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1. WO2002017853 - HYALURONAN-BASED ANTIADHESION COMPOSITIONS, THEIR PREPARATION AND USE

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[ EN ]

HYALURONAN-BASED ANTIADHESION COMPOSITIONS,
THEIR PREPARATION AND USE.

Technical Field
The present invention relates to biocompatible solid compositions based on hyaluronan and derivatives thereof, and methods of making and using them in preventing post surgical adhesions.
Background Art
Hereinafter, the term "hyaluronan" is deemed to be interchangeable with and is largely used instead of the term "hyaluronic acid" as suggested by Balazs, et al. "Nomenclature Of Hyaluronic Acid", Biomedical Journal, vol. 235(3), 1986, p.903). The abbreviation HA heretofore used for hyaluronic acid is hereinafter used to refer to hyaluronan (and hyaluronic acid).
Post surgical adhesions are among the most common complications occurring after any type of surgery. Any trauma to a tissue is normally followed by healing which may lead to the formation of undesirable collagenous scars connecting two separate tissues at the surgical site providing that the distance between these tissues is small enough. Such events as excessive bleeding, inflammation and intimate contact between tissues substantially increase the probability of adhesion formation at the site.
Post surgical adhesion formation is extremely undesirable since it can cause numerous clinical complications such as chronic pain and functional disorders and in many cases will require re-operation. Several approaches have been suggested to prevent or minimize the incidence of post surgical adhesion formation. One approach involves isolating the damaged tissue and separating it from any adjacent tissue with a biocompatible fluid, gel, or film. The first use of HA in the form of a concentrated solution or a film for prevention of adhesion formation in healing of flexor tendons and conjunctival wounds was described by Balazs et al. , in Hyaluronic Acid and Matrix Implantation, 1971, ed. E. A. Balazs, Biotrics, Inc. , Arlington MA, p.p. 1-6). The effect of HA and derivatives thereof on tendon repair has also been evaluated in other numerous publications (R. S. Onge, C. Weiss, J. L. Denlinger, and E. A. Balazs. "A Preliminary Assessment of Na-Hyaluronate Injection into "No Man's Land" for Primary Flexor Tendon Repair", Clinical Orthopaedics and Related Research, 1980, No. 146, January-February, p.p. 351-357; C. Weiss, H. J. Levy, J. L. Denlinger, J. M. Suros, and H. E Weiss, "The Role of Na-Hylan in Reducing Postsurgical Tendon Adhesions", Bulletin of the Hospital for Joint Diseases Orthopaedic Institute, 1986, v. 46, No. 1, p.p. 9-15; C. Weiss, J. M. Suros, A. Michalow, J. L. Denlinger, M. Moore, and W. Tejero, "The Role of Na-Hylan in Reducing Postsurgical Tendon Adhesions: Part 2", Bulletin of the Hospital for Joint Diseases Orthopaedic Institute, 1987, v. 47, No. 1, p.p. 31-39). The use of absorbable HA based membranes and gels to reduce post surgical adhesion formation is also described in J. W. Burns, "Prevention of Post Surgical Adhesion Formation with Sodium Hyaluronate Based Products" in Wound Healing, III, Conference Proceedings, January 1992, Orlando, Florida, Technology Management Group.
An essentially different method for preventing surgical adhesions is described in US Patent No. 5,140,016. The method consists of a preliminary coating of the tissues involved in surgery by an aqueous solution of a biocompatible polymer such as HA. It is stated in the patent (col.5, lines 65-68; col.8, lines 1-14) that such pretreatment noticeably decreases the occurrence and severity of post surgical adhesions. There are a number of other publications describing various substances and methods for decreasing the formation of post surgical adhesions.
The analysis of the prior art reveals that the prevention of adhesions after surgery by using anti-adhesion barriers is used more often than other methods. However, the success of the method is determined by such factors as the residence time of the barrier material at the site of the surgery, the permeability of the barrier to the cells and proteins normally occurring during the reparative process, and the biocompatibility of the material used as a barrier.
The last mentioned factor is of extreme importance since any tissue reaction toward the material may cause the formation of additional adhesions. For this reason, and because of its exceptional biocompatibility HA is becoming the material of choice. However, when used as an aqueous solution, HA will disappear from the desirable site rather fast and significant elimination of the adhesions is rarely achieved. Insoluble gels of HA may be efficient in some surgical procedures in which the surgery is done in a more or less "closed compartment" which can accommodate and contain the gel for a substantial period of time, e.g. , a space between a tendon and a tendon sheath. When a membrane made of HA or a derivative thereof is used as a barrier for adhesion control, in most cases it is impossible to maintain the slippery membrane at the site and, as a result, adhesion prevention is not achieved. The method of adhesion prevention disclosed in the U.S. Patent No. 5, 140, 016 which uses our HA solution for pretreatment of the tissues before manipulation though it decreases the frequency of adhesions nevertheless makes manipulation of the tissues very difficult because pf the extreme slipperiness of HA solutions.
Therefore, biocompatible materials capable of providing reliable and efficient reduction, or elimination of post surgical adhesions is highly desirable.
Disclosure of Invention
The processes by which the hereinabove described products are obtained will now be discussed in detail.
In order to obtain a compressed film, a sample of a hyaluronate in salt or acid form from any source is dissolved in water. The molecular weight of HA can be as low as 5xl04, but generally speaking, the higher the molecular weight the better the product. Thus, when using a natural hyyaluronan, the lower limit for the molecular weight is around 0.5 million, with no upper limit. Any natural hyaluronan, no matter how high its molecular weight, will suffice for this invention. When using a hylan A fluid, the molecular weight can be as low as 0.5 million and up to 20 - 25xl06. With a hylan B gel, molecular weight is not a meaningful parameter as the gel is an infinite network.
The concentration of hylan or HA in the starting solution can vary from 0.05 % by weight to 10% by weight. When the concentration is below the lower limit, a useful film can not be obtained because of its very low integrity in saline; but the molecular weight is also a factor because the higher the molecular weight, the lower the concentration can be. When the concentration is more than 10% from a high molecular weight fiber source, the solution becomes so viscous that it is difficult to handle. The combination of molecular weight and the starting concentration of hylan or HA substantially affects the dilution behavior of the films by affecting the rate of swelling and the integrity of the films in saline on or into the tissues where it is implanted over time.
We have found that the force used during the pressing step is another parameter which can be used to control the dissolution pattern of the hylan films. An increase in the applied force during the pressing procedure results in an increase in the film integrity in saline on or into the tissues where it is implanted. An increase in the temperature during pressing results in an accelerated rate of swelling.
The compositions according to the invention, as more fully described hereinafter include compressed films formed from varying concentrations, amounts and molecular weights of HA and hylans; hylan B (cross-linked, insoluble hylan) of varying concentrations and amounts, both homogenized (slurry) and non-homogenized; hylan A and B mixtures and layers thereof in the form of "sandwiches"; hylan A and other polysaccharides such as glycosaminoglycans, dextrans, proteins such as collagen, elastin and others; hylan A with various pharmaceutical agents, such as anti-inflammatory agents (steroids, NSAIDS), antibiotics (gentamycin, streptomycin), analgesics (morphine), growth factors (rh-gcsf, fgf, pdgf), anesthetics (novocaine, lidocaine), and weak acids including ascorbic acid and polylactic acid.
The compositions according to the invention are compressed films having a thickness of 0.1 - 1.0 mm, preferably 0.2 - 0.5 mm made from a hyaluronan, of either bacterial or tissue origin, hylan A (fluid) and hylan B (gel). The molecular weight of the starting material used in the process for the compositions varies over a very broad range, but greater than about 3xl05. The compositions can be in the free acid form or preferably, in the form, of a salt. The salt can, for example, be the sodium, calcium, acetylcholine and hexamethonium salts or any other physiologically acceptable salt. The sodium salt is the preferred salt.
In another embodiment, the films according to the invention can have incorporated therein additional substances to take advantage of their particular properties in addition to the adhesion prevention properties of the compressed films. Thus, the films can have incorporated therein glycosaminoglycans such as heparin; antineoplastic agents such as carmustine, 5-fluorouracil and vinblastine; and other therapeutic agents.
Among the polysaccharides that can be incorporated into the films heparin is a known antithrombotic and antigrowth agent. The inclusion of such an agent in an adhesion preventing HA film or hylan gel film will enhance the action of the film or gel. This is because when an adhesion typically forms, the first process to occur is that fibrinogen present in the surrounding tissue is converted to fibrin. Anything that can inhibit the formation of fibrin or slow down the migration of leucocytes, and thus the onset of adhesion formation will therefore necessarily inhibit the formation of adhesions. Thus, in addition to enhancing the adhesion prevention properties of the compositions of the invention, a hyaluronan or hylan film or gel and heparin composition can also be said to be a drug delivery system for heparin.
The antineoplastic agents can be incorporated into the films for a similar purpose. Thus, for example, when performing cancer surgery, the film and antineoplastic agent functions both to prevent post surgical adhesions and also to deliver the antineoplastic agent directly to tlie situs of the cancer where it is likely to be more effective than if it were introduced to the body intravenously.
When a material such as polylactic acid ("PLA")is incorporated into the films according to the invention, it has been shown that the PLA slows down or retards the dissolution of the gel/film barrier meaning the residence time of the film is increased so as to render its anti-adhesion properties more efficacious.
Additionally, other drugs can also be incorporated into the films of the invention. Among these are antibiotics - to prevent infection at the surgical site; anti-inflammatory agents to prevent inflammation at the surgical site and hormones such as human growth factor. All of these materials perform their usual pharmacological functions at the same time that the film per se is acting to prevent post surgical adhesion formation. Thus, they too can be said to be drug delivery systems.
The process for making the HA, hylan A and hylan B film compositions according to the invention involves first preparing a homogeneous aqueous solution of the starting polymer including, if necessary, the other materials described above, i.e. , heparin, antineoplastic, PLA, etc. of molecular weight as stated above, and at a concentration (by weight) of 0.05 - 10%, preferably 0.5 - 10% and placing the solution into a mold where it is allowed to settle in order to remove any air bubbles. Thereafter, the solution is subjected sequentially to freeze drying or drying at any other temperature up to about 65 °C to remove essentially all of the water and then compression to form the films. These steps will now be described in detail. The solution is placed in a suitable vacuum apparatus for freeze drying and first cooled to about -15°C, where it is held for about 1-3 hours, preferably about 2 hours after which vacuum is applied to reduce the temperature to about -30 °C. These conditions are maintained for an appropriate time that depends on the temperature in order to remove most, but not all of the water from the solution. This is what is called the primary drying phase. Next, heat is introduced to the environment, first to about 0°C for about 180-220 minutes, preferably about 200 minutes and then to about 20° to 50°C for about 400-600 minutes. There next follows what is considered the secondary drying phase for the purpose of removing interstitial water remaining behind after the primary phase. This is accomplished by reducing the vacuum for about 120 minutes or longer after which the dried disc is removed from the mold.
The disc is now subjected to compression at room temperature (about 20 °C). Typically, a disc of about 6 in2 or larger is compressed between two stainless steel or tantalum plates under a force of between 500 to 15,000 pounds, or 10-450 lbs/in2. After the compression step, the preparation of the film is complete and a film having a thickness of about 0.1 - 1.5, preferably 0.2 - 0.5 mm is formed. In order to use these films in surgical procedures, they must be sterile and low in endotoxin content. Therefore, the process described above must be carried out under aseptic conditions with sterile starting materials. Alternatively, the films may be sterilized by standard sterilization procedures. Now the film is ready for use in any suitable surgical procedure. That is, the surgeon will typically cut a piece of the film of the desired thickness to the desired size and place it where needed at the site of the surgery prior to closing the site. While typically, a flat piece of film is placed in an open surgical site, it is also possible to use the film in other ways; e.g., by rolling up a small piece of the film and inserting it into a cannula which may then be injected into an arthroscopic surgical site through tubes or cannula or other appropriate delivery systems.
The process for preparing the films of the invention can also be performed by drying the solution under non-freeze drying conditions, for example, at temperatures of up to about 65 °C, after which the compression is effected as described above.
The present invention is described in more detail in the following examples. These examples are given merely by way of illustration and are not intended to limit the invention as set forth in the claims.
Therefore, to summarize, the present invention provides hyaluronan based solid compositions and salts thereof for preventing post surgical adhesions;

provides hylan based solid compositions for preventing post surgical adhesions. As used herein, the term hylan includes both hylan A which is a fluid and hylan B which is a gel;
provides hyaluronan and/or hylan based compositions further comprising other components for controlling the properties of the composition for preventing post surgical adhesions; and provides methods of making and using these compositions.
Brief Description of Drawings
Fig. 1 illustrates the dissolution experiment described in Example 2.
Fig. 2 illustrates the swelling rate of a pressed and non-pressed films
produced from a high molecular weight hylan.
Fig. 3 illustrates the difference in the swelling rate of films that pressed
at room temperature (20°C), 60°C and 80°C.
Fig. 4 illustrates the swelling rates of films made of hylan and a
hylan/heparin mixture as described in Example 6.
Fig. 5 illustrates the effect of the molecular weight of the starting
material on the integrity of the films in excess of saline as
described in Example 11.
Fig. 6 illustrates % elongation at the breakage point for films made of
hyaluronans and hylans having different molecular weights and
concentrations as described in Example 11.
Fig. 7 illustrates a comparison of the force necessary to punch through a
dry film and a wet film made of the same material as described in
Example 11.
Best Mode for Carrying Out the Invention
The best mode (or modes) for carrying out the invention are set forth in the following examples:
EXAMPLE 1
This example illustrates a typical procedure for making a compressed film according to the present invention.

Hylan A sodium salt was prepared from rooster combs according to the method described in U.S. Patents 4,713,448 and 5,099,013. The preparation was sterile. The procedure described below was carried out under aseptic conditions.
A 2% aqueous solution of this hylan was prepared by mixing appropriate amounts of hylan and water with slow rotation over a long enough period of time, typically 6-9 days to obtain a uniform solution. 10 ml of the solution were placed into a small (50-mm diameter) plastic Petri dish and kept covered for between 2 to 48 hours to let the solution settle and to let any air bubbles escape from the solution. Any large air bubbles were removed using a pipet. After a visual inspection confirming the absence of bubbles, the solution was freeze-dried in a lyophilizer (FTS Kinetics, model TD2CT5302) over a period of about 10-16, preferably 17 hours. The completion of the freeze-drying process was monitored by thermocouples inserted into the sample.
A highly porous sponge-like solid retaining its disk shape was obtained. The process for the sponge preparation is considered successful if the foam does not contain dense collapsed inclusions or, in other words, no holes or major indentations. The sponge-like disk was compressed using 2 stainless steel plates (6 in2) designed in such a way that the material was horizontally pressed between the two polished surfaces while lateral or side to side movement was restrained. The compression was done in a Carver Laboratory Press (model C), under a load of 12,000 - 14,000 lbs. for 30-60 seconds. The stability of this film was ascertained by appropriate microbiological methods.
A thin flexible film with a thickness of 0.30 mm was obtained. This film is characterized by its ability to strongly adhere to any wet surface and its ability to quickly absorb any fluid present on the surface.
EXAMPLE 2
This example illustrates a method for evaluating the dissolution characteristics of the compressed films according to the invention. In order to determine the dissolution rate of the film, a disk 18.5 mm in diameter was cut out of the film and placed between 2 disks of 25 mm diameter, made of the following materials: nylon (thickness 90 μm; pore size 20 μm; MSI, Westboro MA); nylon mesh (thickness 70 μm; pore size 70 μm); or polypropylene mesh (thickness 420 μm; pore size 297 μm; both from Spectrum Laboratories, Laguna Hills, CA).

The three-layer sandwich was put into a special holder which was made from a modified Sykes-Moore chemotaxis chamber (Bellco Glass, Inc. , Nineland, ΝJ). The chamber consisted essentially of an aluminum threaded cylindrical compartment with a lip on the bottom which supported the rubber ring gasket over which the mesh sandwich followed by another rubber gasket was secured. The gaskets and the sandwich assembly were fixed in the chamber by a threaded aluminum ring. The chamber assembly was situated into a glass Coplin staining jar (Wheaton) which was a square glass jar with a tip-resistant base and a plastic screw cap. The relative dimensions of the jar and the chamber were such that the chamber could maintain a fixed position in a diagonal plane of the jar approximately in the middle of its height. 50 ml of 0.15 M aqueous sodium chloride (physiological saline) was added to the jar with the chamber in it. The jar was placed on the top of an orbital shaker (Lab-Line, model # 3520). The speed of the shaker was constant at 60 rev/min, and the dissolution experiment was carried out at ambient temperature (18-22°C) continuously which was interrupted only for brief moments of sampling. The samples of saline were taken by a pipet from the top of the jar in an amount 1 ml per sample at predetermined time points. The hylan concentration in saline was determined by the carbazole method and the amount of the film substance that went into the solution was calculated. The dissolution curve shows the percent amount of polymer which went into solution with time. A comparison of the three nylon or polypropylene barriers showed that the most reproducible results were obtained with the use of the mesh with 70 um pore size. The dissolution curve for the film obtained in Example 1 is shown in Figure 1. In order to compare the dissolution characteristics of the films of various thicknesses and compositions, a parameter named "dissolution factor" (DF) was introduced which represented the ratio of total weight of a film expressed in milligrams of HA to number of days required for dissolution of 50% of the original film (DF-50) and 80% of the original film (DF-80). The lower the DF, the slower the film is dissolved. For the film illustrated in Example 1, DF-50 was 12.7 mg/day and DF-80 was 8.0 mg/day.
EXAMPLE 3
This Example illustrates the preparation of a film thinner than the product of Example 1 prepared according to the invention.
The procedure described in Example 1 was repeated with the exception that a 1 % aqueous solution of hylan A was used as the starting material. A uniform membrane was obtained with an apparent thickness 0.12 mm. The following values of DF were calculated from the dissolution curve DF-50; 9.1 mg/day, DF-80; 4.3 mg/day. A comparison of this membrane with that of Example 1 showed that rates of dissolution for the 50% point were close to one another but not the rates for the 80% point. What this demonstrates is that the residence time for the antiadhesion material in a surgical site can be easily controlled by changing the thickness of the membrane.
EXAMPLE 4
This Example illustrates the preparation of a membrane made of a combination of high and medium molecular weight hylan A. An aqueous solution of a medium molecular weight hylan A (intrinsic viscosity 850 cc/g which corresponds to molecular weight of about 3.7xl05), concentration 1 % was prepared and mixed in a weight ratio of 2.7 : 1 with a 1 % solution of the high molecular weight hylan A used in Example 2. A sample of 5.0 g of the mixture was used to prepare a membrane as described in Example 1. Thus, the membrane contained 27 % of high MW and 73 % of medium MW hylan. The values of DF found from the dissolution curve were 24.5 mg/day for DF-50 and 9.0 mg/day for DF-80. A comparison of these values with the corresponding values for the membranes of Examples 1 and 2 showed that the membrane of this Example had the highest rate of dissolution.
Thus, the rate of dissolution and, accordingly, the residence time of the membrane can be suitably controlled by using combinations of high and low molecular weight polymers of various compositions.
EXAMPLE 5
This Example illustrates the proportion of membranes produced from hylan A salts other than the sodium salt such as calcium, acetylcholine, and hexamethonium. The following example describes the procedure for the preparation of a calcium hylan membrane.
100 ml of a 1 % aqueous solution of sodium hylan similar to that used in Example 1 , were placed into a dialyzing tubing, OD 10mm (Spectra/Por® 4, Baxter) and dialyzed against 1 of 0.1N hydrochloric acid for 24 hours at 5°C, after which the acid was replaced by the same amount of fresh acid of the same concentration and the dialysis was continued for 72 hours in the cold room. Then the tubing with the acidic form of hylan was dialyzed against several changes of distilled water until the pH of the dialyzing fluid was about 7.0 to remove the excess free acid from the tubing. After that, dialysis was continued against an aqueous solution of calcium chloride containing 13.9 g per liter. The concentration of hylan and calcium chloride in corresponding solutions and the ratio of these two solutions in dialysis were such that about a 100-fold excess of Ca++ to polymer (in equivalent weights) was established during dialysis. The dialysis continued with H2O to remove excess calcium chloride, after which the film was prepared as described in Example 1.
EXAMPLE 6
This Example illustrates the preparation of a film made of a combination of a high molecular weight hylan A and heparin.
An aqueous solution of a high molecular weight hylan A (molecular weight of about 6xl06), concentration 2% was prepared and mixed in a weight ratio of 4 : 1 with a 2% solution of heparin. 10.0 g of the mixture were used to prepare a film as described in Example 1. Thus, the film contained by weight, 80% of high MW hylan and 20% of heparin. A strong film with an apparent thickness of 0.24 mm was obtained. The swelling rate was tested and compared to the hylan film described in Example 1. Two additional
mixtures of high MW hylan and heparin were prepared in a similar fashion. The ratios of these mixtures were 1: 1 and 1:4. : films.
EXAMPLE 7
This Example illustrates the preparation of a membrane made of water-insoluble hylan B (gel). 10.0 g of a gel slurry were used to prepare a film as described in Example 1. The gel can be used in the slurry form (homogenized onto the particles of any size) or in a large piece.
EXAMPLE 8
This Example illustrates a membrane made of mixtures of hylan A fluids and hylan B gels.
Three different mixtures of hylan A and hylan B were prepared in weight ratios of 1:4, 1:1 and 4:1. 10 cc of each mixture were loaded into a petri dish and processed as described in Example 1.
EXAMPLE 9 This example illustrates the preparation of a film from 1 % hylan A which is then imbibed with various concentrations of poly-lactic acid.
The procedure described in Example 1 was repeated with the exception that the films were not pressed. Four solutions of Lactide/Glycolide Polymer (hereinafter referred to as PLA) (DL5050, Medisorb Technologies International, LP) in tetrahydrofuran were prepared having final PLA concentrations of 0.1 %, 1.0%, 5%, and 10% weight percent. The PLA solutions were poured into separate petri dishes with covers to reduce evaporation. In the next step, two pieces of the membrane were dipped in each of the solutions and then dried while being suspended under a fume hood. The duration of the two steps were about 1 minute for dipping and about 1-4 hours for the drying. The other set of the films were pressed at 12,000 lbs for 1 minute at around room temperature, i.e. , 20 °C prior to dipping. The dipping and drying procedures were then carried out in a similar manner to the procedures for the non-pressed films. Both sets of the films were pressed again and tested for dissolution as described in Example 2. It was found that an increase in the concentration of PLA and pressing the film prior to dipping significantly affects the dissolution time of a film. It appears that the lowest concentration of PLA tested and dipping without prior pressing produces the slowest dissolving film.
EXAMPLE 10
This example illustrates the preparation of a film made of hylan A that was imbibed with various concentrations of poly-lactic acid and then pressed at different temperatures. The procedure described in Example 9 was repeated with the exception that the films were pressed at 20 °C and 80 °C. The dissolution time proved to be slightly longer for the films pressed at 80°C.
EXAMPLE 11
This example illustrates materials and methods for various test methods. Molecular weights of the samples were determined by PAGE and/or intrinsic viscosity tests. Seven physical characteristics of the test film formulations were measured as follows.
Thickness: A Micrometer was used to determine the thickness of the films. Using a pair of forceps, the thickness at the center of the membrane and at 4 random points were measured, for a total of 5 measurements on each film, and averaged.

Swell test: The amount of saline absorbed by a film over a certain period of time was determined as follows. Saline (0.9%, 15 ml) was added to each 60 mm petri dish containing a test sample. While the films were hydrating, their weight was recorded after 1, 5, 15, 30, 60, 90, 120, and 180 minutes. After each period, the saline was discarded and the weight of the sample was recorded. The percent swell is graphically represented as weight vs. time and shown in Fig. 2.
Tensile Strength and Strain at Failure: Tests were carried out with a Texture Analyzer (Model TA-Xti2, Texture Technologies Corp. , Stone Ridge, NY) connected to a PC. For the Tensile Strength test, film samples were clamped in the jaws of the testing device. The samples were torn at the rate 0.5 mm/sec. Each test produced 3 data points - percent elongation (change in length as percentage of the initial length), time required for breakage, and the distance that the upper clamp traveled at breakage.
For the Strain at Failure test, the film samples were secured in a way such that the middle portion of the film was accessible from above and below. The Texture Analyzer was programmed to push a 1/8-inch ball probe through the film at the rate of 0.5mm/sec. In the second part of the test, the film was wetted with 1 ml of saline and the procedure was repeated after a delay of 60 seconds. The data from the above mentioned tests are shown in Table 1 and Table 2.
EXAMPLE 12
Antineoplastic drug delivery from hylan films
The sustained delivery of antineoplastic drugs was investigated by making a solution of hylan A in water (20 mg / ml), lyophilizing the hylan fluid to create a sponge-like solid (300μl of fluid to create a sponge 1 cm2). Various antineoplastic drugs, i.e., carmustine, 5-fluorouracil and vinblastin were then loaded, in 400 mg doses into the sponge-like solid using appropriate buffers. The samples were then dried under vacuum and pressed for 30 seconds at 389 psi in a hydraulic press. As a control, Whatman filter paper, 1 cm2, was loaded with the drug in the same manner.
Hylan A films, which were loaded with drugs, as described above, were placed in vials filled with 10 ml of saline. A barrier between the films and the saline was created using a 0.45 μm filter. Samples were agitated to assure proper mixing. An 0.5ml sample of saline was removed for testing at 0.25, 0.5 1.0, 2.0, 4.0, 6.0, and 24 hours. Immediately after removal, the saline was replenished with fresh saline. The samples were analyzed by reading the optical density peak at the appropriate wavelength for each drug. Drugs loaded into the films consistently exhibited a significantly slower release rate as compared to control suggesting that the films may be used as a time release drug delivery system.



EXAMPLE 13
This example illustrates the results of using sterile hylan films in the rat cecal abrasion model.
36 female Sprague Dawley rats (225-250 g body weight) were used to evaluate the efficacy of hylan A films in preventing peritoneal adhesions. A separate group of 12 such rats was used as a control group. Surgically created defects were produced on the abdominal wall and the proximal end of the cecum of test and control rats. The 36 test rats received 1 cm2 hylan A film implants. Seven days after surgery the animals were sacrificed and evaluated for the presence of adhesions between the peritoneal defect and the cecal surface in both groups of rats. Of 36 rats that received the film, only 1 rat was found to have adhesion formation. Indices of adhesion formation in control animals were 67%. The difference between the occurrence of adhesion in animals with films and the control animals was significant at the level of p < 0.005.
EXAMPLE 14
This example illustrates the results of using hylan films in the rat liver abrasion model;
Sterile hylan A film was effective in preventing adhesion formation in the rat liver abrasion model. The films were found to adhere to the liver surface without the need for sutures. 92% percent of the animals did not develop adhesions (compare to 8% in the control group). The wound at the surface of the liver and on the mesenthelium were completely healed, indicating that hylan A membranes do not interfere with the wound healing process.
EXAMPLE 15
This example illustrates the results of using hylan A films in the rabbit uterine horn model.
The ability of hylan A film to reduce the development of postsurgical adhesions was investigated in this model. Hylan A film reduced the occurrence of adhesions compared with the control group (hylan A 19.8 + 4.3 % vs. control 46 + 5.9%). No adverse effects were observed with hylan A films.
EXAMPLE 16
This example illustrates the procedure for the preparation of the hylan B gel-hylan A fluid combined multilayered films.
Two different ways of producing multilayered films have been developed. One way to make a 3 -layered film (gel-fluid-gel or fluid-gel fluid), is to lyophilize each layer separately and then press them separately using a press with less than 5,000 lbs of pressure. Then, all three layers are arranged one atop the other in the desired sequence and pressed together at 12,000 lbs for 3 to 5 minutes. As a result, a number of layered films was produced with an average thickness of 0.44 mm for the gel-fluid-gel configuration and an average thickness of 0.50 mm for fluid-gel-fluid configuration.
The other way of accomplishing this is to skip the first pressing step and simply press all three layers together. Sandwiches made up of different glycosaminoglycan or hylan materials in various concentration may have applications preventing adhesions in specific types of surgery.