Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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PROCESS FOR INSOLUBILIZING N-CARBOXYALKYL
~ERIVATIVES OF CHITOSAN
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BACKGROUND :)F THE INVENTION
Field of the Invention
The present invention relates to a rr~ethod of temporarily insolubilizing
10 films and gels of N-carboxyalkyl derivatives of chitosan to provide materials which can
have varied degradation times for various biomedical applications such as adhesion
prevention. More particularly, the method relates to an annealing process in which the
material is insolubilized by exposing film to temperatures between 50C and 200C for
various lengths of time.
ûescriPtion of the Prior Art
Chitin and chitosan (partially deacetylated chitin) are well known biocompatiblematerials whose preparation has been described in U.S. Patent 2,040,880, which issued
on May 19, 1936. A derivative of chitosan, N,O-carboxymethyl chitosan, and its
20 produ~tion ha~ been described in U.S. Patent 4,6i9,995~ which issued to E.R. Hayes
in Octooer of 1986~ The preparation of another chitosan derivative, N-carboxybutyl
chitosan, is described in U.S. Patent 4,835,265.
The uses of chitin, chitosan, and other polysaccharides in biomedical applications
is most evident in wound dressings. Materlals for use in wound dressing applications
25 are disclosed in U.S. Patents 3,632,754, 4,532,134, 4,659,700, 4,572,g06, 4,378,017,
foreign patents G8 2026~16, EP 0200574 and publications WO 86/009t2 and
WO 87/07618. Others have addressed the problem of adhesion prevention utilizing
biodegradable materials. U.S. Patent 4,603,69~, which issued August 5,1986 to Ikada
et al, discloses the use of an absorbable polyester polymer~ (: hitin and Chitosan can
3Q be absorbed by hydrolysis in vivo.
Co-pending applicatlon Serial No~ 07/644,758, filed on ~anuary 24, 1991 (U~S~
Patent 5,Q93,319) and assisned to the assignee of the present invention, teaGhes the
use of chitosan and derivatives thereof for adhesion prevention. None of these patents
or patent applications, teach a simple way of insolubilizing films and gels from35 derivatives of chitin for intemal application to vary degradation times~
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As described in co-pending application Serial No. 07/644,758, scar tissue results
from the organization of fibrinous exudate on tissue surfaces due to the infliction of
trauma or inflammation. Vital tissues such as blood vessels, or organs including the
kidney, liver, and intestines are coated with mucous or serous membranes so that they
can function independently of each other. Examples of the mucous or serous
membranes are the body wall pleura and the organ pleura in the thoracic cavity and the
parietal peritoneum and mesentery in the abdominal cavity, each protecting the
corresponding organs. Surgical trauma or inflammation in those portions of the body
coated with serous membranes could result in the build up of fibrinous exudate
10 regardless of the size of the affected part. This ultimately causes the creation of
organized fibrin many times referred to as scarring or adhesions. Such adhesionsbetween these tissues may be observed in all tissues of the body, not just thosementioned above~ Scarring between tissues can lead to severe pain, decreased
function, and even permanent loss of motility.
Adhesions occur in the orthopedics field where conditions such as acute or
chronic arthritis, suppurative, rheumatoid, gonorrheal, or tuberculous arthritis,
or traumatic injuries at the joint, such as fracture or sprain, result in ankylotic diseases
wherein the surface of the bones constituting the joint adhere to ~ach other and thereby
restrict the mobility of the joint. In addition, congenital radioulnar syntosis, wherein a
2Q spoke bone and an ulna adhere together, is difficult to remedy by a surgical operation,
since the separated bones frequently re-adhere. Adhesions are also prominent in
tendon surgery. In this instance, there is a general tendency towards adhesion
betNeen the tendon and the surrounding sheath or other surrounding tissue during an
immobilization period following the operation.
More recently, there has been increased interest in the prevention of the
"laminectomy membrane" which forms following spinal laminectomy procedures. The
laminectomy membrane is a well oryanized mass of fibrinous tissue which replaces the
bone that was`'removed at the laminectomy~ This fibrinous mass binds the dura to the
overlying muscles~ This causes narrowing of the spinal canal which places pressure
30 on the cauda equina or nerve roots~ This scar tissue formation rnay require reoperation
which is tedious and dangerous leading to the possibility of dural tears and the damage
to the emergent nerve roots resulting in motor weakness, sensory change, and painful
paresthesia~
SUBSTlTUTE S~E~
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Num0rous papers have been published on the various treatments to prevent scar
tissue build up. Treatments such as liquid paraffln, camphor oil, chondroitin sulfate,
and urea exhibit an insufficient effect since they function only temporarily. Other
prophylactic treatments such as silicone membranes, gutta percha, or poly (tetra-
5 fluoroethylene) membranes have been used to serve as barriers to adhesion formation.
However, these materials are inert and will, therefore, remain in the body and many
times be recogni2ed as a foreign body. Therefore, a second operation may be
necessary to remove the barrier material.
SUMMARY OF THE INVENTION
It is an object of the invention to provide a process for producing modified
N~arboxyalkyl derivatives of chitosan for the prevention of fibrinous exudate build-up,
which degrades in vivo in various predetermined time periods~
It is a further object of the invention to provide a slmple annealing process for
insolubilizing N-carboxyalkyl derivatives of chitosan.
Accordingly, these and related objects are achieved by a process for insolubilizing
films of N-carboxyalkyl denvatives of chitosan. The N-carboxyalkyl derivative
of chitosan is dissolved in a neutral pH aqueous solution or a slightly acidic solution.
20 The solution is then cast and dried to form a thin clear film. Alternatively, the
solution is poured into a mold (for example a petri dish) and Iyophilized to forrn
a sponge-type film with varying dimensions depending on the mold, the solution
concentration, and the solution volume. The resulting films are insolubiîized by an
annealing process in which they are exposed to temperatures between 50C and
25 200C for various periods of time depending on the desired length of insolubilizattori
time, and uitimately bioresorption time.
rnese and other objects and advantages of the presen~ invention will become
apparent from the following detailed description which discloses several embodtments
of the invention~ It is to be understood that the examples used are for the purposes of
30 illustration only, and not as a deflnition of the inventton.
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DESCRIPTION OF THE PREFERRED EMBODIMENT
The biodegradable polymer films to be used to inhibit fibrin fomlation and
organization are materials which will eventually revert to the gel or solution state
5 and ultimately be resorbed and metabolized by the body. As taught in co-pending
application Serial Number 07/644,758, these materials include amino N-carboxyalkyl
derivatives of chitosan. Chitosan is a partially deacetylated chitin defined for the
purposes herein as being greater than 50% deacetylated.
The N~arboxyalkyl derivatives used in the present invention are water soluble
10 polymers which have not been crosslinked to form insoluble materials. Specific
examples of these materials are N-carboxymethyl chitosan, N~arboxybutyl chitosan,
N,O carboxymethyl chitosan, and N,O-carboxybutyl chitosan, N-carboxy-ethyl chitosan,
N,O-carboxyethyl chltosan, N,O-carboxypropyl chitosan, sulfated N-carboxyalkyl
chitosan and N-carboxypropyl chitosan. However, these materials can be temporarily
15 insolubilized to form substances which will begin to degrade in a period of from 2-5
days to up to one year in vitto. It has been found that exposure of films of these
materials to heat from 50C to 200C for various time periods varies the insolubilization
time and ultimately the bioresorption time. In general, the higher the temperature of
exposure and the longer the time of exposure, the greater the time to solubilize the film.
20 In order to vary the degradation time, the temperature and the length of time of heat
exposure are varied from 50 - 200C anywhere from 20 minutes to 24 hours but could
be as little as 10 minutes and preferably 10 minutes to ~ hours.
These materials are prepared from natural products or by ferrnentation methods
~;as described in U.S. Patents 4,835,265 and 4,619,995. The molecular weight of the
25 b~odegradable N-carboxyalkyl derivatives of chitosan for use in the present invention
preferably range from 1,0Q0 daltons to 3,000,000 daltons.
In the preferred embociiment, these polymers would be in the form of a film,
sponge, or woven sheet which will break down into visco-elasffc matenals. Examples
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of these would be the use of N,~carboxymethyl chitosan or N-carboxy-butyl chitosan
~30 films insolubilized by annealing with heat as described herein. While the exact
-~ mechanism which causes the insolubillzation of the materials is unclear, it is postulated
that either a dehydration mechanism, or a crystallization mechanism or a combination
of the two is causing this phenomena. Additives such as anff-thrombogenic materiais,
such as heparin or the like, may be added to the films before insolubilizing.
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As described in co-pending application 07/644,758, it is also possible to form
these materials into a viscous gel for injection into the affected location to prevent
fibrinous buildup. This gel could be used for applications where a shorter ln vlvo
residence time is desired and/or the location would best be suited for prevention
5 of adhesions with a material in this form. The gels may be formed by placing the
insolubilized films in a sterile aqueous media and heating the solution to increased
temperatures until the film hydrates and swells to form a gelatinous mass.
Again, as with the film, the degree of insolubilization of the viscous gels
can be varied by varying the temperature of the aqueous solution and the extent of time
10 the film is allowed to remain in the solution.
The present invention has the added advantage that the insolubilization step canbe done during the heat or steam sterilization of the device, if desired. For example,
the film may be insolubilized during the autoclave sterilization of the device at a
temperature greater than 1 20C for a standard autoclave cycle.
The invention will now be described in further detail with reference being made to
the following examples. It should, however, be recognized that the examples are given
as being illustrative of the present invention and are not intended to define the spirit and
spirit thereof.
20 Exam~le 1
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One gram of medical grade N,O-carboxymethyl chitosan (NOVA Chern, Nova
Scotia) was dissolved in 100 cc of purified water. The resulting 1% solution wasthen flltered through a series of cellulose membranes (12, 8, 0.45 micrometer) to
remove all insoluble matter. The solution was then recirculated over and through a 0.5
25 micron tangential flow membrane which was specially treated with polypeptides specific
for removal of pyrogen and other hydrophobic impurities (Catalog number 4200 -
AlerChek, Portland, ME) for a period of approximately one hour on tangenttal flow ultra-
flltration device (Filtron Non~ood, MA). The resumng solution was then rendered ~
of all low molecular weight impurities by extensive dialysis with water via a 300K
; 30 rnolecular weight cutoff membrane (Filtron - Norwood, MA) on the same ultra-filtration
device.
Films were then formed from this solution by both casting (allowing evaporation)on a non-stick surface such as a glass petrii dish, or a plece of mylar fi1m and by freeze
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drying by adding 300 ml of the solution to a 90 rnm diameter disposable petri dish and
Iyophilized in a tray dryer for 72 hours a~ -50C. The resulting films (either cast or
freeze dried~ were then insolubilized by placing the film into an oven at 121C for 20
minutes (correlating to a standard autoclave cycle). Both films were placed in aqueous
media and dissolved within 5 days. Based on the dissolution which did occur, the film
would dissolve in approximately two months.
Example 2
Three films were prepared as descnbed above in Example 1 by casting in a petri
dish. Each was exposed to a different temperature for a period of 30 minutes. The first
was heated at 76C, the second at 121C and the third.at 168C. One film was cast
and not treated. This served as a control.
The four films were placed in ~ cc of PBS and placed in an incubator at 37C.
At various times, the samples were removed and obseNed yisually for solubility.
Followirig this the incubation medium was removed from the dish and analyzed forsoluble product via size exclusion chromatography. The control film was completely
solubilized within one day. The film at 76C indicated complete solubility between 1-2
days. The 121 C treated film indicated complete solubility at 4-5 days, while the film
treated at 168C indicated no evidence of solubility up to 14 days~
ExamPle ~
Three films were prepared as described above in Example 1 with the exception
that the temperature of annealing was kept constant at 121C and the exposure time
was varied between 15 minutes and 23 hours. Again, a non-treated film ser~Jed as a
control. All films were evaluated as mentioned above.
The film treated at 15 minutes indicated complete solubility in 2-3 days, the film
treated for 60 minutes indicated solubility through 14 days (complete solubility had not
yet been attairied) while the film treated at 2 hours indicated no solubility in an excess
of 14 days.
Thus the degradation of the films in wvo could be lengthened by increasing the
annealing time and temperature. For a typical orthopedic application, a film which
would degrade within 14 days is desirable and may be attained by annealing at 121C
for about 60 minutes~
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The material of the present invention may be used in a nnethod for preventing
adhesions between soft intemal body tissue by first preparing an aqueous solution of
N-carboxyalkyl derivatives of chitosan, then forming a film from the aqueous solution
by removing water therefrom and then insolubilizing the film by heating the film at a
5 temperature of at least 70C for a minimum of 15 minutes and placing the film between
the tissues after being insolubilized.
While several examples of the present invention have been described, it is obvious
that many changes and modifications may be made thereunto, without departing from
the spirit and scope of the invention.
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