Note: Descriptions are shown in the official language in which they were submitted.
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~.nO~ FOR REDUCING OR PREVENTING POST-8URGICAL
ADHESION FORMATION USING 5-LIPOXYGENA8E INHIBITOR8
FIELD OF THE lNV~llON
The present invention relates to 5-lipoxygenase (5-LO)
inhibitors and their use thereof in a method for reducing
or preventing post-operative adhesion formation between
tissue, e.g., organ, surfaces in a body cavity.
BACKGROUND OF THE lNV~:N'l'lON
Adhesion formation, in particular following peritoneal
surgery, is a major source of postoperative morbidity and
mortality. Appendectomy and gynecologic surgery are the
most frequent surgical procedures implicated in clinically
significant adhesion formation. The most serious
complication of intraperitoneal adhesions is intestinal
obstruction; in addition, adhesions are associated with
chronic or recurrent pelvic pain and infertility in
females.
The pathogenesis of adhesion formation is complex and
not entirely understood. The first step is believed to
involve excess fibrin deposition to form a scaffold.
Organization of the fibrin scaffold by cellular elements,
including fibroblasts and mesothelial cells, then follows.
Various approaches for the prevention of adhesion
formation have been actively explored [diZerega, G.S. &
Rodgers, K.E., "Prevention of Postoperative Adhesions," in
"The Peritoneum," diZerega, G.S. & Rodgers, K.E., eds.,
Springer-Verlag, New York, pp. 307-369 (1992)]. In
general, the treatments fall into three categories:
prevention of fibrin deposition in the peritoneal exudate,
reduction of local tissue inflammation; and removal of
fibrin deposits.
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Therapeutic attempts to prevent fibrin deposition
include peritoneal lavages to dilute or wash away fibrinous
exudate, surgical techniques to minimize tissue ischemia
and introduction of barriers to limit apposition of healing
serosal surfaces. Although the use of agents affecting
coagulation of the fibrinous fluid has also been proposed,
results obtained to date suggest that the use of
procoagulants in areas of substantial bleeding may actually
promote adhesion formation [Elkins, T.E., "Can a
Pro-Coagulant Substance Prevent Adhesions?" in "Treatment
of Post-Surgical Adhesions," diZerega, G.S. et al., eds.,
Wiley-Liss, New York, pp. 103-112 (1990)].
Physical barriers have been used in attempts to
prevent adhesion formation by limiting tissue apposition
during the critical period of peritoneal healing, thereby
minimizing the development of fibrin matrix between tissue
surfaces. Barrier agents which have been employed include
both merhAnical barriers and viscous solutions. Mixed
results have been obtained using a barrier comprising a
thin sheet of expanded poly-tetrafluoroethylene; in any
event, such a membrane is less than ideal, as it must be
sutured into place and is nonabsorbable. While an
absorbable barrier (for example, a barrier made of oxidized
regenerated cellulose) would be preferable, not all studies
have demonstrated the efficacy of such barriers in
preventing adhesions. Liquid barriers have also been
considered for use in preventing adhesions; for example,
chondroitin sulfate and carboxymethyl cellulose have both
shown some promise in animal models. In addition, solution
of dextran 70 (molecular weight = 70,000) has been the
subject of a number of clinical studies. Not all clinical
evaluations of 32% dextran 70 have found a therapeutic
effect, however, and the clinical use of the solution is
also associated with clinically important side effects.
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Anti-inflammatory drugs have been evaluated for their
effects on postoperative adhesion formation, as they may
limit the release of fibrinous exudate in response to
inflammation at the surgical site. Two general classes of
these drugs were tested: cortico-steroids and nonsteroidal
anti-inflammatory drugs. The results of corticosteroid use
in animal studies have generally not been encouraging, and
clinical use of corticosteroids is limited by their other
pharmacologic properties. While experimental evaluations
of nonsteroidal anti-inflammatory drugs in postoperative
adhesion formation show promise [Rodgers, K.E.,
"Nonsteroidal anti-inflammatory drugs (NSAIDs) in the
treatment of Postsurgical adhesion," in "Treatment of Post-
Surgical Adhesions," diZerega, G.S. et al., eds.,
Wiley-Liss, New York, pp. 119-129 (1990)~, clinical
evaluations of these drugs for adhesion prevention is
needed.
The third approach explored to date involves the
removal of fibrin deposits. Although proteolytic enzymes
(e.g., pepsin, trypsin and papain) should theoretically
augment the local fibrinolytic system and limit adhesion
formation, these enzymes are rapidly neutralized by
peritoneal exudates rendering them virtually useless for
adhesion prophylaxis. While various fibrinolytics (for
example, fibrinolysin, streptokinase and urokinase) have
been advocated, a potential complication to the clinical
use of these enzymes in postoperative therapy is excessive
bleeding resulting from their administration. Topical
application of a recombinant tissue plasminogen activator
(rt-PA) has been shown to reduce adhesion formation in a
variety of animal models; further research is necessary to
develop suitable delivery systems to provide this drug to
the surgical site and identify the postoperative time when
adhesion prevention is feasible.
.
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To date, no single therapeutic approach has proven
universally effective in preventing formation of
postoperative intraperitoneal adhesions. Therefore, there
is a need for compositions and methods which may be used
safely and effectively to prevent adhesion formation in a
variety of different contexts.
OBJECTS OF THE lNV~llON
It is an object of the present invention to provide 5-
lipoxygenase (5-LO) inhibitor-based compositions for use in
preventing or minimizing adhesion formation.
It is another object of the invention to provide a
method for reducing or preventing post-surgical adhesion
formation between tissue surfaces in body cavities which
employ 5-LO inhibitors.
These and other objects of the invention will be
apparent in light of the detailed description below.
SU~qARY OF THE lNV~ 'l'lON
The present invention relates to 5-lipoxygenase (5-LO)
inhibitors and their use in a method for reducing or
preventing adhesion formation between tissue, e.g., organ,
surfaces in body cavities comprising administering to a
subject an effective amount of at least one 5-LO inhibitor,
e.g., phenidone, nordihydroguaiaretic acid (NDGA),
5,8,11,14-eicosatetraynoic acid (EYTA) and Zileuton. The
5-LO inhibitor is preferably administered in conjunction
with a drug delivery system which maintains an effective
concentration of the compound at a site of potential
adhesion formation during the perioperative interval.
Pursuant to another aspect of the present invention,
adhesion formation is minimized or prevented by
administration of at least one 5-LO inhibitor at a site of
potential adhesion formation for a period of time
sufficient to permit substantial tissue repair (e.g.,
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re-epithelialization or mesothelial repair) at the site.
DETAILED DESCRIPTION OF THE I~v~ ON
All literature references, patents and patent
applications cited in this application are incorporated
herein in their entirety.
The inventive composition and method are useful in
minimizing or preventing formation of adhesions between
organ surfaces (not cell-to-cell adhesion), the most common
cause of which is prior surgery. The inventive composition
and method have been shown to be especially effective in
preventing adhesion formation in the peritoneum following
surgery. In addition, the present invention finds utility
in other contexts, e.g., for cardiovascular, orthopedic,
thoracic, ophthalmic, CNS and other uses, where prevention
of the formation of adhesions is a significant concern.
For example, prevention of adhesion formation or drug
loculation during the intraperitoneal administration of
chemotherapeutic agent is contemplated as within the scope
of the present invention. For the purposes of the
following discussion, attention is directed primarily to
description of compositions and methods useful in
inhibiting peritoneal adhesion formation.
The present invention is based on the discovery that
compounds which inhibit 5-lipoxygenase (5-LO) activity are
useful in reducing or preventing formation of adhesions
between tissue surfaces in body cavities following surgical
procedures. The 5-LO enzyme, found primarily in
polymorphonuclear leukocytes (PMNs) and eosinopils, is a
major enzyme involved in second pathway (the "5-LO
pathway") of arachidonate metabolism in which arachidonic
acid is converted to pro-inflammatory products called
leukotrienes (LTs). 5-LO catalyses the stereospecific
oxidation of arachidonic acid to a 5-hydroperoxyeicosa-
tetraenoic acid (5-HPETE) in the initial step towards the
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biosynthesis of leukotrienes. The enzyme contains non-heme
iron in the active site, and the mechanism of the
transformation probably involves an organoiron intermediate
or a dienyl radical which is trapped by molecular oxygen.
For a review of the properties and proposed mechanisms of
5-L0, see Musser and Kreft (1992), "5-Lipoxygenase:
Properties, pharmacology and the quinolinyl (bridged)aryl
class of inhibitors", J. Med. Chem., Vol. 35, pp. 2501-
2524; Batt (1992), "5-Lipoxygenase inhibitors and their
anti-inflammatory activities," Prog. Med. Chem., Vol. 29,
pp. 1-63.
Because leukotrienes and other pro-inflammatory
arachidonic acid metabolites have been implicated in many
inflammatory disease processes such as asthma, rheumatoid
arthritis, inflammatory bowel disease, psoriasis and
glomerulonephritis, compounds which inhibit 5-LO activity
are highly desirable as therapeutic agents. For a review
of the role of 5-LO in inflammatory processes, see for
instance, Batt (1992), "5-Lipoxygenase inhibitors and their
anti-inflammatory activities," Prog. Med. Chem., Vol. 29,
pp. 1-63. Prior to the present invention, however, use of
5-LO inhibitors in preventing post-surgical adhesions was
unknown.
Numerous compounds of vast structural diversity have
been shown to inhibit 5-L0 activity. Classes of 5-LO
inhibitors based on structure include substrate or product
analogs, catechols, phenols, aminophenols, flavinoids,
naphthols, heterocycles such quinones, indazolines, and
benzothiophenes, and hydroxamic acid derivatives of common
NSAIDs. Examples of these classes of compounds are
described in the literature, for instance, in Musser and
Kreft (1992), "5-Lipoxygenase: Properties, pharmacology and
the quinolinyl (bridged)aryl class of inhibitors", J. Med.
Chem., Vol. 35, pp. 2501-2524; Salmon et al. (1990),
"Inhibition of 5-lipoxygenase: development of hydroxamic
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acids and hydroxyureas as potential therapeutic agents,"
Adv. Prost., Thromb., Leukotriene Res., Vol. 21, pp. 109-
112; Riendeau et al. (1989), "Sensitivity of
immunoaffinity-purified porcine 5-lipoxygenase to
inhibitors and activating lipid hydroxyperoxides," Biochem.
Pharmacol., Vol. 38, pp. 2313-2321; Hlasta et al. (1991),
"5-Lipoxygenase inhibitors: the synthesis and structure
activity relationships of a series of 1-phenyl-3-
pyrazolidinones," J. Med. Chem., Vol. 34, pp. 1560-1570.
Compounds which inhibit 5-LO activity assert their effect
through a variety of mec~nisms which include alterations
in cellular metabolism, a direct effect on the enzyme
leading to reduced function or through competitive
inhibition. A recent extensive review describes the
representative structural classes of 5-LO inhibitors. See
Batt (1992), "5-Lipoxygenase inhibitors and their anti-
inflammatory activities," Prog. Med. Chem., Vol. 29, pp. 1-
63.
5-LO enzyme is inhibited by substrate and product
analogues. Acetylenic, methylated, cyclized, or thia-
analogues of arachidonic acid, and cyclopropyl analogues of
LTB4 inhibit 5-LO. One compound described further below,
5,8,11,14-eicosatetraynoic acid (ETYA), is a competitive
inhibitor of CO and LO. Anderson et al. (1992) "EYTA, a
pleotrophic membrane-active arachidonic acid analogue
affects multiple signal transduction pathways in cultured
transformed mammalian cells," Clin. Biochem., Vol. 25, pp.
1-9. See Batt at 6-7.
Catechols and aminophenols have also been shown to
inhibit 5-LO enzyme. Lipophilic catechols, notably
nordihydroguaiaretic acid (NDGA) which is used as an
example below, were more potent than pyrocatechol. The
inactivation of 5-LO enzyme is irreversible, and is
accompanied by oxidation of phenolic compound. The ortho-
dihydroxyphenyl moiety is required for the best potency
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which correlated with overall lipophilicity of the
inhibitor. NDGA and other phenolic compounds have been
shown by electron paramagnetic resonance spectroscopy to
reduce the active-site iron from Fe-(III) to Fe-(II).
Electron-poor, less easily oxidized catechols form stable
complexes with the active-site iron atom. See Batt at 8-11
and references cited therein.
A large number of catechol-containing compounds have
been reported to inhibit a variety of 5-LOs. Many of these
are natural products or synthetic analogues, such as
gossypol, caffeic acid and derivatives, and a wide variety
of other ortho-dihydroxyphenyl compounds. In general, most
inhibitors of 5-L0 are somewhat less potent against 12-L0,
and are often significantly less potent as inhibitors of
C0. Potency is often roughly correlated with
lipophilicity. Many reports have appeared over the last
decade dealing with 5-L0 inhibition by flavinoids. The
most studied compound is quercetin, ~-Tocopherol (vitamin
E) represents another class of phenolic lipophilic
antioxidants, para-substituted by an oxygen atom in a fused
saturated ring which inhibits platelet 12-L0 and soybean
15-L0. A series of related benzoxanthiols potently inhibit
5-L0; replacement of the propyl group by ethyl, butyl or
phenyl maintains this potency. L-651,896 is a compound
from a series of dihydrobenzofuranols. Ibid.
The structure activity relationship (SAR) for L-
651,896 and analogues was examined. There is shape
specificity for 5-L0 inhibition demonstrated by the greater
potency of 6-substituted analogues compared to 4-
substituted compound. RS-43179 (lonapalene) is a selective
5-L0 inhibitor with topical anti-inflammatory activity.
SAR studies showed that lipophilicity plays a strong role,
but if the compounds are too lipophilic (such as with
larger alkoxy groups) activity is reduced. The best
substituent on the fused ring is chloro, although other
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_g_
groups capable of ~-electron donation (other halogens,
methoxy) are also effective. Hydrolysis of at least one of
the ester groups appears essential for activity, since
compounds with increasing hydrolytic stability (pivalate,
benzoate) are less potent. A similar naphthol derivative
from UpJohn is U-66,858 (bunaprolast). Simple 2-
substituted 1-naphthols (DuP 654) are both potent 5-L0
inhibitors and topical anti-inflammatories. SAR studies
showed that various positional isomers are significantly
less potent against 5-L0 than DuP 654, although the C0
inhibition is less sensitive to these changes. Lipophilic
phenols lacking the fused ring system, such as 2,6
dibenzylphenol, are also less potent. Lipophilic
arylmethyl 2-substituents are favored in vivo although even
2 methyl-1 naphthol is selective (but less potent) for 5-
L0. Substitution at the 4-position by electron withdrawing
groups reduces potency, as expected for a compound acting
by redox mechAn;cm. See Batt at 15-19 and references cited
therein.
Heterocyclic naphthol isosteres are also potent 5-L0
inhibitors. Heterocyclic analogues of bunaprolast are
about equipotent with the isocyclic versions. The
hydroxyquinoline N-oxide KF 8940 is a potent inhibitor of
5-L0 and is quite selective with respect to inhibition of
12-L0 and C0. Another heterocyclic inhibitor (L-656,224)
is selective for 5-L0. Like some of the naphthol series,
and alkyl substituent (preferable methyl, ethyl or propyl)
ortho to the hydroxyl is required for activity; the ~-butyl
analogue is less potent, as are analogues with heteroatom-
containing chains at the position. Substitution on the
benzyl group is relatively unimportant, as long as a
carboxyl group is not present. Closely related
benzimidazoles shows similar activity. Ibid.
Phenolic compounds, particularly those with para-
oxygen substituents, are readily oxidized to quinones.
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Likewise, quinones are easily reduced (chemically and
metabolically) to 5-LO-inhibiting hydroquinones. AA861
(docebenone) is one such compound which is a standard 5-LO
inhibitors used in various physiological and
pharmacological studies. The side-chain of docebenone is
required for in vitro activity, but partial or full
saturation of this group has little effect. Replacement of
the methyl groups on the benzoquinone moiety by methoxyls
also give similar activity. See Batt at 19-21 and
references cited therein.
Amino-substituted naphthoquinones and heterocyclic
variants (which are fully conjugated, but non-aromatic) are
5-LO inhibitors. Ibid.
Heterocycles which are fully conjugated, but non-
aromatic, such as phenothiazine, are easily oxidized. This
redox activity, coupled with the lipophilicity and ~-
electron character (which is felt to mimic the arachidonic
acid chain), led to the development of a series of
substituted phenothiazinones such as L-651,392, as
selective 5-LO inhibitors. See Batt at 21-23 and
references cited therein.
Phenoxazine is a potent inhibitor of 5-LO.
Substitution at the l-position by carboxylic acid, ester or
hydroxamic acid decreases potency. Lipophilic substitution
at the 2-position is less destructive. A series of
substituted dihydrothiazines has also been reported.
Substitution on the phenyl group or variation of the benzyl
by alkyl or hydroxyalkyl reduces potency about 10-fold.
Replacement of the phenyl substituent by benzoyl reduces
potency, while reduction of the trisubstituted double bond
completely destroys activity. Ibid.
Phenidone and BW-755c are inhibitors of 5-LO.
Derivatives of phenidone have been reported; C-alkylation
with lipophilic groups alpha to the carbonyl is acceptable,
while N-methylation destroys in vitro activity. A
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--11--
quantitative SAR study of the phenyl ring of BW-755c with
respect to activity shows that electron-donating
substituents are better than electron-withdrawing ones, and
that large substituents are disfavoured, especially in the
ortho position. A-53612 is a ring-expanded version of
phenidone which is selective for 5-LO. A-65620, a perhydro
analogue, is very similar to A-53612, having equal or
slightly reduced potency. The pyridazinones are generally
more potent than the triazinones; enhanced lipophilicity
(by substitution on the l-phenyl or the heterocyclic ring)
increased potency. Acyclic analogues of phenidone, such
pyrazolecarboxylic hydrazides, inhibits 15-LO and are
inactive against CO. A series of indazolinones, such as
ICI 207968, are benzo-fused analogues of phenidone. Some
analogues inhibited CO as well, but ICI 207968 itself is
highly selective (ca.300-fold) for 5-LO. See Batt at 23-27
and references cited therein.
SAR studies indicate that unsubstituted indazolines
inhibit 5-LO, but without selectivity. Substitution on the
l-nitrogen has no effect, while substitution on oxygen or
both nitrogens destroys activity. Phenyl and methyl groups
replacing the pyridylmethyl of ICI 207968 gives greater
potency, but poorer selectivity. Benzyl and heterobenzyl
groups give the best profile; lengthening the link group
beyond one methylene has little effect. The 1-
naphthylmethyl analogue is potent and selective, while the
2-naphthyl analogue has increased CO activity. The 1
naphthylethyl analogue showed good stereoselective action
in that the (R) enantiomer was very selective for 5-LO over
CO. Other compounds containing heteroatom-heteroatom bonds
have been reported as 5-LO inhibitors such as
diphenyldisulphides and substituted analogues, as well as
disulphiram. Ibid.
N-Hydroxyarachidonamides are potent reversible
inhibitors of 5-LO. Alkylation on nitrogen increased the
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inhibitory potency significantly. Placement of the
hydroxamic acid moiety in 5-position gives analogues of 5-
HPETE which also inhibit 5-LO. A series of
aralkylhydroxamic acids, represented by 9-
phenylnonanohydroxamic acid (BMY 30094), inhibits 5-LO.
Small substituents on the phenyl ring (methyl, methoxy,
chloro) has little effect on potency, but larger
substituents (butyloxy) lead to greatly decreased activity.
See Batt at 27-28 and references cited therein.
Hydroxamic acid derivatives of common NSAIDs
(meclofenamic acid, indomethacin, sulindac, and ibuprofen)
inhibit 5-LO with the following potency:
CON(Me)OH~CONHON>CONH(OMe)->COOH. The CO potency ranking
is exactly opposite, although the best 5-LO inhibitors
still possess significant CO activity. Hydroxamic acids,
including many simple ~-aralkylhydroxamic acids, have been
extensively studied and yielded potent 5-LO inhibitors.
The inhibitory activity correlates most strongly with the
over-all lipophilicity. However, hydrophobicity in the
immediate vicinity of the hydroxamic acid, as well as
greater than 12 angstroms away from this moiety, does not
greatly influence potency. The hydroxamic acids have a
large lipophilic group attached to the carbonyl, and a
small alkyl group on nitrogen. SARs are similar to those
observed for the arylacetohydroxamic acids: methyl is
favored on the carbonyl group, the best group linking the
aryl moiety to the nitrogen was CH(Me), and lipophilic
substituents on the phenyl ring, preferably in the para
position, are optimal. Heterocycles such as
benzothiophene, benzofuran, N-methylindole, and
~;hPnzofuran could also serve as the aryl group. See Batt
at 27-32 and references cited therein.
The benzothiophene analogue A64077 (zileuton) is one
of the most interesting 5-LO inhibitors studied to date.
Although the original rationale for 5-LO inhibition by
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hydroxaminacids was iron chelation, this functional group
also contains an N-O single bond which is capable of
oxidation. N-Alkylhydroxylamines are mixed CO/5-LO
inhibitors (approximately equipotent). O-Methylation
increases CO potency and decreases 5-LO potency, while N-
methylation has the opposite effect, and larger N-
substituents decreases activity. Analogues with 7-
substituted 2-naphthyl moieties, exemplified by QA 208-199,
give the best 5-LO potency. See Batt at 32-33 and
references cited therein.
15-HETE inhibits 5-LO. A series of combined 5-LO
inhibitors/LT antagonists were derived from the structure
of 15-HETE. REV 5901A is the best of the series with
respect to Co and 12-LO inhibition. The quinoline could be
replaced by another lipophilic aromatic group, but potency
is decreased (naphthalene is 40-fold less potent, and
substituted phenyl is 5-to-20-fold less active). Pyridines
are active but also less potent; 2-pyridyl is only 4-fold
less active, while 3- and 4-pyridyl are 20-fold weaker.
Ortho- and para- substituted phenylene groups are less
active. See Batt at 33 and references cited therein.
Similar naphthalenemethyloxyphenyl compounds are
potent, selective 5-LO inhibitors which display no
antioxidant or iron chelation properties. Replacement of
the ethyl group by hydrogen or methyl decreases activity,
as does conversion of the methoxy to the hydroxy. Diaryl
2,3-dihydromidazo [2,1-b]thiazoles in which one of the aryl
groups is pyridyl, such as SK&F 86002, are dual CO/5-LO
inhibitors. A related series of diaryl pyrrolo tl,2-
a]imidazoles, represented by SK&F 104351, 104493 and105809, show similar profiles. Tepoxaline (RWJ 20485) is
a hydroxamic acid derivative which inhibits 5-LO. see Batt
at 35 and references cited therein.
As discussed above, a wide variety of agents have been
reported as 5-LO inhibitors. The majority of the series
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appear to be lipophilic reducing agents, including phenols,
partially saturated aromatics, and compounds containing
heteroatom-heteroatom bonds. However, many of these
compounds are not selective 5-LO inhibitors, but often
affect CO and other LOs as well. In vivo systemic activity
for many of these has been in general, disappointing,
probably because of poor bioavailability caused by
lipophilicity and metabolic instability (oxidation, and
conjugation of phenolic compounds). However, the method of
administration outlined in this application, that is local
and slow-release, should overcome some of these
difficulties. A few structural types are selective 5-LO
inhibitors which have shown systemic activity in vivo and
in the clinic. Zileuton appears to be one of the leading
compounds in this category, along with other hydroxamates
such as BW-A4C. Recent selective non-reducing agents such
as Wy-50,295 and the similar ICI compounds such as ICI
216800 also hold promise. See Batt at 32-33 and references
cited therein.
While the present invention is not bound to any
particular theory of operation, it is believed that 5-LO
inhibitors may inhibit adhesion formation between tissue,
e.g. organ, surfaces in body cavities through a variety of
mechAni-cms. For example, 5-LO inhibitors alter arachidonic
acid metabolism, which produces mediators,
e.g.,leukotrienes, of an inflammatory response and thus
reduces inflammation [Anderson et al. (1992) "EYTA, a
pleotrophic membrane-active arachidonic acid analogue
affects multiple signal transduction pathways in cultured
transformed mammalian cells," Clin. Biochem., Vol. 25, pp.
1-9; Miyano and Chiou. (1984), "Pharmacological prevention
of ocular inflammation induced by lens proteinsll,
Ophthalmic Research, Vol. 16, pp. 256-263.]
In addition, leukotrienes (whose synthesis is blocked
by LO inhibitors) are chemotactic for leukocytes tMusser
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and Kreft (1992), "5-Lipoxygenase: Properties, pharmacology
and the quinolinyl (bridged)aryl class of inhibitors", J.
Med. Chem., Vol. 35, pp. 2501-2524; Gulbenkian et al.
(1990), "Anaphylactic challenge causes eosinophil
accumulation in bronchoalveolar lavage fluid of guinea
pigs. Modulation by betamethasone, phenidone,
indomethacin, WEB 2086, and a novel antiallergy agent, SCH
37224," Am. Rev. Res~ir. Dis., Vol. 142, pp. 680-685]. As
an example, L0 inhibitors reduce PMN infiltration
[DeMartino et al. (1989), "The pharmacology of arachidonic
acid-induced rat PMN leukocyte infiltration," Aqent Action,
Vol. 27, pp. 325-327]. As leukocytes are instrumental in
wound repair and lysis of fibrin, these agents may affect
adhesion formation through these actions on leukocyte
chemotaxis.
As is well recognized in the art, however, no one of
these possible mech~n;sms of action of 5-L0 inhibitors
would in and of itself be sufficient to enable one to
predict whether these compounds would have any utility in
reduction of adhesion formation. Indeed, several
properties of 5-L0 inhibitors would suggest that such
compounds would be ineffective at reducing adhesion
formation. For example, fibrinolysis is crucial in
clearance of fibrin deposited after surgical injury. If
the deposition of fibrin is prolonged, then the potential
for adhesion formation is increased. NDGA inhibits the
production of urokinase (a plasminogen activator), which
catalyzes the cleavage of plasminogen to plasmin, a major
fibrinolytic enzyme [Rondeau et al. (1990),
"Nordihydroguaiaretic acid inhibits urokinase synthesis by
phorbol myristate acetate-stimulated LCC-PKl cells," BBA,
Vol. 1055, pp. 165-172]. Inhibition of urokinase would be
expected to promote, not diminish, fibrin deposition.
It is also known in the art that nonsteroidal anti-
inflammatory drugs (NSAIDs) reduce the formation of
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adhesions. NSAIDs are believed to inhibit the formation of
arachidonic acid metabolites through inhibition of
cyclooxygenase (CO). Some of the compounds described above
and in the Examples below are capable of inhibiting CO.
However, NSAIDs and the compounds that inhibit 5-LO have
many distinct biological properties such that a person of
ordinary skill in the art would not have any reason to
believe that 5-LO inhibitors would reduce adhesion
formation. 5-LO inhibitors are distinct from NSAIDs in a
number of ways. For instance, NSAIDs reduce early phase of
sc~ r inflammation. NDGA, however, inhibits both the
early and late phase of ocular inflammation [Miyano and
Chiou. (1984), "Pharmacological prevention of ocular
inflammation induced by lens proteins", Ophthalmic
Research, Vol. 16, pp. 256-263.].
NSAIDs increase urokinase production, however,
lipoxygenase inhibitors do not affect or reduced urokinase
production tChow et al. (1987), "Pharmacological modulation
of plasminogen activator secretion by P388Dl cell line,"
Agents and Actions, Vol. 21, pp. 387-389].
LO inhibitors decrease granuloma formation, however,
NSAIDs have no effect on the formation of granulomas
~Kunkel et al. (1984), "Role of lipoxygenase products of
murine pulmonary granuloma formation," J. Clin. Invest.,
Vol. 74, pp. 514-524].
Additionally, LO inhibitors decrease the arachidonic
acid-induced increase in myeloperoxidase, whereas NSAIDs
have no effect [DiMartino et al. (1989), "The pharmacology
of arachidonic acid-induced rat PMN leukocyte
infiltration," Agents and Action, Vol. 27, pp. 325-327;
Griswold et al. (1989), "Inhibition of inflammatory cell
infiltration by bicyclic imidazoles, SK&F 86002 and SK&F
1004493," Inflammation, Vol. 13, pp. 727-739].
LO inhibitors also reduce eosinophil accumulation,
while NSAIDs had no effect [Gulbenkian et al. (1990),
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"Anaphylactic challenge causes eosinophil accumulation in
bronchoalveolar lavage fluid of guinea pigs. Modulation by
betamethasone, phenidone, indomethacin, WEB 2086, and a
novel antiallergy agent, SCH 37224," Am. Rev. Respir. Dis.,
Vol. 142, pp. 680-685].
In addition, L0 inhibitors had no effect on the
biphasic response to bradykinin, but NSAIDs altered the
response [Calixto and Medeiros. (1991), "Characterization
of bradykinin mediating pertussis toxin-insensitive
biphasic response in circular muscle of the isolate guinea
pig ileum," J. Pharm. Exper. Ther., Vol. 259, pp. 659].
Accordingly, in light of these possible mechanisms of
action of 5-L0 inhibitors, there is no suggestion that 5-L0
inhibitors would in and of itself have any utility in
preventing or reducing post-surgical adhesion formation.
5-L0 inhibitors phenidone, nordihydroguaiaretic acid
(NDGA), 5,8,11,14-eicosatetraynoic acid (EYTA) and Zileuton
have been exemplified below as useful compounds for
reducing or preventing post-surgical adhesion formation.
These structurally unrelated compounds share a common 5-L0
inhibitory effect and other compounds which are also
capable of inhibiting 5-L0 activity are also contemplated
for use in the present invention. In addition to the
compounds described in the references cited in this
application, other non-limiting examples of 5-L0 inhibitors
are described in U.S. Patent Nos. 5,246,948, 5,023,255, and
4,708,964; European Patent Application Nos. 0612 729 A2,
published August 31, 1994 and 0146 348 A2, published June
26, 1985; and W0 95/04055, published February 9, 1995.
In practicing this invention, the preferred 5-L0
inhibitor compounds are those which have little or no
toxicity at the local and systemic level and are suitable
for topical use in animals, including humans. Methods
which may be employed in identifying compounds which
inhibit 5-L0 activity are disclosed, for example, in Batt
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(1992), "5-Lipoxygenase inhibitors and their anti-
inflammatory activities," Prog. Med. Chem., Vol. 29, pp. 1-
63; Riendeau et al. (1989), "Sensitivity of immunoaffinity-
purified porcine 5-lipoxygenase to inhibitors and
activating lipid hydroxyperoxides," Biochem. Pharmacol.,
Vol. 38, pp. 2313-2321; Miyazawa et al. (1985), "Effects of
some non-steroidal anti-inflammatory drugs and other agents
on cycloxygenase and lipoxygenase activities in some enzyme
preparations," Jap. J. Pharmacol., Vol. 38, pp. 199-205.
A representative method for identifying compounds which
inhibit 5-L0 activity is disclosed in the Examples.
Pursuant to the method of the present invention, at
least one 5-Lo inhibitor is maintained in an effective
concentration at the site of potential adhesion formation
for a period of time sufficient to permit substantial re-
epithelialization. The 5-L0 inhibitor is typically
administered over the perioperative interval, which for
purposes of the present invention may include time shortly
prior to surgery through the surgery itself up to some time
after completion of surgery.
The effective therapeutic concentrations of 5-L0
inhibitors is one that minimizes or prevents post-surgical
adhesion formation between tissue surfaces in body
cavities. Typically, the concentrations of 5-L0 inhibitor
which can be administered would be limited by efficacy at
the lower end and the solubility of the compound at the
upper end. In general, the effective therapeutic
concentration of the 5-L0 inhibitors is one that inhibits
5-L0 activity from between about 1 to about 100%,
preferably from between about 10 to about 100%.
The 5-L0 inhibitor may be administered directly
following the surgical procedure in a suitable vehicle, for
example, a solution of saline, 5% DMS0 in saline or 10%
ethanol in saline, to a site at which it is desired to
reduce or prevent adhesion formation. Pursuant to
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preferred embodiments of the present invention, however, at
least one 5-LO inhibitor is administered in a single dose
delivery (for example, prior to skin closure after surgery)
using a drug-delivery system which enables the maintenance
of requisite concentrations of the compound for a period of
time sufficient for re-epithelialization. A suitable
drug-delivery system would itself be essentially non-
inflammatory and non-immunogenic and would permit release
of the 5-LO inhibitor so as to maintain effective levels
thereof over the desired time period.
A large variety of alternatives are known in the art
as suitable for purposes of sustained release and are
contemplated as within the scope of the present invention.
Suitable delivery vehicles include, but are not limited to,
the following: microcapsules or microspheres; liposomes and
other lipid-based release systems; crystalloid and viscous
instillates; absorbable and/or biodegradable mechanical
barriers; and polymeric delivery materials, such as
polyethylene oxide/polypropylene oxide block copolymers
(e.g. poloxamers), poly-orthoesters, cross-linkedpolyvinyl
alcohol, polyanhydrides, polymethacrylate and
polymethacryladmide hydrogels, anionic carbohydrate
polymers, etc.. Useful delivery systems are well known in
the art and are described in, e.g., U.S. Patent No.
4,937,254, the entire disclosure of which is hereby
incorporated by reference.
One particularly suitable-formulation to achieve the
desired near pseudo zero-order release of 5-LO inhibitors
comprise injectable microcapsules or microspheres prepared
from a biodegradable polymer, such as poly(dl-lactide),
poly(dl-lactide-co-glycolide), poly-caprolactone,
polyglycolide, polylactic acid-co-glycolide,
poly(hydroxybutyric acid), a polyortho-ester or a
polyacetal.
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Injectable systems comprising microcapsules or
microspheres of a diameter on the order of about 50 to
about 500 ~m offer advantages over other delivery systems.
For example, they generally use less active agent and may
be administered by paramedical personnel. Moreover, such
systems are inherently flexible in the design of the
duration and rate of separate drug release by selection of
microcapsule size, drug loading and dosage administered.
In addition, such microcapsules can be successfully
sterilized with gamma irradiation.
Microcapsules are systems comprising a polymeric wall
that encloses a liquid or solid core. The capsule wall
usually does not react with the core material; however, it
is designed to provide sufficient strength to enable normal
handling without rupture while being sufficiently thin to
allow a high core to wall volume ratio. The capsule
contents remain within the wall until released by diffusion
or other means that dissolve, melt, break, rupture or
remove the capsule material. Preferably, the capsule wall
can be made to degrade and decompose in suitable
environments while diffusing the core material through the
capsule wall to allow for its slow, prolonged delivery.
The meçh~nism of release in biodegradable
microcapsules is a combination of drug diffusion and
polymer biodegradation. Therefore, the rate and duration
of release are determined by microcapsule size, drug
content and quality, and polymer parameters such as
crystallinity, molecular weight and composition. In
particular, adjustment in the amount of drug released is
generally achieved by modification of capsule wall
thickness, capsule diameter, or both. Detailed information
concerning the design, preparation and use of microspheres
and microcapsules is provided by, e.g., Lewis, D.H.,
"Controlled Release of Bioactive Agents from
Lactide/Glycolide Polymers," in "8iodegradable Polymers as
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Drug Delivery systems," Jason & Langer, eds., pp. 1-41
(1990), the entire disclosure of which is hereby
incorporated by reference. The sustained intraperitoneal
release of dexamethasone using poly(lactide-co-glycolide)
microparticles is described in Hoeckel, M. et al.,
"Prevention of Peritoneal Adhesions in the Rat with
Sustained Intraperitoneal Dexamethasone Delivered by a
Novel Therapeutic System," Annales Chirurgiae et
Gynaecoloqiae, Vol. 76, pp. 306-313 (1987), the entire
disclosure of which is also incorporated by reference.
As is well known to those skilled in the art, various
methods are currently available for preparing
microcapsules, any of which could be employed to provide
formulations in accordance with the present invention.
Biodegradable polymeric materials suitable for preparation
of microcapsules for controlled (i.e., near zero-order)
release would be readily determined through routine
experimentation by those skilled in the art. Moreover,
alternative delivery systems suitable for use in accordance
with the present invention (for example, fibers or
filaments comprising the active agents) based on
biodegradable polymers are also contemplated as within the
scope of the present invention.
An alternative approach for the single-dose delivery
of at least one 5-LO inhibitor involves the use of
biodegradable polymers, such as the ones described above,
in the form of a film. Such films may be produced by
spraying or discharging dispersed liquid droplets
containing the biopolymer and the 5-L0 inhibitor in a
suitable carrier from a pressurized container onto the
targeted site.
Another approach for the single-dose delivery of at
least one 5-LO inhibitor, in accordance with the present
invention, involves the use of liposomes and other lipid-
based delivery systems. The encapsulation of an active
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agent in multilamellar vesicles (or liposomes) is a well
known technique used to assist in target drug delivery and
prolong drug residence. In a typical procedure, a
liposome-forming powdered lipid mixture is added to the
desired quantity of active agent in aqueous solution (e.g.,
phosphate buffered saline) to form a suspension. After a
suitable hydration period, the hydrated suspension is then
autoclaved to provide the liposome-active agent
preparations. A lipid mixture suitable for formation of
liposomes may be prepared from L-alpha-distearoyl
phosphatidylcholine and cholesterol dissolved in
chloroform, to which alpha-tocopherol is added; other
compositions and methods for formation of liposomes would,
however, also be useful for this purpose. The
intraperitoneal administration of liposomes containing
ibuprofen or tolmetin is described in Rodgers, K. et al.,
"Inhibition of Postsurgical Adhesions by Liposomes
Cont~;ning Nonsteroidal Anti-inflammatory Drugs," Int. J.
Fertil., Vol. 35, p. 40 (1990), the entire disclosure of
which is hereby incorporated by reference.
Other lipid-based delivery systems are also
contemplated for use in this invention. One useful system
includes lipid foams such as DepoFoam extended-release
formulations comprising spherical particles bounded by a
single bilayer lipid membrane and each containing numerous
nonconcentric aqueous chambers which encapsulate the active
ingredient (see, e.g, Kim, T.K. et al. (1993) "Extended-
release formulation of morphine for subcutaneous
administration," Cancer Chemother. Pharmacol., Vol. 33,
187; Chatelut, E. et al. (1993) "A slow-release
methotrexate formulation for intrathecal chemotherapy,"
Cancer Chemother. Pharmacol., Vol. 32, 179.] Such lipid
particles are made from nontoxic lipids identical to those
found in cell membranes.
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Another suitable lipid-based delivery system for
deli ering the 5-Lo inhibitors according to the invention
includes emulsion carrier systems based on egg sphinomyelin
and egg phosphatidylcholine. Such emulsion carrier systems
have prolonged blood circulation retention times and were
developed for delivering highly lipophilic drugs. T.
Takino et al. (1994) "Long Circulating Emulsion Carrier
Systems for Highly Lipophilic Drugs," Biol. Pharm. Bull.,
Vol. 17, pp. 121-125.
Yet another suitable approach for single dose delivery
of at least one 5-LO inhibitor in accordance with the
present invention involves the use of crystalloid or
so-called viscous instillates. Crystalloids are known in
the art as water soluble crystalline substances, e.g. NaCl,
capable of diffusing through a semi-permeable membrane.
Solutions of crystalloids, such as saline, are known as
crystalloids, crystalloid solutions or crystalloid
instillates. Crystalloids include, but are not limited to,
phosphate buffered saline, saline or lactated Ringer's
solution. High-molecular-weight viscous carriers used in
admixture with the active agents include, but are not
limited to, the following: dextrans and cyclodextrans;
hydrogels; cross-linked viscous materials, including
viscoelastics and cross-linked viscoelastics;
carboxymethylcellulose; hyaluronic acid, crosslinked
hyaluronic acid, and hyaluronic acid compounded with
orthoesters. While some studies have suggested that the
use of viscous barrier solutions per se may have an
advantageous effect in reducing the incidence of adhesion
formation, it is believed that any such effect is of
limited scope when compared to the combination of at least
one 5-LO inhibitor and carrier. The intraperitoneal
administration of a viscous instillate comprising tolmetin
is described in Abe, H. et al., "The Effect of intra-
peritoneal Administration of Sodium Tolmetin-Hyaluronic
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Acid on the Postsurgical Cell Infiltration In Vivo, " J
Surq. Res., Vol. 49, p. 322 (1990), the entire disclosure
of which is hereby incorporated by reference.
Pursuant to yet another approach, at least one 5-LO
inhibitor is administered in combination with an absorbable
mec-hAn;cal barrier which alone reduces adhesion formation.
As would be readily apparent to one working in the field,
at least one 5-LO inhibitor may be covalently or
non-covalently (e.g., ionically) bound to such a barrier,
or it may simply be dispersed therein. A particularly
suitable vehicle for use in this particular embodiment of
the invention comprises hydroxyethyl starch which is
described in U.S. Patent application Ser. No. 08/482,235,
filed concurrently with this application, entitled
HYDROXY~lnYL STARCH AND USE THEREOF AS AN ABSO~R~RT~
MECHANICAL BARRIER AND INTRACAVITY CARRIER DEVICE by Gere
diZerega (University of Southern California, assignee), and
incorporated by reference in its entirety.
Another suitable m~ch~n;cal barrier for use in this
invention includes oxidized regenerated cellulose which is
available under the designation INTERCEED(TC7) from Johnson
and Johnson Medical, Inc., New Brunswick, New Jersey
[INTERCEED(TC7) Adhesion Barrier Study Group, "Prevention
of postsurgical adhesions by INTERCEED(TC7), an absorbable
adhesion barrier: a prospective, randomized multicenter
clinical study," Fertility and SterilitY, Vol. 51, p. 933
(1989)]. -The use of a mechanical barrier as a carrier to
deliver heparin to traumatized surfaces is disclosed in
Diamond, M. P. et al., "Synergistic effects of
INTERCEED(TC7) and heparin in reducing adhesion formation
in the rabbit uterine horn model," Fertility and Sterility,
Vol. 55, p. 389 (1991) and Diamond, M.P. et al., "Adhesion
reformation: reduction by the use of INTERCEED(TC7) plus
heparin," J. Gyn. Surg., Vol. 7, p. 1 (1991), the entire
disclosures of which are hereby incorporated by reference.
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The invention may be better understood with reference
to the accompanying examples, which are intended to be
illustrative only and should not be viewed as in any sense
limiting the scope of the invention, which is defined
hereinafter in the accompanying claims.
EXAMPLES
Multiple studies to confirm the efficacy of 5-
lipoxygenase inhibitors in the reduction of adhesion
formation after peritoneal surgery were performed. Two
model systems were employed: the sidewall adhesion model
and the uterine horn model. A clear correlation between
results obtained using both of these models and utility in
adhesion prevention has been demonstrated with
INTERCEED(TC7), for which clear clinical efficacy has been
shown and FDA approval for adhesion prevention in
gynecological surgery has been obtained.
In the peritoneal sidewall model, rabbits were pre-
anesthetized with 1. 2 mg/kg acetylpromazine and
anesthetized with a mixture of 55 mg/kg ketamine
hydrochloride and 5 mg/kg xylazine intramuscularly.
Following preparation for sterile surgery, a midline
laparotomy was performed. A 3 x 5-cm area of peritoneum
and transversus abdominis muscle was removed on the right
lateral abdominal wall. The cecum was exteriorized, and
digital pressure was exerted to create subserosal
hemorrhages over all cecal surfaces. The cecum was then
returned to its normal anatomic position. The compound to
be tested was placed in an Alzet miniosmotic pump (Alza
Corporation, Palo Alto, CA, USA) to allow continuous
release of the molecule through the postsurgical interval.
The Alzet miniosmotic pump was placed in the subcutaneous
space and a delivery tube connected the pump with the site
of injury at sidewall. Vehicle was placed in the pump of
control rabbits. The abdominal wall and skin were closed
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in a standardized manner.
After 7 days, the rabbits were sacrificed and the
percentage of the area of the sidewall injury that is
involved in adhesions was determined. In addition, the
tenacity of the adhesion formed was scored use a system as
follows:
0 = No adhesions
1 = mild, easily dissectable adhesions
2 = moderate adhesions; non-dissectable, does
not tear organ
3 = dense adhesions; non-dissectable, tears
when removed
A reduction in the area or the tenacity of the adhesions
would be considered beneficial.
In additional experiments, a rabbit uterine horn model
was employed. This model has been previously shown to
cause severe adhesions in rabbits after surgery [Nishimura,
K. et al., "The Use of Ibuprofen for the Prevention of
Postoperative Adhesions in Rabbits," Am. J. Med., Vol. 77,
pp. 102-106 (1984)]. The rabbits were anesthetized (130
mg/kg ketamine and 20 mg/kg acetylpromazine im) and
prepared for sterile surgery. A midline laparotomy was
performed, and surgical trauma was performed on both
uterine horns by abrading the serosal surface with gauze
until punctate bleeding developed. Ischemia of both
uterine horns was induced by removal of the collateral
blood supply. After traumatization, the abdominal wall was
closed in two layers. The inhibitor to be tested was
delivered as described for the peritoneal sidewall model,
but the tubing was placed over the injured uterine horns.
With the uterine horn model, an initial score to
represent the overall extent of adhesions is given (0 to
4+). The percentage of a surface of the horn involved in
adhesions to various organs are given in the tables below
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the overall adhesion score.
In the model systems employed ~a the examples reported
herein, exemplary 5-lipoxygenase inhibitor compounds
phenidone, NDGA, Zileuton, and ETYA were shown to reduce
the incidence of peritoneal adhesions. In these Examples,
the drug was delivered to the targeted site at a rate of 10
~l/hour. The concentration ranges employed were 0.0001 to
0.5 mg/ml. For purposes of preventing adhesion formation
in accordance with the present invention, it is not
believed that high systemic levels of 5-lipoxygenase
inhibitors would be necessary.
EXAMPLE 1: General 5-LO Activity Assay Procedure
In this Example, a assay procedure is provided to
identify compounds that inhibit 5-LO activity. According
to the procedure as disclosed by Riendeau et al. (1989),
"Sensitivity of immunoaffinity-purified porcine 5-
lipoxygenase to inhibitors and activating lipid
hydroxyperoxides," Biochem. Pharmacol., Vol. 38, pp. 2313-
2321. 5-Lipoxygenase activity is measured from the
increase in absorbance at 235nm following incubation of 5-
LO, isolated from porcine leukocytes, arachidonic acid, ATPand calcium. The standard reaction mixture contains 0.55
M Tris-HCl, pH 7.4, 0.2 mM ATP, 0.4 mM CaCl2, 20 or 27 mM
arachidonic acid (5 ~1 of a 100-fold concentrated solution
in ethanol), 24 ~g/ml phosphatidylcholine, and an aliquot
of the enzyme preparation (5-75 ~1) in a final volume of
0.5 ml. The volume of enzyme is completed to 100 ~1 using
a chromatography elution buffer (50 mM sodium carbonate, pH
10, containing 0.2% sodium deoxycholate, 0.5 mM
dithiothreitol and 1 mM EDTA into 0.5 M Tris). The buffer
solution containing CaCl2 (0.4 M) and phosphotidylcholine
(24 ~g/ml) is filtered through 0.2 ~m Nalgene filters.
The assay reactions are performed in semi-micro
cuvettes (1.4 ml capacity, 100 mm path length and 4 mm
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internal width) and initiated by the addition of the enzyme
to the assay mixture. The reaction mixture is gently mixed
with a Pasteur pipet (15 sec) before recording the
variation in A235 as a function of time at room
temperature.
EXAMPLE 2: Sidewall Model Evaluation of Phenidone
The efficacy of phenidone (Sigma Chemical Co., St.
Louis, MO), a 5-LO inhibitor discussed above, in preventing
adhesion formation was evaluated at two doses in a sidewall
model. The drug was delivered for 7 days at a rate of 10
~l/hr and the animals were sacrificed after 7 days. The
vehicle used was saline. Relative to the control,
phenidone was found to be efficacious in 5 of 6 rabbits at
the high dose and in 4 of 6 rabbits in the lower dose. No
inflammation or precipitation noted at the site of injury.
The results are summarized in Table 1. A student t test
analysis of the data was performed and the results are also
reported in Table 1.
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TABLE 1
TREATMENT % ADHESION
ADHESIONS SCORE
Vehicle 80% 2+
Control
60% 3+
60% 2+
70% 2+
50% 2+
100% 3+
Mean: 70-0% i 17.8%
0.5 mg/ml 40% 2+
Phenidone
10% 1+
80% 2+
30% 1+
10% 1+
0% 0+
Mean-: 28.3% + 29.3%
0.05 mg/ml 70% 1+
Phenidone
100% 3+
0% . 0+
0% 0+
10% 1+
40% 2+
Meanb: 36.7% + 41.3%
a: p = 0.014
b: p = 0.100
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EXAMPL~ 3: Sidewall model evaluation of NDGA
The efficacy of nordihydroguaiaretic acid (NDGA)
(available from Sigma Chemical, St. Louis, MO), a 5-LO
inhibitor discussed above, in preventing adhesion formation
was evaluated at two doses in a sidewall model. The drug
was delivered for 7 days at a rate of 10 ~l/hr and the
animals were sacrificed after 7 days. The vehicle used was
0.1% ethanol in saline, pH 10. 3. Relative to the control,
NDGA appears to be efficacious in 5 of 6 rabbits at the
high dose and in 3 of 5 rabbits in lower dose. No
inflammation or precipitation noted at the site of injury.
The results are summarized in Table 2. A student t test
analysis of the data was performed and the results are also
reported in Table 2.
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TABLE 2
TREATMENT % ADHESION
ADHESIONS SCORE
Vehicle 90% 2+
Control
Died due to bowel
obstruction due to adhesions
100% 3+
90% 3+
40% 3+
100% 2+
Mean: 84.0% + 25.1%
0.3 mg/ml NDGA 30% 2+
30% 2+
0% 0+
60% 1+
0% 0+
0% 0+
Mean: 20.0% + 24.5%
0.03 mg/ml 100% 3+
NDGA
10% ~+
20% 1+
70% 2+
0% 0+
Died on Day 5 P/O
Meanb: 40.0% + 43.0%
a: p = 0.002
b: p = 0.084
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EXAMPLE 4: Sidewall Model evaluation of ETYA
The efficacy of 5,8,11,14-eicosatetraynoic acid (ETYA)
(available from Sigma Chemical, St. Louis, MO), a 5-LO
inhibitor discussed above, in preventing adhesion formation
was evaluated at two doses in a sidewall model. The drug
was delivered for 7 days at a rate of 10 ~l/hr and the
animals were sacrificed after 7 days. The vehicle used was
11.1% ethanol in saline, pH 10.6. Relative to the control,
ETYA was efficacious in the prevention of adhesions in this
rabbit sidewall model. The results are summarized in
Table 3. A student t test analysis of the data was
performed and the results are also reported in Table 3.
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TABLE 3
TREATMENT % ADHESION
ADHESIONS SCORE
Vehicle 40% 3+*
Control
100% 3+*
70% 3+
70% 2+
80% 3+
50% 2+
Mean: 68.3% + 21.4%
0.1 mg/ml ETYA 30% 2+*
Died
0% 0+
0% 0+
5% 1+
10% 0+
Mean: 9.0% + 12.5%
0.01 mg/ml 100% 3+*
ETYA
40% 1+
40% 1+
Died
0% 0+
30% 1+
Mean: 42.0% + 36.0%
a: p = o.000
b: p = 0.169
* Sidewall was inflamed.
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EXAMPLE 5: DUH Model Evaluation of Phenidone
The efficacy of phenidone, the compound exemplified in
Example 2 above, in preventing adhesion formation was
evaluated at two doses in a double uterine horn (DUH)
model. The drug was delivered for 7 days at a rate of 10
~l/hr and the animals were sacrificed after 7 days. The
vehicle used was saline. Statistical analysis was
performed on the overall score of the nonparametric double
uterine horn model data. The data was rank ordered, a rank
value was given and an analysis of variance on the ranks
was performed. The results are summarized in Tables 4 and
5. Relative to the control, phenidone was efficacious in
reducing adhesion formation in this model.
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TABLE 4
TREATMENT OVERALL ADHESION
SCORE
Vehicle Control 3+
3.5+
3.5+
3+
3+
3.5+
0.5 mg/ml 1+
Phenidone
2+
2+
0.5+
1.5+
l+
0.05 mg/ml 2+
Phenidone
2+
l+
1.5+
2+
1.5+
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TABLE S
% Organ Involvement in Uterine
Horn Adhesion
Tre tment Right Horn LeR Hom
Bowel Bl-dder It elf Lefl Bowel Bl~dder llself Right
Cont~l 60 S0 S0 S0 60 S0 40 S0
S0 60 40 S0 S0
S0 40 100 30 S0 40 40 30~
100 40 S0 20 100 40 S0''
S0 60 80 30 S0 S0 60 30
Me D 46.7 60 61.7 41.7 46.7 60 45 41.7
0.5 mg/ml 10 0 10 0 10 0 0 0
rDeDidoDe
0 0 40 40 0 0 40 40
0 0 30 30 0 40 40 30
0 20 0 0 0 0 0
O O 10 0 0 0 0 0~
0 10 0 40 0 10 0 40
Me D 3.3 1.7 18.3 18.3 1.7 8.3 13.3 1~.3
0 O.OS rag/ml 10 10 40 10 10 10 0 10
rDeDidoDe
0 30 10 20 0 30 10
0 0 20 0 0 20 40 0
0 20 20 20 0
0 30 0 30 0 60 0
100 0 10 0 100 0 0 0~
Me n 30 S 23.3 3.3 30 8.3 25 3.3
* Bladder, horn and/or bowel adhered to the sidewall at
the suture for the tube.
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Statistical analysis was performed on the overall
score of the nonparametric data taken from Table 4. The
data was rank ordered and assigned a rank value. Analysis
of the variance of the ranks was then performed and the
resulting student t test results are summarized below.
Treatment Rank orderp value
Control 15.5 + 1.5 ---
0.5 mg/ml 5.5 + 3.50.000
phenidone
0.05 mg/ml 7.5 + 2.7O.OOO
phenidone
BXANP~B 6: DUH Model Evaluation of NDGA
The efficacy of NDGA, the compound exemplified in
Example 3 above, in preventing adhesion formation was
evaluated at two doses in a double uterine horn model. The
drug was delivered for 7 days at a rate of lO,ul/hr and the
animals were sacrificed after 7 days. The vehicle used was
0.1% ethanol in saline, pH 10.3. Statistical analysis was
performed on the overall score of the nonparametric double
uterine horn model data. The data was rank ordered, a rank
value was given and an analysis of variance on the ranks
was performed. The results are summarized in Tables 6 and
7. Relative to the control, NDGA was efficacious in
reducing adhesion formation in this model.
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TABLB 6
TREATMENT OVERALL ADHESION
SCORE
Vehicle Control 3+
3+
3.5+
3.5+
3+
3.5+
0.3 mg/ml NDGA 1.5+
1.5+
1.5+
Infection
2.5+
2+
0.03 mg/ml NDGA 2+
2+
1.5+
1.5+
2.5+
1.5+
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TABL~ 7
% Organ Involvement in Uterine
Horn Adhesion
Treetment Right Hom LeR Hom
Bowel Bladder It elf Left Bowel Bladder It~elf Right
Cootrol 60 0 50 50 60 0 40 50
100 10 40 50 100 10 40 50
Mean 51.7 30 46.7 51.7 51.7 30 45 51.7
0.3 nUlml 0 40 30 0 0 40 0 0
NDGA
0 30 0 30 0 0 0
0 0 10 0 50 0 0 0
INFECllON
0 0 40 30 0 0
0 10 30 20 20 10
Mean 20 18 14 2 30 18 4 2
0 0.03 mg/ml 0 0 20 30 0 0 30 30
NDGA
0 0 30 10 20 0
0 0 40 0 0 0 50 0
0 20 0 40 0 0 0
0 0 30 30 30 0
0 10 30 0 0 10 0 0
Mean 16.7 8.3 18.3 5 16.7 8.3 21.7 5
* Bladder and/or bladder adhered to sidewall at the tube
or suture for the tube.
~5 ** Horn along with bowel and/or bladder adhered to
sidewall at the tube or the suture for the tube.
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Statistical analysis was performed on the overall
score of the nonparametric data taken from Table 6. The
data was rank ordered and assigned a rank value. Analysis
of the variance of the ranks was then performed and the
resulting student t test results are summarized below.
Treatment Rank orderp value
Control 14.5 + 1.5 ---
0.3 mg/ml 5.8 + 2.9O.O00
NDGA
0.03 mg/ml 6. 2 + 2.8O.OOO
NDGA
EXANPLE 7: DUH Model Evaluation of ETYA
The efficacy of ETYA, the compound exemplified in
Example 4 above, in preventing adhesion formation was
evaluated at two doses in a double uterine horn model. The
drug was delivered for 7 days at a rate of 10 ~l/hr and the
animals were sacrificed after 7 days. The vehicle used was
11.1% ethanol in saline, pH 10.6. Statistical analysis was
performed on the overall score of the double uterine horn
model nonparametric data. The data was rank ordered, a rank
value was given and an analysis of variance on the ranks
was performed. The results are summarized in Tables 8 and
9. Relative to the control, ETYA was efficacious in the
reduction of adhesion formation in this model.
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TABLE 8
TREATMENT OVERALL ADHESION
SCORE
Vehicle Control 2.5+
3+
3.5+
3+
3.5+
3+
0.1 mg/ml ETYA 2+
1.5+
l+
1.5+
1.5+
1.5+
0.01 mg/ml ETYA 0;5+
l+
l+
0.5+
l+
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TABLE 9
% Organ Involvement in Uterine
Horn Adhesion
Treatrnent Right Horn Lefl Horn
Bowcl Bhdder It~elf Lefl Bowel Bbdder It~elf Right
Control 40 50 30 40 40 50 30 40
Me~n 40 56.7 43.3 46.7 40 56.7 38.3 46.7
0.1 mg/ml 30 20 20 40 30 0 20 40
ETYA
0 40 0 10 0 0 0
0 0 30 0 10 0 10 0
0 0 20 20 0
0 40 0 30 0 20 0
0 20 10 30 0 0 10
Me~n 18.3 6.7 30 8.3 18.3 3.3 11.7 8.3
0 0.01 mg/ml 0 0 10 0 0 0 10 0
ETYA
0 0 40 0 0 0 40 0
0 0 30 0 0 0 0 0
0 30 0 0 0 0 0
O 0 10 0 0 0 0 0
0 20 0 0 0 20 20 0
Mesn 1.7 3.3 20 0 0 3.3 11.7 0
* Bowel and/or bladder adhered to sidewall at the tube
or suture for the tube.
~5 ** Horn along with bowel and/or bladder adhered to
sidewall at the tube or the suture for the tube.
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Statistical analysis was performed on the overall
score of the nonparametric data taken from Table 8. The
data was rank ordered and assigned a rank value. Analysis
of the variance of the ranks was then performed and the
resulting student t test results are summarized below.
i
Treatment Rank orderp value
Control 15.5 + 1.6 ---
0.1 mg/ml 9.2 + 2.10.000
ETYA
0.01 mg/ml 3.8 + 1.70.000
ETYA
EXANPLE 8: Kinetic DUH Model evaluation of ETYA
The efficacy of ETYA in the rabbit double uterine horn
model was further evaluated in a kinetics study. In this
study, the pump was disconnected (D/C) at various times
after surgery (24, 48, or 72 hours) to determine the time
period of exposure to the drug effective to reduce adhesion
formation. The results are summarized in Tables 10 and 11.
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TABLE 10 .
TR~T~T OVERALL ADHESION
SCORE
Vehicle Control 3+
2.5+
3+
2.5+
3.5+
3.5+
0.1 mg/ml ETYA 1.5+
24 hour D/C
1.5+
1.5+
1.5+
0.5+
0.5+
0.1 mg/ml ETYA 1+
48 hour D/C 21+
l+
l+
2+
0.1 mg/ml ETYA 0.5+
72 hour D/C
1.5+
1.5+
1.5+
1.5+
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TREATMENT OVERALL ADHESION
SCORE
2+
i
O.O1 mg/ml ETYA 1+
24 hour D/C
Infection
0.5+
1.5+
0.5+
1.5+
O.O1 mg/ml ETYA l+
48 hour D/C
l+
l+
1.5+
2+
0.5+
O.O1 mg/ml ETYA 1+
72 hour D/C
0.5+
5+
l+
l+
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TABLE 11
% organ Involvement in Uterine
Horn Adhesion
Trcatment Right Horn Lefl Horn
Bowel Bbdder It elf Left Bowel Bbdder Itself Right
Con rol 50 10 20 30 50 10 40 30
S0 40 40 80 S0 40 40
Me~n S0 33.3 36.7 40 50 33.3 36.7 40
0.1 mg/ml 10 0 20 10 10 0 20 10
El YA
24 D/C
0 0 10 10 30 0
0 30 0 0 0~
0 10 20 10 0 10 10 10
O 10 0 0 0 10 10 0
0 0 0 0 20 0 0 0
Me~n 8.3 6.7 10 3.3 8.3 5 11.7 3.3
0.1 mg/ml 10 0 10 0 10 0 10 0
ETYA 48 D/C
0 20 30 30 0 10 30
0 0 0 30 0 10 0
0 10 0 10 0 0 0
0 20 0 20 0 0 0
0 10
Mesn 20 3.3 13.3 6.7 20 3.3 5 6.7
0.1 mg/ml 0 0 10 10 0 0 0 10
ETYA 72 D/C
0 10 10 10 0
0 20 10 40 0
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Tre~tmcnt Right Hom Left Horn
Bowel Bladder Itself Lefl Bowel Bhdder It~elf Right
0 0 20 10 0 20 20
0 30 10 20 0 30 0 30
0 40 20 20 0 30 20
Me~n 10 8.3 13.3 11.7 10 8.3 16.7 13.3
0.01 mg/rnl 0 0 30 10 0 0 30 10
El'YA 24 D/C
INFECTION
0 0 20 0 0 0 20 0
0 10 10 20 0 10 10 20
O 0 10 0 0 0 0 0
0 10 10 20 0 30 10
Mc~n 4 2 16 8 4 2 18 8
0.01 mg/ml 10 0 0 0 10 0 10 0
ETYA 48 D/C
0 0 10 10 0 0 10
0 0 0 10 0 10 0~
0 0 30 20 0 0 30 20
0 20 30 20 0 20
0 0 30 0 10 0 0 0
Mc~n 10 3.3 10 8.3 11.7 3.3 8.3 8.3
0.01 mg/ml 0 0 20 40 0 0 20 40
El'YA 72 D/C
0 0 20 0 0 0 0 0
~ 10
0 0 20 30 0 0 20 30
0 20 0 20 0 10 0
Lefl Horn Only in Rabbit 20 0 20 0
Me~n 5 2 20 16 7.5 1.7 11.7 13.3
* Bladder and/or bowel adhered to sidewall (either at
the tube or suture).
** Horn and bowel or bladder to sidewall.
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Statistical analysis was performed on the overallscore of the nonparametric data taken from Table 10. The
data was rank ordered and assigned a rank value. Analysis
of the variance of the ranks was then performed and the
resulting student t test results are summarized below.
Treatment Rank order p value
Control 38.5 + 1.6 ---
0.1 mg/ml 18.3 + 10.1 0.000
ETYA ( 24 hr)
0.1 mg/ml 20.2 + 9.4 0.000
ETYA (48 hr)
0.1 mg/ml 25.3 + 9.1 0.002
ETYA ( 72 hr)
0.01 mg/ml 14.5 + 9.6 0.000
ETYA (24 hr)
0.01 mg/ml 17.3 + 9.6 0.000
ETYA ( 48 hr)
0.01 mg/ml 13.9 + 6.2 0.000
ETYA ( 72 hr)
20 E~NPLE 9: Dose Response Study of ETYA in the DUH Model
A dose response study with ETYA was then conducted in
the double uterine horn model to better define the ranges
over which an inhibitor of 5-LO would be efficacious in the
reduction of adhesion formation. The conditions are the
same as described in Example 7. The results are summarized
in Tables 12 and 13.
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TABLE 12
TREATMENT OVERALL ADHESION
SCORE
Vehicle Control 3+
2.5+
3+
3+
3.5+
3.5+
0.1 mg/ml ETYA 1+
1.5+
l+
0.5+
2+
0.5+
0.01 mg/ml ETYA 1.5+
1.5+
1.5+
1.5+
1.5+
0.5+
0.001 mg/ml ETYA 1.5+
2.5+
2.5+
2.5+
0.5+
3+
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TREATMENT OVERALL ADHESION
SCORE
0.000l mg/ml ETYA 3+
l+
3 5+
2+
2+
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TABLE 13
% Organ Involvement in Uterine
Horn Adhesion
Tre tment Right Horn Lefl Horn
Bowel Bl-dder It~elf Lefl Bowel Bl-ddcr It clf Right
Conlrol S0 80 100 0 50 80 S0 0
0 40 0 30 0 40 0
S0 30 60 100 50 30 60 100
Me~n 55 38.3 53.3 35 55 38.3 48.3 46.7
0.1 mg/ml 10 10 20 0 10 0 0 0
El'YA
0 30 20 10 0 30 20
0 30 0 10 0 0 0
0 0 0 0 0 0 20 0
0 50 20 30 0 50 20
0 10 10 0 - O 10 0 0
Me n 10 3.3 23.3 6.7 10 1.7 16.7 6.7
10 0.01 mg/ml 10 0 40 20 10 0 30 20
ETYA
0 30 0 10 0 30 30 10
0 30 10 10 0 40 10
0 40 20 lO 0 40 20
0 0 30 10 0 30 30
0 10 10 0 0 10 0 0
Me~n 6.7 6.7 20 15 6.7 6.7 28.3 15
0.001 mg/ml 0 0 50 0 0 0 50 0
El YA
0 40 20 30 0
0 30 10 30 0
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Tre trnent Right Horn LeR Horn
Bowel Bladder It elf l~fl Bowel Bladder It elf Right
0 0 10 80 60 0
O O 10 0 0 0 10 0
0 50 70 50 30 50
Me n 25 26.7 13.3 8.3 25 26.7 35 8.3
0.0001 rng/rnl 20 40 50 30 20 40 20 30
El'YA
0 10 10 10 0 10 10
0 0 20 20 0 30 20
0 0 30 50 20 0
0 60 0 40 0 0 0
Mc~n 30 25 26.7 16.7 30 25 21.7 16.7
* Bowel and/or bladder adhered to sidewall at the tube
or suture for the tube.
** Horn along with bowel and/or bladder adhered to
sidewall at the tube or the suture for the tube.
Statistical analysis was performed on the overall
score of the nonparametric data taken from Table 12. The
data was rank ordered and assigned a rank value. Analysis
of the variance of the ranks was then performed and the
resulting student t test results are summarized below.
Treatment Rank order p value
Control 25.6 + 2.9 ---
0.1 mg/ml ETYA 7.8 + 5.2 0.000
0.01 mg/ml ETYA 10.4 + 3.5 O.OOo
0.001 mg/ml ETYA 16.8 + 7.5 0.023
0.0001 mg/ml 16.8 + 8.4 0.036
ETYA
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EXAMPLE 10: Sidewall Model Evaluation of Zileuton
The efficacy of Zileuton, a 5-LO inhibitor discussed
above, in preventing adhesion formation was evaluated at
two doses in a sidewall model. The drug was delivered for
7 days at a rate of 10 ~l/hr and the animals were
sacrificed after 7 days. The vehicle used was saline.
Relative to the control, Zileuton was found to be
efficacious at both concentrations tested. No inflammation
or precipitation noted at the site of injury. The results
are summarized in Table 14. A student t test analysis of
the data was performed and the results are also reported in
Table 14.
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TABLE 14
TREATMENT % ADHESION
ADHESIONS SCORE
Vehicle 100% 2+
Control
100% 3+
100% 3+
100% 2+
70% 3+
Died
Mean: 94.0% + 13.42%
0.2 mg/ml 80% 2+
Zileuton
10% 1+
50% 2+
20% 2+
10% 2+
0% 0+
Mean:- 28.3% + 30.6%
0.02 mg/ml 20% 1+
Zileuton
Died
80% 1+
10% 1+
60% 1+
20% 1+
Mean:b 38.0 + 30.3
a: p = 0.002
b: p = 0.005
-
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While the fundamental novel features of the invention
has been shown and described, it will be understood that
various omissions, substitutions and changes in the form
and details illustrated may be made by those skilled in the
art without departing from the spirit of the invention. It
is the intention, therefore, to be limited only as
indicated by the scope of the following claims.
