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WO2009067407A2 - Compositions de métalloprotéinase matricielle activant des endopeptidases pour réduire la pression intraoculaire, et procédés d'utilisation de celles-ci - Google Patents

Compositions de métalloprotéinase matricielle activant des endopeptidases pour réduire la pression intraoculaire, et procédés d'utilisation de celles-ci Download PDF

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Publication number
WO2009067407A2
WO2009067407A2 PCT/US2008/083783 US2008083783W WO2009067407A2 WO 2009067407 A2 WO2009067407 A2 WO 2009067407A2 US 2008083783 W US2008083783 W US 2008083783W WO 2009067407 A2 WO2009067407 A2 WO 2009067407A2
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Prior art keywords
plasmin
acid
compounds
enzyme
mmp
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WO2009067407A3 (fr
Inventor
Gregory L. Mcintire
Alyson Berliner
Norman D. Lafrance
Tyle Praveen
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Bausch and Lomb Inc
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Bausch and Lomb Inc
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
    • A61K38/482—Serine endopeptidases (3.4.21)
    • A61K38/484—Plasmin (3.4.21.7)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents

Definitions

  • the present invention relates to compositions of matrix metalloproteinase (MMP) activating endopeptidases such as plasmin and plasmin derivatives including, but not limited to, microplasmin, miniplasmin, recombinant truncated plasmin, etc., and to methods for the use of these compositions for the treatment of diseases of the eye by the remodeling of extracellular matrix (ECM).
  • MMP matrix metalloproteinase
  • compositions of MMP-activating endopeptidases such as plasmin and plasmin derivatives, alone or in combination, to reduce or eliminate ECM-associated blockage/clogging of the aqueous humor outflow channel through the trabecular meshwork that can produce elevated intraocular pressure and diseases of the eye resulting from such elevated pressure, e.g., glaucoma.
  • the eye can be divided into three chambers, the anterior, the posterior, and the vitreous.
  • the anterior and posterior chambers are filled with aqueous humor, a fluid with an ionic composition similar to blood plasma.
  • the aqueous humor has two primary functions in the eye: to supply nutrients to the structures of the eye that lack a blood supply (the cornea and lens); and, to maintain the pressure within the eye (the intraocular pressure, or lOP) within a normal range.
  • lOP intraocular pressure
  • Both of the roles of the aqueous humor in supplying nutrition and intraocular pressure regulation are critical to normal visual function, and severe damage to the eye can result when either of these functions are compromised.
  • POAG primary open-angle glaucoma
  • TM trabecular meshwork
  • the aqueous humor flows sequentially through the three different layers of the TM: the uveal meshwork, which is a relatively open meshwork of cells; the corneoscleral meshwork, which is a tighter meshwork which offers increased resistance to the outflow of aqueous humor; and, finally, the juxtacanalicular or cribriform meshwork, which is a relatively high fluid-flow- resistant region that consists of cells embedded in a dense extracellular protein matrix (alternatively, extracellular matrix or ECM) adjacent to the cells of Schlemm's canal.
  • ECM extracellular matrix
  • MMPs matrix metalloproteinases
  • interstitial collagenase gelatinase, or type IV collagenase
  • stromelysin or proteoglycanase a group of proteins of particular interest in this process.
  • MMPs matrix metalloproteinases
  • the MMPs are proteolytic enzymes that act to cleave various components of the ECM, i.e., are enzymes that act to increase ECM degradation.
  • An alternative to increasing levels of MMPs in the TM by, for example, microinjection of exogenous MMPs is to increase the levels of these enzymes in the TM region by increasing the conversion of the inactive precursor forms of these enzymes already present in this region of the eye to the ECM-degrading active forms of these enzymes.
  • This conversion from precursor MMPs (synonymously "inactive MMPs” or “inactive precursor MMPs”) to their active forms (synonymously "active MMPs" or "MMPs”) can be accomplished by a variety of MMP-activating endopeptidases, including particularly the MMP- activating endopeptidase plasmin, as is peripherally disclosed in, e.g., U.S. Patent No. 5,260,059 to Acott et al.
  • endopeptidase-based methods of precursor MMP activation suffer from a variety of problems related to the length of time that these endopeptidases are available to activate precursor MMPs, because outflow of aqueous humor, even when reduced by clogging of the TM, can be expected to dilute down or otherwise wash away these endopeptidases from the areas where they are needed to activate precursor MMPs. Specifically, aqueous humor continues to flow out through the TM even when there is clogging in this region.
  • MMP-activating endopeptidase such as plasmin in this region to activate precursor MMPs to MMPs able to remodel ECM will have limited or no efficacy if aqueous humor outflow sufficiently dilutes down or washes away the MMP-activating endopeptidase(s) locally delivered for this purpose.
  • compositions comprising MMP- activating endopeptidases, particularly the MMP-activating endopeptidase plasmin, that extend the time frame during which these endopeptidases are able to activate precursor MMPs in or in the vicinity of the TM, and therefore increase the ability of the active MMPs so produced to dissolve ECM in this region, thereby increasing the outflow of aqueous humor and decreasing potentially dangerously elevated intraocular pressure levels in the eye.
  • MMP-activating endopeptidases particularly the MMP-activating endopeptidase plasmin
  • the present invention is generally directed to compositions comprising MMP-activating endopeptidase(s) and agents that act to extend the time during which these endopeptidases are able to activate precursor MMPs in the eye, particularly precursor MMPs in or in the vicinity of the TM region of the eye, and to methods for delivering these compositions to the desired region(s) of the eye.
  • the present invention is directed to compositions comprising the MMP-activating endopeptidase plasmin, plasmin derivatives, or combinations thereof (referred to collectively as "plasmin compounds") and agents that act to extend the time during which these plasmin compounds are able to activate precursor MMPs in the eye, particularly precursor MMPs in or in the vicinity of the TM region of the eye.
  • These agents comprise retention agents. They can be those that act to increase the retention of the plasmin compounds of the invention in a particular region of the eye, particularly in the TM, or in the vicinity of the TM, thereby maximizing the time in which the plasmin is able to exert its effect on the activation of precursor MMPs in or in the vicinity of the TM.
  • these agents can comprise stabilizing agents that stabilize the activity of these plasmin compounds during storage in vitro prior to their administration in vivo, and, also include stabilizing agents that additionally or separately act to stabilize the activity of these plasmin compounds in vivo.
  • the present invention is directed to methods for relieving intraocular pressure, where these methods comprise the administration of one or more MMP-activating endopeptidase, and particularly the administration of one or more of the plasmin compounds of the invention with one or more of the retention agents of the invention, one or more of the stabilizing reagents of the invention, or a composition comprising one or more of a combination of these three components.
  • These compositions preferably also comprise a pharmaceutically acceptable carrier.
  • administration is preferably to the TM or to the vicinity of the TM, with the methods of such administration described below.
  • Figure 1 provides a schematic drawing of the structure of the human eye as viewed in a sagital section (e.g., front to back).
  • Figure 2 provides a schematic drawing showing the activation pathway of pro-MMPs. Adapted from Circulation Research, 2003, 92, 827.
  • Figure 3 shows plasmin activity in buffers containing 40 mM ⁇ - aminocaproic acid or tranexamic acid.
  • Figure 4 shows plasmin activity in buffers containing 40 mM ⁇ - aminocaproic acid and another additive.
  • Figure 5 shows plasmin activity in buffers containing 40 mM ⁇ - aminocaproic acid and other additives, as indicated.
  • Figure 6 shows plasmin activity in buffers containing 0.4 M y- aminobutyric acid, 0.5 M L-ornithine hydrochloride, or 0.5 M glycylglycine.
  • Figure 7 shows plasmin activity in buffers containing L-lysine hydrochloride or tranexamic acid.
  • Figure 8 shows the effect of ⁇ -aminocaproic acid on plasmin activity in pig vitreous at 37 9 C.
  • Figure 9 shows the effect of diglycine on plasmin activity in pig vitreous at 37 2 C.
  • Figure 10 shows the effects of 5-aminovaleric acid on plasmin activity in pig vitreous at 37 2 C.
  • Figure 11 shows the effects of betaine and streptokinase on plasmin activity in pig vitreous at 37 2 C.
  • Figure 12 shows the effect of TXA on plasmin activity in pig vitreous at 37 2 C.
  • Figure 13 shows the effect of ⁇ -aminocaproic acid on plasmin activity in pig vitreous at 37 2 C.
  • Figure 14 shows the effects of various concentrations of TXA in combination with Tween 80 ® on plasmin activity in pig vitreous at 37 2 C.
  • Figure 15 shows the effect of ⁇ -aminocaproic acid on activity of human plasmin and recombinant truncated plasmin in pig vitreous at 37 2 C.
  • the present invention is directed to compositions comprising at least one MMP-activating endopeptidase, including plasmin, plasmin derivatives, or combinations thereof (collectively "plasmin compounds"), in combination with one or more retention agent, one or more stabilizing agent, or combination of one or more retention agent and one or more stabilizing agent.
  • the retention agents of the invention act to increase the retention of the plasmin compounds of the invention in a particular region, particularly in the TM, or in the vicinity of the TM 1 preferentially by counterbalancing the diluting and or washing-away effects of aqueous humor outflow through the TM.
  • the stabilizing agents of the invention act to stabilize the activity of the plasmin compounds of the invention in vitro, in vivo, or both in vitro and in vivo.
  • the present invention is also directed to methods for relieving intraocular pressure, where these methods comprise the administration of one or more of the MMP-activating endopeptidases of the invention (e.g., the plasmin compounds of the invention) with one or more of the retention agents of the invention, one or more of the stabilizing reagents of the invention, or a composition comprising one or more of a combination of these three components.
  • These compositions preferably also comprise a pharmaceutically acceptable carrier.
  • administration is preferably to the TM or to the vicinity of the TM.
  • administration may be by any method known to the artisan, including, e.g., injection, topical administration, etc.
  • administration may be by topical administration to the anterior surface of the eye (e.g., the cornea) either by directly administering drops to the eye or via application of a composition between the eye lid and the eye wherein the composition can be comprised of surfactants and water (e.g., micelles); surfactants, polymers, and water (e.g, micelles, emulsions); surfactants, oils, and water (e.g., emulsions); phospholipids, oils, and water (e.g., liposomes and/or emulsions); and any other composition as known in the pharmaceutical art (e.g., see Remington, the Science and Practice of Pharmacy, 21 st Ed, part 5 pp 745-888, and Ophthalmic Drug Delivery Systems, 2 nd Ed, Drugs and the Pharmaceutical Sciences, Vo1 130, Section III, pp 281-662) for application to the front surface of the eye.
  • surfactants and water e.g., micelles
  • surfactants, polymers, and water e.
  • administration may be via injection either by peri-ocular injection into the conjunctival space around the eye, into the inner side of the eyelid, or by subscleral injection, intracameral injection, intravitreal injection, or via any other injectable route of administration into the tissues of the eye.
  • administration may be retrograde via Schlemm's canal; that is the injection may be through the sclera into Schlemm's canal with pressure maintained for a length of time necessary to afford MMP activation in the juxticanicular TM.
  • solutions are used for injectable administration, it is conceivable that the formulations discussed above would be suitable for injection as well as particle suspensions, nanoparticle suspensions, polymeric rods carrying drug, implantable devices (e.g., Retisert®) and by iontophoretic administration as known in the art (see Remington).
  • compositions of the present invention may include an anti-inflammatory agent or agents, e.g., soft steroids, hard steroids, NSAIDs, antibacterials, antivirals, etc., including, but not limited to, loteprednol, dexamethasone, triamcinolone acetinide, fluocinolone acetinide, Quixin® (levofloxacin ophthalmic solution) and other fluoroquinalone antibacterial agents (e.g., 1 st , 2 nd , 3 rd , and 4 th generation agents), viroptic® (trifluridine ophthalmic solution) and other antivirals.
  • an anti-inflammatory agent or agents e.g., soft steroids, hard steroids, NSAIDs, antibacterials, antivirals, etc., including, but not limited to, loteprednol, dexamethasone, triamcinolone acetinide, fluocinolone acetinide, Quixin® (lev
  • anti-inflammatory agents such as Zylet® (loteprednol and tobramycin) and Tobradex® (dexamethasone and tobramycin) in combination with and post administration of the MMP activating endopeptidase of the invention.
  • anti-inflammatory agents such as any or all of those included in the PDR for Ophthalmic Medicines (35 th edition, 2007) and those included more broadly in the Physician's Desk Reference (61 st edition, 2007) may be used in conjunction with the MMP activating compounds envisioned within this invention.
  • MMP-activating endopeptidase refers to any endopeptidase capable of activating a matrix metalloproteinase (MMP).
  • MMP matrix metalloproteinase
  • endopeptidases include, but are not limited to, e.g., trypsin, chymotrypsin, elastase, thermolysin, pepsin, and endopeptidase V8 (See Figure 2), and particularly include the “plasmin compounds” of the invention, as described in detail below.
  • endopeptidases include: Trypsin, which cuts after Arg or Lys, unless followed by Pro and is very strict; Chymotrypsin, which cuts after Phe, Trp, or Tyr unless followed by Pro, and cuts more slowly after Asn, His, Met, or Leu; Elastase, which cuts after Ala, GIy, Ser, or VaI, unless followed by Pro; Thermolysin, which cuts before lie, Met, Phe, Trp, Tyr, or VaI, unless preceded by Pro, and sometimes cuts after Ala, Asp, His, or Thr, and is heat stable; Pepsin, which cuts before Leu, Phe, Trp, or Tyr, unless preceded by Pro, and also at others, and is quite nonspecific and works best at pH 2; and Endopeptidase V8, which cuts after GIu.
  • Plasmin compounds refers to the serine proteinase plasmin, as well as to derivatives of plasmin including, but not limited to, miniplasmin, and microplasmin.
  • the plasmin derivatives of the invention include, more generally, any amino acid sequence derived from or related to the plasmin sequence that retains the basic enzymatic function of plasmin.
  • Retention of the basic or core enzymatic function of plasmin may be judged based on the presence within the amino acid sequence of the enzymatic domain of plasmin as defined by its amino acid sequence. Retention of this function may additionally or separately be defined experimentally by measuring "plasmin activity," so that a plasmin compound is selected to provide, e.g., plasmin activity as defined by chromogenic assay using the plasmin substrate S- 2251. Although any assay for plasmin activity is contemplated herein, additional assays include assays for measuring the ability to cause the activation of MMP from its inactive precusor form.
  • Such assays may include, for example, gel electrophoresis methods to determine the amount of conversion of the longer precursor MMP to its cleaved active form.
  • Such an assay is provided in, for example, Takano et al., Am. J. Opthal. (2005) 140(4): 654-660.
  • Other methods for measuring activation include immunoblotting or other techniques; see, e.g., U.S. Patent No. 5,260,059, the contents of which is herein incorporated by reference in its entirety.
  • An additional experimental assay for plasmin activity is end-result- based, i.e., is directed to determining the extent to which a plasmin compound results in the breakdown of ECM, or results in a reduction of intraocular pressure.
  • Non-limiting examples of such assays are provided in U.S. Patent Nos. 5,260,059 and 6,020,181 , the contents of which are herein incorporated by reference in their entireties.
  • the "plasmin compounds” of the invention comprise the serine proteinase plasmin, and also derivatives of plasmin .
  • derivatives of an enzyme encompass variants of the enzyme that still substantially retain the basic enzymatic function of the enzyme. Such variants can be modified forms of the enzyme, such as for example a truncated form wherein one or more amino acid residues or segments of the enzyme molecule are deleted. Such variants also can be a form of the enzyme wherein one or more amino acid residues are substituted, such as by conservative substitutions, or wherein one or more amino acid residues are added to the polypeptide.
  • a derivative of plasmin encompasses a polypeptide that is a fragment or portion thereof that can comprise the enzymatic or catalytic domain or region of plasmin.
  • a derivative of plasmin can further comprise a kringle domain or region of the plasmin molecule.
  • a kringle domain of plasmin is characterized by a triple-loop conformation and comprises about 75-85 amino acid residues with three disulfide bridges.
  • microplasmin which comprises the serine proteinase enzymatic domain of plasmin and a short polypeptide sequence (e.g., comprising about 25-40 amino acid residues) between the enzymatic domain and where it would normally be connected to the kringle-5 domain of plasmin.
  • a derivative of plasmin can be a miniplasmin, which comprises the kringle-5 domain and the enzymatic domain of plasmin.
  • Enzymatically active microplasmin and miniplasmin are obtained from microplasminogen and miniplasminogen precursors by cleavage of the peptide bond at Arg 561 -Val 562 , wherein the amino acid residue numbers correspond to those of human Glu-plasminogen, which has 791 amino acid residues.
  • Microplasminogen and miniplasminogen are disclosed in U.S.
  • Patent Application Publications 2004/0071676 A1 (US provisional application S/N 60/564,472) and 2005/0124036 A1 (US provisional applications S/Ns 60/728,615 and 60/732,588), which are incorporated herein by reference in their entirety.
  • a derivative of plasmin can comprise one or more kringle domains (i.e., one or more kringle-1 , -2, -3, -4, and -5) attached in any order to the enzymatic domain.
  • a derivative of plasmin can be a material known as angiostatin, which comprises only one or more kringle domains of plasmin, without its enzymatic domain, such as 3 to 5 contiguous kringle domains.
  • a derivative of plasmin can be any of the plasmin derivatives recited in International Publication Numbers WO2005/105990 or WO2007/047874, the contents of which are herein incorporated by reference in their entireties.
  • active plasmin can be generated in vivo by co-administration with tissue plasminogen activator, urokinase, streptokinase, or any other plasminogen activator that is acceptable for injection into the body (e.g, nattokinase, etc.). See, e.g., U.S. Patent No. 6,899,877, the contents of which are herein incorporated by reference in its entirety.
  • Plasmin can be produced by activation of plasminogen precursor, which may be obtained from plasma.
  • plasminogen precursor which may be obtained from plasma.
  • plasminogen precursor a method of producing high- purity plasmin is disclosed in U.S. Patent Application Publication 2004/0171103 A1 , which is incorporated herein by reference in its entirety.
  • the starting material, plasminogen can be extracted from Cohn Fraction ll+lll paste by affinity chromatography on Lys-SEPHAROSETM as described by D.G. Deutsch and E.T. Mertz, "Plasminogen: purification from human plasma by affinity chromatography," Science 170(962): 1095-6 (1970).
  • SEPHAROSETM is a trade name of Pharmacia, Inc., New Jersey.
  • lipid and protein impurities and Transmissible Spongiform Encephalopathies ( 1 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.
  • PEG polyethylene glycol
  • the PEG or ammonium sulfate precipitate is removed by depth filtration and the resulting solution placed on a lysine affinity resin column.
  • lysine affinity resin is used generally for affinity resins containing lysine or its derivatives or ⁇ -aminocaproic acids as the ligand.
  • the column can be eluted with a solution having a low pH of approximately 1 to 4.
  • 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 ⁇ -amino acids.
  • Plasminogen in solution is then 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.
  • a plasminogen activator is soluble streptokinase.
  • stabilizers or excipients such as glycerol, ⁇ -amino acids such as lysine, polylysine, arginine, ⁇ -aminocaproic acid and tranexamic acid, and salt can enhance the yield of plasmin.
  • 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.
  • a polishing step for the removal of remaining streptokinase hydrophobic interaction chromatography ("HIC") at low pH is performed (e.g., pH ⁇ 4).
  • HIC hydrophobic interaction chromatography
  • plasmin is formulated as a sterile protein solution by ultrafiltration and diafiltration and 0.22- ⁇ m filtration.
  • 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 which includes at least one amino acid, or a combination thereof.
  • 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.
  • 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.
  • buffer added is typically that which will give the reversibly inactive acidified plasmin solution a pH between about 2.5 to about 4, or between about 3 and about 3.5.
  • a stabilizing or bulking agent to the reversibly inactive acidified plasmin solution obtained as disclosed above.
  • stabilizing or bulking agents are a polyhydric alcohols, 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.
  • 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 0.01 M to about 1 M.
  • Plasmin formulated according to the method disclosed above in buffered acidified water has been found to be very stable. It can be kept in this form for months without substantial loss of activity or the appearance of degradation products of a proteolytic or acidic nature. At 4 0 C, such plasmin is stable for at least nine months. Even at room temperature, such plasmin is stable for at least two months.
  • 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 O 0 C to about -5O 0 C, or preferably from about O 0 C to about -20 0 C, to produce a powder for long-term storage.
  • plasmin or variants thereof can be produced by recombinant technology, and a method of the present invention is applied to such plasmin and variants thereof.
  • the production of recombinant microplasminogen (which can be activated to microplasmin by cleavage of the peptide bond at Arg 561 -Val 562 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 , which is incorporated herein by reference.
  • Plasminogen and miniplasminogen (which also can be activated to miniplasmin by cleavage of the peptide bond at Arg 561 -Val 562 using one of the plasminogen activators disclosed above) in the Pichia pastoris yeast system is disclosed in U.S. Patent Application Publication 2005/0124036 A1 , which is incorporated herein by reference.
  • retention agent refers to agents which increase the retention of the plasmin compounds of the invention in a particular region of the eye, and preferably in the TM, or in the vicinity of the TM. These retention agents can exhibit a variety of properties that increase the retention of the plasmin compounds of the invention in or in the vicinity of the TM, particularly properties to help counter dilution resulting from the active outflow of aqueous humor from the eye through the TM or other channels such as may result from both unintended tearing as well as surgically- induced drainage channels.
  • the "vicinity" of the TM refers generally to any area of the eye in fluidic contact with the anterior-chamber-facing side of the TM.
  • the vicinity of the TM includes, most broadly, any ocular tissue in contact with the aqueous humor within the anterior chamber of the eye.
  • the "vicinity” of the TM also includes the "near vicinity" close to the anterior-chamber-facing side of the TM where aqueous humor outflow is sufficiently high to create more powerful diluting or washing away effects.
  • the retention agent of the invention exhibit more powerful retention properties than need be exhibited further away from this near vicinity to the TM.
  • the retention reagents of the invention may exhibit a variety of properties that increase the retention of the plasmin compounds of the invention in the TM or in the vicinity of the TM.
  • Such properties include, for example, bulking properties that help counter the tendency toward dilution or washing away of the plasmin compounds of the invention in the vicinity of the TM by increasing the effective "bulk" of these compounds, e.g., their hydrodynamic size, and thereby inhibit the ability of these plasmin compounds to penetrate through the TM.
  • the retention reagents of the invention may exhibit binding properties that increase the stability or persistence of the plasmin compounds of the invention in or in the vicinity of the TM, including at the surface of the uveal meshwork, within the uveal meshwork, within the corneoscleral network, within the juxtacanalicular network, etc.
  • the present invention also includes additional methods of administration that may also act to facilitate retention, including, for example, administration to the TM via a retrograde injection.
  • injection into the Schlemm's canal of the MMP activating agent with or without the retention agents described herein is contemplated as useful in pushing agent into the TM from the normal "backside" of the tissue thereby exposing TM to the lytic properties of activated MMPs.
  • a colored or fluorescent tracer could be additionally included in order to observe the progress of such an injection; such inclusion may make the dosing and retention of that dose via the Schlemm's canal possible or easier for the physician/surgeon.
  • paracentesis could also be used either in concert with or precedent to the retrograde injection to lower IOP and avoid a spike in IOP from the reverse flow in the TM during the injection.
  • the retention reagents of the invention may be defined a priori by known bulking or binding characteristics of known compounds. Such a priori definition may also be applied on other bases, since bulking and binding properties are not intended to define the entire universe of the advantageous properties of the binding reagents of the invention.
  • the retention reagents of the invention may be defined by determining experimentally whether a particular reagent increases the retention time of a plasmin compound of the invention in or in the vicinity of the TM.
  • Such retention time in the TM or in the vicinity of the TM may be determined by a variety of methods, including, for example, labeling the plasmin by, e.g., a radioactive label, and determining by radioactivity retention or autoradiography the time course for label loss from the TM, or further by covalently or non-covalently "tagging" the MMP activating endopeptidase (e.g., plasmin) with a dye such as fluorescein which is known to be safe in the tissues of the eye.
  • a dye such as fluorescein which is known to be safe in the tissues of the eye.
  • the retention agent is selected from the group consisting of viscosity modifying compounds, dimensionaliy large compounds such as antibodies, gel forming compounds, micelle forming compounds either classical surfactants or polymeric surfactants, liposome forming compounds such as phosphatidyl choline, other phospholipids and cholesterol, emulsion forming compounds either water-in-oil or oil-in-water emulsions including the oil, the surfactant(s), and the continuous phase solvent (e.g., normally water), a solid dispersion formed from an inert, degradable particle suspension or a suspension of the endopeptidase itself.
  • viscosity modifying compounds dimensionaliy large compounds such as antibodies, gel forming compounds, micelle forming compounds either classical surfactants or polymeric surfactants, liposome forming compounds such as phosphatidyl choline, other phospholipids and cholesterol
  • emulsion forming compounds either water-in-oil or oil-in-water emulsions including the
  • the retention agents of the invention include, but are not limited to, homogeneous solutions of elevated viscosity such as those created by adding soluble polymers (e.g., soluble cellulose derivatives, Human serum albumin, soluble starch, Hyaluronic acid, synthetic polymers such as polyvinylpyrrolidinone (PVP), polyvinylalcohol (PVA), Polyethylene glycol (PEG), etc.) at concentrations sufficient to increase the viscosity of the solution as is known in the art (Remingon). Additionally, high concentrations of mono and disaccharides result in "syrups" which are known to be high in viscosity. Finally, high concentrations of iodinated X-ray contrast agents are also known to be high in viscosity, safe for injection, and dense adding to their efficacy as retention agents.
  • soluble polymers e.g., soluble cellulose derivatives, Human serum albumin, soluble starch, Hyaluronic acid, synthetic polymers such as polyvinylpyrrol
  • the retention agents of the invention further include dimensionally large molecules that would temporarily occlude the TM such that the enzyme would be resident long enough to activate MMPs and hence breakup the extracellular matrix.
  • Such molecules include serum albumin, anti-inflammatory antibodies (e.g., infliximab, etanercept, or adilimumab), anti-VEGF antibodies (e.g., Bevacizumab (Avastin®)), very high molecular weight PEG molecules, and any other soluble, dimensionally large molecule. It should be clear that the endopeptidase would not only activate the pro-MMPs resident near or in the TM but would also lyse large proteins and thus aid in their clearance from the TM with time.
  • the retention agents of the invention further include a special class of compounds which are known to be reverse thermal gel forming agents. That is to say they are solutions at lower temperatures (e.g., room temperature) and yet upon exposure to elevated temperature they form gels, thereby increasing the retention of the enzyme in or near the TM.
  • Such agents are best illustrated by the pluronic surfactants also known as poloxamers, block copolymers of an A-B- A form wherein the A portion is formed from polyethylene glycol while the B portion is formed from polypropylene glycol.
  • the gel forming properties of these agents are described in, e.g., B.K.
  • additional mechanisms of formation including pH shift and ionic strength shift.
  • the retention agents of the invention also include micellar solutions containing the enzyme(s) of interest, with excipients that control pH and ionic strength.
  • Said micellar solution containing the enzyme of interest include those solutions wherein the surfactant composition of the micelle preferably affords a positively charged micelle, more preferably yields a negatively charged micelle, and most preferably results in a non charged (i.e., neutral) micelle; these would be administered to the TM of the eye to increase the "retention" of the active enzyme without significantly inhibiting the activity of the enzyme.
  • micellar solutions containing the enzyme of interest wherein the surfactant composition of the micelle is primarily monomeric surfactant molecules.
  • micellar solutions containing the enzyme of interest wherein the composition of the micelle is primarily polymeric surfactant or surfactants (e.g., Tweens, Spans, Pluronics, Tetronics, Myj, Brij, and polyethylene glycol (PEG)) either alone or in combination with each other or in combination with classical small molecule surfactants.
  • polymeric surfactant or surfactants e.g., Tweens, Spans, Pluronics, Tetronics, Myj, Brij, and polyethylene glycol (PEG)
  • PEG polyethylene glycol
  • the retention agents of the invention can further be comprised of a suspension either containing the enzyme or of the enzyme alone wherein the enzyme can be the solid suspended particle or present in solution in the solution phase.
  • Said suspension either containing the enzyme or of the enzyme alone wherein the enzyme can be the solid suspended particle or present in solution in the solution phase together with excipients to control the pH of the solution phase of the suspension, with excipients to control the osmolality of the solution phase of the suspension, with excipients to control the pH and ionic strength and osmolality of the solution phase of the suspension, and additional excipients which provide stability to the enzyme during changes in pH, or which provide optimal lyophilization of the serine proteinase enzyme including appearance of the freeze dried cake, reconstitution time using water or a mixture of water and nonaqueous solvent or a nonaqueous solvent alone, and preservation of activity of the enzyme can be administered to the TM to enhance the "retention" of the enzyme in the TM and thus activate MMPs and result in increased outflow
  • suspensions it is meant to describe a dispersion of solid particles within a continuous liquid phase.
  • these dispersions require special additives to afford physical stability and particle size control to the suspension such as surfactants and polymers as are well known in the art. Processes for production of such suspensions are well known in the art and are described in various textbooks (i.e, Remington, Martindale), regulatory guidelines (i.e., USP, EP, JP), and the literature including patents and publications all of which are herein embodied in this disclosure.
  • the retention agents of the invention can further be comprised of a liposome solution (including, but not limited to, phosphatidyl choline, other phospholipids and cholesterol, etc.) containing the active enzyme resulting from a frozen liposome solution containing the enzyme or a lyophilized liposome solution containing the enzyme together with excipients to control the pH of the aqueous phase of the liposome solution, with excipients to control the osmolality of the liposome solution, with excipients which provide optimal lyophilization of the serine proteinase enzyme liposome solution including appearance of the freeze dried cake, reconstitution time using water or a mixture of water and nonaqueous solvent or a nonaqueous solvent alone, and preservation of activity of the enzyme, with excipients that stabilize the enzyme to changes in pH, ionic strength, and osmolality.
  • a liposome solution including, but not limited to, phosphatidyl choline, other phospholipids and cholesterol, etc.
  • the enzyme may reside within the liposome, outside of the excluded volume of the liposome or both within and outside the liposome bilayer and further that the term "liposome” may represent unilamellar vesicles, multilamellar vesicles, chocleates, and 'niosome” vesicles where niosomes are known in the art as a nonaqueous core stabilized by a monolayer of phospholipids rather than the traditional bilayer of phospholipids. It is further understood that the composition of the bilayer in the liposome solution is also claimed in the delivery of these serine proteinase enzymes to the vitreous. Further, it is understood that individual particle size of the liposome solutions may vary from less than 80 nm to greater than 1000 nm.
  • the retention agents of the invention can further be comprised of an emulsion such as an oil-in-water emulsion containing the enzyme of interest with excipients to control pH, ionic strength and osmolality, with excipients that stabilize the enzyme to changes in pH and ionic strength, wherein the oil phase stabilizes the enzyme of interest to changes in pH.
  • an emulsion such as an oil-in-water emulsion containing the enzyme of interest with excipients to control pH, ionic strength and osmolality, with excipients that stabilize the enzyme to changes in pH and ionic strength, wherein the oil phase stabilizes the enzyme of interest to changes in pH.
  • the enzyme would most likely be resident in the continuous aqueous phase of these oil-in-water emulsions.
  • the enzyme may also be dosed in water-in-oil emulsions wherein it will be resident in the water pockets suspended in the continuous nonaqueous phase. In that instance, the presence of various excipients within
  • the present invention includes, most generally, emulsions such as water-in-oil, oil-in-water, emulsions including the oil, the surfactant(s), and the continuous phase solvent (e.g., normally water), a solid dispersion formed from an inert, degradable particle suspension or a suspension of the endopeptidase itself, etc., although the present invention is not limited to these specifically stated emulsions and includes other emulsions as would be known to one of ordinary skill.
  • emulsions such as water-in-oil, oil-in-water, emulsions including the oil, the surfactant(s), and the continuous phase solvent (e.g., normally water), a solid dispersion formed from an inert, degradable particle suspension or a suspension of the endopeptidase itself, etc.
  • the retention agents of the invention can further be comprised of a rapidly dissolving tablet containing the enzyme of interest for insertion of the rapidly dissolving tablet into the aqueous humor near the TM.
  • the rapidly dissolving tablets of the invention will also contain excipients to provide properties important in the preparation of tablets including compressibility, lubricity, hardness, and density and excipients that stabilize the enzyme to changes in pH and ionic strength, and excipients that control the release of the active enzyme for periods of minutes to hours.
  • Such tablets are known in the art and comprise Mini Tablets with dimensions of .5 mm ⁇ diameter ⁇ 4 mm and more preferably 1.0 mm ⁇ diameter ⁇ 2 mm and most preferably 1.25 mm ⁇ diameter ⁇ 1.75 mm with length determined by dose (concentration of enzyme) in the tablet mixture. Generally, length is ⁇ 10 mm and more preferably ⁇ 5 mm and most preferably ⁇ 2 mm.
  • the retention agents of the invention can further be comprised of a powder, as a powder mixed with excipients to control pH and ionic strength, as a powder mixed with excipients and suspended in nonaqueous solvents (e.g., mineral oil, vitamin e, silicon oil, perfluorocarbon oils, vegetable oils, peanut oil, safflower oil, glycerin, as a powder mixed with excipients and granulated into particles for administration into the eye, as a powder mixed with excipients and granulated and sieved for administration into the eye via aerosol or suspended in solvents as given above.
  • nonaqueous solvents e.g., mineral oil, vitamin e, silicon oil, perfluorocarbon oils, vegetable oils, peanut oil, safflower oil, glycerin
  • autodegradable enzyme and “autolyzable enzyme” are used interchangeably and mean an enzyme that is capable of breaking down, digesting, degrading, or hydrolyzing its own molecule due to its enzymatic or catalytic activity.
  • physiological pH means pH of about 7.2-7.6.
  • the present invention provides a composition comprising an enzyme that has been preserved at a pH less than about 5 and is subsequently reconstituted in a formulation comprising a material selected from the group consisting of tranexamic acid, ⁇ -aminocaproic acid, analogs of L-lysine other than tranexamic acid and ⁇ -aminocaproic acid, combinations thereof, and mixtures thereof; wherein said enzyme is autodegradable at a pH greater than about 5.
  • combination encompasses, but is not limited to, two or more 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 interpenetrating molecules, or a complex comprising two or more molecules by, e.g., bonding or conformational interaction.
  • the material is tranexamic acid.
  • the material is a combination or mixture of tranexamic acid and ⁇ -aminocaproic acid.
  • the material is an analog of L-lysine other than tranexamic acid and ⁇ -aminocaproic acid.
  • analogs of L-lysine include L-2-amino-3-guanidinopropionic acid, L-citruline, D- citruline, 2,6-diaminoheptanoic acid, ⁇ , ⁇ -dimethyl-L-lysine, ⁇ -methyl-DL-ornithine, ⁇ -benzyloxycarbonyl-L-omithine, (N-d-4-methyltrityl)-L-omithine, N- ⁇ -1 -(4,4- dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl)-D-omithine, p-aminomethylbenzoic acid, and 2-aminoethylcysteine.
  • the material is a mixture of tranexamic acid and an analog of L-lysine other than tranexamic acid and ⁇ -aminocaproic acid.
  • the formulation used in the composition further comprises a compound selected from Group 1 , Group 2, and Group 3; wherein Group 1 consists of L-lysine, L-arginine, L-ornithine (or its pharmaceutically acceptable salts; e.g., L-ornithine hydrochloride), ⁇ -aminobutyric acid, 5- aminovaleric acid, 7-aminoheptanoic acid, glycylglycine, triglycine, N- ⁇ -acetyl-L- arginine, betaine, sarcosine, combinations thereof, and mixtures thereof; Group 2 consists of gelatin, human serum albumin ("HSA"), streptokinase, tPA, uPA, combinations thereof, and mixtures thereof; and Group 3 consists of non-ionic surfactants, glycerin, D-sorbitol, combinations thereof, and mixtures thereof.
  • Group 1 consists of L-lysine, L-arginine, L-or
  • the formulation comprises a mixture of: (1 ) tranexamic acid, ⁇ -aminocaproic acid, or an analog of L-lysine other than tranexamic acid and ⁇ -aminocaproic acid; and (2) a compound of either Group 1 , Group2, or Group 3.
  • the formulation has a pH corresponding approximately to a pH at which said enzyme has the highest activity in a preselected reaction or use.
  • the compound is selected from Group 1. In another embodiment, the compound is selected from Group 2. In still another embodiment, the compound is selected from Group 3. In yet another embodiment, the compound is selected from the group of non-ionic surfactants.
  • Group 1 consists of L-ornithine (or its pharmaceutically acceptable salts; e.g., L-ornithine hydrochloride), y- aminobutyric acid, 5-aminovaleric acid, 7-aminoheptanoic acid, glycylglycine, triglycine, N- ⁇ -acetyl-L-arginine, betaine, sarcosine, combinations thereof, and mixtures thereof; Group 2 consists of gelatin, streptokinase, tPA, uPA, combinations thereof, and mixtures thereof; and Group 3 consists of non-ionic surfactants, glycerin, combinations thereof, and mixtures thereof [0086]
  • the pH of the formulation is in the range from about 6.5 to about 11.
  • the pH of the formulation is in the range from about 6.5 to about 9, or from about 6.5 to about 8.
  • the formulation comprises a buffer having a pH in one of said pH range
  • the pH of the formulation changes by less than about 1 pH unit (or alternatively, less than about 0.5, or about 0.2, or about 0.1 pH unit) when the preserved enzyme is added into the formulation.
  • a composition of the present invention comprises an enzyme that has been preserved at a pH less than about 5 and is subsequently reconstituted in a formulation comprising tranexamic acid and a compound selected from Group 2; wherein said enzyme is autodegradable at a pH greater than about 5.
  • the formulation can have a pH corresponding approximately to a pH at which said enzyme has the highest activity in a preselected reaction or use.
  • a composition of the present invention comprises an enzyme that has been preserved at a pH less than about 5 and is subsequently reconstituted in a formulation comprising: (a) ⁇ -aminocaproic acid; and (b) a compound selected from Group 2; wherein said enzyme is autodegradable at a pH greater than about 5.
  • the enzyme has been preserved at a pH less than about 4 (or alternatively, at a pH in the range from about 2.5 to bout 4, or from about 2.5 to about 3.5).
  • the formulation has a pH corresponding approximately to a pH at which said enzyme has the highest activity in a preselected reaction or use.
  • the concentration of tranexamic acid, ⁇ -aminocaproic acid, or another analog of L-lysine other than tranexamic acid and ⁇ - aminocaproic acid in any formulation disclosed herein is in a range from about 1 ⁇ M to about 500 mM (or alternatively, from about 10 ⁇ M to about 200 mM, or from about 50 ⁇ M to about 100 mM, or from about 50 ⁇ M to about 20 mM).
  • the concentration of the compound in any formulation disclosed herein is in a range from about 0.001 to about 5 weight percent (or alternatively, from about 0.01 to about 4 weight percent, or from about 0.01 to about 2 weight percent).
  • a composition of the present invention comprises an enzyme that has been preserved at a pH less than about 5 and is subsequently reconstituted in a formulation comprising: (a) a mixture of tranexamic acid and ⁇ -aminocaproic acid; and (b) a compound selected from Group 2; wherein said enzyme is autodegradable at a pH greater than about 5.
  • the concentration of each of tranexamic acid and ⁇ - aminocaproic acid in the formulation is in a range from about 1 ⁇ M to about 500 mM (or alternatively, from about 10 ⁇ M to about 200 mM, or from about 50 ⁇ M to about 100 mM, or from about 50 ⁇ M to about 20 mM).
  • the compound is HSA.
  • the compound is gelatin.
  • the compound is a mixture of HSA and gelatin.
  • an additive binds reversibly to certain active regions of an enzyme molecule, thereby preventing it from catalyzing the break down of itself or other molecules of the same enzyme.
  • Different additives may bind to different regions of the enzyme molecule and provide a synergistic inhibiting effect.
  • the present invention provides a method for producing an active enzyme after prolonged storage, the method comprising: (a) storing said enzyme at a pH less than about 5; and (b) adding said enzyme to a formulation that comprises a material selected from the group consisting of tranexamic acid, ⁇ -aminocaproic acid, analogs of L-lysine other than tranexamic acid and ⁇ -aminocaproic acid, combinations thereof, and mixtures thereof; wherein said enzyme is autodegradable at a pH greater than about 5; to produce a formulated enzyme substantially immediately before using the enzyme or carrying out the reaction, wherein said enzyme is autodegradable at pH greater than about 5.
  • a formulation that comprises a material selected from the group consisting of tranexamic acid, ⁇ -aminocaproic acid, analogs of L-lysine other than tranexamic acid and ⁇ -aminocaproic acid, combinations thereof, and mixtures thereof; wherein said enzyme is autodegradable at a pH greater than about 5; to produce
  • the formulation used in the foregoing method further comprises a compound selected from Group 1 , Group 2, and Group 3; wherein Group 1 consists of L-lysine, L-arginine, L-ornithine, ⁇ -aminobutyric acid, glycylglycine, combinations thereof, and mixtures thereof; Group 2 consists of gelatin, HSA, combinations thereof, and mixtures thereof; and Group 3 consists of non-ionic surfactants, glycerin, combinations thereof, and mixtures thereof.
  • Group 1 consists of L-lysine, L-arginine, L-ornithine, ⁇ -aminobutyric acid, glycylglycine, combinations thereof, and mixtures thereof
  • Group 2 consists of gelatin, HSA, combinations thereof, and mixtures thereof
  • Group 3 consists of non-ionic surfactants, glycerin, combinations thereof, and mixtures thereof.
  • the compound is selected from the group of non- ionic surfactants.
  • the non-ionic surfactants are selected from the group consisting of 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® F68, Pluronic® F127, or Pluronic® F108) ), 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 Te
  • the pH of the formulation changes by less than about 1 pH unit (or alternatively, less than about 0.5, or less than about 0.2, or less than about 0.1 pH unit) when the preserved enzyme is added into the formulation.
  • the relative amounts of the enzyme, the material, the compound (when present), and other constituents of the formulation (when present) are thus chosen based on the desired maximum change in the pH of the solution, the enzyme, the type of material, the type of the compound (when present), and the types of other constituents (when present) without difficulty.
  • the concentration of tranexamic acid or ⁇ -aminocaproic acid in any formulation used in any method disclosed herein is in a range from about 1 ⁇ M to about 500 mM (or alternatively, from about 10 ⁇ M to about 200 mM, or from about 50 ⁇ M to about 100 mM, or from about 50 ⁇ M to about 20 mM).
  • the concentration of the compound in any formulation used in any method disclosed herein is in a range from about 0.001 to about 5 weight percent (or alternatively, from about 0.01 to about 4 weight percent, or from about 0.01 to about 2 weight percent).
  • the step of storing of said enzyme is effected at a pH less than about 4.
  • said pH is less than 3.5 or in the range from about 2.5 to about 4, or from about 2.5 to about 3.5, or from about 3 to about 3.5.
  • Recombinant plasmin or variants thereof are acidified and stored at pH less than about 5 (or alternatively less than about 4, or between about 2.5 and about 3.5).
  • the acidified enzyme is reconstituted by adding said enzyme to a formulation having pH corresponding approximately to a pH at which said enzyme has the highest activity in a preselected reaction or use and containing one or more additives disclosed above, to produce a formulated enzyme substantially immediately before using the enzyme or carrying out the reaction.
  • the formulation has a buffering capacity such that the pH of the formulation changes by less than about 1 pH unit upon adding said enzyme.
  • said formulation has a buffering capacity such that the pH of the formulation changes by less than about 0.5 (or alternatively, less than about 0.2, or less than about 0.1) pH unit upon adding said enzyme.
  • the formulation has a pH of about 7.
  • the formulation has a pH in a range from about 7 to about 7.5.
  • the formulation has a pH of about 7.4.
  • the formulation comprises a phosphate buffer or a Tris-HCI buffer (comprising tris(hydroxymethyl)aminomethane and HCI).
  • a Tris-HCI buffer having pH of 7.4 comprises 3 g/l of tris(hydroxymethyl)aminomethane and 0.76 g/l of HCI.
  • the buffer is 10X phosphate buffer saline ("PBS") or 5X PBS solution.
  • 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 pKa of 7.5 at 25 0 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 25°C and pH in the range of about 6.4-7.8; MOPS (3- ⁇ N-morpholino ⁇ propanesulfonic acid) having pK a of 7.2 at 25 0 C 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 25 0 C and pH in the range of about 6.8-8.2; MOBS (4- ⁇ N-morpholino ⁇ butanesul
  • the present invention provides a method for prolonging an activity of plasmin or derivatives thereof in a region of the eye, particularly in the anterior chamber of the eye, and more particularly in the TM or in the vicinity of the TM, the method comprising: (a) providing said plasmin or derivatives thereof that have been preserved at a pH less than about 5; and (b) adding said plasmin or derivatives thereof to a formulation that comprises a material selected from the group consisting of tranexamic acid, ⁇ -aminocaproic acid, analogs of L-lysine other than tranexamic acid and ⁇ -aminocaproic acid, combinations thereof, and mixtures thereof, to produce a formulated plasmin or derivatives thereof; before administering said formulated plasmin or derivatives thereof into a region of the eye, thereby prolonging the activity of plasmin or derivatives thereof in said region of the eye; wherein the post-administering activity is higher than the activity of plasmin or derivatives thereof in
  • the formulation has a pH in the range from about 6.5 to about 11 (or alternatively, from about 6.5 to about 9, or from about 6.5 to about 8).
  • the formulation has a buffering capacity such that a pH of buffered solution of said plasmin or derivatives thereof remains within about 1 pH unit (alternatively, within about 0.5, or 0.2, or 0.1 pH unit) upon adding said plasmin or derivatives thereof.
  • the formulation further comprises a compound selected from Group 1 , Group 2, and Group 3; wherein Group 1 consists of L- lysine, L-arginine, L-ornithine (or its pharmaceutically acceptable salts; e.g., L- ornithine hydrochloride), ⁇ -aminobutyric acid, 5-aminovaleric acid, 7- aminoheptanoic acid, glycylglycine, triglycine, N- ⁇ -acetyl-L-arginine, betaine, sarcosine, combinations thereof, and mixtures thereof; Group 2 consists of gelatin, HSA, streptokinase, combinations thereof, and mixtures thereof; and Group 3 consists of non-ionic surfactants, glycerin, D-sorbitol, combinations thereof, and mixtures thereof.
  • Group 1 consists of L- lysine, L-arginine, L-ornithine (or its pharmaceutically acceptable salts; e.g.,
  • Group 1 consists of L- lysine, L-arginine, L-ornithine (or its pharmaceutically acceptable salts), v- aminobutyric acid, glycylglycine, combinations thereof, and mixtures thereof;
  • Group 2 consists of gelatin, HSA, combinations thereof, and mixtures thereof; and
  • Group 3 consists of non-ionic surfactants.
  • the present invention provides a kit for making an active enzyme or derivatives thereof.
  • the kit comprises: (a) the enzyme or derivatives thereof that have been preserved at a pH less than about 5; and (b) a formulation that comprises a material selected from the group consisting of tranexamic acid, ⁇ -aminocaproic acid, analogs of L-lysine other than tranexamic acid and ⁇ -aminocaproic acid, combinations thereof, and mixtures thereof, provided in a separate container or package.
  • said formulation further comprises a compound selected from Group 1 , Group 2, and Group 3, wherein Group 1 consists of L-lysine, L-arginine, L-ornithine (or its pharmaceutically acceptable salts; e.g., L-ornithine hydrochloride), y- aminobutyric acid, 5-aminovaleric acid, 7-aminoheptanoic acid, glycylglycine, triglycine, N- ⁇ -acetyl-L-arginine, betaine, sarcosine, combinations thereof, and mixtures thereof; Group 2 consists of gelatin, HSA, streptokinase, tPA, uPA, combinations thereof, and mixtures thereof; and Group 3 consists of non-ionic surfactants, glycerin, D-sorbitol, combinations thereof, and mixtures thereof.
  • said formulation has a buffering capacity such that the pH of the formulation remains within about 1 pH unit upon adding said plasmin or derivative
  • the method of the present invention has an advantage of substantially preventing precipitation of said plasmin or derivatives thereof in the desired region of the eye upon administering said plasmin or derivatives thereof.
  • said formulation is made by adding plasmin or derivatives thereof to a solution containing an additive selected from said materials and said compounds, substantially immediately before use.
  • the present invention provides a method for preventing or reducing precipitation of an enzyme administered into a region of a patient, the method comprising: (a) providing the enzyme at a pH of less than about 5; (b) adding said enzyme to a formulation that comprises a material selected from the group consisting of tranexamic acid, ⁇ -aminocaproic acid, analogs of L-lysine other than tranexamic acid and ⁇ -aminocaproic acid, combinations thereof, and mixtures thereof; to produce a formulated enzyme before administering said formulated enzyme into said region of the patient.
  • the formulation has a pH in the range from about 6.5 to about 11 (or alternatively, from about 6.5 to about 9, or from about 6.5 to about 8).
  • the pH of the formulation remains within about 1 pH unit (or alternatively, within about 0.5, or about 0.2, or about 0.1 pH unit) of the original formulation pH.
  • the formulation comprises a buffer.
  • said region of the patient is a vitreous of an eye or a circulatory system.
  • the formulation has a pH of about 7.
  • the formulation has a pH in a range from about 7 to about 7.5.
  • the formulation has a pH of about 7.4.
  • the formulation comprises a phosphate buffer or a Tris-HCI buffer.
  • the formulation comprises 10X phosphate buffered saline ("PBS”) or 5X PBS solution.
  • TXA tranexamic acid
  • ⁇ -ACA ⁇ -aminocaproic acid
  • y- aminobutyric acid 5-aminovaleric acid
  • 7-aminoheptanoic acid glycylglycine
  • triglycine L-omithine hydrochloride
  • N- ⁇ -acetyl-L-arginine L-arginine
  • betaine sarcosine
  • D-sorbitol glycerin, and gelatin.
  • the reconstituted acidified plasmin solution (10 mg/ml in 0.9% NaCI solution) formulated according to the procedure of Example 1 were used.
  • An amount of this reconstituted acidified plasmin solution was added to a 0.9% NaCI solution containing one or more selected additives as shown in Table 2 below, to produce a plasmin formulation containing the additive or additives and a plasmin concentration of 1 mg/ml.
  • An amount of 50 ⁇ l of each of the plasmin formulations was added to a 0.5 ml sample of homogenized young rabbit vitreous, which has a pH of about 8.5. The vitreous samples were observed for any precipitation within 2 hours following addition of plasmin, and the results are shown in Table 3.
  • the reconstituted acidified plasmin solution (10 mg/ml in 0.9% NaCI solution) and the buffered plasmin compositions containing selected additives, formulated according to the procedure of Example 1 were used.
  • An amount of 50 ⁇ l of each of the buffered plasmin compositions was added to a 1.5 ml sample of PBS buffer (pH of about 7.4).
  • the sample was stored at 37 0 C. Aliquots of the sample were collected at time 0, 1 , 3, and 5 hours following addition of plasmin, and analyzed for plasmin activity by chromogenic assay using the plasmin substrate S-2251.
  • S-2251 is a short peptide substrate for plasmin (H-D-Val-L-Leu-L-Lys-p-nitroaniline dihydrochloride, available from Chromogenix-lnstrumentation Laboratory SpA, Milano, Italy). Plasmin hydrolyzes this substrate between the lysine residue and the p-nitroaniline moiety. The method determines the activity of plasmin based on the difference in absorbance (optical density) between the p-nitroaniline formed and the original substrate. The rate of p-nitroaniline formation; i.e., the increase in absorbance per second at wavelength of 405 nm, is proportional to the enzymatic activity of plasmin, and is conveniently measured with a photometer.
  • alternate chromogenic substrates for plasmin also may be used to determine its enzymatic activity, such as S-2390 (H-D-VaI- L-Phe-L-Lys- p-nitroaniline dihydrochloride) or S-2403 (L-Pyroglutamyl-L-Phe-L- Lys-p-nitroaniline dihydrochloride); both are available from Chromogenix- lnstrumentation Laboratory SpA, Milano, Italy.
  • Formulation 1 L-lysine hydrochloride (5 mM), sodium phosphate monobasic (0.185% by weight), sodium phosphate dibasic (0.98% by weight), sodium chloride (0.4% by weight), and water (USP, q.s. to 100% by weight). This solution had a pH of about 7.4 and osmolarity of 308 m ⁇ sm/l.
  • Formulation 2 tranexamic acid (5 mM), sodium phosphate monobasic (0.185% by weight), sodium phosphate dibasic (0.98% by weight), sodium chloride (0.4% by weight), and water (USP, q.s. to 100% by weight). This solution had a pH of about 7.4 and osmolarity of 308 m ⁇ sm/l.
  • Formulation 3 tranexamic acid (100 ⁇ M), sodium phosphate monobasic (0.185% by weight), sodium phosphate dibasic (0.98% by weight), sodium chloride (0.4% by weight), and water (USP, q.s. to 100% by weight). This solution had a pH of about 7.4 and osmolarity of 308 m ⁇ sm/l.
  • Tween 80 ® (also known as Polysorbate 80) is polyoxyethylene sorbitan monooleate surfactant.
  • EXAMPLE 6 Stability of Human Plasmin in Pig Vitreous
  • the reconstituted acidified plasmin solution (10 mg/ml in 0.9% NaCI solution) and the plasmin compositions buffered at pH 3.5 ⁇ 0.3 containing selected additives in 0.9% NaCI solution were used.
  • human-derived plasmin that is to say, active plasmin fractionated from human plasma with all five kringle regions linked in the correct (i.e., native) configuration relative to the active "light" chain was administered through an intracameral injection (i.e., and injection into the anterior chamber of the eye) of active plasmin with 5 mM epsilon aminocaproic acid as a stabilizing agent and 6% trehalose for tonicity to cynomologous monkeys as a single 100 ⁇ g dose in10 ⁇ l at day O, and ocular pressure in mm Hg was measured for up to 26 days post dosing. Results were compared to untreated eyes, that is to say, eyes that were not injected or otherwise treated.
  • Table 7 provides a followup study to the feasibility study of Table 6, using a larger population of 7 monkeys.

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Abstract

L'invention porte sur des compositions de métalloprotéinase matricielle (MMP) activant des endoprotéinases telles que la plasmine et des dérivés de plasmine comprenant, mais sans s'y limiter, la microplasmine, la miniplasmine, la plasmine tronquée recombinante, etc., et sur des procédés pour l'utilisation de ces compositions pour le traitement de maladies de l'oel par le remodelage de la matrice extracellulaire (ECM). Une application particulièrement envisagée est l'utilisation de (MMP) activant des endoprotéinases telles que la plasmine et des dérivés de plasmine, seule ou en combinaison avec un agent de rétention, pour réduire ou éliminer l'obstruction/blocage, associé(e) à l'ECM, du canal de sortie de l'humeur aqueuse par le système trabéculaire qui peut produire une pression intraoculaire élevée et des maladies de l'oel résultant d'une telle pression élevée, par exemple, un glaucome.
PCT/US2008/083783 2007-11-19 2008-11-17 Compositions de métalloprotéinase matricielle activant des endopeptidases pour réduire la pression intraoculaire, et procédés d'utilisation de celles-ci Ceased WO2009067407A2 (fr)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011023805A1 (fr) 2009-08-28 2011-03-03 Thrombogenics N.V. Utilisation de plasmine pour le traitement d'une insuffisance de filtration après une trabéculectomie
WO2013079713A1 (fr) 2011-12-01 2013-06-06 Thrombogenics Nv Amélioration du résultat de la trabéculectomie
RU2484795C2 (ru) * 2011-08-18 2013-06-20 Федеральное государственное учреждение "Межотраслевой научно-технический комплекс "Микрохирургия глаза" имени академика С.Н. Федорова Федерального агентства по высокотехнологичной медицинской помощи" Способ индукции задней отслойки стекловидного тела с помощью миниплазмина
WO2014160281A3 (fr) * 2013-03-14 2015-04-09 Wayne State University Procédé pour améliorer l'administration de composés thérapeutiques à l'œil

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EP1140041A2 (fr) * 1999-01-05 2001-10-10 University of Utah Research Foundation Procedes de traitement d'etats associes a l'accumulation d'un excedent de matrice extracellulaire
US20060257391A1 (en) * 2005-05-11 2006-11-16 Bausch & Lomb Incorporated Non-surgical method for preventing or reducing the rate of the progression of non-proliferative diabetic retinopathy and the treatment of other ocular conditions
US20070196350A1 (en) * 2006-02-22 2007-08-23 Bartels Stephen P Compositions and Methods for Effecting Controlled Posterior Vitreous Detachment

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011023805A1 (fr) 2009-08-28 2011-03-03 Thrombogenics N.V. Utilisation de plasmine pour le traitement d'une insuffisance de filtration après une trabéculectomie
US8920794B2 (en) 2009-08-28 2014-12-30 Thrombogenics Nv Method for treating filtration failure after trabeculectomy surgery
RU2484795C2 (ru) * 2011-08-18 2013-06-20 Федеральное государственное учреждение "Межотраслевой научно-технический комплекс "Микрохирургия глаза" имени академика С.Н. Федорова Федерального агентства по высокотехнологичной медицинской помощи" Способ индукции задней отслойки стекловидного тела с помощью миниплазмина
WO2013079713A1 (fr) 2011-12-01 2013-06-06 Thrombogenics Nv Amélioration du résultat de la trabéculectomie
US9089552B2 (en) 2011-12-01 2015-07-28 Thrombogenics Nv Improving trabeculectomy outcome by administering an anti-placental growth factor antibody
WO2014160281A3 (fr) * 2013-03-14 2015-04-09 Wayne State University Procédé pour améliorer l'administration de composés thérapeutiques à l'œil
JP2016519063A (ja) * 2013-03-14 2016-06-30 ウェイン ステイト ユニバーシティー 治療用化合物の眼への送達を増強する方法
US9730888B2 (en) 2013-03-14 2017-08-15 Wayne State University Method of enhancing delivery of therapeutic compounds to the eye
AU2014243925B2 (en) * 2013-03-14 2018-12-06 Wayne State University Method of enhancing delivery of therapeutic compounds to the eye
JP2019055980A (ja) * 2013-03-14 2019-04-11 ウェイン ステイト ユニバーシティー 治療用化合物の眼への送達を増強する方法

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