Note: Descriptions are shown in the official language in which they were submitted.
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0
CYTOPROTECTIVE COMPOUNDS
Field of the Present Invention
The present invention relates to compositions and
methods for protecting mammalian cells from injury due to
intrinsic membrane lysis, oxidation and/or invasion by destructive
agents. In particular the present invention relates to compositions
and methods for treating against andJor prophylactically
inhibiting the injury causation. Even more particularly, the
present invention relates to bioactive agents and the use thereof
for treating or prophylactically inhibiting phospholipase mediated
injury, injury due to oxidation, and inflammation. In a specific
sense the present invention provides agents for preventing and/or
treating inflammation and cell destruction in mammalian tissue
and for protection and preservation of biologic nmaterial derived
from animals, humans and plants such as food and tissue samples.
In a very specific sense, this invention provides compositions that
are inhibitors of phospholipase and methods of making these
compositions.
Background of the Invention
The base structure of all living organisms is the cell
which is structurally defined by its membranous lipoprotein
envelope. The membranous network that holds the cell together
maintains the ionic balance and provides the receptors for
hormones and neurotransmitters that enable a cell to interact with
its environment. This is pertinent to interaction with neighboring
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0 cells which enable isolated cells, tissues, or whole organisms to
survive as both independent units and as participants in cellular
interactions, in vitro and in vivo.
External factors which govern cell function, renewal,
reproduction and death act via their effects on the phospholipid
bilayer and proteins of the cell membrane. This controls the
receptor-mediated signals and ionic fluxes which govern cell
responsiveness and survival. Damage to the cell membrane with
particular emphasis on lipid peroxidation, membrane oxidation
and the action of phospholipases, affects resistance to injury,
repair and host responses to environmental change and ionic and
osmotic integrity.
Pathological events in a host under clinical
circumstances can result in cellular insult, leading to loss of
membrane integrity. The events are mediated by factors which
digest and destroy cell membrane and propagate an injury by
producing a cascade of cell membrane changes. By interfering
with the cascade of external and internal events involving
membrane integrity and toxic changes which lead to cell death,
injury can be prevented, modified or reversed. This has been a
major role of anti-inflammatory agents in the past.
The most important presently used clinically effective
anti-inflammatory drugs include the corticosteroids and the
non-steroidal anti-inflammatory drugs (NSAIDs). Corticosteroids
inhibit the activity of cell phospholipases among other actions.
NSAIDs inhibit the metabolism by cyclooxygenase of arachidonic
acid released by phospholipases. These drugs act to control
inflammation and to minimize cell injury by regulating the
breakdown of phospholipids. These drugs also affect the action of
the products of phospholipid breakdown leading to the formation
of prostaglandins and leukotrienes which are produced in
increased quantities in inflammation and promote cell dysfunction
and injury.
In addition, cellular and extracellular phospholipases
may be activated by the generation of oxygen free radicals. This
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0 can establish a damaging cycle as phospholipase activation can
release free radicals which, in t~arn, activate more phospholipases.
In this regard, free radicals are produced from the fatty acids
which are released by the action of phospholipases and then
converted to prostaglandins and leukotrienes by cyclooxygenase
and lipoxygenase enzymes with oxygen free radical production as
a by product. Fatty acids and free_radicals are known to be prime
mediators in the cascade of reactions that result in membrane
injury, cell death and inflammation. Phospholipase A2 (PLA2), a
key enzyme in the metabolism of phospholipid, can promote fatty
acid release. PLA2 may be activated by a variety of factors
involving hormonal, neural, metabolic, or immunologic
pathways.
One of the hallmarks of inflammation and cell injury
is the breakdown of cellular membrane phospholipid.
Phospholipids are the major structural building blocks of the cell
mem'orane; they give rise to the barrier-structural and functional
properties of membranes and their integrity is crucial to normal
cell responsiveness and function. Phospholipid changes in cell
membrane integrity, particularly changes in fatty acids at the 2
position, alter the fluidity of cell membranes, cell receptor
function and the availability of cellular contents to the external
environment. The breakdown of phospholipid membranes results
in lysis of cells, produces holes in the cell membrane, affects ion
channels and membrane receptors which destroy cellular integrity
and functional responses.
During inflammation, phospholipases, from whatever
source, that are normally under the control of natural suppressor
systems, are activated to degrade membrane phospholipid which,
in turn, generates oxygen free radicals. PLA2 is a key enzyme
which is activated in inflammation to metabolize substrate
phospholipids and release free fatty acids. These fatty acids (i.e.,
arachidonate) released by PLA2 are converted to potent
biologically active metabolites, lysophospholipids, prostaglandins,
and leukotrienes. These are themselves substrates for other
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0 enzymes leading to the production of thromboxanes, platelet
activating factor and other substances, with the concomitant
generation of oxygen free radicals.
Phospholipases, particularly PLA2, as membrane
targeted enzymes, play an important role since expression of their
activity results in further production of inflammatory mediators
leading to membrane injury which propagates damage within the
cell itself or to adjacent tissue. Thus, the spread of injury from
the initial site to contiguous or distant sites can be promoted by
the activation and/or release of PLA2.
In addition to the intrinsic membrane-related tissue
breakdown via the activation of PLA2, phospholipases, and
particularly PLA2, are part of the normal defense system of the
body. PLA2 is found in human white blood cells (WBCs). WBCs
play a role in resisting infection, but when these cells are
mobilized to ward off injury and infection, PLA2 is released
from adherent and circulating WBCs and produces local tissue
activation which can increase the extent of initial injury. In
addition, WBCs adhere to blood vessel walls where they release
enzymes such as PLA2. WBCs also generate free radicals such as
superoxide, in large quantities, and thus promote damage to the
vascular endothelium, lung alveoli or to tissue sites contiguous
with WBC infiltration or concentration. Where inflammation is
found, WBCs are usually present in abundance and the WBCs
adhere to vascular endothelium, with subsequent release and
activation of PLA2 resulting in damage to vascular integrity
during shock and ischemia. Thus, in spite of being a prime
defense system of the body against infection, WBCs can also
damage the body by propagating injury and inflammation.
A classical description of inflammation is redness and
swelling with heat and pain. Inflammation has been defined as the
reaction of irritated and damaged tissues which still retain vitality.
Inflammation is a process which, at one level, can proceed to cell
death, tissue necrosis and scarring. At another level,
inflammation can be resolved with a return to normalcy and no
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0 apparent injury or with minimal changes, i.e., pigmentation,
fibrosis or tissue thickening with collagen formation related to
healing and scarring.
Microscopically, inflammation has been described as:
(1) atony of the muscle coat of the blood vessel wall; (2)
endothelial adherence of inflammatory cells followed by
migration of these cells from the vascular space into tissue.
The events described above are often mediated by
phospholipase activation, followed by fatty acid release and the
formation of free radicals. Cytokines, secreted by immune cells,
induce PLA2 secretion by their actions on a variety of cells.
Interleukin-6 stimulates hepatocytes to increase PLA2 secretion
many-fold. Interleukin-1 and tumor necrosis factor induce PLA2
secretion by endothelial cells and by chondrocytes. Thus, immune
cell products directly stimulate the hydrolysis of membrane
phospholipids and production of arachidonic acid metabolites by a
variety of target cells, amplifying the inflammatory response.
Alternatively, increased phospholipase activity can
relate to exogenous enzyme released from infecting pathologic
organisms such as viruses, bacteria, Rickettsia, protozoa, and
fungi. These pathogens often possess phospholipases as factors
intrinsic to their infectious activity. In the case of Naegleria
fowleri, a pathogenic amoeba with affinity for the brain,
destruction of brain membranes induced by phospholipases
secreted by Naegleria can occur at sites in the brain distant from
where the organism is localized. In another example toxoplasma
cannot enter the host cell if its PLA2 enzyme is inhibited by a
specific drug. What is needed to treat certain infections,
particularly intracellular pathogens, is an effective PLA2
inhibitor. Such an effective PLA2 inhibitor is particularly needed
in cases of protozoal infections for which there are few effective
antibiotics.
PLA2 is also one of the major toxic components of
snake venom. Bites of certain snakes inject venom containing
PLA2 into the wound, causing toxic and inflammatory responses
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0 which may be lethal. What is needed are inhibitors of PLA2
which may be administered to recipients of snake bites and bites
of other animals.
Pathologic effects of phospholipases may be local,
regional or systemic. These pathologic effects are governed by
the phospholipase enzyme released, the level of albumin, natural
inhibitors of enzyme action, and factors of diffusion, circulation
and tissue vulnerability based on intrinsic inhibitors or the
susceptibility of previously damaged or oxidized membranes or
proteins to phospholipase action.
Inflammation is associated with trauma, infection and
host defense reactions related to direct bacterial or virus killing
by the associated immune responses. In general, immune
responses can be both beneficial, protective or tissue damaging as
can be seen in their role to promote resistance to infection or cure
of infection. Alternatively, immune responses may produce
autoimmune phenomena that result in allergy, i.e., asthma,
urticaria, in graft versus host disease, in glomerular nephritis, in
rheumatic fever, or in lupus and rheumatoid arthritis.
In regard to the current treatment of inflammation,
corticosteroids are effective anti -inflammatory agents, but must
be used cautiously because they are powerful immunosuppressants
and inhibitors of fibroblastic activity necessary for wound healing
and bone repair. In addition, corticosteroids have powerful
hormonal activities and their toxic side effects involve
interference with wound repair and bone matrix formation,
sodium retention, potassium loss, bone demineralization,
decreased resistance to infection, and diabetes. Corticosteroids
also have effects on steroid formation, cataracts, blood pressure,
protein utilization, fat distribution, hair growth and body habitus.
Alternatively, the clinically active NSAIDs, such as aspirin,
indomethacin, ibuprofen, etc., work by inhibiting the conversion
of free fatty acids to prostaglandins. The side effects of NSAIDs
include gastric ulceration, kidney dysfunction and Reye's
Syndrome. Metabolites of prostaglandin can be either damaging
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0 or protective to cells depending on the structure of the
prostaglandin produced or utilized pharmacologically and the
route of administration, cell or tissue effected.
As discussed previously, in conjunction with fatty
acid release, leukotrienes are generated as part of phospholipid
cell membrane mediated injury produced by phospholipase
activation. These leukotrienes produced from membrane
phospholipid breakdown damage tissue through direct toxic
action, effects on ionic channels, and associated free radical
formation. Leukotrienes also damage tissue by indirect effects on
vascular smooth muscle or on the vascular endothelial lining via
effects on platelets, WBCs, or endothelial cells, or secondarily
through effects on constriction of smooth muscle. Leukotrienes
are responsible for smooth muscle constriction leading to
bronchospasm and the asthmatic attacks seen in allergy or
infectious asthma. Thus, there is an ongoing search for
leukutriene inhibitors for clinical application in the treatment of
allergy, asthma and ti ssue injury and inflammation.
Because the phospholipase-activated biochemical
pathway for the formation of prostaglandins and leukotrienes
derived from free fatty acids is branched, inhibition of one
branch of this pathway, as with NSAIDs, can create an imbalance
in these reactive metabolites. This imbalance may actually
aggravate inflammation and promote cell injury as evidenced by
the gastric ulcerative side effects of NSAIDs.
Due to these adverse effects of both steroids and
NSAIDs, there is great clinical interest in identifying
phospholipase inhibiting agents that do not have steroidal or
NSAID side effects, but like corticosteroids modulate the first step
leading to the production of injurious metabolites, fatty acids and
free radicals.
Free radicals, produced by white blood cells, tissue
injury or metabolic processes, are highly reactive chemical
species which, in the case of tissue injury, are most often derived
from respiratory oxygen. Oxygen, while necessary for energetics
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0 of life, is also a toxin which, as the chemically related superoxide,
or as peroxides, can damage tissue instead of supporting it. Free
radicals derived from oxygen are critical to damage produced by
radiation, inflammation, reperfusion tissue injury or through
excess oxygen inhalation or exposure. Free radicals are used by
white blood cells to destroy infecting organisms, but can, under
circumstances of shock, infection and ischemia, damage or
destroy the tissue they were meant to protect. Free radicals,
induced by radiation, oxygen exposure, chemical agents (i.e.,
alkylating agents, dioxin, paraquat) or white blood cell reactions
may damage tissue or be involved in mutational changes
associated with aging, radiation or chemotherapy injury, the
development of cancer, and hyperimmune proliferative disease
such as rheumatoid arthritis. In addition, these reactive chemical
species can, through oxidation of proteins, enhance the
vulnerability of proteins to protease digestion.
The exact pathologic mechanisms of many skin
inflammations, such as atopic dermatitis, are not clear, but
probably involve inflammatory cells which can secrete or respond
to PLA2. Allergic diseases involve tissue mast cells, which can be
primed or triggered by PLA2 for the release of their
inflammatory granule contents, such as histamine. These cells
also release additional PLA2. What is needed are inhibitors of
PLA2 that adequately penetrate skin after topical application and
possess prolonged anti-PLA2 activity.
Previous published studies have demonstrated high
levels of a proinflammatory PLA2 in human herniated vertebral
discs. The isolated enzyme is toxic to dorsal root ganglion cells in
culture and excised sciatic nerve. While not wanting to be bound
by this statement, it is believed that PLA2 may mediate
inflammation and nerve tissue damage in spinal cord injury and in
sciatic nerve inflammation and may also mediate a variety of
neurological inflammatory conditions. Recently, Stephenson et
al., (Neurobiology of Disease 3:51-63 (1996) have observed
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elevated cytosolic PLA2 activity in brains with Alzheimer's disease.
PLA2 also has the capability to induce severe, delayed neurotoxicity syndrome,
including extensive cortical and subcortical injury to forebrain neurons and
fiber pathways, when
injected intracerebroventricularly as described by Clapp et al. (Brain
Research 693:101-111,1995)
which may be referred to for further details. We have also observed that
preparations of PLA2 and
homogenates of human vertebral disks containing extracts of the nucleus
pulposus are inflammatory
when injected into the mouse paw and induce edema. Edema induced by human disk
homogenate
is maximal between 1-3 hrs and remains so for at least 6 hrs. These results
support the hypothesis
that leakage of nucleus pulposus from a herniated disk may promote
inflammation in human disk
disease. Accordingly, what is needed are inhibitors of PLA2 mediated
inflammatory processes.
Such inhibitors should alleviate the inflammation and resultant pain and
discomfort associated with
disk disease and other neurological inflammatory conditions.
Tissues that are excised from animals for subsequent transplantation into
recipients
often display damage following transplantation during reperfusion of ischemic
tissue. Both
ischemia and reperfusion increase PLA2 activity and release leading to
inflammatory processes with
marked activation of the vascular endothelium. These processes decrease the
probability of
successful transplantation thereby increasing the incidence of rejection and
the need for additional
immunosuppressive therapy. Such problems greatly increase morbidity and
mortality, increase the
costs of treatment and insurance, and result in lost time at work. What is
needed are drugs that
will inhibit PLA2 activity and enhance tissue preservation before
transplantation thereby decreasing
ischemia reperfusion injury.
Infections caused by parasites constitute a major public health problem
throughout
the world for humans and animals, annually resulting in significant incidence
of disease,
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0 suffering and death. Parasites such as those that cause malaria and
other protozoal parasites of animals and humans are especially
troublesome. We have found that the PLA2 inhibitor, quinacrine
(mepacrine), significantly reduced molting of larval forms of an
animal filarid. What is needed are new compounds that effectively
inhibit PLA2 activity for application to parasites such as those
causing malaria and other protozoal parasites injurious to animals
and humans.
A previous study by Clay et al. (Third International
Congress: Eicosanoids & Other Bioactive Lipids in Cancer,
Inflammation, & Radiation Repair, Abstract #162) reported that
the product of PLA2 activation, 1-acyl lysophospholipid, which
affects membrane fluidity, accumulates in stored blood and may
be taken up by white blood cells (WBCs) and used to make
platelet activating factor (PAF) thereby "priming" WBCs during
storage and promoting injury during subsequent transfusion. It
has been suggested that increased PLA2 activity may perturb cells
in storage. What is needed are compounds that protect blood cells
and other cells during storage so that these cells will not cause
problems when utilized.
Accordingly, what is needed are compounds and
methods of using these compounds which provide protection
against the deleterious effects of PLA2 activation. These
compounds should be capable of inhibiting PLA2, thereby
decreasing the PLA2 metabolites which are substrates for the
cyclooxygenase, 5-lipoxygenase, 12-lipoxygenase, and other
enzymatic pathways which lead to formation of cyclic
endooperoxides, prostaglandins (such as prostacyclin and
thromboxane), leukotrienes, and platelet activating factor. These
compounds should decrease inflammatory processes and free
radical production in a variety of tissues and cells. They should
be capable of being administered in vivo (topically, orally, by
injection and through other means), ex vivo and in vitro and
should also exhibit low or no toxicity. These compounds should
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0 display different solubilities in lipid and aqueous systems
depending on the mode of application and the desired target.
Summary of the Invention
The present invention provides both lipid and/or
water soluble compounds that are PLA2 inhibitors having
antioxidant properties and/or antiinflammatory properties. This
invention provides bioactive compounds which are oligomers
(dimers, trimers and tetramers, etc.) of fatty moieties that inhibit
PLA2 activity. The terms dimer, trimer, tetramer and pentamer
as used throughout the present description define the number of
fatty moieties present in the particular molecule. That is to say a
dimer has two fatty moieties, a trimer has three, a tetramer has
four, a pentamer has five, etc. The compounds of the present
invention possess at least one double bond to enhance their
anti-inflammatory and cytoprotective or tissue protective effects.
The compounds of the present invention may be used
for treating or prophylactically inhibiting phospholipase mediated
injury and/or injury due to oxidation. In a specific sense, the
present invention provides agents for preventing and/or treating
inflammation and cell destruction in mammalian tissue and for
protection and preservation of biologic material such as cells,
tissues, organs and fluids obtained from animals and humans. The
present invention also provides agents for protection and
preservation of food obtained from animals and plants, and for
cellulose products and wood products obtained from plants. The
compounds of the present invention protect phospholipid cell
membranes, and proteins from the effects of oxidative injury or
aging. These compounds of the present invention also inhibit free
radical reactions and thereby stabilize proteins for maintenance of
biologic half-life, anti-coagulant activity, and food preservation.
More specifically, the present invention provides
pharmacologically active, anti-phospholipase compounds. These
compounds are soluble and/or dispersible in a suitable carrier.
The compounds may exist as oligomers such as dimers, trimers,
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tetra.mers etc., or as combinations thereof. In accordance with the present
invention, the
compounds have at least two fatty moieties and contain at least one
unsaturated double
bond. Each fatty moiety may take a variety of forms, may possess the same or
different
functional groups, may be a different length. In one preferred form, the
compounds of the
present invention may have an acid group or any salt form thereof. The
compounds of the
present invention may also be present in ionized form.
Accordingly, an aspect of the present invention seeks to provide compositions
that inhibit the activity of the enzyme PLA2.
A related aspect of the present invention seeks to decrease levels of products
of the enzymes cyclooxygenase, 5-lipoxygenase, 12-lipoxygenase, prostacyclin
oxycyclase,
thromboxine synthase, and prostaglandin isomerase pathways by inhibiting the
activity of
PLA2.
Another aspect of the present invention seeks to provide methods of
synthesizing these compositions that inhibit the activity of PLA2.
It is also an aspect of the present invention to provide methods of treating
conditions associated with PLA2 activity.
Yet another aspect of the present invention seeks to provide a composition
and treatment for oxidative and free radical damage to cells, tissues and
organs in vitro and
in vivo.
Another aspect of the present invention seeks to provide methods of applying
an effective amount of the compositions to treat inflammation.
Another aspect of the present invention seeks to provide a-composition and
treatment for inflammatory processes.
It is also an aspect of the present invention to provide oral and topical
treatments for arthritis with the compositions of the present invention.
Yet another aspect of the present invention seeks to provide treatment for
pain using the composition of the present invention.
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Another aspect of the present invention seeks to provide oral and
topical treatments comprising administration of an effective amount of the
compositions of the present invention, for a variety of conditions, such
conditions
including, but not limited to, the following: reflex sympathetic dystrophy;
inflammation of the central and peripheral nervous system, diseases related to
inflammation of the nervous system including, but not limited to, Alzheimer's
disease, inflammation of spinal nerves, autonomic nerves and cranial nerves;
inflammatory radiculopathy; back pain including low back pain; myo-fascial
pain
syndromes; inflammation of the connective tissues including meninges, inflamed
and diseased facet joints, herniated disks, diseased disks, torn and injured
annulus
fibrosis, diseases of the joints, ligaments, cartilage and synovial membranes;
mastocytosis; shock including septic shock, anaphylactic shock, anaphylactic
shock resulting from radiocontrast administration, and shock resulting from
bacterial infections; bacterial infections; uremia; autoimmune disorders;
parasitic
infections including, but not limited to, malaria; inflammation including
allergic
inflammation; skin inflammation, itching, and other dermatologic disorders due
to allergic reactions, dry skin, erythema, solar, nuclear and other forms of
radiation, windburn, acne, psoriasis, eczema, reactions to chemicals and
toxins,
contact dermatitis, and reactions to plants including, but not limited to,
poison
ivy, poison oak, poison sumac; bites of insects including, but not limited to,
mosquitos, fire ants, chiggers, ticks, bees, spiders, fleas and flies; bites
of reptiles,
especially venomous reptiles, amphibians, and other animals; contact with
various
animals with venom on their skin such as poisonous frogs; pruritis associated
with local dermatologic or systemic disease; prevention of tissue ischemia
including tissue in vivo and tissue destined for transplantation, prevention
of
ischemia-reperfusion injury, prevention of ischemia-reperfusion injury in the
setting of resuscitation from hypovolemic shock, renal ischemia, myocardial
infarction, angina, and cardiac ischemia; endothelial inflammation,
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cardiotoxicity associated with administration of anti-cancer compositions,
inhibition of coronary or cerebral restenosis following angioplastic or other
vascular procedures, inhibition of platelet activity, especially in vessels
following various procedures such as angioplasty and after insertion of
catheters, shunts and other devices, inhibition of thrombin-activated platelet
aggregation; pulmonary disease including, but not limited to, asthma, cystic
fibrosis, inflammation of the lungs secondary to ischemia of the
gastrointestinal
system, adult respiratory distress syndrome, and other allergic and
inflammatory
reactions of the pulmonary system including inflammation of the tissues of the
upper respiratory system, allegic rhinitis, and respiratory distress syndrome;
inflammation of the gastrointestinal system including, but not limited to,
Crohn's disease, eosinophilic gastroenteritis, peritonitis, ulcerative
colitis, ulcers
of the small bowel and stomach, esophagitis, inflammation of the stomach,
inflammatory bowel disease; ocular inflammation; preservation of whole blood;
preservation of tissues, cells, and organs for transplantation; and protection
of
mitochondria.
Another aspect of the present invention seeks to provide a method
of applying an effective amount of the compositions of the present invention
through injection, topical, oral, or aerosol administration, for the treatment
of
inflammation resulting from the bites of insects, reptiles, amphibians, and
other
animals, especially venomous animals, such as venomous snakes.
Another aspect of the present invention seeks to provide a
composition and method for inhibition of platelet function.
It is an aspect of the present invention to provide a composition
for prevention and treatment for acute and chronic rejection of transplants,
and
for treatment of graft-vs-host disease and autoimmune diseases.
Another aspect of the present invention seeks to provide a
composition for the treatment of neoplastic disease.
It is another aspect of the present invention to provide an
easy to use topical therapeutic composition and topical
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treatment for various forms of arthritis and other inflammatory diseases,
including, but not
limited to, rheumatoid arthritis, inflammatory arthropathies, osteoarthritis,
gout, and lupus.
It is another aspect of the present invention to provide a composition and
treatment for parasitic infections including, but not limited to,
toxoplasmosis, malaria,
Naegleria fowleri, Dilofilaria immitis, nematodes, and pathogenic protozoans
such as
toxoplasma gondii, falciparum malaria, amebiasis, amoeba, and cryptosporidia.
It is another aspect of the present invention to provide the enhanced range of
motion and reduced pain provided to patients with reflex sympathetic dystrophy
following
topical or oral application of the compositions of the present invention.
By way of example, the invention pertains to
A-C4 ~cl D1
~F ~ C2-Dz
E C3-D3
wherein A comprises H, OH, a sugar moiety, an ether, an ester, an amide or
NHz, or an acid
or salt thereof. B is selected from the group consisting of C, -(CH2)õC-, N+,
and (CHZ)õ N+,
wherein n is an integer from 1 to 24, and the (CH2)õ chain may be
functionalized or
non-functionalized. Ct, C2, C3 and C4 may be different and are selected from
the group
consisting of (CH2)õ and (CH2-CH2-O)y, wherein n is an integer from 1 to 24,
the (CH2)n
chain may be functionalized or nonfunctionalized, and y is an integer from 1
to 12. D,, D2
and D3 are selected from the group consisting of fatty acid chains, which may
be different,
and are selected from the group consisting of fatty acid esters consisting of
CH3(CHz),,COO
and fatty acid amides consisting of CH3(CH2)õCONH, wherein n is an integer
from 1 to 32,
at least one of the fatty chains is unsaturated at one or more positions, the
chains may be of
different lengths and may be unsaturated at different locations, and H wherein
only D,, D2 or
D3 may be H. E is selected from the group consisting of H, a fatty acid amide
consisting of
CO(CH2)õCH3 or CO(CH2)õCOOH, wherein n is an integer from 0 to 24 and the
alkyl (CHZ)õ
chain may be functionalized or nonfunctionalized, or is the same as A-C4. F is
selected from
the group consisting of N, NR, P, P===O, CH and CR, wherein R is an alkyl
chain of I to
6 carbons which may be functionalized or non-functionalized, with the proviso
that when F
is N, E is not H, and when F is CH and C4 is -(CHZ)n , wherein n is less than
5, and E is
H, A is not H.
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These and other aspects, features and advantages of the present invention will
become apparent after a review of the following detailed description.
Description of the Preferred Embodiments
Cis-unsaturated, but not saturated fatty acids, inhibit in vitro PLA2
activities
derived from human platelets and human polymorphonuclear leukocytes (PMNs).
PLA2
activity is inhibited by oleic, linoleic, and arachidonic acids to
approximately the same extent
indicating that the presence of a single cis-double bond is as inhibitory as
multiple cis-double
bonds. In contrast, fatty acids containing trans-double bonds or
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0 methyl esters of cis-unsaturated fatty acids are less inhibitory of
PLA2 activity. Thus, it is hypothesized that the preferred
structural characteristics for inhibition of in vitro PLA2 activity
by unesterified fatty acids include at least one double bond. Oleic
acid inhibits in vitro PLA2 activity due to the presence of a single
double bond at the C-9 position. Oxidation of the sarcoplasmic
reticulum of muscle and of phospholipid membranes predisposes
them to phospholipase degradation. Phospholipid membranes that
have been oxidized at particular sites may appear intact and
maintain functional activity, but their oxidation makes them
vulnerable to degradation and destruction by PLA2 or other
phospholipases from endogenous or exogenous sources.
The observations illustrating the enhanced
vulnerability of phospholipid membranes to phospholipase
following oxidative and free radical mediated changes in cell
membranes and/or cisunsaturated fatty acids have been employed
in accordance with the present invention in the design of novel
anti-inflammatory and cytoprotective agents. The present
invention thus provides a biochemical and synthetic organic
approach to controlling the expression of PLA2 enzymes which is
vital to the maintenance of membrane structure.
Although not wanting to be bound by the following
hypothesis, it is believed that the number of available methylene
interrupted unsaturated double bonds is directly related to the
susceptibility of fatty acids to oxidation. This governs the ability
of unsaturated fatty acids to act as anti-oxidants. This property,
in conjunction with the anti-PLA2 activity of the fatty moiety
compounds of the present invention, markedly expands the scope
of the anti-inflammatory and cytoprotective activity of the new
agents disclosed herein. It is the property of the dual action of
those compounds, i.e., their action as PLA2 inhibitors with
varying anti-oxidant activity, that provides the spectrum of anti-
inflammatory activity in model systems that have direct
applicability to cytoprotection and the control of inflammation
and pathophysiology.
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0 A nonfunctional alkyl chain or group is known as a
hydrocarbon group because it contains only C-C and C-H single
bonds. A nonfunctional alkyl chain therefore contains the
maximum possible number of hydrogens per carbon which may
be represented as -CnH2n+,. A functionalized alkyl chain or group
has substituted for one or more hydrogens on the alkyl chain, one
or more atoms or groups of 4toms that have characteristic
chemical behavior. These atoms or groups of atoms that have
characteristic chemical behavior are also known as functional
groups. Included in these functional groups are C=C, OH,
COOH, SO3H, PO3H, NH2, -0-, and halides.
In summary, a single double bond in a fatty moiety
compound is sufficient to inhibit PLA2 activity in vitro and in
situ. The addition of multiple double bonds provides the
additional value of an increase in potent anti-oxidant activity
along with PLA2 inhibitory action. The present invention thus
provides compounds characterized by both anti-PLA2 and
varying anti-oxidant activity to maximize the anti-inflammatory
and cytoprotective action which is the key to the clinical value of
the compounds of the present invention.
In addition to inhibiting PLA2 activity, the
anti-oxidant action of these compounds protects proteins that
become increasingly vunerable to attack by proteases due to
oxidation. Thus, the cytoprotective PLA2 inhibitors of the
present invention, which have anti-oxidant activity as well, have
value both in stabilizing membrane phospholipid and in inhibiting
or preventing protein degradation or denaturation. This suggests
that the compounds of the present invention act to minimize
inflammation at its onset and will also interrupt the inflammatory
process in progress.
The compounds of the present invention block
arachidonic acid release from human polymorphonuclear cells
and endothelial cells, a reaction mediated by cellular and
secretory PLA2 activity. Thus, the compounds of the present
invention are potent, reversible PLA2 inhibitors, and, as such,
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0 these agents inhibit the proinflammatory response of the human
PMN and other inflammatory cells by inhibition of cellular and
secretory PLA2 activity. In addition, the compounds of the
present invention inhibit, to various extents, the free radical
activity in cells and tissues involved in the inflammatory process.
In a general sense, the present invention provides
pharmacologically active, an,ti-phospholipase compounds.
Preferably the compounds of the present invention are water or
lipid soluble antioxidants. The preferred compounds have at least
two fatty moieties and contain at least one unsaturated double
bond. The fatty moieties may be different from each other in
several features including, but not limited to, chemical
composition, functional groups, the degree of unsaturation, and
the length of the hydrocarbon chain. The compounds may also
have at least one organic group, one active acid group or any salt
form or ionized form thereof. The invention contemplates a
variety of configurations including oligomers such as dimers,
trimers, tetramers, and combinations thereof. Several of these
compounds provided in accordance with principles and concepts
of the present invention may be prepared as outlined in the
following specific examples.
The compounds of the present invention are not
generally hydrolyzed by pancreatic enzymes and are different
from glycerol-based compounds in terms of their chemistry and
metabolism. For example, we have observed that two compounds
of the present invention, PX-13 and PX-18, are resistant to
degradation in vitro by the commercially available pancreatic
enzyme preparation, pancreatin, obtained from Sigma Chemical
Company (St. Louis, MO). The resistance of PX-13 and PX-18 to
metabolism by pancreatic enzymes supports their stability after
oral administration.
The oligomers (dimers, trimers, tetramers,
pentamers, etc.) of unsaturated fatty acids and the other
compounds having at least one unsaturated straight chain fatty
radical as described above, affect fundamental membrane
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0 phospholipid reactions of phospholipase-induced degradation and
free radical peroxidation. The data set forth herein confirm with
experimental results that these compounds are potent anti-
inflammatory and cytoprotective agents.
The appropriate dosage of an effective amount of the
unsaturated fatty moiety compounds of the present invention for
treatment of mammals including humans, against phospholipase
mediated injury and/or inflammation should be in the range of
approximately 1 to 75 mg per kg (mg/kg) of body weight and
preferably approximately 2 to 50 mg/kg of body weight, with a
more preferable range of approximately 10 to 40 mg/kg of body
weight, when the compound is administered orally or
intraperitoneally (IP). When administered intravenously the
dosage should be approximately 50% of the oral or IP dosage to
achieve the same level of the drug in the blood. The described
dosage is also be appropriate for prevention of human platelet
aggregation or blood clotting. As is known to those skilled in the
art, therapeutic doses expressed in terms of amounts per kilogram
of body weight or surface area may be extrapolated from
mammal to mammal, including to human beings. The compounds
of the present invention may also be administered in an aerosol
manner, such as an intranasal spray to treat inflammation of the
nasal cavities, nasopharynx and adjacent regions or as inhaled
formulations to treat inflammation of the upper and lower
respiratory system.
The compositions of the present invention may be
formulated for administration in any convenient way by analogy
with other topical compositions adapted for use in mammals.
These compositions may be used in any conventional manner with
the aid of any of a wide variety of pharmaceutical carriers or
vehicles. The compounds of the present invention described above
can be provided as pharmaceutically acceptable formulations
using formulation methods known to those of ordinary skill in the
art. These formulations can be administered by standard routes.
In general, the combinations may be administered by the topical,
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0 transdermal (including ionophoretic administration), buccal, oral,
rectal or parenteral (e. g., intravenous, subcutaneous or
intramuscular) route. In addition, the combinations may be
incorporated into biodegradable polymers allowing for sustained
release of the compound, the polymers being implanted in the
vicinity of where drug delivery is desired, for example, at the site
of a tumor. The biodegradable- polymers and their use are
described, for example, in detail in Brem et al., J. Neurosurg.
74:441-446 (1991)
A pharmaceutically acceptable solvent is one which is
substantially non-toxic and non-irritating under the conditions
used and may be readily formulated into any of the classical drug
formulations such as powders, creams, ointments, lotions, gels,
foams, aerosols, solutions and the like. Particularly suitable
solvents include, but are not limited to, water, ethanol, acetone,
glycerin, propylene carbonate, dimethylsulfoxide (DMSO), and
glycols such as 1,2-propylene diol, i.e., propylene glycol, 1,3-
propylene diol, polyethylene glycol having a molecular weight of
from 100 to 10,000, dipropylene glycol, etc. and mixtures of the
aforementioned solvents with each other.
A topical cream may be prepared as a semi-solid
emulsion of oil in water or water in oil. A cream base
formulation by definition is an emulsion, which is a two-phase
system with one liquid (for example fats or oils) being dispersed
as small globules in another substance (e.g., a glycol-water solvent
phase) which may be employed as the primary solvent. The
cream formulation may contain fatty alcohols, surfactants,
mineral oil or petrolatum and other typical pharmaceutical
adjuvants such as anti-oxidants, antiseptics, or compatible
adjuvants. A typical cream base formulation is as follows:
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Water/glycol mixture 50-99 parts by weight
(15% or more I col)
Fatty Alcohol 1-20
Non-ionic Surfactant 0-10
Mineral Oil 0-10
Typical Pharmaceutical 0-05
Adjuvants
Active In edients 0.0001-10
0
A "classical" ointment is a semi-solid anhydrous composition
which may contain mineral oil, white petrolatLUm, a suitable solvent such as
a glycol and may include propylene carbonate and other pharmaceutically
suitable additives such as surfactants, for example Span''' and Tween', or
wool fat (lanolin), along with stabilizers such as antioxidants and other
adjuvants as mentioned before. Following is an example of a typical
"classical" ointment base:
White Petrolatum 40-94 parts by weight
Mineral Oil 5-20
Gl - col Solvent 1-15
Surfactant 0-10
Stabilizer 0-10
Active In redients 0.0001-10
Additionally, the compounds may be formulated as
oral, parenteral, subcutaneous, intravenous, intraarticular,
intramuscular, intraperitoneal, intralesional and otherwise
systemic compositions. Depending on the intended mode, the
compositions may be in the form of solid, semi-solid, or liquid
dosage forms, such as, for example, tablets, suppositories, pills,
capsules, powders, liquids, suspensions, or the like, preferably in
unit dosage forms suitable for single administration of precise
dosages.
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0 The compositions will include a conventional
pharmaceutical carrier or excipient and a therapeutically effective
amount of a compound of the present invention and, in addition,
may include other medicinal agents, pharmaceutical agents,
carriers, adjuvants, etc.
The amount of active compound administered will, of
course, be dependent on the huxnan or animal subject being
treated, the severity of the affliction, the manner of
administration and the judgment of the prescribing clinician.
Typical compositions contain approximately 0.01-
95% by weight of active ingredient, with the balance one or more
acceptable non-toxic carriers. The percentage of active
ingredient, will, of course, depend upon the dosage form and the
mode of administration.
For solid compositions, conventional non-toxic solid
carriers including, for example, pharmaceutical grades of
mannitol, lactose, starch, magnesium stearate, sodium saccharin,
talcum, cellulose, glucose, sucrose, magnesium carbonate, and the
like may be used. The active compound as defined above may be
formulated as suppositories using, for example, polyalkylene
glycols and propylene glycol, as the carrier. Liquid
pharmaceutically administerable compositions can, for example,
be prepared by dissolving, dispersing, etc. an active compound as
defined above and optional pharmaceutical adjuvants in a carrier,
such as water, saline, aqueous dextrose, glycerol, ethanol, and the
like, to thereby form a solution or suspension. If desired, the
pharmaceutical composition to be administered may also contain
minor amounts of nontoxic auxiliary substances such as wetting or
emulsifying agents, pH buffering agents and the like, for example,
sodium acetate, sorbitan monolaurate, triethanolamine, sodium
acetate, triethanolamine oleate, etc. Actual methods of preparing
such dosage forms are known, or will be apparent, to those skilled
in this art, for example, see Remington's Pharmaceutical Sciences,
Mack Publishing Company, Easton, PA, 15th edition, 1975. The
composition or formulation to be administered will, in any event,
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0 contain a quantity of the active compound(s) in an amount
effective to alleviate the symptcms of the subject being treated.
For oral administration of the compounds of the
present invention, a pharmaceutically acceptable non-toxic
composition is formed by the incorporation of any of the
normally employed excipients, such as pharmaceutical grades of
mannitol, lactose, starch, magnesium stearate, sodium saccharin,
talcum, cellulose, glucose, sucrose, magnesium, carbonate, and the
like. Such compositions take the form of solutions, suspensions,
tablets, pills, capsules, powders, sustained release formulations
and the like. Such compositions may contain 2%-95% active
ingredient, preferably 5%-90%.
Parenteral administration is generally characterized
by injection, either subcutaneously, intramuscularly or
intravenously. Injectables can be prepared in conventional forms,
either as liquid solutions or suspensions, solid forms suitable f or
solution or suspension in liquid prior to injection, or as
emulsions. Suitable excipients are, for example, water, saline,
dextrose, glycerol, ethanol or the like. In addition, if desired, the
pharmaceutical compositions to be administered may also contain
minor amounts of non-toxic auxiliary substances such as wetting
or emulsifying agents, pH buffering agents and the like, such as
for example, sodium acetate, sorbitan monolaurate,
triethanolamine oleate, etc.
Ointments for topical application may be prepared by
incorporating approximately 0.1 to 10% of the compound as an
oil or salt form into an ointment base containing emulsifying
agents such as stearic acid, triethanolamine and/or cetyl alcohol.
The formulation may also include ingredients such as glycerol,
petrolatum, water and preservatives as required.
Water based lotions may contain the compounds of
the present invention as an oil or solid in amounts ranging from
approximately 0.1% to 5.0% by volume. Such lotions may
contain glycerine andlor bentonite as suspending agents as is well
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0 known in the art. The present invention may also be incorporated
into creams.
The compounds may also be incorporated into
classical (one or two phase) or non-classical (aqueous emulsion)
aerosol formulations. Such fonnulations include the compounds
and an appropriate propellant carrier in which the compounds are
dissolved or dispensed. In the classical form the active
ingredients are generally used as an oil dispersion or in solution
in an organic solvent such as ethanol. In the non-classical form
the active ingredient is dissolved in water. In each case the
concentration of the active ingredient in the carrier may be about
approximately 0.1 to 10% by weight or volume.
Of particular advantage is the fact that the
unsaturated straight chain fatty moiety compounds described
above function pharmacologically at the site of inhibitory action
for the arachidonate cascade and preferentially affect stimulus-
induced mobilization of arachidonate. Inhibition of PLA2
depresses the production of both prostaglandins and leukotrienes
in stimulated or inflamed cells. Importantly, the compounds
described above have a much more pronounced effect on
stimulus-induced, than on controlled release of arachidonate
indicating a selective effect on the former. Moreover, when
phospholipids are peroxidized, the polymer compounds described
above are capable of inhibiting the degradation of such lipids by
lysosomal phospholipase C, indicating that these compounds can
protect already damaged (oxidized) membranes.
Thus multiple actions are responsible for the anti-
inflammatory activity of the fatty moiety compounds of the
present invention, and on the basis of inflammatory models, it is
evident that these compounds can effectively rival or replace both
currently available steroids and NSAIDs in the treatment of
inflammation, making the fatty moiety compounds of the present
invention candidates for clinical application and usefulness in
localized and systemic injury and disease.
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0 The fatty moiety compounds described above, by
protecting lipid membranes and possessing anti-oxidant activity,
are potent anti-oxidants for preservation, not only of living cells
and tissues, but their action makes them effective as preservatives
of biological materials from animal, human or plant origin, and
as preservatives of chemical agents subject to oxidative injury.
For purposes of protecting and preserving biological materials
subject to oxidation injury, the fatty moiety compounds of the
present invention may be used at concentrations of approximately
0.1 to 100 M. These molarities are calculated as the molarity
that would be obtained if the drug were dissolved in a weight of
water which is the same as the weight of the biological material to
be preserved. For example, in vitro, anti-oxidant and/or anti-
phospholipase applications, concentrations of from about
approximately 0.1 to about 500 M should be effective.
This invention is further illustrated by the following
examples, which are not to be construed in any way as imposing
limitations upon the scope thereof. On the contrary, it is to be
clearly understood that resort may be had to various other
embodiments, modifications, and equivalents thereof which, after
reading the description herein, may suggest themselves to those
skilled in the art without departing from the spirit of the present
invention andlor the scope of the appended claims.
EXAMPLE 1
Ricinoleic Acid Related Compounds
In one preferred form, the compound may be a
derivative of ricinoleic acid having a structural configuration as
set forth below
il
CH3-(CH2)5- i H-CH2-C H~H-(CH2)7-C-R2
OR1
where R 1 is an alkyl group or acyl group, which may be
functionalized or non-functionalized, which includes an active
acid group or salt form thereof, and R2 is an alkoxy or
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0 alkylamino group which includes one of the fatty moieties. More
particularly, in the foregoing compound, R2 may be
-Y-(CH2)n-(CH= CH-CH2)m-(CH2)X-CH3
or
-Y-CH-(CH2)n-CH3 O
11
(CH2-CH=CH)m-(CH2)x-C- OH
wherein n is an integer from 1 to 18, m is an integer from 1 to 4,
x is an integer from 0 to 12, and Y is -O- or -NH-, or -NR-
wherein R is a functionalized or non-functionalized alkyl group of
1 to 6 carbons.
In one case the R 1 moiety may be obtained by
esterification of the 12-position hydroxy group of ricinoleic acid
with an acid group of a divalent acid such as sebacic acid, fumaric
acid, maleic acid, oxalic acid, succinic acid, or organic moieties
including, but not limited to, ethylenediamine tetraacetic acid
(EDTA) and its analogs and derivatives, and the R2 moiety may
be obtained by esterification of the 1-position carboxy group of
ricinoleic acid with the hydroxyl group of another ricinoleic acid
molecule or of some other fatty alcohol such as, for example,
oleyl alcohol, linoleyl alcohol, linolenyl alcohol, arachidonyl
alcohol or cis-5, 8, 11, 14, 17-eicosapentaenyl alcohol.
Alternatively, the R2 moiety may be obtained by amidification of
the 1-position carboxy group of ricinoleic acid with the amine
group of an unsaturated fatty amine such as oleyl amine, linoleyl
amine, linolenyl amine, arachidonyl amine or cis-5, 8, 11, 14, 17-
eicosapentaenyl amine.
EXAMPLE 2
Alternatively, the compounds of the present invention
may have the generic structural formula as set forth below;
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O
11
CH3-(CH2)5- i H-CH2-CH==CH-(CH2)7-C-R2
0 OR~
however, in this case Ri may be an alkyl group which may be
functionalized or non-functionalized and includes an active acid
group or one of the fatty moieties, and R2 may be either a
hydroxy group or an alkoxy group which includes fatty moieties.
In this alternative case, when R2 is a hydroxy group, Rl may be
derived by esterification of the 12-position hydroxy group of the
ricinoleic acid, with, for example, the acid group of oleic acid,
linoleic acid, linolenic acid, arachidonic acid or cis-5, 8, 11, 14,
17-eicosapentaenoic acid. Thus, in this case, Ri may be a fatty
moiety having one of the following configurations:
0
11
-C-(CH2)n-(CH==CH-CH2)m-(CH2)x--CH3
~ 11 0
-C-CH~H- C-OH
O 0
II II
-C-(CH2)n-C-OH
or
0
11
-C-(CH2)n (CH=CH-CH2)m-CH-(CH2)x-CHs
OR
where n is an integer from 1 to 18, m is an integer from 1 to 4, x
is an integer from 0 to 12, and R is H a fatty moiety or an alkyl
group which may be functionalized or non-functionalized.
The invention contemplates compounds of a variety
of configurations including, for example oligomers including
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0 dimers, trimers, tetramers and combinations thereof. Dimers of
ricinoleic acid are prepared for example, by esterifying the 12-
position hydroxy groups of two molecules of ricinoleic acid with
the carboxy groups of a diacid such as sebacic acid, fumaric acid,
maleic acid, oxalic acid or succinic acid.
A trimer of ricinoleic acid is prepared, for example,
by esterifying the 12-position hydroxy groups of three molecules
of ricinoleic acid with the carboxy groups of a tri-acid such as
cis-aconitic acid.
A tetramer of ricinoleic acid is prepared, for
example, by esterifying the 12-position hydroxy groups of four
molecules of ricinoleic acid with the carboxy groups of a tetra-
acid such as ethylenediaminetetraacetic acid.
Various compounds may be linked together by
esterification through one of the free acid groups. Thus, the acid
groups may be converted to acid chloride groups and reacted with
hydroxy or amine groups of a divalent compound.
In each case, the compounds of the present invention
include at least one unsaturated bond and at least two fatty
moieties. Desirably, each compound may also include at least one
active acid group or any ionized form or salt form thereof. Some
of the preferred acid groups include but are not limited to
sulfonyl, sulfonate, phosphoryl, phosphonate, carboxyl and
carboxylate.
EXAMPLE 3
Synthesis of N,N-Bis(oleic acid-12-oxycarbonylmethyl)-N-N'
bis(carboxylmethyl) ethylenediamine
This compound is represented by the following structural
configuration:
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0
CH3-(CH2)5-CH-CH2-CH=CH-(CH2)7-C
"OH
O
C=0
CH2 O
N-CH2--C'OH
CH2
CH2 /0
N--CH2-C
CH2 OH
C=0
O
~ /%
CH3-(CH2)5-CH-CH2--OH=CH-(CH2)7-C 1, OH
0
To 6.22 g (20.8 mmol) of ricinoleic acid and 4.00 g
of triethylamine in 150 mL THF was added 2.31 g (9.0 mmol) of
ethylenediamine tetraacetic acid dianhydride. After stirring 15
min at 40 C, 20 mL of acetonitrile was added, since
ethylenediamine tetraacetic acid diaiihydride was not dissolved
completely. The solution was stirred for 72 h under reflux and
gradually turned yellow during this time. The solvent was
removed under reduced pressure. The mixture was suspended in
100 mL of ether and extracted twice with 50 mL of saturated
aqueous sodium chloride. After drying the ether solution, the
solvent was removed under reduced pressure. The residue was
suspended in methanol and filtered. The solvent was removed
under reduced pressure. The residual oil was purified by
chromatography on silica gel, e:uted with a gradient of
acetone/methanol (70:30 v/v to 0:100 v/v). N,N-bis(oleic acid-12-
oxycarbonylmethyl)-N-N'-bis(carboxylmethyl) ethylenediamine
was isolated as a yellow solid with a yield of 1.23 g (14 %). This
compound is readily soluble in water.
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0 N,N-Bis(oleic acid-l2-oxycarbonylmethyl)-N-N'
bis(carboxylmethyl) ethylenediamine exhibits an EC50 value of 5
to 10 M in the inhibition of PLA2 activity.
EXAMPLE 4
1, 3-Bis(12-hydroxyoleoylamino)-2-hydroxypropane
This compound is represented by the following structural
configuration:
0 OH
II 1
/CH2 NH-C-(CH2)7-OH=CH-CH2-CH-(CH2)-C Ha
HO-CH 0 OH
CH2 NH-C-(CH2)7-CH=CH-CH2-CH-(CH2)5-CH3
To a solution of 12.60 g (31.9 mmol) of ricinoleic
acid N-hydroxysuccinimide ester and 1.8 g (20 mmol) of
1,3-diamino2-hydroxypropane in 125 mL of methylene chloride
was added a catalytic amount of sodium bicarbonate. The
suspension was stirred for 48 h at 35 C. The mixture was
filtered and the solvent was removed under reduced pressure.
The oil was suspended in water and extracted 3 times with
methylene chloride. The solvent was removed and the yellow
heavy viscous oil was dried in vacuo. The yield was 930 mg.
EXAMPLE 5
1,3-Bis(oleoylamino)-2-hydroxypropane
This compound is represented by the following structural
configuration:
0
11
/CH2 NH-C-(CH2)7-CH~H-(CH2)~Ha
HO-CH\
0
-(CH2)7-CH=CH-(CH23
CH2 NH--C }7 CH
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0
To a solution of 8.60 g (95.4 mmol) of
1,3-diamino-2-hydroxypropane in 400 mL of water was added
200 mL of methylene chloride. The emulsion was cooled to 0 C
and 85.0 g (280 mmol) of 85% oleoyl chloride were added. The
mixture was stirred vigorously for 1 h at 0 C and for 1 h at
room temperature. The resulting precipitate was recovered by
filtration and was dried in vacuo. Reprecipitation from ethanol
provided a white solid with a yield of 31.10 g (53%) and a
melting point of 91-92 C.
EXAMPLE 6
1,3-Bis(oleoylamino)-2 propyl succinic acid monoester
This compound is represented by the following structural
configuration:
O'C~OH
I
CH2
CH2 ~- _
0=C CH2 NH-C (CH2)7-CH=CH (CH2)7 3
O-CH
O
CH2 N H--C-(CH2)~-CH=CH-(CH2) ~H3
To a solution of 505 mg (0.817 mmol) of 1,3-
bis(oleoylamino)-2-hydroxypropane and 140 mg (1.40 mmol) of
succinic anhydride in 20 mL of acetonitrile and 20 mL of THF
was added 5 mL of triethylamine. The suspension was stirred at
35 C. After 15 min succinic anhydride was completely
dissolved. The solvent was removed under reduced pressure after
48 h. The yellow oil was suspended in water and extracted three
times with ether. The solvent was removed under reduced
pressure and the yellow oil 1,3 bis(oleoylamino)-2-propyl
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0 succinic acid monoester was dried in vacuo. The yield was 380
mg (65%).
In addition to the foregoing compounds, some of
which comprise derivatives of ricinoleic acids, additional
compounds are included within the broad scope of the present
invention. Some of these compounds are defined structurally by
the following Examples.
EXAMPLE 7
One such class of compounds is defined by the
following generic formula,
~H3+X
Rj- i -CH2-O-R
CH2-O-R
wherein X is an organic or inorganic anionic moiety such as
bicarbonate, acetate, citrate, succinate, p-toluenesulfonate
(Example 13), or halide such as chloride, fluoride, bromide or
iodide, or phosphate, sulfate and other pharmaceutically
acceptable anions; wherein R1 is - CH2 - O- R, a hydrogen, or an
alkyl group or chain which may be functionalized; and wherein
the R groups may be the same or different and each R group is a
fatty moiety. In this form of the present invention the R groups
may have the following structural formulation:
0
11
--C-(CH2)n (CH=CH-CH2)m-(CH2)x'-CH3
where n is an integer from 1 to 18, m is an integer from 1 to 4, x
is an integer from 0 to 12, and the R groups may be the same or
different.
A method for preparing a particularly preferred
compound having the foregoing structural configuration, p-
___
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0 toluene sulfonic acid salt of tristrioleate, is described in Example
13.
EXAMPLE 8
Generic TES Related Compounds
Alternatively, the compounds of the present invention
may have the generic formula:
A-C4 \ / Ci -Di
/F-B~C2-C2
E C3_ p3
In this structure, A comprises H, OH, a sugar moiety,
an ether, an ester, an amide or NH2, or an acid or salt thereof.
Some of the preferred acids that may be substituted for A include
but are not limited to COOH, SO3H or PO3H.
B is a connecting group selected from the group
consisting of C, -(CH2)nC-, N+, and (CH2)n N+ wherein n is an
integer from 1 to 24, and the -(CH2)n chain may be
functionalized or non-functionalized.
Cl, C2, C3 and C4 are connecting groups selected
from the group -(CH2)n- where n is an integer from 1 to 24,
wherein the -(CH2)n- chain may be functionalized or
nonfunctionalized. C1, C2, C3 and C4 may also be selected from
the group consisting of poly(ethylene oxide) of the formula (CH2-
CH2-O)y wherein y is an integer from 1 to 12. Cl, C2, C3 and
C4 may be the same or different.
D1, D2 and D3 are fatty chains consisting of fatty
acid esters of the form CH3(CH2)n COO or fatty acid amides of
the form CH3(CH2)n CONH wherein n is an integer from 0 to 32.
At least one of the fatty chains is unsaturated at one or more
positions, and D 1, D2 and D3 may be the same or different with
respect to length and degree of unsaturation. D1, D2 or D3 may
also be H provided no more than one of D 1, D2 and D3 is H in
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0 any one compound. In the generic formula listed above, the fatty
acid chains of the molecule may be comprised of (CH2)n wherein
n is an integer from 1 to 32. These fatty acid chains may each be
unsaturated at one or more sites and may be of different lengths
from 1 to 32 carbon atoms.
E is H, or is the same as A-C4, or is a fatty acid
amide of the form CO(CH2)nCH3 or CO(CH2)nCOOH where n is
an integer from 0 to 24. The alkyl (CH2)n chain may be
functionalized or nonfunctionalized.
F is selected from the group consisting of N, NR, P,
P=O, CH or CR, wherein R is an alkyl chain of 1 to 6 carbons
which may be functionalized or non-functionalized.
More specifically, the compounds may also have the formula.
NH-Z-A
~CH2- O-R
C\ H2-O-R
CH2 O-R
wherein the R groups may be the same or different and each R is
a fatty moiety as set forth below,
0
11
-C'-(CH2)n-(CH=CH-CH2)m-(CH2)x-CH3
wherein n is an integer from 1 to 18, m is an integer from I to 4,
and x is an integer from 0 to 12.
In this form of the present invention Z represents a
C1 to C5 aliphatic moiety, which may be functionalized or non-
functionalized, and A represents an organic acid moiety or salt
form thereof or any organic acid radical. It is to be understood
that the acidic groups and the NH groups in the generic structure
described above may be present in ionized form. Some of the
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0 preferred acid groups that may be substituted for A include but
are not limited to COOH, SO3H or PO3H.
EXAMPLE 9
Synthesis of 2-[Tris(oleoyloxymethyl)methylaminoJ-1-
ethanesulfonic acid also called PX-13 or TES Trioleate
To a 250 ml single-neck, round-bottom flask 1.0 g
(4.26 mmoles) of 2-[tris(hydroxylmethyl)methylamino]-1-
ethanesulfonic acid (TES; Aldrich; 99% purity) and 25.0 mL of
anhydrous dimethyl formamide (DMF) were added. The flask
contents were then cooled to 0 C in an ice-water bath. Oleoyl
chloride (Aldrich, technical grade) 5.25 g (17.45 mmoles), was
added dropwise over a 5 minute period. The reaction mixture
was stirred at room temperature for 4 days. We have also
performed this reaction by stirring for a shorter period of time at
elevated temperatures. The DMF was removed at 40-45 C under
reduced pressure. The residue was a viscous oil which was
transferred to a flask containing 200 mL acetone and vigorously
stirred until a slightly cloudy solution formed. This solution was
refrigerated overnight. The precipitate formed was collected by
filtration and thoroughly extracted with distilled acetone (20 mL)
five times. The product was dried in vacuo at room temperature
for 24 h producing a yield of 2.32 g (52%), and has the following
structural configuration:
0
11
CH3(CH2)7CH=CH(CH2)7COCH2
0
II
CH3(CH2)7CH=CH(CH2)7COCH2 ~NHCH2CH2SO3H
0
11
CH3(CH2)7CH=CH(CH2)7COCH2
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0 Another method of synthesizing
2-[Tris(oleoyloxymethyl) methylamino]-l-ethanesulfonic acid is
provided in the following paragraph.
To 49.0 g (219 mmol) of 2-
[tris(oleoyloxymethyl)methylamino]-1-ethanesulfonic acid in 500
mL of CH3CN was added 385 mL (990 mmol) of 85% oleoyl
chloride. The orange-brown suspension was stirred under
nitrogen at 35 C for 36 h. Then 500 mL of acetone were added
into the brown suspension. The reaction mixture was put in the
freezer overnight. The light brown solid was reprecipitated from
ethanol, then from methanol/acetone (approximately 1:9) and
from ethanol again. The white solid was dried in vacuo. The
yield was 152.2 g (70%) and the melting point was 62-64 C.
EXAMPLE 10
Synthesis of N-[Tris(oleoyloxymethyl)methyl]-N-(2-sulfoethyl)
succinic acid monoamide
This compound is represented by the following structural
configuration:
O~C.0OH
iH2 O
11
CH CH2 O-C-(CH2)7-CH =CH-(CH2)7-CH3
t 2
O
0=C H
N -C CH2 O--C-(CH2)7-CH~H-(CH2)~ CHs
CH2 ~ --H
i H2 CH2 O-C-(CH2)7-CH~H-
(CH2)~ 3
HO3S
To 566 mg (0.553 mmol) of 2-[tris(oleoyloxymethyl)
methylamino]-1-ethanesulfonic acid in 10 mL THF was added 410
mg (4.06 mmol) of triethylamine. After 5 min 96 mg (0.960
mmol) of succinic anhydride was added. The yellow solution was
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0 stirred for 18 h under reflux. The solvent was removed under
reduced pressure. The residual oil was purified by
chromatography on silica gel, eluted with an ethyl
acetate/methanol gradient (25:75 vol/vol to 0:100 vol/vol).
N-[tris(oleoyloxymethyl)methyl]-N-(2-sulfoethyl) succinic acid
monoamide was isolated as a yellow heavy viscous oil with a yield
of 240 mg (38 %).
EXAMPLE 11
Synthesis of N-[Tris(oleoyloxymethyl)methyl]-N-(2-sulfoethvl)
tartaric acid monoamide
O'C~OH
HO-CH ~
6H-OH C H 2 O -C - (C H 2) 7-C H =C H-(CH2)7-CH3
O~ ~
N CH2 O-C-(CH2)7-CH==CH-(CH2)7Ha
CHZ ~_ - -CH3
i H2 CH2 O--C (CH2)7-CH~H (CH2)7
HO38
Synthesis of N-[tris(oleoyloxymethyl)methyl]-N-(2-
sulfoethyl) tartaric acid monoamide was accomplished in two
steps. First, reaction of 2-[tris(oleoyloxymethyl)methylamino]-1-
ethanesulfonic acid with diacetyltartaric acid anhydride resulted
in N-[tris(oleoyloxymethyl)methyl]-N-(2-sulfoethyl)-
2,3-diacetyltartaric acid monoamide. This was hydrolyzed with
sodium hydroxide to yield N-[tris(oleoyloxymethyl)methyl]-N-(2-
sulfoethyl) tartaric acid monoamide. The reaction is described in
detail below.
First the synthesis of N-[tris(oleoyloxymethyl)methyl]-N-
(2-sulfoethyl)-2,3-diacetyltartaric acid monoamide was performed
by adding added 1.23 g (11.6 mmol) of triethylamine to 2.07 g
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0 (2.02 mmol) of
2-[tris(oleoyloxymethyl)methylamino]-1-ethanesulfonic acid in
150 mL THF. After 5 min 793 mg (3.66 mmol) of
diacetyltartaric anhydride was added. The yellow solution was
stirred for 18 h under reflux. The solvent was removed under
reduced pressure. The residual oil was purified by
chromatography on silica gel, eluted with an ethyl
acetate/methanol gradient (20:80 vol/vol to 0:100 vol/vol) and a
trace of acetic acid. N-[tris(oleoyloxymethyl)methyl]-N-(2-
sulfoethyl)-2,3-diacetyltartaric acid monoamide was isolated as a
yellow heavy viscous oil with a yield 1.36 g (56 %).
Next, to 1.30 g (1.05 mmol) of
N-[tris(oleoyloxymethyl)methyl]-N-(2-sulfoethyl)-
2,3-diacetyltartaric acid monoamide in 50 mL THF was added 20
mL 0.5 aqueous sodium bicarbonate. After 60 min treatment with
ultrasound the solvent was removed under reduced pressure. The
residual mixture was suspended in water and extracted with ether.
N- [Tris(oleoyloxymethyl)methyl] -N-(2-sulfoethyl) tartaric acid
monoamide was isolated as a yellow heavy viscous oil with a yield
of 1.12 g (92 %).
EXAMPLE 12
Tris(oleoyloxymethyl)methylamine p-Toluene Sulfonic Acid
Tris(hydroxymethyl) aminomethane (Tris, Aldrich)
0.54 g (4.4 mmoles), 5.0 g (17.7 mmoles) of oleic acid (Aldrich),
and 1.26 g (6.6 mmoles) of p-toluenesulfonic acid monohydrate
(Sigma) were mixed in 50 mL of toluene and placed in a 100 mL
round-bottomed single-necked flask equipped with a Dean-Stark
trap and a Teflon-coated magnetic stirring bar. After bubbling
the reaction mixture with N2 gas for 10 minutes, the reaction
mixture was stirred and heated to reflux. The reaction was
continued until a stoichiometric amount of water was recovered
(0.38 ml). After removal of a small amount of undissolved
material by filtration, the toluene was removed by
rotoevaporation to yield a white waxy product. This product was
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0 purified on a silica gel column (Aldrich 230-400 mesh, 2.0 x 55
cm) with 8:2 petroleum ether (bp 60-90 C) - ethyl acetate as
developing solvent. After eluting with developing solvent the top
uncolored layer was carefully removed and the product extracted
with ethyl acetate from the silica gel. The solvent was removed
by rotoevaporation and the product recovered as a
tris(oleoyloxymethyl)methylamine p-toluene sulfonic acid having
the following structural configuration:
NH3 03 S H3
O
11
CH2- 0z)7-CH==CH-(CH2)7--CH3
C-CH2- O-C-(CH2)7-CH==CH-(CH2)7--CH3
\ ~T
CH2- O--C-(CH2)7-CH==CH-(CH2)7--CH3
In this procedure, the amine group of the Tris is protected from
reacting with the fatty acid because it is in the form of a p-toluene
sulfonate salt. Moreover, the p-toluene sulfonic acid acts as a
catalyst for the esterification between the alcohol functions on the
Tris and the fatty acid.
EXAMPLE 13
Generic Formula for PX-18 Related Compounds
These compounds are represented by the following structural
configuration:
/ ' -D'
A-Cg B\
C2-D2
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0 A comprises H, OH, a sugar moiety, an ether, an
ester, an amide or NH2, or an acid group or salt thereof. Some
of the preferred acids include COOH, SO3H, and PO3H.
B is N, NR, P, P=O, CH or CR wherein R is an alkyl
chain of 1 to 6 carbons which may be functionalized or non-
functionalized.
C1, C2 and C3 are connecting groups selected from
the group consisting of -(CH2)n- wherein n is an integer from 1
to 24, and the -(CH2)n- chain may be functionalized or non-
functionalized. C1, C2 and C3 may also be selected from the
group consisting of poly(ethylene oxide) of the formula
(CH2CH2-O)y wherein y is an integer from I to 12. C1, C2 and
C3 may be the same or different.
D 1 and D2 are fatty acid chains consisting of fatty
acid esters of the form CH3(CH2)n COO or fatty acid amides of
the form CH3(CH2)n CONH wherein n is an integer from 1 to 32.
At least one of the fatty chains is unsaturated at one or more
positions, and D1 and D2 may be the same or different with
respect to length and degree of unsaturation.
In the generic formula listed above, the fatty acid
chains of the molecule may be comprised of (CH2)n wherein n is
an integer from 1 to 32. These fatty acid chains may each be
unsaturated at one or more sites and may be of different lengths
from 1 to 32 carbon atoms.
EXAMPLE 14
BES Dioleate, also called PX-18 or 2-[N,N-Bis(2-
oleoyloxyethyl)amino]-l-ethanesulfonic acid
A preferred embodiment of the generic structure in the preceding
Example is represented by the following structural configuration:
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0
11
CH3(CH2)7CH=CH (CH2)7COCH2CH2'
O /NCH2CH2SO3H
0 CH3(CH2)7CH=CH(CH2)7COCH2CH2
In the formula for PX- 18 shown above, the SO3H
acid group may also be in the salt-form or in an ionized form.
N,N-Bis(2-hydroxyethyl)-2-aminoethane sulfonic
acid was prepared (according to Izumi, 1954) by refluxing an
aqueous solution of sodium 2-bromoethanesulfonate with 2
equivalents of diethanolamine for 2 hours. The cooled reaction
mixture was passed over a sulfonic acid resin (Dowex 50) in the
acid form. The eluate, containing product and HBr, was taken to
dryness at reduced pressure and the product was recrystallized
from aqueous alcohol. The yield was 52% and the compound had
a melting point of 153-155 C.
In a I liter round bottomed flask, 16.2 g (0.076 mol)
of N,N-bis(2-hydroxyethyl)-2-aminoethane sulfonic acid were
dissolved in a solution of 60 mL of DMF and 36 mL of triethyl
amine. To this solution 72 g (0.239 mol) of oleoyl chloride (85%
Aldrich) were slowly added under a N2 atmosphere with stirring.
(During the addition of the oleoyl chloride a precipitate is
produced). After all of the oleoyl chloride was added, 300 mL of
acetone or THF was added to the mixture. The reaction mixture
was allowed to stir under a N2 atmosphere for 12 h. The
precipitate was collected by a filtration, and then recrystallized
from CHC13/CH3 CN. A yellowish product (43.6 g) was obtained
in 77% yield. The product was decolorized with charcoal in
CH30, recrystallized from CHC13/CH3CN, and exhibited a
melting point of 133-136 C.
EXAMPLE 15
Topical Cream Formula and Formulation Including TES
Trioleate (4% PX-13)
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0 Ingredients for solution A are 8 g cetyl alcohol; 2 g
oleic acid; 2 g isopropyl myristate 0.5 g paraben; 4 g PX-13.
Ingredients for solution B are 78 ml dH2O (78g); 1 g TRIS.HCI;
3.0 g SDS (sodium lauryl sulfate); 3 g sorbitol; 5 g 1,2
propanediol.
Formulation of Skin Cream
Formulation of solution A: In a 100 mL beaker cetyl
alcohol was heated to melting (approximately 70 C) and the oleic
acid and isopropylmyristate are added and mixed together with
the paraben. When these ingredients are in solution, the PX-13
was added and the temperature maintained at 70 C until solution
occurred.
Formulation of solution B: In 200 mL beaker the
water was brought to boiling and then removed from the hot
plate. TRIS.HCI and SDS were added and stirred until dissolved.
The sorbitol and propanediol were then added with stirring in
succession while maintaining the temperature at approximately
70 Next, solution B was rapidly stirred while solution A was
slowly added. The mixture was thoroughly stirred (for about 5
min). While the mixture was hot (70 C) 25 g was poured into 4
(1 oz) dispensing tubes.
It is understood that the formulation may optionally
contain additives such as lanolin, aloe, herbal extracts, or various
scents such as floral scents. Additionally, the formulation may
optionally contain preservatives, antimicrobials, penetration
enhancers and other skin cream ingredients commonly known to
one skilled in the art.
EXAMPLE 16
Dermatologic Disorders
Studies were conducted with skin creams produced
under GMP conditions containing PX-11 (tricinyl trioleate) and
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0 PX-13 and prepared according to the following recipe using
ingredients known to one of ordinary skill in the art.
TABLE
Base Cream Experimental
d H2O 92.5 % 87.5 %
carbopol 0.3 0.3
glycerin 2.0 2.0
pollex 2.0 2.0
Finsolv 2.0 2.0
PX-11/13 (active) ----- 5.0
Germaben 2E 1.0 1.0
AMP (amine) 0.2 0.2
These products were used with four psoriatic and three atopic
dermatitis volunteers. PX preparations applied topically to
psoriatic lesions twice a day over a three-week period reduced
swelling, diameter and elevation of plaques in all patients. In the
atopic dermatitis patients there was also improvement including
reduced erythema, itching and burning.
Evaluation of PX Compounds in Cream Formulations
Two PX compounds PX-11 and PX-13 were
evaluated in a cream formulation prepared under GMP. The
chemicals were formulated into a bland cream and evaluated in
the following human or mouse skin tests: (1) occluded patch test
for irritation; (2) cell turnover rate; (3) transepidermal water
loss; (4) skin moisturization; and (5) reduction in erythema after
induced sunburn.
The human skin tests were conducted on three
volunteer female subjects, ages 29-32; a cream base with no added
active was included as a control, while the untreated skin served
as an additional basis for evaluation. Sunburn tests were
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0 performed on a total of 30 hairless albino mice. Results of the
studies are summarized below.
1. Occluded patch test. Samples were applied on the subjects'
backs between the scapulae and waist adjacent to the spinal
colunln. Each site was sensitized to the chemicals by removal of
half the stratum corneum barrier by tape stripping. The levels of
irritation on the sensitized sites were measured after 24 hours of
occluded exposure to the creams. The levels of erythema, edema,
vesiculation and blister formation were scored. All samples were
non-irritating.
2. Cell turnover rate: This parameter was measured by
adsorption of dansyl chloride, in which 3% dansyl chloride was
incorporated into petrolatum. This salve was applied over a 2.5
cm diameter circular area in the upper arm and a patch applied.
24 hours later the patch was removed. The intensity of dansyl
chloride fluorescence was indicative of the cell turnover rate in
the epidermis.
It was found that the control cream decreased cell
regeneration time by 35%, i.e., the cells renewed more rapidly
compared with the untreated skin (a skin patch with nothing
applied). The addition of PX samples inhibited the rate of cell
regeneration. In fact, for PX-11, the time to regenerate was
longer than that observed for the untreated skin (see table below).
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0 Days to Cell Renewal % Change in Cell
Untreated Treated Turnover Time
PX- l l 21.5 28.8 +34
PX-13 30.4 21.6 -29
Control 30.4 19.7 -35
Note that the control cream accelerates cell growth but in the
presence of PX-11 and PX-13 cell growth is inhibited. For PX-
11, growth is so severely retarded that the treated skin area
requires significantly longer times for the cells to regenerate.
These results suggest that PX compounds may be useful in the
treatment of psoriasis.
3. Transepidermal water loss. This parameter was measured by
affixing a flow cell on the forearm site, applying the cream,
waiting 20 minutes, then flushing the volume with nitrogen and
noting the moisture content of the effluent gases. It was found
that PX-13 functioned as an occluding moisturizer, reducing the
rate at which water is lost from the epidermal layer. The control
and PX-11 opened the pores and permitted water to escape from
the epidermal layer. PX-11 was 10 times more effective than the
control at promoting water loss.
% Change in Transepidermal Water Loss
PX-l 1 +649.0
PX-13 -8.5
Control +56.5
4. Skin moisturization. Skin moisturization was measured using
20 megahertz ultrasound (the prescnce of moisture produces a
density decrease in the outer skin layers), before and 30 minutes
after product application to the forearm. It was found that PX-11
exhibited good moisturization properties, while PX-13 and the
control were not good moisturizers.
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0 Moisturization Effects
Ultrasound Density Score
Subject PX-11 PX-13 Control
1 3 1 2
2 3 2 1
3 1 -1 -2
Total 7 2 1
7 = Best; 1= Baseline
5.Protection against ultraviolet-induced ervthema. The protection
against ultraviolet-induced erythema (i.e., the burn and irritation
produced after exposure to ultraviolet radiation), was measured
by first irradiating 12 mice with destructive 3130 angstrom (A)
rays, applying the creams, and then evaluating the skin cells for
erythema. PX-11 was significantly better than the base cream in
erythema reduction.
Post-Radiation Ervthema Rankings
Subject PX- i 1 PX-13 Control
1 2 5 3
2 2 5 4
3 2 4 5
Mean 2 4.7 4
1 = Best; 4 = Worst
The results presented in Example 16 suggest that the PX test
compounds were active in modifying human skin cell activity and,
therefore, may impart beneficial effects when used as a skin
cream in a variety of dermatologic and cosmetic conditions.
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0 EXAMPLE 17
Neurological Inflammatory Conditions
While not wanting to be bound by this statement, it is
believed that PLA2-mediates inflammation and nerve tissue
damage in spinal cord injury and sciatic-nerve inflarnmation and
in a variety of central and peripheral neurological inflammatory
conditions. The following experiments demonstrate
antiinflammatory activity of PX compounds.
1. Activity of PX-13 to Inhibit PLA,?-Induced Inflammation in
Mouse Paw Edema Tests
Edema was induced by injection of 1 ug of purified
human disc PLA2 into the mouse footpad. PX-13 was
administered either by gavage or intraperitoneally (IP) 60 min
prior to enzyme challenge. Forty-five min after enzyme
injection, the animals were sacrificed and the control and injected
paws were removed and weighed to assess edema. The results are
presented in the following table.
PX- 13 (mg/kg) Percent Protection
20(gavage) 33%
40 " 55%
60 63%
40 (IP) 48%
PX-13 displays anti-inflammatory activity in this model when
administered by gavage or IP.
2. Protective effects of PX-13 in Cultured Rat Dorsal Root
Ganglion Cells Exposed to Snake Venom and Human Disc PLA2
Primary cultures of rat dorsal root ganglion cells
were used. Cells were washed 3-times with media to remove
serum. Then, PX-13 (20 uM) in HEPES buffer or a buffer
control was applied to the cells. After 10 min of incubation,
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0 purified human disc PLA2 (activity 1 umol/min) was added and
the cells were incubated for an additional 60-90 min. Cells were
then observed, fixed, and photographed 1.5 hrs after addition of
PLA2.
Cells treated with PX-13 (20 uM) alone or PX-13 +
PLA2 treated cells were indistinguishable from control cultures,
whereas, extensive morphologic damage was induced by PLA2
alone within 60 min; considerable cell blebbing and loss of texture
was noted. PX-13 is cytoprotective in this system to a toxic dose
of human disc PLA2. PX-13 also protected against the toxicity
induced by purified snake venom PLA2 used in comparable
amounts (1-3 umols/min/mg for 60 min).
These and other results support the concept that the
high levels of secretory PLA2 in herniated disc conditions can
irritate nerves and may contribute to the generation of low back
pain. These results suggest the potential efficacy of PX
compounds in treating these and related neurological conditions.
3. Inhibitory Effect of PX-13 on Nucleus Pulposus (NP)-Induced
Mouse Paw Edema
Human NP-homogenate was prepared according to
the following protocol and stored frozen until use. Human
vertebral discs were thawed and the tissue pieces were washed
twice with isotonic saline. Tissue samples were homogenized in a
minimum volume of isotonic saline using a Brinkman
homogenizer. This homogenate was filtered through two layers
of cheesecloth and the resultant filtrate was subjected to Dounce
homogenization. This homogenate was again filtered through two
layers of cheesecloth and the filtrate was capable of passing
through a 26-gauge needle; 50 ul of the filtrate was administered
to each mouse paw.
The filtrate contained 1.57 mg/ml of protein and its
PLA2 activity was 12.8% hydrolysis of 10 nmols of E.coli
phospholipid/10 ul filtrate/30 min. incubation at 37 C.
Therefore, each mouse paw received 78.5 ug NP filtrate. This
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0 sample of 78,500 ng total protein contained approximately 4.3 ng
NP PLA2 protein. By comparison, we use 500-2000 ng of highly
purified PLA2 to induce edema in this mouse model; and under
these experimental conditions the maximal edema achieved is
160% of the contralateral paw minus 4-7% edema due to saline
injection alone.
The data are expressed as percent of contralateral
paw and as percent of maximal edema achieved with purified
PLA2. Fifty ul (78.5 ug) was injected into the paw 30 min. after
the mice were treated with PX-13 in HEPES administered IP or
by gavage at 30 or 60 mg/kg. After 3 hrs, the paws were excised
and weighed.
Results:
Percent Percent of* Percent
Edema Maximal
Inhibition
Saline Control 106 2.5 ---- ----
NP-Homogenate 124 3.0 34% ----
alone
PX-13: IP
30 mg/kg 120 2.0 26% 24%
60 mg/kg 115 1.6 17% 50%
PX-13: Gavage
mg/kg 122 5.6 ----- -----
30 60 mg/kg 118 2.0 22% 36%
*calculated as previously described; PLA2 alone = 160% edema -
106% control = 54% (maximal edema).
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Considerable variability or overlap is noted in both 30 mg/kg PX samples;
however,
at 60 mg/kg both IP and gavage administration of PX-13 inhibited inflammation
by
50% and 36%, respectively. These results indicate that nucleus pulposus
extracts are
inflammatory, and PX-13 protects against inflammation induced by nucleus
pulposus
extracts.
4. The lipid soluble (PX-13) and water soluble (PX-18) both inhibit the
purified
human disc type II, PLA,z
Both PX-13 and PX-18 inhibit the purified human disc type II, PLA2.
PLA2 activity was measured as described in Franson et al., Prostaglandins,
Leukotrienes
and Essential Fatty Acids, 43:63-70, 1991, which may be referred to for
further details.
The water soluble compound PX-18, displayed an IC;o of 0.3 M whereas the
lipid
soluble compound PX-13 displayed an IC50 of 2.5 M.
EXAMPLE 19
Ischemia-Reperfusion Injury
1. Small Bowel Transplantation: Ischemia-Reperfusion Injury
PX-13 and mepacrine inhibited PLA2 activity in extracts of rat bowel,
and PX-13 was a more effective inhibitor (by more than 2 log units) of PLA2
exzymatic activity than mepacrine. Both compounds were shown to protect
against
ischemia and reperfusion injury following small bowel transplantation. These
drugs
reduced weight of the tissues after storage for 24 and 48 hours in the
perfusate
solution.
These drugs were also tested for their ability to enhance the preservation
of tissues stored for subsequent transplantation. The small bowel was removed
from
rats, washed free of luminal contents and the tissue stored in University of
Wisconsin
(UW) preservative media 40 uM PX-13 or mepacrine for 48 hrs. The graft was
then
transplanted into rats with or without intravenous (IV) infusion of 20 mg/kg
PX-13
or mepacrine at the time of transplantation. Upon transplantation,
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0 the bowel is immediately injured as evidenced by tissue darkening
within two minutes. With PX-13 or mepacrine added to the
media the gross and microscopic injury to tissue was dramatically
reduced. Animals receiving PX-13 maintained normal bowel
color in contrast to the blackening (necrosis) which occurs within
minutes in the absence of drug. Treatment with the
anti-malarial, anti-PLA2 drug, mepacrine, produced some
protection although not as great as with PX-13. Both PX-13 and
mepacrine inhibit the peroxidation (malondialdehyde formation)
of homogenized rat bowel, an indication that the
membrane-stabilizing, PLA2-inhibitors, also have anti-oxidant
activity.
Recent studies completed in our laboratories
demonstrate that ischemic bowel releases PLA2 into the media in
a time dependent manner. The addition of PX-13 reduced the
enzyme release by more than 80% during 24 hrs of ischemia.
These results indicate that PX-13, which inhibits
PLA2 activity, exerts significant protective effects on tissues
chosen for transplantation. PX-13 and related compounds may
decrease complications resulting from tissue transplantation
thereby increasing successful outcomes and reducing costs. The
use of PX-13 and related compounds, acting as
preservation-enhancing additives, may extend storage time and
enhance the preservation of small bowel grafts and other tissue
destined for transplantation in cold storage bathing solutions.
EXAMPLE 20
Parasitic Infection: protozoal parasites
1. Malaria: lipid soluble PX-13 inhibits in vitro replication of
Plasmodium falciparum.
PX-13 was used to block the incorporation of
3H-hypoxanthine into the DNA, to provide a measure of growth,
of P.falciparum cultures which were resistant to the drugs,
chloroquine or mefloquine, which are normally used
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0 therapeutically. The growth of two strains in RBC cultures were
tested:
The compound PX-13 inhibited the growth of clone
W2 (P.falciparum), which is chloroquine resistant and mefloquine
sensitive with an IC50 of 13 ug/ml. This value for drug resistant
clone W2 compares to control cultures sensitive to both drugs:
IC50 chloroquine = 30 ng/ml vs mefloquine = 1.3 ng/ml.
PX-13 also inhibited the growth of clone D6 (P.
falciparum) which is chloroquine sensitive and mefloquine
resistant, with an IC50 of 17 ug/ml. This value for drug resistant
clone D6 compares to control cultures sensitive to both drugs:
IC50 chloroquine = 1.6 ng/ml; and mefloquine = 4.3 ng/ ml.
PX-13 is active against strains of P. falcinarum that
are resistant to the drugs currently used for therapy. In addition,
the slope of the inhibition curves indicates that PX- 13 acts rapidly
so that it can more rapidly interfere with the replication process
or the disease in progress. These findings are also significant
because the levels of current therapeutics necessary for control
are often toxic. Recent findings indicate that the therapeutic index
of PX-13 (dose for 50% lethality/dose for 50% effectiveness or
LD50/ED50) is a large positive number, or highly favorable
relative to the known toxicity problems for chloroquine,
mefloquine, and other drugs used in the treatment of malaria.
2. Malaria: Water Soluble PX-18 inhibits in vitro rolication of
Plasmodium falciparum.
The water soluble compound PX- 18 inhibited the
same drug-resistant P. falciparum strains as described above. The
IC50 for PX-18 was 1.3 and 2.4 ug/ml. Thus, the water soluble
PX-18 compound appears to be 10-times more effective than the
lipid soluble compound PX-13.
The inhibitory effects of lipid soluble PX-13 and
water soluble PX-18 on the growth of drug sensitive and drug
resistant species of Plasmodium falciparum indicate that these PX
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anti-parasitic drugs.
EXAMPLE 21
Preservation of Whole Blood
The effect of PX-13 and mepacrine on whole blood
cell viability were tested as a function of time of storage. The
results are summarized below.
Percent Protection
LDH Hemoglobin
48 hr 72 hr 48 hr 72hr
A. Cells alone 0 0 0 0
B."+ PX-13 20 uM 22 15 55 25
C. 80 uM 39 41 72 54
D. " " 160 uM 78 72 77 80
E. "+ mepacrine 20 uM 58 27 61 45
Heparinized human blood was centrifuged at 400 x g and the
resulting plasma was removed and replaced with an equal volume
of Dulbecco's medium. The resuspended cells were then
incubated at 37 C for 0-72 hrs. in a shaking water bath. At the
indicated times, a 0.8 ml aliquot was removed and centrifuged at
400 x g to sediment cells. Appearance of lactate dehydrogenase
(LDH) and hemoglobin in the supernate was indicative of cell
injury. PX-13 and mepacrine stabilized cells and decreased the
injury as evidenced by diminished levels of LDH and hemoglobin
in the supernate. This is expressed as percent protection.
The addition of snake venom PLA2 accelerated the
release of both LDH and hemoglobit~ at 24 hrs (not shown).
EXAMPLE 22
Blocking of thrombin-activated platelet aggregation.
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dose of PX-13 inhibited platelet aggregation. Applicants have
also observed that PX-13 inhibited serotonin release by
thrombin-stimulated platelets. This observation supports the
contention that the PX compounds of the present invention may
block release of inflammatory mediators in the coagulation
cascade. This observation also supports the use of PX compounds
to inhibit thrombin-induced platelet aggregation and to act as anti-
clotting agents.
EXAMPLE 23
Treatment for Snake Bite
Animal and human recipients of venomous snake
bites require rapid treatment to alleviate the toxic inflammatory
reactions which may be lethal. The compositions of the present
invention are available in a readily injectable form for
administration to the patient through intramuscular injection. The
compositions of the present invention are stable for periods up to
3 months at room temperature.
Low molecular weight PLA2 is a major toxic
component of snake venoms. In venoms with neurotoxic effects
(i.e. cobra venom), this is mediated by a PLA2 which binds to a
neuronal cells. Snake venom injuries have 3 components: 1)
peripheral and central neurotoxicity (certain venoms), 2) systemic
inflammation, including complement activation, and 3) extensive
local tissue damage, including muscle necrosis and swelling which
can cause distal vascular compromise (compartment syndrome).
The following hypothetical example describes the treatment of a
rattlesnake bite occurring several hours before conventional
medical treatment with an emergency snakebite kit containing a
water-soluble PLA2 antagonist, PX-18, in injectable form.
A patient is bit by a rattlesnake on the upper calf
while backpacking above the tree-line in Colorado. He uses his
snake bite kit to attempt local suction extraction of venom at the
bite site. He applies a tourniquet proximal to the bite. He then
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0 takes out the 2 ml syringe with 22 gauge needle, preloaded with
200 mg of PX-18 in sterile solution, which is contained in the kit.
Following the kit instructions, he injects 1 ml of PX-18
intramuscularly (im) for systemic absorption, in the anterior
thigh. He then injects the remaining 1 ml deep subcutaneously at
the bite site, to attempt to neutralize the local concentration of
venom PLA2. After 30 minutes, he releases the tourniquet and
proceeds to seek medical attention.
EXAMPLE 24
Reflex Sympathetic Dystrophy (RSD)
A patient presents with severe RSD characterized by
persistent pain and limitation of movement at the wrist joint.
Topical application of PX-13 to the wrist in a concentration of
about 4% in the cream formulation described in Example 15
results in immediate pain relief and increased range of motion
within 10 to 15 minutes.
Another patient presents with a two year history of
RSD in the knee joint. This male patient previously received
arthroscopic evaluations. This patient also had limited range of
motion and became addicted to narcotics in an attempt to reduce
pain. A combination of sympathetic blockade and cream
containing PX-13 is topically applied and decreases the patient's
report of pain and provides a full range of motion in the knee
joint. The reduction in pain is immediate and the increased range
of motion occurs within 10 - 20 minutes. This patient, who had
been unemployed for an extended period, eliminated intake of
narcotics and returned to work.
A third patient presented with RSD of the foot and
ankle. This patient had no eversion or inversion of the foot.
Topical application of PX-13 (about 4%) in a cream vehicle
provides immediate and complete pain relief and restores full
eversion and inversion within 20 minutes.
In each of the cases described above, pain relief
analgesia is immediate with no production of anesthesia.
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EXAMPLE 25
Acute Toxicity Studies
An LDSO study was conducted to determine a lethal
dose for PX-13 suspended in HEPES buffer at a pH of 7.4. A
single IP dose of 50, 100, 200 or 400 mg/kg was administered to
mice (n=6 per group). No deaths or gross physical or behavorial
abnormalities were noted after 48 hours in all dosage groups. At
144 hrs one animal in the 400 mg/kg group died. No necropsy
was performed. The scientist conducting this trial noted that
"PX-13 was extremely well tolerated", confirming the results in
the gut reperfusion experiments described in another example
following IV administration of the test compound.
The results indicate that doses of 50 to 200 mg/kg
administered IP to mice do not produce acute toxicity in terms of
death or gross physical or behavorial abnormalities.
EXAMPLE 26
Effects of PX-18 on Histamine Release from Basophils
The protocol to evaluate the effects of PX-18 on
histamine release is described below. Freshly drawn heparinized
blood from donors non-medicated for 3 days is diluted 1:5 with
PBS containing 1% human serum albumin (diluent). Test drugs
are diluted in diluent to 10 times final concentration, and 25 1
per well is added to duplicate wells of round-bottomed microtiter
plates. Generally 3-fold dilutions of drug are examined four or
five times. 200 1 of diluted blood is then added to each well. 5
minutes later, 25 1 of rabbit anti-human IgE (purchased from
Dako) is added per well, to give final concentrations of 1/400 and
1/1200 of antibody. Unstimulated (background release wells)
have 25 l of diluent added. Plates are gently tapped for mixing,
then incubated for 30 minutes. After incubation, plates are cooled
on ice, spun down in a refrigerated centrifuge, and replaced on
ice. 100 41 plasma is removed from each well for assay, and
placed in a 1.5 ml Eppendorf tube containing acetylation reagent.
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0 Following acetylation, the histamine adduct is quantitated by a
sensitive and specific commercial competitive assay, utilizing the
competition of enzyme conjugated acetyl-histamine with sample
analyte for binding to a specific antibody on the solid phase. A
sample of blood is also lysed by repeated freeze/thaw to determine
total blood histamine. Results are expressed as a percentage of
total blood histamine release. Ili an acceptable assay maximal
release is greater than 45% and background release is less than
15%. Experiments are performed in duplicate or triplicate using
separate blood donors.
These experiments demonstrate that PX-18 inhibits
the degranulation of basophils (and probably mast cells) in
response to stimuli such as antibody surface-bound IgE. PX- 18 is
equipotent to epinephrine, a potent anti-anaphylactic drug, and
more efficacious than cromolyn, a prototypic "mast-cell
stabilizer". These results support the use of PX-18 and related
compounds as compounds to treat allergic disease.
EXAMPLE 27
Effects of PX-18 on production of leukotrienes and
prostaglandins by blood under basal and stimulated conditions
The following protocol was employed to evaluate
effects of PX- 18 on production of leukotrienes and prostaglandins
by blood cells under basal conditions, with ionophore stimulation
of cyclooxygenase type I, and with lipopolysaccharide (LPS)
induction of cyclooxygenase type II. Freshly-obtained
heparinized blood from medication-free donors is diluted 1:1 with
RPMI, and aliquoted in 0.6 ml amounts in sterile polypropylene
12 x 75 ml tubes. Test drugs are added at appropriate
concentrations and diluted in saline with 1% human serum
albumin. Control tubes have only c,iluent or drug vehicle added.
Drugs are added five minutes prior to addition of stimulus, at 100
x concentrations, (6 l volumes). This is replicated for 3 racks of
tubes, A thru C. A has no stimulus added, and has a 30 minute
and 6 hr. tube for each drug concentration. B has 20 uM of the
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0 calcium ionophore A23187 added, and is incubated at 37 C for
30 minutes. C has 5 ug/ml of E. coli strain Bort LPS added, and
is incubated at 37 C for 6 hours. At the end each time period,
the appropriate tubes are placed on ice, centrifuged, and 0.4 ml of
plasma carefully decanted into a tube containing formic acid and
BHT to stop all reactions and preserve eicosanoid products.
These tubes are then frozen at -70 C for up to one week, then
applied to Sep-pac mini columns and extracted into methanol.
The methanol extract is aliquoted into 3 tubes and evaporated,
then stored at -70 C until the time of eicosanoid assay.
Measurements include thromboxane (by a sensitive fluorometric
plate-format assay) and either 15 HETE or LTB4 to measure
lipoxygenase products.
The whole blood eicosanoid release experiments
show that PX-18 appears to strongly inhibit the cytokine-inducible
cyclooxygenase II, with less inhibition of the constitutive
cyclooxygenase I enzyme, stimulated by ionophore, and even less
or no inhibition of basal production of prostaglandins. This is an
excellent anti-inflammatory profile as most NSAID-associated
toxicities (i.e., gastric, renal, fluid retention, possibly asthma) are
due to inhibition of constitutive cyclooxygenase I prostaglandin
production, which plays a necessary physiologic role.
cyclooxygenase II is involved in pathologic inflammation.
These studies also demonstrate that production of
lipoxygenase products such as LTC4, is inhibited by PX-18. The
comparison is to MK 592, a selective lipoxygenase inhibitor. PX-
18 does not inhibit either the lipoxygenase or cycloxygenase
enzymes, but rather PLA2 which produces free arachidonic acid,
thus depleting the pool of this enzyme substrate for the
lipoxygenase and cyclooxygenase pathways. Our current
experiments demonstrate that if exogenous arachidonic acid is
added and lipoxygenase and cyclooxygenase metabolites are
measured, the addition of PX-18, unlike indocin or MK 592 does
not inhibit their production. These results support the notion that
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0 PX-18 inhibits PLA2 and not downstream enzymes such as
lipoxygenase and cyclooxygenase.
Additionally, blockade of secretory (s) PLA2 activity
decreases the production of lysophospholipid, the immediate and
rate-limiting precursor for platelet-activating factor (PAF).
Secretory PLA2 as opposed to cytosolic perinuclear membrane-
localized PLA2, seems to be the key enzyme providing substrate
which is acetylated to form PAF. PAF is a potent inflammatory
mediator, a potent neutrophil chemotactic, and a key factor in
tissue injury in such situations as ischemia. PAF is implicated in
inflammatory diseases such as ulcerative colitis, and is unaffected
by therapy with NSAIDs.
The invention has been described in detail with
particular reference to certain embodiments, but variations and
modifications can be made without departing from the spirit and
the scope of the present invention as defined in the appended
claims.
