U.S. patent application number 12/678060 was filed with the patent office on 2010-12-09 for inhibitors of naaa and methods thereof.
This patent application is currently assigned to THE REGENTS OF THE UNIVERSITY OF CALIFORNIA. Invention is credited to Andrea Duranti, Marco Mor, Daniele Piomelli, Giorgio Tarzia, Andrea Tontini.
Application Number | 20100311711 12/678060 |
Document ID | / |
Family ID | 40549611 |
Filed Date | 2010-12-09 |
United States Patent
Application |
20100311711 |
Kind Code |
A1 |
Piomelli; Daniele ; et
al. |
December 9, 2010 |
Inhibitors of NAAA and Methods Thereof
Abstract
Compounds and pharmaceutical compositions are contemplated that
inhibit N-acyl-ethanolamine-hydrolyzing acid amidase (NAAA) to so
increase the concentration of the substrate of NAAA,
palmitoylethanolamide (PEA). NAAA inhibition is contemplated to be
effective to alleviate conditions associated with a reduced
concentration of PEA. Among other uses, various NAAA inhibitors are
especially contemplated as therapeutic agents in the treatment of
inflammatory diseases.
Inventors: |
Piomelli; Daniele; (Irvine,
CA) ; Tarzia; Giorgio; (Pertiano, IT) ; Mor;
Marco; (Ghedi, IT) ; Duranti; Andrea; (Urbino,
IT) ; Tontini; Andrea; (Pesaro, IT) |
Correspondence
Address: |
FISH & ASSOCIATES, PC;ROBERT D. FISH
2603 Main Street, Suite 1000
Irvine
CA
92614-6232
US
|
Assignee: |
THE REGENTS OF THE UNIVERSITY OF
CALIFORNIA
Oakland
CA
|
Family ID: |
40549611 |
Appl. No.: |
12/678060 |
Filed: |
October 10, 2008 |
PCT Filed: |
October 10, 2008 |
PCT NO: |
PCT/US08/79621 |
371 Date: |
August 27, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60979304 |
Oct 11, 2007 |
|
|
|
Current U.S.
Class: |
514/210.02 ;
435/184; 514/345; 514/424; 514/430; 514/432; 514/445; 514/449;
514/460; 514/473 |
Current CPC
Class: |
A61K 31/195 20130101;
A61P 29/00 20180101; A61P 11/06 20180101; A61P 19/02 20180101; A61K
31/337 20130101; A61P 17/00 20180101; A61K 31/365 20130101; A61P
1/04 20180101; C07D 305/10 20130101; A61P 25/00 20180101; A61P
37/08 20180101; A61P 17/06 20180101; A61K 31/165 20130101 |
Class at
Publication: |
514/210.02 ;
435/184; 514/449; 514/430; 514/432; 514/445; 514/460; 514/473;
514/345; 514/424 |
International
Class: |
A61K 31/397 20060101
A61K031/397; C12N 9/99 20060101 C12N009/99; A61K 31/337 20060101
A61K031/337; A61K 31/38 20060101 A61K031/38; A61K 31/382 20060101
A61K031/382; A61K 31/381 20060101 A61K031/381; A61K 31/35 20060101
A61K031/35; A61K 31/34 20060101 A61K031/34; A61K 31/44 20060101
A61K031/44; A61K 31/40 20060101 A61K031/40; A61P 25/00 20060101
A61P025/00 |
Goverment Interests
[0002] This invention was made with government support under NIH
Grant No. DA-12413, awarded by the National Institutes of Health.
The Government may have certain rights in the invention.
Claims
1. A pharmaceutical composition for treatment of a condition
associated with a reduced level of palmitoylethanolamide, or in
which elevation of palmitoylethanolamide levels is therapeutically
desirable, comprising: a compound according to Formula I and a
pharmaceutically acceptable carrier ##STR00004## wherein A is O or
S; B is O, S, or NR.sup.a; R.sub.1 and R.sub.2 are independently H,
halogen, or optionally substituted lower alkyl; n is an integer
between 0 and 3; X is O, S, C(O), NR.sup.b, CHR.sup.b or null; Y is
C(O), C(S), or CHR.sup.c; Z is O, S, NR.sup.d, or CHR.sup.d; V is
optionally substituted lower alkyl or optionally substituted lower
alkenyl; wherein W is aryl, heteroaryl, cycloalkyl,
cycloheteroalkyl, or C(R.sub.3R.sub.4R.sub.5); and wherein Y and V
may optionally form a 5- or 6 membered ring; wherein R.sup.a,
R.sup.b, R.sup.c, and R.sup.d are independently selected from the
group consisting of H, optionally substituted lower alkyl, or
optionally substituted lower thioalkyl; and wherein R.sub.3,
R.sub.4 and R.sub.5 are independently H, optionally substituted
lower alkyl, optionally substituted lower aryl, optionally
substituted lower cycloheteroalkyl, or optionally substituted lower
heteroaryl.
2. The pharmaceutical composition of claim 1 wherein A and B are
O.
3. The pharmaceutical composition of claim 1 wherein X is NR.sup.b
and Y is C(O) or C(S).
4. The pharmaceutical composition of claim 2 or claim 3 wherein Z
is O or CHR.sup.d, V is lower alkyl, and wherein W is aryl or lower
alkyl.
5. The pharmaceutical composition of claim 2 or claim 3 wherein n
is 1, R.sub.1 is H and R.sub.2 is lower alkyl.
6. The pharmaceutical composition of claim 1 wherein A is O, B is O
or NR.sup.a, X is NR.sup.b and Y is C(O) or C(S).
7. The pharmaceutical composition of claim 6 wherein W is aryl or
lower alkyl.
8. A method of treating a patient having a condition associated
with reduced levels of palmitoylethanolamide or in which elevation
of palmitoylethanolamide levels is therapeutically desirable,
comprising administering a pharmaceutical composition comprising a
compound according to Formula I according to claim 1.
9. The method of claim 8 wherein the condition associated with
reduced levels of palmitoylethanolamide includes an inflammatory
component, and wherein administration of the composition reduces
inflammation in the patient.
10. The method of claim 8 wherein the condition is rheumatoid
arthritis, osteoarthritis, asthma, allergic dermatitis, psoriasis,
an inflammatory bowel disease, or a spinal cord injury.
11. The method of claim 8 wherein A is O, B is O or NR.sup.a, X is
NR.sup.b and Y is C(O) or C(S).
12. The method of claim 11 wherein Z is O or CHR.sup.d, V is lower
alkyl, and wherein W is aryl or lower alkyl.
13. The method of claim 11 wherein W is aryl or lower alkyl.
14. A method of inhibiting of N-acylethanolamine-hydrolyzing acid
amidase (NAAA), comprising contacting the NAAA with a compound
according to Formula I ##STR00005## wherein A is O or S; B is O, S,
or NR.sup.a; R.sub.1 and R.sub.2 are independently H, halogen, or
optionally substituted lower alkyl; n is an integer between 0 and
3; X is O, S, C(O), NR.sup.b, CHR.sup.b or null; Y is C(O), C(S),
or CHR.sup.c; Z is O, S, NR.sup.d, or CHR.sup.d; V is optionally
substituted lower alkyl or optionally substituted lower alkenyl;
wherein W is aryl, heteroaryl, cycloalkyl, cycloheteroalkyl, or
C(R.sub.3R.sub.4R.sub.5); and wherein Y and V may optionally form a
5- or 6 membered ring; wherein R.sup.a, R.sup.b, R.sup.c, and
R.sup.d are independently selected from the group consisting of H,
optionally substituted lower alkyl, or optionally substituted lower
thioalkyl; and wherein R.sub.3, R.sub.4 and R.sub.5 are
independently H, optionally substituted lower alkyl, optionally
substituted lower aryl, optionally substituted lower
cycloheteroalkyl, or optionally substituted lower heteroaryl.
15. The method of claim 14 wherein A is O, B is O or NR.sup.a, X is
NR.sup.b and Y is C(O) or C(S).
16. The method of claim 15 wherein Z is O or CHR.sup.d, V is lower
alkyl, and wherein W is aryl or lower alkyl.
17. The method of claim 14 wherein the step of contacting is
performed in vivo using topical administration of the compound,
aerosol administration of the compound, or intrathecal injection of
the compound.
18. The method of claim 14 wherein the compound inhibits the NAAA
at an 1050 of less than 20 microM.
Description
[0001] This application claims priority to our copending U.S.
provisional application with the Ser. No. 60/979,304, which was
filed Oct. 11, 2007, and which is incorporated by reference
herein.
FIELD OF THE INVENTION
[0003] The field of the invention is compositions and methods
relating to inhibition of N-acylethanolamine-hydrolyzing acid
amidase (NAAA), and especially as it relates to treatment and
prevention of pain, inflammation, and other disorders in which
fatty acid ethanolamide modulation is clinically relevant.
BACKGROUND OF THE INVENTION
[0004] While there are numerous compositions and methods known in
the art to treat pain and/or inflammation, numerous difficulties
remain. Most significantly, side effects over long administration
periods and/or higher dosages often prevent successful use of such
drugs. For example, certain COX-2 inhibitors have recently been
implicated in adverse cardiovascular events, while aspirin-type
pain medication often increases the risk of intestinal bleeding. In
other examples, ibuprofen and acetaminophen tend to negatively
impact hepatic function, especially at higher dosages.
[0005] Ethanolamides of long-chain fatty acids (N-acylethanolamines
(NAEs)) are present in numerous lower organisms, higher organisms,
and mammals with a wide variety of functions. For example,
anandamide (a polyunsaturated fatty acid-type NAE), was
demonstrated to have cannabimimetic activity and was reported as
acting as a ligand of TRPV 1 (transient receptor potential
vanilloid type 1). In contrast, saturated and monounsaturated NAEs
are inactive as ligands of cannabinoid receptors. However, such
compounds have been reported to possess a variety of other
biological activities. For example, N-palmitoylethanolamine (PEA)
has anti-inflammatory, anti-nociceptive, immunosuppressive,
neuroprotective, and also antioxidant activity. Interestingly, the
anti-inflammatory action of N-palmitoylethanolamine could be
mediated by activation of peroxisome proliferator-activated
receptor-alpha (PPAR-alpha). In other examples,
N-oleoylethanolamine was shown to be anorexic via PPAR-alpha (see
e.g., The Journal Of Pharmacology And Experimental Therapeutics
(2006), Vol. 318, No. 2, pages 563-570), and N-stearoylethanolamine
to be pro-apoptotic and anorexic.
[0006] NAEs are a substrate of NAAA that catalytically hydrolyze
the NAE to ethanolamine and the corresponding fatty acid.
Remarkably, the catalytic activity of NAAA is significantly
different from a similar enzyme, FAAH (fatty acid amide hydrolase).
Among various other differences, one characteristic trait of NAAA
is its activity optimum at a pH of about 5.0. NAAA also exhibits a
substantial preference for N-palmitoylethanolamine (PEA) over other
NAEs, is activated by TRITON X100.TM. (registered trademark by
Union Carbide; 4-octyl-phenol polyethoxylate) and dithiothreitol
(DTT). Remarkably, NAAA has lower sensitivity to inhibition with
phenylmethylsulfonyl fluoride and methylarachidonyl
fluorophosphonate. While the gene for NAAA has been cloned and the
corresponding polypeptide is relatively well characterized (see
e.g., J Biol Chem (2005), Vol. 280, No. 12, pages 11082-11092), the
functional properties of NAAA in mammals are not well
understood.
[0007] While numerous FAAH inhibitors have been identified in the
literature (see e.g., Eur J Pharmacol (2007), 565(1-3); pages
26-36; J Enz Inhib and Med Chem (2003), 18(1), pages 55-58; Arch
Biochem Biophys (1999), 362(2), pages 191-196), no inhibitors are
currently reported for NAAA. Moreover, as FAAH and NAAA are not
structurally closely related, it is not expected that FAAH
inhibitors will provide significant inhibition of NAAA.
[0008] Therefore, while numerous compositions and methods of
treating and prevention of pain and inflammation are known in the
art, all or almost all of them suffer from one or more
disadvantages. Consequently, there is still a need to provide
improved composition and methods to treat and prevent pain and
inflammation.
SUMMARY OF THE INVENTION
[0009] The present invention is directed to compositions and
methods of inhibiting NAAA using various compounds contemplated and
identified by the inventors. Most advantageously, such compounds
and compositions will be useful in the treatment of conditions
associated with a reduced level of palmitoylethanolamide, and
especially inflammatory diseases.
[0010] In one preferred aspect of the inventive subject matter, a
pharmaceutical composition for treatment of a condition associated
with a reduced level of palmitoylethanolamide comprises a compound
according to Formula I and a pharmaceutically acceptable
carrier
##STR00001##
[0011] wherein A is O or S; B is O, S, or NR.sup.a; R.sub.1 and
R.sub.2 are independently H, halogen, or optionally substituted
lower alkyl; n is an integer between 0 and 3; X is O, S, C(O),
NR.sup.b, CHR.sup.b or null; Y is C(O), C(S), or CHR.sup.c; Z is O,
S, NR.sup.d, or CHR.sup.d; V is optionally substituted lower alkyl
or optionally substituted lower alkenyl; W is aryl, heteroaryl,
cycloalkyl, cycloheteroalkyl, or C(R.sub.3R.sub.4R.sub.5), each of
which may be optionally substituted; in some aspects, Y and V may
form a 5- or 6-membered ring; most typically, R.sup.a, R.sup.b,
R.sup.c, and R.sup.d are independently selected from the group
consisting of H, optionally substituted lower alkyl, or optionally
substituted lower thioalkyl; and R.sub.3, R.sub.4 and R.sub.5 are
independently selected from the group consisting of H, optionally
substituted lower alkyl, optionally substituted lower aryl,
optionally substituted lower cycloheteroalkyl, and optionally
substituted lower heteroaryl.
[0012] Particularly contemplated compounds include those in which A
and B are O, in which X is NR.sup.b and Y is C(O) or C(S), and/or
in which Z is O or CHR.sup.d, V is lower alkyl, and wherein W is
aryl or lower alkyl. Most preferably, n is 1, R.sub.1 is H and
R.sub.2 is lower alkyl, and/or W is aryl or lower alkyl. Further
preferred compounds include those where A is O, B is O or NR.sup.a,
X is NR.sup.b and Y is C(O) or C(S).
[0013] Therefore, a method of treating a patient having a condition
associated with reduced levels of palmitoylethanolamide in a cell,
organ, or body compartment will include a step of administering a
pharmaceutical composition that includes a compound according to
Formula I above. Most typically, the condition includes an
inflammatory component (e.g., rheumatoid arthritis, osteoarthritis,
or asthma), pain, and/or a neurodegenerative aspect. Administration
of the composition is then performed under a protocol and at a
dosage sufficient to reduce the inflammation in the patient. Viewed
from a different perspective, contemplated methods will therefore
also include in which compounds according to Formula I above will
be used to inhibit NAAA. In preferred aspects, the step of
contacting the NAAA is performed in vivo, and/or the compound
inhibits the NAAA at an IC.sub.50 of less than 20 microM.
[0014] Various objects, features, aspects and advantages of the
present invention will become more apparent from the following
detailed description of preferred embodiments of the invention.
BRIEF DESCRIPTION OF THE DRAWING
[0015] FIGS. 1A-1C are exemplary compounds according to the
inventive subject matter and their respective IC.sub.50 values.
[0016] FIGS. 2A-2C are graphs illustrating the effect of
inflammation on the number of infiltrating neutrophils (A), the
levels of endogenous cellular PEA(B), and time course of the effect
of inflammation on PEA (C).
[0017] FIGS. 3A-3B are graphs illustrating the effect of exogenous
PEA on the number of infiltrating neutrophils as a function of PEA
concentration (A), and the number of infiltrating neutrophils in
PPAR-alpha wild-type and null mutant mice (B).
[0018] FIG. 4 is a detail view of a computer model of the active
site of NAAA.
[0019] FIG. 5 is a graph depicting NAAA inhibition by selected
compounds according to the inventive subject matter.
[0020] FIGS. 6A-6C are graphs illustrating the effect of an
exemplary NAAA inhibitor on the number of infiltrating neutrophils
(A), and the quantity of endogenous PEA (B), and the number of
infiltrating neutrophils in PPAR-alpha wild-type and null mutant
mice (C).
[0021] FIG. 7 depicts photomicrographs of tissue sections after
spinal cord injury stained with selected markers of animals treated
with and without an NAAA inhibitor and control.
DETAILED DESCRIPTION
[0022] The present invention is directed to compounds,
compositions, and methods of NAAA inhibition, and especially to
compounds, compositions, and methods suitable for treatment of
various diseases associated with reduced PEA levels in a cell,
organ, or other body structure (or even the entire body). Most
preferably, such modulation will result in treatment and/or
prevention of pain, inflammation, and other disorders in which
abnormal NAE levels are associated with a disorder. In still
further contemplated aspects, the inhibitors and methods according
to the inventive subject matter are also deemed useful for
investigation into mechanisms and pathways in which PEA plays a
regulatory or modulating role.
Contemplated Compounds
[0023] Compounds generally contemplated herein will have a
structure that is effective to inhibit NAAA in competitive,
non-competitive, allosteric, or other manner. Most preferably, the
compounds according to the inventive subject matter will inhibit
NAAA at relatively low concentrations (e.g., IC.sub.50 equal or
less than 50 microM). Among other suitable choices, especially
preferred compounds have a structure according to Formula I
##STR00002##
[0024] in which A is O or S; B is O, S, or NR.sup.a, with R.sup.a
being H, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, halo,
nitro, hydroxy, alkoxy, alkenyloxy, cyano, carboxy, alkoxycarbonyl,
carboxyalkyl, amino, acylamino, alkylamino, dialkylamino,
cycloalkylamino, N-alkyl, N-cycloalkyl, amino, thio, alkylthio, and
haloalkyl (all of which may be optionally substituted); n is an
integer between 0 and 3, wherein the so formed saturated or
unsaturated C1-3 may be optionally and at independent locations
substituted with R.sub.1 and R.sub.2, and wherein R.sub.1 and
R.sub.2 are independently R.sup.a as defined above; Q is as R.sup.a
as defined above; X is O, S, C(O), NR.sup.b, or CHR.sup.b with
R.sup.b being as R.sup.a defined above, or null; Y is C(O), C(S),
or CHR.sup.c with R.sup.c being as R.sup.a defined above, or null;
Z is O, S, NR.sup.d, or CHR.sup.d with R.sup.d being as R.sup.a
defined above, or null; V is an optionally substituted lower alkyl
or an optionally substituted lower alkylene or null; W is H, aryl,
heteroaryl, cycloalkyl, cycloheteroalkyl, or
C(R.sub.3R.sub.4R.sub.5), each of which may be optionally
substituted, wherein R.sub.3, R.sub.4 and R.sub.5 are independently
R.sup.a as defined above; and wherein Y and V may optionally form
an optionally substituted 5-, 6-, or 7-membered ring.
[0025] In especially preferred aspects, A and B are O, and/or X is
NR.sup.b and Y is C(O) or C(S). Additionally, or alternatively, it
is contemplated that Z is O or CHR.sup.d, V is lower alkyl, and
wherein W is aryl or lower alkyl, and/or that n is 1, R.sub.1 is H
and R.sub.2 is lower alkyl. Still further preferred compounds
include those in which A is O, B is O or NR.sup.a, X is NR.sup.b
and Y is C(O) or C(S). In still further contemplated aspects of the
inventive subject matter, X--Y may together form --CH.dbd.CH-- or
CH.dbd.CJ where J is halogen, especially where Z.dbd.--CHR.sup.d--
or null. It is still further contemplated that Y--Z--V may become
Y--V by eliminating Z, or Y, by eliminating Z and V. Similarly,
Z--V--W may become Z--W by eliminating V. Still further
contemplated compounds include all (e.g., acidic or alkaline)
hydrolytic cleavage products of the A=C--B-- bond.
[0026] Still further contemplated compounds include those according
to Formulae II and II as depicted below
##STR00003##
[0027] in which B is O, S, or NR.sup.a, with R.sup.a being H,
alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, halo, nitro,
hydroxy, alkoxy, alkenyloxy, cyano, carboxy, alkoxycarbonyl,
carboxyalkyl, amino, acylamino, alkylamino, dialkylamino,
cycloalkylamino, N-alkyl, N-cycloalkyl, amino, thio, alkylthio, and
haloalkyl (all of which may be optionally substituted); n is an
integer between 0 and 3, wherein the so formed saturated or
unsaturated C1-3 may be optionally and at independent locations
substituted with R.sub.1 and R.sub.2, and wherein R.sub.1 and
R.sub.2 are independently R.sup.a as defined above; Q is as R.sup.a
as defined above; X is O, S, C(O), NR.sup.b, or CHR.sup.b with
R.sup.b being as R.sup.a defined above, or null; Y is C(O), C(S),
or CHR.sup.c with R.sup.c being as R.sup.a defined above, or null;
Z is O, S, NR.sup.d, or CHR.sup.d with R.sup.d being as R.sup.a
defined above, or null; V is an optionally substituted lower alkyl
or an optionally substituted lower alkylene or null; W is H, aryl,
heteroaryl, cycloalkyl, cycloheteroalkyl, or
C(R.sub.3R.sub.4R.sub.5), each of which may be optionally
substituted, wherein R.sub.3, R.sub.4 and R.sub.5 are independently
R.sup.a as defined above; and wherein Y and V may optionally form
an optionally substituted 5-, 6-, or 7-membered ring.
[0028] It should still further appreciated that where stereocenters
are present, all isomers and mixtures thereof are contemplated.
Similarly, where a double bond is present, the orientation of
radicals at each side of the double bond may be cis or trans. FIGS.
1A-1D depict various exemplary preferred compounds according to the
inventive subject matter.
[0029] As used herein, the term "halogen" refers to a fluorine,
bromine, chlorine, or iodine, which is typically covalently bound
to another atom (e.g., carbon). As further used herein, the term
"hydroxyl" refers to a-OH group. As still further used herein, the
term "carbonyl atom" refers to a carbon atom to which three atoms
are covalently bound, wherein one of the three atoms is bound to
the carbon atom via a double bond (which may be partially
delocalized). Thus, particularly contemplated carbonyl atoms
include carbon atoms in an oxo group, an aldehyde group, a
carboxamide group, a carboxamidine group, and a thiocarboxamide
group.
[0030] The term "alkyl" as used herein refers to a cyclic,
branched, or straight hydrocarbon in which all of the carbon-carbon
bonds are single bonds, and the term "lower alkyl" refers to a
cyclic, branched, or straight chain alkyl of one to ten carbon
atoms (e.g., methyl, ethyl, n-propyl, i-propyl, n-butyl, t-butyl,
i-butyl (or 2-methylpropyl), cyclopropylmethyl, i-amyl, n-amyl,
hexyl, etc.). The term "alkylene" as used herein refers to an alkyl
having two hydrogen atoms less than the corresponding alkane (i.e.,
C.sub.nH.sub.2n). For example, suitable alkylenes include methylene
groups, ethylene groups, propylene groups, etc. The term
"cycloalkyl" as used herein refers to a cyclic or polycyclic alkyl
group containing 3 to 15 carbons. For polycyclic groups, these may
be multiple condensed rings in which one of the distal rings may be
aromatic (e.g., indanyl, tetrahydronaphthalene, etc.). The term
"alkaryl" as used herein refer to an alky that is covalently
coupled to an aryl moiety. For example, a benzyl radical is
considered an alkaryl under the definition provided herein.
[0031] Similarly, the term "alkenyl" as used herein refers to an
alkyl in which at least one carbon-carbon bond is a double bond.
Thus, the term "lower alkenyl" includes all alkenyls with one to
ten carbon atoms. The term "cycloalkenyl" as used herein refers to
a cyclic or polycyclic group containing 3 to 15 carbons and at
least one double bond. Likewise, the term "alkynyl" as used herein
refers to an alkyl or alkenyl in which at least one carbon-carbon
bond is a triple bond. Thus, the term "lower alkynyl" includes all
alkynyls with one to ten carbon atoms.
[0032] As still further used herein, the term "alkoxy" refers to
a-OR group, wherein R is lower alkyl, substituted lower alkyl,
acyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl,
heteroarylalkyl, cycloalkyl, substituted cycloalkyl,
cycloheteroalkyl, or substituted cycloheteroalkyl. Similarly, the
term "aryloxy" refers to a-OAr group, wherein Ar is an aryl,
substituted aryl, heteroaryl, or substituted heteroaryl group.
[0033] Furthermore, the term "aryl" refers to an aromatic
carbocyclic group having at least one aromatic ring (e.g., phenyl
or biphenyl) or multiple condensed rings in which at least one ring
is aromatic, (e.g., 1,2,3,4-tetrahydronaphthyl, naphthyl, anthryl,
or phenanthryl). The term "heteroatom" as used herein refers to an
atom other than carbon (e.g., S, O, or N), which can optionally be
substituted with, e.g., hydrogen, halogen, lower alkyl, alkoxy,
lower alkylthio, trifluoromethyl, amino, amido, carboxyl, hydroxyl,
aryl, aryloxy, heterocycle, heteroaryl, substituted heteroaryl,
nitro, cyano, alkylthio, thiol, sulfamido and the like.
[0034] Still further, the term "substituted" as used herein means
that a hydrogen atom that is covalently bound to a group or atom
(or a free electron pair or electron pair of a double bond of an
atom) is replaced by a covalently bound non-hydrogen substituent,
including hydroxyl, thiol, alkylthiol, halogen, alkoxy, amino,
amido, nitro, carboxyl, cycloalkyl, heterocycle, cycloheteroalkyl,
acyl, carboxyl, aryl, aryloxy, heteroaryl, arylalkyl,
heteroarylalkyl, alkyl, alkenyl, alknyl, and cyano.
[0035] The term "prodrug" as used herein refers to a modification
of contemplated compounds, wherein the modified compound exhibits
less pharmacological activity (as compared to the modified
compound) and wherein the modified compound is converted within a
target cell (e.g., B-cell) or target organ/anatomic structure
(e.g., joint) back into the modified form. For example, conversion
of contemplated compounds into prodrugs may be useful where the
active drug is too toxic for safe systemic administration, or where
the contemplated compound is poorly absorbed by the digestive tract
or other compartment or cell, or where the body breaks down the
contemplated compound before reaching its target. Thus, it should
be recognized that the compounds according to the inventive subject
matter can be modified in numerous manners, and especially
preferred modifications include those that improve one or more
pharmacokinetic and/or pharmacodynamic parameter. For example, one
or more substituents may be added or replaced to achieve a higher
AUC in serum.
[0036] On the other hand, and especially where increased solubility
is desired, hydrophilic groups may be added. Still further, where
contemplated compounds contain one or more bonds that can be
hydrolyzed (or otherwise cleaved), reaction products are also
expressly contemplated. Exemplary suitable protocols for conversion
of contemplated compounds into the corresponding prodrug form can
be found in "Prodrugs (Drugs and the Pharmaceutical Sciences: a
Series of Textbooks and Monographs)" by Kenneth B. Sloan (ISBN:
0824786297), and "Hydrolysis in Drug and Prodrug Metabolism:
Chemistry, Biochemistry, and Enzymology" by Bernard Testa, Joachim
M. Mayer (ISBN: 390639025X), both of which are incorporated by
reference herein. Moreover, especially where contemplated compounds
have a higher activity when the compound is metabolized (e.g.,
hydrolyzed, hydroxylated, glucuronidated, etc.), it should be
appreciated that metabolites of contemplated compounds are also
expressly contemplated herein.
[0037] Depending on the particular purpose (e.g., analgesic,
anti-inflammatory), it should be recognized that contemplated
compounds may be combined (in vivo or in a pharmaceutical
formulation or administration regimen) with at least one other
pharmaceutically active ingredient, and especially contemplated
other ingredients include various analgesics (e.g., opioids,
ibuprofen-type drugs, acetaminophen-type drugs, aspirin-type drugs,
etc.) various immunosuppressants and/or anti-inflammatory drugs
(e.g., steroids and NSAIDS), etc. Concentrations of second
pharmaceutically active ingredients are typically at or preferably
below those recommended for stand-alone administration, however,
higher concentrations are also deemed suitable for use herein.
[0038] Therefore, contemplated pharmaceutical compositions will
especially include those in which contemplated compounds (and
additional pharmaceutically active ingredients) are provided with a
suitable carrier, wherein contemplated compounds are preferably
present at a concentration effective to modulate fatty acid
ethanolamide concentration in an organism and/or target organ to a
degree effective to reduce and more preferably to treat signs and
symptoms of a disease associated with an abnormal level in fatty
acid ethanolamide. Viewed from a different perspective,
contemplated compounds are present in a composition in an amount
effective to reduce pain and/or inflammation.
[0039] Depending on the particular use and structure, it is
therefore contemplated that the compounds according to the
inventive subject matter are present in the composition in an
amount between 1 microgram to 1000 milligram, more typically
between 10 microgram to 500 milligram, and most typically between
50 microgram to 500 milligram per single dosage unit. Thus,
preferred concentrations of contemplated compounds in vivo or in
vitro will generally be between 0.1 nM and 500 microM, more
typically between 50 nM and 400 microM, and most typically between
100 nM and 200 microM.
[0040] Furthermore, it should be recognized that all formulations
are deemed suitable for use herein and especially include oral and
parenteral formulations. For example, for oral administration,
contemplated compositions may be in the form of a tablet, capsule,
suspension, or liquid. The pharmaceutical composition is preferably
made in the form of a dosage unit containing a particular amount of
the active ingredient. Examples of such dosage units are tablets or
capsules. The active ingredient may also be administered by
injection as a composition wherein, for example, saline, dextrose
or water may be used as a suitable carrier. In especially preferred
aspects, it is contemplated that the formulation is suitable for
topical administration, administration via aerosol, and for
intrathecal administration. Consequently, especially suitable
formulations may be sterile aqueous solutions for topical spray or
drop administration, or application as a tincture. Alternatively,
suitable topical formulations include creams, ointments, foams,
lotions, emulsions, etc. Furthermore, where the compound is
formulated for intrathecal administration (e.g., in the treatment
of spinal cord injury), it is preferred that the compound is
prepared as an injectable solution, suspension, or emulsion. In
still further contemplated formulations, contemplated compounds may
be formulated for aerosol delivery (e.g., micropowderized, coated
onto a dispersible carrier, dissolved in atomizable solvent,
etc.)
[0041] It should be appreciated that the choice of the particular
formulation and carrier will at least in part depend on the
specific use and type of compound. There are numerous manners of
drug formulation known in the art, and all of those are deemed
suitable for use herein (see e.g., Pharmaceutical Preformulation
and Formulation: A Practical Guide from Candidate Drug Selection to
Commercial Dosage Form by Mark Gibson; Informa HealthCare, ISBN:
1574911201; or Advanced Drug Formulation Design to Optimize
Therapeutic Outcomes by Robert O. Williams, David R. Taft, and
Jason T. McConville; Informa HealthCare; ISBN: 1420043870).
[0042] The amount of therapeutically active compound that is
administered and the dosage regimen for treating a disease
condition with the compounds and/or compositions of this invention
depends on a variety of factors, including the age, weight, sex and
medical condition of the subject, the severity of the disease, the
route and frequency of administration, and the particular compound
employed, and thus may vary widely. However, especially suitable
quantities are provided above, and may therefore allow for a daily
dose of about 0.001 (or even less) to 100 mg/kg body weight,
preferably between about 0.01 and about 50 mg/kg body weight and
most preferably from about 0.1 to 20 mg/kg body weight. Typically,
a daily dose can be administered in one to four doses per day.
[0043] For therapeutic or prophylactic purposes, contemplated
compounds are ordinarily combined with one or more excipients
appropriate to the indicated route of administration. If
administered per os, the compounds may be admixed with lactose,
sucrose, starch powder, cellulose esters of alkanoic acids,
cellulose alkyl esters, talc, stearic acid, magnesium stearate,
magnesium oxide, sodium and calcium salts of phosphoric and
sulfuric acids, gelatin, acacia gum, sodium alginate,
polyvinylpyrrolidone, and/or polyvinyl alcohol, and then tableted
or encapsulated for convenient administration. Such capsules or
tablets may contain a controlled-release formulation as may be
provided in a dispersion of active compound in hydroxypropylmethyl
cellulose. Formulations for parenteral administration may be in the
form of aqueous or non-aqueous isotonic sterile injection solutions
or suspensions. These solutions and suspensions may be prepared
from sterile powders or granules having one or more of the carriers
or diluents mentioned for use in the formulations for oral
administration. The compounds may be dissolved in water,
polyethylene glycol, propylene glycol, ethanol, corn oil,
cottonseed oil, peanut oil, sesame oil, benzyl alcohol, sodium
chloride, and/or various buffers. Other excipients and modes of
administration are well and widely known in the pharmaceutical
art.
Contemplated Uses
[0044] It is generally contemplated that the compounds and
compositions according to the inventive subject matter may be
employed to affect any condition and/or disease associated with
abnormal levels (e.g., deviation of at least 10% relative to
average PEA level in healthy person at the corresponding test site)
of NAEs or where modulation of normal levels of such compounds is
desired for a particular purpose. Thus, and viewed from a different
perspective, contemplated compounds may be used for treatment of
diseases or conditions where elevation of palmitoylethanolamide
levels are therapeutically desirable. Therefore, particularly
contemplated conditions and diseases include those sensitive to
changes of NAEs. For example, contemplated compounds and
compositions may be useful in the prevention and/or treatment of
pain, inflammation, cancer and metabolic diseases. Further
contemplated diseases include disorders of the nervous system, and
especially relating to neuroinflammation, Alzheimer's Disease,
asthma, dermatitis, irritable bowel syndrome (IBS), Crohn's Disease
and appetite disorders.
[0045] Therefore, conditions and diseases to be treated with
contemplated compounds and compositions especially include pain,
inflammation, and neurodegenerative diseases. Among other example,
such diseases may include neuropathic pain, trigeminal neuralgia,
postherpetic neuralgia, diabetic neuropathy, cancer pain, phantom
limb pain, complex regional pain syndrome, and fibromyalgia;
rheumatoid arthritis, ankolysing spondylitis, ulcerative colitis,
tendonitis, psoriasis, Faber's Disease, Crohn's Disesase, rhinitis,
skin allergies, asthma, and autoimmune diseases with inflammatory
components such as multiple sclerosis and other demylenating
disorders; Alzheimer's Disease, traumatic brain injury. Other
conditions and diseases characterizable by abnormal NAE levels and
for which contemplated compounds may be useful include various
metabolic disorders, appetite regulation, and obesity.
[0046] Still further contemplated uses include those in which
compounds and compositions according to the inventive subject
matter are used for inhibition studies in vitro and in vivo to
determine structure-function relationship of a compound with
respect to inhibition of NAAA. Alternatively, or additionally, it
should also be appreciated that contemplated compounds and
compositions may be employed in various methods of inhibiting NAAA
and/or activating or modulating signaling in a pathway in which
PPAR-alpha is a member in signal transduction or other
processing.
Synthesis of Exemplary Contemplated Compounds
[0047] URB783 was prepared from N-Boc-L-serine (1) which was
cyclized in a Mitsunobu reaction to give 2, the deprotection and
salification of which provided tosylate 3. The latter compound was
reacted with 3-phenylpropionyl chloride to afford URB783. URB894
resulted from the reaction between 3 and 4-biphenylcarbonyl
chloride, which in turn was obtained by treating 4-phenylbenzoic
acid with oxalyl chloride.
[0048] (S)-(2-oxooxetan-3-yl)carbamic acid tent-butyl ester (2): To
a stirred solution of dry PPh.sub.3 (72 h under vacuum in the
presence of P.sub.2O.sub.5) (5 mmol) in dry CH.sub.3CN (31 mL),
kept at -50.degree. C. under N.sub.2, dimethylazodicarboxylate (5
mmol) and, after 20 min, a solution of 1 (4.47 mmol) in CH.sub.3CN
(10.4 mL), were added dropwise. The mixture was stirred for 1.5 h
at -50/-35.degree. C. and concentrated. Purification of the residue
by flash-chromatography (cyclohexane/EtOAc 8:2 to 6:4) gave crude
2, which was finally washed and triturated to afford a white solid.
Mp 120-122.degree. C. (Et.sub.2O).
[.alpha.].sub.D.sup.20=-27.degree. (c 0.1, CH.sub.3CN). .sup.1H NMR
is in accordance with literature (Arnold, et al., 1985).
[0049] (S)-2-oxooxetan-3-yl-ammonium-4-toluenesulphonate (3): To a
stirred mixture of 2 (1.34 mmol) and anhydrous p-toluenesulphonic
acid (72 h under vacuum in the presence of P.sub.2O.sub.5) (1.43
mmol), kept at 0.degree. C. under N.sub.2, trifluoroacetic acid (3
mL) was added dropwise in the course of 10 min. The solution was
reacted under stirring at 0.degree. C. for 15 min, allowed to reach
room temperature, and concentrated at a temperature below
30.degree. C. The oily residue was kept under vacuum for 1 h, and
the resulting white solid triturated and washed with dry diethyl
ether, then kept under vacuum for 24 h. Yield 81%. Mp and .sup.1H
NMR are in accordance with literature (Arnold, et al., 1988).
[0050] (S)--N-(2-oxooxetan-3-yl)-3-phenylpropionamide (URB783): To
a stirred mixture of 3 (0.4 mmol) in dry CH.sub.2Cl.sub.2 (2 mL),
kept at 0.degree. C. under N.sub.2, Et.sub.3N (1.59 mmol) and
3-phenylpropionyl chloride (0.6 mmol) were added dropwise. The
mixture was reacted at 0.degree. C. for 30 min and at room
temperature for 2 h, then concentrated. Purification of the residue
by flash-chromatography (cyclohexane/EtOAc 1:1 to 3:7) and
recrystallization gave URB783 as a white solid. Yield 60%. Mp
104-106.degree. C. (acetone/petroleum ether).
[.alpha.].sub.D.sup.20=-13.degree. (c 0.5, MeOH). MS (EI): m/z 219
(M.sup.+), 91 (100). IR (Nujol) 3333, 1832, 1625, 1541 cm.sup.-1;
.sup.1H NMR (CDCl.sub.3) .delta. 2.57 (br s, 1H), 2.99 (t, 2H),
4.34 (t, 1H, J=5 Hz), 4.43 (dd, 1H, J.sub.1=5 Hz, J.sub.2=6.5),
5.14 (m, 1H), 5.96 (br s, 1H), 7.18-7.36 (m, 5H) ppm. .sup.13C NMR
(CDCl.sub.3): .delta. 31.2 (CH.sub.2), 37.7 (CH.sub.2), 58.4 (CH),
66.1 (CH.sub.2), 126.5 (CH), 128.3 (2CH), 128.7 (2CH), 140.2, 168.4
(C.dbd.O), 172.5 (C.dbd.O) ppm.
[0051] (S)--N-(2-oxooxetan-3-yl)biphenyl-4-carboxamide (URB 894):
To a solution of biphenyl-4-carboxylic acid (2.1 mmol) in dry
CH.sub.2Cl.sub.2 (5.3 mL) and dry DMF (1 mL), kept at 0.degree. C.
under N.sub.2, was added oxalyl chloride (0.3 mL, 3.13 mmol). The
mixture was reacted 20 min at 0.degree. C. and 2 h at room
temperature, then concentrated to give crude biphenyl-4-carbonyl
chloride as a light-yellow solid. An amount of his sample (2 mmol)
were dissolved in dry THF (20 mL) and the resulting solution added
dropwise, at 0.degree. C., to a suspension obtained by mixing 3
(1.3 mmol), Et.sub.3N (1.143 mL, 8.2 mmol) and dry THF (3 mL) at
0.degree. C. under N.sub.2. The ensuing mixture was reacted at
0.degree. C. for 30 min and at room temperature for 3 h, then
evaporated. Purification by flash-chromatography (cyclohexane/EtOAc
1:1) and recrystallization gave pure URB894 as an ivory coloured
solid. Yield 46%. Mp 218-220.degree. C. (acetone/petroleum ether;
sealed capillary tube; decomposition of the sample with changing of
colour and shrinking of the mass was noted starting from
146.degree. C.); [.alpha.].sub.D.sup.20=-20.degree. (c 0.55,
CH.sub.3CN). MS (EI): m/z 267 (M.sup.+), 222, 181, 167 (100). IR
(Nujol) 3270, 1827, 1641, 1540 cm.sup.-1; .sup.1H NMR
(d.sub.6-acetone) .delta. 4.53-4.65 (m, 2H), 4.50-4.59 (m, 1H),
7.38-7.55 (m, 3H), 7.70-7.84 (m, 4H), 8.00-8.07 (m, 2H), 8.68 (br
d, 1H, J=7 Hz) ppm. .sup.13C NMR (d.sub.6-acetone): .delta. 58.8,
65.1, 126.9, 127.0, 128.0, 128.1, 129.0, 131.9, 139.7, 144.4,
166.4, 169.2 ppm.
EXAMPLES
[0052] Previous studies by the inventors and others have shown that
the PEA produces rapid broad-spectrum analgesic effects by
activating the nuclear receptor peroxisome proliferator-activated
receptor alpha (PPAR-.alpha.) in both inflammatory and neuropathic
pain models. More recent work has shown that PEA levels are reduced
in inflamed tissues (e.g., synovial fluid from rheumatoid arthritis
and osteoarthritis patients), suggesting that this bioactive lipid
may participate in the modulation of the inflammatory response
and/or contribute to chronic inflammatory states.
[0053] Supporting this possibility, the inventors have found that
restoring PEA levels during inflammation strongly alleviates
inflammation. These results suggest a novel mechanistic strategy to
reduce inflammation and pain by inhibition of PEA degradation to
restore normal PEA levels in inflamed tissues. In the present
application the inventors have developed a class of potent and
selective inhibitors of N-acylethanolamine-hydrolyzing acid amidase
(NAAA), the enzyme responsible for degrading PEA.
Inflammation Reduces the Levels of Endogenous PEA
[0054] The contribution of endogenous PEA to inflammatory processes
remains largely undefined. However, several lines of evidence
suggest that endogenous PEA may participate in inflammation. The
inventors have previously shown that the proinflammatory phorbol
ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), decreases dermal
PEA levels following skin inflammation. These findings raised the
possibility that reduced PEA levels during inflammation may allow
for the progression of the inflammatory process. To further explore
this idea, the inventors evaluated the effects of
carrageenan-induced inflammation on PEA levels in the mouse.
Polyethylene sponges injected with vehicle (10% DMSO in saline) or
carrageenan (1%) were surgically implanted under the dorsal skin of
mice. After 3 days, the mice were sacrificed and the sponges were
removed and analyzed for inflammatory cell infiltration and
cellular PEA content. In carrageenan-treated animals the number of
infiltrating cells (primarily neutrophils) increased by
approximately 3-fold as can be taken from FIG. 2. Here, surgical
implantation of vehicle (v) or carrageenan (c) soaked sponges under
the dorsal skin of Swiss mice for 3 days (A) increased the number
of infiltrating neutrophils and (B) decreased the levels of
endogenous cellular PEA. (C) Time course of the effects of vehicle
(open symbols) or carrageen (closed symbols) on PEA levels **
P<0.01 or *** P<0.001 vs. V, t-test or ANOVA, followed by
Dunnett's post-hoc as appropriate (n=6).
[0055] Further studies revealed (data not shown) that the reduction
of PEA in the inflamed tissue was at least in part due to
suppression of leukocyte expression of
N-acylphosphatidyl-ethanolamine-specific phospholipase D
(NAPE-PLD). NAPE-PLD-deficient mice, which produce PEA through a
compensatory enzymatic route, fail to lower PEA levels in response
to an inflammatory challenge and display a dampened reactivity to
such challenge. Inhibitors of N-acylethanolamine-hydrolyzing acid
amidase (NAAA) prevent the decrease in PEA levels and so blunt the
responses induced by inflammatory stimuli. The anti-inflammatory
effects of this agent are mimicked by exogenous PEA and abolished
by PPAR-alpha deletion. Thus, it should be noted that the results
strongly indicate that PEA activation of PPAR-alpha in leukocytes
serves as an early stop signal that impedes or even inhibits the
progress of inflammation.
Restoring PEA Levels with Exogenous PEA Reduces Inflammation
[0056] As a first step in determining the role of endogenous PEA in
inflammation, the inventors examined the effects of local
application of PEA on inflammatory responses. Polyethylene sponges
injected with vehicle or carrageenan (1%) and either vehicle (10%
DMSO in saline) or various doses of PEA (0.1-50 .mu.g) were
surgically implanted under the dorsal skin of mice. After 3 days,
the mice were killed and the sponges were removed and analyzed for
cell infiltration. PEA dose-dependently reduced the number of
infiltrating cells as shown in FIG. 3 and edema (data not shown) in
wild-type mice, but had no effect in PPAR-.alpha.-null animals.
Here panels (A-B) depict the effect of vehicle (open bars),
carrageenan (filled bars) or PEA (0.1-50 .mu.g) in Swiss mice,
black bars; PEA (50 .mu.g in PPAR-.alpha.-/- mice), scored as the
number of infiltrating inflammatory cells into a polyethylene
sponge (size) injected with vehicle, carrageenan (1%) or PEA,
implanted under the dorsal skin of (A) Swiss mice or (B) wildtype
C57BL6 mice (+/+) or PPAR-.alpha./- mice for 3 days. ** P<0.01
or *** P<0.001 vs. V, ## P<0.01 vs. carrageenan control.
ANOVA, followed by Tukey's or Dunnett's post-hoc as appropriate
(n=6). Thus, it should be appreciated that peripheral inflammation
is associated with decreased PEA synthesis in infiltrating cells,
and that restoring PEA levels by exogenous PEA administration
reduces inflammatory responses.
Design of NAAA Inhibitors
[0057] NAAA belongs to the choloylglycine hydrolase family, which
is a subgroup of the Ntn (N-terminal nucleophile) amino hydrolase
superfamily. These enzymes specialize in the cleavage of linear
amides and have a cysteine, serine or threonine at the first
position of their aminoacidic sequence, which acts as the
nucleophilic agent responsible for the catalytic attack. In the
case of NAAA, the nucleophilic residue is likely Cys131.
Experimental evidence suggests that the native NAAA protein
undergoes a maturation process involving proteolytic cleavage of
the first 130 residues, which gives a protein of 232 amino acids,
where Cys131 becomes N-terminus, an event commonly observed with
other Ntn hydrolases.
[0058] Recently, the structure of Conjugated Bile Acid Hydrolase
(CBAH), a member of the Ntn family, was resolved by X-ray
crystallography. An alignment of the amino acids sequences of NAAA
and CBAH revealed a high degree of sequence homology between the
binding sites of two enzymes. Using the coordinates of CBAH as a
template, a NAAA model was built by comparative modeling. According
to this model as illustrated in FIG. 4, the tetrahedral
intermediate formed through attack of the catalytic nucleophile
cysteine 131 on PEA is stabilized by electrostatic interactions
between the carbonyl oxygen of PEA and the enzyme oxyanion hole,
which is partly formed by the side-chain amide of asparagine 292
and the backbone amide of asparagine 209. In addition, a
hydrophobic pocket lined by tyrosine 151, among other residues, may
accommodate the flexible acyl chain of PEA.
[0059] These predictions were confirmed by site-directed
mutagenesis of these amino acids lining the catalytic side. For
example, replacement of Cys131, Ser133, Asp150, Tyr151 or Asn292
with alanine, completely abolished NAAA activity in vitro, whereas
mutations of peripheral residues had no such effect (data not
shown). Based on these results, the inventors designed a first
series of NAAA inhibitors that included a hydrophobic backbone (to
mimic the aliphatic fatty-acid moiety of PEA) linked to a lactone
head group (to target the active cysteine residue). The inventors
then synthesized and tested a number of compounds, a few of which
inhibited recombinant NAAA with sub-micromolar potencies (FIGS.
1A-C, FIG. 5). The two most potent compounds (URB783 and URB894)
inhibited NAAA with IC.sub.50 values of 420.+-.20 nM and 115.+-.13
nM, respectively (FIGS. 1A-C, FIG. 5).
NAAA Inhibition Restores PEA Levels and Reduces Inflammation
[0060] The inventors evaluated the effects of the NAAA inhibitor
URB783 on inflammation, using the mouse carrageenan model. As
previously shown, carrageenan exposure stimulated cell infiltration
(FIG. 6A) produced edema (data not shown), and significantly
reduced endogenous PEA levels (FIG. 6B). Inclusion of compound
URB783, into the sponge restored PEA to basal levels (FIG. 6B) and
significantly reduced the number of both infiltrating cells (FIG.
6A) and exudates volume (data not shown). Notably, the selective
FAAH inhibitor URB597 had no anti-inflammatory effect in this
model, suggesting that FAAH does not participate in the regulation
of PEA during inflammation. The effects of compound URB783 are
likely to occur through activation of PPAR-.alpha. by endogenous
PEA, as these effects were absent in PPAR-.alpha. null mice (FIG.
6C) and were not reproduced by compound URB818, a chiral analog of
URB783 that does not inhibit NAAA (data not shown).
[0061] More specifically, panels (A,C) illustrate inflammatory cell
counts while panel (B) shows PEA levels in sponges removed from
mice following 3 days of surgical implantation under the dorsal
skin of (A-B) Swiss mice or (C) wildtype C57BL6 mice (+/+) or
PPAR-.alpha.-knockout mice (-/-). Polyethylene sponges (1 cm.sup.3)
were injected with either vehicle (100 .mu.l of water:DMSO (9:1),
V, open bars), carrageenan (1%, filled bars), URB597 (30 .mu.g),
and URB783 (30 .mu.g) as indicated. ** P<0.01 or *** P<0.001
vs. V, ## P<0.01 vs. carrageenan control. ANOVA, followed by
Tukey's or Dunnett's post-hoc as appropriate (n=5-7).
NAAA Assay
[0062] Recombinant NAAA or native rat lung NAAA was incubated at
37.degree. C. for 30 min in 0.2 ml of sodium hydrogen phosphate
buffer (50 mM, pH 5.0) containing 0.1% Triton X-100, 3 mM
dithiothreitol (DTT) and 50 mM heptadecenoylethanolamide as
substrate. The reaction was terminated by the addition of 0.2 ml
cold methanol containing 1 nmol of heptadecanoic acid (HDA, NuChek
Prep, Elysian, Minn.). Samples were analyzed by LC/MS (liquid
chromatography/mass spectrometry). Heptadecanoic acid was eluted on
an XDB Eclipse C18 column isocratically at 2.2 ml/min for 1 min
with a solvent mixture of 95% methanol and 5% water, both
containing 0.25% acetic acid and 5 mM ammonium acetate. The column
temperature was 50.degree. C. ESI was in the negative mode,
capillary voltage was 4 kV, and fragmentor voltage was 100 V. N2
was used as drying gas at a flow rate of 13 liters/min and a
temperature of 350.degree. C. Nebulizer pressure was set at 60 psi.
[M-H]-- was monitored in SIM mode using heptadecanoic acid as
internal standard. Calibration curves were generated using
commercial heptadecenoic acid (Nu-Chek Prep, m/z=267).
Spinal Cord Injury
[0063] In the spinal cord injury (SCI) model, extradural
compression of a section of the spinal cord exposed via a
four-level T5-T8 laminectomy, caused a substantial increase in iNOS
expression in the inflammatory cells as well as in nuclei of
Schwann cells in the white and gray matter of the spinal cord
tissues collected from mice 24 hours after SCI. No iNOS staining
was detected in the spinal cord obtained from sham mice.
Administration of the NAAA inhibitor URB783 after SCI led to a
significant reduction in the expression of iNOS as well as other
markers of inflammation and cell apoptosis induced by this model,
including protease-activated receptor (PAR), nitrotyrosine,
Fas-ligand, Bax, Bcl-2 and Terminal
Deoxynucleotidyltransferase-Mediated UTP End Labeling (TUNEL). A
typical experimental protocol is described below.
[0064] Mice were randomized into 4 groups (n=40 animals/group).
Sham animals were subjected to the surgical procedure except that
the aneurysm clip was not applied and treated locally at the spinal
cord T5-T8 level with vehicle (saline) or 3 (30 mg/mouse) 1 h and 6
h after surgical procedure. The remaining mice were subjected to
SCI (as described below) and treated locally at the spinal cord
T5-T8 level with vehicle (saline) or 3 (30 .mu.g/mouse) 1 h and 6 h
after SCI. The mice from each group were sacrificed at 24 h after
SCI in order to collect samples for the evaluation of the
parameters as described below.
[0065] Mice were anaesthetized using chloral hydrate (400 mg/kg
body weight). We used the clip compression model described by
Rivlin and Tator (Rivlin and Tator, 1978) and produced SCI by
extradural compression of a section of the SC exposed via a
four-level T5-T8 laminectomy, in which the prominent spinous
process of T5 was used as a surgical guide. A six-level laminectomy
was chosen to expedite timely harvest and to obtain enough SC
tissue for biochemical examination. With the aneurysm clip
applicator oriented in the bilateral direction, an aneurysm clip
with a closing force of 24 g was applied extradurally at T5-T8
level. The clip was then rapidly released with the clip applicator,
which caused SC compression. In the injured groups, the cord was
compressed for 1 min. Following surgery, 1.0 ml of saline was
administered subcutaneously in order to replace the blood volume
lost during the surgery. During recovery from anesthesia, the mice
were placed on a warm heating pad and covered with a warm towel.
The mice were singly housed in a temperature-controlled room at
27.degree. C. for a survival period of 10 days. Food and water were
provided to the mice ad libitum. During this time period, the
animals' bladders were manually voided twice a day until the mice
were able to regain normal bladder function. Sham injured animals
were only subjected to laminectomy.
[0066] Immunohistochemical localization of PAR, nitrotyrosine,
FAS-ligand, Bax, Bcl-2 and iNOS. Twenty-four hours after SCI,
nitrotyrosine, a specific marker of nitrosative stress, was
measured by immunohistochemical analysis in the spinal cord
sections to determine the localization of "peroxynitrite formation"
and/or other nitrogen derivatives produced during SCI. At the 24 h
after SCI, the tissues were fixed in 10% (w/v) PBS-buffered
formaldehyde and 8 mm sections were prepared from paraffin embedded
tissues. After deparaffinization, endogenous peroxidase was
quenched with 0.3% (v/v) hydrogen peroxide in 60% (v/v) methanol
for 30 min. The sections were permeabilized with 0.1% (w/v) Triton
X-100 in PBS for 20 min. Non-specific adsorption was minimized by
incubating the section in 2% (v/v) normal goat serum in PBS for 20
min. Endogenous biotin or avidin binding sites were blocked by
sequential incubation for 15 min with biotin and avidin (DBA),
respectively. Sections were incubated overnight with
anti-PAR(Alexis; 1:500 in PBS, v/v), anti-iNOS antibody (1:500 in
PBS, v/v), anti-nitrotyrosine rabbit polyclonal antibody (Upstate,
1:500 in PBS, v/v), with anti-FAS-ligand antibody (Abcam, 1:500 in
PBS, v/v), anti-Bax antibody (Santa Cruz Biotechnology, 1:500 in
PBS, v/v) or with anti-Bcl-2 polyclonal antibody (Santa Cruz
Biotechnology, 1:500 in PBS, v/v). Sections were washed with PBS,
and incubated with secondary antibody. Specific labeling was
detected with a biotin-conjugated goat anti-rabbit IgG and
avidin-biotin peroxidase complex (DBA). To verify the binding
specificity for nitrotyrosine, PAR, iNOS, Bax, and Bcl-2, some
sections were also incubated with only the primary antibody (no
secondary) or with only the secondary antibody (no primary). In
these situations no positive staining was found in the sections
indicating that the immunoreactions were positive in all the
experiments carried out.
[0067] Terminal Deoxynucleotidyltransferase-Mediated UTP End
Labeling (TUNEL) Assay was conducted by using a TUNEL detection kit
according to the manufacturer's instruction (Apotag, HRP kit DBA,
Milan, Italy). Briefly, sections were incubated with 15 mcg/ml
proteinase K for 15 min at room temperature and then washed with
PBS. Endogenous peroxidase was inactivated by 3% H2O2 for 5 min at
room temperature and then washed with PBS. Sections were immersed
in terminal deoxynucleotidyltransferase (TdT) buffer containing
deoxynucleotidyl transferase and biotinylated dUTP in TdT buffer,
incubated in a humid atmosphere at 37.degree. C. for 90 min, and
then washed with PBS. The sections were incubated at room
temperature for 30 min with anti-horseradish peroxidase-conjugated
antibody, and the signals were visualized with diaminobenzidine.
The number of TUNEL positive cells/high-power field was counted in
5 to 10 fields for each coded slide.
[0068] Light microscopy. Spinal cord tissues were taken at 24 h
following trauma. Tissue segments containing the lesion (1 cm on
each side of the lesion) were paraffin embedded and cut into
5-.mu.m-thick sections. Tissue sections (thickness 5 .mu.m) were
deparaffinized with xylene, stained with Haematoxylin/Eosin
(H&E), with methyl green pyronin staining (used to
simultaneously DNA and RNA) and studied using light microscopy
(Dialux 22 Leitz).
[0069] The segments of each spinal cord were evaluated by an
experienced histopathologist. Damaged neurons were counted and the
histopathologic changes of the gray matter were scored on a 6-point
scale (Sirin et al., 2002): 0, no lesion observed, 1, gray matter
contained 1 to 5 eosinophilic neurons; 2, gray matter contained 5
to 10 eosinophilic neurons; 3, gray matter contained more than 10
eosinophilic neurons; 4, small infarction (less than one third of
the gray matter area); 5, moderate infarction; (one third to one
half of the gray matter area); 6, large infarction (more than half
of the gray matter area). The scores from all the sections from
each spinal cord were averaged to give a final score for individual
mice. All the histological studies were performed in a blinded
fashion. FIG. 7 depicts exemplary results from the experiments
described above.
[0070] Thus, specific embodiments and applications of inhibiting
N-acylethanolamine-hydrolyzing acid amidase have been disclosed. It
should be apparent, however, to those skilled in the art that many
more modifications besides those already described are possible
without departing from the inventive concepts herein. The inventive
subject matter, therefore, is not to be restricted except in the
spirit of the appended claims. Moreover, in interpreting both the
specification and the claims, all terms should be interpreted in
the broadest possible manner consistent with the context. In
particular, the terms "comprises" and "comprising" should be
interpreted as referring to elements, components, or steps in a
non-exclusive manner, indicating that the referenced elements,
components, or steps may be present, or utilized, or combined with
other elements, components, or steps that are not expressly
referenced. All extrinsic materials discussed herein are
incorporated by reference in their entirety. Where a definition or
use of a term in an incorporated reference is inconsistent or
contrary to the definition of that term provided herein, the
definition of that term provided herein applies and the definition
of that term in the reference does not apply.
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