WO1995014488A1 - Compositions and methods for the treatment of dry eyes and other ocular disorders - Google Patents

Abstract

An ophthalmic composition comprising a non-conjunctival, epithelial-derived secretion(s), component(s), or analog(s) thereof is provided. Typically, the non-conjunctival, epithelial-derived secretion will comprise pulmonary surfactant or a component(s) thereof, such as a surfactant-associated protein. The composition typically will further comprise an ophthalmically-acceptable carrier and also can contain a variety of additives, buffers, medicants, and the like.

Description

COMPOSITIONS AND METHODS FOR THE TREATMENT OF DRY EYES AND OTHER OCULAR DISORDERS

CROSS-REFERENCE TO RELATED APPLICATOKS

The present application is a continuation-in-part of U_SSN 08/157,560, filed November 24, 1993, which is incorporated _by reference in its entirety for all purposes.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to ophthalmic compositions and methods for the treatment of dry eyes (keratoconjunctivitis sicca) and other ocular disorders. More particularly, this invention relates to a ophthalmic compositions and methods comprising a non-conjunctival, epithelial-derived secretion particularly suitable for the alleviation of ocular disorders.

2. Description of the Background Art

"Dry eyes" is a general term referring to a broad category of diseases involving abnormalities of the tear film. Approximately 400,000 people in the United States alone currently suffer from this affliction. In general, dry eye syndrome is a condition that is characterized by a decrease in the production of aqueous tears and/or tear film dysfunction, ultimately leading to ocular surface disease. Untreated, this disease can cause permanent structural damage to the cornea and, in some cases, visual loss, and constitutes one of the major reasons for patients to seek ocular medical care. The symptoms associated with dry eyes are often exacerbated in subjects using contact lenses. In some cases, contact lens intolerance is caused in part or in total by the condition of dry eyes and the symptoms thereof. The morbidity associated with dry eyes is related to changes of the ocular surface that give rise to a spectrum of clinical abnormalities, including punctate erosions, corneal filaments, mucous plaques, epithelial defects, and in some cases, corneal ulcers. The symptoms of this disease often include redness, irritation, tearing, burning, stinging, foreign body sensation, blurred vision, and mucous discharge, all of which may be exasperated by exposure to environmental irritants.

Various compositions for treating dry eye have been used. Although methods for the treatment of dry eyes include preservation of existing tears, stimulation of tear production ( see, e. g. , Shoenwald et al., US Patent 4,820,737 (1989)), anti-inflammatory therapy, diminution of tear viscosity, hormone treatment, and surgical intervention, the use of topical tear substitutes (artificial tears) represents the main treatment in the management of dry eyes and probably represents the ophthalmic medication most frequently utilized. Currently available artificial tears, used in the treatment of dry eyes and other pathologies related to the inefficient secretion or synthesis of tear constituents, consist primarily of various aqueous-based solutions, with or without preservatives. These artificial tear solutions provide relatively poor coverage and lubricating properties compared to normal, natural tear film. As such, frequent reapplication is necessary.

The employment of aqueous solutions of inert, substituted, cellulose ethers such as methyl cellulose has also been utilized. Other substituted cellulose ethers such as hydroxyethyl cellulose, hydroxypropyl methyl cellulose, and hydroxypropyl cellulose have been subsequently utilized as polymeric components in artificial tear formulations. Each of these polymerics imparts high viscosities to the tear formulations, even when employed in relatively low concentrations. It has been this impartation of high viscosity to the formulations which has been believed to prolong retention time of the tear substitute in the fornices and over the preocular surface. However, it has been subsequently demonstrated that the ocular retention time is not a direct function of the vehicle viscosity. Further, the use of highly viscous polymeric solutions also results in unpleasant side effects to the user. For example, insufficient lubrication of the lids and the tendency for encrustations to form at the lid margins produces irritation and discomfort.

Hydrophilic polymerics such as polyvinyl alcohol and polyvinyl pyrrolidone, combining good film-generating properties with relatively low viscosities in aqueous solutions, have been utilized. Such formulations, however, remain less than satisfactory inasmuch as they do not provide good wetting properties. Compositions consisting essentially of phospholipids and/or hyaluronic acids have also been proposed as potential therapeutic agents for treating ocular disorders. Glonek et al., US Patent No. 4,914,088; Hills et al., WO 91/12808. However, the existence and extent of clinical efficacy of these compositions remains doubtful.

To date, therefore, artificial tear substitutes have proved, at best, marginally effective in the treatment of dry eyes. For these reasons it would be desirable to provide an improved tear substitute that mimics more closely the composition and physical properties of natural tears, that is easy to use, that provides a sustained period of relief from dry eyes symptoms, and that permits use of contact lenses by subjects.

SUMMARY OF THE INVENTION

The subject invention provides improved compositions and methods for the treatment of ocular disorders, including dry eyes. The invention comprises correcting deficiencies in the tear film by topical application of an ophthalmic composition to the ocular surface of the eye. This ophthalmic composition comprises a non-conjunctival, epithelial-derived secretion. In some embodiments, the composition will further comprise an ophthalmically-acceptable carrier. Examples of epithelial-derived secretions include pulmonary surfactants, either natural or synthetic analogs thereof. Natural pulmonary surfactants include surfactant-associated protein, for example, SP-A, SP-B, SP-C, and SP-D. One embodiment of this invention utilizes Survanta®, Exosurf Neonatal , or Infasurf® as the epithelial-derived secretion.

The compositions of the present invention, topically applied, provide relief for aqueous-deficient, lipid-deficient, and/or mucus-deficient eyes and are effective also therapeutically in situations where protein, carbohydrate, and/or lipid abnormalities exist. In addition, the compositions can be used intraocularly in anterior or posterior procedures, for example, as lubricants during cataract surgery or vitrectomy procedures.

Some ophthalmic compositions of the present invention are similar to the physiological composition of the normal tear film. The compositions usually exhibit an increased tear break up time, enhanced film stability, and improved wettability and viscosity. In addition, many of the ophthalmic compositions provided herein are believed to be compatible with the use of contact lenses.

Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims.

BRIEF DESCRIPTION OF THE FIGURES Fig. 1 compares tear break-up time (TBUT) for Survanta™, Hypotears PF™ and no treatment. Fig. 2 compares TBUT for Exosurf Neonatal™,

Hypotears PF™ and no treatment.

Fig. 3 compares TBUT for Survanta™ and Hypotears PF™ at different times after treatment.

Fig. 4 shows values of TBUT at different dosages of Survanta™.

DESCRIPTION OF SPECIFIC EMBODIMENTS 1. Definitions and General Parameters

Unless otherwise stated, the following terms used in the specification and claims have the meanings given below: "Epithelial" or "epithelium" refers to tissue that forms the surface of an organ or organism and serves to enclose and protect the other tissues of the organ or organism. "Conjunctival" refers to the ocular conjunctiva; the mucous membrane covering the anterior surface of the eye and lining the lids. "Non-conjunctival" refers to epithelial other than the conjunctiva. "Secretio (s)" refers to those molecular substance(s) that result from, leave, or separate from, a cell or bodily fluid. Typically, a secretion is produced by a cell or gland and has chemical and/or physical properties different from the body from which is produced. The term "secretion" also includes derivative compositions obtained from a naturally occurring secretion. Derivative compositions may contain only a single component from the naturally occurring secretion, for example, a surfactant protein. However, derivative compositions do not include compositions in which lipids and/or glycosaminoglycans, such as hyaluronic acid are the only active component(s) . The term "secretion" also includes analogs of naturally occurring secretions or derivative compositions, as described above.

The term "analog" refers to molecular substance(s) that have structural or biologically functional properties similar to the naturally occurring secretion or derivative composition for use in an ophthalmic composition. Typically, the analog is synthetically constructed. For example, a surfactant protein, such as SP-A, that is synthesized by recombinant DNA techniques is an analog of the naturally occurring SP-A protein. The recombinant protein likely shares the primary amino acid sequence and most of the biological properties of the naturally occurring protein, but may differ in certain respect, such as in post-translational modifications.

"Pulmonary surfactant" refers to those molecular substances forming a layer over alveolar surfaces and tending to stabilize alveolar volume by reducing surface tension and altering the relationship between surface tension and surface area. As used herein, "surfactant" refers to a compound having the ability to reduce surface tension on a surface and/or reduce surface tension at an air/liquid interface. Procedures for determining the surfactant activity of substances described herein can be found in Sarin et al., European patent publication No. 0,413,956, published 1991. The term "pulmonary surfactant" is used generically to encompass derivative compositions and analogs. As discussed above in connection with "secretions", derivative compositions do not include compositions consisting essentially of lipids and/or glycosaminoglycans. Natural pulmonary surfactants are prepared from lavage of mammalian lungs (e. g. , porcine or bovine) or washes of tissue extracts therefrom, or from amniotic fluid. The components can be isolated from the wash by centrifugation or lyophilization and resuspended in an aqueous solution at a concentration effective for opthalmic use. Components of a natural pulmonary surfactant include phospholipids ( e. g. , dipalmitoyl phosphatiditylycholine, unsaturated phosphatidylcholine, phosphatidyl-glycerol, phosphatidylethanolamine, and phosphatidylinositol) , neutral lipids ( e. g. , triglycerides, cholesterol, cholesterol esters, steroids, terpenes) , proteins, glycoproteins, glycosaminoglycans (e. g. , heparin sulfate, heparin, chondroitin sulfate, hyaluronate) , anti-proteases , anti-oxidants , ions and water. The proportions (w/v) of the major components are about 80-95% phospholipid, 2-10% neutral lipid, and 5-10% protein. Trace components or components not contributing to ophthalmic activity are sometimes removed from the natural pulmonary surfactant. Artificial surfactants are prepared by mixing components (particularly lipids) from commercially available purified sources. Artificial surfactants sometimes omit, or contain reduced proportions of, protein components.

"Surfactant-associated protein" refers to a specific class of proteins found in pulmonary surfactant. Pulmonary surfactant contains at least four families of surfactant- associated proteins, SP-A, SP-B, SP-C, and SP-D. These substances appear to be important for the adsorption of phospholipids to an air-liquid interface. They also serve as regulators of surfactant phospholipid metabolism, for example, by promoting uptake of phospholipids by certain cells.

"Glycoproteins" refer to proteins with covalently linked oligosaccharides. The covalent protein-carbohydrate linkage may be N-glycosidic (N-glycans) or O-glycosidic (0- glycans) . The polypeptide chains generally carry a number of short heterosaccharide chains, and these, in turn, almost always include N-acetylhexosamines and hexoses. The last member of the chain is very often sialic acid or L-fucose. The oligosaccharide chains are often branched, and rarely contain more than 15 monomers, although the number of saccharide chains on a polypeptide varies widely. In addition, glycoprotein- containing solutions generally have high viscosities. "Glycosaminoglycans" or "mucopolysaccharides" refer to long, unbranched polysaccharide chains composed of repeating disaccharide units wherein one of the two sugar residues in the repeating disaccharide is always an amino sugar (e. g. , N- acetylglucosamine or N-acetylgalactosamine) . Four main groups of glycosaminoglycans have been distinguished by their sugar residues, the type of linkage between these residues, and the number and location of sulfate groups: (1) hyaluronic acid; (2) chondroitin sulfate and dermatan sulfate; (3) heparin sulfate and heparin; and (4) keratin sulfate. "Proteoglycans" refer to glycosaminoglycans (mucopolysaccharides) covalently bound to core proteins.

The term "lipid" encompasses a heterogeneous group of fats and fat-like substances that are usually water-insoluble. Lipids usually contain aliphatic hydrocarbons as a major constituent. Lipids may be positively charged, negatively charged, or neutral. Charged lipids include phospholipids such as phosphoglycerides, phosphoglycolipids, phosphodiol lipids, phosphosphingolipids, phosphatidyl-glycerols, phosphatidylinositols, diphosphatidylglycerols phosphatidylsugars, and plas alogen analogs. See generally, Lehninger, Biochemistry (2d ed. Worth, N.Y.), at pp. 279-306). Neutral lipids include triglycerides, cholesterol esters, waxes, isoprenoids, and cholesterol.

"Therapeutically- or pharmaceutically-effective amount" as applied to the compositions of the instant invention refers to the amount of composition sufficient to induce a desired biological result. That result can be alleviation of the signs, symptoms, or causes of an ocular disorder, such as dry eyes, or any other desired alteration of an ophthalmic biological system.

"Ophthalmically- or therapeutically-acceptable carrier" refers to a carrier medium which does not interfere with the effectiveness of the ophthalmic biological activity of the active ingredients and which is not toxic to the host or patient.

The term "water-soluble" means that a composition exhibits a solubility of at least about 50 μg/ml and usually at least about 100 μg/ml in aqueous solvents. The compositions of the invention may or may not be water soluble, depending on their components. For example, a composition with a high percentage of lipids is likely to be insoluble.

2. The Tear Film

The preocular tear film is a complex layer that covers the anterior surface of the eye, where it increases the optical resolution of the eye, inhibits microbiological contamination, prevents dehydration of the cornea, and provides a source of oxygen to the cornea. The tear film comprises three distinct layers - the lipid, aqueous, and mucous layers - from its anterior to posterior, respectively.

The lipid layer is approximately 0.1 μm thick and is secreted primarily by the Meibomian glands. This layer is comprised of esters, triacyl glycerols, free sterols, sterol esters, and fatty acids, and behaves as a film that is essentially independent of the aqueous layer underneath. The lipid layer appears to participate in preventing evaporation from the aqueous phase. In addition, it has been suggested that the non-polar nature of the lipid layer prevents surface contamination of the tear film with highly polar skin lipids, which could lead to rapid dissolution of the tear film.

The bulk of the tear film consists of the aqueous layer which is approximately 7 μm thick and is secreted by the lacrimal glands. As many as 60 proteins are present in this layer, in addition to glycoproteins, glucose, urea, and electrolytes. The mucous layer, secreted by the goblet cells of the conjunctiva and often described as a component of the tear film, is intimately associated with the corneal and conjunctival epithelium and is approximately 0.05 μm thick. The primary function of this layer is to coat the surface of the corneal epithelium and render it wettable by the aqueous tears.

3. Drv Eves "Dry eyes" refers to conditions in which the eye may be deficient in either the aqueous or mucous layers of the tear film, including, for example, as in keratoconjunctivitis sicca, hypovitaminosis A, Stevens-Johnson syndrome, ocular pemphigoid, extensive trachoma, Sjogren's syndrome (i.e., dry eyes related to rheumatoid arthritis or other connective tissue disease) , and after chemical burns.

In addition, disturbances of the lipid composition can cause "dry eyes". This disturbance can result from an inadequacy of the quantity of secretion from the Meibomian glands or from an inadequacy in the quality of the secretion. Regardless of the cause of the disturbance, the compromised lipid layer fails to act as an adequate barrier against evaporation of the aqueous portion of the tear film, thus resulting in one form of the condition known as dry eyes. This form of dry eyes is often associated with forms of conjunctivitis, traumatized bulbar and palpebral conjunctiva, and related lid disease, as well as external ocular surface disease. It also may be due to decreased tear production resulting from diseases or inflammation of the lacrimal gland. Dysfunction of the lacrimal gland is frequently associated with aging.

Dry eyes can also occur as a result of treatment with drugs. Many drugs have been described as having an effect on lacrimation. (See Crandall and Leopold (1979) Ophthalmology 86:115.) For example, the following drugs have been reported to decrease tear flow: atropine and scopolamine ( see Erickson (1960) Stanford Med. Bull . 18:34); antihistamines (see Chang (1977) J. Am. Optom. Assoc. 48:319); practolol (see Garner and Rahi (1976) Br. J. Ophthalmol. 65:414); and nitrous oxide, halothane, and enflurane (see Krupin et al. (1977) Arch. Ophthalmol . 95:107).

4. The Ophthalmic Composition

According to one aspect of the present invention, there is provided an ophthalmic composition comprising a non- conjunctival, epithelial-derived secretion. These compositions are capable of altering the surface tension of the tear layer, prolonging tear break-up time, and enhancing lubricating properties. In addition, these compositions will be compatible with topical ocular application. In another aspect of the invention, substantially pure conjunctival, epithelial-derived secretion(s) are provided; in particular, cultured cells from isolated lacrimal or goblet cells can be used.

For this invention, "epithelial-derived secretions" refer to naturally-occurring secretions, including those secretions of nasal, oral, respiratory, gastrointestinal, mammary, and female reproductive tract origin. See, e. g. , Baraniuk et al. (1990) J. All . Clin . Immunol . 86:620-627;

Kaliner (1991) Am. Rev. Respir. Dis . 144:S52-S56; Kim et al. (1991) Am. .Rev. Respir. Dis . 144:S10-S14; Lamblin et al. Eur. Respir. J. 5:247-256; Peden et al. (1991) Am. .Rev. Respir. Dis . 143:545-552; Raphael et al. (1988) Am. Rev. Respir. Dis . 138:413-420; and Sheehan et al. (1991) Am. .Rev. .Respir. Dis . 144:S4-S9. These secretions typically originate as a combination of transudates from the vascular system and glycoconjugates derived from secretory cells and glandular elements ( e. g. , goblet cells and/or serous or seromucous glands) . However, the present invention also contemplates the use of epithelial-derived secretion(s) that are either synthetically or genetically produced, for example, using recombinant DNA and molecular biology techniques, or cultured, for example, from isolated lacrimal, goblet, or other epithelial cells, including cultured epithelial cell supernatant, as well as whole cell extracts, homogenates, and other cellular fractions of cultured epithelial cells. In addition, combinations of epithelial-derived secretions derived from more than one source can be employed in the compositions and methods described herein.

Epithelial-derived secretions typically occur as complex mixtures and may include inorganic ions, lipids, secreted proteins, chemical mediators, and other products from plasma cells, mast cells, basophils, epithelial cells, and other cells within the epithelial mucosa. Epithelial-derived secretions need not necessarily be purified prior to utilization in the ophthalmic compositions of the present invention.

In some embodiments of the present invention, pulmonary surfactant is utilized as the epithelial-derived secretion. Pulmonary surfactants usually contain one or more of four proteins, commonly referred to as SP-A, SP-B, SP-C, and SP-D that are thought to be important for normal surfactant function in vivo. (See Weaver and Whitsett (1988) Semin . Perinatol . 12:213.). In addition, pulmonary surfactant consists of phospholipids, in particular, of dipalmitoyl phosphatidylcholine and smaller amounts of unsaturated phosphatidylcholine and phosphatidylglycerol. Other proteins and peptides found in pulmonary surfactant include mucus glycoproteins, surface muco-substance, surfactant hypophase (produced by Clara or type II alveolar cells) , lysozyme, lactoferrin, secretory piece, regulatory neuropeptides, fibronectin, and the like. See, e. g. , Jeffrey (1987) Eur. J". Respir. Dis . 71 Supp. 153:34-42.

Other preferred compositions of the invention consist essentially of the specific protein component(s) of pulmonary surfactant, i.e., the surfactant-associated proteins, as the epithelial-derived secretion. Examples of these surfactant- associated proteins include the hydrophobic, low molecular weight, surfactant-associated proteins commonly known as SP-B, SP-C, and SP-D, as well as the larger molecular weight, hydrophilic surfactant-associated protein known as SP-A. (See Weaver and Whitsett (1991) Biochem. J. 273:249-264.) SP-A, a glycoprotein of 228 amino acids, is synthesized in type II alveolar cells and is associated with intracellular and extracellular lamellar bodies. The charge and size heterogeneity of SP-A varies among species. (See Weaver and Whitsett supra.) A number of important functions have been attributed to SP-A. Although initial evidence suggested this protein facilitated the adsorption to and spreading of dipalmitoyl phosphatidylcholine at the air-liquid interface, studies have now suggested that its major contribution towards surface activity is to enhance the effects of SP-B and/or SP-C. (See Possmayer (1988) Am. Rev. Respir. Dis . 138:990-998.) SP-B has a molecular weight of 18,000 under nonreducing conditions and a molecular weight of 5000-8000 under reducing conditions. (See Hawgood et al. (1987) Am. J. Physiol . 257:L13.) The reduced form of SP-B consists of 79 amino acids and has a high cysteine content (7 cysteines in a total of 79 residues) . Two regions of 23 and 24 amino acids with a high content of both hydrophobic and cationic residues are possible sites for interaction with the lipid and aqueous layers of the tear film.

SP-C has a molecular weight of 4500-8000 under both reducing and nonreducing conditions. (Supra . ) The monomeric form of SP-C consists of 35 amino acids and has 2 palmitoyl groups covalently liked to the polypeptide chain. (See Cursted et al. (1990) Proc. Natl . Acad. Sci . USA 87:2985.) A continuous hydrophobic domain of 23 residues with a high valine content probably plays an important role in the interaction with the lipid layer by forming a membrane-spanning domain. SP-B and SP-C can be obtained following the procedures of Oosterlaken-Dijksterhuis et al. (1991) Biochemistry 30:10965. A fourth surfactant-associated protein, SP-D, has recently been identified in rat bronchoalveolar lavage fluid. SP-D consists of a charge train of proteins (M_r 43,000 pi 6-8) that forms disulfide-bonded trimers. SP-D is similar to SP-A in that it contains a bacterial collagenase-sensitive domain, hydroxyproline, asparagine-linked oligosaccharide and lectin- like activity, but differs in the presence of hydroxylysine and hydroxylysine glycosides. (See Persson et al. (1989) Biochemistry 28:6361-6367; Persson et al. (1988) Biochemistry 27:8576-8585; and Persson et al. (1990) J. Biol . Chem. 265:5755-5760) .

These surfactant-associated proteins can be employed individually or in any combination. Preferred combinations includes SP-B and/or SP-C. In one preferred embodiment, Survanta®, a natural bovine lung extract containing phospholipids, neutral lipids, fatty acids, and surfactant- associated proteins (beractant, available from Ross Laboratories, Columbus, OH) to which colfosceril palmitate, palmitic acid and tripalmitin are added, is utilized as the surfactant-associated protein. The resulting composition provides 25 mg/ml phospholipids, 0.5-1.75 mg/ml triglycerides, 1.4-3.5 mg/ml free fatty acids, and less that 1.0 mg/ml proteins. It is suspended in 0.9% aqueous sodium chloride (volume/volume) and contains no preservatives. Survanta® contains both SP-B and SP-C as protein components, but lacks SP-A. Other embodiments will employ other commercially or non- commercially available substances having the physical and physiological properties of Survanta®, including but not limited to, the protein-free synthetic lung surfactant Exosurf Neonatal™ (comprising colfosceril palmitate, cetyl alcohol, and tyloxapol, available from Burroughs Wellcome Co., Research Triangle Park, NC) .

In addition, fragments of surfactant-associated proteins and analogs thereof can be utilized to produce the compositions described herein. See, e. g. , Sarin et al., EPA 413,956 (1991). These fragments and analogs can be naturally isolated, chemically synthesized and/or recombinantly produced. The fragments or analogs will usually share a biologically activity of the naturally occurring protein from which they derive. For example, fragments are usually capable of lowering surface tension on the surface of a liquid in which they are dispersed. In some embodiments, these surfactant-associated protein fragments and analogs thereof will be combined with lipid(s) .

Artificial or synthetic pulmonary surfactant, synthesized from mixtures of synthetic compounds that may or may not be constituents of natural surfactant comprises another type of epithelial-derived secretion according to the present invention. These synthetic or artificial pulmonary surfactant can be reconstructed in vitro from surfactant-associated proteins or fragments or analogs thereof, synthesized using recombinant DNA and molecular biology techniques or isolated from natural or cultured sources, and from mixtures of phospholipids and neutral lipids. See, e. g. , Benson et al. PCT Publication No. WO89/04326 and Schilling et al. British Patent No. 2,181,138. More specifically, synthetic polypeptide-phospholipid complexes can be utilized in the compositions and methods described herein. Phospholipids useful in forming synthetic pulmonary surfactants by admixture with polypeptides are well known in the art. See, e. g. , Notter et al. (1987) Clin . Perinatology 14:433-79. Suitable natural isolated, chemically synthesized and/or recombinant proteins and peptides for use according to the present invention (and isolative and recombinant techniques for obtaining them) are disclosed in the following publications: Weber European patent publication No. 0,335,133, published 1988; Tanaka U.S. patent No. 4,338,301; Tanaka U.S. Patent No. 4,397,839; Takei et al. U.S. Patent No. 4,603,124; Hawgood et al. (1987) Proc. Natl . Acad. Sci . USA 84:66-70; Jacobs et al. (1987) J^". Biol . Chem. 262:9808-9811; Floros et al. (1986) J. Biol . Chem. 19:9029-9033; Glasser et al. (1987) Proc. Natl . Acad. Sci . USA 84:4007-4011; Whitsett et al. (1986) Pediatric Res . 20:460-467; Whitsett et al. (1986) Pediatric Res . 20:744-749; Schilling et al. PCT Publication No. WO86/03408; Whitsett (1991) U.S. Patent No. 5,013,720; Clement U.S. Patent No. 4,826,821; Jackson (1989) U.S. Patent No. 4,861,756; and Whitsett PCT Publication No. WO86/06943. Other synthetic pulmonary surfactants include the compositions comprising the SP18 monomer-related polypeptide described in Cochrane et al. (1992) U.S. Patent No. 5,164,369 admixed with a phospholipid. Modified natural surfactants also may find use in the compositions described herein. Modified natural surfactants generally are prepared by e. g. , separating lipids from proteins in natural surfactant obtained form lung minces or alveolar lavage, followed in some cases by selective addition (or removal) of certain compounds, and suspension procedures designed to restore the desired surface properties. For example, early studies have demonstrated that protein-depleted lipid extracts of natural pulmonary surfactant have the ability to reduce the surface tension of a pulsating bubble to near 0 mN/m at minimum bubble size and to promote lung expansion and survival of surfactant-deficient prematurely delivered animals. See, e. g. , Yu et al. PCT Publication No. WO 92/04906. A further example of an epithelial-derived secretion which can be utilized to prepare the ophthalmic compositions of the present invention is mucus. Mucus is the clear, viscid secretion of mucous membranes and consists of mucin and various inorganic salts suspended in water. Mucin is a secretion, comprising proteoglycans and glycoproteins, from the goblet cells of the intestine, the submaxillary glands, and other mucous glandular cells.

Other embodiments of this invention will employ various components of epithelial-derived secretions. In addition to water and inorganic ions, epithelial-derived secretions typically include various proteins, glycoproteins, mucoproteins, proteoglycans, glycosaminoglycans, and the like, immunoglobulins (e. g. , IgG, IgM, IgA, secretory component), albumin, lactoferrin, transferrin, glucose, glycogen, "intrinsic factor", inflammatory mediators ( e. g. , histamine, prostaglandins, bradykinin, and leukotriene) and various organ- specific enzymes ( e. g. , pepsin, lysozyme, amylase, RNAase, DNAase, peroxidase, disaccharidases, dipeptidases, enterokinase, ATPase, alkaline phosphatase, and trypsin) . The various components can be either synthesized or isolated from natural sources. Preferred components include proteoglycans and glycosaminoglycans.

The epithelial-derived secretions or components thereof of the present invention can be combined with an ophthalmically-acceptable carrier. Such carriers can be aqueous or non-aqueous ophthalmically-acceptable sterile liquids. Suitable non-aqueous liquid media include the physiologically acceptable oils such as silicone oil, USP mineral oil, white oil, and vegetable oil, for example, corn oil, peanut oil, and the like. Some embodiments of this invention will utilize an organic solvent such as propylene glycol, either alone or in combination with an aqueous solution, as the carrier. In other embodiments, instead of using a liquid medium, the surfactant-associated proteins can be suspended in an ointment such as lanolin, petrolatum, and other known ointments.

The ophthalmically-acceptable carrier is typically an aqueous solution. In some embodiments, the carrier will comprise a substantially isotonic solutions which is defined for present purposes as containing 270-310 milliosmoles/ kilogram of solute. Other embodiments will employ a hyperosmolar solution, for example, 5% sodium chloride solutions. See PDR for Ophthalmology (1993), p. 15. The tonicity adjusting agent is employed to bring the final solution tonicity within the stated ranges if not already there due to contributions of the other ingredients. Generally, the tonicity adjusting agent is an ionic salt, such as sodium chloride.

In certain embodiments, the ophthalmic composition also will contain a variety of other materials to adjust pH (typically to a pH between about 5.5 to 8.5, preferably between about 6.5 to 7.5), preserve the treatment system, and the like. Suitable buffering agents include alkali metal phosphates, such as sodium and potassium phosphate, or mixtures thereof. These buffering agents are generally present in amounts of from about 0.1 to about 1.0 percent weight/volume. Some embodiments will employ two or more buffering agents. Preservative agents which can be used include benzalkonium chloride in a concentration range of from 1:15,000 to 1:30,000; chlorobutanol in a concentration range of from 0.3% to 0.8%; thimerosal in a concentration range of 0.001% to 0.003%; and phenyl mercuric nitrate in a concentration range of from 1:60,000 to 1:80,000 (volume/volume) . Additional suitable preservatives include sorbic acid, ethylenediaminetetraacetic acid (EDTA) , and other ophthalmically-acceptable preservatives. Other agents may be added to increase viscosity, promote suspension and/or improve ocular compatibility, such as methyl cellulose in an amount of from 0.1% to 0.7%, or poly(vinyl alcohol) in an amount of from about 0.4% to 2.0% (volume/volume) or non-ionic surfactants, such as polyoxyalkylene oleic esters of sorbitol anhydrides, and the like. These and other additive materials, such as medicants (pharmaceutically active agents, such as compounds for the treatment of glaucoma, conjunctivitis, inflammatory ocular conditions, or red eye) , stabilizers, lubricants, vasoconstricting (i.e., decongesting) agents are known in the art.

Additionally, in some embodiments of this invention, the ophthalmic composition will further comprise glycosaminoglycan, hyaluronic acid, or other conventional agents for anterior segment surgery, such as Occucoat® (2% hydroxylpropyl methyl cellulose, available from Storz Ophthalmics, Inc., St. Louis, MO), Viscoat® (sodium chondroitin sulfate-sodium hyaluronate solution, available from Alcon Surgical, Inc., Fort Worth, TX) , Healon® and Healon® GV (sodium hyaluronate, available from Kabi Pharmacia Ophthalmics, Inc., Monrovia, CA) , Amvisc® and Amvisc® Plus (sodium hyaluronate, available from Alcon Surgical, Inc.) and AMO® Vitrax® solution (sodium hyaluronate, Allergan Medical Optics, Irvine, CA) .

5. Formulation and Administration

Formulation of the ophthalmic compositions of the present invention may be effected in any convenient manner known to the art, such as by simply admixing the desired amount of the specified ingredients, and providing the necessary amount of liquid carrier to provide the necessary dilution. On a volume/volume basis, the epithelial-derived secretion in the ophthalmic composition will generally comprise between about 2 and 100%, more preferably, between about 10 and 50%, of the composition. The amounts of the active compound within these ranges, dissolved in a suitable ophthalmically-acceptable carrier, have been demonstrated to effectively increase tear break-up time and alleviate the symptoms of dry eyes, as described further below.

The method of application of the ophthalmic compositions of the instant invention to the subject is by topical application of a therapeutically- or pharmaceutically- effective amount of the treatment composition to the ocular surface. Conventional means for dispensing the treatment composition are suitable. For example, application of the treatment composition may be by spray dispenser, eye drop, and the like. Alternatively, slow or extended-release systems, including, collagen shields, for example, Bio-Cor® Collagen Corneal Shields (a clear, pliable, thin film fabricated from porcine scleral tissue, available from Bausch and Lomb) , other biological-based systems, systems employing liposomes, and polymeric delivery systems, can be utilized with the compositions described herein to provide a continuous source of the ophthalmic composition to the eye. Examples of these delivery systems are described in "Ophthalmic Drug Delivery Systems", Mitra (ed.), Marcel Dekker, Inc.: New York, NY (1993).

If the treatment composition is in the form of an aqueous solution or emulsion, a single drop from a conventional eyedropper is satisfactory. Preferred dosage comprises application of one or more drops several times daily. The preferred dosage is believed to be capable of providing relief for periods of time at least several fold greater than that of commercial formulations, dependent upon the subject.

The compositions of the present invention, instilled drop-wise in the eye, swell the tear meniscus and, with normal blinking, the tear meniscus becomes thoroughly admixed with the tear film. The flooded tear meniscus returns to its steady- state size and the superficial lipid layer is reestablished over the aqueous layer. Moreover, the ophthalmic compositions of the present invention interact favorably with both the superficial lipid layer and the underlying mucous layer, enhancing film stability.

The compositions are frequently packaged with labelling indicating their suitability for formulating medicaments for ophthalmic use. The label provides information as to the formulation of the compositions and/or the recommended ophthalmic treatment regime.

The compositions of the present invention can lower the surface tension of the tear film by increasing the film pressure of the lipid without interfering with the integrity thereof, the film pressure of an insoluble film such as lipid over water being defined as the difference between the surface tension of the pure water surface and that of the lipid-covered water surface.

Further, the compositions of the present invention do not affect adversely the clarity of the aqueous tear layer. Moreover, the compositions of this invention are compatible with the use of contact lenses. Finally, the compositions do not contribute to hydrophobic contamination of the mucus layer, but on the contrary, are capable of forming a hydrophilic layer in the absence of a functional mucus layer, having all of the functional properties of a normal mucus layer. The compositions of the present invention, topically applied, provide relief for both aqueous-deficient, lipid- deficient, and mucus-deficient eyes, provide highly effective lipid-masking and scavenging layers, and are effective also therapeutically in situations where protein, carbohydrate, and/or lipid abnormalities exist. In addition, the compositions can be used as lubricants for intra-ocular lenses and for post-cataract surgery.

In order that the invention described herein can be more fully understood, the following examples are set forth. It should be understood that these examples are for illustrative purposes only, and are not to be construed as limiting this invention in any manner.

EXPERIMENTAL

I. Compositions

The epithelial-derived secretion or component(s) thereof can be applied directly to the ocular surface or can be diluted with a suitable ophthalmically-acceptable carrier. Such carriers are generally known in the art. Typically an aqueous-base system wherein the carrier includes a buffer system to provide a suitable pH, a viscolyzer to provide suitable viscosity for eye comfort, an antibacterial agent, and a chemical preservative is used. An example of such a carrier is Dacriose which is a sterile ophthalmic irrigating solution from Iolab (Claremont, CA) , containing water, sodium phosphate, potassium chloride, sodium hydroxide, disodium ethylenediamine tetraacetic acid, sodium chloride, and benzoalkonium chloride.

II. Absence of Side-Effects

A trial was performed to assess possible side effects from ocular administration to New Zealand white rabbits of Survanta™ or Exosurf Neonatal™ Two groups of four rabbits received one drop of 1:10 or 1:100 dilutions of Survanta™ or Exosurf Neonatal™ three times daily for a period of four weeks. The contralateral eyes of each animal received diluent only three times daily over the same period of time, as a control. Parameters measured included swelling or injection of the conjunctiva, clouding of the cornea, cells and flare in the anterior chamber, reddening of the iris, and clouding of the lens, using a standard rating scale. Corneal thickness (pachymetry) and intraocular pressure (i.o.p.) were also monitored. After four weeks of receiving drops, the rabbits were followed for two additional weeks without drugs. At the end of this period, four rabbits were euthanized and the eyes processed for histological studies. No adverse effects or pathology due to topical administration of either Survanta™ or Exosurf Neonatal™ were observed at any time in any animal in this study based on the clinical and histological parameters investigated. The remaining four rabbits continued to receive two doses of either Survanta™ or Exosurf Neonatal™ per day without side effects. III. Human Clinical Trials Tests

A. Evaluation of Tear Break-Up Time

The normal tear film maintains an uninterrupted surface for a finite but adequate period of time to both protect the eye and to permit clear vision without blinking. Blinking restores and maintains the tear film. Certain conditions, such as forcibly restraining blinking, can cause the tear film to become discontinuous. The measurement of the time (in seconds) required for the tear film to evidence areas of discontinuity when blinking is suspended, is known as tear break-up time (BUT) . Measurement of TBUT is a standard screening test for assessing the efficacy of therapeutics for treating tear film disorders (see Lemp, Diagnosis and Treatment of Tear Deficiencies in Clinical Ophthalmology (eds., Duane, T.D. & Jagger E. , Harper & Rowe, 1988)).

In the prior art, BUT has been measured by adding fluorescein, a fluorescent dye, and observing the stained tear film with light passed through a cobalt blue filter. Other workers have employed optical imaging (for example, as described in Norn "Tear Film Break Up Time. A Review" in Holly (1986) riie Preocular Tear Film, pp. 52-54 and Glonek et al. (1990) U.S. Patent No. 4,914,088). This test is performed with an ophthalmometer without the use of fluorescein dye. For each subject, a baseline BUT value is established. Larger BUTs represent a more stable tear film. The effectiveness of ophthalmic compositions is investigated by comparison of BUT for various test solutions and controls versus a baseline BUT value for each eye tested. To evaluate BUT for a given test solution, the test solution is added to the eye by depressing the lower eyelid and placing one standard drop of the test solution into the inferior fornix (the space between the lower eyelid and the eye) . The eyelid is then released, the subject is asked to blink at five second intervals, and one minute is allowed for the tear film to stabilize. The BUT measurement is then made with the ophthalmometer.

To obtain baseline data, the BUT test is performed prior to the addition of a test solution and then following the use of a test solution. Where multiple test solutions are used on a single subject on a given day, a recovery (waiting) time is required between trials. The recovery procedure involves irrigation (flushing) of the front surface of the eye with sterile saline. Following irrigation, a recovery time of 20 minutes is provided to allow for stabilization of the tear film. If BUT is not consistent with initial baseline findings, further recovery time is provided until BUT times have returned to normal.

B. Break-up Time Results

The ophthalmic compositions of the present invention can be tested as described below.

50%, 10%, and 2% solutions of Survanta® in Dacriose were prepared. Full strength (100%) or diluted solutions of Survanta® were dropped in the eyes of two healthy men and compared with one another with respect to tear break-up time. Full strength Dacriose and Alcon Tears Naturale Free served as the controls. Table 1 lists the tear break-up times which were determined.

TABLE 1 TEAR BREAK-UP TIME (sec)

Composition Person 1 Person 2

Control 7, 5 12, 15 Dacriose

Control

Alcon Tears - 20

Naturale Free

2% Survanta™ >35¹ 15, 15

10% Survanta™ 15, 16 >18¹, >18¹, 21

50% Survanta™ 20, 22 >29^X, >30¹

100% Survanta™ >30¹ >25¹

The symbol ">" denotes that the person blinked. As shown by Table 1, tear break-up time using Survanta™ increased by approximately seven-fold when compared to tear break-up time using Dacriose and approximately two-fold when compared to Alcon Tears Naturale Free. As also indicated by Table 1, the subjects often blinked before the actual tear break-up time was recorded. Therefore, the actual tear break¬ up times are longer than shown in Table 1 in those instances. Based on these promising results and the absence of side-effects from animal trials, a larger-scale 40-human patient clinical trial was conducted at the Anheuser-Busch Eye Institute under Institutional Review Board (IRB) approval. A first group consisting of 10 patients with dry eye (untreated tear break up time (TBUT) < 10 sec) and a second group consisting of 10 patients without dry eye (TBUT ≥ 10 sec) each received Survanta™ in the right eye. Third and fourth groups, consisting of 10 patients with and without dry eye, each received Exosurf Neonatal™ in the right eye. At least 5 patients in each group were treated with Survanta™ or Exosurf Neonatal™ at a dilution of 1:10; the remaining patients received Survanta™ or Exosurf Neonatal™ at dilution of 1:25, 1:50, or 1:100 to determine the efficacious dose range. The left eye of each patient was used first to measure TBUT in the absence of treatment, and then after about a 30 min interval, to measure TBUT after treatment with HypoTears PF™. Hypotears PF™ is a mixture of polyvinyl alcohol and Lipiden™ polymeric vehicle (polyethylene glycol 400 dextrose, EDTA, and water) manufactured by CibaVision.

TBUT was measured using a slit lamp after staining the eye with fluorescein (using the procedure described in the specification, at p. 19, second paragraph) . Fig. 1 compares TBUT (sec) from 14 patients receiving Survanta™ at a 1:10 dilution, Hypotears PF™ or no treatment. Similar data comparing 10 patients receiving Exosurf Neonatal™ at a dilution of 1:10, Hypotears PF™ or no treatment are shown in Fig. 2. These data support a number of conclusions.

Treatment with Survanta™ at a 1:10 dilution results in a significant increase in TBUT values, 2.4 to 3.5 fold greater than Hypotears PF™ or no treatment (p < 0.01). Treatment with Exosurf Neonatal™ at a 1:10 dilution significantly increases TBUT values by a factor of 1.6 to 2.0 compared with Hypotears PF™ or no treatment (p < 0.01). The increased TBUT values from treatment with Survanta™ are significantly greater than those from Exosurf Neonatal™ (p < 0.05). In addition, retention of increased TBUT was noted up to 25 min after treatment with Survanta™ (see Fig. 3) , in contrast to treatment with Hypotears PF™, after which TBUT returned to baseline within minutes.

In the dose-response study, dilutions of Survanta™ at 1:25, 1:50, and 1:100 were equally effective in promoting increased TBUT (see Fig. 4) , suggesting that low doses of a component or group of components in Survanta™ achieve full efficacy.

None of the patients in the trial reported having experienced any side effects from administration of Survanta™. The majority of patients reported increased comfort from treatment with Survanta™ compared with treatment with Hypotears PF™. The significantly increased values of TBUT for both Survanta™ and Exosurf Neonatal™ indicate a rapid and substantial response to the administration of a pulmonary surfactant in a type of human clinical trial predictive of efficacy in treatment of ocular disorders characterized by human tear film deficiencies, such as dry eye disease. The significantly greater efficacy of the natural pulmonary surfactant, Survanta™, over the protein-free synthetic pulmonary surfactant, Exosurf Neonatal™, suggests that surfactant-associated protein B and/or C, which are present in Survanta™ but not Exosurf Neonatal™ is/are among the active components of Survanta™.

C. Measurement of Visual Blur Time

Any liquid added to the tear film would be expected to cause a blur until the tear film returns to a form providing optimal optical characteristics. The test described below is intended to quantify blur time as a consequence of addition of a test solution.

To qualify a subject prior to the addition of a test solution, the subject is directed to a standard vision testing chart to determine if the subject's habitual tear film results in a visual blur with a measurable visual blur time (VBT) . A subject qualifies if the habitual tear film does not produce a measurable VBT, i.e., in the absence of a test solution, VBT is expected to be zero.

The test solution is added to the eye by depressing the lower eyelid and placing one standard drop into the inferior fornix. The eyelid is released, the subject's attention is directed to a standard vision testing chart, and the subject is asked to blink at five second intervals until the blur resolves. The time required for the blur to resolve, in seconds, is recorded as the VBT. This time may vary from several seconds to minutes. Times of less than 30 seconds are desirable.

IV. Rose Bengal Staining

Rose bengal is a vital dye that stains injured epithelial cells. It stains dry, desiccated epithelium, while those cells are still in situ. This test involves the application of 1% Rose Bengal to the eye. The amount of staining is then rated according to the list of ratings of Kaufman et al. (1963) Archives of Ophthalmology 69:926.

V. Other Testing Procedures The compositions of the present invention can also be evaluated using other testing procedures well known in the art. Examples of these protocols include the Snellen visual acuity test, the Schirmer test (see Lamberts (1987) "Physiology of the Tear Film" in TΛe Cornea, S olin and Thoft, Eds., Little Brown and Company: Boston, pp. 38-52) , conjunctival cytology, contrast sensitivity, and evaluations of patient comfort and blink rate.

All publications and patent applications cited above are incorporated herein by reference in their entirety for all purposes.

It is to be understood that the above description is intended to be illustrative and not restrictive. Many embodiments will be apparent to those of skill in the art upon reviewing the above description. The scope of the invention should, therefore, be determined not with reference to the above description, but should instead be determined with reference to the appended claims, along with the full scope of equivalents to which such claims are entitled.

As will be clear to those skilled in the art from the above, the invention includes a number of general concepts which can be expressed as follows. One general aspect of the invention is the use of a natural pulmonary surfactant for manufacture of a medicament for treating ophthalmic diseases, such as dry eye. A second feature of the invention is the use of active component(s) of a pulmonary surfactant for the same purpose. Preferred components include surfactant-specific proteins B and C. In some uses, active components are formulated in a medicament with phospholipids and/or neutral lipids in proportions and amounts simulating the concentrations of the same in a natural pulmonary surfactant. In other uses, active components of a natural pulmonary surfactant are used to supplement conventional ophthalmic compositions. Yet another aspect of the invention, is the use of a natural pulmonary surfactant as a source for purifying active components suitable for manufacture of a medicament for treating ophthalmic disease.

Claims

WHAT IS CLAIMED IS:

1. An ophthalmic composition comprising a non- conjunctival, epithelial-derived secretion and an ophthalmically-acceptable carrier.

2. The composition of claim 1, wherein the non- conjunctival, epithelial-derived secretion comprises a pulmonary surfactant.

3. The composition of claim 2, wherein the nonconjunctival, epithelial-derived secretion comprises a natural pulmonary surfactant.

4. The composition of claim 3, wherein the pulmonary surfactant comprises a surfactant-associated protein or a fragment thereof capable of reducing surface tension on a liquid in which the fragment is dispersed.

5. The composition of claim 4, wherein the surfactant-associated protein is selected from the group consisting of SP-A, SP-B, SP-C, and SP-D.

6. The composition of claim 4, wherein the non- conjunctival, epithelial-derived secretion consists essentially of a surfactant associated protein selected from the group consisting of SP-A, SP-B, SP-C, and SP-D.

7. The composition of claim 5, wherein the non- conjunctival, epithelial-derived secretion further comprises a component selected from the group consisting of phospholipids, proteoglycans, and glycosaminoglycans.

8. The composition of claim 1, wherein the secretion is water-soluble.

9. A method for treating ocular disorders, the method comprising administering to the eye a therapeutically effective amount of composition comprising a non-conjunctival, epithelial-derived secretion.

10. The method of claim 9, further comprising the step of diluting the composition with an ophthalmically- acceptable carrier.

11. The method of claim 10, wherein the non- conjunctival, epithelial-derived secretion comprises a pulmonary surfactant.

12. The method of claim 11, wherein the pulmonary surfactant comprises a surfactant-associated protein or a fragment thereof capable of reducing surface tension on a liquid in which the fragment is dispersed.

13. The method of claim 12, wherein the surfactant- associated protein is selected from the group consisting of SP- A, SP-B, SP-C, and SP-D.

14. The method of claim 13, wherein the composition consists essentially of a surfactant-associated protein selected from the group consisting of SP-A, SP-B, SP-C, and SP- D.

15. The method of claim 13, wherein the non- conjunctival, epithelial-derived secretion further comprises a component selected from the group consisting of phospholipids, proteoglycans, and glycosaminoglycans.

16. The method of claim 15, wherein the ocular disorder is a deficiency in the ocular tear film.

17. A process for preparing an ophthalmic solution, the process comprising: providing a non-conjunctival, epithelial-derived secretion; and mixing at least one component of the secretion with an ophthalmically-acceptable carrier.

18. The process of claim 17 further comprising the step of purifying the component from the secretion before the mixing step.

19. The process of claim 18 wherein the non- conjunctival, epithelial-derived secretion comprises a pulmonary surfactant.

20. The process of claim 19, wherein the pulmonary surfactant comprises a surfactant-associated protein or a fragment thereof capable of reducing surface tension on a liquid in which the fragment is dispersed.

21. The process of claim 20, wherein the component comprises a surfactant-associated protein selected from the group consisting of SP-A, SP-B, SP-C, and SP-D.

22. The process of claim 21, wherein the component is selected from the group consisting of phospholipids, glycosaminoglycans, and proteoglycans.