 Syrosingopine 7407.407 nM -0.4 -0.1 0.3
-0.1 -0.1 1.1 -0.3 -0.2 0.8 Syrosingopine 2469.136 nM -0.1 0.0 0.5
-0.1 -0.1 1.1 0.0 -0.1 2.2 Syrosingopine 823.045 nM 0.0 0.0 0.0 0.0
0.0 0.0 Terconazole 28000 nM -0.9 -1.2 1.3 -1.1 -0.9 0.8 -0.8 -1.0
1.2 Terconazole 9333.333 nM -0.4 -0.2 0.5 -0.2 -0.3 1.2 -0.5 -0.3
0.5 Terconazole 3111.111 nM -0.1 0.0 0.2 0.0 0.0 -0.1 0.0 0.3
Terconazole 1037.037 nM 0.0 0.0 0.0 0.0 0.0 0.0
Trimethylcolchicinic 44000 nM -0.1 -0.3 5.2 -0.1 -0.3 4.9 -0.1 -0.3
5.1 Acid Trimethylcol- 14666.667 nM 0.0 -0.2 6.3 0.0 -0.2 7.9 0.0
-0.2 15.0 chicinic Acid Trimethylcol- 4888.889 nM 0.0 0.0 0.0 -0.1
3.7 0.0 -0.1 12.0 chicinic Acid Trimethylcol- 1629.63 nM 0.0 0.0
0.0 0.0 0.0 0.0 chicinic Acid
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