The present invention relates to the use of plasminogen and plasmin as agents for enhancing healing of tympanic membrane perforations or other wounds, and for reducing scars or necrotic tissue forming during wound healing. The invention also relates to a method for screening of compounds which enhance wound healing by evaluating the healing of tympanic membrane perforations in an animal model.

SUMMARY OF THE INVENTION

The present invention provides a new method for improving the healing of tympanic membrane perforations, or minimizing scar formation during healing, by administration of plasminogen.

Accordingly, the invention provides a method of promoting healing of a tympanic membrane perforation in a subject in need of such treatment, which comprises administering to the subject a composition containing an effective amount of plasminogen for promoting healing of the tympanic membrane perforation. Preferably, the subject is a human, and the plasminogen is human plasminogen. The composition may further comprise a pharmaceutically acceptable carrier, and can be in the form of an aqueous solution, a gel, a lotion, a balm, a powder, a paste, a bandage, a wound dressing, or another suitable delivery vehicle. The plasminogen can be administered topically or systemically. In the case of topical administration, the administered composition may comprise from about 0.05 mg to about 10 mg plasminogen, preferably from about 0.5 to about 5 mg plasminogen. The composition may promote healing by accelerating the healing of the perforation, reducing necrotic tissue, and reducing the formation of scar tissue in the wound area. In one embodiment, the plasminogen administration is repeated at least once, preferably at least once every day.

The invention also provides a method of reducing scar formation from a healing wound in a subject in need of such treatment, which comprises administering to the subject a composition containing an effective amount of plasminogen for reducing scar formation. The plasminogen can, for example, reduce fibrin deposition. The subject is preferably a human subject, and the plasminogen is preferably human plasminogen. In one embodiment, the plasminogen is administered topically, and the composition can comprise from about 0.5 mg to about 5 mg plasminogen per square centimeter wound area.

The invention also provides for a method of accelerating wound healing in a patient in need of such treatment, which comprises administering to the patient a composition containing an effective amount of plasminogen to promote healing of the wound. Optionally, the wound is a chronic wound. The subject may be a human subject, in which case the plasminogen is preferably, although not necessarily, human plasminogen. In case the plasminogen is administered topically, it may be administered so that from about 0.5 mg to about 5 mg plasminogen is applied per square centimeter wound area.

The invention also provides for a method of accelerating wound healing in a patient in need of such treatment, which comprises administering to the patient a composition containing an effective amount of plasmin to promote healing of the wound. The wound may, optionally, be a chronic wound. In one embodiment, the subject is a human subject, and the plasmin is human plasmin. If the composition is administered by topical administration, the composition can comprise, for example, from about 0.005 mg to about 0.5 mg plasminogen per square centimeter wound area.

The invention also provides for a method of reducing necrotic tissue formation in a healing wound in a subject in need of such treatment, which comprises administering to the subject a composition containing an effective amount of plasminogen for reducing necrotic tissue formation. Optionally, the plasminogen reduces fibrin deposition. In one embodiment, the subject is a human subject, and the plasminogen is human plasminogen. If administered topically, the composition may be applied in an amount corresponding to, for example, from about 0.5 mg to about 5 mg plasminogen per square centimeter wound area.

The invention also provides a method of identifying an agent that is useful in promoting wound healing, which method comprises: (i) administering a test agent to an animal having a tympanic membrane perforation; (ii) evaluating at least one of the extent of healing, necrotic tissue formation, and scar formation of the tympanic membrane perforation; (v) comparing the extent of healing, necrotic tissue formation, or scar formation to a control value; and (vi) selecting any test agent for which the extent of healing is higher or the necrotic tissue or scar formation lower than the control value as an agent useful in promoting wound healing. The animal can, for example, be a wild-type animal or a transgenic animal lacking endogenous expression of plasminogen, preferably selected from a mouse or a rat. In one embodiment, the test agent is administered by topical administration. The control value may be, for example, the extent of healing or scar or necrotic tissue formation in a second animal, to which the test agent has not been administered.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to healing of perforated tympanic membranes, and to healing of wounds. The invention is also applicable to diseases and conditions characterized by degeneration or poor healing of extracellular matrix structures, particularly the keratinized tissue such as, e.g., the tympanic membrane. Other abnormal wound healing processes include diabetic ulcers, keloids, hypertrophic scars, and the application of skin substitutes.

The invention is based, in part, on the discovery that in the absence of plasminogen, the wound healing process does not progress properly, showing that plasminogen plays a pivotal role in the healing of wounds, particularly in the case of tympanic membrane perforations (see Examples and Table 1 (see Original Patent)). As shown by the Examples, healing of tympanic membrane perforations was dramatically altered and abnormal in mice lacking plasminogen as compared to wild-type controls and other transgenic models (see Table 1). In plasminogen deficient mice, tympanic membrane perforations did not heal properly during the 144-day test period during which healing was monitored, resulting in an abnormal tympanic membrane. It was also found that plasminogen-deficient mice reverted to a normal healing process following injection of human plasminogen. (see Examples 2 and 5), and that wild-type rats displayed enhanced tympanic membrane healing after administration of plasminogen (Example 6). In addition, experiments showed that uPA, but not tPA, may also play some role in normal wound healing, in that uPA appeared to be needed for the removal of fibrin depositions (Example 3 and 4). However, in no animal model was the lack of normal healing more apparent than in plasminogen-deficient mice, indicating that plasminogen plays a more essential and probably different role during wound healing than uPA. Without being bound to any specific theory, the present findings supports that the inability to proper healing may be caused by a defect in the inflammatory response, keratinocyte migration, or matrix remodeling events during the healing process.

As demonstrated by the results in Table 1 (see Original Patent), uPA, tPA, and plasminogen play distinct roles in the wound healing process, with plasminogen playing a pivotal role. In addition, as shown in the Examples, removal of necrotic tissue and fibrin are critically dependent on the presence or administration of plasminogen/plasmin rather than tPA or uPA.

Accordingly, plasminogen could be used in the treatment of wound healing diseases, as well as for acceleration strategies for tympanic membrane and wound healing, treatment of conditions or diseases affecting healing of disrupted epidermal tissues, methods for reducing scar formation or necrotic tissue accumulation or formation, as well as screening methods for drugs to be used in such treatments. The administration of plasminogen can also minimize scar formation or necrotic tissue accumulation in tympanic membranes or other epidermal tissues during wound healing. For example, plasminogen can be applied in conjunction with plastic surgery to reduce the appearance of, or prevent, the formation of residual scars, fibrin deposits, or necrotic tissue. Also, plasminogen can be applied onto ulcers or burns to improve healing.

In addition, the method of the invention can be used for improving wound healing in conditions of local or systemic deficiency of plasminogen, or to improve the healing of non- or slow-healing wounds. Notably, restoration of plasminogen weeks after injury can diminish the accumulated extracellular matrix and restart normal healing, thus showing that plasminogen can be applied for treatment of chronic wounds such as ulcerations and bedsores.

Alternatively, plasmin itself could be used in the treatment of wound healing diseases, as well as for acceleration strategies for tympanic membrane and wound healing, treatment of conditions or diseases affecting healing of disrupted epidermal tissues, or methods for reducing scar formation or necrotic tissue accumulation or formation. The administration of plasmin can also minimize scar formation or necrotic tissue accumulation in tympanic membranes or other epidermal tissues during wound healing. For example, plasmin can be applied in conjunction with plastic surgery to reduce the appearance of, or prevent, the formation of residual scars, fibrin deposits, or necrotic tissue. Also, plasmin can be applied onto ulcers, bedsores, or burns to improve healing.

The compositions of the invention can be administered topically, by injection, or by intravenous infusion. Preferably, although not necessarily, the administration is local, i.e., in some proximity to the wound. When the plasminogen or plasmin is administered by injection (e.g., intravenously, subcutaneously, intramuscularly), it is advantageously prepared as a solution of the material in a pharmaceutically acceptable liquid, such as, e.g., isotonic saline. In a preferred embodiment, plasminogen or plasmin is administered locally to attain a high concentration, for example, at least 200 .mu.g/ml plasminogen or at least 20 .mu.g/ml plasmin, in the perforation or wound area. For topical administration, the plasminogen or plasmin composition may, for example, be part of a gel, lotion, balm, paste, spray, powder, bandage, or wound dressing. For accelerating healing of a tympanic membrane perforation, the composition is preferably administered via the outer ear canal, by, e.g., a spray delivered to the area of the perforation, or by adding plasminogen or plasmin solution drop-wise. Devices for delivering compositions by spray are known in the art, and is described in, e.g., U.S. Pat. No. 6,027,712. Strategies such as administering the plasminogen/plasmin by spray, as well as by gel or paste, or a topically administered solution, can also be used for treating wounds in the oral cavity. For acceleration of wound healing, plasminogen or plasmin can be present in a wound dressing applied onto the wound, from which it is transferred to the wound area. In another embodiment, the composition comprises a compound that mimics plasminogen activity or plasmin activity, such as a plasminogen/plasmin fragment or mutant, or another molecule with similar activity.

The invention also provides for screening methods to identify compounds useful for improving wound healing in an animal model. The screening method is preferably based on a tympanic membrane perforation animal model which provides detailed functional description with respect to wound healing, thereby enabling efficient screening, identification and characterization of new chemical entities, pharmacological effects, and novel drug targets involved in the healing of tympanic membrane perforations and other wound healing diseases. Accordingly, a wound can be inflicted in a wild-type or plasminogen-deficient animal, and a test compound administered to the animal via a predetermined route. Wound healing in the animal and/or plasminogen levels in the wound area can then be compared to a control value such as, e.g., wound healing in a wild-type animal or an animal to which the test agent has not been administered, to evaluate whether the test compound improved the rate of healing or reduced necrotic tissue or scar formation. In one embodiment, the animal is a knock-out mouse lacking one or both alleles of plasminogen. As a control model, or for further testing of the agent identified, a model in which wild-type components of the plasminogen activation system is normally expressed, or, alternatively, a transgenic animal model in which one or more wild-type proteins have been replaced with the corresponding human or non-human homologs, can be used.
 

Claim 1 of 8 Claims

1. A method of promoting healing of a tympanic membrane perforation comprising administering to a subject in need of such treatment, a composition containing 1 to 10 mg/ml plasminogen directly onto the tympanic membrane perforation.

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