U.S. patent application number 10/847897 was filed with the patent office on 2004-12-02 for heterocyclic compounds and uses thereof.
This patent application is currently assigned to Ambit Biosciences Corporation. Invention is credited to Grotzfeld, Robert M., Lai, Andiliy G., Lockhart, David J., Mehta, Shamal Anil, Milanov, Zdravko V., Patel, Hitesh K..
Application Number | 20040242673 10/847897 |
Document ID | / |
Family ID | 33479880 |
Filed Date | 2004-12-02 |
United States Patent
Application |
20040242673 |
Kind Code |
A1 |
Lockhart, David J. ; et
al. |
December 2, 2004 |
Heterocyclic compounds and uses thereof
Abstract
The invention provides compounds having the thiophene or furan
skeleton and uses thereof. The invention provides methods of using
the compounds and/or compositions in the treatment of a variety of
diseases and unwanted conditions in subjects. Kits comprising the
compounds of the invention are also provided. The compounds and
compositions disclosed herein are preferably used in the treatment
of neurodegenerative diseases, cardiovascular diseases,
proliferative diseases, and visual disorders. In particular,
methods and compositions for the treatment of stroke are disclosed
herein.
Inventors: |
Lockhart, David J.; (Del
Mar, CA) ; Patel, Hitesh K.; (Encinitas, CA) ;
Mehta, Shamal Anil; (San Diego, CA) ; Milanov,
Zdravko V.; (San Diego, CA) ; Grotzfeld, Robert
M.; (Carlsbad, CA) ; Lai, Andiliy G.; (San
Diego, CA) |
Correspondence
Address: |
WILSON SONSINI GOODRICH & ROSATI
650 PAGE MILL ROAD
PALO ALTO
CA
943041050
|
Assignee: |
Ambit Biosciences
Corporation
San Diego
CA
|
Family ID: |
33479880 |
Appl. No.: |
10/847897 |
Filed: |
May 17, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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60471425 |
May 16, 2003 |
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60480289 |
Jun 20, 2003 |
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60488178 |
Jul 16, 2003 |
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60488172 |
Jul 16, 2003 |
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60480475 |
Jun 20, 2003 |
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60516610 |
Oct 30, 2003 |
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60516651 |
Oct 30, 2003 |
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60516616 |
Oct 30, 2003 |
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Current U.S.
Class: |
514/448 |
Current CPC
Class: |
A61K 31/519 20130101;
A61P 17/06 20180101; A61P 19/06 20180101; A61P 35/00 20180101; A61P
29/00 20180101; Y02A 50/465 20180101; A61P 17/18 20180101; A61P
3/10 20180101; A61P 15/06 20180101; A61P 19/02 20180101; A61P 7/06
20180101; A61P 37/04 20180101; A61P 5/24 20180101; A61P 9/00
20180101; A61P 43/00 20180101; A61P 7/04 20180101; A61P 15/00
20180101; A61P 37/08 20180101; A61P 9/08 20180101; A61P 29/02
20180101; A61P 25/16 20180101; A61P 3/14 20180101; A61P 27/02
20180101; Y02A 50/401 20180101; A61P 13/12 20180101; A61P 19/10
20180101; A61P 21/04 20180101; A61P 9/10 20180101; A61P 11/06
20180101; A61P 37/06 20180101; Y02A 50/411 20180101; A61P 33/02
20180101; A61P 37/02 20180101; A61K 31/381 20130101; A61P 35/02
20180101; A61P 1/02 20180101; A61P 9/04 20180101; A61P 25/00
20180101; A61P 25/28 20180101; A61P 39/06 20180101; A61P 13/08
20180101; A61K 31/00 20130101; A61P 25/02 20180101; A61P 35/04
20180101; A61P 1/04 20180101; A61P 27/06 20180101; A61P 25/08
20180101; A61P 7/02 20180101; A61P 19/08 20180101; A61P 11/00
20180101; A61P 31/04 20180101; A61P 33/06 20180101; A61P 5/14
20180101; A61P 25/14 20180101; Y02A 50/30 20180101; A61P 9/12
20180101; A61P 9/06 20180101 |
Class at
Publication: |
514/448 |
International
Class: |
A61K 031/381 |
Claims
1. A method of treating a disease comprising administering to a
subject in need thereof an effective amount of a compound of
formula I 4or a pharmaceutically acceptable salt thereof, wherein
R.sub.1, R.sub.2, R.sub.3, and R.sub.4 are independently selected
from the group consisting of hydrogen, halogen, alkyl, cycloalkyl,
aryl, heteroaryl, NR.sub.5C(O)R.sub.7, C(O)NR.sub.5R.sub.6,
C(O)R.sub.7 and C(O)OR.sub.7, wherein R.sub.5, R.sub.6, and R.sub.7
are independently selected to be hydrogen, alkyl, cycloalkyl or
aryl, and where R.sub.5 and R.sub.6 or R.sub.5 and R.sub.7 together
can optionally form a 3, 4, 5, 6, or 7 membered ring optionally
having one or more degrees of substitution.
2. The method of claim 1 wherein the compound is at least one of a
compound selected from the compounds 781118, 781222, 781196,
781300, 781326, 781352, 781378, 781560, 779974, 781456, 781534,
781482, 781586, 781612, 781794, 781716, 781898, 781924, 783042, and
783120.
3. A method of treating a disease comprising administering to a
subject in need thereof an effective amount of a compound of
formula II 5or a pharmaceutically acceptable salt thereof, wherein
R.sub.10 and R.sub.11 are independently selected from the group
consisting of hydrogen, halogen, alkyl, cycloalkyl, aryl,
heteroaryl, NR.sub.5C(O)R.sub.7, C(O)NR.sub.5R.sub.6, C(O)R.sub.7
and C(O)OR.sub.7, wherein R.sub.5, R.sub.6, and R.sub.7 are
independently selected to be hydrogen, alkyl, cycloalkyl or aryl,
and where R.sub.5 and R.sub.6 or R.sub.5 and R.sub.7 together can
optionally from a 3, 4, 5, 6, or 7 membered ring optionally having
one or more substitutions.
4. The method of claim 1 wherein the compound is at least one of a
compound selected from the compounds 781372 and 783146.
5. A method of treating a disease comprising administering to a
subject in need thereof an effective amount of a compound of
formula III 6or a pharmaceutically acceptable salt thereof, wherein
R.sub.1, R.sub.2, R.sub.3, and R.sub.4 are independently selected
from the group consisting of hydrogen, halogen, alkyl, cycloalkyl,
aryl, heteroaryl, NR.sub.5C(O)R.sub.7, C(O)NR.sub.5R.sub.6,
C(O)R.sub.7 and C(O)OR.sub.7, wherein R.sub.5, R.sub.6, and R.sub.7
are independently selected to be hydrogen, alkyl, cycloalkyl or
aryl, and where R.sub.5 and R.sub.6 or R.sub.5 and R.sub.7 together
can optionally form a 3, 4, 5, 6, or 7 membered ring optionally
having one or more degrees of substitution.
6. The method of claim 1 wherein the compound is at least one of a
compound selected from the compounds 781508, 781768, 781742,
781872, 782002, 781040, 781092, 781248, 781170, 781144, 781274,
781638, 781664, 781690, 781820, 781846, 781404, 781950, and
781976.
7. The method of claim 1, 3, or 5 wherein said disease is a
PDE6-related condition.
8. The method of claim 1, 3, or 5 wherein said disease is a
condition wherein a beneficial effect is obtained by modulating
PDE6 or any of its subunits.
9. The method of claim 1, 3, or 5 wherein said disease is a
condition wherein a beneficial effect is obtained by modulating
PDE6D.
10. The method of claim 1, 3, or 5 wherein said disease is related
to an aberrant or undesired phosphodiesterase activity.
11. The method of claim 1 wherein said disease is stroke.
12. The method of claim 11 wherein said PDE6 modulating compound is
acutely administered to a subject following a stroke.
13. The method of claim 11 wherein said PDE6 modulating compound is
prophylactically administered to a subject to prevent a stroke.
14. The method of claim 1, 3, or 5 wherein said disease is at least
one of a disease selected from cancer, impaired vision, an
oxidative stress disorder, inflammation, and multiple
sclerosis.
15. The method of claim 1, 3, or 5 wherein said disease is at least
one of a disease selected from cerebrovascular accident,
neurodegenerative disease, and cardiovascular disease
16. The method of claim 15 wherein said cerebrovascular accident is
a stroke.
17. The method of claim 15 wherein treatment of said cardiovascular
disease decreases the frequency and/or the severity of damage of
myocardial infarction
18. The method of claim 15 wherein said neurodegenerative disease
is at least one of an ischemic stroke, Alzheimer's disease,
diabetic peripheral neuropathy, multiple sclerosis, amyotrophic
lateral sclerosis, Huntington's disease and Parkinson's
disease.
19. The method of claim 1, 3, or 5 wherein said compound stimulates
blood vessel growth, bone growth, or immune system.
20. A pharmaceutical composition comprising a pharmaceutically
acceptable excipient and a compound of formula I 7or a
pharmaceutically acceptable salt thereof, wherein R.sub.1, R.sub.2,
R.sub.3, and R.sub.4 are independently selected from the group
consisting of hydrogen, halogen, alkyl, cycloalkyl, aryl,
heteroaryl, NR.sub.5C(O)R.sub.7, C(O)NR.sub.5R.sub.6, C(O)R.sub.7
and C(O)OR.sub.7, wherein R.sub.5, R.sub.6, and R.sub.7 are
independently selected to be hydrogen, alkyl, cycloalkyl or aryl,
and where R.sub.5 and R.sub.6 or R.sub.5 and R.sub.7 together can
optionally form a 3, 4, 5, 6, or 7 membered ring optionally having
one or more degrees of substitution.
21. The pharmaceutical composition of claim 20 wherein the compound
is at least one of a compound selected from the compounds 781118,
781222, 781196, 781300, 781326, 781352, 781378, 781560, 779974,
781456, 781534, 781482, 781586, 781612, 781794, 781716, 781898,
781924, 783042, and 783120.
22. A pharmaceutical composition comprising a pharmaceutically
acceptable excipient and a compound of formula II 8or a
pharmaceutically acceptable salt thereof, wherein R.sub.10 and
R.sub.11 are independently selected from the group consisting of
hydrogen, halogen, alkyl, cycloalkyl, aryl, heteroaryl,
NR.sub.5C(O)R.sub.7, C(O)NR.sub.5R.sub.6, C(O)R.sub.7 and
C(O)OR.sub.7, wherein R.sub.5, R.sub.6, and R.sub.7 are
independently selected to be hydrogen, alkyl, cycloalkyl or aryl,
and where R.sub.5 and R.sub.6 or R.sub.5 and R.sub.7 together can
optionally form a 3, 4, 5, 6, or 7 membered ring optionally having
one or more substitutions.
23. The pharmaceutical composition of claim 22 wherein the compound
is at least one of a compound selected from the compounds 781372
and 783146.
24. A pharmaceutical composition comprising a pharmaceutically
acceptable excipient and a compound of formula III 9or a
pharmaceutically acceptable salt thereof, wherein R.sub.1, R.sub.2,
R.sub.3, and R.sub.4 are independently selected from the group
consisting of hydrogen, halogen, alkyl, cycloalkyl, aryl;
heteroaryl, NR.sub.5C(O)R.sub.7, C(O)NR.sub.5R.sub.6, C(O)R.sub.7
and C(O)OR.sub.7, wherein R.sub.5, R.sub.6, and R.sub.7 are
independently selected to be hydrogen, alkyl, cycloalkyl or aryl,
and where R.sub.5 and R.sub.6 or R.sub.5 and R.sub.7 together can
optionally form a 3, 4, 5, 6, or 7 membered ring optionally having
one or more degrees of substitution.
25. The pharmaceutical composition of claim 24 wherein the compound
is at least one of a compound selected from the compounds 781508,
781768, 781742, 781872, 782002, 781040, 781092, 781248, 781170,
781144, 781274, 781638, 781664, 781690, 781820, 781846, 781404,
781950, and 781976.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Application No. 60/471,425 filed May 16, 2003, U.S. Provisional
Application No. 60/480,289 filed Jun. 20, 2003, U.S. Provisional
Application No. 60/488,178 filed Jul. 16, 2003, U.S. Provisional
Application No. 60/488,172 filed Jul. 16, 2003, U.S. Provisional
Application No. 60/480,475 filed Jun. 20, 2003, U.S. Provisional
Application No. 60/516,610 filed Oct. 30, 2003, U.S. Provisional
Application No. 60/516,651 filed Oct. 30, 2003 and U.S. Provisional
Application No. 60/516,616 filed Oct. 30, 2003 all of which are
incorporated herein by reference.
INTRODUCTION
[0002] Phosphodiesterase 6 delta (PDE6D) was originally identified
as a regulatory (non-catalytic) subunit of the enzyme PDE6. PDE6 is
expressed exclusively in photoreceptor cells, and plays a critical
role in retinal phototransduction. (Stryer, L. (1991) J. Biol.
Chem. 266:10711-14; (Florio, S. K. et al. (1996) J. Biol. Chem.
271:24036-47. The PDE6 holoenzyme exists as both
membrane-associated and-soluble forms, and only the
membrane-associated form is active in phototransduction.
Importantly, only the soluble form contains the PDE6D subunit.
PDE6D regulates the subcellular localization and thus the activity
of PDE6, and the release of PDE6 from membranes is mediated by
PDE6D. PDE6D has been observed to reduce light-induced cGMP
hydrolysis in rod outer segments (Cook et al., J. Biol. Chem
276(7):5248-5255 (2001)), presumably by removing the PDE6
holoenzyme from the membrane. PDE6D solubilizes PDE6 by binding
specifically to prenylated peptide sequences near the C-termini of
the PDE6A and PDE6B subunits. PDE6D is referred to in the
scientific literature by a several designations, including PDE
delta, PDE.delta., PDE6 delta, PDE6.delta., PDE6D and PDED.
[0003] PDE6D interacts specifically with a host of important cell
signaling proteins through their post-translational modification
with isoprenoid intermediates, which are products of the
cholesterol biosynthetic pathway. One such modification is called
prenylation and involves the attachment of phospholipids to
proteins after translation, particularly the large class of
GTP-binding proteins. Prenyl groups are important for proper
cellular localization and trafficking. The availability of
isoprenoid intermediates for prenylation of GTP-binding proteins
has been associated with various cardiovascular, inflammatory,
cancerous and neurological diseases. A reduction in the amount of
available prenyl groups has been associated with improved
endothelial function, decreased oxidative stress, decreased
inflammation and increased neuroprotective effects.
[0004] Due to the possible role of PDE6 in several diseases, there
is a need to develop PDE6 modulators.
SUMMARY OF THE INVENTION
[0005] The present invention provides methods and compositions for
treating and preventing diseases, in particular PDE6-realated
diseases. The compounds of the invention, having the thiophene or
furan structure, can be delivered alone or in combination with
additional agents, and are used for the treatment and/or prevention
of diseases. Accordingly, in one aspect, the invention is directed
to methods for treating or preventing such diseases in a subject in
need thereof. The methods comprise administering to the subject a
pharmaceutically effective amount of a compound having the
thiophene or furan skeleton.
[0006] In another aspect, the invention provides methods for
modulating the activity of phosphodiesterase 6D (PDE6D) with a
compound having the thiophene or furan skeleton.
[0007] The compounds and compositions of the invention modulate the
activity of PDE6D, and hence are useful as therapeutic or
prophylactic agents for conditions and diseases caused by or
aggravated by the localization and function of prenylated proteins,
for example, cardiovascular diseases, such as arteriosclerosis,
hypertension, arrhythmia (e.g. ischemic arrhythmia, arrhythmia due
to myocardial infarction, myocardial stunning, myocardial
dysfunction, arrhythmia, and the like), angina pectoris, cardiac
hypertrophy, myocardial infarction, heart failure (e.g. congestive
heart failure, acute heart failure, cardiac hypertrophy, etc.);
renal diseases, such as, diabetes mellitus, diabetic nephropathy,
ischemic acute renal failure, acute renal failure, and the like;
cerebrovascular diseases, such as ischemic stroke, hemorrhagic
stroke, and the like; and cerebro ischemic disorders, such as
disorders associated with cerebral infarction, disorders caused
after cerebral apoplexy as sequelae, or cerebral edema.
[0008] In another aspect, the present invention provides
compositions and methods for treating and preventing
neurodegenerative conditions and diseases. These compounds
disclosed herein can be delivered alone or in combination with
additional agents, and are used for the treatment and/or prevention
of neurodegenerative conditions and diseases such as those
resulting from ischemic strokes. The neurodegenerative conditions
and disease can be ischemic stroke, basal ganglia or Parkinson's
disease, epilepsy or brain or spinal cord ischemia or trauma;
Alzheimer's disease, diabetic peripheral neuropathy, multiple
sclerosis, amyotrophic lateral sclerosis, traumatic brain injury,
spinal cord injury, Huntington's disease, heart failure (e.g.
congestive heart failure, acute heart failure, cardiac hypertrophy,
etc.) renal diseases, ischemic stroke, traumatic brain injury, or
Parkinson's disease. The thiophene-containing and furan-containing
compounds of the invention are preferably administered in a
pharmaceutical composition containing a pharmaceutically acceptable
excipient.
[0009] In yet another aspect, the present invention provides
methods and compositions for the rehabilitation of a subject with a
central nervous system disorder, such as stroke and traumatic brain
injury. Accordingly, in one embodiment, the invention is directed
to methods for treating or preventing neurodegenerative conditions
or diseases in a subject in need thereof, or, optionally preventing
further progression of the disease. The thiophene-containing and
furan-containing compounds can be administered for a length of time
necessary to allow for the recovery from the neurodegenerative
condition or disease, such as, for example, from about 1 month to
about 3 months to about a year or more if necessary. The
neurodegenerative condition or disease can be ischemic stroke,
basal ganglia or Parkinson's disease, epilepsy or brain or spinal
cord ischemia or trauma, Alzheimer's disease, diabetic-peripheral
neuropathy, multiple sclerosis, amyotrophic lateral sclerosis,
traumatic brain injury, spinal cord injury, or Huntington's
disease.
[0010] These and other aspects of the present invention will become
evident upon reference to the following detailed description. In
addition, various references are set forth herein which describe in
more detail certain procedures or compositions, and are
incorporated by reference in their entirety.
BRIEF DESCRIPTION OF THE FIGURES
[0011] FIG. 1 shows thiophene-containing compounds that are
illustrative of formula I, as described below, along with the
corresponding compound identification numbers.
[0012] FIG. 2 shows thiophene-containing compounds that are
illustrative of formula II, as described below, along with the
corresponding compound identification numbers.
[0013] FIG. 3 shows furan-containing compounds that are
illustrative of formula III, as described below, along with the
corresponding compound identification numbers.
DETAILED DESCRIPTION
[0014] I. Definitions
[0015] Unless otherwise stated, the following terms used in this
application, including the specification and claims, have the
definitions given below. It must be noted that, as used in the
specification and the appended claims, the singular forms "a," "an"
and "the" include plural referents unless the context clearly
dictates otherwise. Definition of standard chemistry terms may be
found in reference works, including Carey and Sundberg (1992)
"Advanced Organic Chemistry 3.sub.rdEd." Vols. A and B, Plenum
Press, New York. The practice of the present invention will employ,
unless otherwise indicated, conventional methods of mass
spectroscopy, protein chemistry, biochemistry, recombinant DNA
techniques and pharmacology, within the skill of the art.
[0016] The terms "effective amount" or "pharmaceutically effective
amount" refer to a nontoxic but sufficient amount of the agent to
provide the desired biological, therapeutic, and/or prophylactic
result. That result can be reduction and/or alleviation of the
signs, symptoms, or causes of a disease, or any other desired
alteration of a biological system. For example, an "effective
amount" for therapeutic uses is the amount of the compound having
the thiophene or furan skeleton as disclosed herein per se or a
composition comprising the compound required to provide a
clinically significant decrease in a disease. An appropriate
effective amount in any individual case may be determined by one of
ordinary skill in the art using routine experimentation.
[0017] By "pharmaceutically acceptable" or "pharmacologically
acceptable" is meant a material which is not biologically or
otherwise undesirable, i.e., the material may be administered to an
individual without causing any undesirable biological effects or
interacting in a deleterious manner with any of the components of
the composition in which it is contained.
[0018] The term "pharmaceutically acceptable salt" of a compound
means a salt that is pharmaceutically acceptable and that possesses
the desired pharmacological activity of the parent compound. Such
salts, for example, include:
[0019] (1) acid addition salts, formed with inorganic acids such as
hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,
phosphoric acid, and the like; or formed with organic acids such as
acetic acid, propionic acid, hexanoic acid, cyclopentanepropionic
acid, glycolic acid, pyruvic acid, lactic acid, malonic acid,
succinic acid, malic acid, maleic acid, fumaric acid, tartaric
acid, citric acid, benzoic acid, 3-(4-hydroxybenzoyl)benzoic acid,
cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic
acid, 1,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid,
benzenesulfonic acid, 2-naphthalenesulfonic acid,
4-methylbicyclo-[2,2,2]oct-2-ene-1-carboxylic acid, glucoheptonic
acid, 4,4'-methylenebis-(3-hydroxy-2-ene-1-carboxylic acid),
3-phenylpropionic acid, trimethylacetic acid, tertiary butylacetic
acid, lauryl sulfuric acid, gluconic acid, glutamic acid,
hydroxynaphthoic acid, salicylic acid, stearic acid, muconic acid,
and the like;
[0020] (2) salts formed when an acidic proton present in the parent
compound either is replaced by a metal ion, e.g., an alkali metal
ion, an alkaline earth ion, or an aluminum ion; or coordinates with
an organic base. Acceptable organic bases include ethanolamine,
diethanolamine, triethanolamine, tromethamine, N-methylglucamine,
and the like. Acceptable inorganic bases include aluminum
hydroxide, calcium hydroxide, potassium hydroxide, sodium
carbonate, sodium hydroxide, and the like. It should be understood
that a reference to a pharmaceutically acceptable salt includes the
solvent addition forms or crystal forms thereof, particularly
solvates or polymorphs. Solvates contain either stoichiometric or
non-stoichiometric amounts of a solvent, and are often formed
during the process of crystallization. Hydrates are formed when the
solvent is water, or alcoholates are formed when the solvent is
alcohol. Polymorphs include the different crystal packing
arrangements of the same elemental composition of a compound.
Polymorphs usually have different X-ray diffraction patterns,
infrared spectra, melting points, density, hardness, crystal shape,
optical and electrical properties, stability, and solubility.
Various factors such as the recrystallization solvent, rate of
crystallization, and storage temperature may cause a single crystal
form to dominate.
[0021] Where chiral centers occur in the compounds having the
R.sub.1, R.sub.2, R.sub.3 and R.sub.4 moieties as defined above,
the invention includes the enantiomeric compounds resulting from
the chiral center as well as racemic mixtures thereof.
[0022] As used herein, the term "halogen" includes fluorine,
chlorine, bromine, and iodine.
[0023] The term "alkyl" as used herein refers to a substituted or
unsubstituted straight, branched, or cyclic hydrocarbon chain
fragment or radical, preferably containing between about one and
about twenty carbon atoms, more preferably between about one and
about ten carbon atoms (e.g., methyl, ethyl, n-propyl, iso-propyl,
cyclopropyl, n-butyl, iso-butyl, tert-butyl, cyclobutyl, adamantyl,
noradamantyl and the like). Straight, branched, or cyclic
hydrocarbon chains having ten or fewer carbon atoms will also be
referred to herein as "lower alkyl". The hydrocarbon chains may
further include one or more degrees of unsaturation, i.e., one or
more double or triple bonds (e.g., vinyl, propargyl, allyl,
2-buten-1-yl, 2-cyclopenten-1-yl, 1,3-cyclohexadien-1-yl,
3-cyclohexen-1-yl and the like. Alkyl groups containing double
bonds such as just described will also be referred to herein as
"alkylenes".
[0024] The term "aryl" as used herein refers to cyclic aromatic,
hydrocarbon chains having twenty or fewer carbon atoms, e.g.,
phenyl, naphthyl, biphenyl and anthracenyl. One or more carbon
atoms of the aryl group may also be substituted with, e.g., alkyl;
aryl; heterocycle; formyl; halogen; nitro; cyano; hydroxyl, alkoxyl
or aryloxyl; thio or mercapto, alkyl-, or arylthio; amino,
alkylamino, arylamino, dialkyl-, diaryl-, or arylalkylamino;
aminocarbonyl, alkylaminocarbonyl, arylaminocarbonyl,
dialkylaminocarbonyl, diarylaminocarbonyl or
arylalkylaminocarbonyl; carboxyl, or alkyl- or aryloxycarbonyl;
carboxaldehyde, or aryl- or alkylcarbonyl; iminyl, or aryl- or
alkyliminyl; sulfo; alkyl- or arylsulfonyl; hydroximinyl, or aryl-
or alkoximinyl; ureido; or thioureido. In addition, two or more
alkyl or heteroalkyl substituents of an aryl group may be combined
to form fused aryl-alkyl or aryl-heteroalkyl ring systems (e.g.,
tetrahydronaphthyl). Substituents including heterocyclic groups
(e.g., heterocycleoxy, heteroaryloxy, and heteroaralkylthio) are
defined by analogy to the above-described terms.
[0025] As used herein, "aliphatic" includes alkanes, olefins
(alkenes or alkyldienes), and alkynes.
[0026] Alicyclic includes substituted or unsubstituted
cycloparaffins (saturated), cycloolefins (unsaturated with two or
more double bonds), and cycloacetylenes (cyclynes) with at least
one triple bond. Non-limiting examples include cyclopropane,
cyclohexane, cyclopentane, cyclopentadiene, and
cycloctatetraene.
[0027] Aromatic refers to substituted or unsubstituted unsaturated
cyclic hydrocarbons of one or more rings and includes aryl
structures typified, but not limited to, phenyl, naphthalyl,
phenanthrenyl, and anthracenyl. Non-limiting aromatic examples
include 6 membered (typified by benzene) as well as 5 membered
(typified by furan, thiophene, pyrrole, and indole) rings.
[0028] Heterocycle refers to the presence of at least one
non-carbon atom in a cyclic structure. Non-limiting examples
include the presence of a nitrogen, oxygen, and sulfur atom to
result in heterocyclic rings including, but not limited to, phenyl,
naphthyl, pyridinyl, pyrimidinyl, pyrazinyl, pyridazinyl, pyrrolyl,
imidazolyl, pyrazolyl, thienyl, furyl, tetrahydrofuryl, isoxazolyl,
isothiazolyl, quinolinyl, isoquinolinyl, indolyl, benzimidazolyl,
benzofuranyl, benzoxazolyl, benzisoxazolyl, benzpyrazolyl,
benzothiofuranyl, cinnolinyl, pterindinyl, phthalazinyl,
naphthypyridinyl, quinoxalinyl, quinazolinyl, purinyl andindazolyl;
wherein such phenyl, naphthyl or heterocyclic group is optionally
substituted with one to five groups selected from the group
consisting of a C1-6 branched or unbranched alkyl, phenyl,
naphthyl, heterocycle selected from the group hereinabove
described, C1-6 branched or unbranched alkyl which is optionally
partially or fully halogenated, cyclopropyl, cyclobutyl,
cyclopentanyl, cyclohexanyl, cycloheptanyl, bicyclopentanyl,
bicyclohexanyl, bicycloheptanyl, phenyl C1-5 alkyl, naphthyl C1-5
alkyl, halo, hydroxy, cyano, C1-3 alkyloxy which may optionally be
partially or fully halogenated, phenyloxy, naphthyloxy,
heteraryloxy wherein the heterocyclic moiety is selected from the
group hereinabove described, nitro, amino, mono- or
di-(C1-3)alkylamino, phenylamino, naphthylamino, heterocyclylamino
wherein the heterocyclyl moiety is selected from the group
hereinabove described, NH.sub.2C(O), a mono- or di-(C1-3)alkyl
aminocarbonyl, C1-5 alkyl-C(O)-C1-4 alkyl, amino-C1-5 alkyl,
mono-or di-(C1-3)alkylamino-C1-5 alkyl, amino-S(O)2, or di-(C1-3)
alkylamino-S(O)2; or a fused aryl selected from the group
consisting of benzocyclobutanyl, indanyl, indenyl, dihydronaphthyl,
tetrahydronaphthyl, benzocycloheptanyl and benzocycloheptenyl, or a
fused heterocyclic moiety selected from the group consisting of
cyclopentenopyridine, cyclohexanopyridine, cyclopentanopyrimidine,
cyclohexanopyrimidine, cyclopentanopyrazine, cyclohexanopyrazine,
cyclopentanopyridazine, cyclohexanopyridazine,
cyclopentanoquinoline, cyclohexanoquinoline,
cyclopentanoisoquinoline, cyclohexanoisoquinoline,
cyclopentanoindole, cyclohexanoindole, cyclopentanobenzimidazole,
cyclohexanobenzimidazole, cyclopentanobenzoxazole,
cyclohexanobenzoxazole, cyclopentanoitnidazole,
cyclohexanoimidazole, cyclopentanothiophene and
cyclohexanothiophene, wherein the fused aryl or fused heterocyclic
ring is substituted with 0 to 3 groups independently selected from
phenyl, naphthyl and heterocyclic moiety selected from the group
consisting of pyridinyl, pyrimidinyl, pyrazinyl, pyridazinyl,
pyrrolyl, imidazolyl, pyrazolyl, thienyl, furyl, isoxazolyl, and
isothiazolyl, C1-6 branched or unbranched alkyl which is optionally
partially or fully halogenated, halo, cyano, C1-3 alkyloxy which is
optionally partially or fully halogenated, phenyloxy, naphthyloxy,
heterocyclyloxy wherein the heterocyclic moiety is selected from
the group hereinabove described, nitro, amino, mono- or di-(C1-3)
alkylamino, phenylamino, naphthylarnino, heterocyclylamino wherein
the heterocyclic moiety is selected from the group hereinabove
described, NH.sub.2C(O), a mono- or di-(C1-3)alkyl aminocarbonyl,
C1-4 alkyl-OC(O), C1-5 alkyl-C(O)-C1-4 branched or unbranched
alkyl, an amino-C1-5 alkyl, or mono- or di-(C1-3)alkylamino-C1-5
alkyl; or c) cycloalkyl selected from the group consisting of
cyclopentanyl, cyclohexanyl, cycloheptanyl, bicyclopentanyl,
bicyclohexanyl and bicycloheptanyl, wherein the cycloalkyl may
optionally be partially or fully halogenated and which may
optionally be substituted with one to three C1-3 alkyl groups; or
azetidinyl, pyrrolidinyl, piperidinyl, morpholino, piperazinyl,
hexahydroazepinyl or octahydroazocinyl.
[0029] All of the above described aliphatic, carboxyalkyl,
carbalkoxyalkyl, alkoxy, alicyclic, aryl, aromatic, and
heterocyclic moieties may, of course, also be optionally
substituted with 1-3 substituents independently selected from halo
(fluoro, chloro, bromo or iodo), alkyl, and alkoxy.
[0030] Sulfonyl refers to the presence of a sulfur atom, which is
optionally linked to another moiety such as an aliphatic group, an
aromatic group, an aryl group, an alicyclic group, or a
heterocyclic group. Aryl or alkyl sulfonyl moieties have the
formula --SO.sub.2R', and alkoxy moieties have the formula --O--R',
wherein R' is alkyl, as defined above, or is aryl wherein aryl is
phenyl, optionally substituted with 1-3 substituents independently
selected from halo (fluoro, chloro, bromo or iodo), lower alkyl
(1-6C) and lower alkoxy (1-6C).
[0031] II. Compounds
[0032] The present invention is directed to thiophene-containing
and furan-containing compounds and methods for their use as drugs.
The compounds described herein may be used to treat a variety of
diseases. Preferably, the compounds described herein are used in
the treatment of PDE6-related conditions. In certain embodiments,
the compounds of the present invention preferably modulate PDE6 and
have minimal effects on HMG CoA reductase. In certain other
embodiments, the compounds described herein are used in conditions
wherein a modulation of PDE6 is desired with minimal effects on HMG
CoA reductase.
[0033] Those of skill in the art will recognize that the compounds
described herein may exhibit the phenomena of tautomerism,
conformational isomerism, geometric isomerism and/or optical
isomerism. It should be understood that the invention encompasses
any tautomeric, conformational isomeric, optical isomeric and/or
geometric isomeric forms of the compounds described herein, as well
as mixtures of these various different forms. For example, the
compounds of the present invention comprise several chiral atoms
and it is intended that the present invention encompass all
possible stereoisomers and racemic mixtures thereof Accordingly,
the compounds described herein may be administered in their
entantiomerically pure forms or as a mixture of enantiomers, such
as a racemic mixture.
[0034] It will also be appreciated that in many instances the
thiophene-containing and furan-containing compounds may metabolize
to produce active compounds. The use of active metabolites is also
within the scope of the present invention.
[0035] In one aspect, the invention provides compounds having the
thiophene skeleton, or a pharmaceutically acceptable salt of such a
compound. The thiophene containing compound have the general
formula I: 1
[0036] wherein R.sub.1, R.sub.2, R.sub.3, and R.sub.4 are
independently selected from the group consisting of hydrogen,
halogen, alkyl, cycloalkyl, aryl, heteroaryl, NR.sub.5C(O)R.sub.7,
C(O)NR.sub.5R.sub.6, C(O)R.sub.7 and C(O)OR.sub.7, wherein R.sub.5,
R.sub.6, and R.sub.7 are independently selected to be hydrogen,
lower alkyl, cycloalkyl or aryl, and where R.sub.5 and R.sub.6 or
R.sub.5 and R.sub.7 together can optionally form a 3, 4, 5, 6, or 7
membered ring optionally having one or more degrees of
substitution. Thus, R.sub.5 and R.sub.6 or R.sub.5 and R.sub.7
together can optionally form substituted or unsubstituted
aziridino, substituted or unsubstituted piperidino, substituted or
unsubstituted morpholino, substituted or unsubstituted imadazolyl,
substituted or unsubstituted pyridyl, or substituted or
unsubstituted pyrrolidino rings.
[0037] The thiophene-containing compounds illustrative of formula I
are shown in FIG. 1 along with the corresponding compound
identification numbers.
[0038] In another aspect of the invention, thiophene containing
compounds of formula II, or a pharmaceutically acceptable salt of
such a compound, are provided: 2
[0039] wherein R.sub.10 and R.sub.11 are independently selected
from the group consisting of hydrogen, halogen, alkyl, cycloalkyl,
aryl, heteroaryl, NR.sub.5C(O)R.sub.7, C(O)NR.sub.5R.sub.6,
C(O)R.sub.7 and C(O)OR.sub.7, wherein R.sub.5, R.sub.6, and R.sub.7
are independently selected to be hydrogen, alkyl, cycloalkyl or
aryl, and where R.sub.5 and R.sub.6 or R.sub.5 and R.sub.7 together
can optionally form a 3, 4, 5, 6, or 7 membered ring optionally
having one or more substitutions. Thus, R.sub.5 and R.sub.6 or
R.sub.5 and R.sub.7 together can optionally form substituted or
unsubstituted aziridino, substituted or unsubstituted piperidino,
substituted or unsubstituted morpholino, substituted or
unsubstituted imadazolyl, substituted or unsubstituted pyridyl, or
substituted or unsubstituted pyrrolidino ring.
[0040] The thiophene-containing compounds illustrative of formula
II are shown in FIG. 2 along with the corresponding compound
identification numbers.
[0041] In one aspect, the invention provides compounds having the
furan skeleton, or a pharmaceutically acceptable salt of such a
compound. The furan containing compound have the general formula
III: 3
[0042] wherein R.sub.1, R.sub.2, R.sub.3, and R.sub.4 is as defined
above for formula I.
[0043] The furan-containing compounds illustrative of formula III
are shown in FIG. 3 along with the corresponding compound
identification numbers.
[0044] The invention also provides prodrug forms of the
above-described compounds, wherein the prodrug is metabolized in
vivo to produce a derivative as set forth above. Indeed, some of
the above-described derivatives may be a prodrug for another
derivative or active compound. The invention further provides for
the optical isomers of the compounds disclosed herein, especially
those resulting from the chiral carbon atoms in the molecule. In
additional embodiments of the invention, mixtures of enantiomers
and/or diastereoisomers, resulting from a single preparative step,
combination, or interconversion are provided.
[0045] III. Methods of Synthesis
[0046] The compounds of the invention comprise thiophene-containing
and furan-containing compounds, as described above. The compounds
can be obtained from commercial sources, such as Aldrich Chemical
Co. (Milwaukee, Wis.), Sigma Chemical Co. (St. Louis, Mo.), or
Maybridge (Cornwall, England), or the compounds can be synthesized;
The compounds of the present invention, and other related compounds
having different substituents identified by any of the methods
described above can be synthesized using techniques and materials
known to those of skill in the art, such as described, for example,
in March, ADVANCED ORGANIC CHEMISTRY 4.sup.th Ed., (Wiley 1992);
Carey and Sundberg, ADVANCED ORGANIC CHEMISTY 3.sup.rd Ed., Vols. A
and B (Plenum 1992), and Green and Wuts, PROTECTIVE GROUPS IN
ORGANIC SYNTHESIS 2.sup.nd Ed. (Wiley 1991). Starting materials for
the compounds of the invention may be obtained using standard
techniques and commercially available precursor materials, such as
those available from Aldrich Chemical Co., Sigma Chemical Co,
Lancaster Synthesis (Windham, N.H.), Apin Chemicals, Ltd. (New
Brunswick, N.J.), Ryan Scientific (Columbia, S.C.), and Maybridge.
Starting materials useful for preparing compounds of the invention
and intermediates thereof are commercially available or can be
prepared by-well-known synthetic methods (see, e.g., Harrison et
al., "Compendium of Synthetic Organic Methods", Vols. 1-8 (John
Wiley and Sons, 1971-1996); "Beilstein Handbook of Organic
Chemistry," Beilstein Institute of Organic Chemistry, Frankfurt,
Germany; Feiser et al., "Reagents for Organic Synthesis," Volumes
1-21, Wiley Interscience; Trost et al., "Comprehensive Organic
Synthesis," Pergamon Press, 1991; "Theilheimer's Synthetic Methods
of Organic Chemistry," Volumes 1-45, Karger, 1991; March, "Advanced
Organic Chemistry," Wiley Interscience, 1991; Larock "Comprehensive
Organic Transformations," VCH Publishers, 1989; Paquette,
"Encyclopedia of Reagents for Organic Synthesis," 3d Edition, John
Wiley & Sons, 1995).
[0047] The procedures described herein for synthesizing the
compounds of the invention may include one or more steps of
protection and deprotection (e.g., the formation and removal of
acetal groups). Examples of protecting groups can be found in
Greene and Wuts, Protective Groups in Organic Chemistry, 3.sup.rd
Ed., 1999, John Wiley & Sons, NY and Harrison et al.,
Compendium of Synthetic Organic Methods, Vols. 1-8, 1971-1996, John
Wiley & Sons, NY. Representative amino protecting groups
include, but are not limited to, formyl, acetyl, trifluoroacetyl,
benzyl, benzyloxycarbonyl ("CBZ"), tert-butoxycarbonyl ("Boc"),
trimethylsilyl ("TMS"), 2-trimethylsilyl-ethanesulfonyl ("SES"),
trityl and substituted trityl groups, allyloxycarbonyl,
9-fluorenylmethyloxycarbonyl ("FMOC"), nitro-veratryloxycarbonyl
("NVOC") and the like. Representative hydroxyl protecting groups
include, but are not limited to, those where the hydroxyl group is
either acylated (e.g., methyl and ethyl esters, acetate or
propionate groups or glycol esters) or alkylated such as benzyl and
trityl ethers, as well as alkyl ethers, tetrahydropyranyl ethers,
trialkylsilyl ethers (e.g., TMS or TIPPS groups) and allyl
ethers.
[0048] In addition, the synthetic procedures can include various
purifications, such as column chromatography, flash chromatography,
thin-layer chromatography (TLC), recrystallization, distillation,
high-pressure liquid chromatography (HPLC) and the like. Also,
various techniques well known in the chemical arts for the
identification and quantification of chemical reaction products,
such as proton and carbon-13 nuclear magnetic resonance (.sup.1H
and .sup.13C NMR), infrared and ultraviolet spectroscopy (IR and
UV), X-ray crystallography, elemental analysis (EA), HPLC and mass
spectroscopy (MS) can be used as well. Methods of protection and
deprotection, purification and identification and quantification
are well known in the chemical arts.
[0049] IV. Activity of Thiophene and Furan Compounds
[0050] In another aspect, the compounds of the invention can be
screened for their ability to bind to PDE6D and to modulate the
activity of the protein, i.e. identify compounds that increase
(stimulate) or decrease (inhibit) the function and/or activity of
PDE6D polypeptides or fragments, portions, or analogs thereof. The
methods may be performed in vitro or in vivo as described in detail
in the copending patent application U.S. Ser. No. ______, entitled
"Pyrrole compounds and uses thereof," Docket No. 30583-715.201,
filed on May 17, 2004. Briefly, one method for identifying whether
a thiophene-containing or furan-containing compound can bind and
modulate the activity of PDE6D comprises providing an indicator
composition comprising a PDE6D polypeptide or fragment, portion, or
analog thereof, contacting the indicator composition with one or
more of the compounds disclosed above (a potential PDE6D activator
or inhibitor), and determining the effect of the compound on PDE6D
activity in the indicator composition to identify a compound that
stimulates or inhibits the activity or function of the target. The
methods are preferably used to identify stimulators and inhibitors
for use in the treatment or prevention of diseases as disclosed
herein.
[0051] In some embodiments, a compound affects the function and/or
activity of PDE6D such that it may be administered to a subject,
preferably human, in need of a change in the function and/or
activity of PDE6D. The invention thus provides for the treatment of
a disease or undesirable condition mediated by insufficient or
unwanted, or in the alternative excess, PDE6D activity, including
the binding of PDE6.D to its binding partner(s) or its association
with other protein(s), particularly prenylated proteins. The
compounds of the invention are expected to include those useful for
the modulation of cellular signaling cascades mediated by PDE6D as
well as those for the treatment or prevention of cancer and sacral
agenesis.
[0052] In another aspect of the present invention, compounds and
compositions that are useful for preventing and treating conditions
associated with ischemic cell death, such as myocardial infarction,
stroke, glaucoma, and other neurodegenerative diseases are
provided. Neurodegenerative conditions are characterized by the
dysfunction and death of neurons, leading to the loss of functions
mediated by the brain, spinal cord and the peripheral nervous
system. Other examples of chronic neurodegenerative diseases
include diabetic peripheral neuropathy, multiple sclerosis,
amyotrophic lateral sclerosis, traumatic brain injury, spinal cord
injury, Huntington's disease and Parkinson's disease. Normal brain
aging is also associated with loss of normal neuronal function and
may entail the depletion of certain neurons.
[0053] The term "stroke" broadly refers to the development of
neurological deficits associated with impaired blood flow to the
brain regardless of cause. Potential causes include, but are not
limited to, thrombosis, hemorrhage and embolism. Thrombus, embolus,
and systemic hypotension are among the most common causes of
cerebral ischemic episodes. Other injuries may be caused by
hypertension, hypertensive cerebral vascular disease, rupture of an
aneurysm, an angioma, blood dyscrasias, cardiac failure, cardiac
arrest, cardiogenic shock, septic shock, head trauma, spinal cord
trauma, seizure, bleeding from a tumor, or other blood loss.
Depending on the area of the brain that is damaged, a stroke can
cause coma, paralysis, speech problems and dementia. Some of the
major causes of cerebral infarction are vascular thrombosis,
cerebral embolism, hypotension, hypertensive hemorrhage, and
anoxia/hypoxia. The compounds disclosed herein are useful in the
treatment of stroke.
[0054] In accordance with the present invention, the compounds
disclosed herein are useful for preventing and treating conditions
associated with ischemic cell death, such as myocardial infarction,
stroke, glaucoma, and other neurodegenerative conditions. Various
neurodegenerative conditions which may involve apoptotic cell
death, include, but are not limited to, Alzheimer's Disease, ALS
and motor neuron degeneration, Parkinson's disease, peripheral
neuropathies, Down's Syndrome, age related macular degeneration
(ARMD), traumatic brain injury, spinal cord injury, Huntington's
Disease, spinal muscular atrophy, and HIV encephalitis. The
thiophene-containing or furan-containing compounds can be used in
methods and compositions for imparting neuroprotection and for
treating neurodegenerative diseases.
[0055] V. Biological Activity
[0056] The compounds of formulas I, II and III can be used in a
pharmaceutical composition for the prevention and/or the treatment
of a condition selected from the group consisting of arthritis
(including osteoarthritis, degenerative joint disease,
spondyloarthropathies, gouty arthritis, systemic lupus
erythematosus, juvenile arthritis and rheumatoid arthritis), common
cold, dysmenorrhea, menstrual cramps, inflammatory bowel disease,
Crohn's disease, emphysema, acute respiratory distress syndrome,
asthma, bronchitis, chronic obstructive pulmonary disease,
Alzheimer's disease, organ transplant toxicity, cachexia, allergic
reactions, allergic contact hypersensitivity, cancer (such as solid
tumor cancer including colon cancer, breast cancer, lung cancer and
prostrate cancer; hematopoietic malignancies including leukemias
and lymphomas; Hodgkin's disease; aplastic anemia, skin cancer and
familiar adenomatous polyposis), tissue ulceration, peptic ulcers,
gastritis, regional enteritis, ulcerative colitis, diverticulitis,
recurrent gastrointestinal lesion, gastrointestinal bleeding,
coagulation, anemia, synovitis, gout, ankylosing spondylitis,
restenosis, periodontal disease, epidermolysis bullosa,
osteoporosis, atherosclerosis (including atherosclerotic plaque
rupture), aortic aneurysm (including abdominal aortic aneurysm and
brain aortic aneurysm), periarteritis nodosa, congestive heart
failure, myocardial infarction, stroke, cerebral ischemia, head
trauma, spinal cord injury, neuralgia, neurodegenerative disorders
(acute and chronic), autoimmune disorders, Huntington's disease,
Parkinson's disease, migraine, depression, peripheral neuropathy,
pain (including low back and neck pain, headache and toothache),
gingivitis, cerebral amyloid angiopathy, nootropic or cognition
enhancement, amyotrophic lateral sclerosis, multiple sclerosis,
ocular angiogenesis, corneal injury, macular degeneration,
conjunctivitis, abnormal wound healing, muscle or joint sprains or
strains, tendonitis, skin disorders (such as psoriasis, eczema,
scleroderma and dermatitis), myasthenia gravis, polymyositis,
myositis, bursitis, burns, diabetes (including types I and II
diabetes, diabetic retinopathy, neuropathy and nephropathy), tumor
invasion, tumor growth, tumor metastasis, corneal scarring,
scleritis, immunodeficiency diseases (such as AIDS in humans and
FLV, FIV in cats), sepsis, premature labor, hypoprothrombinemia,
hemophilia, thyroiditis, sarcoidosis, Behcet's syndrome,
hypersensitivity, kidney disease, Rickettsial infections (such as
Lyme disease, Erlichiosis), Protozoan diseases (such as malaria,
giardia, coccidia), reproductive disorders (preferably in
livestock) and septic shock (preferably arthritis, fever, common
cold, pain and cancer) in a mammal, preferably a human, cat,
livestock or a dog, comprising an amount of a compound of formula
I, II, or III or a pharmaceutically acceptable salt thereof
effective in such prevention and/or treatment optionally with a
pharmaceutically acceptable carrier.
[0057] The ability of one or more compounds of formulas I, II, and
III to bind to phosphodiesterase 6D (PDE6D) and modulate the
activity of PDE6D can be readily determined using methods well
known to those skilled in the art. For example, a compound can be
contacted (in vitro or in vivo) with cells that express the
protein, after which phenotypic changes in the cell culture can be
scored as compared to control cells that were not exposed to the
compound.
[0058] Biological Target--PDE6 Delta (PDE6D)
[0059] PDE type 6 family members are associated with retinal
phototransduction (Stryer, L, et al., J. Biol. Chem. 266:10711-14
(1991)). In phototransduction, photoreceptor cells absorb light to
trigger a nerve signal via activation of an intracellular cascade
of biochemical reactions leading to cGMP hydrolysis by PDE6.
Decreases in cGMP result in closure of a membrane-bound cGMP-gated
cation channel in the photoreceptor cell to generate a nerve
signal. The dark state of the cell is recovered by PDE6
deactivation, guanylcyclase activation, and restoration of cGMP
levels.
[0060] PDE6 is a tetrameric protein made up of two catalytic
subunits (alpha and beta) and two inhibitory (gamma) subunits.
Release of the gamma subunits from the PDE6 complex is mediated by
transducin, which activates the enzyme. Reassociation of the gamma
subunits is mediated by recoverin, which deactivates the enzyme.
While PDE6 is associated primarily with disk membranes of outer rod
segments in retinal cells, a soluble form of the enzyme contains a
fourth (delta) subunit (Florio, S. K. et al., J. Biol. Chem.
271:24036-47 (1996)).
[0061] The PDE6 holoenzyme exists as both membrane-associated and
soluble forms, and only the membrane-associated form is active in
phototransduction. Importantly, only the soluble form contains the
PDE6D subunit. PDE6D regulates the subcellular localization and
thus the activity of PDE6, and the release of PDE6 from membranes
is mediated by PDE6D. Indeed, PDE6D has been observed to reduce
light-induced cGMP hydrolysis in rod outer segments (Cook et al.,
J. Biol. Chem. 276(7):5248-5255.(2001)), presumably by removing the
PDE6 holoenzyme from the membrane. PDE6D solubilizes PDE6 by
binding specifically to prenylated peptide sequences near the
C-termini of the PDE6A and PDE6B subunits. In one embodiment, the
invention provides for treatment of visual impairment disorders,
particularly those associated with the phototransduction signaling
cascade through the modulation PDE6's participation in cGMP
hydrolysis.
[0062] The PDE6 delta (PDE6D) is a 17 kDa subunit that has not been
found in association with membrane bound PDE6 but has been observed
to solubilize membrane-bound PDE6. This release of PDE6 from the
rod membrane appears to be via delta subunit binding to the
C-terminal portion of PDE6 and is thought to reduce the likelihood
of PDE6 activation by membrane-bound transducing. The delta subunit
is also hypothesized as providing another level of enzyme
regulation.
[0063] The human PDE delta gene product (PDE6D) has been recognized
as a chaperone for the catalytic PDE alpha and beta subunits.
Prenylated PDE alpha and beta subunits have been found to be bound
by PDE6D and to be solubilized from membranes possibly as a
regulatory mechanism in the-visual cascade. Retinitis pigmentosa
(RP) is a hereditary retinal dystrophy characterized by impaired
dark adaptation and severe reductions in visual acuity. The RP gene
has been identified as the retinitis pigmentosa GTPase regulator
(RPGR) protein, which has been observed to have binding affinity
for members of the PDE6 family even in the absence of prenylation
(Linari M, et al., Proc. Natl. Acad. Sci. USA 96:1315-1320 (1999)).
PDE6D interacts with the retinitis pigmentosa GTPase regulator
(RPGR) in a thermosensitive fashion. Interaction is abolished by
mutations in the RCC1-domain of RPGR. In one embodiment, the
invention provides for treatment of RP through the modulation of
PDE6 interaction with RPGR and other molecules involved in the
phototransduction cascade.
[0064] PDE6D is expressed in many cell types and has a general role
in regulating the intracellular localization and transport of
prenylated proteins, including H-Ras, Rheb, Rho6, Rac, Rap, and
PDE6 (Hanzal-Bayer et al. EMBO J. 21(9):2095-2106 (2002) and Linari
et al. Proc. Natl. Acad. Sci., USA 96(4):1315-1320). The role of
PDE6D in regulating the membrane localization of these prenylated
proteins is still unclear, but it has been proposed that PDE6D
delivers proteins from endomembranes (endoplasmic reticulum and
Golgi) to trafficking structures that ensure correct delivery to
the ultimate membrane compartment.
[0065] PDE6D interacts with GTPases, a large super-family of
proteins that play a major role at the cell membrane as molecular
switches, active when GTP-bound and inactive when GDP-bound. The
majority of these GTPases have a covalently attached prenyl group
for anchorage to the intracellular side of the cell membrane. They
function by shuttling between the membrane-anchored form and a free
cytosolic form. The delta subunit can bind the isoprenylated region
of the small Rab13 GTPase and displace it from the plasma membrane.
(Marzesco et al., J. Biol. Chem. 273(35):22340-22345 (1998)). In
addition, PDE6D is capable of interacting with the C-terminal
regions of both the Ras and Rap GTPases and regulating their
association with the plasma membrane. For Ras binding PDE6D
requires a prenylated region of the C-terminus. (Nancy et al., J.
Biol. Chem. 277(17):15076-15084 (2002)).
[0066] PDE6D has also been observed to interact with H-Ras, Rheb,
Rho6 and Ga(il) and suggested as a transport factor for prenylated
proteins, including subunits of PDE and small GTP-binding proteins.
(Hanzal-Bayer et al. EMBO J. 21(9):2095-2106 (2002)). cGMP
PDE-specific inhibitors which act on PDE6 and PDE5 include
zaprinast, desmethylsildenophil, vinopocetine, milrinone,
amnrinone, pimobendan, cilostamide, enoximone, peroximone,
vesnarinone, rolipran, R020-1724, and dipyridamole. The compounds
of the invention may be administered to treat medical disorders or
diseases attributable to errant intracellular transport of
prenylated proteins and/or GTP-binding proteins.
[0067] The biosynthetic pathways for prenyl groups (e.g. farnesyl
and geranyl-geranyl) and cholesterol are overlapping and both
require HMG-CoA reductase activity. PDE6D can also be considered an
important component of the prenylation pathway since it regulates
the transport and localization of prenylated proteins. Prenylated
proteins have critical roles in signal transduction (e.g. Ras), and
there is evidence suggesting a role for prenylated proteins in
neurotoxicity (Liao J. K. J. Clinical Investigation, 110: 285-288
(2002)). Thus, compounds that bind to PDE6D and modulate its
activity should perturb directly the localization and function of
prenylated proteins, and be useful in the prevention and treatment
of conditions and diseases.
[0068] PDE6D has been observed to reduce light-induced cGMP
hydrolysis in rod outer segments (Cook et al., J. Biol. Chem.
276(7):5248-5255 (2001)). The delta subunit interacts directly with
the prenylated C-terminal regions of two G-protein coupled
rhodopsin kinases, GRK1 and GRK7 that are specific to
photoreceptors. (Zhang et al., J. Biol. Chem. 279(1):407-413
(2004)). Rhodopsin kinases phosphorylate membrane photoreceptors to
regulate phototransduction. In one embodiment, the invention
provides for a method of treatment of visual impairment through
modulation of PDE6 interaction with rhodopsin kinases and other
molecules involved in the phototransduction signaling cascade.
[0069] PDE6D has also been found to interact with other proteins
absent post-translational prenylation. For example, PDE6D interacts
with the unprenylated region of the retinitis pigmentosa GTPase
regulator (RPGR) protein. (Linari et al., Proc. Natl. Acad. Sci.,
USA 96(4):1315-1320 (1999)). In addition, the delta subunit can
interact with two members of the GTPase subfamily known as Arl
proteins or the ARF (ADP-ribosylation factor)-like proteins. PDE6D
interacts with the Arl2 and Arl3 proteins independent of any
post-translational modification (Hanzal-Bayer et al., EMBO J.
21(9):2095-2106 (2002) and Linari et al., FEBS Letter 458:55-59
(1999)). Based on their structure-function studies, Hanzal-Bayer et
al., have posited that the delta subunit is a transport factor for
membrane bound prenylated proteins, such as the GTP binding
molecules, and Arl2/3 serves as the mediator of the delta subunit
in the release and/or uptake of prenylated proteins. In one
embodiment, the invention provides a method of treating a
PDE6-related and/or GTP-binding protein-related disorder through
the modulation of Arl2/3 molecule activity. The use of the term
"PDE6" and references to its activities and/or modulation, herein,
is intended to also include activity and/or modulation of PDE6D
without an interaction with PDE6.
[0070] VI. Clinical Uses of Compounds
[0071] The compounds of formulas I, II and III may be used to treat
a variety of diseases and unwanted conditions. Preferably, the
compounds described herein are used in the treatment of
PDE6-related conditions. Diseases that may be treated with the
compounds described herein include, but are not limited to,
cerebral accident (or cerebrovascular accident, including stroke),
inflammation (including inflammation due to autoimmune diseases),
multiple sclerosis, blood vessel growth (angiogenesis), bone
formation/bone growth, immune system stimulation, acute coronary
syndromes (including myocardial infarction, non-Q-wave myocardial
infarction and unstable angina), and cardiovascular disease. In one
aspect, the compounds of the invention may be used to reduce the
likelihood of stroke or cardiovascular disease, and to decrease
damage following brain and/or heart infarction or other trauma. In
another aspect, the compounds may be used to reduce the severity of
damage caused by stroke or cardiovascular disease in a subject.
Non-limiting examples of the benefit provided by the compounds
include decreased brain and/or heart infarction. The use of the
compounds described herein is not intended to be limited to
PDE6-related conditions or modulation of PDE6; the present
compounds can be used to treat other conditions and modulate other
biological targets.
[0072] The term "PDE6-related condition" as used herein refers to a
condition in which directly or indirectly modulating the activity
and/or production of a PDE6 molecule, respectively, is desirable.
This modulation includes modulation of one or more molecules in the
upstream or downstream signaling cascades of PDE6. For example, a
PDE6-related condition may involve over-production or unwanted
production of one or more prenylated PDE6 subunits, such as PDE6D,
prenylated PDE6.alpha. or PDE6.beta., or other chemical messengers
of cell signaling pathways associated with phototransduction
(including responses to and expression of PDE6 alpha and PDE6
beta).
[0073] In some embodiments, the methods of the present invention
employ a PDE6 modulating compound. The term "PDE6 modulating
compound" as used herein and its grammatical conjugations refer to
a compound that preferably modulates PDE6, for example by binding
to PDE6, preferably by binding to PDE6D. For example, a PDE6
modulating compound may modulate one or more prenylated PDE6
subunits, such as PDE6D, prenylated PDE6.alpha. or PDE6.beta., or
other chemical messengers of cell signaling pathways associated
with phototransduction (including responses to and expression of
PDE6 alpha and PDE6 beta). In some embodiments the term "preferable
modulation" and its grammatical conjugations refers to a specific
modulation of PDE6. In other embodiments, the term refers to
preferable modulation of PDE6 with minimal modulation of HMG Co A
reductase. The term "minimal modulation" refers to essentially no
modulation, but does not require a complete lack of modulation; it
refers to essentially no observable or measurable activity. In
treatment scenarios, "minimal modulation" refers to modulation that
is not sufficient to produce a therapeutic and/or prophylactic
benefit in a condition caused by the activity that is not being
modulated.
[0074] Modulating the activity of a PDE6 molecule includes
reducing, increasing, or stabilizing the activity of these
molecules. Reducing the activity of PDE6 is also referred to as
"inhibiting" the molecule. The term "inhibits" and its grammatical
conjugations, such as "inhibitory," is not intended to require
complete inhibition in PDE6 activity. Such reduction is preferably
by at least about 50%, at least about 75%, at least about 90%, and
more preferably by at least about 95% of the activity of the
molecule in the absence of the inhibitory effect, e.g., in the
absence of an inhibitor. Most preferably, the term refers to an
observable or measurable reduction in activity. In treatment
scenarios, preferably the inhibition is sufficient to produce a
therapeutic and/or prophylactic benefit in the condition being
treated. The phrase "does not inhibit" and its grammatical
conjugations does not require a complete lack of effect on the
activity. For example, it refers to situations where there is less
than about 20%, less than about 10%, and preferably less than about
5% of reduction in PDE6 activity in the presence of an inhibitor
such as a compound of the invention.
[0075] The PDE6-modulating agents of the invention can be
administered to a mammalian subject to treat a disorder by
modulating the binding of PDE6D to prenylated GTPases, thereby
modulating GTPase-dependent signal transduction pathways. The
disruption of GTPase-dependent pathways contributes to a variety of
medical conditions, such as vascular hyperplasia, thrombin-induced
cell death, the pathogenesis and progression of bladder cancer,
chronic inflammatory disease, endothelial dysfunction in
cardiovascular disease, cardiac hypertrophy, a change in cerebral
blood flow to ischemic regions of the brain, phagocytosis of
amloid-beta fibrils in Alzheimer's disease patients,
immunodeficiency disorders and increased free radical production in
aortic vascular smooth muscle cells.
[0076] PDE6-related conditions can include neurodegenerative
diseases, including ischemic stroke, basal ganglia or Parkinson's
disease, epilepsy or brain or spinal cord ischemia or trauma;
Alzheimer's disease, dementia, diabetic peripheral neuropathy,
multiple sclerosis, amyotrophic lateral sclerosis, traumatic brain
injury, spinal cord injury, Huntington's disease, heart failure
(e.g. congestive heart failure, acute heart failure, cardiac
hypertrophy, etc.) or renal diseases.
[0077] PDE6-related conditions can include visual impairment
disorders, including macular degeneration, amblyopia, Blepharitis,
Bietti's Crystalline Dystrophy, corneal disease, diabetic eye
disease, glaucoma, histoplasmosis, and retinitis pigmentosa.
PDE6-related conditions can include cardiovascular-related
conditions, including atherosclerosis, myocardial infarction,
congestive heart failure, ischemic-reperfusion injury and other
vascular inflammatory conditions. PDE6-related conditions can also
include proliferative disorders, including cancers, e.g., leukemia,
melanoma, Non-Hodgkins Lymphoma, as well as bladder, breast, colon,
endometrial, head and neck, lung, ovarian, prostate and rectal
cancers.
[0078] PDE6-related conditions can also include neurological
deficits that develop from a stroke-induced impairment of blood
flow to the brain regardless of cause. Potential causes include,
but are not limited to, thrombosis, hemorrhage and embolism.
Thrombus, embolus, and systemic hypertension are among the most
common causes of cerebral ischemic episodes. Other injuries may be
caused by hypertension, hypertensive cerebral vascular disease,
rupture of an aneurysm, an angioma, blood dyscrasias, cardiac
failure, cardiac arrest, cardiogenic shock, septic shock, head
trauma, spinal cord trauma, seizure, bleeding from a tumor, or
other blood loss.
[0079] In one embodiment, the PDE6-modulating agent modulates the
activity small GTP binding protein Rho in its role in cell
proliferation. It has been reported that Rho proteins are more
abundant in tumor bladders than in non-tumor bladders and
upregulated in ovarian carcinomas (Kamai T, et al., Clin. Cancer
Res. July; 9(7):2632-41 (2003) and Horiuchi A, et al., Lab Invest.
June 2003; 83(6): 861-70 (2003)).
[0080] A disruption in Rho GTP binding activity has been shown to
have the neuroprotective effect of increasing cerebral blood flow
to ischemic regions of the brain (Laufs U, et al., J. Clin. Invest.
106(1):15-24 (2000)). The study demonstrated that under absent or
decreased rho-dependent actin cytoskeleton stress fiber formation,
eNOS was upregulated and the severity of cerebral ischemia was
decreased. An embodiment of the invention provides for the
treatment of ischemic stroke by modulation of Rho by a compound of
the invention.
[0081] Researchers have reported that the cardiac hypertrophy,
which requires intracellular oxidation may be reduced by
statin-induced inhibition of post-translational modification of the
small G proteins of the Rho family (Takemoto M, et al., J. Clin.
Invest. 108(10):1429-37 (2001)). Takemoto M, et al. observed that
an inhibition of the Rho isoprenylation produced an
intracellular-antioxidant effect and inhibit cardiac hypertrophy.
One embodiment of the invention provides for the treatment of
cardiac hypertrophy by modulation of Rho by a compound of the
invention.
[0082] In another embodiment, the PDE6-modulating agent modulates
the activity of the small GTP binding protein Rac in its role in
Alzheimer's disease. Rac has been observed to participate in the
phagocytosis of amyloid-beta fibrils from extracellular senile
plaques. (Kitamura Y, et al, J. Pharmacol. Sci. 92(2):115-23
2003)).
[0083] The compounds I, II, and III of the invention can be used to
treat a variety of diseases and unwanted conditions, including, but
not limited to, cerebral accident (or cerebrovascular accident,
including stroke), inflammation (including inflammation due to
autoimmune diseases), multiple sclerosis, blood vessel growth
(angiogenesis), bone formation/bone growth, immune system
stimulation, acute coronary syndromes (including myocardial
infarction, non-Q-wave myocardial infarction and unstable angina),
and cardiovascular disease. In one embodiment, the compounds of the
invention can be used to reduce the likelihood of stroke or
cardiovascular disease, and to decrease damage following brain
and/or heart infarction or other trauma. In another embodiment, the
compounds can be used to reduce the severity or damage caused by
stroke or cardiovascular disease in a subject. Non-limiting
examples of the benefit provided by the compounds include decreased
brain and/or heart infarction.
[0084] VII. Formulations, Routes of Administration, and Effective
Doses
[0085] The compounds of formulas I, II, and III are preferably used
to prepare a medicament, such as by formulation into pharmaceutical
compositions for administration to a subject using techniques
generally known in the art. A summary of such pharmaceutical
compositions may be found, for example, in Remington's
Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa. The
compounds of the invention can be used singly or as components of
mixtures. Preferred forms of the compounds are those for systemic
administration as well as those for topical or transdermal
administration. Formulations designed for sustained and/or delayed
release are also with the scope of the invention.
[0086] Such pharmaceutical compositions can be used to treat
PDE6-related conditions, as described in detail above. If necessary
or desirable, the modulator or inhibitor may be administered in
combination with other therapeutic agents. The choice of
therapeutic agents that can be co-administered with the
compositions of the invention will depend, in part, on the
condition being treated.
[0087] The modulators may be administered per se or in the form of
a pharmaceutical composition wherein the active compound(s) is in
an admixture or mixture with one or more pharmaceutically
acceptable carriers, excipients or diluents. Pharmaceutical
compositions for use in accordance with the present invention may
be formulated in conventional manner using one or more
physiologically acceptable carriers compromising excipients and
auxiliaries, which facilitate processing of the active compounds
into preparations that can be used pharmaceutically. Proper
formulation is dependent upon the route of administration chosen.
The modulators useful in the present invention can be delivered to
the patient using a number of routes or modes of administration,
including oral, buccal, topical, rectal, transdermal, transmucosal,
subcutaneous, intravenous, and intramuscular applications, as well
as by inhalation.
[0088] Methods for the preparation of compositions comprising the
compounds of the invention include formulating the derivatives with
one or more inert, pharmaceutically acceptable carriers to form
either a solid or liquid. Solid compositions include, but are not
limited to, powders, tablets, dispersible granules, capsules,
cachets, and suppositories. Liquid compositions include solutions,
emulsions, or a solution containing liposomes, micelles, or
nanoparticles comprising a compound as disclosed herein.
[0089] Compounds of this invention may also be integrated into
foodstuffs, e.g, cream cheese, butter, salad dressing, or ice cream
to facilitate solubilization, administration, and/or compliance in
certain patient populations.
[0090] The compounds of the invention may be labeled isotopically
(e.g. with a radioisotope) or by another other means, including,
but not limited to, the use of chromophores or fluorescent
moieties, bioluminescent labels, or chemiluminescent labels. The
compositions may be in conventional forms, either as liquid
solutions or suspensions, solid forms suitable for solution or
suspension in a liquid prior to use, or as emulsions. Suitable
excipients or carriers are, for example, water, saline, dextrose,
glycerol, alcohols, aloe vera gel, allantoin, glycerin, vitamin A
and E oils, mineral oil, propylene glycol, PPG-2 myristyl
propionate, and the like. Of course, these compositions may also
contain minor amounts of nontoxic, auxiliary substances, such as
wetting or emulsifying agents, pH buffering agents, and so
forth.
[0091] For oral administration, the compounds can be formulated
readily by combining the active compound(s) with pharmaceutically
acceptable carriers well known in the art. Such carriers enable the
compounds of the invention to be formulated as tablets, including
chewable tablets, pills, dragees, capsules, lozenges, hard candy,
liquids, gels, syrups, slurries, powders, suspensions, elixirs,
wafers, and the like, for oral ingestion by a patient to be
treated. Such formulations can comprise pharmaceutically acceptable
carriers including solid diluents or fillers, sterile aqueous media
and various non-toxic organic solvents. Suitable excipients are, in
particular, fillers such as sugars, including lactose, sucrose,
mannitol, or sorbitol; flavoring elements, cellulose preparations
such as, for example, maize starch, wheat starch, rice starch,
potato starch, gelatin, gum tragacanth, methyl cellulose,
hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose,
and/or polyvinyl pyrrolidone (PVP). If desired, disintegrating
agents may be added, such as the cross-linked polyvinyl
pyrrolidone, agar, or alginic acid or a salt thereof such as sodium
alginate. The compounds may also be formulated as a sustained
release preparation.
[0092] Dragee cores can be provided with suitable coatings. For
this purpose, concentrated sugar solutions may be used, which may
optionally contain gum arabic, talc, polyvinyl pyrrolidone,
carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer
solutions, and suitable organic solvents or solvent mixtures.
Dyestuffs or pigments may be added to the tablets or dragee
coatings for identification or to characterize different
combinations of active compound doses.
[0093] Pharmaceutical preparations that can be used orally include
push-fit capsules made of gelatin, as well as soft, sealed capsules
made of gelatin and a plasticizer, such as glycerol or sorbitol.
The push-fit capsules can contain the active ingredients in
admixture with filler such as lactose, binders such as starches,
and/or lubricants such as talc or magnesium stearate and,
optionally, stabilizers. In soft capsules, the active compounds may
be dissolved or suspended in suitable liquids, such as fatty oils,
liquid paraffin, or liquid polyethylene glycols. In addition,
stabilizers may be added. All formulations for oral administration
should be in dosages suitable for administration.
[0094] Aqueous suspensions may contain a compound of this invention
with pharmaceutically acceptable excipients, such as a suspending
agent (e.g., methyl cellulose), a wetting agent (e.g., lecithin,
lysolecithin and/or a long-chain fatty alcohol), as well as
coloring agents, preservatives, flavoring agents, and the like.
[0095] For injection, the inhibitors of the present invention may
be formulated in aqueous solutions, preferably in physiologically
compatible buffers such as Hank's solution, Ringer's solution, or
physiological saline buffer. Such compositions may also include one
or more excipients, for example, preservatives, solubilizers,
fillers, lubricants, stabilizers, albumin, and the like. Methods of
formulation are known in the art, for example, as disclosed in
Remington's Pharmaceutical Sciences, latest edition, Mack
Publishing Co., Easton P. These compounds may also be formulated
for transmucosal administration, buccal administration, for
administration by inhalation, for parental administration, for
transdermal administration, and rectal administration.
[0096] In addition to the formulations described previously, the
compounds may also be formulated as a depot preparation. Such long
acting formulations may be administered by implantation or
transcutaneous delivery (for example subcutaneously or
intramuscularly), intramuscular injection or use of a transdermal
patch. Thus, for example, the compounds may be formulated with
suitable polymeric or hydrophobic materials (for example as an
emulsion in an acceptable oil) or ion exchange resins, or as
sparingly soluble derivatives, for example, as a sparingly soluble
salt.
[0097] In some embodiments, pharmaceutical compositions comprising
compounds of the present invention exert local and regional
anti-inflammatory effects when administered topically or injected
at or near particular sites of inflammation. For example, ocular
allergic, inflammatory and/or autoimmune conditions can be
effectively treated with ophthalmic solutions, suspensions,
ointments or inserts comprising one or more compounds of the
present invention. Allergic, inflammatory and/or autoimmune
conditions of the ear can be effectively treated with otic
solutions, suspensions, ointments or inserts comprising one or more
compounds of the present invention. Allergic, inflammatory and/or
autoimmune conditions of the skin and skin structures can be
effectively treated with skin ointments comprising one or more
compounds of the present invention in an oleaginous hydrocarbon
base, an anhydrous absorption base, a water-in-oil absorption base,
an oil-in-water water-removable base and/or a water-soluble base.
Gastrointestinal allergic, inflammatory and/or autoimmune
conditions can be effectively treated with orally- or rectally
delivered solutions, suspensions, ointments, enemas and/or
suppositories comprising one or more compounds of the present
invention. Respiratory allergic, inflammatory and/or autoimmune
conditions can be effectively treated with aerosol solutions,
suspensions or dry powders comprising one or more compounds of the
present invention.
[0098] For example, for treating inflammatory and/or autoimmune
conditions, a cream comprising a compound of the invention may be
topically applied to the affected site, for example, sites
displaying red plaques or dry scales in psoriasis, or areas of
irritation and dryness in dermatitis. As another example, for
treating inflammatory bowel disease, a suppository formulation of a
compound disclosed herein can be used. In such embodiments, the
active ingredient produces a benefit locally at or near the site of
application, rather than systemically, by modulating PDE6, e.g.,
PDE6D.
[0099] Direct topical application, e.g., of a viscous liquid, gel,
jelly, cream, lotion, ointment, suppository, foam, or aerosol
spray, may be used for local administration, to produce for example
local and/or regional effects. Pharmaceutically appropriate
vehicles for such formulation include, for example, lower aliphatic
alcohols, polyglycols (e.g., glycerol or polyethylene glycol),
esters of fatty acids, oils, fats, silicones, and the like. Such
preparations may also include preservatives (e.g., p-hydroxybenzoic
acid esters) and/or antioxidants (e.g., ascorbic acid and
tocopherol). See also Dermatological Formulations: Percutaneous
absorption, Barry (Ed.), Marcel Dekker Incl, 1983.
[0100] In some preferred embodiments, the compounds of the present
invention are delivered in soluble rather than suspension form,
which allows for more rapid and quantitative absorption to the
sites of action. In general, formulations such as jellies, creams,
lotions, suppositories and ointments can provide an area with more
extended exposure to the compounds of the present invention, while
formulations in solution, e.g., sprays, provide more immediate,
short-term exposure.
[0101] The formulations also may comprise suitable solid or gel
phase carriers or excipients that increase penetration or help
delivery of inhibitory compounds of this invention across the
permeability barrier of the skin. Many of these
penetration-enhancing compounds are known in the art of topical
formulation. Examples of such carriers and excipients include
humectants (e.g., urea), glycols (e.g., propylene glycol and
polyethylene glycol), alcohols (e.g., ethanol), fatty acids (e.g.,
oleic acid), surfactants (e.g., isopropyl myristate and sodium
lauryl sulfate), pyrrolidones, glycerol monolaurate, sulfoxides,
terpenes (e.g., menthol), amines, amides, alkanes, alkanols,
ORGELASE, calcium carbonate, calcium phosphate, various sugars,
starches, cellulose derivatives, gelatin, other polymers and water.
In some embodiments, the pharmaceutical compositions will include
one or more penetration enhancers such as water, methanol, ethanol,
2-propanol, dimethyl sulfoxide, decylmethyl sulfoxide,
tetradecylmethyl sulfoxide, 2-pyrrolidone, N-methyl-2-pyrrolidone,
N-(2-hydroxyethyl)pyrrolidone, laurocapram, acetone,
dimethylacetamide, dimethylformamide, tetrahydrofurfuryl alcohol,
L-.alpha.-amino acids, anionic surfactants, cationic surfactants,
amphoteric surfactants, nonionic surfactants, fatty acids, fatty
alcohols, clofibric acid amides, hexamethylene lauramide,
proteolytic enzymes, .alpha.-bisabolol, d-limonene, urea,
N,N-diethyl-m-toluamide, and the like.
[0102] In some embodiments, the pharmaceutical compositions will
include one or more antimicrobial preservatives such as quaternary
ammonium compounds, organic mercurials, p-hydroxy benzoates,
aromatic alcohols, chlorobutanol, and the like.
[0103] Pharmaceutical compositions suitable for use in the present
invention include compositions wherein the active ingredients are
present in an effective amount, i.e., in an amount effective to
achieve therapeutic and/or prophylactic benefit in at least one of
a PDE6-related condition. The actual amount effective for a
particular application will depend on the condition or conditions
being treated, the condition of the subject, the formulation, and
the route of administration, as well as other factors known to
those of skill in the art. Determination of an effective amount of
a PDE6 modulator is well within the capabilities of those skilled
in the art, in light of the disclosure herein, and will be
determined using routine optimization techniques.
[0104] In therapeutic use, the compounds of the invention are
administered to a subject at dosage levels of from about 0.05 mg/kg
to about 10.0 mg/kg of body weight per day. For a human subject of
approximately 70 kg, a dosage of from 40 mg to 600 mg per day may
be used as a non-limiting example. Preferred doses include about 1
mg/kg, about 2.5 mg/kg, about 5 mg/kg, and about 7.5 mg/kg. Lower
or higher doses than those disclosed herein may be used, as
required. Such dosages, however, may be altered depending on a
number of variables, not limited to the activity of the compound
used, the condition to be treated, the mode of administration, the
requirements of the individual subject, the severity of the
condition being treated, and the judgment of the practitioner. The
foregoing ranges are merely suggestive, as the number of variables
in regard to an individual treatment regime is large, and
considerable excursions from these recommended values are not
uncommon.
[0105] The effective amount for use in humans can be determined
from animal models. For example, a dose for humans can be
formulated to achieve circulating, liver, topical and/or
gastrointestinal concentrations that have been found to be
effective in animals.
[0106] The effective amount when referring to an inhibitor of the
invention will generally mean the dose ranges, modes of
administration, formulations, etc., that have been recommended or
approved by any of the various regulatory or advisory organizations
in the medical or pharmaceutical arts (e.g. FDA, AMA) or by the
manufacturer or supplier.
[0107] In some embodiments, administration of compounds of the
present invention may be intermittent, for example administration
once every two days, every three days, every five days, once a
week, once or twice a month, and the like. In some embodiments, the
amount, forms, and/or amounts of the different forms may be varied
at different times of administration.
[0108] VIII. Kits/Articles of Manufacture
[0109] For use in the therapeutic applications described herein,
kits and articles of manufacture are also within the scope of the
invention. Such kits can comprise a carrier, package, or container
that is compartmentalized to receive one or more containers such as
vials, tubes, and the like, each of the container(s) comprising one
of the separate elements to be used in a method of the invention.
Suitable containers include, for example, bottles, vials, syringes,
and test tubes. The containers can be formed from a variety of
materials such as glass or plastic.
[0110] For example, the container(s) can comprise one or more
thiophene-containing or furan-containing compounds of the
invention, optionally in a composition or in combination with
another agent as disclosed herein. The container(s) optionally have
a sterile access port (for example the container can be an
intravenous solution bag or a vial having a stopper pierceable by a
hypodermic injection needle). Such kits optionally comprising a
thiophene-containing or furan-containing compound with an
identifying description or label or instructions relating to its
use in the methods of the present invention.
[0111] A kit of the invention typically may comprise one or more
additional containers, each with one or more of various materials
(such as reagents, optionally in concentrated form, and/or devices)
desirable from a commercial and user standpoint for use of a
thiophene-containing or furan-containing compound of the invention.
Non-limiting examples of such materials include, but not limited
to, buffers, diluents, filters, needles, syringes; carrier,
package, container, vial and/or tube labels listing contents and/or
instructions for use, and package inserts with instructions for
use. A set of instructions will also typically be included.
[0112] A label can be on or associated with the container. A label
can be on a container when letters, numbers or other characters
forming the label are attached, molded or etched into the container
itself; a label can be associated with a container when it is
present within a receptacle or carrier that also holds the
container, e.g., as a package insert. A label can be used to
indicate that the contents are to be used for a specific
therapeutic application. The label can also indicate directions for
use of the contents, such as in the methods described herein.
[0113] The terms "kit" and "article of manufacture" may be used as
synonyms.
EXAMPLES
[0114] Having now generally described the invention, the same will
be more readily understood through reference to the following
examples. The examples are offered for illustrative purposes only,
and are not intended to limit the scope of the present invention in
any way. Efforts have been made to ensure accuracy with respect to
numbers used (e.g., amounts, temperatures, etc.), but some
experimental error and deviation should, of course, be allowed
for.
Example 1
The Affinity of the Thiophene-Containing Compounds for PDE6D
[0115] The T7 phage displaying human PDE6D and CLB2 were obtained
following the procedure of WO 01/18234, published Mar. 15, 2001,
and U.S. patent application Ser. No. 10/115,442, "Phage Display
Affinity Filter and Forward Screen, filed Apr. 2, 2002. Briefly, a
T7 phage-display-based affinity chromatography procedure was used
where atorvastatin was chemically coupled to a biotinylated linker
moiety, which enabled the attachment of atorvastatin to
streptavidin-coated magnetic beads. The atorvastatin-coated
magnetic beads were used as an affinity matrix to probe the human
proteome for atorvastatin-binding proteins. T7 phage display
libraries that broadly cover the human proteome were mixed with the
atorvastatin affinity matrix and non-binding clones were removed by
washing. Atorvastatin-binding clones were eluted by incubating the
affinity matrix with soluble atorvastatin. The phage eluate was
then amplified by growth in E. coli and the affinity enrichment
procedure was repeated. After four rounds of affinity enrichment,
predominant atorvastatin-binding clones emerged and was identified
by DNA sequencing as human PDE6D and CLB2. The binding of
atorvastatin to PDE6D and CLB2 was further validated by competition
binding assays, where the Kd's for the interactions between soluble
atorvastatin (non-immobilized, no linker moiety) and the proteins
were determined to be 65 nM and 500 nM for PDE6D and CLB2,
respectively.
[0116] The dissociation constants, Kd's, for the interaction
between the thiophene compounds and the novel targets were obtained
following the procedure of the co-pending patent applications U.S.
Ser. Nos. 10/115,442, "Phage Display Affinity Filter and Forward
Screen," filed Apr. 2, 2002 and 60/480,587, "Protein Family
Profiling Tool and Methods," filed Jun. 20, 2003. To measure the Kd
values, the T7 phage displaying human PDE6D or CLB2 were incubated
with an atorvastatin-coated affinity matrix in the presence of
various concentrations of a soluble (non-immobilized) thiophene
compounds of the invention, as described in detail above. Soluble
thiophene compounds that bind PDE6D and/or CLB2 prevent binding of
PDE6D and/or CLB2 phage to the affinity matrix; hence, fewer phage
are recovered in the phage eluate in the presence of an effective
competitor than in the absence of an effective competitor. The Kd
for the interaction between the soluble thiophene compound
(competitor) molecule and PDE6D or CLB2 is equal to the
concentration of soluble competitor molecule that causes a 50%
reduction in the number of phage recovered in the eluate compared
to a control sample lacking soluble competitor. Table 1 displays
the .mu.M Kd of certain thiophene compounds for PDE6D.
1 TABLE 1 Thiophene Kd PDE6D Compound (nM) 781560 830 779974 348
781456 567 783172 469 783146 1675
Example 2
Efficacy Studies of Compounds in the Rodent MCAo Model
[0117] Overview. The purpose of this study is to evaluate the
efficacy of the compounds described herein in the rat Middle
Cerebral Artery Occlusion (MCAo) model induced by
electrocoagulation of the middle cerebral artery and tandem carotid
artery occlusion according to Chen et al., Stroke, 17:4 (1986).
Infarct Volume comparison to control vehicle volume will be used
for evaluating efficacy of the compound(s). Small numbers of
animals (n=5/group) will be used to test for potential efficacy in
reducing the infarct size after 48 hours.
[0118] Methods
[0119] Animal Preparation. Forty-five male, adult, Sprague-Dawley
rats (Taconic Farms), weighing 300-350 g, will be used for the
study. Animals are housed in a standard animal facility and fed
with commercial rodent chow ad libitum. Extra animals will be
ordered to allow for proper randomization and mortality from the
surgical procedures. The rats will be approximately 10 weeks of age
when they arrive at the laboratory.
[0120] All animals will be housed for 7 days prior to surgery for
acclimation purposes. At the end of the training period, animals
will be randomized and assigned to different groups. Animals will
be given a unique identification number by tail marking. The
animals will be identified by tail number and cage cards.
[0121] Surgical Preparation. Middle Cerebral Artery Occlusion
(MCAo). Male Sprague Dawley rats (300-350 grams,) are anesthetized
with an intramuscular 4 ml/kg "cocktail" of Ketamine (25 mg/ml),
Xylazine (1.3 mg/mL) and Acepromazine (0.33 mg/mL). In general, the
common carotid arteries are exposed through a ventral midline
cervical incision in the neck. The temporalis muscle is bisected
and reflected through an incision performed midway between the eye
and the eardrum canal. A 3 mm burr hole is made at the junction of
the zygomatic arch and the squamos bone such that the bifurcation
of the frontal and parietal branch of the middle cerebral artery is
exposed. The right middle cerebral artery is permanently occluded
using electrocoagulation directly below the bifurcation of the
frontal and parietal branch and superior to the rhinal vein on the
MCA, the common carotid arteries are temporarily occluded using
atraumatic aneurysm clips for one hour. Body temperature is
maintained at 38.degree. C..+-.1 throughout the entire procedure.
Animals are euthanized at various time periods after MCA occlusion
and the brains are removed for histological analysis.
[0122] Morphometric Analysis. Forty-eight hours after the induction
of focal ischemia, rats will be deeply anesthetized with CO.sub.2
and decapitated. The brain is removed and placed in ice chilled
(.about.4.degree. C.) saline for fifteen minutes. Seven 2.0 mm
coronal slices are cut using a brain-cutting matrix and incubated
in two percent 2, 3, 5 triphenyltetrazolium chloride (TTC) for 20
minutes at 37.degree. C. Slices are removed, washed in saline and
put into 10% formalin for 24 hours before tissue analysis. (TTC is
an established marker for functional mitochondrial enzymes and
produces a visible deep red color within normal tissue. Ischemic
tissue, lacking mitochondrial activity, remains unstained and
appears white. This is a standard method for use in image analysis
of the sliced brain and quantification of the ischemic area after
MCAo.)
[0123] Infarct Volume Measurement. Using the Image Pro-Plus imaging
system, a total of 14 images per brain of both the frontal and
posterior side of each slice are analyzed through digital analysis.
The total infarct volume is calculated for the left hemisphere
using the equation below. Digitizing and computation is done under
blinded conditions. 1 Volume ( mm 3 ) = area ( mm 2 ) per side No .
of sides analyzed .times. 14 mm
[0124] The total infarct volumes are calculated for each animal and
subsequent group means are determined as volume of area (mm.sup.3).
To account for tissue shrinkage and possible edema indirect method
of infarct volume is calculated using the formula:
Total Contralateral Hemisphere Volume-Total Infarcted Hemisphere
Volume (mm.sup.3)
[0125] Body Temperature and Weight. The body temperatures of all
animals are monitored throughout the surgery and maintained near
normal values (37-38.degree. C). The body weight of all animals are
measured and documented before surgery and at the end of the study
immediately before euthanation.
[0126] Clinical Observations. Animals will be observed daily for
abnormal behavior, including but not limited to circling, lethargy,
respiratory wheezing or discharge, hair loss, and early death. All
abnormalities will be recorded. Likewise, normal behavior will also
be noted as normal.
[0127] Statistical Analysis. All data are expressed as mean.+-.SEM.
Infarct volume will be analyzed using a two tailed t-test and
Dunnett's multiple comparisons. Behavioral tests, temperature, and
body weight are analyzed using one-way ANOVA followed by post hoc
Bonferroni's Multiple Comparison Test. A p value of .ltoreq.0.05 is
considered a statistically significant difference.
[0128] Experimental Design
[0129] Experimental Groups and Compound Administration. One or more
compounds, selected from the compounds described herein, will be
tested. Fifteen animals will be used to test a compound using three
groups of animals, n=5 animals per group. One group will receive 1
mg/kg body weigh, the second group will receive 10 mg/kilogram body
weight and the third group will receive vehicle only at the same
time points as the test article via intraperitoneal injections
beginning 2 hours pre-MCAo, 3 hours and 24 hours post MCAo
[0130] All animals are treated with the compound(s)/vehicle(s) in a
blinded random investigation. Table 2 provides a summary of an
Experimental Design for testing one of the compounds described
herein.
2 TABLE 2 Treatment Paradigm Group n Test articles Dose Route Time
Period 1 5 Vehicle No TX IP 2 Hours Pre 3, 24 Hours Post 2 5
Compound 1 mg/kg IP 2 Hours Pre Body weight 3, 24 Hours Post 3 5
Compound 10 mg/kg IP 2 Hours Pre Body weight 3, 24 Hours Post
[0131] It is anticipated that it will take approximately 5 weeks to
complete the technical work of this study at the rate of 2
compounds, using 30 animals screened per week.
Example 3
Efficacy Studies of Compounds in the Rodent MCAo Model
[0132] The purpose of this study is to further evaluate the
efficacy of the compound described herein. Infarct volume of
compound-treated animals in comparison to control volume will be
used for evaluating efficacy of the compound described herein.
[0133] Methods
[0134] Animal Preparation. One Hundred and twelve male, adult,
Sprague-Dawley rats (Taconic Farms), weighing 300-350 g, will be
used for the study. Animals are housed in a standard animal
facility and fed with commercial rodent chow ad libitum. Extra
animals will be ordered to allow for proper randomization and
mortality from the surgical procedures. The rats will be
approximately 10 weeks of age when they arrive at the
laboratory.
[0135] All animals will be housed for 7 days prior to surgery for
acclimation purposes. At the end of the training period, animals
will be randomized and assigned to different groups. Animals will
be given a unique identification number by tail marking. The
animals will be identified by tail number and cage cards.
[0136] Surgical Preparation. Middle Cerebral Artery Occlusion
(MCAo). Male Sprague Dawley rats (300-350 grams,) are anesthetized
with an intramuscular 4 ml/kg "cocktail" of ketamine (25 mg/ml),
xylazine (1.3 mg/mL) and acepromazine (0.33 mg/mL). In general, the
common carotid arteries are exposed through a ventral midline
cervical incision in the neck. The temporalis muscle is bisected
and reflected through an incision performed midway between the eye
and the eardrum canal. A 3 mm burr hole is made at the junction of
the zygomatic arch and the squamos bone such that the bifurcation
of the frontal and parietal branch of the middle cerebral artery is
exposed. The right middle cerebral artery is permanently occluded
using electrocoagulation directly below the bifurcation of the
frontal and parietal branch and superior to the rhinal vein on the
MCA, the common carotid arteries are temporarily occluded using
atraumatic aneurysm clips for one hour. Body temperature is
maintained at 38.degree. C..+-.1 throughout the entire procedure.
Animals are euthanatized at various time periods after MCA
occlusion and the brains are removed for histological analysis.
[0137] Morphometric Analysis. Forty-eight hours after the induction
of focal ischemia, rats will be deeply anesthetized with CO.sub.2
and decapitated. The brain is removed and placed in ice chilled
(.about.4.degree. C.) saline for fifteen minutes. Seven 2.0 mm
coronal slices are cut using a brain-cutting matrix and incubated
in two percent 2, 3, 5 triphenyltetrazolium chloride (TTC) for 20
minutes at 37.degree. C. Slices are removed, washed in saline and
put into 10% formalin for 24 hours before tissue analysis. (TTC is
an established marker for functional mitochondrial enzymes and
produces a visible deep red color within normal tissue. Ischemic
tissue, lacking mitochondrial activity, remains unstained and
appears white. This is a standard method for use in image analysis
of the sliced brain and quantification of the ischemic area after
MCAo.).
[0138] Infarct Volume Measurement. Using the Image Pro-Plus imaging
system, a total of 14 images per brain of the posterior side of
each slice are analyzed through digital analysis. The total infarct
volume is calculated for the left hemisphere using the equation
below. Digitizing and computation is done under blinded conditions.
2 Volume ( mm 3 ) = area ( mm 2 ) per side No . of sides analyzed
.times. 14 mm
[0139] The total direct infarct volumes are calculated for each
animal and subsequent group means are determined as volume of area
(mm.sup.3).
[0140] Body Temperature and Weight. The body temperatures of each
animal are monitored throughout the surgery and maintained near
normal values (37-38.degree. C.). Animal's body weights are
measured and documented before surgery and at the end of the study
immediately before euthanation.
[0141] Clinical Observations. Animals will be observed daily for
abnormal behavior, including but not limited to circling, lethargy,
respiratory wheezing or discharge, hair loss, and early death. All
abnormalities will be recorded. Likewise, normal behavior will also
be noted as normal.
[0142] Statistical Analysis. All data are expressed as mean.+-.SEM.
Infarct volume will be analyzed using a two tailed t-test and
Dunnett's multiple comparisons. Behavioral tests, temperature, and
body weight are analyzed using one-way ANOVA followed by post hoc
Bonferroni's Multiple Comparison Test. A p value of .ltoreq.0.05 is
considered a statistically significant difference.
[0143] Experimental Design
[0144] Experimental Groups and Compound Administration. A number of
animals will undergo MCAo using subcutaneous administration of all
test articles. One or more compounds, selected from the compounds
disclosed herein, at one predetermined dose will be compared to a
control or vehicle and assigned to three different time schedules
of administration relative to the time of MCAo.
[0145] The time schedules for administration of all treated groups
will be divided as follows:
[0146] a. 2 hrs pre, 3 hrs post, & 25 hrs post
[0147] b. 1 hr,3 hrs, & 25 hrs post
[0148] c. 3 hrs, 6 hrs, & 25 hrs post
[0149] All animals are treated with the compound(s)/vehicle(s) as a
blinded random investigation.
Example 4
Efficacy Studies of Compounds in the Rodent MCAo (Tamura) Model
[0150] The Tamura model of MCAo will be utilized to determine the
effect of one or more of the compounds described herein in
alleviating the neurological deficit demonstrated as motor skill
performance. Behavioral assessment as well as infarct volume of
compound treated animals in comparison to control (vehicle) volume
animals will be used for evaluating efficacy of the compound(s)
over a 7 day period.
[0151] Methods
[0152] Animal Preparation. Eighty, adult, Sprague-Dawley rats
(Taconic Farms), weighing 300-350 g, will be used for the study.
Animals are housed in a standard animal facility and fed with
commercial rodent chow ad libitum. Extra animals will be ordered to
allow for proper randomization and mortality from the surgical
procedures. The rats will be approximately 10 weeks of age when
they arrive at the laboratory.
[0153] All animals will be housed and handled for behavioral
assessment for 7 days prior to surgery for acclimation purposes. At
the end of the training period, animals will be randomized and
assigned to different groups. Animals will be given a unique
identification number by tail marking. Tail number and cage cards
will identify the animals.
[0154] Surgical Preparation. Middle Cerebral Artery Occlusion
(MCAo), Tamura Model. Focal cerebral infarcts are made by permanent
occlusion of the proximal right middle cerebral artery using a
modification of the method of Tamura et al. Male Sprague-Dawley
rats (300-350 g) are anesthetized with 2% isoflourane in 50%
Air/50% O.sub.2, and is maintained with 1-1.5 % isoflourane. The
temporalis muscle is bisected and reflected through an incision
made midway between the eye and the eardrum canal. The proximal MCA
is exposed through a subtemporal craniectomy without removing the
zygomatic arch and without transecting the facial nerve. The artery
is then occluded by microbipolar coagulation from just proximal to
the olfactory tract to the inferior cerebral vein, and is
transected. Body temperature is maintained at 38.degree. C..+-.1
throughout the entire procedure. Animals are euthanatized at
various time periods after MCA occlusion and the brains are removed
for histological analysis.
[0155] Cerebral Blood Flow Measurement. Blood Flow Monitoring will
take place in one group of forty animals to determine the effects
of the three experimental compounds selected from the compounds
disclosed herein on cerebral blood flow at certain times in
relation to the MCAo. A Perimed Laser Doppler System 500 will be
used to determine percent changes in cerebral blood before MCAo,
sixty minutes post MCAO and then days 1 and 5 after MCAo.
[0156] Animals are briefly anesthetized with isoflourane and a
primed flow probe is inserted through a burr hole made
approximately at the following stereotaxic coordinates on the
ipsilateral side of the MCAo: -1.0 millimeters to bregma and +6 mm
lateral to the midline. The tip of the probe is inserted 1 mm and
fixed in place using cyanoacrylate glue. Recordings are made over a
5-minute period. The muscle flap and skin is sutured with running
4-0 silk and animals returned to their cages. The results of the
groups treated with the 3 test compounds will be compared to
Vehicle treated rats.
[0157] Behavioral Analysis. Two behavioral tests will be performed,
the limb placing and body swing test. This test will be performed
immediately after surgery and days 1, 3, 7 after MCAo.
[0158] Limb Placing. The limb placing tests are divided into both
forelimb and hindlimb tests. For the forelimb-placing test, the
examiner holds the rat close to a tabletop and scores the rat's
ability to place the forelimb on the tabletop in response to
whisker, visual, tactile, or proprioceptive stimulation. Similarly,
for the hindlimb placing test, the examiner assesses the rat's
ability to place the hindlimb on the tabletop in response to
tactile and proprioceptive stimulation. Separate sub scores are
obtained for each mode of sensory input and added to give total
scores (for the forelimb placing test: 0=normal, 10=maximally
impaired; for the hindlimb placing test: 0=normal; 6=maximally
impaired). Typically, there is a slow and steady recovery of limb
placing behavior during the first month after stroke.
[0159] Body Swing Test. The animal is held approximately 1 inch
from the base of its tail. It is then elevated to an inch above a
surface of a table. The animal is held in the vertical axis,
defined as no more than 10.degree. to either the left or the right
side. A swing is recorded whenever the animal moves its head out of
the vertical axis to either side. Before attempting another swing,
the animal must return to the vertical position for the next swing
to be counted. Thirty total swings are counted. A normal animal
typically has an equal number of swings to either side. Following
focal stroke, the animal tends to swing to the contralateral side.
There is a slow spontaneous recovery of body swing during the first
month after stroke.
[0160] Morphometric Analysis--Brain Perfusion. Seven days after the
induction of focal ischemia, rats will be deeply anesthetized with
a ketamine, and xylazine cocktail. The animals are then perfused
transcardially with normal saline (with heparin 2 unit/ml) followed
by 4% paraformaldehyde for infarct volume measurement (H&E
staining). Brains are removed and stored in 10% formalin to be
sectioned and stained with Hematoxylin and Eosin.
[0161] Infarct Volume Measurement. Using the Image Pro-Plus imaging
system, a total of 7 images per brain of the posterior side of each
slice are analyzed through digital analysis. The total direct
infarct volume is calculated for the left hemisphere using the
equation below. Digitizing and computation is done under blinded
conditions. 3 Volume ( mm 3 ) = area ( mm 2 ) per side No . of
sides analyzed .times. 7 mm
[0162] The total infarct volumes are calculated for each animal and
subsequent group means are determined as volume of area (mm.sup.3).
To account for tissue shrinkage and possible edema, the indirect
method of infarct volume is calculated using the formula:
Total Contralateral Hemisphere Volume-Total Infarcted Hemisphere
Volume (mm3)
[0163] Body Temperature and Weight. All animals' body temperature
are monitored throughout the surgery and maintained near normal
values (37-38.degree. C.). Animal's body weights are measured and
documented before surgery and at the end of the study immediately
before euthanation.
[0164] Clinical Observations. Along with the behavioral assessment
animals will be observed daily for abnormal behavior, including but
not limited to circling, lethargy, respiratory wheezing or
discharge, hair loss, and early death. All abnormalities will be
recorded. Likewise, normal behavior will also be noted as
normal.
[0165] Statistical Analysis. Behavioral scores and body weight are
analyzed by two-way repeated measures analysis of variance (ANOVA;
treatment X time). Infarct volume is analyzed by one-way ANOVA. A p
value of .ltoreq.0.05 is considered a statistically significant
difference.
[0166] Experimental Design
[0167] Experimental Groups and Compound Administration. A number of
animals will undergo MCAo using subcutaneous administration of all
test articles. One or more compounds selected from the compounds
disclosed herein, at predetermined doses will be compared to a
control or vehicle and assigned to two different time schedules of
administration relative to the time of MCAo. The time schedules for
administration of all treated groups will be divided as
follows:
[0168] a. 3 hrs, 6 hrs, & 25 hrs and days 2-7 b.i.d. post
[0169] b. 6 hrs, 9 hrs & 25 hrs and days 2-7 b.i.d. post
[0170] All animals are treated with the compound(s)/vehicle(s) as a
blinded random investigation.
Example 5
Preparation of Tablets
[0171] The compound of FIGS. 1, 2, or 3 (10.0 g) is mixed with
lactose (85.5 g), hydroxypropyl cellulose HPC-SL (2.0 g),
hydroxypropyl cellulose L-HPC, LH-22 (2.0 g) and purified water
(9.0 g), the resulting mixture is subjected to granulation, drying
and grading, and the thus obtained granules are mixed with
magnesium stearate (0.5 g) and subjected to tablet making, thereby
obtaining tablets containing 10 mg per tablet of the compound.
Example 6
Administering to a Subject
[0172] The tablet prepared in Example 5 is provided to a subject at
time 0. One tablet every 24 h is provided for a period of one week.
After administration of the third tablet, the subject is exposed to
a neurodegenerative event. The treated subject exhibits symptoms of
neurological disorder that are less severe compared to the subject
that was not treated.
[0173] All publications, patents, and patent applications mentioned
in this specification are herein incorporated by reference to the
same extent as if each individual publication or patent application
was specifically and individually indicated to be incorporated by
reference.
[0174] Having now fully described this invention, it will be
appreciated by those skilled in the art that the same can be
performed within a wide range of equivalent parameters,
concentrations, and conditions without departing from the spirit
and scope of the invention and without undue experimentation.
[0175] While this invention has been described in connection with
specific embodiments thereof, it will be understood that it is
capable of further modifications. This application is intended to
cover any variations, uses, or adaptations of the invention
following, in general, the principles of the invention and
including such departures from the present disclosure as come
within known or customary practice within the art to which the
invention pertains and as may be applied to the essential features
hereinbefore set forth.
* * * * *