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Description  |
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The invention relates to a method for inhibiting post-surgical adhesion
formation.
BACKGROUND OF THE INVENTION
Adhesion formation is a major post-surgical complication with no practical
solution. The incidence of adhesion formation following surgery approaches
100 percent, according to some sources, with a clinically significant
complication rate of about 5 to 10 percent, depending on the type of
surgery. Among such complications are bowel obstruction, infertility, and
pain. Occasionally, adhesions necessitate a second operative procedure to
remove the adhesion, which may in turn further aggravate the problem.
Because of the seriousness of the problem, much medical research has been
performed in efforts to find ways to combat adhesions. See, for instance,
Stangel et al., "Formation and Prevention of Postoperative Abdominal
Adhesions", the Journal of Reproductive Medicine, Vol. 29, No. 3, March
1984 (pp. 143-156), and diZerega, "The Cause and Prevention of
Postsurgical Adhesions", published by Pregnancy Research Branch, National
Institute of Child Health and Human Development, National Institutes of
Health, Building 18, Room 101, Bethesda, Md. 20205. Among the approaches
that have been tried for preventing post-surgical adhesion are the
following:
Systemic administration of ibuprofen (e.g., see Singer, U.S. Pat. No.
4,346,108);
Parenteral administration of antihistamines, corticosteroids, and
antibiotics;
Intraperitoneal administration of dextran solution and of
polyvinylpyrrolidone solution; and
Systemic administration of oxyphenbutazone, a non-steroidal
anti-inflammatory drug that acts by inhibiting prostaglandin production.
Corticosteroids have been administered intraperitoneally as well as
systemically in efforts to prevent adhesions. (See the Stangel et al.
article, cited above, on p. 147, as well as the articles cited therein.)
Some studies have questioned the efficacy of corticosteroids in adhesion
prevention. In high doses, these materials may suppress the immune system
and interfere with wound healing. Therefore, the use of corticosteroids
does not seem to be an acceptable solution to the post-operative adhesion
problem.
BRIEF SUMMARY OF THE INVENTION
The invention comprises a method for inhibiting the formation of
post-surgical adhesions in mammals (including humans) which method
comprises the topical locally effective administration of an effective
amount of a composition including as an active ingredient tissue
plasminogen activator ("t-PA") to the injured tissue surface site of
surgical trauma, for a period of time sufficient to inhibit the formation
of post-surgical adhesions.
PRIOR ART
Kapur et al., in "Prevention of Reformation of Peritoneal Adhesions", Arch.
Surg., Vol. 105, Nov. 1972 (pp. 761-764), disclose the systemic
administration of proteolytic enzymes from carica papaya to combat
adhesions.
Singh, in U.S. Pat. No. 3,912,704, suggests that a protease inhibitor
isolated from horse urine may be useful in preventing the formation of
post surgical adhesions.
The use of streptokinase, streptodornase, and urokinase in preventing
adhesions has been reported. Administration was by a single
intraperitoneal dose. The references are:
Ascherl et al., PREVENTION OF INTRAPERITONEAL ADHESIONS WITH A
FIBRINOLYTIC, Medwelt, 34, No. 13/83, pp. 410-415;
Mund-Hoym et al., PREVENTION OF POSTOPERATIVE ADHESIONS-AN ANIMAL STUDY,
Geburtsh, u. Franenheilk, 44 (1984), pp. 463-467; and
Minju et al., ANIMAL STUDIES ON THE PREVENTION OF ADHESION AND ADHESIVE
INTESTINAL OBSTRUCTION AFTER ABDOMINAL OPERATIONS IN RATS, Acta Acadeniae
Medicinae Wuhan, 3, (2), pp. 77-83.
(The publication dates of the Ascherl et al. and Minju et al. articles are
not known. The translations have the dates "7/18/85" printed on the title
pages. It is possible that these two publications are not prior art to
applicants.)
Human fibrinolysin has been evaluated, either alone or combined with other
medicaments, to combat post-surgical adhesions. See Gazzaniga et al.,
Arch. Surg. Vol. 110, pp. 429-432 (1975), and references cited therein.
Other authors have reported that fibrinolytic agents have either not been
successful in preventing adhesions, or the risks associated with their use
were too high. In this connection, see, for instance, the following
articles:
Holtz, "Prevention of Postoperative Adhesions", The Journal of Reproductive
Medicine, Vol. 24, No. 4, April 1980, pp. 141-146, esp. p. 144; and
Rivkind et al., Eur. Surg. Res. (Switzerland), 1985, Vol. 17, No. 4, pp.
254-8.
Buckman et al., in "A Unifying Pathogenetic Mechanism in the Etiology of
Intraperitoneal Adhesions", Journal of Surgical Research, Vol. 20, No. 1,
January 1976 (pp. 1-5), theorized that post-surgical adhesion formation in
the peritoneal cavity was associated with trauma or ischemia induced
reduction in plasminogen activator activity.
DETAILED DESCRIPTION OF THE INVENTION
The pharmacologically active composition employed in this invention is
tissue plasminogen activator. t-PA is a product that is normally present
in the body, where it plays a role in the lysis of thrombi. There is some
evidence that in its "storage mode" in the body, t-PA exists in the single
stranded form. t-PA can also exist in a double stranded form. Both forms
have a high affinity for fibrin, a component of thrombi. Upon being bound
to fibrin, the single stranded form of t-PA appears to be converted to the
double stranded form. Both the single stranded and double stranded forms
of t-PA can be used in this invention. Natural t-PA is glycosolated and
contains associated carbohydrates or oligosaccharides at the glycosylated
sites. Both the degree of glycosolation and the number of and specific
nature of the associated carbohydrates or oligosaccharides are believed to
vary normally, and can also vary with the t-PA employed in this invention.
t-PA can be isolated from human tissue, and more recently has been produced
using recombinant DNA technology. See, for instance, Goeddel et al., UK
Patent Application GB 2 119 804 A, published Nov. 23, 1983, Gill, European
Patent Application No. 0 174 835, published Mar. 19, 1986, and Mori et
al., European Application No. 0 100 982, published Feb. 22, 1984, for
various methods of isolating t-PA from tissue or producing it by r-DNA
techniques.
In accordance with the process of the invention, the active agent is
applied topically to the site of surgical trauma in effective amounts for
a period of time sufficient to inhibit the formation of post-surgical
adhesions. It is preferably administered before significant wound healing
has begun. It is preferred and most convenient to administer the active
agent in a single dose application during the surgery prior to closing the
surgical field. In most cases, the time of administration of a single dose
would be just prior to closing; however, in some cases it may be desired
to administer the composition earlier during the surgical procedure. In
some situations it may be desired to administer the active agent
continually over a period of time, as would be the case if the active
agent were administered by a catheter or in a sustained release
formulation. The specification, below, describes some methods that can be
used to determine the optimum periods of administration when the mode of
application is continual, as by catheterization (in the animal models
used, an implanted osmotic mini-pump was used--this procedure is analogous
to catheterization in a human). However, the most convenient mode of
administration is via a single dose application of the active agent prior
to closing the surgical field (as indicated above, in most cases this time
of administration would be just prior to closing).
By the term "topically", is meant that the t-PA is administered
non-systemically to the surface of the tissue (internal or, in some cases,
external) to be treated. The treatment is intended to be "locally
effective", that is, the treatment is intended to affect the tissue
treated or adjacent or neighboring tissue. The term "site of surgical
trauma" is meant to include the site of tissue that has been injured in
any way, and includes, for example, tissue sites that have undergone
incision, excision, drying, suturing, fulgeration, cauterization,
abrasion, contusion, manipulation, laceration, anastomosis, prosthetic
surgery, curettage, orthopedic surgery, neurosurgery, cardiovascular
surgery, or plastic or reconstructive surgery. "Site of surgical trauma"
also includes tissue that is adjacent to the injured tissue. In some
cases, effective treatment may be obtained simply by the topical
application of t-PA to tissue near that which has been surgically
manipulated.
The method of the invention is useful in any surgical procedure in which it
is desired to inhibit the formation of post-surgical adhesions. It is thus
broadly useful in all types of surgery in which adhesion formation can be
a complication. For instance, the invention is useful in abdominal
surgery, in gynecological surgery, in thoracic surgery, in cardiovascular
surgery, in orthopedic surgery affecting tendons, ligaments, etc., in
neurological surgery affecting the dura mater, and the like.
The t-PA may be administered to the site of surgical trauma by any
convenient mode such as, for example, by lavage, by catheter, by coating
directly on the site in a salve, ointment, gel, cream, aqueous surface
active composition, emulsion, suspension, film, or foam, or by any other
convenient mode. The site can be contacted directly, as by applying a
salve, ointment, gel, etc., or in some cases the medicament can be
introduced to a site near the site of trauma and natural migration of
fluids will serve to carry the medicament to the desired site. Such
natural migration of fluids can occur, for instance, intraperitoneally, in
response to peristaltic contraction of the intestines.
The t-PA is ordinarily administered in a sterile formulation in a
pharmaceutically acceptable carrier or vehicle such as phosphate buffered
saline ("PBS"), isotonic saline, purified water, an organic carrier (which
may be in an aqueous solution or suspension) such as a proteoglycan, for
example a glycosaminoglycan such as hyaluronic acid or a derivative
thereof (such as a pharmaceutically acceptable salt or ester thereof) or a
similar polysaccharide such as chitosan or a derivative thereof, a lipid,
for example, a phospholipid micelle or vesicle (the lipid may simply be a
mixture of a phospholipid in water), dextran, a cellulosic material,
polymers such as polyacrylamide or p-dioxanone, lactide, and/or glycolide
based absorbable polymers, (the polymer may be in the form of
microcapsules or it may be incorporated in a salve- or ointment-like
formulation or a gel or gel-like composition), or in an aqueous solution
of a surface active agent such as a polyoxyethylene-polyoxypropylene block
copolymer or a sorbitan fatty acid ester-polyoxyethylene ether.
Sterilization of the formulation may be accomplished in the usual ways,
including aseptic preparation, filtration, exposure to gamma radiation,
autoclaving, and the like.
In one aspect of the invention, the t-PA is contained in a controlled or
sustained release carrier that is capable of releasing the active drug for
a period of at least [one] a few hours (e.g., one to three hours) and up
to about seven days. However, the preferred and most convenient mode of
administration is via a single dose application, which is usually made at
the conclusion of the surgical procedure prior to closing. The preferred
vehicle for such single dose applications is a gel-like or salve-like
composition such as an aqueous solution or suspension of a material such
as a phospholipid, hyaluronic acid or a derivative thereof (such as sodium
hyaluronate), polyacrylamide, a cellulosic material, or the like.
Methods for incorporating drugs in phospholipid carriers are known in the
art. For instance, one procedure for encapsulating a drug in a
phospholipid vesicle is the following:
a lipid or mixture of lipids such as lecithin or other phospholipid, which
may be mixed with cholesterol or other lipoid substance, is dissolved in a
substantially water-immiscible organic solvent such as diethyl ether; and
an aqueous phase containing the material to be encapsulated (in this case,
t-PA) is added to the lipid solution, and the mixture is agitated as by
exposing it to ultrasonic sound waves (sonicated). Preferably, the organic
solvent is removed during sonication, as by use of heat or vacuum or both,
although in some cases the solvent can be removed after the sonication.
This procedure typically produces a unilamellar vesicle.
Another procedure for producing a phospholipid vesicle (in this case a
multilamellar vesicle "MLV") containing a medicament is to form a film of
dry lipid, as by evaporating the solvent from an organic solvent solution
containing a lipid to form a film on the walls of the vessel containing
the solution, and then stirring in the aqueous phase containing the t-PA
to be encapsulated. (The evaporation can be done by spray drying or by
vacuum evaporation, or by any other convenient method.) Free
unencapsulated t-PA can be separated from MLV's by centrifugation at,
e.g., 12,000 rpm.
The vesicle containing the t-PA may be dehydrated, as by freeze drying,
after preparation, in order to enhance long term storage stability. The
aqueous vesicle suspension can be reconstituted just prior to use by
adding sterile phosphate buffered saline, sterile water, or the like.
The use of multilamellar vesicles of comparatively large size (e.g., from
about 1 to about 10 microns) appears to be preferable in order to increase
the dwell time of the vesicle containing the t-PA in the peritoneal cavity
(or other body cavity). It is also preferred to use a pure or synthetic
phosphatidylcholine in which the fatty acid moieties in the
phosphatidylcholine are derived from a single fatty acid, in preparing the
vesicle instead of natural lecithin, which is ordinarily a mixture of
compounds. The fatty acid moieties in the liposomes are usually derived
from C.sub.12 to C.sub.24 fatty acids, and preferably from C.sub.14 to
C.sub.20 unsaturated fatty acids.
The following U.S. patents describe the preparation, by various procedures,
of phospholipid vesicles containing various medicaments:
______________________________________
Rahman No. 3,993,754
Lenk et al. No. 4,522,803
Baldeschwieler et at.
No. 4,310,505
Mezei et al. No. 4,485,054
Gersonde et al. No. 4,452,747
Kelly No. 4,356,167
Papahadjopoulos et al.
No. 4,241,046
Suzuki et al. No. 4,016,100
Sache et al. No. 4,239,754
MacDonald No. 4,532,089
______________________________________
See also Callahan et al., European Patent Application No. 0126580,
published Nov. 28, 1984, and Gregoriadis, "The Carrier Potential of
Liposomes In Biology and Medicine", New England Journal of Medicine, Vol.
295, pp. 704-710 and pp. 765-770 (Sept. 23 and 30, 1976).
The foregoing are incorporated herein by reference as general procedures
which can be utilized for the incorporation of t-PA in liposomes.
Other procedures for containing drugs in phospholipids (micelles or
liposomes) are described in Sears, U.S. Pat. Nos. 4,426,330 and 4,145,410,
and Sears et al., U.S. Pat. No. 4,298,594, the disclosures of which are
incorporated herein by reference.
It is not essential that the t-PA active ingredient used in the invention
be encapsulated in an inside compartment of the carrier as will normally
be the case when the carrier is a phospholipid vesicle. In some cases it
is acceptable for the t-PA to be dissolved or otherwise distributed more
or less evenly throughout the carrier.
The t-PA is administered to the site of surgical trauma in effective
quantities for a period of time sufficient to inhibit the formation of
post-surgical adhesions, which Period varies from patient to patient and
with the type of surgical trauma encountered. It has been found that in
many cases the t-PA need be administered only during the initial stages of
the wound healing process, and therefore the duration of the
administration in such cases may be only a few hours, e.g., as short as
from about one to three hours. In other cases, the duration of
administration may be from about one or two and up to five days, and in
some cases up to seven days or more, post-operatively. The examples below
illustrate procedures for determining the order of magnitude of effective
quantities of the drug and the period of time over which the drug is
administered for effective results.
The following studies use rabbit models to illustrate the adhesion
inhibition effectiveness of the topical administration of t-PA to the site
of surgical trauma:
STANDARDIZED SURGICAL INJURY
New Zealand white female rabbits (1.8-2.0 kg) underwent midline laparotomy
using acelepromazine and ketamine anaesthesia. A 3.times.5 cm flap of
parietal peritoneum (about 1 mm thick) was sharply dissected from the
right lateral peritoneal side-wall. The serosal surface of the adjacent
large bowel was abraided with a scalpel until punctate bleeding developed.
This area between the excised parietal peritoneum and adjacent large bowel
serosa was then used for evaluating the efficacy of t-PA for adhesion
prevention. A second incision of parietal peritoneum covering the same
total area (about 15 cm.sup.2) was performed in some of the rabbits
1.5-2.0 cm inferior to the initial test site along the right lateral
peritoneal side-wall. Abrasion of the adjacent large bowel serosa was
performed as described above for the treatment site. This second area was
used as a non-treated control site to determine the effectiveness of the
surgical procedure in producing adhesions, and the response to vehicle
controls.
Alzet mini pumps (model 2ML1, 2 ml volume, pumping rate 10 microliters per
hour, Alza, Palo Alto see U.S. Pat. No. 3,995,631) containing phosphate
buffered saline with varying doses of tissue plasminogen activator
(Genentech, South San Francisco, Calif.--the Genentech t-PA employed was
predominantly double stranded t-PA) were sewn into the right dorsal
subcutaneous space with Vicryl sutures placed 3-5 mm from each end of the
pump. The polyethylene catheter tip leading from the pump into the
peritoneal cavity was placed 2-3 mm over the injury test site. The
catheter was secured by two 300 Vicryl sutures placed outside the site of
injury. A similar pump and catheter system containing only Ringer's
lactate was implanted in the middle portion of the inferior (control) test
site.
Seven days after the day of abrasion, the rabbits were sacrificed by
pentobarbital overdose. The extent of adhesions was evaluated as follows:
1. No adhesions
2. Filmy adhesions (separable)
3. Mild adhesions (not separable--covering up to about 35% of the test
area)
4. Moderate adhesions (not separable--covering about 35 to 60% of the test
area)
5. Severe adhesions (not separable--covering greater than about 60% of the
test area)
The evaluation ratings set forth above are useful in the context of
comparing the efficacy of various means for inhibiting the formation of
adhesions. A rating of "1" is the objective, since clinical complications
can result from even mild adhesions, although such complications are
considered to be more likely to occur with severe adhesions than with mild
or moderate adhesions.
EXAMPLE 1
In the first series of experiments, nine rabbits were used. Four were
vehicle controls in which the pumps contained isotonic buffer alone, and
the other five were used to evaluate the efficacy of t-PA to combat
post-surgical adhesion as described above. The mini pump in each t-PA test
rabbit contained two ml of isotonic buffer containing 5 mg per ml of
essentially pure t-PA. The results of the experiments are shown below in
Table I:
TABLE I
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Rabbit No. t-PA or Control
______________________________________
Adhesion Evaluation
1 Control 5
2 " 5
3 " 4
4 " 5
5 t-PA 1*
6 " 1*
7 " 1
8 " 1*
9 " 1
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*Three of the rabbits exhibited bleeding at the site of the trauma; two
bled lightly and the other moderately to severely.
EXAMPLE 2
In order to try to better define the threshold dosage rate in this
experimental model, similar experiments were carried out with the mini
pump, using different concentrations of t-PA per pump. Table II, below,
sets forth the dosage rates and responses in this series of experiments:
TABLE II
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mg t-PA
Per ml of
Adhesion
Rabbit No. Buffer Evaluation
______________________________________
1 3 1
2 3 1
3 1.5 1
4 1.5 1
5 1.5 1
6 0.5 4
7 0.5 4
8 0.15 1
9 0.15 1
10 0.15 3
11 0.05 1
12 0.05 3
13 0.05 4
14 0.015 5
15 0.015 5
16 0.015 4
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These results indicate that the threshold dosage (for this Genentech t-PA)
at which significant beneficial anti-adhesion properties are obtained is
between 0.015 and 0.05 mg t-PA/ml of buffer. (The results observed with
rabbit Nos. 6 and 7 appear to be anomalous.) No bleeding was encountered
with any of these rabbits.
Since no bleeding was encountered with any of the rabbits in the dosage
rate studies, it appears that in this model and mode of administration, a
safe upper limit of dosage that would not result in significant bleeding
would be found in the dosage rate administered to Rabbit Nos. 1 and 2 (in
which the concentration of t-PA was 3 mg/ml).
EXAMPLE 3
In order to determine the time period over which the anti-adhesion agent is
preferably administered in order to have a significant anti-adhesion
effect in the particular animal model and mode of administration used, the
following series of experiments were carried out:
The pumps contained 10 mg of t-PA (5 mg/ml), and the catheter delivering
the treatment solution to the site of the surgical trauma was disconnected
2, 3, and 5 days post-operatively. The rabbits were sacrificed 7 days
post-operatively, and evaluated as above. The results are displayed in
Table III, below.
TABLE III
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Post-Op Day
Catheter
Rabbit No. Disconnected
Evaluation
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l 2 1
2 2 1
3 2 1
4 3 1
5 3 1
6 3 1
7 5 1
8 5 4
9 5 1
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EXAMPLE 4
In this experiment, t-PA obtained from Damon Biotech, Needham Heights,
Mass. This t-PA was predominantly single stranded t-PA. The experiment was
similar to that described above in Example 2, except that a vehicle
control site was used in each rabbit, as explained above in the
Standardized Surgical Injury section. The results were as follows:
TABLE IV
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mg t-PA per Evaluation
Rabbit No
ml buffer t-PA Site Vehicle Control Site
______________________________________
1 0.5 1 5
2 " 1 5
3 " 1 1
4 " 1 5
5 " 3 4
6 0.15 1 1
7 " 1 1
(Bleeding)
8 " 1 1
9 " 3 1
10 " 1 1
11 0.05 1 3
12 " 1 3
13 " 1 1
14 " 1 4
15 " 1 3
16 0.015 1 1
17 " 3 4
18 " 1 1
19 " 1 5
20 " 1 5
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The Damon Biotech t-PA appears to be more active, on a weight basis, than
the Genentech t-PA.
Urokinase and streptokinase were both evaluated for anti-adhesion activity
in the rabbit model described above, and both were found to have no
anti-adhesion activity.
It is relevant to note that when the vehicle control site is in the same
rabbit as the test site, migration of fluid in the peritoneal cavity can
carry some of the medicament from the treatment site to the vehicle
control site. Therefore, it is possible that some vehicle control sites
could have received small amounts of t-PA owing to migration or
circulation of fluid within the peritoneal cavity. However, if any of the
untreated control sites did receive some of the active medicament by such
fluid migration, it would have been significantly less than that received
at the treatment site in the same rabbit. Therefore, differences in
results between the treatment sites and the control sites in the same
rabbit can confidently be interpreted as being caused by the adhesion
inhibition effect of the t-PA.
An effective dose of a topically applied drug is normally expressed in
terms of concentration of the drug in the carrier, coupled with the number
of times per day the drug is applied. In the present invention, the
effective dose will be dependent upon factors such as nature of specific
t-PA used, nature of vehicle, nature of tissue to be treated, type of
trauma, and mode of delivery (i.e., continuous delivery by catheter or a
one-time application in a vehicle such as a gel, ointment, salve, or
controlled release vehicle). Therefore, no hard and fast rule can be
formulated that will apply in all cases, and experiments analogous to
those reported in Examples 2 and 4 will have to be performed in order to
precisely define the threshold dosage for specific vehicle systems, for
specific modes of delivery, etc. It is well within the ability of the
person skilled in the art to carry out the necessary experiments to
determine threshold dosages and periods of time over which the t-PA should
be administered for best results, after having read this disclosure.
The t-PA active ingredient is administered to the site of surgical trauma
topically. Such topical administration can be by spraying, lavage,
dripping on the site, by catheter administration, or the like. The exact
method of administration chosen is not critical, as long as an effective
dose is administered over the appropriate period of time, which can be
determined by a series of experiments analogous to that described above in
Example 3.
Referring to the question of the effective dose of t-PA in accordance with
this invention, while no hard and fast numbers can be presented that will
be applicable to all cases, the examples presented above can be referred
to as a guide to determine the order of magnitude of t-PA to employ. Thus,
a concentration of t-PA in the vehicle of at least about 0.015 milligrams
of t-PA per milliliter of total vehicle or carrier can be expected to
exhibit anti-adhesion activity in most cases.
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