WO2009073457A1

WO2009073457A1 - Methods and compositions for the rescue of a filtering bleb

Info

Publication number: WO2009073457A1
Application number: PCT/US2008/084600
Authority: WO
Inventors: Steven Bartels; Gregory L. Mcintire
Original assignee: Bausch and Lomb Inc
Current assignee: Bausch and Lomb Inc
Priority date: 2007-11-30
Filing date: 2008-11-25
Publication date: 2009-06-11
Anticipated expiration: 2010-05-30

Abstract

A method for the rescue of a failing or failed filtering bleb in an eye of a patient at a time when scar tissue has formed, the method comprises: (a) providing a composition comprising plasmin or an enzymatically equivalent derivative thereof; and (b) administering said composition into said filtering bleb. Compositions including plasmin or an enzymatically equivalent derivative thereof are used in such a method. These compositions can also include gelling agents, bioadhesives, medicaments, anesthetics, or a combination thereof.

Description

METHODS AND COMPOSITIONS FOR THE RESCUE OF A FILTERING BLEB

BACKGROUND

[001] The present invention relates to methods and compositions for the rescue of a filtering bleb. The method can cause an increase in flow via the bleb, improved control of intraocular pressure, and prevention or treatment of vision loss in people at risk of retinal neuropathy.

[002] A major risk of patients who suffer from glaucoma is elevated intraocular pressure (lOP). Elevated IOP can cause damage to the optic nerve and eventual blindness. The aqueous humor is the fluid in the anterior chamber of the eye. This fluid is continuously refreshed in that new fluid is produced by the eye's ciliary body and drained back into the bloodstream through the trabecular meshwork and uveoscleral tissue. When the aqueous humor does not drain properly, fluid builds up and IOP becomes elevated.

[003] While there is no cure for glaucoma, treatment to control IOP can slow the progression of the disease. One such treatment is glaucoma filtering surgery (GFS), one such procedure is known as "trabeculectomy". In this surgery, a fistula is created between the anterior chamber of the eye and the subconjunctival space creating a "filtering bleb".

[004] Tissue plaminogen activator (tPA) has been used for a variety of therapies related to the eye. With respect to glaucoma surgery, tPA has been used to reduce fibrin formed immediately after trabeculectomy surgery (A. Szymanski, A. Gierek-Lapinska, M. Koziak & S. Gierek-Kalicka, "Corneal endothelial permeability in protection glaucoma filter bleb with tissue plasminogen activator," Int. Ophthalmol., 19: 13-17 (1995)). It has also been used immediately after secondary surgeries, for example cataract extraction or penetrating keratoplasty (see M. F. Smith et al. "Use of Tissue Plasminogen Activator to Revive Blebs Following Intraocular Surgery", Arch. Opthamol., 119:809-812 (2001)), again to reduce fibrin.

[005] Cairns (J. E. Cairns, "Trabeculectomy: Preliminary Report of a New Method," Am. J. Opthalmol., 66: 673-679 (1968)) described the technique of trabeculectomy and this general technique is still used today. For this surgical technique to be optimally successful not only must it produce adequate outflow of aqueous humor but also excessive external drainage, or leaking, due to holes in the filtering bleb and obstruction of outflow due to post-operative scarring must be avoided. External drainage of aqueous through a hole in the conjunctiva presents a serious problem in that the interior of the eye gets exposed to the environment resulting in complications such as hypotony and infection. It has been suggested that a leaking bleb can be treated with a wide variety of techniques. A review of these techniques is found at Column 2 of U.S. Patent 6,503,892.

[006] Scarring, such as at the level of the episclera adjacent to scleral flap, can cause filtering bleb failure for the opposite reason: inadequate drainage. Several treatments to reduce post-operative scarring have been proposed. Antifibrotic agents can be administered at the time of surgery or shortly thereafter. The antifibrotic agents that have been suggested include tPA, 5- fluorouracil (5-FU)₁ mitomycin C (MMC), and anti-TGFβ antibodies. However, in spite of the immediate use of these agents post-surgery, long-term failure due to scar tissue formation in the filtering bleb is still a major clinical problem. Needle revision or ocular massage can be attempted to repair a failure of this type and thus rescue the filtering bleb but the results are often not satisfactory and often must be repeated. P. G. Mardelli et al., "Slit-lamp Needle Revision of Failed Filtering Blebs Using mitomycin-C," Ophthalmol., 103:1946-1955 (1996); M. F. Smith et al., "Use of Tissue Plasminogen Activator to Revive Blebs Following Intraocular Surgery," Arch. Ophthalmol, 119:809-812 (2001) (cited above).

[007] There is a continuing need to improve the drainage of a filtering bleb at a time after surgery when scar tissue may have formed.

SUMMARY

[008] In general, the present invention provides a method for the rescue of a failing or failed filtering bleb in an eye of a patient at a time when scar tissue has formed, the method comprising:

(a) providing a composition comprising plasmin-like material or an enzymatically equivalent derivative thereof; and

(b) administering said composition into said filtering bleb.

[009] The composition comprising plasmin or an enzymatically equivalent derivative thereof (hereinafter simply "composition comprising plasmin-like material-like material") is administered in a manner such that the scar tissue is degraded, the flow of aqueous humor is reestablished and IOP is lowered.

[0010] The composition comprising plasmin-like material can be administered as either a single treatment or as an intermittent treatment, it is easily administered in the physician's office and is an improvement over current practices that frequently include additional surgery and/or increased chronic, daily medical therapy. One method of administering the composition comprising plasmin-like material according to the present method is subconjunctival injection. Subconjunctival injection of the composition comprising plasmin-like material into a filtering bleb can lead to the degradation of accumulated extracellular matrix components and thereby reduce the resistance to outflow of aqueous humor and improve control of intraocular pressure.

[0011] The described method is carried out at a time after surgery when scar tissue may have formed. This is normally well after surgery. The attending physician can often visually observe the formation of scar tissue. Another indication of scar formation can be an increase of IOP. Typically, scar formation occurs weeks or even months after surgery although the present method is useful, at the discretion of the attending physician, at any time that scarring is believed to have occurred.

[0012] Other aspects of the invention include compositions including plasmin or an enzymatically equivalent derivative thereof and other components. In one aspect, a composition of the present invention comprises plasmin or an enzymatically equivalent derivative thereof and a gelling agent. In another aspect, a composition of the present invention comprises plasmin or an enzymatically equivalent derivative thereof and a bioadhesive. The gelling agent or the bioadhesive assists in containing the composition at or near the site of injection and can reduce side effects. The gelling agent and the bioadhesive can be used in combination. In another aspect of the invention, a composition comprises plasmin or an enzymatically equivalent derivative thereof and a medicament. Useful medicaments include anti-inflammatory agents. In another aspect of the invention, a composition comprises plasmin or an enzymatically equivalent derivative thereof and an anesthetic.

DETAILED DESCRIPTION

[0013] Plasmin, a serine proteinase, is the principal fribrinolytic enzyme in mammals, and has the important function of breaking down in-vivo blood clots. It can be derived from the inactive precursor plasminogen, which circulates in plasma. Circulating plasminogen can be activated, for example in vivo, by plasminogen activators, such as tissue plasminogen activator ("tPA") or urokinase, which cleave a single-chain plasminogen molecule at the Arg⁵⁶⁰ - VaI⁵⁶¹ peptide bond, producing active plasmin. Plasminogen can also be activated by the bacteria-derived enzyme streptokinase. However, when a plasminogen activator is administered to a patient, it still must encounter the plasminogen in order to generate active plasmin, and the magnitude of the effectiveness of these activators still depends on the inherent in-vivo level of plasminogen. Thus, according to one embodiment of the present method, a precursor and an activator can be admixed just prior to administration or administered sequentially. When administered sequentially, the precursor can be administered first followed by the activator or the activator can be administered first followed by the precursor, for example by making two separate injections, thereby forming the active plasmin in situ. In another embodiment, the precursor and the activator can be coadministered. Plasmin precursors (plasminogen) can be coadministered with plasmin activators such as tPA, urokinase or bacteria-derived enzyme streptokinase, as. mentioned above.

[0014] Plasmin and its variants that are useful in the present method include various forms that retain enzymatic proteolytic activity such as human-derived plasmin, recombinant plasmin, or combinations thereof.

[0015] As used herein, the term "enzymatically equivalent derivative" of plasmin means an enzyme that can be derived from plasmin and has a proteolytic function similar to that of plasmin. A derivative of plasmin can be a fragment or a variant of plasmin that has a proteolytic function similar to that of plasmin. A derivative of plasmin can be microplasmin comprising the enzymatic domain of plasmin and a short amino acid sequence (e.g., comprising about 20- 40 amino acid residues) at the amino terminus of the enzymatic domain, miniplasmin comprising the enzymatic domain attached to the kringle-5 domain of plasmin, or other truncated forms of plasmin that comprise the enzymatic domain and one or more kringle domains of plasmin having retained lysine- binding property. A variant of plasmin can be generated from a molecule of plasmin by deleting, substituting, or adding one or more amino acid residues. Such substitution can be, for example, a conservative substitution. Enzymatically active microplasmin and miniplasmin can be obtained from microplasminogen and miniplasminogen precursors by cleavage of the peptide bond at Arg⁵⁶¹-Val⁵⁶², wherein the amino acid residue numbers correspond to those of human Glu-plasminogen, which has 791 amino acid residues. Microplasmin is disclosed in, for example, U.S. Patent 4,774,087; and miniplasmin is disclosed in, for example, U.S. Patent Application Publications 2005/0118158A1 of Pakola et al, published 2 June 2005 and 2005/0124036A1, published 9 June 2005 of Suslio et al. The contents of these documents are incorporated herein by reference.

[0016] In one aspect, derivatives useful in the present method include truncated plasmins comprising the enzymatic domain of plasmin attached at its amino terminus to kringle-1, kringle-2, kringle-3, kringle-4, or kringle-5 domain of plasmin, or combinations of these kringles. In one embodiment, two or more kringle domains can be attached in any order to the amino terminus of the enzymatic domain. A kringle domain of plasmin is characterized by a triple-loop conformation and comprises about 75-85 amino acid residues with three disulfide bridges. In still another embodiment, the truncated plasmin comprises two or more, but fewer than five, kringle domains attached in any order to the amino terminus of the enzymatic domain. An example of the enzymatic domain attached at its amino terminus to kringle-1 is described in World Patent Organization Application, "Recombinantly Modified Plasmin," PCT/US2006/040940, of Scudri et al published 26 April 2007, (US 60/728,615 20.10.2005; US 60/732,588 02.11.2005) which is incorporated herein in its entirety. [0017] The enzymatically equivalent derivative of plasmin can be a combination compound wherein the enzymatically active plasmin moiety can be associated with or otherwise attached to another useful material. The other useful material can be, without limitation, a carrier molecule to facilitate the delivery of the combination. The term "combination" encompasses, but is not limited to, two or more molecules or fragments of molecules attached, attracted, held, or adhered together by bonds (hydrogen bonding, ionic bonding, physical (such as by van der Waals force) or chemical adsorption, covalent bonding, or organometallic interaction), two inter-penetrating molecules, or a complex comprising two or more molecules by, e.g., bonding or conformational interaction.

[0018] In the composition comprising plasmin-like material, the concentration of plasmin, or one of its enzymatically equivalent derivatives, can range from about 10^'4 to about 10, or from about 10^"4 to about 5, or from about 10^"3 to about 5, or from about 10^"2 to about 5, or from about 10^'2 to about 2, or from about 10^'2 to about 1 percent by weight.

[0019] The composition comprising plasmin-like material can be injectable and can be in a form of a liquid. For example, the composition comprising plasmin-like material can comprise sterile saline solution. The composition comprising plasmin-like material can further comprise a compound that has a function of stabilizing plasmin or its enzymatically equivalent derivatives. Such a compound is hereinafter referred to as a "plasmin stabilizing agent," which has a capability of slowing the rate of autodegradation of plasmin or its derivative in a solution; in particular, when the solution has a near neutral pH (e.g., from about 6.5 to about 8.5). The concentration of the plasmin stabilizing agent can be in the range from about 0.001 to about 50 weight percent depending upon the nature of the stabilizer (e.g., small molecule lysine or lysine analog vs "solvent" type stabilizers like glycerol or glycerine). Alternatively, small molecule stabilizers of lysine or lysine analog-type can be present from about 0.01 to about 20, or from about 0.01 to about 10, or from about 0.01 to about 5, or from about 0.01 to about 4 weight percent). Such plasmin stabilizing agents can be selected from the group consisting of tranexamic acid, ε-aminocaproic acid, L-lysine, analogs of L-lysine, L-arginine, L-ornithine, γ-aminobutyric acid, glycylglycine, diglycine, , combinations thereof, and mixtures thereof. Non-limiting examples of analogs of L-lysine include L-2-amino-3-guanidinopropionic acid, L-citruline, D-citruline, 2,6- diaminoheptanoic acid, ε,ε-dimethyl-L-lysine, α-methyl-DL-omithine, δ- benzyloxycarbonyl-L-ornithine, (N-d-4-methyltrityl)-L-ornithine, N-δ-1-(4,4- dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl)-D-omithine, p-aminomethylbenzoic acid, and 2-aminoethylcysteine. These types of plasmin stabilizers are detailed in WO/2007/078761A2 of Jani et al, "Method for Prolonging Activity of Autodegradable Enzymes and Compositions Thereof," 12 July 2007, (60/749806, 16 December 2005; 11/601 ,389, 17 November 2006) which is incorporated herein in its entirety.

[0020] "Solvent" type stabilizers such as gelatin, human serum albumin ("HSA"), glycerin, glycerol, and other generally nonionic molecules or large polymer (protein) stabilizers must be present at higher concentrations to afford stability in solution at or near neutral pH (e.g., from about 6.5 to about 8.5). The concentration of these plasmin stabilizing agents can be in the range from about 0.1 to about 50 weight percent, alternatively, these types of stabilizers can be present from about 0.1 to about 40, or from about 0.1 to about 20, or from about 1 to about 10 weight percent. These stabilizers can be used in combination and in combination with lysine or lysine analog stabilizers mentioned hereinabove. Their optimal concentration ranges were also disclosed hereinabove.

[0021] The composition comprising plasmin-like material can further comprise a non-ionic surfactant. Surfactants can provide a number of functions. For example, a surfactant can be used to reduce the tendency of the composition to adhere to containers and other materials such a tubing and the like. Useful non- ionic surfactants include polysorbates (such as polysorbate 80 (polyoxyethylene sorbitan monooleate), polysorbate 60 (polyoxyethylene sorbitan monostearate), polysorbate 20 (polyoxyethylene sorbitan monolaurate), commonly known by their trade names of Tween® 80, Tween® 60, Tween® 20), poloxamers (synthetic block polymers of ethylene oxide and propylene oxide, such as those commonly known by their trade names of Pluronic®; e.g., Pluronic® F127 or Pluronic® F108; Poloxomer® 188) , or poloxamines (synthetic block polymers of ethylene oxide and propylene oxide attached to ethylene diamine, such as those commonly known by their trade names of Tetronic®; e.g., Tetronic® 1508 or Tetronic® 908, etc., other nonionic surfactants such as Brij®, Myrj®, and long chain fatty alcohols (i.e., oleyl alcohol, stearyl alcohol, myristyl alcohol, docosohexanoyl alcohol, etc.) with carbon chains having about 12 or more carbon atoms (e.g., such as from about 12 to about 24 carbon atoms). Such compounds are delineated in Martindale, 34^th ed., pp. 1411-1416 (Martindale, "The Complete Drug Reference," S. C. Sweetman (Ed.), Pharmaceutical Press, London, 2005) and in Remington, "The Science and Practice of Pharmacy," 21^st Ed., pp. 291-291 and the contents of chapter 22, Lippincott Williams & Wilkins, New York, 2006). The concentration of a non-ionic surfactant, when present, can be in the range from about 0,001 to about 5 weight percent (or alternatively, from about 0.01 to about 4, or from about 0.01 to about 2, or from about 0.01 to about 1 weight percent).

[0022] Plasmin can be produced by activation of plasminogen precursor, which may be obtained from plasma. For example, a method of producing high- purity plasmin is disclosed in U.S. Patent Application Publication 2004/0171103 A1 , Bradley et al, published 2 September 2004 which is incorporated herein by reference in its entirety. The starting material, plasminogen, can be extracted from Cohn Fraction IMII paste by affinity chromatography on Lys- SEPHAROSE™ as described by D.G. Deutsch and E.T. Mertz, "Plasminogen: purification from human plasma by affinity chromatography," Science 170(962): 1095-6 (1970). (SEPHAROSE™ is a trade name of Pharmacia, Inc., New Jersey.)

[0023] Following the extraction of plasminogen from the Cohn Fraction ll+lll paste, lipid and protein impurities and Transmissible Spongiform Encephalopathies ("TSE") contaminants are reduced by precipitation with the addition of polyethylene glycol ("PEG"), in a range of about 1 to about 10 percent weight/volume or the addition of about 80 to about 120 g/l ammonium sulfate. The PEG or ammonium sulfate precipitate is removed by depth filtration and the resulting solution placed on a lysine affinity resin column. The phrase "lysine affinity resin" is used generally for affinity resins containing lysine or its derivatives or ε-aminocaproic acid as the ligand. The column can be eluted with a solution having a low pH of approximately 1 to 4.

[0024] The protein obtained after elution from the affinity column is generally at least 80 percent plasminogen. The purified plasminogen is then stored at low pH in the presence of simple buffers such as glycine and lysine or ω-amino acids.

[0025] Plasminogen in solution can then be activated to plasmin by the addition of a plasminogen activator, which may be accomplished in a number of ways including but not limited to streptokinase, urokinase, tissue plasminogen activator ("tPA"), or the use of urokinase immobilized on resin and use of streptokinase immobilized on resin. In one embodiment, the plasminogen activator is soluble streptokinase. The addition of stabilizers or excipients such as glycerol, ω-amino acids such as lysine, polylysine, arginine, ε-aminocaproic acid and tranexamic acid, and salt can enhance the yield of plasmin.

[0026] Plasmin can be purified from unactivated plasminogen by affinity chromatography on resin with benzamidine as the ligand and eluted preferably with a low pH solution (e.g., pH < 4, or alternatively pH between about 2.5 and about 4). This step can remove essentially all degraded plasmin as well as the majority of the streptokinase. [0027] As a polishing step for the removal of remaining streptokinase, hydrophobic interaction chromatography ("HIC") at low pH is performed (e.g., pH < 4). Following the HIC step, plasmin is formulated as a sterile protein solution by ultrafiltration and diafiltration and 0.22-μm filtration.

[0028] The eluted plasmin from such polishing step can be buffered with a low pH (e.g., pH < 4), low buffering capacity agent. The low pH, low buffering capacity agent typically comprises a buffer of either an amino acid, a derivative of at least one amino acid, an oligopeptide that includes at least one amino acid, or a combination thereof. In addition, the low pH, low buffering capacity agent can comprise a buffer selected from acetic acid, citric acid, hydrochloric acid, carboxylic acid, lactic acid, malic acid, tartaric acid, benzoic acid, serine, threonine, methionine, glutamine, alanine, glycine, isoleucine, valine, alanine, aspartic acid, derivatives, and combinations thereof. The concentration of plasmin in the buffered solution can range from about 0.01 mg/ml to about 50 mg/ml of the total solution. The concentration of the buffer can range from about 1 nM to about 50 mM. Of course, these ranges may be broadened or narrowed depending upon the buffer chosen, or upon the addition of other ingredients such as additives or stabilizing agents. The amount of buffer added is typically that which will give the reversibly inactive acidified plasmin solution a pH less than 5.0; preferably between about 2.5 to about 4, and still more preferably between about 3 and about 3.5. Inactive acidified plasmin compositions including a bulking agent, such as a carbohydrate, can be optionally lyophilized at a temperature in a range, for example, from about O⁰C to about -5O⁰C, or preferably from about O⁰C to about -2O⁰C₁ to produce a powder for long-term storage.

[0029] The acidified plasmin or variants thereof, produced from plasma or by recombinant technology, can be reconstituted by adding the enzyme to a formulation having a near neutral pH, to produce the composition comprising plasmin-like material substantially immediately before using the enzyme.

[0030] It may be advantageous to add a stabilizing or bulking agent to the reversibly inactive acidified plasmin solution obtained as disclosed above. Non- limiting examples of such stabilizing or bulking agents are polyhydric alcohols such as those detailed in US 5,879,923 (Mar 9, 1999) Stable Plasmin Solution, which is incorporated herein its entirety, pharmaceutically acceptable carbohydrates, salts, glucosamine, thiamine, niacinamide, and combinations thereof. The stabilizing salts can be selected from the group consisting of sodium chloride, potassium chloride, magnesium chloride, calcium chloride, and combinations thereof. Sugars or sugar alcohols may also be added, such as glucose, maltose, mannitol, sorbitol, sucrose, lactose, trehalose, and combinations thereof. Other carbohydrates that may be used are polysaccharides, such as dextrin, dextran, glycogen, starches, carboxymethylcellulose, derivatives thereof, and combinations thereof. Concentrations of a carbohydrate added to add bulk to the reversibly inactive acidified plasmin solution can be in a range from about 0.2 percent weight/volume ("% w/v") to about 20% w/v. Concentrations for a salt, glucosamine, thiamine, niacinamide, and their combinations can range from about 0.001 M to about 1 M.

[0031] Plasmin or variants thereof can be produced by recombinant technology. For example, the production of recombinant microplasminogen (which can be activated to microplasmin by cleavage of the peptide bond at Arg⁵⁶¹-Val⁵⁶² using one of the plasminogen activators disclosed above) in the Pichia pastoris yeast system is disclosed in U.S. Patent Application Publication 2004/0071676 A1 , to Collen et al, published 15 April 2004, which is incorporated herein by reference. Plasminogen and miniplasminogen (which also can be activated to miniplasmin by cleavage of the peptide bond at Arg⁵⁶¹-Val^56z using one of the plasminogen activators disclosed above) in the Pichia pastoris yeast system are disclosed in U.S. Patent Application Publication 2005/0124036 A1 , to Susilo, cited previously, which is incorporated herein by reference.

[0032] The composition comprising plasmin-like material used in the present method can have a pH of about 7. Alternatively, the composition comprising plasmin-like material can have a pH in a range from about 5 to about 8, preferably 6.5 to 7.5 and still more preferably about 7.0 about 7.4.

[0033] The composition comprising plasmin-like material can further comprise a buffer, such as a phosphate buffer or a Tris-HCI buffer (comprising tris(hydroxymethyl)aminomethane and HCI). For example, a Tris-HCI buffer having pH of 7.4 comprises 3 g/l of tris(hydroxymethyl)aminomethane and 0.76 g/l of HCI. In yet another aspect, the buffer is 10X phosphate buffer saline ("PBS") or 5X PBS solution. [00343 Other buffers also may be found suitable or desirable in some circumstances, such as buffers based on HEPES (N-{2- hydroxyethyl}peperazine-N'-{2-ethanesulfonic acid}) having pK_a of 7.5 at 25 'C and pH in the range of about 6.8-8.2; BES (N,N-bis{2-hydroxyethyl}2- aminoethanesulfonic acid) having pK_a of 7.1 at 25⁰C and pH in the range of about 6.4-7,8; MOPS (3-{N-morpholino}propanesulfonic acid) having pK_a of 7.2 at 25⁰C and pH in the range of about 6.5-7.9; TES (N-tris{hydroxymethyl}- methyl-2-aminoethanesulfonic acid) having pK_a of 7.4 at 25⁰C and pH in the range of about 6.8-8.2; MOBS (4-{N-morpholino}butanesulfonic acid) having pK_a of 7.6 at 25⁰C and pH in the range of about 6.9-8.3; DIPSO (3-(N,N-bis{2- hydroxyethyl}amino)-2-hydroxypropane) ) having pK_a of 7.52 at 25⁰C and pH in the range of about 7-8.2; TAPSO (2-hydroxy-3{tris(hydroxymethyl)methylamino}- 1-propanesulfonic acid) ) having pK_a of 7.61 at 25⁰C and pH in the range of about 7-8.2; TAPS ({(2-hydroxy-1 ,1-bis(hydroxymethyl)ethyl)amino}-1- propanesulfonic acid) ) having pK_a of 8.4 at 25⁰C and pH in the range of about 7.7-9.1; TABS (N-tris(hydroxymethyl)methyl-4-aminobutanesulfonic acid) having pK_a of 8.9 at 25⁰C and pH in the range of about 8.2-9.6; AMPSO (N-(1 ,1- dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic acid) ) having pK_a of 9.0 at 25⁰C and pH in the range of about 8.3-9.7; CHES (2- cyclohexylamino)ethanesulfonic acid) having pK_a of 9.5 at 25⁰C and pH in the range of about 8.6-10.0; CAPSO (3-(cyclohexylamino)-2-hydroxy-1- propanesulfonic acid) having pK_a of 9.6 at 25⁰C and pH in the range of about 8.9-10.3; or CAPS (3-(cyclohexylamino)-1 -propane sulfonic acid) having pK_a of 10.4 at 25°C and pH in the range of about 9.7-11.1. [0035] Excessive deposition of matrix components resulting in substantial alteration of normal tissue architecture, is considered to be the determining factor for tissue fibrosis and scar formation. The conjunctival scarring response is thought to be greatly influenced by growth factors such as transforming growth factor beta (TGF-β) in the surgical wound. The complex healing process following surgery includes proliferation, migration and differentiation of fibroblasts and synthesis of extracellular matrix (ECM) components. These components include fibronectin, laminin and collagen produced by human Tenon's capsule and episcleral fibroblasts. These are believed to be the key cells involved in the subconjunctival healing process and which become activated by TGF-β. The histopathology of post trabeculectomy scar tissue is described in Meitz et al., "Histopathology of Episcleral Fibrosis After Trabeculectomy With and Without Mitomycin C," Graefe's Arch. Clin. Exp. Opthalmol. 234:364-368 (1996). See also Francis et al., "Histopathologic Features of Conjunctival Filtering Blebs," Arch. Ophthalmol. 123:166-170 (2005).

[0036] Tissue directly adherent to the scleral flap seems to be the major cause of obstruction of the outflow from the filtering bleb. Thus, according to the present method, the composition comprising plasmin-like material is preferably administered to this tissue. The composition can be administered subconjunctival^ directly over the glaucoma filtering site or under the scleral flap.

[0037] Administration by injection can be accomplished by known techniques. Reference is made to the Mardelli et al article cited above. The composition comprising plasmin-like material can be drawn into a syringe and a small gauge (e.g. 25-32 gauge) needle attached. In the event that the plasmin is created just prior to administration, the plasmin precursor can be drawn first into the syringe and then the activator or vice versa. A topical anesthetic can be applied to the ocular surface and the surrounding area can be sterilized using, for example, povidone-iodine.

[0038] The composition containing plasmin can also contain an injectable anesthetic. Useful injectable anesthetics include bupivacaine hydrochloride, chloroprocaine HCI, etidocaine HCI, lidocaine HCI, mepivacaine HCI, prilocaine HCI, procaine HCI, and tetracine HCI.

[0039] A lid speculum can be used to expose thescarred area. The needle containing the composition comprising plasmin-like material is then caused to enter the conjunctiva a short distance from the desired site of the injection. The plasmin containing composition is injected at the desired site and then spread within subconjunctival space, for example by using a sterile cotton swab. Typically, an amount from about 25 μl to about 200 μl of a composition comprising about 0.25-5 IU of plasmin or derivatives thereof per 50 μl of formulation can be administered. Alternatively, a composition can comprise about 0.001-50 mg/ml (or about 0.2-20 mg/ml, or about 0.2-10 mg/ml, or about 0.5-8 mg/ml) of plasmin or derivatives thereof. Such administration of plasmin or derivatives thereof may be repeated to achieve a substantially full effect upon assessment of the treatment results and recommendation by a skilled medical practitioner. [0040] The composition comprising plasmin-like material can also include a bioadhesive. The term "bioadhesive" are compounds which exhibit general or specific adhesion to one or more components of the surfaces to which the composition is administered. One type of useful bioadhesive is a mucoadhesive. The term "mucoadhesive" means a natural or synthetic component, including macromolecules, polymers, and oligomers, or mixtures thereof, that can adhere to a mucous membrane. Specific useful bioadhesives and/or mucoadhesives are well known in the art and are described in a number of references, for example United States Patent 7,244,768 B2 of Bakhtt et al, published 17 July 2007, at CoI 9 line 19-64; United States Patent 7,244,709 of Quay et al, published 17 July 2007 at CoI 24 line 54 to CoI 27 line 30; the disclosures of which are hereby incorporated by reference.

[0041] The composition comprising plasmin-like material can also include a gelling agent. The composition can include a combination of a gelling agent and a bioadhesive. One useful gelling agent is an agent that is thermo-responsive so that the composition is easily injectable at room temperature but then gels at body temperature. This facilitates containing the composition at the injection site. This can result in reduced side effects and can allow for lower concentrations for the active plasmin agent. One such composition is described in United States Patent Publication 2007/0231 , 352A1 published 4 October 2007. This reference describes useful thermo-responsive polymers, for example at paragraph 038. This reference also discloses useful bioadhesives, for example at paragraph 036. The entire disclosure of this reference is hereby incorporated by reference. The use of in-situ gel forming agents in opthalmic compositions is described in: Nanjawade et al, "Review: In Situ-Forming Hydrogels for Sustained Opthalmic Drug Delivey", J. Controlled Release, 122(2): 119-134(2007).

[0042] The composition comprising plasmin-like material can also include a medicament. The medicament can be, without limitation, an anti-inflammatory agent. In one embodiment, such an anti-inflammatory agent can be selected from the group consisting of non-steroidal anti-inflammatory drugs ("NSAIDs"); peroxisome proliferator-activated receptor ("PPAR") ligands, such as PPARα, PPARδ, or PPARy ligands; combinations thereof; and mixtures thereof.

[0043] Non-limiting examples of the NSAIDs are: aminoarylcarboxylic acid derivatives (e.g., enfenamic acid, etofenamate, flufenamic acid, isonixin, meclofenamic acid, mefenamic acid, niflumic acid, talniflumate, terofenamate, tolfenamic acid), arylacetic acid derivatives (e.g., aceclofenac, acemetacin, alclofenac, amfenac, amtolmetin guacil, bromfenac, bufexamac, cinmetacin, clopirac, diclofenac sodium, etodolac, felbinac, fenclozic acid, fentiazac, glucametacin, ibufenac, indomethacin, isofezolac, isoxepac, lonazolac, metiazinic acid, mofezolac, oxametacine, pirazolac, proglumetacin, sulindac, tiaramide, tolmetin, tropesin, zomepirac), arylbutyric acid derivatives (e.g., bumadizon, butibufen, fenbufen, xenbucin), arylcarboxylic acids (e.g., clidanac, ketorolac, tinoridine), arylpropionic acid derivatives (e.g., alminoprofen, benoxaprofen, bermoprofen, bucloxic acid, carprofen, fenoprofen, flunoxaprofen, flurbiprofen, ibuprofen, ibuproxam, indoprofen, ketoprofen, loxoprofen, naproxen, oxaprozin, piketoprolen, pirprofen, pranoprofen, protizinic acid, suprofen, tiaprofenic acid, ximoprofen, zaltoprofen), pyrazoles (e.g., difenamizole, epirizole), pyrazolones (e.g., apazone, benzpiperylon, feprazone, mofebutazone, morazone, oxyphenbutazone, phenylbutazone, pipebuzone, propyphenazone, ramifenazone, suxibuzone, thiazolinobutazone), salicylic acid derivatives (e.g., acetaminosalol, aspirin, benorylate, bromosaligenin, calcium acetylsalicylate, diflunisal, etersalate, fendosal, gentisic acid, glycol salicylate, imidazole salicylate, lysine acetylsalicylate, mesalamine, morpholine salicylate, 1-naphthyl salicylate, olsalazine, parsalmide, phenyl acetylsalicylate, phenyl salicylate, salacetamide, salicylamide o-acetic acid, salicylsulfuric acid, salsalate, sulfasalazine), thiazinecarboxamides (e.g., ampiroxicam, droxicam, isoxicam, lornoxicam, piroxicam, tenoxicam), ε-acetamidocaproic acid, S-(5'-adenosyl)-L- methionine, 3-amino-4-hydroxybutyric acid, amixetrine, bendazac, benzydamine, α-bisabolol, bucolome, difenpiramide, ditazol, emorfazone, fepradinol, guaiazulene, nabumetone, nimesulide, oxaceprol, paranyline, perisoxal, proquazone, superoxide dismutase, tenidap, zileuton, their physiologically acceptable salts, combinations thereof, and mixtures thereof.

[0044] The anti-inflammatory agent can be a PPAR-binding molecule. In one embodiment, such a PPAR-binding molecule can be a PPARa-, PPARδ-, or PPARy-binding molecule. In another embodiment, such a PPAR-binding molecule can be a PPARα, PPARδ, or PPARy agonist. Such a PPAR ligand binds to and activates PPAR to modulate the expression of genes containing the appropriate peroxisome proliferator response element in its promoter region.

[0045] PPARy agonists can inhibit the production of TNF-α and other inflammatory cytokines by human macrophages (C-Y. Jiang et a\., Nature, Vol. 391 , 82-86 (1998)) and T lymphocytes (A. E. Giorgini et al., Horm. Metab. Res. Vol. 31 , 1-4 (1999)). More recently, the natural PPARY agonist 15-deoxy-Δ- 12,14-prostaglandin J2 (or "15-deoxy-Δ-12,14-PG J2"), has been shown to inhibit neovascularization and angiogenesis (X. Xin et al., J. Biol. Chem. Vol. 274:9116-9121 (1999)) in the rat cornea. Spiegelman et al., in U.S. Patent 6,242,196, disclose methods for inhibiting proliferation of PPARγ-responsive hyperproliferative cells by using PPARy agonists; numerous synthetic PPARy agonists are disclosed by Spiegelman et al., as well as methods for diagnosing PPARy-responsive hyperproliferative cells. Ail documents referred to herein are incorporated by reference. PPARs are differentially expressed in diseased versus normal cells. PPARy is expressed to different degrees in the various tissues of the eye, such as some layers of the retina and the cornea, the choriocapillaris, uveal tract, conjunctival epidermis, and intraocular muscles (see, e.g., U.S. Patent 6,316,465).

[0046] In one aspect, a PPARy agonist used in a composition or a method of the present invention is a thiazolidinedione, a derivative thereof, or an analog thereof. Non-limiting examples of thiazolidinedione-based PPARy agonists include pioglitazone, troglitazone, ciglitazone, englitazone, rosiglitazone, and chemical derivatives thereof. Other PPARy agonists include Clofibrate (ethyl 2- (4-chlorophenoxy)-2-methylpropionate), clofibric acid (2-(4-chlorophenoxy)-2- methylpropanoic acid), GW 1929 (N-(2-benzoy!phenyl)-O-{2-(methyl-2- pyridinylamino)ethyl}-L-tyrosine), GW 7647 (2-{{4-{2- {{(cyclohexylamino)carbonyl}(4-cyclohexylbutyl)amino}ethyl}phenyl}thio}-2- methylpropanoic acid), and WY 14643 ({{4-chloro-6-{(2,3- dimethylphenyl)amino}-2-pyrimidinyl}thio}acetic acid). GW 1929, GW 7647, and WY 14643 are commercially available, for example, from Koma Biotechnology, Inc. ( Seoul , Korea ). In one embodiment, the PPARy agonist is 15-deoxy-Δ-12, 14-PG J2. Non-limiting examples of PPAR-α agonists include the fibrates, such as fenofibrate and gemfibrozil. A non-limiting example of PPAR-δ agonist is GW501516 (available from Axxora LLC₁ San Diego, California or EMD Biosciences, Inc., San Diego, California).

[0047] Tables 1-26 show non-limiting examples of compositions comprising plasmin, which can be used in the practice of the methods for rescuing a filtering bleb, as described above. Table 1

Table 2

Table 3

Table 4

Table 5

Table 6

Table 7

Table 8

Table 9

Table 10

Table 11

Table 12

Table 13

Table 14

Table 15

Table 16

Table 17

Table 18

Table 19

Table 20

Table 21

Table 22

Table 23

Table 24

Table 25

Table 26

[0048] Each of these exemplary compositions further comprises an ophthalmically acceptable carrier. In some embodiments, such a carrier comprises sterile saline solution or an ophthalmically acceptable buffer or oil.

[0049] While specific embodiments of the present invention have been described in the foregoing, it will be appreciated by those skilled in the art that many equivalents, modifications, substitutions, and variations may be made thereto without departing from the spirit and scope of the invention as defined in the appended claims.

Claims

WHAT IS CLAIMED IS:

1. A method for the rescue of a failing or failed filtering bleb in an eye of a patient at a time when scar tissue has formed, the method comprising:

(a) providing a composition comprising plasmin or an enzymatically equivalent derivative thereof; and

(b) administering said composition into said filtering bleb in an effective amount and at an effective frequency for improving performance of said filtering bleb.

2. The method according to claim 1 wherein said administering is by subconjunctival injection.

3. The method according to claim 1 wherein said administering is to tissue directly adherent to a scleral flap.

4. The method according to claim 1 wherein an amount from about 25 μl to about 200 μl of said composition is administered.

5. The method according to claim 1 wherein said composition is provided by creating said plasmin or an enzymatically equivalent derivative thereof by admixing a plasmin precursor or precursor for an enzymatically equivalent derivative with a plasmin activator just prior to administration.

6. The method according to claim 5 wherein said precursor comprises plasminogen.

7. The method according to claim 5 wherein said activator comprises soluble streptokinase.

8. The method according to claim 5 wherein said composition further includes a plasmin stabilizing agent.

9. The method according to claim 1 wherein said plasmin or enzymatically equivalent derivative is selected from the group consisting of plasmin, microplasmin or miniplasmin derived from either human plasma or from recombinant technology, and combinations thereof.

10. The method according to claim 1 wherein the concentration of plasmin, or its enzymatically equivalent derivatives in the composition, is from about 10^'4 to about 10 percent by weight.

11. The method according to claim 1 wherein the composition has a pH in a range from about 5 to about 8.

12. The method according to claim 1 wherein the composition further comprises a buffer.

13. The method according to claim 1 wherein the composition further includes a plasmin stabilizing agent.

14. The method according to claim 13 wherein said plasmin stabilizing agent is selected from the group consisting of tranexamic acid, ε-aminocaproic acid, L-lysine, analogs of L-lysine, L-arginine, L-ornithine, γ-aminobutyric acid, glycylglycine, digiycine, gelatin, human serum albumin ("HSA"), glycerin, combinations thereof, and mixtures thereof.

15. The method according to claim 1 wherein said composition comprising plasmin-like material or its enzymatically equivalent derivative further includes a bioadhesive.

16. The method according to claim 15 wherein said bioadhesive is a mucoadhesive.

17. The method according to claim 1 wherein said composition further includes a gelling agent.

18. The method according to claim 1 wherein said composition further includes a bioadhesive and a gelling agent.

19. The method according to claim 1 wherein said composition further includes a medicament.

20. The method according to claim 19 wherein said medicament is an antiinflammatory agent.

21. The method according to claim 1 wherein said composition further includes an injectable anesthetic.

22. A composition comprising plasmin or an enzymatically equivalent derivative thereof and a gelling agent

23. A composition comprising plasmin or an enzymatically equivalent derivative thereof and a bioadhesive.

24. A composition comprising plasmin or an enzymatically equivalent derivative thereof and a medicament.

25. A composition comprising plasmin or an enzymatically equivalent derivative thereof and an injectable anesthetic.