WO2015157681A1

WO2015157681A1 - Microparticles for wound treatment

Info

Publication number: WO2015157681A1
Application number: PCT/US2015/025389
Authority: WO
Inventors: Hobart W. HARRIS
Original assignee: Vitruvian Medical Devices Inc
Current assignee: Vitruvian Medical Devices Inc
Priority date: 2014-04-11
Filing date: 2015-04-10
Publication date: 2015-10-15
Anticipated expiration: 2016-10-11

Abstract

Disclosed herein are devices, methods, and compositions for treating a wound in a mammalian subject. Treating a wound involves administering to the mammalian subject a therapeutic amount of a composition. The composition comprises microparticles composed of a biocompatible material. In some embodiments, the composition also comprises a sterile fibrin sealant.

Description

TITLE

[0001] Microparticles for Wound Treatment BACKGROUND OF THE INVENTION

Field of the invention

[0002] The invention relates to devices, compositions, and methods for treating a wound in a mammalian subject. In particular, the invention relates to decreasing hernia recurrence and preventing incisional hernia formation.

Description of the Related Art

[0003] Ventral hernias are areas of weakness in the anterior abdominal wall muscle. A hernia is generally an abnormal protrusion or bulge of an organ through muscle or tissue in the body. There are several different types of ventral hernias, including inguinal (groin), umbilical, and incisional (post-surgical) hernias. Hernias of the anterior abdominal wall are extremely common and cause considerable patient discomfort and disability, resulting in more than 1.1 million reparative surgeries each year in the United States. Many patients experience complications with hernia repair surgeries, including chronic post-operative pain and recurrence of the hernia. In fact, hernia repair surgery fails in up to 40% of cases depending on the type of hernia, patient and surgical technique. Furthermore, incisional hernias complicate 10-15% of all open abdominal operations. Recurrent hernias and the formation of incisional hernias following abdominal operations are both caused by inadequate wound healing from an operation.

[0004] The current state of the art for repairing hernias involves placing a prosthetic material or mesh in the area of the hernia defect to reinforce the weakened muscle. Sutures or tacks are used to initially fix the mesh in place, and the body's healing reaction to the prosthetic material is responsible for the long-term durability of the repair. There are two basic types of hernia mesh, synthetic and biologic. Synthetic meshes are polymeric materials that can be further sub-divided into permanent versus absorbable (biodegradable) varieties. While permanent meshes remain in the patient's body forever, the absorbable meshes dissolve over time. Alternatively, biologic meshes are derived from collagen-rich tissues (including skin, intestinal submucosa, and pericardium) of cadavers, pigs, cows and horses. Synthetic prosthetics have the advantages of durability, low cost and over fifty years of clinical experience. However, they can also cause bowel obstructions, fistulas and chronic pain, and are not approved for use when operating in a contaminated field. In contrast, biologic meshes are made of naturally occurring proteins, promote the ingrowth of the patient's own cells, and are resistant to infection. Yet, there are concerns about the long-term efficacy of biologic meshes and they are 20-30 times more expensive than the synthetic alternatives. Recently, a novel method of preventing hernias by applying a mixture of silver particles and a fibrin sealant to sites of abdominal incision was disclosed in PCT/US2013/030213, incorporated herein in its entirety by reference. However, silver particles remain in the body permanently, and silver has been associated with certain non- life -threatening complications. There is a need for more varied, effective, and efficient methods of preventing and treating incisional hernias.

SUMMARY OF THE INVENTION

[0005] Disclosed herein are devices, methods, and compositions useful for treatment of a wound in a mammalian subject. In an embodiment, a device for administering a composition for treating a wound in a mammalian subject includes a cartridge containing microparticles composed of a biocompatible material, a cartridge containing thrombin, and a cartridge containing fibrinogen. In a further embodiment, the device also includes an applicator to combine the microparticles, thrombin, and fibrinogen to create a mixture that is administered to a site of abdominal incision in the mammalian subject. In some embodiments, the applicator combines the thrombin and fibrinogen into a pre -mixture, and combines the pre- mixture with the microparticles to create the mixture. In other embodiments, the cartridges and/or the applicator are sterilized by one of any number of methods, including gamma radiation, vapor heating, solvent treatment, or detergent treatment. In some embodiments, the microparticles, thrombin, and/or fibrinogen are sterilized by vapor, heating, solvent treatment, ethylene oxide treatment, or detergent treatment.

[0006] In an embodiment, the applicator includes a nozzle for delivering an aerosol spray. In another embodiment, the applicator includes a syringe. In another embodiment, the applicator is configured to deliver the composition by applying individual drops of the mixture to the site of abdominal incision. In a different embodiment, the applicator is configured to deliver the composition by applying a continuous stream of the mixture to the site of abdominal incision. In some embodiments, the composition is in the form of a spray, powder, liquid, gel, fibrin sheet, or solid strip. In other embodiments, the composition is formulated to be administered by subcutaneous injection, subcutaneous application, or topical administration. [0007] In some embodiments, the composition comprises any microparticles composed of a biocompatible material. In further embodiments, the biocompatible material is

polypropylene, polytetrafluoroethylene (PTFE), expanded PTFE (ePTFE), polyglycolic acid (PGA), polyglycolide-cotrimethylene carbonate (PGA-TMC), PGA-caprolactone, poly (lactic-co-glycolic) acid (PLGA), polyglactin 910, polydioxanone, poly-4-hydroxybutyrate (P4HB) or combinations thereof. In an embodiment, the composition is a combination of fibrinogen, thrombin, and microparticles comprising silver, polypropylene,

polymethylmethacrylate (PMMA), silver-coated PMMA, polylactic-co-glycolic acid (PLGA, 85/15), PLGA (porous), and poly-4-hydroxybutyrate (P4HB). In further embodiment, the biocompatible material is biodegradable.

[0008] In some embodiments, the microparticles are spherical, and each microparticle has a diameter between 5 and 1000 microns. In other embodiments, each microparticle is coated with silver or titanium. In other embodiments, each microparticle includes a hollow center.

[0009] In some embodiments, the composition includes a sterile fibrin sealant. In further embodiments, the sterile fibrin sealant includes thrombin and fibrinogen. In some embodiments, the composition is formulated as a pharmaceutical composition.

[0010] In another embodiment, a kit for treating a wound in a mammalian subject includes a sterile container containing microparticles composed of a biocompatible material, a sterile container containing a fibrin sealant, and instructions for administering the contents of the sterile container to a site of abdominal incision in a mammalian subject.

[0011] A method for treating a wound in a mammalian subject includes administering to the mammalian subject a therapeutic amount of the composition of microparticles composed of a biocompatible material. In an embodiment, the administering includes applying the composition to a site of abdominal incision in the mammalian subject. In another

embodiment, the method further comprises applying a hernia mesh to a site of abdominal incision in the mammalian subject. In some embodiments, the composition includes a sterile fibrin sealant, which may comprise thrombin and fibrinogen. In further embodiments, the sterile fibrin is in the form of a liquid, a powder, a lyophilized powder, or a frozen liquid. In some embodiments, the composition is administered as a dosage of 1 ml of fibrin sealant per 2 cm² surface area at the site of abdominal incision. BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

[0012] These and other features, aspects, and advantages of the present invention will become better understood with regard to the following description, and accompanying drawings, where:

[0013] FIG. 1 shows the incidence of anatomic hernias in rats treated with various microparticles, according to one embodiment.

[0014] FIG. 2 shows the anatomic hernia size in rats treated with various microparticles, according to one embodiment.

DETAILED DESCRIPTION OF THE INVENTION

[0015] Disclosed herein are devices, methods, and compositions for treating a wound in a mammalian subject. In some embodiments, these devices, methods, and compositions are used to improve the results of hernia repair surgeries and to prevent incisional hernias from occurring. These technologies are based on the idea that generating increased native tissue will improve wound healing and decrease initial and recurrent incisional hernia formation.

[0016] In the Examples, the effects of applying biodegradable and inert microparticles to a subject for myofascial wound healing are analyzed. In an embodiment, the analyzed effects include tensile strength of the repair, and hernia formation. The Examples demonstrate that microparticles can be used to prevent and treat incisional hernias. Administering

microparticles to sites of abdominal incisions is a novel approach for treating wounds.

Definitions

[0017] Terms used in the claims and specification are defined as set forth below unless otherwise specified.

[0018] The term "hernia" refers to a protrusion or bulge of an organ or the fascia of an organ that projects from the normal contours of the body. The term "incisional hernia" refers to a hernia defined by a protrusion of the abdomen, generally caused by inadequate healing of a surgical wound.

[0019] The term "microparticle" refers to particles that are generally (but not limited to) between 0.1 and 100 μιη in diameter, composed of any of a variety of materials. In some embodiments, microparticles are between 0.1 and 1000 μιη in diameter. In other

embodiments, microparticles are smaller than 0.1 μιη in diameter or larger than 1000 μιη in diameter. In other embodiments, microparticles are between 100 and 250 μιη in diameter. In some embodiments, microparticles are spherical in shape. In other embodiments, microparticles are oblong in shape. In some embodiments, microparticles do not comprise metals.

[0020] The term "biocompatible" refers to a compatibility of a particular material with a living host. A living host can include a cell line, a tissue sample, or an organism. In an embodiment, biocompatibility means that the material is not toxic to the host.

[0021] The term "ameliorating" refers to any therapeutically beneficial result in the treatment of a disease state, e.g., a wound, an incisional hernia, or other disease state, including prophylaxis, lessening in the severity or progression, remission, or cure thereof.

[0022] The term "mammal" as used herein includes both humans and non-humans and includes but is not limited to humans, non-human primates, canines, felines, murines, bovines, equines, and porcines. The term "patient" or "subject" refers to a cell, tissue, or organism, human or non-human, whether in vivo, ex vivo, or in vitro, male or female. The patient or subject can be a human patient or a non-human.

[0023] The term "sufficient amount" means an amount sufficient to produce a desired effect, e.g., an amount sufficient to modulate protein aggregation in a cell.

[0024] The term "therapeutically effective amount" is an amount that is effective to prevent or ameliorate a wound, an incisional hernia, or a symptom of a disease. A therapeutically effective amount can be a "prophylactically effective amount" as prophylaxis can be considered therapy.

[0025] The term "treatment" refers to a method of ameliorating, healing, or preventing a condition in a subject.

[0026] It must be noted that, as used in the specification and the appended claims, the singular forms "a," "an" and "the" include plural referents unless the context clearly dictates otherwise.

Compositions

[0027] Disclosed herein are compositions for treating a wound in a mammalian subject, the composition comprising microparticles composed of a biocompatible material. In some embodiments, the composition includes bioactive microparticles combined with a fibrin- based tissue sealant.

[0028] The microparticles can be composed of any biocompatible material. In an

embodiment, the biocompatible material is biodegradable or completely resorbable. In some embodiments, permanent microparticles include polypropylene (Prolene),

polytetrafluoroethylene (PTFE), and expanded PTFE (ePTFE). In other embodiments, biodegradable microparticles include polyglycolide-cotrimethylene carbonate (PGA-TMC) (example ratios including but not limited to 67/33 and 75/25), polyglycolic acid (PGA), PGA-caprolactone, PGA-poly (lactic-co-glycolic) acid aka poly lactide-co-glycolide;

poly(lactic-co-glycolic acid) PLGA ( example ratios including but not limited to 85/15, 75/25 and 50/50), polyglactin 910, polydioxanone (poly-/?-dioxanone), and poly-4-hydroxybutyrate (P4HB) (PolyMicrospheres, division of Vasmo, Inc. (Indianapolis, IN)). In some

embodiments, the microparticles are composed of a combination of any of the above materials.

[0029] In one embodiment, the microparticles are spherical. In a further embodiment, each microparticle has a diameter between 0.1-1000 μιη. In other embodiments, each microparticle has a diameter between 5-1000 μιη, 0.1-500 μιη, 100-250 μιη, or 10-100 μιη. In some embodiments, the microparticles are coated with a metal, such as silver or titanium. In other embodiments, the microparticles have a hollow center.

[0030] In one embodiment, the microparticles are combined with a sterile fibrin sealant. In an embodiment, the microparticles are present in the composition at a concentration between 0- 2500 mg/mL of sterile fibrin sealant. In further embodiments, the microparticles are present at a concentration of 25, 50, 75, 100, 200, 250, 300, 400, 500, 1000, 1500, 2000, or 2500 mg/mL of sterile fibrin sealant. In one embodiment, the fibrin sealant is composed of thrombin and fibrinogen (TISSEEL and ARTISS, Baxter (Deerfield, IL); EVICEL, Ethicon BioSurgery). Fibrinogen is a protein that forms a clot when combined with thrombin.

Thrombin is a specific protease that transforms fibrinogen into fibrin.

[0031] The fibrin sealant can be sterilized by one of a variety of methods, including but not limited to, a vapor heating method, a solvent treatment process, or a detergent treatment process. The fibrin sealant can be prepared in a liquid form (such as a gel), or powder form, including lyophilized powder or frozen liquid. In some embodiments, the fibrin sealant is combined with the microparticles prior to application of the resulting composition to a site of abdominal incision. In other embodiments, the fibrin sealant is applied to the site of abdominal incision prior to application of the microparticles, or the microparticles are applied to the incision site before the fibrin sealant is applied. In embodiments wherein the fibrin sealant comprises thrombin and fibrinogen, the thrombin and fibrinogen can be combined and mixed before further combination with the microparticles, all prior to application of the resulting composition to a site of abdominal incision. In other embodiments, the thrombin, fibrinogen, and microparticles are all combined and mixed simultaneously prior to application of the resulting composition to the abdominal incision site. In other embodiments, the microparticles are combined with the thrombin before subsequent combination with the fibrinogen, or the microparticles are combined with the fibrinogen before subsequent combination with the thrombin.

[0032] In an embodiment, the fibrin sealant is manufactured from pooled human plasma and provided as a single use kit including two packages. The first package contains one vial of Sealant Protein X (SPX) and one vial of Thrombin. The second package contains a sterile spray application device and Microparticle Applicator Cartridge. In an embodiment, the two components (SPX and Thrombin) are generally mixed and applied topically. In a further embodiment, the SPX and Thrombin components appear as white to slightly yellowish opaque masses when frozen and as clear to slightly opalescent and colorless to slightly yellowish solutions when thawed. In one embodiment, the components contain no

preservatives.

[0033] In an embodiment, SPX is provided as a sterile solution, pH 6.7-7.2, which mainly includes a concentrate of human fibrinogen. In a further embodiment, SPX is composed of any combination of the following active ingredients: concentrate of human fibrinogen (65- 105 mg/ml), aprotinin (synthetic, 2250-3750 KlU/ml), and Factor XIII (45-70 μg/ml), and other ingredients: human albumin, histidine, tri-sodium citrate, niacinamide, polysorbate 80, and water for injection. In some embodiments, SPX does not include Factor XIII.

[0034] In an embodiment, thrombin is provided as a sterile solution, pH 6.8-7.2, which contains purified human thrombin that activates clotting of the final combined product. In a further embodiment, thrombin is composed of any combination of the following active ingredients: human thrombin (500-625 IU/ml), and calcium chrloride (36-44 μιηοΐ/ml), and other ingredients: human albumin, mannitol, sodium chloride and water for injection.

[0035] In one embodiment, cryoprecipitate (the starting material for SPX), and cryo-poor plasma (the starting material for the production of thrombin) are both made from pooled human plasma obtained from US licensed plasma collection centers. In a further embodiment, SPX is manufactured from pooled Human Source plasma and thrombin is manufactured from pooled Human Source or recovered plasma.

[0036] The compositions of microparticles described herein can be formulated in

pharmaceutical compositions. These compositions can comprise, in addition to one or more of the microparticles, a pharmaceutically acceptable excipient, carrier, buffer, stabiliser or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other material can depend on the route of administration, e.g. oral, intravenous, cutaneous or subcutaneous, nasal, intramuscular, intraperitoneal routes.

[0037] The pharmaceutical compositions for administration can be in solid or liquid form. Liquid pharmaceutical compositions generally include a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline solution, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol can be included. In some embodiments, the pharmaceutical composition is in the form of a spray, powder, liquid, gel, fibrin sheet, or solid strip. In an embodiment, the pharmaceutical composition is formulated to be administered by topical administration or subcutaneous injection.

[0038] For intravenous, cutaneous or subcutaneous injection, injection at the site of affliction, or topical administration, the active ingredient can be in the form of a parenterally acceptable aqueous solution which is pyrogen- free and has suitable pH, isotonicity and stability. Those of relevant skill in the art are well able to prepare suitable solutions using, for example, isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection, Lactated Ringer's Injection. Preservatives, stabilisers, buffers, antioxidants and/or other additives can be included, as required.

[0039] Administration is preferably in a "therapeutically effective amount" or

"prophylactically effective amount" (as the case can be, although prophylaxis can be considered therapy), this being sufficient to show benefit to the individual. The actual amount administered, and rate and time-course of administration, will depend on the nature and severity of the wound or hernia being treated. Prescription of treatment, e.g. decisions on dosage etc., is within the responsibility of general practitioners and other medical doctors, and typically takes account of the disorder to be treated, the condition of the individual patient, the site of delivery, the method of administration and other factors known to practitioners. Examples of the techniques and protocols mentioned above can be found in Remington's Pharmaceutical Sciences, 16th edition, Osol, A. (ed), 1980.

[0040] A composition can be administered alone or in combination with other treatments, either simultaneously or sequentially dependent upon the condition to be treated.

Methods

[0041] Disclosed herein are methods for treating a wound in a mammalian subject, the method comprising administering to the mammalian subject a therapeutic amount of the composition described above (including a pharmaceutical composition), the composition comprising microparticles composed of a biocompatible material. In an embodiment, this composition is administered as an adjuvant to current hernia repair surgical techniques and materials. For example, the composition can be administered to treat or prevent an incisional hernia. In another embodiment, the composition further comprises the fibrin sealant as described above.

[0042] In some embodiments, the composition is administered by applying the composition to a site of abdominal incision in the mammalian subject. The method of administration can be via any application method, including applying the composition by dripping or continuous stream. In some embodiments, the composition is applied to a surface area of 1 cm², 2 cm², 3 cm², 4 cm², 5 cm², 6 cm², 7 cm², 8 cm², 9 cm², 10, cm² 11 cm², 12 cm², 13 cm², 14 cm², or 15 cm² or more. In an embodiment, the composition is administered at a dosage ratio of 1 ml of fibrin sealant per 2 cm² surface area at the site of abdominal incision. In further

embodiments, the composition is administered at a dosage ratio of 1 ml fibrin sealant per 10 cm² surface area.

[0043] In some embodiments, the composition is applied by dripping, and the tip of the applicator is kept as close to the tissue surface as possible without touching the tissue during application. Individual drops of the composition are applied to the incision, creating a 2 cm- wide band. The drops are allowed to separate from each other and from the tip of the applicator.

[0044] In other embodiments, the composition is applied by a continuous stream, and the tip of the applicator is kept approximately 1-2 cm above the tissue surface during application. A slow and continuous stream of the composition is applied to the incision following a zig-zag path to create a 2 cm-wide band of material.

[0045] In some examples, the composition is an adjuvant to current surgical techniques for closing the abdominal wall following open surgery. The composition is applied to the surface of the re-approximated myofascial incision after suture closure is complete. In some embodiments, the composition is not injected into the peritoneal cavity. In another embodiment, the composition is administered by dripping in short bursts (0.1-0.2 ml) or as a continuous stream onto the sutured myofascial incision to produce an even layer that covers the entire incision. Standard surgical techniques are used for closing the myofascial layer of the abdominal wall, including placing a permanent or absorbable suture using a continuous or interrupted technique, prior to the application of the composition. In further embodiments, excess fluid is removed from the site of application.

[0046] In some embodiments, a hernia mesh is affixed to the site of abdominal incision at the time the composition is applied. Once the hernia mesh has been affixed, the composition can be applied in a manner that coats the local surgical field including the hernia mesh, prior to wound closure. In some embodiments, the amount of the composition administered is based on the surface area to be covered.

Devices and Kits

[0047] Disclosed herein are devices and kits for administering the compositions described above to treat a wound in a mammalian subject.

[0048] In some embodiments, the device includes at least: a cartridge containing

microparticles composed of a biocompatible material, and a cartridge containing a fibrin sealant. In further embodiments, the device also includes a cartridge containing thrombin, a cartridge containing fibrinogen. In further embodiments, the device also includes an applicator configured to combine the microparticles, thrombin, and fibrinogen to create a mixture, and the applicator is also configured to deliver the mixture to a site of abdominal incision in a subject. In some embodiments, any or all of the cartridges, applicator, microparticles, fibrinogen, and thrombin are sterilized before use. Sterilization can be performed by using any sterilization technique, including gamma radiation, ethylene oxide treatment, vapor heating, solvent treatment, and detergent treatment.

[0049] In certain embodiments, the applicator is configured to combine the thrombin and fibrinogen into a pre-mixture before combining the pre-mixture with the microparticles to create the mixture for administration. In other embodiments, the applicator is configured to deliver the composition by applying individual drops of the mixture to the site of abdominal incision, or deliver the composition by applying a continuous stream of the mixture to the site of abdominal incision.

[0050] In some embodiments, the applicator is a syringe. The barrel portion (cartridge) of the syringe device can be a single, double, or triple barrel, such that a plunger is configured to push material (such as microparticles, thrombin, and fibrinogen) through one, two, or three barrels simultaneously, or in a specific order. In these embodiments, as material is pushed through the barrel or barrels, some or all of the material within the barrels can be combined and mixed together before being administered by the syringe. In certain embodiments, the microparticles can be contained in one cartridge, while the fibrin sealant (thrombin and fibrinogen) is contained in another cartridge. The plunger is configured to push these materials through the barrels, and the microparticles and fibrin sealant are combined before being pushed out of the device to be applied to the site of abdominal incision.

[0051] In other embodiments, the syringe is configured to push material from the barrel or barrels into a separate cartridge that holds another material, causing all of the material to be mixed before being delivered to an incision site. For example, the syringe can be configured to push thrombin and fibrinogen, both in different barrels, into a cartridge containing microparticles. When the thrombin and fibrinogen are pushed into the cartridge containing microparticles, the thrombin, fibrinogen, and microparticles are all mixed and combined in the cartridge to form a composition, and the plunger is further configured to push the composition out of the device to apply to the abdominal incision site.

[0052] In embodiments, the applicator is a nozzle that is configured to deliver the composition by an aerosol spray. In further embodiments, the cartridges of the nozzle device are configured to combine and mix the microparticles, thrombin, and fibrinogen in various combinations and order prior to administering the aerosol spray to a site of abdominal incision. For example, the cartridges of the nozzle device can be configured to combine thrombin and fibrinogen into a mixture, and subsequently add microparticles to the mixture. Or, the microparticles, fibrinogen, and thrombin can all be mixed simultaneously.

[0053] In one embodiment, the kit for administering a composition for treating a wound in a mammalian subject includes at least: a sterile container containing microparticles composed of a biocompatible material, a sterile container containing a fibrin sealant, and instructions for administering the contents of the sterile container to a site of abdominal incision in a mammalian subject. In a further embodiment, the thrombin and fibrinogen components of the fibrin sealant are packaged separately in sterile containers. In some embodiments, the kit includes an applicator device for administering the composition. In a further embodiment, the applicator device includes a syringe or a nozzle.

EXAMPLES

[0054] Below are examples of specific embodiments for carrying out the present invention. The examples are offered for illustrative purposes only, and are not intended to limit the scope of the present invention in any way. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should, of course, be allowed for. [0055] Example 1 shows the analysis of different dose-response relationships in order to determine the optimal dose of microparticles in a rodent model of ventral abdominal wall incision healing and strength, Example 2 focuses on analyzing the impact of microparticles on treating incisional hernias in rats, and Example 3 shows the impact of varying the types of microparticles used to reduce the incidence of incisional hernias from forming.

[0056] The practice of the present invention will employ, unless otherwise indicated, conventional methods of protein chemistry, biochemistry, recombinant DNA techniques and pharmacology, within the skill of the art. Such techniques are explained fully in the literature. See, e.g., T.E. Creighton, Proteins: Structures and Molecular Properties (W.H. Freeman and Company, 1993); A.L. Lehninger, Biochemistry (Worth Publishers, Inc., current addition); Sambrook, et al, Molecular Cloning: A Laboratory Manual (2nd Edition, 1989); Methods In Enzymology (S. Colowick and N. Kaplan eds., Academic Press, Inc.);

Remington's Pharmaceutical Sciences, 18th Edition (Easton, Pennsylvania: Mack Publishing Company, 1990); Carey and Sundberg Advanced Organic Chemistry 3^rd Ed. (Plenum Press) Vols A and B(1992).

Example 1: Ventral abdominal wall incision healing and strength analysis

[0057] This Example describes a method of determining the optimal dose of microparticles to augment myofascial wound healing as measured by tensile strength of the incision and incisional hernia formation. Treatment doses reflect the volume of synthetic mesh products currently in use. Four concentrations of microparticles; 0, 25, 250 and 2,500 mg/ml fibrin tissue sealant are analyzed. Sprague-Dawley rats (male, 250-300 g) undergo a modified incisional hernia model. Specifically, the animals are placed under isoflurane anesthesia, the ventral abdominal wall hair shaved with electric clippers and the surgical field prepared with 70% alcohol. A ventral abdominal wall incisional hernia is produced in a rat by making a full-thickness, 3-cm x 6-cm paramedian skin incision that has been raised through the avascular, prefascial plane, thereby separating the skin incision from the underlying fascial wound-healing environment. The 1 :2 ratio of flap length to width is maintained to prevent ischemia of the skin flap. After the 5 -cm full-thickness midline laparotomy incision is made, the intestines are briefly manipulated and the myofascial incision is closed with two interrupted 5-0 plain catgut sutures placed 5 mm from the cut myofascial edges and -1.5 cm from the cranial and caudal ends of the midline laparotomy incision. The incision can also be closed with a continuous 4-0 vicryl suture, or two rapidly absorbing 5-0 plain catgut sutures placed -1.5 cm from either end of the incision. The skin flap is then closed using a running 4-0 vicryl suture. A 5-cm, full-thickness, midline laparotomy incision is made.

[0058] Animals in the treatment groups have 0 - 2,500 mg microparticles/ml of sterile fibrin tissue sealant (TISSEEL®, Baxter Healthcare Corp., Hayward, CA) topically applied to their sutured myofascial incisions before skin closure. The treatment is administered by volume per surface area; i.e. 1 ml/10 cm² or 0.5 ml/5-cm myofascial incision, covering a total surface area of 5 cm². The skin flap is then closed with a continuous 4-0 vicryl suture to prevent intestinal evisceration. Animals in the control group have an equal volume of sterile saline (0.5 ml) applied to their sutured myofascial incisions before skin closure.

[0059] Immediately post-op, 0.4 ml of bupivacaine 0.25% is infused subcutaneously around the abdominal incision. The rats are observed every 2 minutes until awake and resuming normal activity; rats are then returned to individual cages and monitored twice daily. If the rat fails to display appropriate postoperative progression (fails to respond to tactile stimulus, displays moribund behavior or has overt bleeding) between 2 minute intervals, it is euthanized immediately. At 12 and 18 hours post-operation, 0.05 mg/kg buprenorphine is injected subcutaneously.

[0060] On day 28 all animals are euthanized and the abdominal wall muscle analyzed as detailed below. In addition, the number of animals who develop hernias, as defined as separation of the fascia- fascia interface >2 mm is recorded and the total area of their hernias (fascial borders traced and area determined via NIH software) measured. An additional section of tissue that includes the wound-healing interface along with normal adjacent tissue is taken for histology. Finally, biopsies are taken of the fascia-fascia interface and immediately snap frozen in liquid nitrogen for biochemical analyses.

[0061] Tensiometric analysis. Mechanical testing of the abdominal wall fascial strips is performed within 6 h of necropsy. Stretch loading is used to facilitate mechanical characterization of the fascia-fascia interface using an Instron Tensiometer (model

MicroTester®; Instron Corporation, Canton, MA), including measures of yield strength, yield energy, yield extension, stiffness, and force extension curves for each fascial strip. The fascial strips are mounted into the load frame via pneumatic graspers, preloaded to 0.1 Newtons with the gauge length measured between the grips. The load frame applies testing loads to the fascial strips until mechanical tissue disruption occurs. The anatomic location of the tissue break is noted for each specimen. Force and tissue deformation data are simultaneously captured via computer and data analysis performed using Bluehill® Software (Instron Corporation, Canton, MA). Failure of the specimen is defined at the yield point, rather than at the point of ultimate tissue disruption.

[0062] Histology. Fresh biopsies of the abdominal wall fascia-fascia interface are fixed in formalin, embedded in paraffin, sectioned, and stained with hematoxylin and eosin or trichrome. Tissue sections are analyzed by two independent pathologists blinded to the various treatment groups. A stronger tissue sample is an indication that an optimal concentration of microparticles was used for that sample.

[0063] Analysis of experimental results identifies optimal microparticle dose ranges for use in the present invention.

Example 2: Treating incisional hernias

[0064] This Example describes a method of determining the impact of microparticles on treating incisional hernias in rats using the optimal dose determined in Example 1. Sprague- Dawley rats (male, 250-300 g) undergo the modified incisional hernia model described above anticipating that >85% of animals treated with saline (controls) will develop hernias within 28 days.

[0065] On day 28 those animals with detectable incisional hernias are placed under isoflurane anesthesia for a second time, the ventral abdominal wall shaved and prepared with 70% alcohol, the previous skin incision re-opened, the acquired hernia sac surgically resected, and the fascia defect measured before the edges of the hernia defect re- approximated with 2 interrupted 5-0 plain catgut sutures placed 5 mm from the cut myofascial edges and -1.5 cm from the cranial and caudal ends of the midline laparotomy incision, respectively. Immediately after the hernia is repaired, the animals are randomly assigned to a treatment group versus a sham control group. Rats in the treatment groups have their fascial closure (hernia repair) reinforced with either synthetic mesh or microparticles. To reinforce the hernia repair with mesh, a 5 cm x 1 cm strip of mesh is centered over the fascial incision and attached using 0.5 ml fibrin sealant before closing the skin with a continuous 4-0 vicryl suture. To reinforce the hernia repair with microparticles, the optimal dose of microparticles dispersed in fibrin sealant (0.5 ml/5-cm myofascial incision) is applied to the fascial repair before closing the skin with a continuous 4-0 vicryl suture. The strips used to reinforce the hernia repair include absorbable synthetic meshes (Vicryl mesh, Ethicon Inc., Somerville, NJ; BioA™ mesh, W.L. Gore & Associates, Inc., Flagstaff, AZ), (Phasix™ mesh, C.R. Bard, Inc. [Davol], Warwick, RI and permanent, synthetic meshes (Ultrapro™, Ethicon; plain prolene mesh, Bard [Davol]). The particles used to reinforce the hernia repair include biodegradable microparticles [polyglycolic acid (PGA), polyglactin 910, polyglycolide-cotrimethylene carbonate (PGA-TMC, 67/33 and 75/25), poly (lactic-co- glycolic) acid (PLGA 85/15, 75/25 and 50/50), PGA-caprolactone and polydioxanone] and inert microparticles (polypropylene, titanium, PTFE, ePTFE, and PMMA). Animals in the sham control group have an equal volume of sterile saline (0.5 ml) applied to their hernia repairs.

[0066] On day 56 (28 days after repair of the incisional hernia) all animals are euthanized and the abdominal wall muscle analyzed as detailed in Example 1 for hernia recurrence, tensile strength and histology.

[0067] Statistical analysis. The Student t test is used to determine differences in tensiometric mechanical measurements. The Fischer exact test is used to determine differences in the incidence of incisional hernias. Values are reported as the mean ± standard deviation. P values of < 0.05 are considered significant. The results show that using microparticles to treat incisional hernias is at least as successful as synthetic mesh for wound healing.

Example 3: Microparticles reduce the incidence of incisional hernias

[0068] This Example describes a method of determining the impact of microparticles on reducing the incidence of incisional hernia formation in rats by using varying microparticles. Sprague-Dawley rats (male, 250-300 g) underwent an incisional hernia model. The animals were placed under isoflurane anesthesia, the ventral abdominal wall hair was shaved with electric clippers, and the surgical field was prepared with chlorhexidine or betadine. A 3-cm, full-thickness, upper (epigastric) midline ventral skin incision was made, followed by a 5-cm midline laparotomy incision through which the intestines were briefly manipulated before the myofascial incision was closed with two interrupted 5-0 plain catgut sutures placed 5 mm from the cut myofascial edges and approximately 1.5 cm from the cranial and caudal ends of the midline laparotomy incision, respectively.

[0069] Ten (10) animals were randomly assigned to each of eight treatment groups and a sham control group before closing the skin flap with a continuous 3-0/4-0 prolene/vicryl suture to prevent intestinal evisceration. Animals in the treatment groups received a dose of microparticle composition with a total particle surface area of 11.44 cm² mixed within a fibrin tissue sealant (1 ml/10 cm² or 0.5 ml/5-cm myofascial incision) topically applied to their sutured myofascial incisions before skin closure using a subcuticular suture. The microparticle composition included a combination of fibrinogen, thrombin, and

microparticles including silver, polypropylene, polymethylmethacrylate (PMMA), silver- coated PMMA, polylactic-co-glycolide (PLGA, 85/15), PLGA (porous), and poly-4- hydroxybutyrate (P4HB). The total particle surface area of 11.44 cm² is the equivalent of using 500 mg/kg or 250 mg/ml of 250 μ silver microparticles. Animals in the sham control group received an equal volume of sterile saline (0.5 ml) applied to their sutured myofascial incisions before skin closure using a subcuticular suture. The tissue sealant (Tisseel ®, Baxter Biosurgery, Deerfield, IL) was mixed with apyrogenic microparticles to create final delivered concentrations as listed in Table 1 (below). After 28 days (+/- 3 days), the animals in each group were inspected for a visible hernia, and euthanized while under anesthesia. The abdominal wall was excised and the abdominal wall muscle was evaluated for a hernia defect (anatomic hernia). Samples from the wound-healing interface were fixed in formalin and paraffin-embedded in preparation for histology. The results showed that using microparticles in combination with fibrin tissue sealant is a feasible approach to preventing incisional hernias.

[0070] Table 1: Microparticle compositions and dosing

[0071] Various biodegradable and permanent microparticles can reduce the incidence of incisional hernias. Figure 1 shows the incidence of anatomic hernias in rats treated with various microparticles. Fisher's exact test was performed. * denotes a p value of 0.0004 between control and silver microparticles. There was no statistically significant difference between control and the other microparticles tested, however important trends were observed. P values were: control versus polypropylene 230 micron: 0.3034, control versus

polypropylene 110 micron: 0.1409, control versus PLGA 85/15: 0.582, control versus PLGA porous: 0.3034, control versus P4HB: 1.0, control versus PMMA: 0.582, and control versus silver-PMMA: 0.582. Silver microparticles were found to be extremely effective at reducing the incidence of anatomic hernias.

[0072] Figure 2 shows the anatomic hernia size in rats treated with various microparticles. Hernia sizes of various treatment groups were compared. A Kruskal-Wallis test was performed, with a p value of 0.0654. Dunn's post-test demonstrated a significant difference between control versus silver, * denotes a p value of <0.05. It was concluded that all of the hernias formed were essentially of the same size.

[0073] While the invention has been particularly shown and described with reference to a preferred embodiment and various alternate embodiments, it will be understood by persons skilled in the relevant art that various changes in form and details can be made therein without departing from the spirit and scope of the invention.

[0074] All references, issued patents and patent applications cited within the body of the instant specification are hereby incorporated by reference in their entirety, for all purposes.

Claims

1. A device for administering a composition for treating a wound in a mammalian subject, the device comprising:

a cartridge containing microparticles composed of a biocompatible material; a cartridge containing thrombin; a cartridge containing fibrinogen; and an applicator configured to combine the microparticles, thrombin, and fibrinogen to create a mixture, and the applicator further configured to deliver the mixture to a site of abdominal incision in the mammalian subject.

2. The device of claim 1, wherein the applicator comprises a syringe.

3. The device of claim 1, wherein the applicator comprises a nozzle configured to deliver an aerosol spray.

4. The device of any of claims 1-3, wherein the applicator is further configured to combine the thrombin and fibrinogen into a pre-mixture, and wherein the applicator is further configured to combine the pre-mixture with the microparticles to create the mixture.

5. The device of any of claims 1-4, wherein the applicator is further configured to deliver the composition by applying individual drops of the mixture to the site of abdominal incision in the mammalian subject.

6. The device of any of claims 1-4, wherein the applicator is further configured to deliver the composition by applying a continuous stream of the mixture to the site of abdominal incision in the mammalian subject.

7. The device of any of claims 1-6, wherein the biocompatible material is

polypropylene, polytetrafluoroethylene (PTFE), expanded PTFE (ePTFE), polyglycolic acid (PGA), polyglycolide-cotrimethylene carbonate (PGA-TMC), PGA-caprolactone, poly (lactic-co-glycolic) acid (PLGA), polyglactin 910, polydioxanone, poly-4-hydroxybutyrate (P4HB) or combinations thereof.

8. The device of any of claims 1-7, wherein the biocompatible material is biodegradable.

9. The device of any of claims 1-8, wherein each microparticle is spherical, and wherein each microparticle has a diameter between 0.1 and 1000 microns.

10. The device of any of claims 1-9, wherein each microparticle is coated with silver or titanium.

11. The device of any of claims 1-10, wherein each microparticle comprises a hollow center.

12. The device of any of claims 1-11, wherein the composition comprises a spray, powder, liquid, gel, fibrin sheet, or solid strip.

13. The device of any of claims 1-12, wherein the composition is formulated to be administered by subcutaneous injection or topical administration.

14. The device of any of claims 1-13, wherein any of the cartridges or the applicator is sterilized.

15. The device of claim 14, wherein the sterilization involves gamma radiation, vapor heating, solvent treatment, or detergent treatment.

16. The device of any of claims 1-15, wherein any of the microparticles, thrombin, or fibrinogen is sterilized by vapor heating, solvent treatment, ethylene oxide treatment, or detergent treatment.

17. A method for treating a wound in a mammalian subject, the method comprising administering to the mammalian subject a therapeutic amount of a composition, the composition comprising microparticles composed of a biocompatible material.

18. The method of claim 17, wherein the administering comprises applying the composition to a site of abdominal incision in the mammalian subject.

19. The method of any of claims 17-18, wherein the method further comprises applying a hernia mesh to a site of abdominal incision in the mammalian subject.

20. The method of any of claims 17-19, wherein the administering comprises applying individual drops of the composition to a site of abdominal incision in the mammalian subject.

21. The method of any of claims 17-19, wherein the administering comprises applying a continuous stream of the composition to a site of abdominal incision in the mammalian subject.

22. The method of any of claims 17-21, wherein the composition is administered to treat or prevent an incisional hernia.

23. The method of any of claims 17-22, wherein the biocompatible material is polypropyelene, polytetrafluoroethylene (PTFE), expanded PTFE (ePTFE), polyglycolic acid (PGA), polyglycolide-cotrimethylene carbonate (PGA-TMC), PGA-caprolactone, PGA-poly (lactic-co-glycolic) acid (PLGA), polyglactin 910, polydioxanone, poly-4-hydroxybutyrate (P4HB) or combinations thereof.

24. The method of any of claims 17-23, wherein the biocompatible material is biodegradable.

25. The method of any of claims 17-24, wherein each microparticle is spherical, and wherein each microparticle has a diameter between 5 and 1000 microns.

26. The method of any of claims 17-25, wherein each microparticle is coated with silver or titanium.

27. The method of any of claims 17-26, wherein each microparticle comprises a hollow center.

28. The method of any of claims 17-35, wherein the composition comprises a spray, powder, liquid, gel, fibrin sheet, or solid strip.

29. The method of any of claims 17-28, wherein the composition is formulated to be administered by subcutaneous injection or topical administration.

30. The method of any of claims 17-29, wherein the composition further comprises a sterile fibrin sealant.

31. The method of claim 30, wherein the sterile fibrin sealant comprises thrombin and fibrinogen.

32. The method of any of claims 30-31 , wherein the fibrin sealant is sterilized by a vapor heating method.

33. The method of any of claims 30-31 , wherein the fibrin sealant is sterilized by a solvent treatment process or a detergent treatment process.

34. The method of any of claims 17-33, wherein the microparticles are sterilized by ethylene oxide treatment.

35. The method of any of claims 30-33, wherein the sterile fibrin sealant is a liquid, a powder, a lyophilized powder, or a frozen liquid.

36. The method of any of claims 17-27, wherein the composition is administered as a dosage of 1 ml of fibrin sealant per 2 cm² surface area at the site of abdominal incision.

37. A kit for treating a wound in a mammalian subject, comprising:

a sterile container containing microparticles composed of a biocompatible

material;

a sterile container containing a fibrin sealant; and

instructions for administering the contents of the sterile container to a site of abdominal incision in a mammalian subject.

38. A composition for treating a wound in a mammalian subject, the composition comprising microparticles composed of a biocompatible material.

39. The composition of claim 38, wherein the biocompatible material is polypropyelene, polytetrafluoroethylene (PTFE), expanded PTFE (ePTFE), polyglycolic acid (PGA), polyglycolide-cotrimethylene carbonate (PGA-TMC), PGA-caprolactone, PGA-poly (lactic- co-glycolic) acid (PLGA), polyglactin 910, polydioxanone, poly-4-hydroxybutyrate (P4HB) or combinations thereof.

40. The composition of any of claims 38-39, wherein the biocompatible material is biodegradable.

41. The composition of any of claims 38-40, wherein each microparticle is spherical, and wherein each microparticle has a diameter between 5 and 1000 microns.

42. The composition of any of claims 38-41, wherein each microparticle is coated with silver or titanium.

43. The composition of any of claims 38-42, wherein each microparticle comprises a hollow center.

44. The composition of any of claims 38-43, wherein the composition further comprises a sterile fibrin sealant.

45. The composition of claim 44, wherein the sterile fibrin sealant comprises thrombin and fibrinogen.

46. The composition of any of claims 44-45, wherein the sterile fibrin sealant is a liquid, a powder, a lyophilized powder, or a frozen liquid.

47. A pharmaceutical composition, comprising a therapeutically effective amount of the composition of any of claims 38-46.

48. The pharmaceutical composition of claim 47, wherein the composition is

administered as a dosage of 1 ml of fibrin sealant per 2 cm² surface area at the site of abdominal incision.

49. The pharmaceutical composition of claim 47, wherein the pharmaceutical

composition comprises a spray, powder, liquid, gel, fibrin sheet, or solid strip.

50. The pharmaceutical composition of any of claims 47-49, wherein the pharmaceutical composition is formulated to be administered by subcutaneous injection or topical administration.