WO2003028751A1 - Glycoproteine et/ou glycomacropeptide contenant une preparation ophtalmique - Google Patents

Glycoproteine et/ou glycomacropeptide contenant une preparation ophtalmique Download PDF

Info

Publication number
WO2003028751A1
WO2003028751A1 PCT/US2002/031657 US0231657W WO03028751A1 WO 2003028751 A1 WO2003028751 A1 WO 2003028751A1 US 0231657 W US0231657 W US 0231657W WO 03028751 A1 WO03028751 A1 WO 03028751A1
Authority
WO
WIPO (PCT)
Prior art keywords
accordance
ophthalmic preparation
glycoprotein
glycomacropeptide
ophthalmic
Prior art date
Application number
PCT/US2002/031657
Other languages
English (en)
Inventor
Edward J. Ellis
Xiaomi Tong
Original Assignee
Vista Scientific Llc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Vista Scientific Llc filed Critical Vista Scientific Llc
Publication of WO2003028751A1 publication Critical patent/WO2003028751A1/fr
Priority to US10/813,998 priority Critical patent/US20040214754A1/en
Priority to US10/814,001 priority patent/US20040220089A1/en

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/20—Milk; Whey; Colostrum
    • 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
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals

Definitions

  • the present invention relates to ophthalmic preparations and more specifically relates to ophthalmic preparations for use as a tear film supplement, wherein the preparation includes at least one of a glycoprotein and a glycomacropeptide component.
  • the tear film as including three distinct layers and as being a three-layered, aqueous-dominated tear film.
  • One of the layers comprises a mucin layer which serves primarily to render the hydrophobic ocular surface hydrophilic, so that the aqueous layer comprising the bulk of the tear film will spread evenly over the eye.
  • This gel has its highest concentration of mucin at the epithelial surfaces of the cornea and conjunctiva, and the mucin concentration gradually decreases farther out into the tear film.
  • the presence of mucin remains significant for the structure, stability and function of the entire tear film.
  • Recent studies of the tear film using laser interferometry and confocal microscopy might be including the entire gel layer in indicating that the human tear film is 30 to 40 microns thick, more than four times thicker than earlier estimates.
  • tear film abnormalities are commonly designated by focus on a specific deficiency, such as an aqueous tear deficiency, kerato-conjunctivitis sicca (KCS), a mucin deficiency, a lipid abnormality, an impaired lid function, or an epitheliopathy.
  • KCS kerato-conjunctivitis sicca
  • ocular surface disease is expressed in ocular symptoms, such as dryness, grittiness, burning, soreness or scratchiness, with variation among individuals. These symptoms can also be exacerbated by factors such as environmental conditions and contact lens wear. The combination of varying clinical signs and symptoms has also been termed dry eye syndrome.
  • Ointments are generally anhydrous preparations based on mixtures of white petrolatum and mineral oil. Because these formulations are greasy and cause blurred vision, they are not widely used other than in cases of severe symptoms, and are mostly limited to application at night just before sleeping. Emulsion based formulations for treating dry eye symptoms have emerged over the past ten years.
  • One approach has been disclosed in a series of U.S. patent Nos.: 5,578,586; 5,371,108; 5,294,607; 5,278,151; 4,914,088, all of which are herein incorporated by reference in their entirety. These patents teach the methods and compositions for reducing evaporation of the aqueous layer from the surface of the eye.
  • the method comprises applying an admixture of a charged phospholipid and a non-polar oil over the eye, preferably in the form of a finely divided oil-in-water emulsion.
  • a charged phospholipid and a non-polar oil over the eye, preferably in the form of a finely divided oil-in-water emulsion.
  • Another approach is described in U.S. patent Nos. 4,818,537 and 4,804,539, incorporated herein by reference in their entirety, where liposome compositions in the form of emulsions are claimed to provide enhanced retention on ocular surfaces and thereby alleviate the symptoms of dry eye.
  • Solid devices, in the form of ocular inserts have been utilized for longer term symptomatic relief of dry eye. These devices are placed in the eye and slowly dissolve or erode to provide a thickened tear film. Often patients find these devices difficult to insert and once in place, they tend to be uncomfortable.
  • the patent literature reveals one dated reference to the use of mucin in sterilized, preserved and stable solutions.
  • U.S. patent No. 4,438,100 incorporated herein by reference in its entirety, describes mucin-containing solutions for application to sensitive mucous membranes of the oral cavity, the nasal system and the eye.
  • the mucins utilized in this invention are non human mammalian mucins selected from the group consisting of buccal and gastrointestinal mucins.
  • the source of their mucins is mucus, a mature and complex secretion containing a mixture of various mucin molecules as well as other proteins and associated contaminants of secretion.
  • the present application relates to ophthalmic preparations for use as a tear film supplement. More specifically, the invention relates to an aqueous formulation to be instilled into the eye, or in which to pre soak or store an object to be inserted into the eye, such as a contact lens, an ointment, or a solid device to be inserted into the conjunctival sac.
  • the preparations disclosed are utilized for the treatment of disorders such as keratoconjunctivitis sicca or dry eye syndrome.
  • the preparations of this invention are also effective for the relief of symptoms of eye irritation, such as those caused by dry environmental conditions or by contact lens wear.
  • the present application relates to ophthalmic compositions including at least one glycoprotein and a glycomacropeptide component, as well as to methods for their preparation and storage.
  • the application also relates to a method of treating the eye by topically applying the composition of the present invention, when indicated, to provide lubrication and protection of the ocular surface, for the relief of dryness and discomfort symptoms, such as experienced in patients with dry eye and following traumatic injury or surgery, and when indicated to achieve the other effects mentioned above.
  • the compositions of the present invention are provided as buffered, sterile aqueous solutions.
  • the subject compositions may be unpreserved (provided in a unit dose format) or may be preserved.
  • the glycoproteins and glycomacropeptides are isolated from mammalian milk or milk byproducts. In another preferred embodiment the glycoproteins and glycomacropeptides are isolated from bovine milk. In yet another preferred embodiment the glycoproteins and glycomacropeptides are derived from dairy whey, a byproduct of cheese making.
  • other ingredients commonly employed in ophthalmic formulations are utilized to provide a balance of physiologically acceptable properties, depending on whether the final product is a solution, ointment, gel or solid. However, it will be understood that the glycoproteins and glycomacropeptides can be derived from a number of other sources so long as the materials are suitable for the intended use described herein.
  • Glycroproteins are of great interest as components of mammalian membranes. Structurally they are composed of relatively short carbohydrate sequences covalently linked to a protein core.
  • the glycoproteins of the cell surface appear to function in recognition, intercellular adhesion and modulation of certain intracellular transport event. That one characteristic phenotypic expression of tumorigenic transformation is an alteration in cell surface glycoproteins strongly implies an involvement of glycoproteins in at least several cell surface functions. It is believed that the glycoproteins and glycomacropeptides of this invention leave biological activity and many stimulate the production tears and in particular the stimulation of mucus. Lipid globules in milk are enclosed in a membrane which is derived directly from the apical plasma membrane of mammary epithelial cells.
  • MFGM milk fat globule membrane
  • glycoproteins With bovine MFGM, the presence of five to eight major glycoproteins have been detected with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS- PAGE). The molecular weight of these glycoproteins can range from about 30,000 to 40,000 daltons up to about 250,000 daltons. The more abundant glycoproteins have molecular weights in the 60,000 to 125,000 dalton range. A mucoprotein has also been isolated from bovine milk and was found to have a molecular weight of about 123,000 daltons.
  • SDS- PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis
  • glycoproteins isolated form bovine milk and milk byproducts can have carbohydrate contents, as a percentage of the protein weight, of 10% to 50% or even higher.
  • carbohydrate contents as a percentage of the protein weight, of 10% to 50% or even higher.
  • the water solubility or compatibility of the glycoproteins increases with increasing carbohydrate content, hi fact, Kobylka et al back in 1972 reported the isolation and characterization of six major glycoproteins from the bovine milk fat globule membrane. The molecular weights of these are listed below.
  • the glycoproteins When isolated from milk fat globules, the glycoproteins may exist as a complex. This complex may contain mucin associated with one or more other glycoproteins and other components such as lipids, phospholipids, lipoproteins and oligosaccharides. These complexes can have an apparent molecular weight of from about 200,000 to 500,000 daltons or greater, hi the practice of this invention either the glycoproteins themselves or the complex of glycoproteins can be utilized.
  • Bovine milk and milk byproducts also contain naturally occurring glycomacropeptides. The most notable is a sixty-four residue glycopeptide with a relatively high molecular weight. Glycomacropeptides are also produced by cleaving the peptide linkages of kappa casein. The cleavage process can be carried out either chemically through hydrolysis or enzymatically utilizing chymosin. The resulting glycomacropeptides structurally are composed of carbohydrate moities covalently linked to relatively short peptide sequences. The molecular of theses glycomacropeptides is generally in the 3,000 to 15,000 daltons and more typically in the 6,000 to 10,000 dalton range.
  • Mucins refer to a family of glycoproteins of high molecular weight, secreted or expressed by goblet and nongoblet epithelial cells of mucosal tissues. These mucins predominate in the formation of mucus, a highly hydrated gel of particular structure and function. Mucins are heavily glycosylated high molecular weight glycoproteins with predominantly O-linked oligosaccharide side chains. Mucin molecules are generally above 200Kda, with carbohydrate composing 50% or more of their dry weight. At least nine distinct mucin genes have been identified (MUC1, 2, 3, 4, MUC5AC, MUC5B, MUC6, 7 and 8). Furthermore, each of these is produced in various forms in different tissues.
  • MUC1 the so-called “epithelial mucin”
  • epithelial mucin is a membrane-spanning mucin found in abundance in epithelial tissues.
  • MUC1 is known by various other names, such as polymorphic epithelial mucin or episialin.
  • MUCl has a molecular weight in the 125 to 225KDa range, which is low when compared to the other types of mucin.
  • the secretory mucins MUC2 and MUC5AC have been detected (via transcripts at the nucleic acid level) from conjunctival isolates, and only MUC5AC has been localized to conjunctival goblet cells.
  • the transmembrane mucin MUCl is associated with the cell membranes of the entire corneal and conjunctival epithelial surface, except the goblet cells.
  • the transmembrane mucin MUC4 is associated with the cell membranes of the entire conjunctival epithelial surface, except the goblet cells.
  • the secreted ocular mucins are relatively large molecules, and have a significant role in the gel-forming nature of the tear film.
  • the visco elasticity of the tear film derives from the specific structure and gel-forming properties of the ocular mucins, and allows the tear film to absorb the shear force of the blink, which would otherwise irritate and damage the ocular surface.
  • the transmembrane mucin serves more as a protective layer on the actual cellular surface of the ocular epithelium, functioning to directly protect and lubricate the ocular surface, as well as to anchor the highly hydrated gel (mucus) of the tear film gel-forming mucins, thereby assisting in the spreading and stability of the tear film over the ocular surface.
  • Stratified layers of the above mucins are known to form over the surface of mucosal membranes, such as in the gut, affecting the flow and interaction of the protective layer and its contents with the cellular surface of the epithelium.
  • a difficiency in one type of mucin would therefore be expected to affect the lubricating, protective, barrier and other functions of the other mucins at the mucosal surface.
  • the goblet cell density is not significantly reduced, indicating that MUC5AC is most likely still able to be produced normally, in quantities sufficient to be spread over the entire ocular surface.
  • localized early ocular surface changes resulting from dryness such as that revealed by fluorescein or rose bengal staining, can be seen in the epithelia of the corneal and conjunctival surfaces. This localized damage to the ocular surface indicates that even marginal dryness might have a significant effect on the presence of functional MUCl on the surface of the ocular epithelium.
  • MUCl Since one of the proposed functions of MUCl is to help the other, more abundant gel-forming ocular mucins adhere to the ocular surface, a paucity of MUCl might significantly affect the stability of the tear film, even in the presence of an abundance of MUC5AC secreted by the conjunctival goblet cells.
  • impression cytology the more severe ocular surface changes resulting from dryness, exhibited in the process of squamous metaplasia, are also seen to occur initially in localized areas. These more pathological localized surface changes are further evidence for a critical protective role of MUCl.
  • glycoproteins and glycomacropeptides described in this invention act to protect and lubricate the ocular surface, as in the role of the natural transmembrane mucins, MUCl and MUC4, which is expressed by the entire surface epithelium of the conjunctiva and cornea.
  • the lubrication and protection of the ocular surface is enhanced, in order to slow the progression, and associated development of symptoms, of changes to the ocular surface epithelium, such as decreased tear film stability, increased staining with fluorescein sodium or rose bengal, decreased goblet cell density and the development of squamous metaplasia seen with ocular surface disease.
  • the property of viscosity in the preferred embodiment is primarily targeted to assist in retention of the invention in the eye at the ocular surface, as well as for lubrication and comfort associated with instillation. Viscosity is not the physical property which gives the mucin formulation of this invention its "mucomimetic" function.
  • This invention primarily protects and lubricates the ocular surface and interacts with the gel-forming secreted mucins of the tear film, thereby enhancing the spreading of the tear film, and by default of instillation adds to the tear film volume and hydration of the ocular surface.
  • the "mucomimetic" effects of this invention protect the ocular surface from dryness and absorb shear forces of the blink, and assist the eye's own secreted gel forming mucins (predominantly MUC5) in maintaining their viscoelastic properties and ensuing structure and stability of the tear film, thereby slowing or preventing the changes to the ocular surface seen in dry eye conditions.
  • glycoproteins and glycomacropeptides of this invention are preferentially isolated from bovine milk or milk byproducts. Most preferably the glycoproteins and glycomacropeptides are isolated from dairy whey.
  • MilcinTM refers to the glycoproteins and glycomacropeptides isolated from milk or milk byproducts.
  • MilcinTM will be used to describe glycoproteins and glycomacropeptides isolated from dairy whey.
  • the material MilcinTM is utilized as the active ingredient in ophthalmic preparations. Recovery and purification of glycoproteins and glycomacropeptides can be carried out utilizing standard methods known in the art.
  • the scientific literature reveals a number of techniques for characterizing the various types of glycoproteins and glycomacropeptides. These techniques include, but are not limited to, chromatographic techniques or gel electrophoresis, particularly SDS- PAGE, followed by direct protein staining (e.g. silver staining) or immunohistochemical staining (e.g. Western blotting or Northern blotting), immunoprecipitation techniques, amino acid analysis, carbohydrate determination, lectin binding probes, light scattering, scanning electron microscopy, mass spectrometry, protein nitrogen content and ash.
  • chromatographic techniques or gel electrophoresis particularly SDS- PAGE
  • direct protein staining e.g. silver staining
  • immunohistochemical staining e.g. Western blotting or Northern blotting
  • immunoprecipitation techniques amino acid analysis, carbohydrate determination, lectin binding probes, light scattering, scanning electron microscopy, mass spectrometry, protein nitrogen content and ash.
  • glycoprotein and/or glycomacropeptide in an ophthalmic formulation can vary greatly depending on the product type.
  • the glycoprotein and/or glycomacropeptide concentration would vary from about 0.0001% to 5.0% by weight.
  • the glycoprotein and/or glycomacropeptide level could vary from about 0.1% to about 10.0% by weight
  • the glycoprotein and/or glycomacropeptide level could range to about 90% or greater by weight.
  • concentration might be varied, depending on such factors as the severity of the dry eye condition being treated, to enhance particular properties of the glycoprotein and/or glycomacropeptide solution.
  • exemplary ophthalmic compositions include a glycoprotein and/or glycomacropeptide from any number of the exemplary sources described herein before.
  • other solution components may be employed as required:
  • Cellulose derivatives are commonly used to increase viscosity. Specific cellulose derivatives include: hydroxypropylmethylcellulose, carboxymethylcellulose, methylcellulose, hydroxyethylcellulose, etc. Some polysaccharides may also be utilized to increase the viscosity of ophthalmic solutions and include xanthan, scleroglucan, carrageenans, tragacanth gum, hyaluronic acid etc. Other viscosifiers that are useful include polyvinylpyrrolidone, polyvinyl alcohol, polyethyleneoxide, polyacrylic acid and crosslinked polyacrylic acid. Generally, viscosifiers are present in the amount of 0.1 to 0.75 % by weight of the solution.
  • Any pharmaceutically acceptable buffer system may be utilized and include phosphates, borates, citrates, acetates and carbonates in amounts necessary to produce a pH of about 6.0 to about 8.0.
  • the tonicity of the ophthalmic solutions described here can be adjusted to either hypotonic, isotonic or hypertonic relative to nonnal tears by use of generally used materials know to the art.
  • Sodium and potassium chloride are widely used to adjust tonicity.
  • Other agents include dextrose, mannitol, sorbitol and urea.
  • Water binding compounds aid in retaining moisture on the ocular surface and include glycerin, propylene glycol, polyethylene glycol.
  • Certain compounds are useful to promote surface wetting, whether it be the ocular surface or the surface of a contact lens.
  • One category that is preferred is the polyoxamers. These polyethyleneoxide-polypropyleneoxide-polyethyleneoxide block copolymer are available from BASF. Other compounds include the Tetronics®, reverse Pluronics® and the reverse Tetronics®, also available from BASF.
  • compositions of this invention may include a preservative in an effective amount.
  • Preservatives known to the art include alkyldimethyl benzylammonium chloride (BAK), chlorhexidene gluconate (CHG), polyhexamethylene biguanide (PHMB), other polyquats and sorbic acid.
  • BAK alkyldimethyl benzylammonium chloride
  • CHG chlorhexidene gluconate
  • PHMB polyhexamethylene biguanide
  • the subject compositions may also include a co-preservative and or chelating agent, such as ethylenediaminetetraacetic acid (EDTA) and its salts.
  • EDTA ethylenediaminetetraacetic acid
  • OTHER ADDITIVES h some cases it may be beneficial to include other components in an ophthalmic solution. These include specific ions, such as Ca++, Zn++ and Mg++, Cu++, selenium, vitamins, such A, C and E, to promote ocular health.
  • the compositions described in this invention may also be utilized as vehicles for drug delivery. Drugs often used in the eye include anti-glaucoma compounds, anti-inflammatory agents and anti-infective agents.
  • this invention finds particular utility as lubricating eye drops, i.e., an artificial tear solution, a tear fluid supplement, a delivery vehicle for topical ophthalmic drug application, hi most of these applications, the compositions of this invention are provided in a buffered, sterile aqueous solution. Typically, these solutions have a viscosity from about 1 to 100 cps. As a solution the compositions of this invention are dispensed in the eye in the form of an eye drop.
  • compositions described in this invention may also be formulated as viscous liquids, i.e., viscosities from several hundred to several thousand cps, gels or ointments, h these applications the mucin component would be dispersed or dissolved in an appropriate vehicle such as Lubragel, GRR Lubricating Jelly or Karajel, all trademarked products of United-Guardian, Inc., Hauppauge, NY.
  • an appropriate vehicle such as Lubragel, GRR Lubricating Jelly or Karajel, all trademarked products of United-Guardian, Inc., Hauppauge, NY.
  • compositions of this invention may also be formulated as solid ocular inserts that dissolve or erode over time when placed in the cul-de-sac of the eye.
  • Swelling-controlled release devices would consist of mucin homogeneously dispersed in a glassy polymer such as a water soluble cellulosic.
  • a glassy polymer such as a water soluble cellulosic.
  • Erodible devices would again consist of glycoprotein and/or glycomacropeptide homogeneously dispersed in a polymer matrix.
  • glycoprotein and/or glycomacropeptide is released by a chemical reaction (hydrolysis) that results in solubilization of the matrix polymer, usually at the surface of the device.
  • the matrix material is a polyanhydride, poly(ortho ester), polylactic acid or a polyglycolic.
  • glycoprotein and/or glycomacropeptide may be chemically modified or crosslinked to act as its own “matrix", where the glycoprotein and/or glycomacropeptide comprises the entire, or nearly entire, device, thus providing the maximum amount of glycoprotein and/or glycomacropeptide available to the eye.
  • the glycoprotein and/or glycomacropeptide disclosed herein may be incorporated into contact lens soaking and conditioning solutions as well as lubricating eye drops for contact lens wearers.
  • the glycoproteins and/or glycomacropeptides may be utilized in drug delivery.
  • the most common and convenient method for delivery of ocular drugs is by way of topical eye drops.
  • the solution vehicles employed are quickly diluted by the tear fluid and drain from the eye in a matter of minutes. This short residence time hinders the absorption and hence the bioavailability of the drug in the eye.
  • the short residence time is overcome by greatly increasing the concentration of the drug to improve bioavailability. This often leads to significant undesirable side effects due to the systemic actions of many of the ocular drugs currently prescribed.
  • ophthalmic drugs for example timolol and pilocarpine
  • they will associate with the negatively charged polymer particles through electrostatic interaction.
  • the hydrated particles are microporous, the drug can be absorbed into the matrix.
  • an ophthalmic solution of this type is placed in the eye, the hydrated polymer particles adhere to the mucosal surface, providing extended residency time. During this residence the drug is released from the hydrated polymer particles, thus providing for a more efficient local delivery to the eye.
  • glycoproteins and/or glycomacropeptides of the present invention are considered "bioadhesive" given their functions in association with the plasma membrane of mammaiy epithelial cells. Given this information one would expect the glycoproteins and/or glycomacropeptides of this invention to act in a similar manner to the crosslinked carboxy-functional polymers as an ophthalmic drug delivery vehicle. In practice, the glycoproteins and/or glycomacropeptides of this invention provide superior retention time due to their ability to interact not only with the epithelial surface but also with the natural mucins in the tear film.
  • glycoprotein and glycomacropeptide containing ophthalmic preparations are merely illustrative of glycoprotein and glycomacropeptide containing ophthalmic preparations and the examples should not be considered as limiting the scope of this invention in anyway.
  • Whey is the by product of cheese manufacturing which begins with milk and can be divided into two processes, where the major one involves enzyme treatment of milk and the minor one involves bacteria treatment.
  • Sweet whey accounts for about 80% of whey produced and is an inexpensive and readily available source of glycoproteins and glycomacropeptides.
  • This example outlines a process for recovering glycoproteins and glycomacropeptides from dairy whey.
  • sweet dairy whey is the starting material, although acid whey can also be utilized since they both contain the glycoproteins and glycomacropeptides of this invention.
  • glycoproteins and glycomacropeptides derived from dairy whey The following table describes the physical and chemical properties of the glycoproteins and glycomacropeptides derived from dairy whey.
  • glycoproteins and glycomacropeptides are referred to as "MilcinTM", a trademark of Vista Scientific.
  • the glycoproteins and glycomacropeptides described in Example 3 are complexes composed essentially of the protein with physically associated lipids. This complex exists in aqueous solution as a hydrocoUoid.
  • a 1% by weight of MilcinTM Lot 4 (Example 3) dispersion was prepared in borate buffer, pH 7.2 and osmolality of 300.
  • Particle size of the MilcinTM hydrocoUoid was then determined by dynamic light scattering utilizing a Protein Solutions DynaPro MS/X instrument equipped with a Peltier temperature control device. The resulting data was analyzed with the Dynamics version 5.26.38 software provided by the manufacturer. Results were obtained using the regularization method at a grid size of 100. The following histogram was generated by the data.
  • the bulk of the MilcinTM hydrocoUoid has a hydrodynamic radius of about 40nm to about 60nm. There is a small amount of material that has a hydrodynamic radius about lOOnm.
  • SDS-PAGE was utilized to separate the glycoproteins and glycomacropeptides contained in the MilcinTM complex as a function of molecular weight. This was accomplished by rendering a negative charge in the proteins through their interactions with sodium dodecylsulfate (SDS). When placed in the gel and an electric field applied, the proteins migrate as a function of their charge to molecular weight. By providing all the proteins with the same charge with SDS the proteins migrate through the gel as a function of molecular weight only, and became distributed in the bands according to molecular weight. Known amounts of protein molecular weight standards were run concurrently to demonstrate molecular weight position.
  • SDS sodium dodecylsulfate
  • the resulting gels were first oxidized then stained with a Schiff reagent to develop only the glycoprotein bands (stains glycoproteins pink). After removing the pink stain, the gel is restained with coomassie blue stain. The coomassie blue reagent reveals only the protein bands (stans proteins blue). In this manner both the glycoprotein and other proteins that are present in the MilcinTM material are identified.
  • the following diagram presents the molecular analysis of a number of MilcinTM samples (see Example 3). It can be seen that the lots of MilcinTM, are for the most part, composed of glycosylated protein.
  • the molecular weight of these glycosylated proteins range from about 2000 daltons (glycomacropeptides) to about 200,000 daltons (mucin).
  • the bulk of the MilcinTM composition appears to be glycoprotein in the molecular weight range of about 20,000 to 100,000 daltons.
  • the following example illustrates a method for extracting the lipids from MilcinTM.
  • a sample of MilcinTM Lot 4 (see example 3) was rigorously extracted in the following manner. Approximately 1.0 gm of MilcinTM (Example 3 Lot 4) was extracted with methanol for one hour and the solids filtered. The solids were then resuspended in 15ml of chloroform and extracted for one hour and the solids filtered. The solids were then resuspended in 15ml of hexanes and extracted for one hour. The solids were filtered and dried in vacuum for one hour. The resulting sample was lipid free.
  • EXAMPLE 7 A sample of MilcinTM Lot 4 (see example 3) was rigorously extracted in the following manner. Approximately 1.0 gm of MilcinTM (Example 3 Lot 4) was extracted with methanol for one hour and the solids filtered. The solids were then resuspended in 15ml of chloroform and extracted for one hour and
  • Glycoproteins and glycomacropeptides are proteins, or protein fragments, that contain carbohydrate side groups.
  • the amount of carbohydrate can vary widely from
  • Glycoproteins and glycomacropeptides typically contain N-linked and O-linked oligosaccharides. This example presents the analysis of the extracted MilcinTM of
  • Example 6 for both neutral monosaccharides and sialic acid content.
  • a sample of the lipid extracted MilcinTM of Example 6 was submitted to:
  • the NeuAc value was measured from the injection of 12.5% of the hydrolysate and the NeuGc value was measured from the injection of 25% of the hydrolysate. From the above values it was calculated that there was 0.346 ⁇ g sialic acid/8 ⁇ g sample, or 4.3%> sialic acid.
  • glycoproteins and glycomacropeptides of this invention as isolated from dairy whey contain a small amount of lipids that are complexed with the protein.
  • the following example illustrates the extraction of lipids from MilcinTM and the subsequent identification of those extracted lipids by liquid chromatography (TLC).
  • MilcinTM contains about 10 to 20%) by weight of loosely bound lipids, that is, those extractable by methanol.
  • These lipids are primarily alkyl esters, fatty acids, polar esters and triglycerides.
  • the lipids extracted from MilcinTM in Example 6 were subjected to analysis by gas chromatography.
  • a series of lipids, Tripalmitate, Phosphotidylchohne dimyristate, Cholesterol oleate and Arachidic acid in a ratio of 1 :10:3 :30, [based on fatty acid content], were subjected to transmethylation.
  • the resulting fatty acid methyl esters were run on High Resolution Gas Chromatography and yielded the FAME standards shown below.
  • the retention time for each of the peaks on the H-P 1 column is proportional to the molecular weight of the FAME. Integration of these peaks showed a quantitative recovery of the starting lipids as their fatty acid methyl esters.
  • a similar extract and transesterification of the lipids recovered in Example 6 gave the FAME profile shown below.
  • the glycoproteins and glycomacropeptides of this invention would be expected to contain common metal ions.
  • DCP direct current plasma
  • the technique is essentially an atomic absorption technique where the spectral emission is formed in a plasma between the anode and cathode of a coupled electrode.
  • the technique is quantitative. MilcinTM Lot 4 (see Example 3) was dissolved in de-ionized water at a concentration of l,000ppm. Nitric acid was added (lppm). Proper DCP procedure was followed, including a two-point calibration (lOppm, Oppm) for multi-element analyses. The results are tabulated below and reflect the averaging of six measurements for each metal.
  • the measured concentration is the concentration of the metal in a l,000ppm solution of pure MilcinTM Lot 4.
  • glycoproteins and glycomacropeptides of the invention illustrates the use of the glycoproteins and glycomacropeptides of the invention as the active ingredients in an ophthalmic solution for the treatment of dry eye signs and symptoms.
  • This example illustrates the ocular compatibility of the glycoproteins and glycomacropeptides of this invention utilizing an in vitro transepithelial permeability assay.
  • MilcinTM The irritation potential of MilcinTM was evaluated at a level of 1.05% in an ophthalmic solution.
  • Example 12 The following solution formulations were prepared utilizing the compounding procedure detailed in Example 12. The finished solutions were transferred aseptically into sterilized bottles.
  • the solutions described in the table above were subjected to the following experiments to determine potential eye irritation of the solutions.
  • the experimental methods follow the procedure developed by R. Tchao, which is described in "Trans- Epithelial Permeability of Fluorescein In Vitro as an Assay to Determine Eye Irritants", Progress in In Vitro Toxicology, Volume 6, 1988, pages 271-283 (Mary Ann Liebert, Inc. Publishers, New York), the disclosure of which is incorporated herein by reference.
  • the Tchao technique is described as a method of determining potential eye irritation of a substance by correlating damage to a mono-layer of Madin-Darby Canine Kidney (MDCK) cells with damage to corneal epithelial cells.
  • MDCK Madin-Darby Canine Kidney
  • the amount of fluorescein passing through the cell mono-layer is a function of permeability of the cell mono-layer. Higher cell mono-layer permeability indicates greater damage to the cell junctions from application of a test solution thereto, whereas lower cell mono-layer permeability indicates less damage to the cell junctions from application of the test solution.
  • MDCK cells are obtained from ATCC, and maintained in minimum essential medium (MEM) supplemented with 10%> bovine calf serum with iron supplementation (Hyclone, Utah). Stock cultures are passaged weekly using trypsin and EDTA. Cultures are used before passage 50. For the test, 0.5ml of a cell suspension containing 2 x 10 E5 cells are seeded in Millicell HA 13mm inserts (Millipore, Bedford, MA). The inserts are placed in 24-well plates and fed with 0.5ml medium. Two days after seeding the cells, the media both inside and outside the inserts are replaced with fresh media. On day 6 after seeding, the inserts are used for testing the solutions. It has been shown that the resistance developed by a confluent MDCK monolayer is about 600 ohms/cm 2 .
  • Each insert is rinsed with Hanks Balanced Salt Solution (HBSS) 3 x 1 ml using a 10 ml syringe without needle.
  • HBSS Hanks Balanced Salt Solution
  • Each test solution 0.5 ml
  • Triplicate inserts are used for each test solution.
  • the 24-well plate with inserts and test solutions are placed in a humidified incubator at 37°C for 30 minutes.
  • Each series of triplicates is handled sequentially to allow exact timing of the treatment.
  • each insert is individually rinsed with HBSS 5 x 1 ml using the 10 ml syringe, and is placed in a fresh 24-well plate containing 0.5 ml HBSS in each well.
  • the assay can measure the effect of 50 ⁇ g/ml SDS, and the effect on the permeability of the monolayer is linearly proportional to the concentrations of SDS from 50 - 250 ⁇ g/ml. Fluorescence units (arbitrary) of each test solution is plotted against test solutions.
  • results are expressed as % of SDS response, and comparisons with the HBSS response. Generally, if the solution is 20% of the SDS response, the solution may be a mild irritant.
  • the results of the in vitro irritation potential testing are presented below along with the results for the positive and negative controls.
  • the positive control 250 ppm of sodium dodecyl sulfate (SDS) is known to cause noticeable irritation when instilled in the human eye.
  • the negative control Hank's balanced salt solution (HBSS) is known not to elicit any adverse reaction when instilled in the human eye.
  • MilcinTM based ophthalmic solutions should be completely compatible with the ocular environment.
  • Example 12 was prepared by the detailed process given in Example 12 except that half the batch was autoclaved and half the batch was not (steps 1 through 8 only in Example 12).
  • Example 6 illustrates the use of lipid free glycoproteins and glycomacropeptides in an ophthalmic solution.
  • the extracted glycoproteins and glycomacropeptides recovered in Example 6 were utilized as the active ingredients in the ophthalmic solution formulation described below:
  • Example 12 was prepared by the detailed process given in Example 12. The finished solution was tested and the following physical properties were generated.
  • the example illustrates the preparation of a preserved ophthahnic solution utilizing the glycoproteins and glycomacropeptides of this invention.
  • Lubragel ® MS available from United-Guardian, h e. was chosen as the gel base.
  • Lubragel ® MS is composed of polyglycerolmethacrylate and propylene glycol preserved with parabens.
  • a 2% MilcinTM (Lot 6) in Lubragel ® MS was prepared by thoroughly mixing the MilcinTM into the gel base to form a uniform dispersion. The resulting gel was slightly hazy.
  • This example illustrates the use of the glycoproteins and glycomacropeptides of this invention in contact lens solutions.
  • the contact lens solution base utilized in this example was RENUTM MultiPlus, manufactured and sold by Bausch & Lomb.
  • RENUTM is a multi-purpose solution for the storage and care of soft hydrogel contact lens.
  • the following formulation was prepared by adding 0.5% by weight of MilcinTM to RENUTM.
  • MilcinTM was thoroughly dispersed in the RENUTM by vigorous stirring for one hour.
  • the resulting solution was slightly hazy with a pH of 7.1 and an osmolality of 292mOsm/kg. It is expected that the addition of MilcinTM to a soft contact lens solution will result in improved lubricity and comfort to the lens wearer.
  • the following example illustrates the use of glycoproteins and glycomacropeptides as an erodible ocular insert to provide long lasting treatment of dry eye.
  • Approximately 50mg of MilcinTM Lot 3 (see Example 3) was placed in a micro ICBr press. The press was heated to about 80°C and then tightened to compress the MilcinTM under heat and pressure. The KBr press was allowed to cool to room temperature and the sample was removed.
  • the MilcinTM was in the form of a solid disk about 5mm in diameter and about 2mm in thickness. The MilcinTM disk was placed in water and slowly eroded over about two hours.
  • This example illustrates the use of the glycoproteins and the glycomacropeptides of this invention as a component in an allergy relief solution.
  • the particular ingredient for allergy relief chosen was olopatadine hydrochloride.
  • Patanol ® is a commercially available solution containing 0.1% by weight of olopatadine.
  • the other solution components are sodium chloride, a phosphate buffer system and benzalkonium chloride as a preservative.
  • Patanol ® is an isotonic solution with a pH of about 7.
  • a solution was prepared by adding 1.0% by weight of MilcinTM Lot 6 (see Example 3) into Patanol ® solution which is manufactured and sold by Alcon Pharmaceuticals.
  • the MilcinTM was compatible in the Patanol ® solution and is expected to provide improved lubricity and comfort to the patient.
  • This example illustrates the use of the glycoproteins and glycomacropeptides of this invention in an antibacterial ophthalmic solution with activity against a broad spectrum of gram-postive and gram-negative ocular pathogens.
  • the antibacterial agent chosen was ciprofloxacin hydrochloride.
  • Ciloxan ® is a commercially available solution containing 0.3%) ciprofloxacin.
  • the other solution ingredients are sodium acetate, mannitol, edetate disodium and benzalkonium chloride.
  • Ciloxan ® is an isotonic solution with a pH about 4.5 that is manufactured and sold by Alcon Pharmaceuticals.
  • a solution was prepared by adding 1.0% by weight of MilcinTM Lot 6 (see Example 3) into Ciloxan ® solution.
  • the MilcinTM was compatible in the Ciloxan solution and is expected to provide improved lubricity and comfort to the patient.
  • This example illustrates the use of the glycoproteins and glycomacropeptides of this invention in an antibiotic and steroid combination ophthalmic solution.
  • the antibiotic agent chosen was tobramycin and the steroid chosen was dexamethasone.
  • Tobradex ® is a commercially available solution containing 0.3% by weight tobramycin and 0.1 % by weight of dexamethasone.
  • the other solution ingredients are hydroxethylcellulose, sodium chloride, sodium sulfate, Tyloxapol ® , edetate disodium and benzylalkonium chloride as the preservative.
  • Tobradex ® is manufactured and sold by Alcon Pharmaceuticals.
  • a solution was prepared by adding 1.0% by weight of MilcinTM Lot 6 (see Example 3) into Tobradex ® solution. The MilcinTM is expected to provide improved lubricity and comfort to the patient.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Epidemiology (AREA)
  • Zoology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Veterinary Medicine (AREA)
  • Immunology (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Cell Biology (AREA)
  • Developmental Biology & Embryology (AREA)
  • Virology (AREA)
  • Medicinal Preparation (AREA)
  • Marine Sciences & Fisheries (AREA)

Abstract

La présente invention se rapporte à une formulation aqueuse destinée à être instillée dans l'oeil, ou dans laquelle il est possible de prétremper ou de conserver un objet destiné à être inséré dans l'oeil, tel qu'une lentille de contact, un onguent, ou un dispositif solide devant être inséré dans le sac conjonctival. Les préparations selon l'invention sont utilisées pour le traitement d'un trouble du film lacrymal et de la surface oculaire connu sous le nom de kératoconjonctivite sèche ou syndrome de l'oeil sec. En général, les préparations selon la présente invention sont aussi d'une grande efficacité pour soulager les symptômes de l'irritation oculaire, tels ceux causés par un environnement sec ou par le port de lentilles de contact. Conformément à la présente invention, la préparation ophtalmique contient un composant de mucine, analogue à celui trouvé dans la surface oculaire humaine normale, et, dans un mode de réalisation exemplaire et préféré, la glycoprotéine et/ou le glycomacropeptide sont dérivés d'un lait de mammifère, de préférence bovin.
PCT/US2002/031657 2001-10-03 2002-10-03 Glycoproteine et/ou glycomacropeptide contenant une preparation ophtalmique WO2003028751A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
US10/813,998 US20040214754A1 (en) 2001-10-03 2004-03-30 Ophthalmic preparation containing glycomacropeptide
US10/814,001 US20040220089A1 (en) 2001-10-03 2004-03-30 Ophthalmic preparation containing glycoprotein

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US32691201P 2001-10-03 2001-10-03
US60/326,912 2001-10-03

Related Child Applications (2)

Application Number Title Priority Date Filing Date
US10/813,998 Continuation-In-Part US20040214754A1 (en) 2001-10-03 2004-03-30 Ophthalmic preparation containing glycomacropeptide
US10/814,001 Continuation-In-Part US20040220089A1 (en) 2001-10-03 2004-03-30 Ophthalmic preparation containing glycoprotein

Publications (1)

Publication Number Publication Date
WO2003028751A1 true WO2003028751A1 (fr) 2003-04-10

Family

ID=23274275

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2002/031657 WO2003028751A1 (fr) 2001-10-03 2002-10-03 Glycoproteine et/ou glycomacropeptide contenant une preparation ophtalmique

Country Status (1)

Country Link
WO (1) WO2003028751A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005097159A1 (fr) * 2004-03-30 2005-10-20 Milcin Therapeutics Llc Preparation ophtalmique contenant de la glycoproteine

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6281192B1 (en) * 1999-03-01 2001-08-28 Vista Scientific Llc Mucin containing ophthalmic preparations
US20020045582A1 (en) * 1997-12-31 2002-04-18 Alexey L. Margolin Stabilized protein crystals formulations containing them and methods of making them
US6429194B1 (en) * 1999-03-01 2002-08-06 Vista Scientific Llc Mucin containing ophthalmic preparation

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020045582A1 (en) * 1997-12-31 2002-04-18 Alexey L. Margolin Stabilized protein crystals formulations containing them and methods of making them
US6281192B1 (en) * 1999-03-01 2001-08-28 Vista Scientific Llc Mucin containing ophthalmic preparations
US6429194B1 (en) * 1999-03-01 2002-08-06 Vista Scientific Llc Mucin containing ophthalmic preparation

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
CHU ET AL.: "Isolation and characterization of porcine milk lactoferrin", AM. J. VET. RES., vol. 54, no. 7, July 1993 (1993-07-01), pages 1154 - 1159, XP002962722 *
FUJIHARA ET AL.: "Lactoferrin suppresses loss of corneal epithelial integrity in a rabbit short-term dry eye model", J. OCUL. PHARMACOL. THER., vol. 14, no. 2, April 1998 (1998-04-01), pages 99 - 107, XP002962723 *
HWANGBO ET AL.: "Purification and characterization of novel whey glycoprotein WGP-88 which binds to a monoclonal antibody to PAS-4 glycoprotein in the bovine milk fat globule membrane", BIOSCI. BIOTECHNOL. BIOCHEM., vol. 61, no. 9, September 1997 (1997-09-01), pages 1568 - 1574, XP002962721 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005097159A1 (fr) * 2004-03-30 2005-10-20 Milcin Therapeutics Llc Preparation ophtalmique contenant de la glycoproteine

Similar Documents

Publication Publication Date Title
EP1156809B1 (fr) Preparations ophtalmiques contenant de la mucine
US6429194B1 (en) Mucin containing ophthalmic preparation
US11878041B2 (en) Compositions and methods for treating the eye
WO2005097157A1 (fr) Preparation ophtalmique contenant du glycomacropeptide
WO2005097159A1 (fr) Preparation ophtalmique contenant de la glycoproteine
US6565861B1 (en) Artificial tear formulation
EP1551417B1 (fr) Formulation lacrymale a trois couches
WO1994004155A1 (fr) Utilisation de la dehydroepiandrosterone pour le traitement de troubles oculaires
AU2019311849B2 (en) Compositions and methods for treating the eye
WO2003028751A1 (fr) Glycoproteine et/ou glycomacropeptide contenant une preparation ophtalmique
AU2022263530A1 (en) Compositions and methods for treating the eye
RU2795913C2 (ru) Композиции и способы лечения глаз
RU2793736C2 (ru) Композиции и способы лечения глаз
RU2792627C2 (ru) Композиции и способы лечения глаз
US11969451B2 (en) Compositions and methods for treating the eye
EP3824877A1 (fr) Compositions et procédés pour le traitement de l' il
US8877494B2 (en) Human corneal epithelial cell line PTA-120527
US6274160B1 (en) Methods of use of histamine H2 agonists in treating dry eye
EP3449907A1 (fr) Composition ophtalmique de nanoparticules pour le traitement de troubles ou de maladies oculaires
WO2005037248A1 (fr) Preparations contenant une glycoproteine et/ou un glycomacropeptide destinees a des usages medicaux
US20040126419A1 (en) Artificial tear formulation
WO1995014488A1 (fr) Compositions et procedes de traitement des yeux secs et d'autres troubles occulaires

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BY BZ CA CH CN CO CR CU CZ DE DM DZ EC EE ES FI GB GD GE GH HR HU ID IL IN IS JP KE KG KP KR LC LK LR LS LT LU LV MA MD MG MN MW MX MZ NO NZ OM PH PL PT RU SD SE SG SI SK SL TJ TM TN TR TZ UA UG US UZ VN YU ZA ZM

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): GH GM KE LS MW MZ SD SL SZ UG ZM ZW AM AZ BY KG KZ RU TJ TM AT BE BG CH CY CZ DK EE ES FI FR GB GR IE IT LU MC PT SE SK TR BF BJ CF CG CI GA GN GQ GW ML MR NE SN TD TG US

121 Ep: the epo has been informed by wipo that ep was designated in this application
WWE Wipo information: entry into national phase

Ref document number: 10813998

Country of ref document: US

Ref document number: 10814001

Country of ref document: US

122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: JP

WWW Wipo information: withdrawn in national office

Country of ref document: JP