Note: Descriptions are shown in the official language in which they were submitted.
lZ92946
METHOD FOR INHIBITING POST-SURGICAL ADHESION FORMATION aY
THE TOPICAL ADMINISTRATION OF NON-STEROIDAL
ANTI-INFLAMMATORY DRUG
The ~nvention relates to a me~hod for inhibiting
post-surgical adhesion formation.
~ackaround of the Invention
AdhQsion formation is a ma30r post-surgical complication
wlth no ~ract~cal solution, The incidence o~ adhQsion
rormation ~ollowing sur~ery a~proaches 100 per aent,
ac¢ording ~o ~ome sources, with a clln~cally signif~cant
¢ompliaation rate oS about 5 to 10 per cent, de~end~ng on
the type of surgery, Among such complications are bowel
ob~truction, infsrtility, and pain. Oc¢as~onally, adhe6ions
nQcQssitate a ~econd operative procedure to remove the
adhesion, wh~ch may in turn further aggravate the problem.
Because o~ the serious~ess of the problem, much medical
: 25 research has been performed in efforts to ~ind ways to
combat adhesions. See, for instance, Stangel et al.,
~Formation and Preventisn of Postoperative Abdominal
Adhesions", the Journal of Reproductive M~dicine, Vol. 29,
No. 3, March 19~4 (pages 143-156), and diZerega, "The Cause
and Prevention of Postsurgical Adhesion~", published by
Pregnancy ~e~earch Branch, National Institute of Child
Health and Human ~evelopment, National In~titutes of Health,
Building 18, Room 101, Bethesda, MD 20205.
Among the approaches that have been tried for preventing
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post-surgical adhesion are the following:
Systemic administration of ibuprofen (e.g., see Singer,
U.S. Patent 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, anon-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 page 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 u~e of corticosteroids does not seem to be
an acceptable solution to the post-operative adhesion
problem.
on the basis of the results of animal studies and limited
human clinical studies, the systemic administration of
non-steroidal anti-inflammatory agents such as ibuprofen
(usually in combination with other medicaments such as
antibiotics) appears to be the most efficacious
pharmacological means now known to reduce the incidence of
post-surgical adhèsions. An objection to this means is
that relatively large amounts of the drug must be
administered over a period of several days, thereby
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subjecting the patient to the significant risk of
experiencing adverse side effects. Also, this means has
been shown to be effective only in a limited number of
types of surgical procedures, e.g., gynecological
surgery. As reported by Nishimura, Nakamura, and diZerega
(Journal of Surgical Research 36, 115-124, February 1984),
the minimum effective dose of systemically administered
ibuprofen to inhibit postsurgical adhesion formation after
abrasion or ischemia of the uterine horn of rabbits, is 70
mg/kg/day, administered once a day for at least 3 and
preferably for 5 days post-operatively, with an additional
dose 1 hour before surgery. In a similar series of
experiments, Siegler et al. (Fertility and Sterility 4,
No. 1, July 1980, pages 46-49) found an effective dose of
systemically administered ibuprofen to be about 21
mg/kg/day, administered three times daily tin three 7
mg/kg doses) for two days post-operatively, with the
initial in~ect~on being given 30 minutes before surgery.
The authors also reported that the best results were found
in two rabbit~ that were each inadvertently given three
extra 7 mg/kg doges. In order to deter adhesion formation
in surgery to try to cure infertility, Corson et al. (The
Journal of Reproductive Medicine, Vol. 29, No. 3, pages
143-156, March 1984) recommend a regimen including
systemically administered ibuprofen, 400 mg per dose three
to four doses per day, starting the night of surgery and
continuing to the fifth postoperative day. A6suming that
the average woman weighs about 48 kg 1110 pounds), this is
a recommended do~age of 25 to 33 mg/kg/day. Singer, in
U.S. Patent No. 4,346,108, recommends a dosage of from
about 2.5 to 50 mg/kg/day in ~ingle or divided doses, for
ibuprofen administered systemically to combat adhesions.
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Brief SummarY of the Invention
The invention comprises a method for inhibitin~ the
formation of post-surgical adhesions in mammals (including
humans) which method comprises the topical administration
of a non-steroidal anti-inflammatory drug ("NSAID") to the
site of surgical trauma, over t~e critical wound healing
period.
The Prior Art
The Singer patent, and the journal articles by Nishimura
et al., Siegler et al., and Corson et al., all cited
above, disclose the 6ystemic administration of ibuprofen
to combat adhesion formation,
Lenk et al., in U. S, Patent No. 4,522,803, at column 17,
lines 67 et seg,, disclose phospholipid vesicles
containing anti-inflammatory agents. Only steroid
anti-inflammatory agents are specifically disclosed. No
anti-adhesion utility is disclosed.
Systemic administration of oxyphenbutazone to combat
adhesions has been proposed. See, for example, Kapur et
al., "Prevention of Reformation of Peritoneal Adhesions~,
Arch. Surg., Vol. 105, Nov. 1972, Pages 761 - 764.
Detailed Description of the Invention
The pharmacologically active compositions that are employed in
this invention are the non-steroidal
anti-inflammatory drugs such as ibuprofen, tolmetin,
indomethacin, sulindac, suprofen, oxyphenbutazone, and
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s
pharmaceutically acceptable salts or esters thereof.
NSAID's comprise a recognized class of compositions.
In accordance with the process of the invention, the
active agent i8 applied topically to the site of surgical
trauma in effective amounts for at least two and up to
about seven days after the operation, to thereby inhibit
the formation of post-surgical adhesions. By the term
~topically~, is meant that the NSAID is administered
non-systemically to the surface of ehe tissue (internal
or, in some cases, external) to be treated, for local
effect. 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, abrasion, contusion,
laceration, anastomosis, manipulation, prosthetic surgery,
curettaqe, orthopedic surgery, neurosurgery,
cacdiovascular surgery, or plastic or recon~tructive
surgery, "Site of surgical trauma~ also includes tissue
that is ad3acent to the injured tissue.
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.
The NSAID 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, or foam, or by any
other convenient mode. The site can be contacted
directly, a~ by applying a salve, or i~ 60me ca6es the
medicament can be introduced to a site near the site of
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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 NSAID 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 such
as a lipid, for example, a phospho}ipid micelle or
vesicle, dextran, polymers (especially p-dioxanone,
lactide, and/or glycolide based absorbable polymers),
which may be in the form of microcapsules or which may be
incorporated in a salve- or ointment-like formulation, or
in an agueous solution of a surfactant such as a
polyoxyethylene-polyoxypropylene block copolymer or a
~orbitan fatty acid ester-polyoxyethylene ether.
5terilization of the formulation may be accomplished in
the usual ways, including aseptic preparation, filtration,
exposure to gamma radiation, autoclaving, and the like.
In one preferred aspect of the invention, the NSAID is
contained in a controlled release carrier that is capable
of relea~ing the active drug for a period of at least one,
preferably at least two, and up to about seYen days. The
mode of delivery of the NSAID is preferably continually
over the critical wound healing period, such as will be
the case when the drug is applied to the site of surgical
trauma in a single dose via a controlled releage carrier,
or continually via a catheter.
A general procedure for preparing a polymeric microcapsule
containing a drug, and which is applicable to
incorporating NSAID' 8 in polymeric microcapsules, is the
followi~.g:
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1. The drug and the polymer are dissolved in a volatile
organic solvent;
2. The solvent containing the drug and polymer is dispersed
in water with a dispersing agent:
3. The organic solvent is evaporated from the dispersion
product of step 2, either by mild heating or by vacuum
evaporation, or by a combination of the two; and
4. The resulting microcapsules are recovered from the aqueous
dispersion by customary procedures such as filtration,
centrifugation, or the like, usually coupled with one or
more washing steps.
This procedure is illustrated for this invention by Examples 1,
2, 9, and 10, below:
Exam~le 1
(a) To a small vial was added 9.39 grams of
poly~lactide-co-glycolide-65:35) (a 65/35, by weight,
copolymer of laceide and glycolide having an inherent
viscosity in chloroform of 0.5 dlJg), 0.75 gram refined
sesame seed oil, 1.140 grams ibuprofen, and 40 milliliters
of dichloromethane. The solution obtained wa~ added to
400 milliliters of aqueous 5% wt/vol poly(vinyl alcohol)
(Air Products Vinol Grade 523) in a one liter resin kettle
equipped with a mechanical stirrer and vacuum take off,
which was being cooled in an ice/water bath and stirred at
500 rpm. After allowing 10 minutes for emulsification,
vacuum was slowly applied by means of an aspirator to an
absolute pressure of 500 mm of mercury over 1.5 hours.
The vacuum was then maintained for an additional 19.5
hours (to remove the dichloromethane solvent), at which
time vacuum and stireing wa~ stopped and the contents of
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the flask was poured into a one liter beaker and diluted
to 800 milliters total volume with water. (An alternative
method for removing the dichloromethane solvent is to heat
at 30C. for two hours at atmospheric pressure.) The
contents of the beaker were added to centrifuge tubes and
they were centrifuged at about 1000 rpm for two minutes.
The liquid was deranted, fresh water was added, and
centrifugation was repeated. This procedure was repeated
one more time, and after the third wash, the last trace~
of water were removed by freeze drying. The dried sample
was a free flowing white powder which weighed 2.03 grams.
The powder was examined at lOOX and was found to contain
microcapsules which ranged in size from about l to 10
microns. No free drug crystals were noted. Subsequent NMR
analysis indicated that 8.6 wt. % ibuprofen was present.
(b) To make control microcapsules, the foregoing procedure
i8 repeated without adding the ibuprofen.
~c) Microcapsules produced as described above, both with
and without ibuprofen, are disperged in an aqueous
dispersion of lecithin in proportions of 1.5 grams of
microcapsules per 50 milliliters of 0.05 weight per cent
aqueous lecithin. After freeze drying, 1.4 grams of
microcapsules are obtained which contain about 1.4 weight
per cent of lecithin.
- ExamPle 2
To a small vial was added 1.50 grams of
poly(lactide-co-glycolide - 65:35), 0.50 gram ibuprofen,
and 5 milliliters of dichloromethane. The resulting
solution was added to 50 milliliters of 3~ aqueous
poly(vinyl alcohol) solution which was being cooled in an
ice/water bath and stirred at 500 rpm. Vacuum was applied
as before and after washing and freeze-drying, l.lO0 grams
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of microcapsules was obtained. The microcapsules ranged
in size from 10 to 120 microns and were found to contain
17.1% by weight ibuprofen by NMR.
ExamPle 3
Ipuprofen/poly(lactide-co-glycolide-65:35) microcapsules
(Example 2) which were found to contain 17.1~ by weight
ibuprofen (NMR) were investigated. In five separate 4
ounce amber jars was placed 100 milligrams of
microcapsules and 100 milliliters of pH 7.27 phosphate
buffer. The caps were tightly closed and the jars
incubated at 37 C., with no agitation. After 15
minutes, 1 day, 2 days, 7 days, and 14 days, a jar was
removed, the microcapsules collected by filtration, washed
well with water, dried under vacuum, and analyzed for
ibuprofen content by NMR~ The results ~how~ in the
attached table ~ndicate that approximately 50% of the drug
wa~ relea6ed in the fir~t 7 days and the remainder was
Z0 completely released by day 14. (Negligible polymer weight
lo~s occurred over the 14 day period.)
Table I
In-Vitro ~elease of
Ibuprofen From Poly(lactide-co-glycolide) Microcapsules
Sample Time WT % IBF REMAINING
(~ays) ~ of TOT CAPSULE WT)
__________________________________________________________
1 0 17.0
2 1 14.3
3 2 13.6
4 7 10.0
14 0.0
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Methods for incorporating drugs in lipid caLriers 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 an organic solvent such as
diethyl ether; and
an aqueous phase containing the material to be encapsulated (in
this case, an NSAID) 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 u6e of heat or vacuum or both, although in
some caseg 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 601ution
containing a lipid to form a film on the walls of the
vessel containing the solution, and then stirring in the
agueous phase containing the NSAID to be encapsulated.
(The evaporation can be done by spray drying or by vacuum
evaporation, or by any other convenient method.) Free
unencapsulated NSAID can be separated from MLV~s by
centrifugation at, e. g., 12,000 rpm.
Preferably, the vesicle containing the NSAID is
dehydrated, as by freeze drying, after preparation, in
order to insure long term storage stability. The agueous
vesicle suspension can be reconstituted just prior to use
by adding sterile phosphate buffered saline, sterile
water, or the like.
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The use of multilamellar vesicles of comparatively large
size (that is, greater than 1 micron, 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
NSAID in the peritoneal cavity (or other body cavity). It
i8 also preferred to~use a pure or synthetic
phosphatidylcholine in which the fatty acid moieties in
- the phosphatidylcholine molecule are derived from a single
fatty acid, in preparing the vesicle instead of natural
lecithin, which is ordinarily a mixture of compounds,
Example 13, below, illustrates the preparation of a
multilamellar vesicle containing an NSAID.
The following United States patents describe the
preparation, by various procedur~s, of phospholipid
vesicles containing various medicaments:
Lenk et al. No. 4,522,803
~alde~chwieler 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
Papahadjopoulo~ 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
Rahman et al. No. 3,993,754
See also Callahan et al., European Patent Application No.
0126580, published November Z8, 1984, and Gregoriadis, "The
Carrier Potential of Lipo~omes In Biology and Medicine", New
England Journal of Medicine, V~l. 295, pp. 704-710 and pp.
765~770 (Sept. 23 and 30, lg76).
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.
The foregoing are incorporated herein by reference a~ general
proeedure~ which can be utilized for the incorporation of
~AID's ln lipo~omes.
S Oth-~ procodu~s ~or aontaining drug~ in ~ho~p~olipid0
~micelles or liposomes) ~ and which are applicable to NSAID~s,
are described in Sears, U.S. Patent Nos. 4,426,330 and
4;1~5,410, and ~ears et al., U.S. P~tent No. 4,298,594 .
It is not essential that the NSAID medicament u6ed in the
invention be encapsulated in an inside aompartment or
compartments of the carrier as will normally be the case when
the carrier i~ a phospholipid vesicle. In some ca6es it i8
a¢co~table for tho NSAID to be d~ssolYed or otherwise
di~tributed more or le~s evenly ~hroughout the carrler.
Thé non-steroidal anti-inflammatory drug i8 delivered to the
~ite of ~urgiaal trauma in effe¢tive quantitie~ over the
¢ritical wound healing period ~whi¢h period varies from patlent
to patient and with the type of ~urgical trauma encountered,
but i8 u~ually from about two to five days, and in some case~
up to seven days or more, post-operatively.) The example6
below illustrate the order of magnitude of effective quantities
: 25 of the drug and the period of time over which the drug is
administered for effective results.
The following ~tudies u~e rabbit models to illustratQ the
adhesion inhibition effectiveness of the topical admini~tration
of a non-steroidal anti-inflammatory drug to the site of
~urgical trauma:
,
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ExamPle 9
New Zealand white female rabbits (1.8-2.0 kg) underwent midline
laparotomy using acelepromazine and ketamine anaesthesia. A
3x5 centimeter abrasion was produced over the right-lateral
peritoneal side-wall by ~craping the surface peritoneum with a
scalpel until punctate bleeding developed over the entire 3x5
centimeter area. A second abrasion covering the same total
area (15 cm2) using the same technique was developed 1.5-2.0
centimeters inferior to the initial site along the
right-lateral peritoneal side-wall. This second site was used
as an untreated control. The serosal surface of the large
bowel adjacent to the peritoneal abrasion sites was also
similarly abraded.
Ibuprofen contained in poly~lactide-co-glycolide-65:35)
microcapsule~ produced as described in Example 1, were
~uspended in 15 we~ght per cent aqueous poly(vinyl pyrrolidone)
"PVP" (GAF Povidone C-15). The proportions were 15 grams of
microcapsules per 100 grams of the PVP solution.
The lecithin-containing microcapsules were used in half of the
experiments. The suspension was dripped on the wound in an
amount such that about 40 milligrams of ibuprofen
(about 470 milligrams of microcapsules) was applied to the
wound site. One control was the PVP solution containing
ibuprofen-free microcapsules, and the other was the PVP
solution containing ibuprofen-free microcapsules with lecithin.
Seven days after the day of abrasion, the rabbitg were
sacrificed by pentobarbital overdose. The extent of adhesions
was evaluated as follows:
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1. No adhesions
2. Filmy adhesions (separable)
3. Mild adhesions (not separable - covering up to about 35%
of the test area)
4. Moderate adhegions (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. However, it should be
mentioned that ultimately only a rating of ~'1" can be
considered to be completely acceptable. Clinical complications
can result from even mild adhesions, although such
complications are considered to be more likely to occur with
severe adhe~ions than with mild or moderate adhegions.
The following Table II presentg the results:
Table II
Evaluation
Ibuprofen Untreated
Rabbit No. IbuProfen Lecithin Treatment Controls
1 yes no 4 4
2 yes no 4 5
3 yes no 1 1 *
4 yes yes 3 5
yes yes 4 5
6 yes yes 3 5
7 no no 5 5
8 no no 5 5
9 no no 5 5
no yes 5 5
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11 no yes 5 5
12 no yes 5 s
____________________________________________________________
* ~he surgical procedure followed in this experiment induces
adhesion formation in about 87 per cent of untreated rabbits.
Evidently this rabbit was one of the approximately 13 per cent
that do not develop adhesions.
________________________________
The results indicate some anti-adhesion activity exhibited by
the microcapsules containing ibuprofen.
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
NSAID treatment site to the vehicle control site. Therefore,
it i8 possible that the untreated (by "untreated" is meant no
actiYe medicament) control ~ites of rabbits Nos. 1-6 in Table
II and analogous untreated control sites reported below could
2~ have received small amounts of NSAID owing to migration or
circulation of fluid within the peritoneal cavity. However, if
any of the untreated control sites did receive some o 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 NSAID.
ExamDle 5
In order to carry out dose response studies and time response
studies, miniature osmotic pump8 (Alzet mini pumps, model 2MlL
or model 2002 - these pumps are described in Higuchi et al.,
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-16-
.
U.S. Patent No. 3,995,631) were used to de,liver the
antl-adhe~lon agent ~n a continuou~ stream at a very low,
controlled Slow rate, to the s~te o~ the surgical trauma in the
te~t rabbits over a period of time, up to ~even days. The
mini-pumps therefore deliver the medicament in a manner
analogou~ to a catheter delivery modQ.
New Zealand white ~emale rabbits (1.8 to 2.0 kg) underwent
midline laparotomy u6ing acelepromazine and ketamine
anaesthesia. A 3 x 5 cm flap of parietal peeitoneum (about 1
mm thic~) was sharply dissected from the right lateral
peritoneal side-wall, The serosal surf'ace of the ad~acent
large bowe? was abraided with a scalpel until punctate bleeding
developed. This area between the excised parietal peritoneum
and ad~acent large b~wel serosa was then used for evaluating
th~ e~fica¢y o~ the te~t medicament ~or adhe~ion inhib~tion. A
~econd oxais~on o~ parie~al peritoneum co~er~ng the ~ame total
area ~about 15 ¢m ) wa~ ~qr~ormed 1.5 to 2.0 cm inferior to,
the initial to~t site along the right lateral peritoneal
s~de-wall. Abrasion of the ad3acent larqe bowel serosa was
p-r~orm-~ a~ de~aribe~ above'for the tr--tm-nt 8ite . Thls
~-cond area wa~ u~ed to dotermine the e~fectiveness ot the
6uryical procedure in producing adhe6ions and the response to
~ vehicle controls.
: 25
Alzet mini pumps containing ibuprofen dissolved in pho&phate
bufered saline ("PBS") were sewn into the right dorsal
subcutaneous space of the test rabbi,t with VICRYL (Polyglactin
910) sutures placed 3 to 5 millimeters from each end of the,
pump. The polyethylene catheter tip leadihg from the pump into
the pe~itoneal cavity of ,each rabbit was placed 2 to 3
,millimeter8 over the in3ury test 8ite. The catheter was
securëd in place by two 3/0 VICRYL 6uture8 which did not
, , involve the site of the injury. A similar ~ump and catheter
,
, *Trademark,
ET~-694 , , '
A
,
lZ9Z9~6
-17-
6ystem containing PBS vehicle only was implanted in the middle
portion of the inferior (vehicle control) abrasion site.
Two different mini pumps were used in these experiments. The
first (a 2 ml pump~ had a pumping rate of 10 microliters per
hour and the second (a 0.2 ml pump) had a pumping rate of 0.5
microliter per hour. Each pump contained phosphate buffered
saline (pH 7.2, unless otherwise indicated), 2 milliliters in
the larger pump and 0.2 milliliter in the smaller pump. The
control pumps contained phosphate buffered saline alone, and
the treatment pumps contained either 20 milligrams of ibuprofen
(2 ml pump) or 2 milligrams of ibuprofen (0.2 ml pump).
The results seven days after the operation were as shown below
in Table III. The evaluation procedure was the same as the one
de~cribed in Example 4.
Table III
Evaluation
Ibuprofen Vehicle
Rabbit No. PumP Size Treatment Control
1 0.2 ml 5 5
2 0.2 ml 4 5
3 0.2 ml 4 5
4 2 ml 4 5
2 ml 2 5
6 2 ml 1 5
The smaller of the two pumps gave a slight positive
response in two of three rabbits, whereas the larger pump
gave significant positive responses in two out of three
rabbits and a 61ight positive re6ponse in the other.
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ExamPle 6
In order to try to better define the threshold dosage rate
for ibuprofen ("IBF") in this experimental model, similar
experiments were carried out with the two sizes of pumps,
using different concentrations of IBF per pump. Table IV,
below, sets forth the concentrations of IBF per pump and
responses in this serie~ of experiments:
Table IV
Evaluation
Concentration of Vehicle
Rabbit No. pumP Size IBF, mo~ml Treatment Control
1 0.2 ml 10 1 4
2 0,Z ml 10 4 5
3 0.2 ml 10 5 5
4 2 ml 10 3 5
2 ml 10 4 4
6 2 ml 10 3 5
7 0.2 ml 3 4 5
8 0.2 ml 3 5 5
9 0.2 ml 3 4 5
2 ml 3 3 5
11 2 ml 3 5 5
12 2 ml 3 4 5
13 0.2 ml 1 5 5
14 0.2 ml 1 5 5
0.2 ml 1 1 4
16 2 ml 1 5 5
17 2 ml 1 5 5
18 2 ml 1 4 5
19 0.2 ml 0.3 5 5
0.2 ml 0.3 5 5
21 0.2 ml 0.3 5 5
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--19--
22 2 ml 0.3 5 5
23 2 ml 0.3 5 5
24 2 ml 0.3 4 5
This series of experiments indicates that, in this model, the
threshold dosage at which significant anti-adhesion effects
began to be noted occurred when pumps containing concentrations
between 1 and 3 milligrams of IBF per milliliter were used. No
significant difference was noted here between the two different
sized pumps.
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 NSAID 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 a6 a 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 this Example 6 will have to be performed in order to
precisely define the threshold dosage for each different NSAID,
for specific vehicle system6, and for specific modes of
delivery. It is well within the ability of the,person skiIled
in the art to carry out the necessary experiments to determine
threshold dosages, after having read this disclosure.
Exam~le 7
In order to determine the time period over which the
anti-adhesion agent must be administered in order to have
a significant anti-adhesion effect, the following series
:: :
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of experiments were carried out (using the experimental
procedure described above in Example 5):
The 2 ml pump was used containing 10 mg/ml of IBF, and the
catheter delivering the treatment solution to the site of
the surgical trauma was disconnected 1, 2, 3, 4, and 5
days post-operatively. The rabbits were sacrificed 7 days
post-operatively, and evaluated a~ above. The results are
displayed in Table V, below.
Table V
Post-Op Day Evaluation
RabbitCatheter Ibuprofen Vehicle
No.Disconnected Treatment Control
1 1 5 5
2 1 4 4
3 1 3
4 2 4 4
2 5 5
6 2
7 3 4
8 3 1 3
9 3 1 3
4 4 5
11 4 3
12 4
13 5 3 5
14 5 4 5
With ibuprofen, in this model, it appears that
administration of the NSAID via the 2 ml mini pump in PBS
for at least three days i~ required in order for the drug
to have significant anti-adhe6ion effects, With other
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NSAID~s and/or other delivery modes, and with other types
of surgical trauma, it is reasonable to expect that the
minimum period of time will differ. In any given case,
the minimum period of time over which the NSAID must be
administered can be determined by experiments analogous to
that described in this Example 7. As a general rule, in
most cases, a minimum of one day is recommended, and in
some cases, longer periods (e. g., up to 5 to 7 days or
longer) may be required,
The NSAID 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
ef~ective do~e is administered over the critical wound
healing period, which can be determ~ned by a series of
experiments analogous to that deficribed above in Example
7. The NSAID should be administered immediately
post-operatively, that i8, before wound healing has begun
ExamDle 8
By a procedure analogous to that described above in
Example 5, the 2 ml Alzet mini pump was used to deliver
suprofen to t~e test site in the rabbits, The
concentration of suprofen was 2.5 mg/ml of phosphate
buffered saline (pH = 7.4). The rabbits were sacrificed 7
days post-operatively, and evaluated as above. The
results are set forth in Table VI, below:
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Table VI
2.5 m~/ml Su~rofen
Evaluation
Suprofen Vehicle
Rabbit No. Treatment Control
1 3 4
2 4 3
3 3 5
4 4 5
4 4
6 3 5
7 3 4
ExamPle 9
To a small vial was added 2,78 grams of
poly(lactide-co-glycolide - 65:35), 0,30 gram suprofen,
and 8 milliliter~ of dichloromethane. The resulting
601ution was added to 120 milliliters of 3~ aqueous
poly(vinyl alcohol) solution which was heated to 30 C.
while being mechanically stirred at 500 rpm. After two
hours, the microcapsules were isolated as described in
Example 1, and after washing and freeze-dryinq, 1.61 grams
of microcapsules were obtained. The microcapsules ranged
in size from >10 to 250 microns and were found to
contain 7.0% by weight suprofen by NMK.
ExamPle 10
To a small vial was addéd 1.25 grams of poly~D,L-Lactic
acid), 0,12 grams refined ~esàme seed oil, 0,152 gram
ibuprofen, and 3 milliliters of dichloromethane, The
resulting solution was added to 30 milliliters of 3%
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a~ueous poly(vinyl alcohol) solution whic~ was being
cooled in an ice/water bath and stirred at 5~0 rpm.
Vacuum was applied as described in Example 1, and after
washing and freeze-drying, 1.12 grams of microcapsules
were obtained. The microcapsules ranged in size from 10
to 75 microns and were found to contain 8.7~ by weight
ibuprofen by NMR.
ExamPle 11
By a procedure similar to that described above in Example
5, the 2 ml mini pump was used to deliver varying
quantities of suprofen sodium (i. e., the sodium salt of
suprofen) to the rabbit model. The quantities of
medicament contained in the phosphate buffered saline (pH
= 6.9) varied from 3,0 mg/ml down to 0.03 mg/ml, The
rabbit~ were sacrificed seven days post-operatively and
evaluated as above, The results are set forth below in
Table VII:
Table VII
Suprofen, Dosage
ResDonse Studies
ConcentrationEvaluation
of Suprofen,Vehicle
Rabbit No. ma~mlTreatment Control
1 3
2 3
3 3
4 3 4 4
6 1 4 5
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8 1 3 4
~ 1 1 5 *
1 1 3
11 0.3 1 5
12 0.3 3 4
13 0.3 3 5
14 0.3 3 3
0.3 3 4
16 0.1 3 3
17 0.1 3 9
18 0.1 3 5
19 0.1 1 5
0.1 5 5
21 0.03 5
Z2 0.03
23 0.03 4 4
24 0.03 1 4
0.03 4 5
__________ _____________________________ ________________
* This rabbit exhibited slight bleeding. (In many cases,
in this model, when bleeding occurs no adhesions
develop.)
__________________ _________ ____________________________
This series of experiments indicates that the threshold
dosage at which significant anti-adhesion effects begin to
be noted in this model with suprofen sodium as the NSATD
occurs when pumps containing a concentration of about 0.1
mg~ml of drug are used. Thus, on a weight basis, suprofen
appears to be slightly more active than ibuprofen.
: 35
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ExamPle 12
By a procedure analogous to that described above in
Example 5, the 2 milliliter mini pump was used to deliver
varying amounts of tolmetin. (The sodium dihydrate salt
of tolmetin was used.) The concentrations of drug
contained in the phosphate buffered saline (pH = 7.4)
varied from 3.0 mg/ml down to 0.01 mg/ml. The treatment
pumps contained the concentrations of tolmetin displayed
below in Table VIII, and the control pumps contained
vehicle only. The rabbits were sacrificed 7 days
post-operatively, with results being di6played below in
Table VIII:
Table VIII
Tolmetin, ~o~age
ResDonse Studie~
Concentration Evaluation
of Tolmetin, Vehicle
Rabbit No. ma/ml_ Treatment Control
1 3
2 3
3 3 l 5
4 3
3 1 3
6 l 1 4
7 1 1 5
8 1 3 5
9 1 l 3
1 Thi8 rabbit died **
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11 0.3 3 3
12 0.3 1 3 *
13 0.3 1 3
14 0.3 3 5
0.3 3 4
16 0.1 3 5
17 0.1 1 5
18 0.1 1 5
19 0.1 1 5
200.1 This rabbit died (snuffles)***
21 0.03
22 0.03 1 5
23 0.03 3 s
24 0.03 1 5
0.03 1 4
26 0.01 5 5
27 0.01 1 5
28 0.01 1 5
29 0.01 1 4
0.01
25--________________________________
* Slight bleeding occurred.
** The cause of death was unknown, but there was no
suggestion that it was drug-related.
*** "Snuffles" is a viru~-cau~ed upper re~piratory
disease that affects rabbits. There i~ no sugge~tion that
it was drug-related.
~: _____________________________
As can be seen from the data presented in Table VIII,
tolmetin appears to be more effective. on a weight basis.
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than either ibuprofen or suprofen in this rabbit model
experiment. The thre6hold dosage, a~ indlcated by the
re~ult~ trom thia experiment, wa~ appar~ntly 1Q~8 than the
dosage admln~stered to Rabblt Nos. 26 - 30, wherein the
S concentration of drug wa~ 0.01 mg/ml.
ExamPle 13
~he following is a typical preparstion of a lipo~ome (MLV)
containing an NSAID:
(In this preparation, all materiais and aquipment used are
sterile and pyrogen-free)
a) P~e~arati~n o~ l~P~d ~lm
L-alpha-di6tearoyl phosphatidylcholine ("DSPC"), 1.21 gm.,
and 0.29 gm. cholesterol ~molar ratio of DSPC to
cholestero,l is 2:1), are dis~olved in 45 ml. of
chloroform. ~he re~ulting solution i8 div~ded into nine 5
ml portions, and each ~uch portion is placed in a lOO ml
~la~k. The 601vent is evaporated from each fla~k using a
rotary vacuum evaporator. Sterility i8 maintained by
attaching a 0.22 micron Millex filter to the air intake of
: 25 the evaporator prior to flask removal. Starile septa are
placed on the flasks after solvent evaporation. The
surface of each septum is ~iped with 70% alcohol, and a 19
gauge sterile needle affixed to a 0.,22 micron filter is
pa~sed through each septum. All flasks are then placed in
a large vacuum desiccator and kept there ove~night. ~ach
flask contains about 167 mg. of lipid.
*Trademark
.:
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.
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b) Pre~aration of NSAID solution
The sodium salt of ibuprofen (Na-IBF), 0.202 gm., is
dissolved in 40 ml of sterile, pyrogen-free water. The
solution is then passed through a 0.22 micron Millex
filter.
c) Pre~aration of MLV containina Na-IBF
3.9 Ml of the Na-IBP solution is injected into each flask
containing lipid film. The flasks are vortex-stirred for
40 to 60 minutes in a 65 C. water bath under nitrogen.
(The nitrogen purge is first passed through a 0.22 micron
filter.) To the contents of each flask are added sterile,
pyrogen-free phosphate buffered saline, and the flask6 are
centrifuged for 6 to 10 minutes at 15,000 rpm. This
wa~hing procedure i~ repeated for a total of five times to
remove unencapsulated ~SAID. The contents of the flasks
are then combined and PBS (5mM P04 in 0.15 NaCl) is
added to a total of 3Z ml. The liposome suspension thus
produced comprises MLV's of about 1 micron in size
~uspended in the PBS. It is storage stable for a period
of several months, but for long term storage is preferably
dehydrated and remixed with sterile, pyrogen-free water
just before use.
To prepare drug-free controls, the procedure is repeated
substituting pure water for the water/Na-IB~ ~olution.
The procedure is repeated using ta) the free-acid form of
IBF, and (b) the sodium salt of tolmetin. Similar result~
are obtained and liposome MLV's containing IB~ or sodium
tolmetin are produced.
MLV's containing IBP or tolmetin, and produced as
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described herein, were found to have particle sizes of
about 1-1/2 to 2 microns (by optical microscope) and about
5 to 8 microns (by Coulter counter).
Analogous procedures would be employed to produce vesicles
from phosphatidylcholines in which the fatty acid moieeies
were derived from other fatty acids, e.g., C12 to C24
fatty acids. C14 to ~20 saturated fatty acids are
preferred.
ExamDle 14
This experiment evaluated the eficacy of a
liposome/ibuprofen combination to combat post-surgical
adhesions. The procedure used was similar to that
described above in Example 4, except that the untreated
control sites in those rabbits that were given the
liposome/ibuprofen treatment were on the left-lateral
peritoneal side-wall instead of being sited 1.5 to 2.0
centimeters inferior to the treatment sites in the
right-lateral peritoneal side wall, and the wounding
procedure described in Example 5 was used.
The liposomes used in this example were DSPC/cholesterol
MLV~s (L-alpha-distearoyl phosphatidylcholine/cholesterol
multilamellar vesicles), prepared in a manner analogous to
that described above in Example 13. The treatment mixture
consisted of 31 milliliters containing 1200 milligrams of
MLV and 35 milligrams of the free acid form o ibuprofen,
suspended in 5mM phosphate buffered saline. The vehicle
control had the same composition, except that the
ibuprofen was omitted. Each rabbit received 10 ml of
suspension, which amounted to 3.5 mg o ibuprofen per
rabbit. The treatment and vehicle control suspensiong
were dripped on the traumatized ~ites, as described above
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in Example 4. Three rabbits received the~
liposome/ibuprofen treatment on the right side-wall site
with the other site being untreated, and three rabbits
received a vehicle control treatment (i. e., liposome
without ibuprofen) on the right side-wall site with no
treatment on the other site. The rabbits were sacrificed
7 days post-operatively, and evaluated as described
above. The results are displayed below in Table IX:
Table IX
LiDosomeJIbuProfen Studies
Rabbit Site
No.IbuProfen Treated Untreated
1 yes
2 yes
3 yes 1 4
4 no 2 4
no 5 5
6 no 1 5
The experiment was repeated in exactly the same fashion,
;~ with the results as displayed below in Table X:
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Table X
Rabbit Site
No.IbuProfen Treated Untreated
1 yes
2 yes
3 yes 1 4
4 no 5 4
no 3 4
6 no 1 1*
_ ___________________________
~ This rabbit exhibited ~light bleéding.
________________________________________________________
In the experiments reported above in Tables IX and X, the
rabbits receiving treatment by the ibuprofen/liposome
combination were virtually free of adhesions, even on the
sides that received no direct application of medicament.
This is considered to be indicative of the fact that the
medicament can migrate in the peritoneal cavity as a
result of circulation of peritoneal fluid. Also, by gross
observation, no tissue reaction or granulomas were found
at the treated sites.
Exam~le l5
In this series of experiments, the procedure of ~xample 14
was repeated, with the following modifications:
The liposomes that were used were DSPC~cholesterol MLV~s
prepared as described above in Example 13, and which
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contained Na-IBF. The control MLV~s (two batches -
"MLV-l" and "MLV-2") contained no drug. The
drug-containing lipo60mes were mixed with the control
liposomes in varying proportions to obtain MLV's that
contained varying amounts of NSAID. In Table XI, below,
there is di6played the compositions of the drug-containing
and tbe control liposomes:
-
Table XI
LiDosome ComPosition
Vol, Lipid, Total Lipid, Drug, Drug/Lipid,
ml mq/ml mq ma/ml (bY weiqht~
Control
MLV-l 50 33 1675 N/A N/A
MLV-2 50 34 1710 N/A N/A
20 IBUPROFEN
MLV 50 33 1675 0.98 0.029
(49 gm)
In Table XII, below, there is displayed the proportions of
the drug-containing and control liposomes that were mixed
together and then administered to the test rabbits in the
manner described above in Example 14, along witb the
results of the evaluations of the rabbits seven days post
operatively:
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Table XII
Na-lBF/MLV Evaluations
Rabbit IBF Control Evaluation
No. MLV, MLV, Treatment Control
ml ml Site Site
1 10 0
2 10 0 3 3
3 10 0
4 3 7
3 7 1 4
6 3 7 1 5
7 3 7 1 4
8 1 9
ZO 9 1 9
1 9
11 1 9
1~ 0.3 9.7 1 3
13 0.3 9.7 1 3
14 0 10 4 5
0 10 5 3
16 0 10 3 3
17 0 10 4 5
18 0 10 3 3
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Exam~le 16
By a pro¢edure analogous to that described above in
Example 14, aqueous ¢ompositions including a surface
at1vo ~g-nt and t~e f roo aoid ~o~m o~ tolmetln we~e
applied to the treatment sitQs in the rabbit model. The
a~ueous compositions comprised various concenteations of
"Tween 80", an ethoxylated sorbitan mono-oleate, in triple
distilled water, and tolmetin at a concentration of either
10 1 or 2 mg~ml. The 601utions were sterilized by pa~sing
them through a 0.22 micron filter. The tables below
display the concentration of tolmetin and Tween 80, and
the evaluation~ of the adhesions seven days
post-operatively. In each case, 10 ml of the solution was
tr1~pod on the treatmen~ s~to.
Table XIII
5 Wt, ~ Tween 80 *
~ ma/ml Tol~,e~
a~gi_~Q~ Evaluation
Treatment Contr~l
Site _Site
1 1 5
3 1 1 ~Bleeding)
4 1 ,
5 (Control*) 3 3
6 1 -3
7 1 4
8 3 5
9 '
: 10 (Control*) ' 4 3
;*Trademark
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* The control rabbits received no treatment.
___________________________________
Table XIV
20 Wt. % Tween 80
2 ma/ml Tolmetin
Rabbit No. Evaluation
Treatment Control
Site Site
1 1 5
2 3 5
3 1 4
4 3
5 (Control*) 4 4
6 1 3
7 3 5
10 (Control*) 5 5
____________________________________________________________
: ~ No Treatment
______________
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Table XV
20 Wt. ~ Tween 80
2 mqJml Tolmetin
Rabbit No.Evaluation
Treatment Control
Site Site
1 1 5
2 1 1 (Bleeding)
3 1 5
4 1 5
1 4
6 1 9
7 1 4
Table XVI
1 ma/ml Tolmetin Sodium (1
No Tween
Rabbit No~valuation
Treatment Control
Site Site
1 3 5
2 4 3
3 1 3
4 3 4
3 3
6 4 3
7 1 3
8 3 5
9 (~Control(2)) 4 3
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____________________________________________________________
(1) The sodium salt was used because the free acid
form is guite insoluble in pure water.
(2) Water only; no Tolmetin
____________________________________________________________
The free acid form of tolmetin, which i6 guite insoluble
in water, was maintained in agueou~ solution or colloidal
lo suspension by the surface active agent.
Referring again to the question of the effective dose of
NSAID 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 drug to
employ in certain cases. In pho~hate buffered saline
admini~tered continually via the Alzet osmotic mini pump,
the following dosages were found to be effective:
ibuprofen, 3 mg~ml, at a rate of 0.5
microliter/hr. ~from EX.6):
suprofen, 0.1 mg/ml, at a rate of 10
microliters~hr. (from Ex. 11); and
tolmetin, 0.01 mg/ml, at a rate of 10
microliters~hr. (from Ex. 12~.
Expres6ed in terms o mg~kq/day, and in term~ of
mg~day/cm2 of traumatized tissue, these numbers are the
ollowing:
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ibuprofen - 18 x 10 3 mg/~g/day
2.4 x 1- 3 mg/day/cm2
suprofen - 12 x 10 3 mg~kg/day
1.6 x 10 3 mg/day/cm2
tolmetin - 1.2 x 10 3 mg/kg/day
0.16 x 10-3 mg/day/cm2
These calculation~ are carried out as follow6 (using the
ibuprofen numbers as illustrative): 0.5 microliter/hr. of
golution containing 3 mg/ml equals 0.5 x 10 6 liter/hr.
x (3 mg/10 3 liter). This reduces to
0.5 x 3 x 10 6/10 3 3 1.5 x 10 3 mg/hr.
~his equals 36 x 10 3 mg/day or 18 x 10 3 mg/kg/day
(each rabbit weighs about 2 kg).
The area of traumatized tissue i8 about 15 cm2, 80 the
~0 dose expressed in terms of ~g/day/cm2 = 36/15 x 10 3
mg/day/cm2, or 2.4 x 10 3 mg/day/cm2.
Compared with the minimum recommended dose of 2.5
mgikg/day (from the Binger patent cited above) for
Z5 6ystemically ad~inistered ibuprofen, the effective dose
for topically administered ibuprofen may be two to three
orders of magnitude lower. Obviously, this greatly
reduced do6age significantly reduces the chances for
; undesired side effects.
The minimu~ effective dose using an MLV or an a~ueous
composition containing a surface active agent as a carrier
has apparently not been approached. Howevèr, it is noted
from Example 15, Rabbit Nos. 12-13, that a total dose of
about 0.3 mg of ibuprofen in an MLV carrier ~as
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effective. This number was calculated as follows:
0.98 mg/ml x 0.3 ml/0.7 ml, eguals 0.0278 mg/ml. 10 ml of
this preparation was admini~tered, 80 the total dosage was
0.3 mg or 0.15 mg/~g or 0.02 mg/cm2. It would appear
that an even lower concentration would be effective in
this ~odel.
In the Tween 80 solutions, the smallest amount of tolmetin
applied was 10 mg (from the data presented in Table
XIII). It would appear that an even smaller dosage
administered in this manner would be effective.
A reasonable extrapolation of the data presented herein ifi
that a minimum effective concentration for an NSAID
preparation applied in a ~ingle dose would be between
about 0.025 mg and 5 mg of N5AID per ml of total vehicle.
(By "total vehicle" i6 meant an orqanic vehicle such as
MLV or Tween 80 plus diluent ~uch as water or PB5.)
5imilarly, for an N5AID preparation that is administered
continuou~ly, as by catheter, minimum effective
concentrations of N5AID in total vehicle will usually be
found within the range of from about 0.01 to about 10
mg~ml.
ExamDle 17
In this series, the uterine horn of New Zealand wbite
female rabbits wa~ used as the model for adhesion
development. It is believed that the trau~a induced in
this type of surgical procedure i~ more apt to produce
severe adhesion~ than any trau~a ordinarily associated
with surgery, and therefore thi~ i~ a very severe te~t for
evaluating the efficacy of a medica~ent in inhibiting the
formation of po~t-~urgical adhe~ions.
ETH-694
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The rabbits were anaesthesized using acelepromazine and
ketamine, and then underwent a lower median laparotomy
incision. Then, the 6erosal surface of both uterine horns
were abraded by grasping them with a gauze surgical sponge
and pulling them away from the uterus until punctate
bleeding developed.
Immediately after the uterine horns were traumatized as
described above, varying quantities of a MLV liposome
Iprepared by a procedure analogous to that described above
in ~xample 13) containing the sodium salt of tolmetin were
dripped on the traumatized site, and the rabbits were then
closed. Seven days post-operatively, the rabbits were
sacrificed and the development of adhesions was evaluated,
with the results discussed below.
The lipo~omes were ~u~pended in PBS, at a concentration of
40 mg. of liposome per ml of suspension and 1.46 mg. of
tolmetin per ml of ~uspension.
The results of the evaluation were as follows, with the
quantities indicated being the volume of liposome
suspen~ion dripped on the site of surgical trauma:
10 ml - Scant adhesion
3 ml - Scant adhesion, but a few more than with
the 10 ~1 dose. The adhesions were filmy
1 ml - Mild adhesions
Control- Ino medicament) - Severe adhesions:
essentially unable to open the rabbit
without tearing adhesions which developed
between the uterus and the bowel and
,
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between the uterus and the anterior
peritoneal wall.
The above-described study was repeated several times with
the liposome/tolmetin combination, with es6entially the
same results.
When the free acid form of tolmetin contained in 15
milliliters of aqueous Tween 80 (5 wsight per cent Tween
80, 1 mg/ml of tolmetin) was substituted for the
tolmetin/liposome suspension in the double uterine horn
model study, the formation of post-surgical adhe6ions was
substantially prevented.
ETH-694
