,
Voilà un article proposant un traitement du KC.
C'est très compliqué.. 
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Il faudrait peut-être l'envoyer à un médecin du comité scientifique afin d'avoir son avis
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http://www.pharmcast.com/Patents/Yr2002/September2002/090302/6444791_Keratoconus090302.htm
et le texte:
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Title: Methods and compositions for the treatment of keratoconus using protease inhibitors
United States Patent: 6,444,791
Issued: September 3, 2002
Inventors: Quay; Steven C. (Edmonds, WA)
Assignee: K-Quay Enterprises, LLC (Edmonds, WA)
Appl. No.: 695774
Filed: October 24, 2000
Abstract
Compositions and methods for treating corneal diseases mediated by elevated protease activity include ocular administration of protease inhibitors. One or more protease inhibitors selected from an aspartic, serine, cysteine, or metallo-protease inhibitor are administered to an ocular fluid, surface, or tissue, preferably by topical administration, to inhibit proteolytic activity associated with a corneal disease or condition, for example keratoconus. Antiproteolytic formulations of the invention may include carriers that prolong the retention and/or enhance delivery of the protease inhibitor. These formulations can also include other therapeutic agents such as antiinflammatory or antibiotic drugs. In preferred aspects of the invention, antiproteolytic formulations are administered during periods of closed eye tear production. Also provided within the invention are implant devices for corneal delivery of a protease inhibitor.
DESCRIPTION OF THE SPECIFIC EMBODIMENTS
The instant invention provides useful methods and compositions for treating or preventing corneal disease in a mammalian patient. The methods of the invention involve ocular administration of an antiproteolytic effective amount of a protease inhibitor in an opthalmically acceptable carrier. The protease inhibitor is preferably selected from an aspartic, serine, cysteine, or metallo-protease inhibitor, obtained from a natural source or produced in a native (i.e., wild-type amino acid sequence) or modified (e.g., by amino acid substitution, insertion, deletion, truncation or extension, or by activation, fusion or conjugation with other proteins or chemical moieties) by recombinant or synthetic techniques known in the art.
According to the methods of the invention, a protease inhibitor formulation is administered to an ocular fluid, surface, or tissue, preferably by topical administration, in an antiproteolytic amount effective to substantially inhibit a proteolytic activity associated with the corneal disease or condition to be treated.
By substantial inhibition of proteolytic activity is meant that administration of the protease inhibitor formulation yields at least about a 10% reduction of proteolytic activity, preferably at least a 20% reduction, compared to a relevant baseline or control value at an ocular target site, for example within the extracorneal fluid, corneal epithelium, corneal stroma, Bowman's layer, or the vitreous humor. Preferably, administration of the protease inhibitor yields approximately a 30-50% reduction of proteolytic activity, more preferably greater than about a 50% reduction, and in some preferred aspects yields effective neutralization of proteolytic activity in a treated sample corresponding to a reduction of between approximately 85% and 100% of the proteolytic activity measured for the relevant baseline or control sample.
As used herein, proteolytic activity refers to a quantitative digestive activity of a target protease against a protein (e.g., collagen, elastin, fibronectin) or glycoprotein (e.g., a proteoglycan or glycosaminoglycan) substrate. Target proteases as herein defined include proteolytic enzymes that exhibit aberrantly high levels of expression or activity (e.g., attributable to structural changes that increase substrate binding or otherwise enhance digestion kinetics, or that render the protease more susceptible to activation from a proenzyme to an active form), or whose regulation (e.g., by metabolic turnover, protease inhibition or other mechanisms) is impaired in association with a corneal disease or condition to be treated, for example keratoconus or corneal infections. As further defined herein, target proteases are amenable to regulatory inhibition by exogenously administered protease inhibitors.
Proteases that may be successfully targeted for inhibition by the compositions and methods of the invention include, but are not limited to, acid esterases, acid phosphatases, acid lipases, cathepsins (e.g., cathepsin B and G), collagenases, elastases, tryptases, chymases, kinins, kalikreins, tumor necrosis factors, chymotrypsins, stromelysins, and matrix metalloproteases (e.g., gelatinase A or MMP-2, gelatinase B or MMP9, MMP1, MMP 8, and MMP 13).
Antiproteolytic activity may be determined by, e.g., various quantitative, in vitro or in vivo assays, for example by enzymatic and/or immunological assays using extracorneal fluid samples, samples from keratocyte culture media, or corneal tissue samples, as described herein below and as otherwise known in the art. Alternatively, antiproteolytic activity may be determined by other indicia, for example by quantitative changes in morphological or ultrastructural features attributable to proteolytic activity that are amenable to prevention or inhibition using the compositions and methods of the invention. Exemplary indicia in this context include quantitative changes in the extent of fragmentation of Bowman's layer, fragmentation of the epithelial cell basement membrane, and/or fibrillation of the anterior corneal stroma. These indicia can be readily compared between treated samples and relevant control samples, for example by histopathological computer-aided image analysis that resolves percentages of optical field areas occupied by proteolytically altered versus normal histological structures (e.g, fragmented versus non-fragmented areas of Bowman's layer).
The extent of antiproteolytic activity elicited by the compositions and methods of the invention (i.e., for determining efficacy and calibrating dosages) can be determined by a variety of assays that compare relevant test and control samples, as detailed in the examples below. Suitable in vitro test and control samples include cultured, normal and keratoconic keratocytes, respectively, each treated with an antiprotease formulation of the invention. Using these samples, protease inhibition can be measured at selected time points, for example, by assaying target protease-inhibitor complex formation, rates or levels of protein digestion attributed to the target protease, morphological indicia as noted above, and other parameters consistent with the quantitative values sought.
Alternate quantitative inhibition assays can be conducted using, e.g., cultured keratocytes or corneal tissues taken from subjects with keratoconic and normal corneas to provide, respectively, test and control samples for in vitro assays. For quantitative determination of in vivo protease inhibition, test and control samples may include extracorneal fluid or corneal tissue samples taken from subjects (e.g., a human or non-human mammal such as a rabbit) following administration of a protease inhibitor formulation (test sample), and following administration of a placebo comprising, e.g., a selected carrier without the protease inhibitor (control sample). Often, test and control samples will be provided by bilateral administration of test and control treatments to an individual patient. Other suitable test and control samples will be determined by those skilled in the art according to the objectives and methods of the chosen assay, as exemplified herein below.
Protease inhibitors that are useful within the invention are any of the inhibitors, their analogs, recombinantly modified variants, proteolytically active fragments, derivatives, or salts, which can inhibit target proteases as defined above. Preferably, the inhibitor is a protein or peptide of sufficient molecular size for use within the formulations described herein that provide for enhanced absorption, retention and delivery of the inhibitor at a site of treatment. In various preferred embodiments, the protease inhibitor may be selected from an aspartic, serine, cysteine, or metallo-protease inhibitor. Useful inhibitors may be derived from a natural source or produced in a native or modified form by recombinant or synthetic techniques known in the art. In more detailed aspects of the invention, protease inhibitors bind with one or more proteases that exhibit increased levels of expression or activity, or aberrant regulation, leading to pathogenic protein or glycoprotein degradation and/or morphologic changes associated with a corneal disease or condition to be treated.
As noted above, preferred protease inhibitors include native or modified aspartic, serine, cysteine, or metallo-protease inhibitors. Exemplary inhibitors in this context include .alpha.1-antiprotease (alp1, formerly known as .alpha.1-antitrypsin), .alpha.2-macroglobulin (.alpha.2-M), secretory leucocyte protease inhibitor (SLP1, formerly known as mucus proteinase inhibitor and antileukoprotease), .beta.1-antigellagenase, .alpha.2-antiplasmin, serine amylyoid A protein, al -antichymotrypsin (.alpha.1-Achy), cystatin C, inter-.alpha.-trypsin inhibitor, elafm, elastinal, aprotinin, phenylmethyl sulfonyl fluoride, the cysteine protease inhibitor E-64, the cathepsin B-trypsin inhibitor leupeptin, and the metalloprotease inhibitors TIMP-1, TIMP-2, and 1, 10-phenanthroline.
A particularly preferred protease inhibitor for use within the compositions and methods of the invention is .alpha.2-macroglobulin. This inhibitor is a high-molecular-weight (718 kD), homotetrameric glycoprotein implicated as a regulator of degradation for certain extracellular matrix components and other macromolecules. Unlike many other protease inhibitors, .alpha.2-macroglobulin is not highly specific for a preferred target protease, and is not particularly fast acting. Instead, .alpha.2-macroglobulin inhibits proteases from all four major classes and is considered to be relatively slow in its activity. Consistent with these properties, the mechanism of action by .alpha.2-macroglobulin is also unique. When this protease inhibitor reacts with a target protease, proteolytic cleavage in the "bait region" of the inhibitor occurs, leading to a conformational change and trapping of the protease. A covalent bond is then formed between the protease and .alpha.2-macroglobulin. The protease-inhibitor complex is ultimately cleared from the circulation by a receptor-mediated mechanism.
Another preferred protease inhibitor for use within the compositions and methods of the invention is .alpha.-1 protease inhibitor (alp1), a major protease inhibitor in human plasma synthesized mainly by parenchymal liver cells. Alp1 is a glycoprotein of 53 kDa that forms a 1:1 complex with its target enzyme, leukocyte elastase. In addition to this primary target, alp1 inhibitor also inhibits chymotrypsin, cathepsin G, trypsin, plasmin, and thrombin. It is present in most body fluids, as well as in many tissues and cells. Alp1 has been demonstrated in all three layers of normal cornea as well as in the tears and aqueous humor.
As noted above, useful compositions within the invention include formulations of antiprotease salts, derivatives and complexes. As used herein, the term pharmaceutically acceptable salts, derivatives and complexes retain the desired biological activity of the corresponding native antiprotease, and exhibit minimal undesired toxicological effects. Nonlimiting examples of useful antiprotease salts are acid addition salts formed with inorganic acids (for example, hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid, and the like), and salts formed with organic acids such as acetic acid, oxalic acid, tartaric acid, succinic acid, malic acid, ascorbic acid, benzoic acid, tannic acid, pamoic acid, alginic acid, polyglutamic acid, naphthalenesulfonic acid, naphthalenedisulfonic acid, and polygalacturonic acid; base addition salts formed with polyvalent metal cations such as zinc, calcium, bismuth, barium, magnesium, aluminum, copper, cobalt, nickel, cadmium, sodium, potassium, and the like, or with an organic cation formed from N,N-dibenzylethylene-diamine, Dglucosamine, ammonium, tetraethylammonium, or ethylenediamine; or combinations of acid and base addition salts.
Pharmaceutically acceptable derivatives and complexes of protease inhibitors include native or modified inhibitors that are chemically modified (e.g., by addition of stabilizing or otherwise finctional chemical moieties), truncated, conjugated (e.g., to a second protein, peptide or carrier) or recombinantly modified (e.g. by site directed mutagenesis using a cDNA encoding the inhibitor to introduce substitute, or delete non-critical amino acid residues), which retain desired biological activity of the corresponding native antiprotease.
Particularly useful in this context are protease inhibitor analogs, which comprise recombinantly modified variants and proteolytically active fragments of native inhibitors. These analogs preferably exhibit at least 80% amino acid identity, more preferably 95% or greater amino acid similarity, as compared to the amino acid sequence of the corresponding native inhibitor, as determined by conventional sequence alignment and comparison methods.
Alignment of amino acid sequences and calculation of percent identity between the aligned sequences is routine in the art. Such routine alignments include the introduction of gaps and employ other widely known conventions to account for sequence additions, deletions, conservative substitutions, etc. Briefly, conventional sequence comparison methods involve alignment of the compared sequences to yield the highest possible alignment score, which is readily calculated according to well known methods based on the number of amino acid or nucleotide matches.
Antiprotease analogs preferably share substantial amino acid sequence identity (e.g., at least 75%, preferably 80%, and more preferably 95% or greater sequence identity) with a "reference sequence" of a corresponding native inhibitor protein or active polypeptide fragment thereof. As used herein, this reference sequence is a defined sequence used as a basis for a sequence comparison. Generally, a reference sequence is at least 20 amino acid residues in length, frequently at least 25 amino acid residues in length, and often at least 50 amino acid residues in length. Since analog and native fragment polypeptides may each (1) comprise a sequence (ie., a portion of the complete native sequence) that is similar between the two polypeptides, and (2) may further comprise a sequence that is divergent between the two polypeptides, sequence comparisons between two (or more) polypeptides are typically performed by comparing sequences of the two polypeptides over a "comparison window" to identify and compare local regions of sequence similarity. A "comparison window", as used herein, refers to a conceptual segment of at least 20 contiguous amino acid residues wherein a polypeptide sequence may be compared to a reference sequence of at least 20 contiguous amino acid residues and wherein the portion of the polypeptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) of 20 percent or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences.
Optimal alignment of sequences for aligning a comparison window may be conducted by the local homology algorithm of Smith & Waterman, (Adv. Appl. Math. 2:482, 1981, incorporated herein by reference), by the homology alignment algorithm of Needleman & Wunsch, (J. Mol. Biol. 48:443, 1970, incorporated herein by reference), by the search for similarity method of Pearson & Lipman, (Proc. Natl. Acad. Sci. USA 85:2444, 1988, incorporated by reference), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Dr., Madison, Wis., incorporated herein by reference), or by inspection, and the best alignment (i.e., resulting in the highest percentage of sequence similarity over the comparison window) generated by the various methods is selected. The term "sequence identity" means that two polypeptide sequences are identical (i.e., on an amino acid-by-amino acid) over the window of comparison. The term "percentage of sequence identity" is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical amino acid residues occur in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (ie., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. The terms "substantial identity" as used herein denotes a characteristic of a polypeptide sequence, wherein the polypeptide comprises a sequence that has at least 80 percent sequence identity, preferably at least 95 percent sequence identity, more usually at least 99 percent sequence identity as compared to a reference sequence over a comparison window of at least 20 amino acid residues, frequently over a window of at least 25-50 amino acid residues, wherein the percentage of sequence identity is calculated by comparing the reference sequence to the analog sequence which may include modifications (e.g., deletions, substitutions, or additions) which total 20 percent or less of the reference sequence over the window of comparison. The reference sequence may be a subset of a larger sequence.
In addition to these polypeptide sequence relationships, protein analogs and peptide fragments of the invention are also typically selected to have conservative relationships with corresponding, native reference proteins and polypeptides. Preferably, residue positions which are not identical differ by conservative amino acid substitutions. Conservative amino acid substitutions refer to the interchangeability of residues having similar side chains. For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulfur-containing side chains is cysteine and methionine. Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine. Abbreviations for the twenty naturally occurring amino acids used herein follow conventional usage (Immunology--A Synthesis (2nd ed., E. S. Golub & D. R. Gren, eds., Sinauer Associates, Sunderland, Mass., 1991), incorporated herein by reference). Stereoisomers (e.g., D-amino acids) of the twenty conventional amino acids, unnatural amino acids such as .alpha.,.alpha.-disubstituted amino acids, N-alkyl amino acids, lactic acid, and other unconventional amino acids may also be suitable components for polypeptides of the present invention. Examples of unconventional amino acids include: 4-hydroxyproline, .gamma.-carboxyglutamate, .epsilon.-N,N,N-trimethyllysine, .epsilon.-N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, .omega.-N-methylarginine, and other similar amino acids and imino acids (e.g., 4-hydroxyproline). Moreover, amino acids may be modified by glycosylation, phosphorylation and the like.
For practicing the methods of the invention, the precise amounts of protease inhibitors to be administered and the frequency and duration of treatment will depend on the status of the corneal condition or disease to be treated, and on other factors such as the patient's state of health and weight, the mode of administration, the nature of the formulation, etc. These factors will vary such that specific regimens can be established by those skilled in the art to maximize efficacy of treatment. Ordinarily, the antiprotease is administered in a dosage of between approximately 0.2 .mu.g/ml and 1.0 mg/ml. Preferably, the inhibitor is present in a concentration of about 0.1-1.0 .mu.g/ml, more preferably at a concentration of about 0.5 .mu.g/ml. Exemplary formulations for .alpha.1-antitrypsin will comprise the inhibitor at approximately the same range of concentrations, with the most preferred concentration being between about 1.0 and 5.0 .mu.g/ml. The administration schedule can range from a continuous infusion, to once or twice a day, up to 6 or more administrations a day, with dose levels and administration protocols being selected by the health professional. Administration onto the concave surface of contact lenses before insertion into the eye is an effective method of enhancing the residence time for the solution in contact with the cornea.
Thus, treatments according to the invention can be in the form of a one time dose, e.g., in the context of sustained delivery and long-term delivery formulations described below. Alternatively, multiple administrations may be indicated and, under certain circumstances, continuous treatments may be selected.
In a preferred aspect of the invention, compositions comprising a protease inhibitor are administered during periods of closed eye tear production (e.g., during a patient's sleep periods). This method greatly enhances antiproteolytic results, yielding prolonged inhibition of proteolytic processes in corneal tissues (e.g., as demonstrated by reduction in the activity of specific target protease(s)), and long-term inhibition of histopathological changes, such as fragmentation of Bowman's layer. Administration of the antiprotease compositions of the invention during periods of closed eye tear production greatly enhances the antiproteolytic efficacy of these compositions compared to the efficacy achieved by antiprotease administration during periods of reflex tear production, although the latter use is effective and within the scope of the invention. This is due in part to the prolonged retention of the antiprotease composition attributable to a reduction in tear flushing between closed eye and reflex tear periods. This enhanced efficacy is also attributable to fundamental differences in the processes and regulation of proteolysis that characterize the closed eye, versus reflex tear environments. The protease inhibitor compositions and pharmaceutical formulations of the invention can also be administered during a period that is concurrent with or closely preceding a medical procedure or other event anticipated to produce a risk of proteolytic injury, for example following eye surgery or during bacterial infection. Thus, methods are provided which involve administration of an antiproteolytic composition concurrent with, or within an antiproteolytic effective period preceding or following, a surgical procedure or infection, whereby the administration reduces or eliminates risk of deleterious proteolytic responses normally associated with the procedure or infection.
Within the methods of the invention, formulations comprising a protease inhibitor, a mixture of a plurality of protease inhibitors, or a mixture of one or more protease inhibitors combined with a second therapeutic agent (e.g., an antibiotic, antiviral or antiinflammatory drug) can be administered by a variety of routes, including via topical administration (using, e.g., drops, gels, creams or microparticles as carriers), injection (e.g., via hypodermic or pneumatic introduction into the cornea or vitreous humor).
Preferred methods of the invention involve coordinate (e.g., simultaneous or closely contemporaneous to yield coordinate treatment) administration of a plurality of antiprotease proteins, analogs, salts, or derivatives, or administration of formulations comprising multiple protease inhibitors that may be admixed or complexed. Practice of these methods reduces abnormal proteolytic mechanisms attending a targeted corneal disorder (e.g., keratoconus) at combinatorial antiproteolytic levels that exceed antiproteolytic levels observed when either of the coordinately administered protease inhibitors are administered alone. This inhibition, as when other compositions and methods of the invention are employed, reduces proteolytic activity in extracorneal fluid (tears), and in corneal tissues (as determined by both enzymatic and histopathological assays). In preferred embodiments, a multispecific protease inhibitor (i.e., an inhibitor which targets multiple protease species), such as .alpha.2-M, SLP1 and alp1, is coordinately administered with another multispecific inhibitor, or, alternatively, a multispecific inhibitor is coordinately administered with an oligospecific or specific inhibitor (the latter types of inhibitors represented, e.g., by .beta.1-antigellagenase, .alpha.2-antiplasmin, serine amylyoid A protein, .alpha.1-antichymotrypsin (.alpha.1-Achy), cystatin C, inter-a-trypsin inhibitor, elafin, elastinal, aprotinin, phenylmethyl sulfonyl fluoride, leupeptin, and the metalloprotease inhibitors TIMP-1, TIMP-2, and 1, 10-phenanthroline). Using these combinatorial compositions and treatment methods, the invention achieves effective inhibition against multiple proteases (and/or their pathogenic effects) involved in a particular corneal disease process. Thus, the methods and compositions of the invention provide antiproteolytic effects against a broad range of proteases, including but not limited to, acid esterases, acid phosphatases, acid lipases, cathepsins, collagenases, elastases, tryptases, chymases, kinins, kalikreins, tumor necrosis factors, chymotrypsins, stromelysins, and matrix metalloproteases, thereby alleviating or preventing the targeted corneal disease or condition.
Additional preferred methods of the invention involve coordinate administration of an antiprotease and an antibiotic, or administration of formulations comprising both a protease inhibitor and an antibiotic. Practice of these methods reduces abnormal proteolytic mechanisms attending a targeted corneal disorder (e.g., keratoconus) and also secondarily reduces proteolytic effects attributed to bacterial infection. Useful antibiotics may be any opthalmically acceptable antibiotic indicated for treatment of an ocular bacterial infection, including, but not limited to fluoroquinolones (e.g., ofloxacin, norfloxacin, ciprofloxacin), gentamicin, and pilocarpine.
Other medications useful in these combinatorial treatment methods include steroidal antiinflammatories, such as corticosteroids, and nonsteroidal antiinflammatories, for example aspirin, ibuprofen, indomethacin, fenoprofen, mefenamic acid, flufenamic acid, and sulindac. Many other combinatorially effective medicaments useful for coordinate ophthalmic treatment within the methods of the invention will be apparent to the skilled practitioner.
Typically, the protease inhibitors, analogs, salts, derivatives, and coordinately administered therapeutic agents of the invention will be administered in the form of a pharmaceutical composition, ie., dissolved or suspended in a physiologically acceptable carrier, preferably an aqueous carrier. A variety of aqueous carriers may be used, e.g., water, buffered water, 0.4% saline, 0.3% glycine and the like. Many other suitable carriers are known in the art and readily formulated with the subject therapeutic agents, including biologically compatible gels, creams, microparticulate solutions and the like suitable for topical administration. The pharmaceutical compositions may be sterilized by conventional, well known sterilization techniques. The resulting formulations may be packaged for use or filtered under aseptic conditions and lyophilized, the lyophilized preparation being combined with a sterile aqueous solution or other carrier prior to administration.
In other embodiments of the invention, the protease inhibitors, analogs, salts, derivatives, and coordinately administered therapeutic agents of the invention are prepared with carriers that protect the compound against rapid elimination from the ocular environment, such as are routinely used in controlled release devices and formulations (e.g., implants and microencapsulated delivery systems). Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, colla-gen, polyorthoesters, and polylactic acid. Such formulations and methods for their preparation will be apparent to those skilled in the art.
In a particularly preferred aspect of the invention, a novel delivery device is provided in the form of an ocular implant adapted for corneal delivery of an effective amount of an antiprotease. The device has a concave inner surface that conforms to an external surface of the cornea, i.e., which is similar in size and shape to the inner surface of a contact lens, and is applied externally to the cornea of a patient suffering from a corneal disease or condition. The device serves as a carrier to deliver to the cornea an antiproteolytically effective amount of a protease inhibitor. Preferably, the device is comprised of a gas-permeable, biocompatible polymer, such as ethylene vinyl acetate, polyanhydride, polyglycolic acid, collagen, polyorthoester, or polylactic acid. The entire body, or at least an inner surface, of the device is coated or impregnated with the protease inhibitor. The device is disposable and provided in sterile packaging, to be implanted by the patient and worn for a selected treatment period, preferably for the full duration of a period of closed eye tear production (e.g., overnight) for maximum therapeutic efficacy.
Liposomal suspensions may provide useful, pharmaceutically acceptable carriers for formulating antiproteolytic compositions of the invention. These may be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811 (incorporated herein by reference). For example, liposome formulations may be prepared by dissolving appropriate lipid(s) (such as stearoyl phosphatidyl ethanolamine, stearoyl phosphatidyl choline, arachadoyl phosphatidyl choline, and cholesterol) in an inorganic solvent that is then evaporated, leaving behind a thin film of dried lipid on the surface of the container. An aqueous solution of the protease inhibitor is then introduced into the container. The container is swirled by hand to free lipid material from the sides of the container and to disperse lipid aggregates, thereby forming the liposomal suspension.
The pharmaceutical compositions for use within the invention may also contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH-adjusting and buffering agents, tonicity-adjusting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, etc. The concentration of the antiprotease in these formulations can vary widely, i.e., from less than about 0.05%, usually at least about 0.5%, to as much as 15 or 20% by weight, and will be selected primarily by fluid volumes, viscosities, etc., in accordance with the particular mode of administration selected.
In preferred methods within the invention, mammalian subjects, including human patients, suffering from protease-mediated corneal disorders are treated by administering to the patient a pharmaceutical or therapeutic composition comprising an effective amount of one or more antiproteases, or a pharmaceutically acceptable derivative or complex thereof, in a pharmaceutically acceptable carrier or diluent.
The active antiprotease is included in the pharmaceutically acceptable carrier or diluent in an amount sufficient to deliver to a patient an antiinflammatory effective amount without causing serious adverse side-effects in the patient treated. The active compound is preferably administered to achieve peak concentration of the antiprotease in tear fluid or corneal tissue of the patient within about 1-4 hours after administration. Concentration of the antiprotease in pharmaceutical compositions and devices of the invention will depend on such factors as absorption, distribution, inactivation, degradation, and flushing of the antiprotease, as well as other factors known to those of skill in the art. It is to be noted that dosage values will also vary with the severity of the condition to be alleviated. It is to be further understood that for any particular subject, specific dosage regimens may be adjusted over time according to the professional judgment of the person administering or supervising the administration of the compositions, and that the concentration ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the invention.
In view of the description and examples provided herein, the methods of the invention are shown to be useful for treating a wide variety of inflammatory conditions and diseases. In particular, the methods of the invention can be employed to treat keratoconus and other corneal disease or conditions characterized by aberrantly high proteolytic activity or damage attributable to aberrant proteolytic processes. Other conditions indicative of treatment using the compositions and methods of the invention include, but are not limited to, corneal ulcers, allergic conditions, bacterial infection, viral infection, corneal injury and post-surgical wound healing.
The pharmaceutical formulations administered within the methods of the invention must be opthalmically acceptable. In general, compounds and formulations with a therapeutic index of at least 2, preferably at least 5-10, more preferably greater than 10, are opthalmically acceptable. As used herein, the therapeutic index is defined as the EC50/IC50, wherein EC50 is the concentration of compound that provides 50% inhibition of a target proteolytic activity (e.g., proteolysis by a specific protease, or histopathologic change attributed to proteolysis) compared to a relevant control, and IC50 is the concentration of compound that is toxic to 50% of target cells (e.g., keratocytes in an in vitro toxicity assay). In this context, cellular toxicity can be measured by direct cell counts, trypan blue exclusion, or various metabolic activity studies such as 3 H-thymidine incorporation, as known to those skilled in the art.
Claim 1 of 6 Claims
What is claimed is:
1. A method for treating keratoconus in a mammalian subject comprising administering to an ocular surface of the subject a protease inhibitor in an opthalnically acceptable carrier, wherein said protease inhibitor is administered in an antiproteolytic effective amount and duration effective to alleviate a symptom of keratoconus in the subject, wherein said protease inhibitor is selected from the group consiting of .alpha.2-macroglobulin (.alpha.2-M) and .alpha.1-protease inhibitor (alp1).
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mais je dois bien admettre qu'on en ressent le manque!
