CONJUGATES OF POLYUNSATURATED FATTY ACIDS AND AMINE-CONTAINING
COMPOUNDS AND USES THEREOF
FIELD AND BACKGROUND OF THE INVENTION
The present invention, in some embodiments thereof, relates to chemical conjugates
and more particularly, to conjugates of a fatty acid and a therapeutically active agent, which
can be used as COX-2 inhibitors and optionally also as 5-LOX inhibitors, for treating
inflammation.
Inflammation is a self-defensive reaction aimed at eliminating or neutralizing injurious
stimuli, and restoring tissue integrity. Inflammation is mediated by hormone-like compounds
called prostaglandins, which cause inflammation and pain. Cyclooxygenases (COXs) are
enzymes responsible for forming prostaglandins in the body. Non-steroidal anti-inflammatory
drugs (NSAIDs), which inhibit COX, are effective at reducing inflammation, but cause
unwanted side effects.
There are two cyclooxygenase (COX) enzymes at work in the body, COX-1 and COX-
2. The COX-1 enzyme is expressed in most tissues, and produced widely throughout the
body, and is necessary for a variety of important internal housekeeping functions, such as
protecting the stomach lining, maintaining renovascular function and platelet aggregation, and
is involved in the regulation of day-to-day cellular and metabolic activities such as
maintaining stomach lining integrity, regulating blood flow within the kidneys and balancing
platelet function. In contrast, COX-2 enzyme is an "inducible" isoform, expressed in response
to a variety of pro-inflammatory stimuli and found in the brain, male and female reproductive
organs, kidneys and in bone-forming cells called osteoblasts. Unlike COX-1, COX-2
expression is usually minimal, but when activated, COX-2 regulates prostaglandin production
primarily within inflammatory cells. This inflammatory response is a vital part of healing and
repairing.
Although NSAIDs are effective, they inhibit both COX-2 and COX-1. This is
problematic because COX-1 inhibition interferes with important functions such as the repair
and maintenance of stomach lining, and may therefore result in varying degrees of gastric
ulcerations, perforations or obstructions in one-third to almost one-half of patients
administered with COX-1 inhibiting NSAIDs. Hence, there has been considerable interest in
the development of selective COX-2 inhibitors, such as the COX-2 inhibitors celecoxib and
rofecoxib.
Omega-3 fatty acids, such as those found in fish oils, have been recommended for
managing chronic inflammatory conditions given their ability to alter prostaglandin production
and to yield measurable changes in certain disease parameters in rheumatoid arthritis patients.
Clare Curtis and colleagues have reported that omega-3 fatty acids (and not other fatty acids)
dose-dependently inhibited production of COX-2 expression without affecting COX-1
expression in an in vitro model, and inhibited degradation of aggrecan, a hallmark process of
arthritic conditions.
Flavonoids are a class of plant-derived chemicals that have been investigated for anti¬
inflammatory effects. Five flavonoids, genistein, kaempferol, quercetin, resorcinol and
resveratrol have been reported to produce dose-dependent decreases in TGF-a-induced COX-2
activity, with quercetin being the most potent [Mutoh et al. Jpn J Cancer Res. 2000 Jul;
91(7):686-91].
Resveratrol has also been reported by researchers from Cornell Medical College to
inhibit COX-1 and COX-2 activity in mammary and oral epithelial cells [Subbaramaiah et al.,
J Biol Chem 1998, 273:21875-21882; Zewczuk et al. J Biol Chem. 2004 May 21;279
(21):22727-37]. Another study has reported that resveratrol inhibits COX-2 expression in
mouse macrophages without affecting COX-1 protein expression [Martinez & Moreno,
Biochem Pharmacol 2000, 59:865-870]. However, an additional study found no effect of
resveratrol on COX-2 induction in mouse skin [Jang & Pezzuto, Cancer Lett 1998, 134:81-
89].
Inflammation occurs in pathologically vulnerable regions of the Alzheimer's disease
(AD) brain. In the AD brain, damaged neurons and neurites and highly insoluble amyloid b
peptide deposits and neurofibrillary tangles provide potential stimuli for inflammation. Thus,
animal models and clinical studies, although still in their infancy, suggest that inflammation in
the AD brain significantly contributes to AD pathogenesis.
In Parkinson's disease, postmortem examination reveals a loss of dopaminergic
neurons in the substantia nigra associated with a massive astrogliosis and the presence of
activated microglial cells. Recent evidence suggests that the disease may progress even when
the initial cause of neuronal degeneration has disappeared, suggesting that toxic substances
released by the glial cells may be involved in the propagation and perpetuation of neuronal
degeneration [Hirsch et al., Ann N Y Acad Sci. 2003 Jun;991 : 14-28]. Glial cells can release
deleterious compounds such as proinflammatory cytokines (TNF-a, II-1b, IFN-g), which may
act by stimulating nitric oxide production in glial cells, or which may exert a more direct
deleterious effect on dopaminergic neurons by activating receptors that contain
intracytoplasmic death domains involved in apoptosis. The anti-inflammatory drugs
pioglitazone, a PPAR-g agonist, and the tetracycline derivative minocycline, have been shown
to reduce glial activation and protect the substantia nigra in an animal model of the disease
degeneration has disappeared, suggesting that toxic substances released by the glial cells may
be involved in the propagation and perpetuation of neuronal degeneration [Breidert et al. Proc.
Natl. Acad. Sci. USA 2002, 98: 14669-14674].
Inflammation is an essential part of the functioning of a normal lung. Tiny areas of
inflammation, typically via IgE antibodies, occur thousands of times a day in order to combat
the viruses, bacteria and pollutants to which lungs are exposed. Normally, none of this
activity produces any obvious symptoms. However, asthmatics react excessively to some
factors, leading to aggravated inflammation throughout the small and medium airways. It is
thought that asthmatics over-produce unique IgE antibodies in response to these factors.
International Patent Application PCT/IL2007/001592 (published as WO 08/075366)
describes conjugates of fatty acids with amines and uses thereof in inhibiting cyclooxygenase
enzymes and treating inflammations.
U.S. Patent No. 4,933,324 discloses a prodrug comprising a fatty acid carrier such as
4,7, 10,13, 16,19-docosahexa-enoic acid covalently bound to a neuroactive drug such as
dopamine.
U.S. Patent No. 5,300,665 discloses a process for preparing fatty acid esters of
hydroxyalkylsulfonates and fatty acid amides of aminoalkylsulfonates.
Additional background art includes U.S. Patent No. 4,218,404, U.S. Patent No.
4,443,475, U.S. Patent No. 7,034,058, and International Patent Application
PCT/IN2009/000382 (published as WO 2010/004579).
Recently, a therapeutic role of dual inhibitors of COX and 5-LOX has been suggested.
For a detailed discussion in this regard see, Martel-Pelletier et al., Ann Rheum Dis 2003 62:
501-509. While both the conventional NSAIDs and the selective COX-2 inhibitors primarily
exert their activity by reducing the production of PGs induced in the inflammatory process, in
recent years, it has been clarified that PG synthesis is only one part of the arachidonic acid
pathway, this precursor being a substrate that gives rise to many other lipid mediators, such as
the LTs and the LXs.
Leucotrienes themselves have a major role in the development and persistence of the
inflammatory process, and it is now clear that PGs and LTs have complementary effects,
whereas the production of LXs can counteract the inflammatory actions of LTs. In view of
these concepts, it has been suggested that blocking both LT and PG production might have
synergistic effects and achieve optimal anti-inflammatory activity. In addition, taking into
account the roles of LTB4 and cysteinyl LTs (against which neither selective nor non¬
selective NSAIDs are effective, in the inflammatory process, dual inhibition of the COX and
5-LOX pathways could produce a wider spectrum of anti-inflammatory effects. Dual
inhibition of COX and 5-LOX may limit the vascular changes seen during inflammation and
leucocyte induced GI damage.
SUMMARY OF THE INVENTION
The invention provides some structural and functional features of chemical conjugates
of a therapeutically active agent and a hydrophobic moiety, which impart to the conjugates an
efficient and selective COX-2 inhibitory activity, and optionally also a 5-LOX inhibition
activity. Some of the currently disclosed conjugates employ known anti-inflammatory drugs
conjugates to hydrophobic moieties such as unsaturated fatty acids, while some employ
medical food agents.
According to an aspect of some embodiments of the present invention there is
provided a chemical conjugate comprising a first moiety and a second moiety covalently
linked therebetween, wherein the second moiety is derived from docosa-4,7,10,13,16,19-
hexaenoic acid, and wherein the first moiety is derived from a therapeutically active agent or
a derivative thereof, each independently having a functional group for forming a covalent
bond with the second moiety, with the proviso that the first moiety is not hydroxyproline, the
chemical conjugate being a cyclooxygenase-2 (COX-2) inhibitor.
According to some embodiments of the invention, the chemical conjugate is further
capable of inhibiting 5-lipoxygenase (5-LOX) inhibitor.
According to some embodiments of the invention, the functional group is selected from
the group consisting of hydroxy, amine, carboxy and amide.
According to some embodiments of the invention, the first moiety and the second
moiety are covalently bound via a bond selected from the group consisting of an amide bond
and an ester bond.
According to some embodiments of the invention, the therapeutically active agent is
an anti-inflammatory agent.
According to some embodiments of the invention, the therapeutically active agent is a
cyclooxygenase (COX) inhibitor.
According to some embodiments of the invention, the therapeutically active agent is a
non-steroidal anti-inflammatory drug (NTHE).
According to some embodiments of the invention, the therapeutically active agent is
selected from the group consisting of: 5-hydroxy-indol-3-yl-acetic acid, 2-amino-nicotinic
acid, salicyclic acid, mesalazine and quercetin.
According to an aspect of some embodiments of the present invention, there is
provided a chemical conjugate comprising a first moiety and a second moiety covalently
linked therebetween, wherein the second moiety is derived from g-linolenic acid, and wherein
the first moiety is derived from a therapeutically active agent or a derivative thereof, each
independently having a functional group for forming a covalent bond with the second moiety,
with the proviso that the first moiety is not hydroxyproline or taurine, the chemical conjugate
being a cyclooxygenase-2 (COX-2) inhibitor.
According to some embodiments of the invention, the chemical conjugate is further
capable of inhibiting 5-lipoxygenase (5-LOX).
According to some embodiments of the invention, the functional group is selected from
the group consisting of hydroxy, amine, carboxy and amide.
According to some embodiments of the invention, the first moiety and the second
moiety are covalently bound via a bond selected from the group consisting of an amide bond
and an ester bond.
According to some embodiments of the invention, the therapeutically active agent is
an anti-inflammatory agent.
According to some embodiments of the invention, the therapeutically active agent is a
cyclooxygenase (COX) inhibitor.
According to some embodiments of the invention, the therapeutically active agent is a
non-steroidal anti-inflammatory drug (NTHE).
According to some embodiments of the invention, the therapeutically active agent is
selected from the group consisting of salicyclic acid and mesalazine.
According to an aspect of some embodiments of the present invention, there is
provided a chemical conjugate comprising a first moiety and a second moiety covalently
linked therebetween, wherein the second moiety is derived from a fatty acid, and wherein the
first moiety is derived from a food-grade or a derivative thereof each independently having a
functional group for forming a covalent bond with the second moiety.
According to some embodiments of the invention, the chemical conjugate is further a
5-lipoxygenase (5-LOX) inhibitor.
According to some embodiments of the invention, the functional group is selected from
the group consisting of hydroxy, amine, carboxy and amide.
According to some embodiments of the invention, the first moiety and the second
moiety are covalently bound via a bond selected from the group consisting of an amide bond
and an ester bond.
According to some embodiments of the invention, the fatty acid is docosa-
4,7,10,13,16,1 9-hexaenoid acid.
According to some embodiments of the invention, the fatty acid is g-linolenic acid.
According to some embodiments of the invention, the food additive is selected from
the group consisting of quercetin, curcumin and resveratrol
According to an aspect of some embodiments of the present invention there is provided
a chemical conjugate comprising a first moiety and a second moiety covalently linked
therebetween, wherein the first moiety is derived from a compound selected from the group
consisting of 5-hydroxy-indol-3-yl-acetic acid, 2-amino-nicotinic acid, salicyclic acid,
mesalazine, quercetin and resveratrol, and wherein the second moiety is derived from a fatty
acid.
According to some embodiments of the invention, the first moiety and the second
moiety are covalently linked therebetween via a bond selected from the group consisting of an
ester bond and an amide bond.
According to some embodiments of the invention, the fatty acid is docosa-
4,7,10,13,16,1 9-hexaenoic acid.
According to some embodiments of the invention, the fatty acid is g-linolenic acid.
According to some embodiments of the invention, the first moiety is derived from a
compound selected from the group consisting of salicyclic acid and mesalazine.
According to an aspect of some embodiments of the present invention there is
provided a chemical conjugate selected from the group consisting of:
N-(docosa-4,7,l 0,13,1 6,19-hexaenoyl)-5-hydroxy-indol-3-yl-aceticacid (MWL004);
N-(docosa-4,7, 10,13,16,19-hexaenoyl)-2-amino-nicotinic acid (MWL005);
N-(docosa-4,7, 10,13,16,19-hexaenoyl)-2-amino-phenyl-acetic acid (MWL006);
N-oleoyl-5-hydroxy-indol-3-yl-acetic acid (MWL007);
N-oleoyl-2-amino-nicotinic acid (MWL008);
O-oleoyl-salicylic acid (MWL009);
O-(docosa-4,7,10,13,16,19-hexaenoyl)-salicylic acid (MWL013);
O-(y-linolenoyl)-salicylic acid (MWL014);
N-(y-linolenoyl)-5-amino-salicylic acid (MWL015);
N-(docosa-4,7, 0,13,16,19-hexaenoyl)-5-amino-salicylic acid (MWL016);
N-oleoyl-5-amino-salicylic acid (MWL017); and
N-docosa-4,7, 0,13,16,19-hexaenoyl-taurine (MWL002).
According to an aspect of some embodiments of the present invention there is
provided a N-docosa-4,7, 10,13,16,19-hexaenoyl-taurine.
According to an aspect of some embodiments of the present invention there is
provided a pharmaceutical composition comprising the chemical conjugate as described
herein and a pharmaceutically acceptable carrier.
According to some embodiments of the invention, the pharmaceutical composition is
packaged in a packaging material and identified in print, in or on the packaging material, for
use in the treatment of an inflammatory disease or disorder.
According to an aspect of some embodiments of the present invention there is
provided a chemical conjugate as described herein, for use in the treatment of an
inflammatory disease or disorder.
According to an aspect of some embodiments of the present invention there is
provided a use of the chemical conjugate as described herein in the manufacture of a
medicament for the treatment of an inflammatory disease or disorder.
According to an aspect of some embodiments of the present invention there is
provided a method of treating an inflammatory disease or disorder, the method comprising
administering to a subject in need thereof an effective amount of the chemical conjugate as
described herein.
According to some embodiments of the invention, the inflammatory disease or
disorder is selected from the group consisting of Alzheimer's disease, Parkinson's disease,
asthma, osteoarthritis, dermatitis, rheumatoid arthritis, pain associated with inflammation,
primary dysmenorrhea, Crohn's disease and ulcerative colitis.
According to some embodiments of the invention, the conjugate inhibits COX-2
activity.
According to some embodiments of the invention, the conjugate further inhibits 5-
LOX activity.
According to some embodiments of the invention, the conjugate does not inhibit
COX-1 activity.
According to some embodiments of the invention, by conjugating DHA with specific
amino acids, the compound's inhibition efficacy is significantly improved. The conjugates
were built and synthesized to inhibit COX-2 in selective and reversible inhibition, with
inhibition lasting only for a very short period. This unique mechanism of action exerts enough
selective inhibition of COX-2 while maintaining an optimal COX-l/COX-2 ratio so that the
body has the necessary levels of COX-2 enzyme to generate proper amounts of AA
metabolites to maintain normal body functions.
According to some embodiments of the invention, the conjugate selectively inhibits
COX-2 while maintaining an optimal balance of COX/LOX enzymes to maintain normal
body functions.
Unless otherwise defined, all technical and/or scientific terms used herein have the
same meaning as commonly understood by one of ordinary skill in the art to which the
invention pertains. Although methods and materials similar or equivalent to those described
herein can be used in the practice or testing of embodiments of the invention, exemplary
methods and/or materials are described below. In case of conflict, the patent specification,
including definitions, will control. In addition, the materials, methods and examples are
illustrative only and are not intended to be necessarily limiting.
BRIEF DESCRIPTION OF THE DRAWINGS
Some embodiments of the invention are herein described, by way of example only,
with reference to the accompanying drawings. With specific reference now to the drawings in
detail, it is stressed that the particulars shown are by way of example and for purposes of
illustrative discussion of embodiments of the invention. In this regard, the description taken
with the drawings makes apparent to those skilled in the art how embodiments of the invention
may be practiced.
In the drawings:
FIG. 1 is a graph showing the average COX-2 inhibition molecular activity index
(MAI) of COX-2 of fatty acid amide derivatives formed by attaching exemplary aminecontaining
compounds to oleic acid (1), linoleic acid (2), a-linolenic acid (3), arachidonic acid
(4), eicosa-5,8,l l,14,17-pentaenoic acid (5) and docosa-4,7,10,13,16,19-hexaenoic acid (6).
FIGs. 2A-D present results from a representative oocyte expressing hERG channels
before and after injection with MWL002 (15 m M). FIG. 2A presents currents before
injection. FIG. 2B presents currents 5 minutes after injection. FIG. 2C presents voltage
activation curves for the oocyte measured in FIGs. 2A and 2B. Presented are normalized
currents at -130 mV that were initiated by the indicated voltage. FIG. 2D presents currents
during 6-minute-long measurements prior and post injection of MLW002, as indicated by the
gray bar. Currents were initiated by a 150-ms-long pulse to +40 and measured at -130 mV,
with 15 seconds interpulse intervals.
FIG. 3 is a bar graph presenting average changes in expressed hERG currents
following internal and external exposure to MWL002, and demonstrating no sensitivity of the
channels to the tested conjugates.
FIG. 4 presents comparative plots showing the effect of MWL-001 and of ibuprofen
on paw swelling volume in paw edema in vivo studies.
FIGs. 5A-B present the effect of MWL-001 and of 5-ASA on body weight (FIG. 5A)
and on MPO activity (FIG. 5B) in UC-diseased rats.
FIGs. 6A-B present images of untreated (FIG. 6A) and of 5-ASA-treated (FIG. 6B,
right) and MWL-001 -treated (FIG. 6B, left) ulcerated colon segment.
FIGs. 7A-B are bar graphs showing the effect of MWL-001 on the PGE2 production
(FIG. 7B) and the levels of TNFa (FIG. 7B) in CIA mice.
FIGs. 8A-B present comparative plots showing the arthritis score (FIG. 8A) and paw
thickness (FIG. 8B) of ibuprofen-treated and MWL-002-treated CIA mice.
FIG. 9 presents the pharmacokinetic profile of MWL001 and DHA (it's metabolite)
after oral administration in rats.
FIG. 10 presents the effect of MWL001 administered bolus I.V. on the rat QT and
QTc intervals.
FIG. 11 presents the effect of quinidine (QND) on rat QT, QTc and heart rate.
FIG. 12 presents the TNF-a colon level of mice at four days after the administration of
DNBS.
FIG. 13 presents the IL-6 colon level of mice at four days after the administration of
DNBS.
FIG. 14 shows the representative immunolocalization of TGF-b expression in the
colon tissues of mice on day four after the administration of DNBS.
FIG. 15 shows the representative immunolocalization of CD25 expression in the colon
tissues of mice on day four after the administration of DNBS.
FIG. 16 shows the representative immunolocalization of CD4 expression in the colon
tissues of mice on day four after the administration of DNBS.
FIG. 17 presents effect of MWL001 and dexamathasone (DEX) on ear thickness of
mice at eighteen hours after sensitization.
FIG. 18 presents the ear weight of mice at eighteen hours after sensitization.
FIGs. 19 A-D representative hematoxylin/eosin-stained sections of mice ear tissues;
A-control B-D sensitized with Oxazolone; B-with vehicle; C with MWOOl and D with DEX.
DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION
The present invention, in some embodiments thereof, relates to chemical conjugates
and more particularly, to conjugates of a fatty acid and a therapeutically active agent, which
can be used as COX-2 inhibitors for treating inflammation.
Before explaining at least one embodiment of the invention in detail, it is to be
understood that the invention is not necessarily limited in its application to the details set forth
in the following description or exemplified by the Examples. The invention is capable of other
embodiments or of being practiced or carried out in various ways.
As discussed hereinabove, currently sought agents for treating inflammation are
selective inhibitors of COX-2, which desirably do not inhibit COX-1. Dual inhibitors of COX
and 5-LOX are also sought for obtaining a wider scope of anti-inflammatory activity.
The there-dimensional (3D) structure of the two enzymes, COX-1 and COX-2,
determined by X-ray diffraction, shows that while the active site in both enzymes consists of a
long narrow hydrophobic channel extending from the membrane-binding domain (the lobby)
to the core of the catalytic domain, yet the COX 2 active site is about 20 % larger and has a
slightly different form as compared with that of COX-1.
These size and shape differences are caused mainly by two changes in the amino acid
sequences of the isoenzymes. In one case, Ile-523 in COX 1 is replaced by a valine in COX-
2, a change which opens up a small hydrophilic side pocket off the main channel; appreciably
increasing the longer side chain of Ile-523. In addition, Ile-434 in COX-1 is also replaced by
valine in COX-2, allowing a neighboring residue Phe-5 18 to swing out of the way, increasing
further access to the side cavity. In another case, His-513 in COX-1, which can interact with
polar moieties, is replaced by Arg in COX-2, thus changing the interaction of the side pocket
with its chemical environment. The hydrophilic side pocket of the COX 2 active site is
defined by residues Tyr-355, Val-523, His-90, Gin- 192 and Arg-513.
The differences between COX-1 and COX-2 are further discussed in, for example,
Dannhardt and, Kiefer, Eur J Med Chem. 2001 Feb;36(2): 109-26.
Currently, more than 500 COX-2-specific inhibitors have been designed. The main
structural features of these compounds are the absence of the carboxylate group, characteristic
of classical NSAIDs, and generally, the presence of a sulfonate (S0 2) or sulfonamide (SO-
2NH2) moiety, which can interact with Arg-513 in the hydrophilic side pocket of the COX 2
active site. Although the majority of these compounds were discovered before the structure of
COX-2 was dissolved, crystallographic data can now be used to rationally design selective
inhibitors.
The present inventor has previously disclosed, in WO 08/075366, conjugates of
various fatty acids and amine-containing compounds, and uses thereof in the treatment of
inflammation.
Triggered by the knowledge obtained thus far on the active site of COX-2 and its
structural differences from the active site of COX-1, the present inventor has now used in
silico analyses in order to better define the structural features required for fatty acid-containing
conjugates to selectively inhibit COX-2 and thereby serve as efficient anti-inflammatory
agents.
The present inventors have focused on therapeutically active agents, mostly such
agents that are naturally-occurring agents, as defined herein, and/or are determined as foodgrade
or Generally Recognized As Safe (GRAS) substances and even edible substance, as
these defined herein, and have tested in silico the binding of conjugates of these agents with
various fatty acids to COX-2. The chemical structures of exemplary substances are presented
in Table 1 below. The fatty acids used in these studies were selected as exhibiting
pharmacological benefits on their own, being of the family of Omega-3 fatty acids.
Table 1

20
2 1

As described in detail in the Examples section that follows, the present inventors have
uncovered that the degree of unsaturation in the fatty acid moiety has a substantial effect on
the COX-2 binding of the studied conjugates, with conjugates comprised of a fatty acid having
six double bonds exhibiting the best scores.
The pharmacological properties and therapeutic activity of exemplary compounds
according to embodiments of the invention can be established in in vitro and in vivo studies, as
further detailed hereinafter.
Embodiments of the present invention therefore generally relate to conjugates of a
therapeutically active agent and a fatty acid.
More specifically, embodiments of the present invention relate to conjugates of a
therapeutically active agent and a highly unsaturated fatty acid (e.g., DHA or linolenic acid),
which are shown herein to have a superior therapeutic activity as compared to conjugates
containing other fatty acids (e.g., mono-unsaturated or di-unsaturated fatty acids, having one
or two double bonds, respectively). Further embodiments of the invention relate to conjugates
of a therapeutically active agent and a fatty acid, both of which are derived from naturallyoccurring
substances, and hence can be categorized as food-grade or GRAS substances.
The chemical conjugates described herein are advantageously prepared by a one-step
chemical synthesis, via a single bond conjugation. The chemical conjugates described herein
exhibit high selectivity towards COX-2 inhibition, with IC 0 values being 200 to 500 folds
lower than COX-1. Some of the tested conjugates advantageously exhibit a dual COX and 5-
LOX inhibition activity.
According to one aspect of embodiments of the present invention there is provided a
chemical conjugate comprising a first moiety and a second moiety covalently linked
therebetween.
According to some embodiments of the present invention, the first moiety is derived
from a therapeutically active agent or a derivative thereof, and the second moiety is derived
from a fatty acid.
According to some embodiments of the present invention, the first moiety is an active
agent or a derivative thereof, or a food additive or a derivative thereof and the second moiety
is derived from a fatty acid.
By "derived from" it is meant that the moiety in the chemical conjugate is the portion
of the substance forming the conjugate which remains upon the conjugation reaction with the
other substance forming the conjugate. The phrase "derived from" further encompasses a
portion of therapeutically active agent which possess most but not all of the structural features
of the therapeutically active agent. For example, (5-hydroxy- lH-indol-3-yl)-acetic acid is a
moiety derived from indomethacin.
As used herein throughout, the phrase "therapeutically active agent" relates to an agent
that exhibits a beneficial pharmacological effect when administered to a subject. Exemplary
therapeutically active agents include, but are not limited to, agents that exhibit anti¬
inflammatory activity, agents that exhibit anti-proliferative activity, anti-oxidants, antithrombogenic
agents, anti-platelet agents, anti-coagulants, antimicrobial agents, analgesics,
and vasoactive agents, as well as metabolites and biological substances such as, but not
limited to, amino acids, nicotinic acid, and the like.
A derivative of a therapeutically active agent includes a substance, which has
essentially the same structural features of the therapeutically active agent, yet is modified at
one or more positions so as to possess a desired functional group. For example, a derivative
of a therapeutically active agent can include an agent modified to include a hydroxy group, an
amine group, a carboxy group and/or an amide group.
In some embodiments, the therapeutically active agent and the fatty acid are
covalently linked therebetween via a bond formed between a functional group of the
therapeutically active agent and the carboxylic group of the fatty acid.
The active agents may be therapeutically active agents or food-grade or food additive
according to embodiments of the present invention therefore possess a functional group that
serves for conjugating these agents to the fatty acid.
Suitable functional groups include an amine group, which forms an amide bond upon
conjugation to a fatty acid, and a hydroxy group, which forms an ester bond upon conjugation
to a fatty acid.
The hydroxy and amine functional groups can be present per se or may form a part of
an amide or carboxy groups of the therapeutically active agent.
Accordingly, the therapeutically active agent and the fatty acid are linked
therebetween via an amide bond or an ester bond. Corresponding moieties include a
therapeutically active agent or a derivative thereof, possessing a -NR- group or a -O- group,
as the first moiety, and a fatty acid possessing a -C(=0)- group, as the second moiety.
However, other functional groups and bonds formed thereby are contemplated. These
include, but are not limited to, thiol, which forms upon conjugation with a fatty acid a
thioester; carbamate, thiocarboxy, phosphonyl, phosphinyl, phosphoryl, phosphoramide,
sulfate, sulfonate, sulfonamide, alkoxy, aryloxy, thioalkoxy, thioaryloxy, imine and halo.
It should be noted that the amine, hydroxy and thiol groups can be present in the
therapeutically active agent either per se or can form a part of a ring, e.g., a heteroalicyclic or
an heteroaromatic ring, or of a functional group such as amide, imine, ether, thioether,
carboxy, thiocarboxy, carbamate, thiocarbamate and the like, as these terms are defined
herein.
The functional group can be present in the therapeutically active agent or can be
generated therein, so as to form a derivative of the therapeutically active agent.
In some embodiments, the active agent is an amino acid. Any of the currently known
amino acids is contemplated, including the 2 1 naturally-occurring amino acids and non
naturally-occurring amino acids, and any derivatives thereof.
In some embodiments, the therapeutically active agent is an anti-inflammatory agent.
Exemplary anti-inflammatory agents include, but are not limited to, steroidal anti¬
inflammatory agents and non-steroidal anti-inflammatory agents.
In some embodiments, the therapeutically active agent is a non-steroidal anti¬
inflammatory agent.
Representative examples of non-steroidal anti-inflammatory agents include, without
limitation, aspirin, celecoxib, diclofenac, diflunisal, etodolac, fenoprofen, flurbiprofen,
ibuprofen, indomethacin, ketoprofen, ketorolac, meclofenamate, mefenamic acid,
nabumetone, naproxen, oxaprozin, oxyphenbutazone, phenylbutazone, piroxicam, rofecoxib,
sulindac and tolmetin.
More generally, examples of non-steroidal anti-inflammatory agents include, without
limitation, oxicams, such as piroxicam, isoxicam, tenoxicam, sudoxicam, and CP- 14,304;
salicylates, such as aspirin, disalcid, benorylate, trilisate, safapryn, solprin, diflunisal, and
fendosal; acetic acid derivatives, such as diclofenac, fenclofenac, indomethacin, sulindac,
tolmetin, isoxepac, furofenac, tiopinac, zidometacin, acematacin, fentiazac, zomepirac,
clindanac, oxepinac, felbinac, and ketorolac; fenamates, such as mefenamic, meclofenamic,
flufenamic, niflumic, and tolfenamic acids; propionic acid derivatives, such as ibuprofen,
naproxen, benoxaprofen, flurbiprofen, ketoprofen, fenoprofen, fenbufen, indopropfen,
pirprofen, carprofen, oxaprozin, pranoprofen, miroprofen, tioxaprofen, suprofen,
alminoprofen, and tiaprofenic; pyrazoles, such as phenylbutazone, oxyphenbutazone,
feprazone, azapropazone, and trimethazone. Mixtures of these non-steroidal anti¬
inflammatory agents may also be employed, as well as the dermatologically acceptable salts
and esters of these agents. For example, etofenamate, a flufenamic acid derivative, is
particularly useful for topical application.
Representative examples of steroidal anti-inflammatory drugs include, without
limitation, corticosteroids such as hydrocortisone, hydroxyltriamcinolone, alpha-methyl
dexamethasone, dexamethasone-phosphate, beclomethasone dipropionates, clobetasol
valerate, desonide, desoxymethasone, desoxycorticosterone acetate, dexamethasone,
dichlorisone, diflorasone diacetate, diflucortolone valerate, fluadrenolone, fluclorolone
acetonide, fludrocortisone, flumethasone pivalate, fluosinolone acetonide, fluocinonide,
flucortine butylesters, fluocortolone, fluprednidene (fluprednylidene) acetate,
flurandrenolone, halcinonide, hydrocortisone acetate, hydrocortisone butyrate,
methylprednisolone, triamcinolone acetonide, cortisone, cortodoxone, flucetonide,
fludrocortisone, difluorosone diacetate, fluradrenolone, fludrocortisone, diflurosone diacetate,
fluradrenolone acetonide, medrysone, amcinafel, amcinafide, betamethasone and the balance
of its esters, chloroprednisone, chlorprednisone acetate, clocortelone, clescinolone,
dichlorisone, diflurprednate, flucloronide, flunisolide, fluoromethalone, fluperolone,
fluprednisolone, hydrocortisone valerate, hydrocortisone cyclopentylpropionate,
hydrocortamate, meprednisone, paramethasone, prednisolone, prednisone, beclomethasone
dipropionate, triamcinolone, and mixtures thereof.
Exemplary active agents from which the first moiety in the chemical conjugates
described herein is derived include, but are not limited to, indomethacin, nicotinic acid
(including derivatives thereof such as 2-amino nicotinic acid, 2-amino benzoic acid, and 2-
aminophenyl acetic acid), salicyclic acid, mesalazine, quercetin, curcumin and resveratrol.
In some embodiments, the agent is a food-grade therapeutically active agent.
The phrase "food grade" or "food additive" is used herein to describe substances that
are generally safe for human consumption by virtue of being generally recognized as safe
(GRAS) or by passing standard safety tests, and thus qualify for use as food additives. This
phrase describes those substances that are known to exhibit a therapeutic effect, either as
nutritional supplements or as therapeutically active agents, as described herein.
The phrase "generally recognized as safe" or GRAS, as used herein, is meant in the
same manner which is defined, for example, under sections 201(s) and 409 of the U.S. FD&C
Act. The U.S. law states that any substance that intentionally contacts food or added to food
is a food additive, that is subject to premarket review and approval by FDA, unless the
substance is generally recognized, among qualified experts, as having been adequately shown
to be safe under the conditions of its intended use, or unless the use of the substance is
otherwise excluded from the definition of a food additive. GRAS substances are
distinguished from food additives by the type of information that supports the GRAS
determination, that it is publicly available and generally accepted by the scientific community,
but should be the same quantity and quality of information that would support the safety of a
food additive.
Since the qualification to a food additive (food-grade) or GRAS category can be
obtained through a process of applying, testing and qualifying to the requirements of the
various official food and drug authorities, the present embodiments are meant to encompass
all relevant substances and their derivatives which are to become food-grade and GRAS in the
future, as well as those which already qualify as food-grade and GRAS.
In some embodiments, the therapeutically active agent is a naturally-occurring
substance.
By "naturally-occurring" it is meant that the substance is found in natural plants or
animals. Naturally-occurring substances can be obtained by extracting the substance from the
plant or animal it is found in, or can be synthetically prepared.
The second moiety in the chemical conjugates described herein is derived from a fatty
acid.
As commonly used in the art, a fatty acid is comprised of a hydrocarbon chain which
terminates with a carboxylic acid group. The hydrocarbon chain can be unbranched and
saturated, branched and saturated, unbranched and unsaturated or branched and unsaturated.
In some embodiment, the fatty acid is an unsaturated fatty acid having one or more
unsaturated bonds (e.g., double bonds) in its hydrocarbon chain.
In some embodiments, the hydrocarbon chain is unbranched.
In some embodiments, the hydrocarbon chain comprises from 5 to 29 carbon atoms,
rendering the fatty acid being of 6 to 30 carbon atoms in length.
In some embodiments, the fatty acid is of 16 to 22 carbon atoms in length.
In some embodiments, the fatty acid has at least 3 double bonds in its hydrocarbon
chain. In some embodiments, the fatty acid has at least 4 double bonds in its hydrocarbon
chain. In some embodiments, the fatty acid has at least 5 double bonds in its hydrocarbon
chain. In some embodiments of the invention, the fatty acid has at least 6 double bonds in its
hydrocarbon chain. The configuration of the double bonds in the hydrocarbon chain, namely
cis or trans, can be the same or different.
In some embodiments, the unsaturated fatty acid is an all-cis unsaturated fatty acid.
In some embodiments, the fatty acid is an omega-3-fatty acid, as this term is widely
recognized in the art.
Exemplary fatty acids that are advantageously used in the context of embodiments of
the present invention include, but are not limited to, a//-cw-7,10,13-hexadecatrienoic acid, allc/
s-9,12,15-octadecatrienoic acid (a-Linolenic acid (ALA)), all-cis-6,9, \2,\5-
octadecatetraenoic acid (Stearidonic acid (SDA)), //- -l l,14,17-eicosatrienoic acid
(Eicosatrienoic acid (ETE)), all-cis-%,\ 1,14,17-eicosatetraenoic acid (Eicosatetraenoic acid
(ETA)), //- -5,8,l l,14,17-eicosapentaenoic acid (Eicosapentaenoic acid (EPA)), all-cis-
7,10,13,16,19-docosapentaenoic acid (Docosapentaenoic acid (DPA), Clupanodonic acid),
a//-cw-4,7,10,13,16,19-docosahexaenoic acid (Docosahexaenoic acid (DHA), all-cis-
9,12,15,18,21-tetracosapentaenoic acid (Tetracosapentaenoic acid) and all-cis-
6,9,12,15, 18,21-tetracosahexaenoic acid (Tetracosahexaenoic acid (Nisinic acid)).
In some embodiments, the fatty acid is a//-cw-4,7,10,13,16,19-docosahexaenoic acid
(Docosahexaenoic acid (DHA).
In some embodiments, the fatty acid is linolenic acid.
According to some embodiments of the present invention, the first moiety is derived
from a therapeutically active agent or a derivative thereof, as described herein, whereas
hydroxyproline is excluded and the second moiety is derived from docosa-4,7,10,13,16,19-
hexaenoic acid (DHA).
According to some embodiments of the present invention, the first moiety is derived
from a therapeutically active agent or a derivative thereof, whereas hydroxyproline or taurine
are excluded as described herein, and the second moiety is derived from linolenic acid.
According to some embodiments of the present invention, the first moiety is derived
from a food-grade therapeutically active agent, as defined herein, and the second moiety is
derived from a fatty acid, as defined herein.
According to some embodiments of the present invention, the first moiety is derived
from 5-hydroxy-indol-3-yl-acetic acid, 2-amino-nicotinic acid, salicyclic acid, mesalazine,
quercetin or resveratrol, or from any derivative thereof, and the second moiety is derived from
a fatty acid.
In some embodiments, the first moiety is derived from 5-hydroxy-indol-3-yl-acetic
acid (e.g., derived from indomethacin), including derivatives thereof as exemplified in Table 1
and in Example 1 that follows.
Exemplary compounds according to some embodiments of the present invention
include, but are not limited to:
N-(docosa-4,7, 10,13,16,1 9-hexaenoyl)-5 -hydroxy-indol-3 -yl-acetic acid (MWL004),
and derivatives thereof;
N-(docosa-4,7, 10,13,16,19-hexaenoyl)-2-amino-nicotinic acid (MWL005);
N-(docosa-4,7, 10,13,16,1 9-hexaenoyl)-2-amino-phenyl-acetic acid (MWL006);
N-oleoyl-5-hydroxy-indol-3 -yl-acetic acid (MWL007);
N-oleoyl-2-amino-nicotinic acid (MWL008);
O-oleoyl-salicylic acid (MWL009);
O-(docosa-4,7,10,13,16,19-hexaenoyl)-salicylic acid (MWL013);
O-(y-linolenoyl)-salicylic acid (MWL014);
N-(Y-linolenoyl)-5-amino-salicylic acid (MWL015);
N-(docosa-4,7, 10,13,16,1 9-hexaenoyl)-5-amino-salicylic acid ( L016);
N-oleoyl-5-amino-salicylic acid (MWL017); and
N-docosa-4,7, 10,13,16,19-hexaenoyl-taurine (MWL002).
The chemical conjugates described herein can be in a form of a pharmaceutically
acceptable salt, a prodrug, a solvate or a hydrate thereof.
The phrase "pharmaceutically acceptable salt" refers to a charged species of the parent
compound and its counter ion, which is typically used to modify the solubility characteristics
of the parent compound and/or to reduce any significant irritation to an organism by the
parent compound, while not abrogating the biological activity and properties of the
administered compound.
As used herein, the term "prodrug" refers to an agent, which is converted into the
active compound (the active parent drug) in vivo. Prodrugs are typically useful for facilitating
the administration of the parent drug. They may, for instance, be bioavailable by oral
administration whereas the parent drug is not. The prodrug may also have improved
solubility as compared with the parent drug in pharmaceutical compositions. Prodrugs are
also often used to achieve a sustained release of the active compound in vivo. An example,
without limitation, of a prodrug would be the chemical conjugate, having one or more
carboxylic acid moieties, which is administered as an ester (the "prodrug"). Such a prodrug is
hydrolysed in vivo, to thereby provide the free compound (the parent drug). The selected
ester may affect both the solubility characteristics and the hydrolysis rate of the prodrug.
The term "solvate" refers to a complex of variable stoichiometry (e.g., di-, tri-, tetra-,
penta-, hexa-, and so on), which is formed by a solute (the NO-donating compound) and a
solvent, whereby the solvent does not interfere with the biological activity of the solute.
Suitable solvents include, for example, ethanol, acetic acid and the like.
The term "hydrate" refers to a solvate, as defined hereinabove, where the solvent is
water.
The term "alkyl", as used herein, describes a saturated aliphatic hydrocarbon including
straight chain and branched chain groups. In some embodiments, the alkyl group has 1 to 20
carbon atoms. Whenever a numerical range; e.g., "1-20", is stated herein, it implies that the
group, in this case the alkyl group, may contain 1 carbon atom, 2 carbon atoms, 3 carbon
atoms, etc., up to and including 20 carbon atoms. In some embodiments, the alkyl is a lower
alkyl having 1 to 3 carbon atoms. The alkyl group may be substituted or unsubstituted, as
indicated herein.
The term alkenyl, as used herein, describes an alkyl, as defined herein, which contains
a carbon-to-carbon double bond.
The term alkynyl, as used herein, describes an alkyl, as defined herein, which contains
carbon-to-carbon triple bond.
The term "cycloalkyl" describes an all-carbon monocyclic or fused ring (i.e., rings
which share an adjacent pair of carbon atoms) group where one or more of the rings does not
have a completely conjugated pi-electron system. The cycloalkyl group may be substituted or
unsubstituted, as indicated herein.
The term "aryl" describes an all-carbon monocyclic or fused-ring polycyclic ( i.e.,
rings which share adjacent pairs of carbon atoms) groups having a completely conjugated pielectron
system. The aryl group may be substituted or unsubstituted, as indicated herein.
The term "alkoxy" describes both an -O-alkyl and an -O-cycloalkyl group, as defined
herein.
The term "aryloxy" describes an -O-aryl, as defined herein.
Each of the alkyl, cycloalkyl and aryl groups in the general formulas herein may be
substituted by one or more substituents, whereby each substituent group can independently
be, for example, alkyl, cycloalkyl, alkoxy, aryl and aryloxy, carbonyl, aldehyde and carboxy,
depending on the substituted group and its position in the molecule.
The term "halide" or "halo" describes fluorine, chlorine, bromine or iodine.
The term "haloalkyl" describes an alkyl group as defined herein, further substituted by
one or more halide.
The term "S-sulfonamide" describes a -S(=0 ) 2-NR'R" group, with R' as defined
herein and R" being as defined herein for R'.
The term "N-sulfonamide" describes an R'S(=0) 2-NR"- group, where R' and R" are
as defined herein.
The terms "S-sulfonamide" and "N-sulfonamide" are collectively referred to herein as
sulfonamide.
The term "thiocarbonyl" as used herein, describes a -C(=S)-R' group, with R' as
defined herein.
The term "carbonyl" as used herein, describes a -C(=0)-R' group, with R' as defined
herein.
The term "hydroxyl" or "hydroxy" describes a -OH group.
The term "thiohydroxy" or "thiol" describes a -SH group.
The term "thioalkoxy" describes both an -S-alkyl group, and a -S-cycloalkyl group, as
defined herein.
The term "thioaryloxy" describes both an -S-aryl and a -S-heteroaryl group, as defined
herein.
The term "sulfoxide" describes a -S(=0)R' group with R' being hydrogen, alkyl,
cycloalkyl or aryl, as defined herein.
The term "phosphonate" describes a -P(=0)(OR')(OR") group, with R' and R" as
defined herein.
The term "sulfonate" describes a -S(=0 ) 2-R' group, where R' is as defined herein.
The term "acyl halide" describes a -(C=0)R"" group wherein R"" is halide, as defined
hereinabove.
The term "C-thiocarboxylate" describes a -C(=S)-OR' group, where R' is as defined
herein.
The term "C-carboxylate" describes a -C(=0)-OR' group, where R' is as defined
herein.
The term "O-thiocarboxylate" describes a -OC(=S)R' group, where R' is as defined
herein.
The term "N-carbamate" describes an R"OC(=0)-NR'- group, with R' and R" as
defined herein.
The term "O-carbamate" describes an -OC(=0)-NR'R" group, with R' and R" as
defined herein.
The term "O-thiocarbamate" describes a -OC(=S)-NR'R" group, with R' and R" as
defined herein.
The term "N-thiocarbamate" describes an R"OC(=S)NR'- group, with R' and R" as
defined herein.
The term "S-dithiocarbamate" describes a -SC(=S)-NR'R" group, with R' and R" as
defined herein.
The term "N-dithiocarbamate" describes an R"SC(=S)NR'- group, with R' and R" as
defined herein.
The term "C-amide" describes a -C(=0)-NR'R" group, where R' and R" are as
defined herein.
The term "N-amide" describes a R'C(=0)-NR"- group, where R' and R" are as
defined herein.
The terms "N-amide" and "C-amide" are collectively referred to herein as amide.
The term "amine" describes a -NR'R" group, with R' and R" as described herein.
The term "heteroaryl" describes a monocyclic or fused ring ( i.e., rings which share an
adjacent pair of atoms) group having in the ring(s) one or more atoms, such as, for example,
nitrogen, oxygen and sulfur and, in addition, having a completely conjugated pi-electron
system. Examples, without limitation, of heteroaryl groups include pyrrole, furane,
thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrimidine, quinoline,
isoquinoline and purine.
The term "heteroalicyclic" or "heterocyclyl" describes a monocyclic or fused ring
group having in the ring(s) one or more atoms such as nitrogen, oxygen and sulfur. The rings
may also have one or more double bonds. However, the rings do not have a completely
conjugated pi-electron system. Representative examples are piperidine, piperazine,
tetrahydrofurane, tetrahydropyrane, morpholino and the like.
As discussed hereinabove, the chemical conjugates as described herein, were designed
and practiced so as to selectively inhibit COX-2.
Accordingly, in some embodiments, the chemical conjugates described herein are
identified as COX-2 inhibitors.
The phrase "COX-2 inhibitor" describes a compound (e.g., a chemical conjugate as
described herein) which is capable of substantially inhibiting an activity of COX-2, whereby
the phrase "selective COX-2 inhibitor" describes a compound has an inhibitory activity
towards COX-2 which is substantially higher than its inhibitory activity towards COX-1.
In some embodiments, the chemical conjugates described herein are characterized as
an inhibitory activity towards COX-2 which is higher by at least 100-folds than its inhibitory
activity towards COX-1.
In some embodiments, the chemical conjugates described herein are characterized by
an inhibitory activity towards COX-2 which is 100-folds, 200 folds, 300-folds, 400-folds,
500-folds or 1000-folds or more than the inhibitory activity towards COX-1.
Methods for determining an inhibitory activity of a compound towards COX-1 and
COX-2 are well known in the art. Exemplary methods are described in the Examples section
that follows.
In some embodiments, the chemical conjugates described herein are characterized by
an inhibitory activity towards 5-LOX.
In some embodiments, the chemical conjugates described herein are advantageously
characterized by a dual effect of inhibiting both COX (e.g., COX-2) and 5-LOX.
Methods for determining an inhibitory activity of a compound towards 5-LOX are
well known in the art.
Data demonstrating such an inhibition activity has been obtained for exemplary
conjugates as described herein, yet is not shown herein.
As noted hereinabove, inhibition of the 5-Lipoxygenase (5-LOX) pathway reduces the
production of Leukotriene B4 (LTB4), a potent chemoattractant molecule of white blood cells
which can cause additional inflammation at the site of injury. Elevated LTB4 has been shown
to contribute to gastric damage in mucosal lesions. Accordingly, a dual inhibition effect of
COX and 5-LOX reduces AA metabolites, but allows the body to maintain pools of these
necessary AA metabolites to perform essential functions.
As further noted hereinabove, agents exhibiting a dual inhibition effect of COX and 5-
LOX are highly potent in treating a wider spectrum of inflammatory conditions.
Accordingly, according to an aspect of embodiments of the present invention, the
chemical conjugates described herein are identified for use in the treatment of an
inflammatory disease or disorder.
According to an aspect of embodiments of the invention there is provided a method of
treating an inflammatory disease or disorder, which is effected by administering to a subject
in need thereof a therapeutically effective amount of a chemical conjugate as described
herein.
According to an aspect of embodiments of the present invention there is provided a
use of any of the chemical conjugates described herein as a medicament.
In some embodiments, the medicament is for treating an inflammatory disease or
disorder.
Exemplary inflammatory disease or disorder that are treatable by the chemical
conjugates described herein include, but are not limited to, Alzheimer's disease, cortical
dementia, vascular dementia, muli-infract dementia, pre-senile dementia, alcoholic dementia,
senile dementia, memory loss or central nervous damage resulting from stroke, ischemia or
trauma, multiple sclerosis, Parkinson's disease, Huntington's disease, epilepsy, cystic fibrosis,
arthritis diseases such as osteoarthritis, rheumatoid arthritis, spondyloarthopathies, gouty
arthritis, systemic lupus erythematosus, and juvenile arthritis fever, periarteritis;
gastrointestinal disorders such as inflammatory bowel disease, Chron's disease, gastritis,
irritable bowel syndrome, ulcerative colitis, cardiovascular disorders such as myocardial
ischemia, reperfusion injury to an ischemic organ; angiogenesis, asthma, bronchitis, menstrual
cramps, premature labor, tendinitis, bursitis, an autoimmune disease, an immunological
disorder, systemic lupus erythematosus, inflammatory disorders of the skin such as psoriasis,
eczema, burns and dermatitis; neoplasia, an inflammatory process in a disease, pulmonary
inflammation, a central nervous system disorder, migraine headaches, allergic rhinitis,
respiratory distress syndrome, endotoxin shock syndrome, a microbial infection, a bacterialinduced
inflammation, a viral induced inflammation, a urinary disorder, a urological disorder,
endothelial dysfunction, organ deterioration, tissue deterioration, adhesion and infiltration of
neutrophils at the site of inflammation, thyroiditis, aplastic anemia, Hodgkin's disease,
sclerodoma, rheumatic fever, myasthenia gravis, sarcoidosis, nephrotic syndrome, Behcet's
syndrome, polymyositis, hypersensitivity, conjunctivitis, gingivitis and swelling occurring
after injury.
In some embodiments, the inflammatory disease or disorder that are treatable by the
chemical conjugates described herein include, but are not limited to, Alzheimer's disease,
Parkinson's disease, asthma, osteoarthritis, dermatitis, rheumatoid arthritis, pain associated
with inflammation, primary dysmenorrhea, Crohn's disease and ulcerative colitis.
The chemical conjugates described herein can be administered via local administration
or systemically, e.g., orally, rectally, intravenously, intraventricularly, topically, intranasally,
intraperitoneally, intestinally, parenterally, intraocularly, intradermally, transdermally,
subcutaneously, intramuscularly, transmucosally, by inhalation and/or by intrathecal catheter.
In some embodiments of the invention, the chemical conjugates described herein are
administered orally or intravenously, and optionally topically, transdermally or by inhalation,
depending on the condition and the subject being treated.
In some embodiments, the inflammatory disease or disorder that are treatable by the
chemical conjugates described herein is a skin or mucosal disease or disorder, or is manifested
by skin or mucosal ailments. In these embodiments, the chemical conjugate can be
administered topically and accordingly is formulated for topical application, as detailed
hereinbelow.
In any of the methods and uses described herein, the chemical conjugate can be
utilized either per se or being formulated into a pharmaceutical composition which further
comprises a pharmaceutically acceptable carrier.
Hence, according to still another aspect of the present invention, there are provided
pharmaceutical compositions, which comprise one or more of the chemical conjugates
described above and a pharmaceutically acceptable carrier.
As used herein a "pharmaceutical composition" refers to a preparation of one or more
of the chemical conjugates described herein, with other chemical components such as
pharmaceutically acceptable and suitable carriers and excipients. The purpose of a
pharmaceutical composition is to facilitate administration of a compound to an organism.
Hereinafter, the phrase "pharmaceutically acceptable carrier" describes a carrier or a
diluent that does not cause significant irritation to an organism and does not abrogate the
biological activity and properties of the administered compound. Examples, without
limitations, of carriers are: propylene glycol, saline, emulsions and mixtures of organic
solvents with water, as well as solid (e.g., powdered) and gaseous carriers.
Herein the term "excipient" refers to an inert substance added to a pharmaceutical
composition to further facilitate administration of a compound. Examples, without limitation,
of excipients include calcium carbonate, calcium phosphate, various sugars and types of
starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.
Techniques for formulation and administration of drugs may be found in
"Remington's Pharmaceutical Sciences" Mack Publishing Co., Easton, PA, latest edition,
which is incorporated herein by reference.
Pharmaceutical compositions of the present invention may be manufactured by
processes well known in the art, e.g., by means of conventional mixing, dissolving,
granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing
processes.
Pharmaceutical compositions for use in accordance with the present invention thus
may be formulated in conventional manner using one or more pharmaceutically acceptable
carriers comprising excipients and auxiliaries, which facilitate processing of the agents
described herein into preparations which, can be used pharmaceutically. Proper formulation
is dependent upon the route of administration chosen.
According to some embodiments, the pharmaceutical composition is formulated as a
solution, suspension, emulsion or gel.
According to some embodiments, the pharmaceutical composition further includes a
formulating agent selected from the group consisting of a suspending agent, a stabilizing
agent and a dispersing agent.
For injection, the agents described herein may be formulated in aqueous solutions,
preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution,
or physiological saline buffer with or without organic solvents such as propylene glycol,
polyethylene glycol.
For transmucosal administration, penetrants are used in the formulation. Such
penetrants are generally known in the art.
For oral administration, the agents described herein can be formulated readily by
combining the chemical conjugates with pharmaceutically acceptable carriers well known in
the art. Such carriers enable the agents described herein to be formulated as tablets, pills,
dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for oral ingestion
by a patient. Pharmacological preparations for oral use can be made using a solid excipient,
optionally grinding the resulting mixture, and processing the mixture of granules, after adding
suitable auxiliaries if desired, to obtain tablets or dragee cores. Suitable excipients are, in
particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose
preparations such as, for example, maize starch, wheat starch, rice starch, potato starch,
gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium
carbomethylcellulose; and/or physiologically acceptable polymers such as
polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as crosslinked
polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.
Also oral compositions may comprise at least one flavorant such as, but not limited to,
wintergreen oil, oregano oil, bay leaf oil, peppermint oil, spearmint oil, clove oil, sage oil,
sassafras oil, lemon oil, orange oil, anise oil, benzaldehyde, bitter almond oil, camphor, cedar
leaf oil, marjoram oil, citronella oil, lavendar oil, mustard oil, pine oil, pine needle oil,
rosemary oil, thyme oil, and cinnamon leaf oil.
Dragee cores are 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, 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 agent doses.
Pharmaceutical compositions, which 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 may contain the active ingredients in admixture
with filler such as lactose, binders such as starches, lubricants such as talc or magnesium
stearate and, optionally, stabilizers. In soft capsules, the agent(s) 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 the chosen route of administration.
For buccal administration, the compositions may take the form of tablets or lozenges
formulated in conventional manner.
For administration by inhalation, the agents described herein are conveniently
delivered in the form of an aerosol spray presentation (which typically includes powdered,
liquified and/or gaseous carriers) from a pressurized pack or a nebulizer, with the use of a
suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane
or carbon dioxide. In the case of a pressurized aerosol, the dosage unit may
be determined by providing a valve to deliver a metered amount. Capsules and cartridges of,
e.g., gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of
the chemical conjugate and a suitable powder base such as, but not limited to, lactose or
starch.
The agents described herein may be formulated for parenteral administration, e.g., by
bolus injection or continuous infusion. Formulations for injection may be presented in unit
dosage form, e.g., in ampoules or in multidose containers with optionally, an added
preservative. The compositions may be suspensions, solutions or emulsions in oily or
aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or
dispersing agents.
Pharmaceutical compositions for parenteral administration include aqueous solutions
of the agents described herein in water-soluble form. Additionally, suspensions of the agents
may be prepared as appropriate oily injection suspensions and emulsions (e.g., water-in-oil,
oil-in-water or water-in-oil in oil emulsions). Suitable lipophilic solvents or vehicles include
fatty oils such as sesame oil, or synthetic fatty acids esters such as ethyl oleate, triglycerides
or liposomes. Aqueous injection suspensions may contain substances, which increase the
viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran.
Optionally, the suspension may also contain suitable stabilizers or agents, which increase the
solubility of the agents to allow for the preparation of highly concentrated solutions.
Alternatively, the chemical conjugates may be in powder form for constitution with a
suitable vehicle, e.g., sterile, pyrogen-free water, before use.
The chemical conjugates described herein may also be formulated in rectal
compositions such as suppositories or retention enemas, using, e.g., conventional suppository
bases such as cocoa butter or other glycerides.
The pharmaceutical compositions herein described may also comprise suitable solid of
gel phase carriers or excipients. Examples of such carriers or excipients include, but are not
limited to, calcium carbonate, calcium phosphate, various sugars, starches, cellulose
derivatives, gelatin and polymers such as polyethylene glycols.
By selecting the appropriate carrier and optionally other ingredients that can be
included in the composition, as is detailed hereinbelow, the compositions of the present
invention may be formulated into any form typically employed for topical application.
Hence, the compositions of the present invention can be, for example, in a form of a cream, an
ointment, a paste, a gel, a lotion, a milk, a suspension, an aerosol, a spray, a foam, a shampoo,
a hair conditioner, a serum, a swab, a pledget, a pad, a patch and a soap.
Ointments are semisolid preparations, typically based on petrolatum or petroleum
derivatives. The specific ointment base to be used is one that provides for optimum delivery
for the active agent chosen for a given formulation, and, preferably, provides for other desired
characteristics as well (e.g., emolliency). As with other carriers or vehicles, an ointment base
should be inert, stable, nonirritating and nonsensitizing. As explained in Remington: The
Science and Practice of Pharmacy, 19th Ed., Easton, Pa.: Mack Publishing Co. (1995), pp.
1399-1404, ointment bases may be grouped in four classes: oleaginous bases; emulsifiable
bases; emulsion bases; and water-soluble bases. Oleaginous ointment bases include, for
example, vegetable oils, fats obtained from animals, and semisolid hydrocarbons obtained
from petroleum. Emulsifiable ointment bases, also known as absorbent ointment bases,
contain little or no water and include, for example, hydroxystearin sulfate, anhydrous lanolin
and hydrophilic petrolatum. Emulsion ointment bases are either water-in-oil (W/O)
emulsions or oil-in-water (O/W) emulsions, and include, for example, cetyl alcohol, glyceryl
monostearate, lanolin and stearic acid. Preferred water-soluble ointment bases are prepared
from polyethylene glycols of varying molecular weight.
Lotions are preparations that are to be applied to the skin surface without friction.
Lotions are typically liquid or semiliquid preparations in which solid particles, including the
active agent, are present in a water or alcohol base. Lotions are typically preferred for
treating large body areas, due to the ease of applying a more fluid composition. Lotions are
typically suspensions of solids, and oftentimes comprise a liquid oily emulsion of the oil-inwater
type. It is generally necessary that the insoluble matter in a lotion be finely divided.
Lotions typically contain suspending agents to produce better dispersions as well as
compounds useful for localizing and holding the active agent in contact with the skin, such as
methylcellulose, sodium carboxymethyl-cellulose, and the like.
Creams are viscous liquids or semisolid emulsions, either oil-in-water or water-in-oil.
Cream bases are typically water-washable, and contain an oil phase, an emulsifier and an
aqueous phase. The oil phase, also called the "internal" phase, is generally comprised of
petrolatum and/or a fatty alcohol such as cetyl or stearyl alcohol. The aqueous phase
typically, although not necessarily, exceeds the oil phase in volume, and generally contains a
humectant. The emulsifier in a cream formulation is generally a nonionic, anionic, cationic or
amphoteric surfactant. Reference may be made to Remington: The Science and Practice of
Pharmacy, supra, for further information.
Pastes are semisolid dosage forms in which the bioactive agent is suspended in a
suitable base. Depending on the nature of the base, pastes are divided between fatty pastes or
those made from a single-phase aqueous gels. The base in a fatty paste is generally
petrolatum, hydrophilic petrolatum and the like. The pastes made from single-phase aqueous
gels generally incorporate carboxymethylcellulose or the like as a base. Additional reference
may be made to Remington: The Science and Practice of Pharmacy, for further information.
Gel formulations are semisolid, suspension-type systems. Single-phase gels contain
organic macromolecules distributed substantially uniformly throughout the carrier liquid,
which is typically aqueous, but also, preferably, contain an alcohol and, optionally, an oil.
Preferred organic macromolecules, i.e., gelling agents, are crosslinked acrylic acid polymers
such as the family of carbomer polymers, e.g., carboxypolyalkylenes that may be obtained
commercially under the trademark Carbopol™. Other types of preferred polymers in this
context are hydrophilic polymers such as polyethylene oxides, polyoxyethylenepolyoxypropylene
copolymers and polyvinylalcohol.; cellulosic polymers such as
hydroxypropyl cellulose, hydroxyethyl cellulose, hydroxypropyl methylcellulose,
hydroxypropyl methylcellulose phthalate, and methyl cellulose; gums such as tragacanth and
xanthan gum; sodium alginate; and gelatin. In order to prepare a uniform gel, dispersing
agents such as alcohol or glycerin can be added, or the gelling agent can be dispersed by
trituration, mechanical mixing or stirring, or combinations thereof.
Sprays generally provide the active agent in an aqueous and/or alcoholic solution
which can be misted onto the skin for delivery. Such sprays include those formulated to
provide for concentration of the active agent solution at the site of administration following
delivery, e.g., the spray solution can be primarily composed of alcohol or other like volatile
liquid in which the active agent can be dissolved. Upon delivery to the skin, the carrier
evaporates, leaving concentrated active agent at the site of administration.
Foam compositions are typically formulated in a single or multiple phase liquid form
and housed in a suitable container, optionally together with a propellant which facilitates the
expulsion of the composition from the container, thus transforming it into a foam upon
application. Other foam forming techniques include, for example the "Bag-in-a-can"
formulation technique. Compositions thus formulated typically contain a low-boiling
hydrocarbon, e.g., isopropane. Application and agitation of such a composition at the body
temperature cause the isopropane to vaporize and generate the foam, in a manner similar to a
pressurized aerosol foaming system. Foams can be water-based or hydroalcoholic, but are
typically formulated with high alcohol content which, upon application to the skin of a user,
quickly evaporates, driving the active ingredient through the upper skin layers to the site of
treatment.
Skin patches typically comprise a backing, to which a reservoir containing the active
agent is attached. The reservoir can be, for example, a pad in which the active agent or
composition is dispersed or soaked, or a liquid reservoir, patches typically further include a
frontal water permeable adhesive, which adheres and secures the device to the treated region.
Silicone rubbers with self-adhesiveness can alternatively be used. In both cases, a protective
permeable layer can be used to protect the adhesive side of the patch prior to its use. Skin
patches may further comprise a removable cover, which serves for protecting it upon storage.
Examples of pharmaceutically acceptable carriers that are suitable for pharmaceutical
compositions for topical applications include carrier materials that are well-known for use in
the cosmetic and medical arts as bases for e.g., emulsions, creams, aqueous solutions, oils,
ointments, pastes, gels, lotions, milks, foams, suspensions, aerosols and the like, depending on
the final form of the composition.
Representative examples of suitable carriers according to the present invention
therefore include, without limitation, water, liquid alcohols, liquid glycols, liquid
polyalkylene glycols, liquid esters, liquid amides, liquid protein hydrolysates, liquid alkylated
protein hydrolysates, liquid lanolin and lanolin derivatives, and like materials commonly
employed in cosmetic and medicinal compositions.
Other suitable carriers according to the present invention include, without limitation,
alcohols, such as, for example, monohydric and polyhydric alcohols, e.g., ethanol,
isopropanol, glycerol, sorbitol, 2-methoxyethanol, diethyleneglycol, ethylene glycol,
hexyleneglycol, mannitol, and propylene glycol; ethers such as diethyl or dipropyl ether;
polyethylene glycols and methoxypolyoxyethylenes (carbowaxes having molecular weight
ranging from 200 to 20,000); polyoxyethylene glycerols, polyoxyethylene sorbitols, stearoyl
diacetin, and the like.
Pharmaceutical compositions for topical application as described herein can be
identified also as cosmetic or cosmeceutic products.
Pharmaceutical compositions suitable for use in context of the present invention
include compositions wherein the active ingredients are contained in an amount effective to
achieve the intended purpose. More specifically, a therapeutically effective amount means an
amount of a chemical conjugate as described herein effective to prevent, alleviate or
ameliorate symptoms of a physiological disorder associated with oxidative stress (such as
tobacco-associated damage) or prolong the survival of the subject being treated.
Determination of a therapeutically effective amount is well within the capability of
those skilled in the art, especially in light of the detailed disclosure provided herein.
For any chemical conjugate or an additional agent utilized in the methods and uses of
the invention, the therapeutically effective amount or dose can be estimated initially from
activity assays in animals. For example, a dose can be formulated in animal models to
achieve a circulating concentration range that includes the IC50 as determined by activity
assays (e.g., the concentration of the test agent, which achieves a half-maximal reduction in
cell death upon exposure to cigarette smoke). Such information can be used to more
accurately determine useful doses in humans.
Toxicity and therapeutic efficacy of the agents described herein can be determined by
standard pharmaceutical procedures in experimental animals, e.g., by determining the EC50,
the IC50 and the LD50 (lethal dose causing death in 50 % of the tested animals) for a subject
compound. The data obtained from these activity assays and animal studies can be used in
formulating a range of dosage for use in human.
The dosage may vary depending upon the dosage form employed and the route of
administration utilized. The exact formulation, route of administration and dosage can be
chosen by the individual physician in view of the patient's condition. (See e.g., Fingl et al.,
1975, in "The Pharmacological Basis of Therapeutics", Ch. 1 p.l).
Depending on the severity and responsiveness of the condition to be treated, dosing
can also be a single administration of a slow release composition described hereinabove, with
course of treatment lasting from several days to several weeks or until cure is effected or
diminution of the disease state is achieved.
The amount of a composition to be administered will, of course, be dependent on the
subject being treated, the severity of the affliction, the manner of administration, the judgment
of the prescribing physician, etc.
Compositions of the present embodiments may, if desired, be presented in a pack or
dispenser device, such as an FDA (the U.S. Food and Drug Administration) approved kit,
which may contain one or more unit dosage forms containing the active agent. The pack may,
for example, comprise metal or plastic foil, such as, but not limited to a blister pack or a
pressurized container (for inhalation). The pack or dispenser device may be accompanied by
instructions for administration. The pack or dispenser may also be accompanied by a notice
associated with the container in a form prescribed by a governmental agency regulating the
manufacture, use or sale of pharmaceuticals, which notice is reflective of approval by the
agency of the form of the compositions for human or veterinary administration. Such notice,
for example, may be of labeling approved by the U.S. Food and Drug Administration for
prescription drugs or of an approved product insert. Compositions comprising an agent as
described herein, formulated in a compatible pharmaceutical carrier may also be prepared,
placed in an appropriate container, and labeled for treatment of an indicated condition, as is
detailed herein.
Thus, according to an embodiment of the present invention, the pharmaceutical
composition is packaged in a packaging material and identified in print, in or on the
packaging material, for use in the treatment of an inflammatory disease or disorder, as
described herein.
In any of the compositions, methods and uses described herein, the chemical
conjugates can be utilized in combination with an additional therapeutically active agent. In
some embodiments, the additional therapeutically active agent is an anti-inflammatory agent.
In some embodiments the additional active agent is a COX-2 inhibitor and/or a 5-LOX
inhibitor.
It is to be noted that some of the compounds encompassed by embodiments of the
invention have been described in the art. Such compounds are excluded from the scope of
embodiments of the invention that relate to chemical conjugates per se, yet are included in
embodiments of the invention that relate to the use of such conjugates as exhibiting beneficial
anti-inflammatory activity.
As used herein the term "about" refers to ± 10 %.
Throughout this application, various embodiments of this invention may be presented
in a range format. It should be understood that the description in range format is merely for
convenience and brevity and should not be construed as an inflexible limitation on the scope
of the invention. Accordingly, the description of a range should be considered to have
specifically disclosed all the possible subranges as well as individual numerical values within
that range. For example, description of a range such as from 1 to 6 should be considered to
have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to
4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example,
1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.
Whenever a numerical range is indicated herein, it is meant to include any cited
numeral (fractional or integral) within the indicated range. The phrases "ranging/ranges
between" a first indicate number and a second indicate number and "ranging/ranges from" a
first indicate number "to" a second indicate number are used herein interchangeably and are
meant to include the first and second indicated numbers and all the fractional and integral
numerals therebetween.
As used herein, the term "treating" includes abrogating, substantially inhibiting,
slowing or reversing the progression of a condition, substantially ameliorating clinical or
aesthetical symptoms of a condition or substantially preventing the appearance of clinical or
aesthetical symptoms of a condition.
It is appreciated that certain features of the invention, which are, for clarity, described
in the context of separate embodiments, may also be provided in combination in a single
embodiment. Conversely, various features of the invention, which are, for brevity, described in
the context of a single embodiment, may also be provided separately or in any suitable
subcombination or as suitable in any other described embodiment of the invention. Certain
features described in the context of various embodiments are not to be considered essential
features of those embodiments, unless the embodiment is inoperative without those elements.
Various embodiments and aspects of the present invention as delineated hereinabove
and as claimed in the claims section below find experimental support in the following
examples.
EXAMPLES
Reference is now made to the following examples, which together with the above
descriptions illustrate some embodiments of the invention in a non limiting fashion.
EXAMPLE 1
In Silico Studies
The COX-2-inhibition ability of the compounds set forth in Table 1 containing a fatty
acid moiety attached to an amine moiety via an amide bond was determined in silico, in order
to determine the effect of the type of a fatty acid and the type of a therapeutically active agent
containing an amine functional group (hereinafter also referred to as "an amine-containing
compound") on COX-2 inhibition.
Experimental Methods
A set of conjugates deriving from all possible combinations of 6 fatty acids and 13
amine-containing compounds, as depicted in Table 2 below, attached to one another via an
amide bond was screened using procedures analogous to those described, for example, in WO
2009/090613 and in Falah et al., Bioinformation 3(9):389-393 (2009). The fatty acids, aminecontaining
compounds, and the conjugates derived therefrom, are shown in Table 2.
The chemical structures of the chemical conjugates are depicted in Table 1 in the
specification.
Table 2: Compound Nos. for exemplary compounds containing an amine moiety
attached to a fatty acid moiety
Results
The molecular activity index (MAI) of each conjugate was calculated in a COX-2
inhibition model, as detailed hereinabove. The MAI values of the abovementioned conjugates
set forth in Table 1 are summarized in Table 3 below, with the conjugates being ranked by
magnitude of the MAI value. MAI values above zero were considered to be zero.
MAI values for the 13 conjugates associated with each fatty acid were averaged to
obtain an average MAI value for each of the 6 fatty acids. The results are presented in FIG. 1.
As shown in FIG. 1, DHA exhibited the average MAI with the highest magnitude,
followed closely by EPA. As further shown therein, the magnitude of the average MAI values
of the fatty acids was correlated to the number of double bonds in the fatty acid.
These results indicate that DHA is particularly suitable for forming COX-2 inhibitors.
As shown in Table 3 below, analysis of the COX-2 inhibition activity correlation to the
amine-containing compounds tested, reveals that 4 of the 10 compounds having the largest
MAI values (i.e., compounds 65, 78, 52 and 39 in Tables 1 and 2) comprised a 4,5,6,7-
tetrahydro-isoxazolo[4,5-c]pyridin-3-ol moiety, 3 of the 10 compounds having the largest
MAI values (i.e., compounds 64, 51 and 77) and 4 of the top 11 compounds (i.e., compounds
64, 51, 77 and 38) comprised a 3a,4,5,6,7,7a-hexahydroisoxazolo[4,5-c]pyridin-3-ol moiety,
and 2 of the 10 compounds having the largest MAI values (i.e., compounds 55 and 68) and 6
of the top 17 compounds (i.e., compounds 55, 68, 16, 29, 3 and 42) comprised an indol-3-ylacetic
acid moiety.
As further shown in Table 3 below, of the 25 compounds with the largest MAI values,
four compounds comprised a 5-methoxy-2-methyl-indol-3-yl-acetic acid moiety, and five
compounds comprised a 3-amino-phenyl-acetic acid moiety.
These results indicate that certain amine-containing moieties are particularly suitable
for forming COX-2 inhibitors.
Table 3 : Molecular activity index for compounds of FIG. 1 (ranked by magnitude)
-576.5 64
-570.6 5 1
-558.0 77
-545.1 68
-526.9 39
-512.0 59
-510.7 38
-427.0 16
-427.0 29
-395.5 3
-389.1 72
-360.1 33
-358.3 42
-352.2 47
-329.1 2 1
-328.2 60
-325.8 34
-324.9 20
-304.2 67
-299.3 76
-296.9 8
-291.1 43
-290.8 69
-263.3 74
-261.5 75
-257.3 4 1
-253.6 54
-247.8 62
-247.8 26
-245.5 49
-244.2 56
-242.0 73
-235.1 7
-229.5 48
-219.7 6 1
-210.6 46
-204.9 66
-204.9 70
-201.0 25
-188.5 40
-188.5 44
-186.7 13
-178.2 23
-176.4 53
-176.4 57
-171.1 17
-170.7 50
-167.7 15
-162.7 27
-162.7 3 1
-160.9 22
-160.3 12
-156.5 10
-152.4 2
-152.2 1
-152.2 5
-150.5 4
-146.6 14
-146.6 18
-140.2 63
-135.3 30
-132.6 9
-127.3 24
-124.8 35
-121.0 11
- 114.6 28
-108.8 36
-64.3 37
0 7 1
0 58
0 45
0 6
0 19
0 32
EXAMPLE 2
Following the In Silico data presented in Example 1 hereinabove, exemplary
conjugates according to embodiments of the invention were designed and successfully
synthesized.
Materials and Methods:
Solvents and reagents were obtained from commercial suppliers and were used without
further purification.
1H NMR spectra were recorded in DMSO-D or CDC13 on a Bruker WM300
spectrometer. Chemical shifts are given in p.p.m. relative to tetramethylsilane (1H).
Thin layer chromatography (TLC) was performed on Merck Silica Gel 60 F2 4 plates.
Column chromatography was performed using Merck silica gel 60.
Generalprocedures:
A general synthetic pathway for preparing conjugates of fatty acids and therapeutically
active agents linked therebetween via an amide bond, according to some embodiments of the
present invention, involves a condensation reaction of a fatty acid and an amine-containing
compound.
A general synthetic pathway for preparing conjugates of fatty acids and therapeutically
active agents linked therebetween via an ester bond, according to some embodiments of the
present invention, involves a condensation reaction of a fatty acid and an alcohol-containing
compound which does not contain an amine group.
Thus, according to a representative synthetic pathway, a desired conjugate is typically
prepared, according to embodiments of the invention, by placing a corresponding fatty acid in
a dry solvent such as dichloromethane or tetrahydrofuran, and adding dimethylaminopyridine
(DMAP) and N N'-dicyclohexylcarbodiimide (DCC). The mixture is stirred at 0 °C for about
20 minutes, and a corresponding amine-containing compound or alcohol-containing
compound is then added at an amount equimolar to the amount of fatty acid, and stirred at
ambient temperature for about 20 hours. The solid residue is removed by filtration and the
solvent is removed by evaporation. The desired conjugate is purified by being dissolved in a
solvent such as n-hexane, removing the undissolved solid, and removing the solvent by
evaporation.
Using the general procedure described above, a variety of conjugates according to
embodiments of the present invention were prepared, as is detailed hereinbelow.
Synthesis of N-docosa-4,7,10,13,16,19-hexaenoyl-5-hydroxy-indol-3-yl-acetic acid
(MWL004):
MWL004
1 gram (3.04 mmol) of all-cis-DHA (a//-cw-docosa-4,7,10,13,16,19-hexaenoic acid)
was dissolved in 50 ml dry dichloromethane under an inert atmosphere, and 0.48 gram (3.39
mmol) dimethylaminopyridine (DMAP) and 0.88 gram (4.26 mmol) N. -
dicyclohexylcarbodiimide (DCC) were then added. The mixture was stirred at 0 °C for 20
minutes, and 0.58 gram (3.04 mmol) of 5-hydroxy-indol-3-yl-acetic acid was added and
stirred at ambient temperature for 20 hours. The solid residue was removed by filtration and
the dichloromethane was removed by evaporation. The solid-oil mixture was dissolved in nhexane,
the undissolved solid was removed, and the hexane was then removed by
evaporation. 2.33 grams of N-docosa-4,7,1 0,1 3,16,1 9-hexaenoyl-5-hydroxy-indol-3-yl -acetic
acid was obtained as a dark brown solid.
1H-NMR (CDC13) : 1.02(t, 3H, CH3), 2.09(q, 2H, CH CH 2.29(m, 2H, CH C¾-
CH), 2.47(t,_2H, CH2CH2-CH) 2.69(m, 10H, CH2), 3.59(s, 2H, CH2COOH), 5.37-3.58(m,
13H, All cis, and OH-Aromatic), 6.50(d, 1H, Aromatic), 7.39(s, 1H), 7.46(s, 1H, Aromatic),
7.60(d, 1H, Aromatic).
Synthesis of N-(docosa-4,7,10,13,16,19-hexaenoyl)-2-amino-nicotinic acid
(MWL005):
MWL005
N-(docosa-4,7,10,13,16,19-hexaenoyl)-2-amino-nicotinic acid (MWL005) was
synthesized according to the general procedures described hereinabove, wherein the fatty acid
was all-cis-OHA and the amine-containing compound was 2-amino-nicotinic acid.
1H-NMR (CDCI3): 1.07(t, 3H, CH3), 2.00(q, 2H, CH2CH3), 2.23-2.5 l(m, 4H,
CH CH ), 2.79(m, 10H, CH2), 5.35(m, 12H, All-cis), 6.86-7.25(m, 3H, Aromatic), 9.47(1H,
NH-CO).
Synthesis of N-(docosa-4, 7,10,13,16,19-hexaenoyl)-2-amino-phenyl-acetic acid
(MWL006):
MWL006
N-(docosa-4,7,10,13,16,19-hexaenoyl)-2-amino-phenyl-acetic acid (MWL006) was
synthesized according to the general procedures described hereinabove, wherein the fatty acid
was all-cis-OHA and the amine-containing compound was 2-amino-phenyl-acetic acid.
To a solution of 20 ml dry THF, 0.5 gram (1.52 mmol) DHA, and 0.45 ml
triethylamine 0.144 ml of triethylchloroformate were added. A white solid immediately
precipitated. The solution was stirred for another 15 minutes at room temperature, and the
white solid was thereafter filtered out. The THF solution was then added to 230 mg of a 2-
amino-phenyl-acetic acid in THF. The clear solution was then stirred at room temperature for
additional 16 hours. The THF was removed and the resulting yellow powder was taken in
dichloromethane and washed twice with water, dried over sodium sulfate and evaporated to
dryness to afford 350 mg of a pale yellow solid (Yield: 49 %).
1H-NMR(CDC13) : 1.1 (t, 3H, CH3), 2.05(q, 2H, CH CHQ. 2.23-2.5 l(m, 4H, CH CH
2.69(m, 10H, CH^), 3.72(s, 2H, CH^-COOH), 5.42(m, 12H, All-cis), 7.01-7.32(m, 4H,
Aromatic).
Synthesis of 3',4',5,7-tetrahydroxy-flavone-3-yl docosa-4,7,10,13,16,19-hexaenoate
(MWL011):
MWL011
3",4',5,7-tetrahydroxy-flavone-3-yl docosa-4,7, 10, 13, 16,19-hexaenoate (MWL01 1) is
synthesized according to the general procedures described hereinabove, wherein the fatty acid
is DHA and the alcohol-containing compound is 3',4',3,5,7-pentahydroxy-flavone (quercetin).
Synthesis of O-(docosa-4,7,10,13,16,19-hexaenoyl)-salicylic acid (MWL013):
MWL013
O-(docosa-4,7,10,13,16,19-hexaenoyl)-salicylic acid (MWL013) was synthesized
according to the general procedures described hereinabove, wherein the fatty acid is DHA and
the alcohol-containing compound is salicylic acid.
To a solution of 20 ml dry THF, 0.5 gram (1.52 mmol) DHA, and 0.45 ml
triethylamine, 0.144 ml triethylchloroformate were added. A white solid immediately
participated. The solution was stirred for additional 15 minutes at room temperature,
followed by filtration of the white solid. The THF solution was then added to 210 mg
salicylic acid in THF. The clear solution was then stirred at room temperature for additional
22 hours. The THF was thereafter removed and the resulting solid was taken in
dichloromethane and washed twice with water, dried over sodium sulfate and evaporated to
dryness to afford 420 mg (0.93 mmol) of a white solid (yield: 62 %).
1H-NMR (CDC13) : 1.02 (t, 3H, CH3), 2.01 (q, 2H, CH CH 2.23-2.51 (m, 4H,
CH CHA 2.70 (m, 10H, CHj), 5.35 (m, 12H, All-cis), 7.75-8.25 (m, 4H, Aromatic).
Synthesis of N-(docosa-4,7,10,13,16,19-hexaenoyl)-5-amino-salicylic acid
(MWL016):
MWL016
N-(docosa-4,7,10,13,16,19-hexaenoyl)-5-amino-salicylic acid (MWL016) was
synthesized according to the general procedures described hereinabove, wherein the fatty acid
is DHA and the amine-containing compound is 5-amino-salicylic acid (mesalazine).
1H -NMR(CDCl3): 1.03 (t, 3H, CH3), 2.00 (q, 2H, CH CH . 2.23-2.51 (m, 4H,
CH CH 2.74 (m, 10H, CH ), 5.39 (s, 1H, OH-Aromatic), 5.39 (m, 12H, All-cis), 7.25 (d,
1H, Aromatic), 7.79 (1H, Aromatic) , 8.30 (s, 1H, Aromatic).
Synthesis of -docosa-4, 7,10,13,16,19-hexaenoyl-pyrrol-3-yl-acetic acid (MWL066):
N-docosa-4,7,10,13,16,19-hexaenoyl-pyrrol-3-yl-acetic acid (MWL066) was
synthesized according to the general procedures described hereinabove, wherein the fatty acid
is DHA and the amine-containing compound is pyrrol-3-yl-acetic acid.
1H-NMR (CDCI3): 0.99 (t, 3H, CH3), 2.00 (q, 2H, CH CH , 2.29 (q, 2H, CH CH
2.31 (q, 2H, CH2-CH2), 2.64 (m, 10H, CH^), 3.55 (s, 1H, CH2-COOH), 5.40 (m, 12H, all cis),
6.00 (d, 1H, Aromatic), 6.79 (1H, Aromatic) 7.00 (d, 1H, Aromatic).
Synthesis of N-docosa-4, 7,10,13,16,19-hexaenoyl-indol-3-yl-acetic acid (MWL068):
MWL068
N-docosa-4,7,1 0,1 3,1 6,19-hexaenoyl-indol-3 -yl-acetic acid (MWL068) was
synthesized according to the general procedures described hereinabove, wherein the fatty acid
is DHA and the amine-containing compound is indol-3-yl-acetic acid.
'H-NMRtCDCls): 1.02 (t, 3H, CH3), 2.09 (q, 2H, CH CH . 2.29 (m, 2H, CH CH -
CH), 2.47 (t,_2H, CH2CH2-CH) 2.69 (m, 10H, CHg), 3.59 (s, 2H, CH2COOH), 5.37-3.58 (m,
12H, All cis), 6.50 (m, 1H, Aromatic), 7.39 (s, 1H), 7.46 (m, 1H, Aromatic), 7.90 (m, 1H,
Aromatic).
Synthesis of N-docosa-4, 7,10,13,16,19-hexaenoyl-5-methoxy-2-methyl-indol-3-ylacetic
acid (MWL072):
MWL072
N-docosa-4,7, 10,13,16,1 9-hexaenoyl-5-methoxy-2-methyl-indol-3 -yl-acetic acid
(MWL072) was synthesized according to the general procedures described hereinabove,
wherein the fatty acid is DHA and the amine-containing compound is 5-methoxy-2-methylindol-
3 -yl-acetic acid.
1H-NMR (CDCI3): 1.07 (t, 3H, CH3), 1.98 (q, 2H, CH CH3 2.30 (s, 3H, CH3), 2.25
(m, 2H, CH2CH2-CH), 2.40 (t,_2H, CH2CH2-CH) 2.69 (m, 10H, CH^), 3.54 (s, 2H,
CH2COOH), 3.85 (s, IH, 0-CH 3), 3.53 (m, 12H, All cis), 6.70 (d, IH, Aromatic), 7.05 (s, IH,
Aromatic), 7.32 (d, IH, Aromatic)..
Synthesis of N-(docosa-4, 7,10,13,16,19-hexaenoyl)-3-amino-phenyl-acetic acid
(MWL073):
N-(docosa-4,7,10,13,16,19-hexaenoyl)-3-amino-phenyl-acetic acid (MWL073) was
synthesized according to the general procedures described hereinabove, wherein the fatty acid
is DHA and the amine-containing compound is 3-amino-phenyl-acetic acid.
1H-NMR(CDC13) : 1.02 (t, 3H, CH3), 2.05 (q, 2H, CH C¾ ) 2.33-2.37 (dd, 4H, CH2-
CH2), 2.65 (m, 10H, C ), 3.70 (s, 2H, CH -COOH), 5.35 (m, 12H, All-cis), 7.00-7.75 (m,
4H, Aromatic).
Synthesis of N-docosa-4,7,10,13,16,19-hexaenoyl-piperidine-3-carboxylic acid
(MWL074):
MWL074
N-docosa-4,7,10,13,16,19-hexaenoyl-piperidine-3-carboxylic acid (MWL074) was
synthesized according to the general procedures described hereinabove, wherein the fatty acid
is DHA and the amine-containing compound is piperidine-3-carboxylic acid (nipecotic acid).
'H-NMR(CDC13) : 1.00 (t, 3H, CH3), 1.18-2.21 (m, 6H, CH2-Pipiridine Ring), 2.25 (q,
2H, CH^CHs), 2.33-2.37 (dd, 4H, CH2-CH2), 2.40 (m, 1H, CH-Pipiridine Ring), 2.65 (m,
10H, CH2), 3.70-3.44 (m, 2H, CH2-Pipiridine Ring), 5.35 (m, 12H, All-cis).
Synthesis of N-docosa-4, 7,10,13,16,19-hexaenoyl-l,2,3,6-tetrahydropyridine-4-
carboxylic acid (MWL07S):
MWL075
N-docosa-4,7, 10,13,16,1 9-hexaenoyl- 1,2,3 ,6-tetrahydro-pyridine-4-carboxylic acid
(MWL075) was synthesized according to the general procedures described hereinabove,
wherein the fatty acid is DHA and the amine-containing compound is 1,2,3,6-
tetrahydropyridine-4-carboxylic acid (isoguvacine).
1H-NMR(CDC13) : 1.09 (t, 3H, CH3), 2.02 (q, 2H, CH2-CH3), 2.1 1 (m, 2H, CH2-
Pipiridine Ring), 1.18-2.21 (m, 6H, CH2-Pipiridine Ring), 2.33-2.37 (dd, 4H, CH2-CH2), 2.65
(m, 10H, CH2), 3,56 (t, CH2, Pipiridine Ring), 3.95 (d, 2H, CH2- Pipiridine Ring), 5.38 (m,
12H, All-cis), 7.34 (d, 1H, CH- Pipiridine Ring).
Synthesis of 5-(N-docosa-4, 7,10,13,16,19-hexaenoyl-aminomethyl)-4,5-
dihydroisoxazol-3-ol (MWL076):
MWL076
5-(7V-docosa-4,7,10,13,16,19-hexaenoyl-aminomethyl)-4,5-dihydroisoxazol-3-ol
(MWL076) is synthesized according to the general procedures described hereinabove,
wherein the fatty acid is DHA and the amine-containing compound is 5-(aminomethyl)-4,5-
dihydroisoxazol-3-ol (4,5-dihydromuscimol).
Synthesis of 5-(docosa-4, 7,10,13,16,19-hexaenoyl)-3a,4,5,6, 7, 7a-hexahydroisoxazolo[
4,5-c]pyridin-3-ol (MWL077):
MWL077
5-(docosa-4,7, 10,13,16,19-hexaenoyl)-3a,4,5,6,7,7a-hexahydro-isoxazolo[4,5-
c]pyridin-3-ol (MWL077) is synthesized according to the general procedures described
hereinabove, wherein the fatty acid is DHA and the amine-containing compound is
3a,4,5,6,7,7a-hexahydro-isoxazolo[4,5-c]pyridin-3-ol.
Synthesis of 5-(docosa-4, 7,10,13,16,19-hexaenoyl)-4,5,6, 7-tetrahydro-isoxazolo[4,5-
cJpyridin-3-ol (MWL078):
5-(docosa-4,7, 10,13,16,1 9-hexaenoyl)-4,5,6,7-tetrahydro-isoxazolo[4,5-c]pyridin-3 -ol
(MWL078) is synthesized according to the general procedures described hereinabove,
wherein the fatty acid is DHA and the amine-containing compound is 4,5,6,7-tetrahydroisoxazolo[
4,5-c]pyridin-3-ol.
Synthesis of 4-(3,5-dihydroxystyryl)phenyl docosa-4,7,10,13,16,19-hexaenoate
(MWL080):
MWL080
4-(3,5-dihydroxystyryl)phenyl docosa-4,7,10,13,16,19-hexaenoate (MWL080) is
synthesized according to the general procedures described hereinabove, wherein the fatty acid
is DHA and the alcohol-containing compound is 3,5,4'-trihydroxystilbene (resveratrol).
Synthesis of 0-(y-linolenoyl)-salicylic acid (MWL014):
MWL014
O-(y-linolenoyl)-salicylic acid (MWL014) is synthesized according to the general
procedures described hereinabove, wherein the fatty acid is g-linolenic acid (GLA) and the
alcohol-containing compound is salicylic acid.
Synthesis of N-(y-linolenoyl)-5-amino-salicylic acid (MWL015):
MWL015
N-(Y-linolenoyl)-5-amino-salicylic acid (MWL015) is synthesized according to the
general procedures described hereinabove, wherein the fatty acid is g-linolenic acid (GLA)
and the amine-containing compound is 5-amino-salicylic acid (mesalazine).
S nthesis ofN-oleoyl-5-hydroxy-indol-3-yl-acetic acid (MWL007):
MWL007
N-oleoyl-5-hydroxy-indol-3-yl-acetic acid (MWL007) was synthesized according to
the general procedures described hereinabove, wherein the fatty acid was oleic acid and the
amine-containing compound was 5-hydroxy-indol-3-yl-acetic acid.
H-NMR CDC ): 0.89 (t, 3H, CH3), 1.29-1.31 (s, 20H, CH2), 1.60 (t, 2H, N-CONCH
2-CH ), 2.201 (m, 4H, CH-CH^), 2.43 (t, 2H, NCOCHA 3.58 (s, 2H, CH COOH 5.49
(dd, 2H, CH-CH-cis), 7.33 (s, 1H, NCHCH2-Ring), 7.42-7.55 (m, 3H, Aromatic).

We Claim:
1. A docosa-4,7, 1 0,13,16, 19-hexaenoic acid linked to a hydroxyproline for use in the
treatment of dermatitis.
2. A method of synthesizing I-docosa-4,7, 1 0, 13,16, 19-hexaenoyl-4-hydroxy-pyrrolidine-2carboxylic
acid comprising:
mixing tetrahydrofurane with decosahexanoic acid;
adding triethylcloroformate;
adding triethylamine;
stirring and filtering;
adding a solution of hydroxyproline and NaOH in water;
adding strong acid;
adding hexane;
collect organic layer; and drying
3. The method according to claim 2, wherein the step of stirring following the addition ofthe
hydroxyproline and NaOH in water solution, is for at 12 hours.
4. The method according to claim 2, wherein the step of drying is over anhydrous sulfate.
5. A chemical conjugate comprising a first moiety and a second moiety covalently linked
therebetween, wherein said second moiety is derived from docosa-4,7,10,13,16,19-hexaenoic
acid, and wherein said first moiety is derived from a therapeutically active agent or a
derivative thereof, each independently having a functional group for forming a covalent bond
with said second moiety, with the proviso that said first moiety is not hydroxyproline, the
chemical conjugate being a cyclooxygenase-2 (COX-2) inhibitor.
6.The chemical conjugate of claim 5, being further a 5-lipoxygenase (5-LOX) inhibitor.
7. The: chemical conjugate ofanyone ofclaims 5 and 6, wherein said functional group is
selected from the group consisting ofhydroxy, amine, carboxy and amide.
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8. The chemical conjugate of anyone of claims 5 to 7, wherein said first moiety and said
second moiety are covalently bound via a bond selected from the group consisting ofan
amide bond and an ester bond.
9. The chemical conjugate of anyone of claims 5 to 8, wherein said therapeutically
active agent is an anti-inflammatory agent.
10. The chemical conjugate of anyone of claims 5 to 9, wherein said therapeutically
active agent is a cyclooxygenase (COX) inhibitor.
11. The chemical conjugate of anyone of claims 5 to 10, wherein said therapeutically
active agent is a non-steroidal anti-inflammatory drug (NSAID).
12. The chemical conjugate of anyone of claims 5 to 11, wherein said therapeutically
active agent is selected from the group consisting of: 5-hydroxy-indol-3-yl-acetic acid, 2amino-
nicotinic acid, salicyclic acid, mesalazine and quercetin.
13. A chemical conjugate selected from the group consisting of:
N-( docosa-4, 7,10, 13,16, 19-hexaenoyl)-5-hydroxy-indol-3-yl-aceticacid (MWL004);
N-( docosa-4, 7,10,13,16, 19-hexaenoyl)-2-amino-nicotinic acid (MWL005);
N -( docosa-4, 7,10,13,16, 19-hexaenoyl)-2-amino-phenyl-acetic acid (MWL006);
N-oleoyl-5-hydroxy-indol-3-yl-acetic acid (MWL007);
N-oleoyl-2-amino-nicotinic acid (MWL008);
O-oleoyl-salicylic acid (MWL009);
0-(docosa-4, 7,10,13,16, 19-hexaenoyl)-salicylic acid (MWLOI3);
O-(?-linolenoyl)-salicylic acid (MWL014);
N-(?-linolenoyl)-5-amino-salicylic acid (MWLOI5);
N-( docosa-4, 7,10,13,16, 19-hexaenoyl)-5-amino-salicylic acid (MWLOI6);
N-oleoyl-5-amino-salicylic acid (MWLOI7); and
N-docosa-4,7,10,13,16,19-hexaenoyl-taurine (MWL002).
14. A chemical conjugate comprising a first moiety and a second moiety covalently linked
therebetween, wherein said second moiety is derived from ?-linolenic acid, and wherein said
79
first moiety is derived from a therapeutically active agent or a derivative thereof, each
independently having a functional group for forming a covalent bond with said second
moiety, with the proviso that said first moiety is not hydroxyproline or taurine, the chemical
conjugate being a cyclooxygenase-2 (COX-2) inhibitor.
15. The chemical conjugate of claim 14, being further a 5-lipoxygenase (5-LOX) inhibitor.
16. The chemical conjugate of claim 14, wherein said functional group is selected from the
group consisting ofhydroxy, amine, carboxy and amide.
17. The chemical conjugate claim 14, wherein said first moiety and said second moiety are
covalently bound via a bond selected from the group consisting of an amide bond and an ester
bond.
18. The chemical conjugate of claim 14, wherein said therapeutically active agent is an antiinflammatory
agent, a cyc100xygenase (COX) inhibitor, a non-steroidal anti-inflammatory
drug (NSAID) or selected from the group consisting of salicyclic acid and mesalazine.
19. A chemical conjugate comprising a first moiety and a second moiety covalently linked
therebetween, wherein said first moiety is derived from a compound selected from the group
consisting of 5-hydroxy-indol-3-yl-acetic acid, 2-amino-nicotinic acid, salicyclic acid,
mesalazine, quercetin and resveratrol, and wherein said second moiety is derived from a fatty
acid.
20. The chemical conjugate of claim 19, wherein said first moiety and said second moiety
are covalently linked therebetween via a bon selected from the group consisting ofan ester
bond and an amide bond and the fatty acid is docosa-4,7,10,13,16,19-hexaenoic acid or?linolenic
acid.
21. A pharmaceutical composition comprising the chemical conjugate of any of claims 3-20
and a pharmaceutically acceptable carrier.
22. The conjugate ofanyone of claims 3-21 for use in treating an inflammatory disease or
disorder selected from the group consisting ofAlzheimer's disease, Parkinson's disease,
80
asthma, osteoarthritis, rheumatoid arthritis, pain associated with inflammation, primary
dysmenorrhea, Crohn's disease and ulcerative colitis.