TREATMENT OF OCULAR INFLAMMATORY CONDITIONS WITH INTERLEUKIN-10

Abstract: A method is provided for treating an ocular inflammatory condition, particularly stromal keratitis induced by Herpes Simplex Virus Type 1 (HSV-1). The method comprises administering a therapeutically effective amount of interleukin-10 to an area within the ocular region of a mammal. Pharmaceutical compositions for the treatment of ocular inflammation, including topical solutions, are also provided.

Description: TREATMENT OF OCULAR INFLAMMATORY CONDITIONS WITH INTERLEUKIN-10 FIELD OF THE INVENTION This invention relates to the use of interleukin- 10 (IL-10) to treat ocular inflammatory conditions, particularly stromal keratitis induced by Herpes Simplex Virus Type 1 (HSV-1). BACKGROUND OF THE INVENTION Inflammatory diseases of the eye can be initiated by viral, bacterial, fungal, or parasitic infection and by autoimmunity. (See, e.g., Focal Points (1992) published by the American Academy of Opthalmology). Common complications of ocular inflammation include corneal scarring and perforation, glaucoma, neovascularization of the cornea and retina, retinal scarring and detachment, cataract, optic nerve damage and scarring of orbital and eyelid tissues. (See, e.g., Wakefield, D. and Lloyd, A., Cytokine 4:1, 1992). It is important to minimize inflammation in tissues such as the clear, avascular comea because the infiltration of leukocytes and blood vessels can lead to severe vision loss, and repair by corneal transplantation has a very poor prognosis when this occurs. One particularly damaging kind of ocular inflammatory condition is stromal keratitis induced by herpes simplex virus type 1 (HSV-1) or less commonly by HSV type 2. Corneal inflammation due to HSV is particularly severe and can persist for years even when treated. A common sequela is corneal vascularization and scarring. Corneal transplantation to restore vision has a high failure rate due to increased rejection rates and recurrences of HSV infection. (See Barney N.P. and Foster, C.S., Cornea 13:232, 1994). Diagnosis of necrotizing stromal keratitis is made by clinical findings and may be confirmed via virus isolation and serology while that of immune medicated stromal keratitis is made by clinical findings, serology, and possibly histomorphologic study. (See Liesegang, T.J. Mayo Clin. Proc. 63:1092, 1988; Wilhelmus, K.R., et al. Ophthalmology 101: .883, 1994; Barron, et al., Ophthalmology 101 :1871 , 1994). Currently, HSV induced stromal disease is treated by topically applied corticosteroids with an antiviral cover, usually trifluridine or acyclovir (See, e.g., Wilhelmus, K.R. et al., Ophthalmology. 707:1883, 1994). However, corticosteroid therapy may prolong and possibly worsen the disease as well as introduce other effects such as enhancement of viral replication, cataracts, glaucoma, corneal melting, secondary infection, and corticosteroid dependence. (See Liesegang, T.J., Mayo Clin. Proc. 63:1092, 1988). Thus, there remains a great need to develop more effective methods to treat ocular inflammatory conditions such as herpes stromal keratitis. SUMMARY OF THE INVENTION This invention fills the foregoing needs by providing a method for treating ocular inflammatory conditions in a mammal, comprising administering a therapeutically effective amount of interleukin-10 to an area within the ocular region of said mammal. Pharmaceutical compositions for the treatment of ocular inflammation, including topical solutions, are also provided. The term "ocular region" refers to components of the eye including the cornea, sclera, choroid, ciliary body, iris, retina, conjunctiva, orbital tissue, eyelids, nasolacrimal drainage apparatus, and optic nerve, lnterleukin-10 has the advantage of reducing inflammation without compromising clearance of the infecting virus from the eye. BRIEF DESCRIPTION OF THE FIGURES This invention can be more readily understood by reference to the accompanying figures, in which: Figures 1A and 1B are graphical representations showing the effect of local IL-10 on local delayed-type hypersensitivity (DTH) response to HSV-1 antigen in mice previously infected with HSV by corneal viral innoculation . In Fig. 1A, recombinant murine interleukin-10 (rmlL-10) (28 ng) was inoculated into the ear with the viral challenge antigen (1X). A second IL-10 inoculum (55 ng) was given 12 h later (2X). In Fig. 1B, IL-10 was preincubated with anti-IL-10 monoclonal antibody or control IgG before being inoculated at the DTH test site simultaneously with viral antigen and 12 h after antigen challenge and was reduced in the presence of active, but not antibody- neutralized IL-10. Ear swelling was measured 24 h after antigen challenge. The ear swelling response in naive mice was 13 ± 3 X 10™ 4 inches. There were three to four mice per group. Figure 2 is a graphical representation showing the effect of rmlL-10 on development of herpes stromal keratitis and demonstrates reduced corneal opacification as judged by biomicroscopy. Mice (eight per group) were given 1 μl intracorneal injections of IL-10 (5 ng per injection) 4 h before and again on days 2 and 5 relative to the time of HSV-1 (RE) corneal infection. Additionally, 110 ng IL-10 was given i.p. at the time of viral innoculation and again 3 days later. The controls were given saline in place of IL-10. ( * ) indicates the treated group was significantly (p < 0.05) different from the control group. Figures 3A and 3B are photomicrographs of corneas removed 28 days after HSV-1 innocluati i. Four corneas per group were examined in two independent experiments. Cross sections of corneas from saline-treated mice (Fig. 3A) and rm I L- 10 treated mice (Fig. 3B) showed marked differences in inflammation, scarring, and neovascularization. H & E stain. Original magnification is x200 for Fig. 3A, x100 for Fig. 3B. Figures 4A and 4B are graphical representations showing the effect of rmlL-10 on systemic DTH sensitization. Mice infected by corneal innoculation with HSV-1 were treated with IL-10 as described in the description of Figure 2 above, or given saline (control). DTH testing was performed on day 6 post¬ infection and ear swelling was measured 24 h later and demonstrated that ear swelling in IL-10 treated mice was not statistically different from saline-treated controls. There were three to four mice per group. Figures 4A and 4B depict two independent experiments. Figure 5 is a graphical representation showing the effect of rmlL-10 on HSV-1 titers in the eye. Mice infected on the cornea with HSV-1 were treated with IL-10 as described in the description of Figure 2 above or given saline. On the days indicated, four animals in each group were killed and the individual eyes were excised and titrated for infectious virus content demonstrating that viral titers in eyes treated with IL-10 were progressively reduced comparable to control animals. (•) IL-10 treated; (o) controls. Figure 6 is a graphical representation showing the effect of rmlL-10 on cytokine synthesis in HSV-1 infected corneas. Mice infected on the comea with HSV-1 were treated with IL-10 as described in the description of Figure 2 above or given saline. Ten days after infection the corneas were excised and individually evaluated by enzyme-linked immunosorbent assay for IL-1α, IL-2, and IL-6 and showed selective downregulation of host IL-2 and IL-6, but not IL-1α. Figures 7A and 7B are graphical representations showing the effect of rmlL-10 on spontaneous synthesis of IL-6 and IL-1α by excised mouse corneal buttons. Normal corneas were removed from BALB/c mice, and pools of three corneas each were incubated in the presence or absence of the indicated amount of IL-10. In Fig. 7A, IL-6 levels in the supernatant were assayed after 12 h of incubation. In Fig. 7B, corneal button lysates were monitored for IL-1α after 4 h of incubation. Host corneal IL-6 was significantly inhibited while host corneal IL-1α was not reduced. ( * ) indicates a significant reduction (p < 0.05) relative to the control as assessed by Student's Mest. Each of the above figures was originally published in Tumpey et al., J. Immunol. 753:2258 (1994). Additional evidence for IL-10 inhibitors of HSK in humans is illustrated by two additional publications. In "lnterleukin-10 Inhibition of HLA-DR Expression in Human Herpes Stromal Keratitis" by Boorstein, et al., (Ophthalmology; 707:1529-1535 (1994), profound reduction of HLA-DR antigen expression in human stromal keratitis was observed following incubation of diseased human corneas with recombinant human IL-10 (rhlL-10)at a dose of 100 units per ml. A second study entitled "lnterleukin-10 Modulation of lnterleukin-8 and Monocyte Chemotactic Protein- 1 Secretion in Human HSK" by Boorstein, etal. (Invest. Ophthalmol Vis Sci, 36 (4):S147, 1995) showed that rhlL-10 is a potent inhibitor of IL-8 (80% inhibition) and MCP-1 (50% inhibition) leukocyte chemotaxins in human corneas affected with HSV-1 stromal keratitis when used at a dose of 100 units/ml. DETAILED DESCRIPTION OF THE INVENTION All references cited herein are hereby incorporated in their entirety by reference. As used herein, "interleukin-10" or "IL-10" is defined as a protein which (a) has an amino acid sequence of mature IL-10 (e.g., lacking a secretory leader sequence) as disclosed in U.S. Patent No. 5,231,012 and (b) has biological activity that is common to native IL-10. For the purposes of this invention both glycosylated (e.g. produced in eukaryotic cells such as CHO cells) and unglycosylated (e.g., chemically synthesized or produced in E. coli) IL-10 are equivalent and can be used interchangeably. Also included are muteins and other analogs, including the Epstein-Barr Virus protein BCRF1 (viral IL-10), which retain the biological activity of IL-10. IL-10 suitable for use in the invention can be obtained from culture medium conditioned by activated cells secreting the protein, and purified by standard methods. Additionally, the IL-10, or active fragments thereof, can be chemically synthesized using standard techniques known in the art. See Merrifield, Science 233:341 (1986) and Atherton et al., Solid Phase Peptide Synthesis: A Practical Approach, 1989, 1. R.L. Press, Oxford. See also U.S. Patent No. 5,231,012. Preferably, the protein or polypeptide is obtained by recombinant techniques using isolated nucleic acid encoding the IL-10 polypeptide. General methods of molecular biology are described, e.g., by Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor, New York, 2d ed., 1989, and by Ausubel et al., (eds.) Current Protocols in Molecular Biology, Green/Woley, New York (1987 and periodic supplements). The appropriate sequences can be obtained using standard techniques from either genomic or cDNA libraries. Polymerase chain reaction (PCR) techniques can be used. See, e.g., PCR Protocols: A Guide to Methods and Applications, 1990, Innis et al., (Ed.), Academic Press, New York, New York. Libraries are constructed from nucleic acid extracted from appropriate cells. See, e.g., U.S. Patent No. 5,231,012, which discloses recombinant methods for making IL-10. Useful gene sequences can be found, e.g., in various sequence databases, e.g., GenBank and BMPL or nucleic acid and PIR and Swiss-Prot for protein, c/o Intelligenetics, Mountain View, California, or the Genetics Computer Group, University of Wisconsin Biotechnology Center, Madison, Wisconsin. Clones comprising sequences that encode human IL-10 have been deposited with the American Type Culture Collection (ATCC), Rockville, Maryland, under Accession Nos. 68191 and 68192. Identification of other clones harboring the sequences encoding IL-10 is performed by either nucleic acid hybridization or immunological detection of the encoded protein, if an expression vector is used. Oligonucleotide probes based on the deposited sequences disclosed in U.S. Patent No. 5,231,012 are particularly useful. Oligonucleotide probes sequences can also be prepared from conserved regions of related genes in other species. Alternatively, degenerate probes based on the amino acid sequences of IL-10 can be used. Standard methods can be used to produce transformed prokaryotic, mammalian, yeast or insect cell lines which express large quantities of the polypeptide. Exemplary E. coli strains suitable for both expression and cloning include W3110 (ATCC Bi, 27325), X1776 (ATCC No. 31244). X2282, and RR1 (ATCC Mp/ 31343). Exemplary mammalian cell lines include COS-7 cells, mouse L cells and CHP cells. See Sambrook (1989), supra and Ausubel et al., 1987 supplements, supra. Various expression vectors can be used to express DNA encoding IL-10. Conventional vectors used for expression of recombinant proteins in prokaryotic or eukaryotic cells may be used. Preferred vectors include the pcD vectors described by Okayama etal., Mol. Cell. Biol. 3:280 (1983); and Takebe et al., Mol. Cell. Biol. 8:466 (1988). Other SV40-based mammalian expression vectors include those disclosed in Kaufman et al., Mol. Cell. Biol. 2:1304 (1982) and U.S. Patent No. 4,675,285. These SV40-based vectors are particularly useful in COS-7 monkey cells (ATCC No. CRL 1651), as well as in other mammalian cells such as mouse L cells. See also, Pouwels et al., (1989 and supplements) Cloning Vectors: A Laboratory Manual, Elsevier, New York. The IL-10 may be produced in soluble form, such as a secreted product of transformed or transfected yeast, insect or mammalian cells. The peptides can then be purified by standard procedures that are known in the art. For example, purification steps could include ammonium sulfate precipitation, ion exchange chromatography, gel filtration, electrophoresis, affinity chromatography, and the like. See Methods in Enzymology Purification Principles and Practices (Springer- Verlag, New York, 1982). Altematively, IL-10 may be produced in insoluble form, such as aggregates or inclusion bodies. The IL-10 in such a form is purified by standard procedures that are well known in the art. Examples of purification steps include separating the inclusion bodies from disrupted host cells by centrifugation, and then solubilizing the inclusion bodies with chaotropic agent and reducing agent so that the peptide assumes a biologically active conformation. For specifics of these procedures, see, e.g. Winkler et al., Biochemistry 25:4041 (1986), Winkler et al., Bio/Technology 3:9923 (1985); Koths etal., and U.S. Patent No. 4,569,790. The nucleotide sequences used to transfect the host cells can be modified using standard techniques to make IL-10 or fragments thereof with a variety of desired properties. Such modified IL-10 can vary from the naturally- occurring sequences at the primary structure level, e.g., by amino acid, insertions, substitutions, deletions and fusions. These modifications can be used in a number of combinations to produce the final modified protein chain. The amino acid sequence variants can be prepared with various objectives in mind, including increasing serum half-life, facilitating purification or preparation, improving therapeutic efficacy, and lessening the severity or occurrence of side effects during therapeutic use. The amino acid sequence variants are usually predetermined variants not found in nature, although others may be post-translational variants, e.g., glycosylated variants or proteins which are conjugated to polyethylene glycol (PEG), etc. Such variants can be used in this invention as long as they retain the biological activity of IL-10. Modifications of the sequences encoding the polypeptides may be readily accomplished by a variety of techniques, such as site-directed mutagenesis (Gillman etal., Gene 8:81 (1987)). Most modifications are evaluated by routine screening in a suitable assay for the desired characteristics. For instance, U.S. Patent No. 5,231,012 describes a number of in vitro assays suitable for measuring IL-10 activity. Preferably, human IL-10 is used for the treatment of humans, although viral IL-10, or IL-10 from some other mammalian species, could possibly be used. Most preferably, the IL-10 used is recombinant human IL-10. The preparation of human and mouse IL-10 is described in U.S. Patent 5,231,012. The cloning and expression of viral IL-10 (BCRF1 protein) from Epstein-Barr virus has been disclosed by Moore et al., Science 248:1230 (1990). (See also intemational patent application WO 91/09127 and U.S. Patent 5,368,854.) When referring to IL-10, active fragments thereof, analogs and homologs are included. Active fragments, analogs and homologs to IL-10 include those proteins, polypeptides, or peptides which possess one or more various characteristic IL-10 activities. Any of these proteinaceous entities can be glycosylated or unglycosylated. Examples of IL-10 activity include inhibition or substantial reduction of the level of IL-2, lymphotoxin, IL-3, or GM-CSF. IL-10 activity also includes inhibition of cytokine production by activated macrophages, e.g., IL-1, IL-6, and TNF-α. For examples of procedures and assays to determine IL-10 activity, see United States Patent No. 5,231,012. This patent also provides proteins having IL-10 activity and production of such proteins including recombinant and synthetic techniques. To prepare pharmaceutical compositions including polypeptide IL-10, the polypeptide is admixed with a pharmaceutically acceptable carrier or excipient which is preferably inert. A pharmaceutical carrier can be any compatible non-toxic substance suitable for delivery of the polypeptide to a patient. Preparation of such pharmaceutical compositions is known in the art; see, e.g., Remington's Pharmaceutical Sciences, and U.S. Pharmacopeia: National Formulary, Mack Publishing Company, Easton, PA (1984); Avis etal. fedsJ (1993) Pharmaceutical Dosage Froms: Parenteral Medications: Dekker, New York; and Lieberman et al. (eds.) (1990) Pharmaceutical Dosage Forms: Disperse Systems Dekker, New York. Ocular preparations, in particular, may employ conventional eye drops or ointments or employ the use of preservatives such as bezylkonium chloride or ethylenediaminetetraacetic acid (EDTA) to enhance penetration; mucoadhesive polymers including hyaluronic acid to prolong drug contact time; and microparticles/nanoparticles and liposomes as drug delivery particulates. Particularly applicable may be protein absorption enhancers that render the cornea permeable to proteins and small peptides that include but are not limited to azone, cetrimide, cvtochalasin B, EDTA, taurocholate, and taurodeoxycholate. (See, e.g., Aldrich Catalogue Handbook of Fine Chemicals, 1994-95 Edition, Milwaulee, Wisconsin). For general reference materials on ocular preparations, reference can be made, e.g., to Lee, Pharm. Int., 6:135 (1985); Hecht, etal., in Modern Pharmaceutics (Banker and Rhodes, eds.) Marcel Dekkar, N.Y. (1979); Maurice, Ophthalmol. Clin., 20:21(1980); Chiou, et al., Pharmacol. Ther., 77:269 (1982); Lee, et al., J. Ocul. Pharmacol., 267 (1986); Camber, et al., (1989), Curr. Eye Res., 8:563; Marsh, et al , Exp. Eye Res., 77:43 (1971); Ramselaar, et al., Curr. Eye Res., 7:947 (1988); Norn, Acta Ophthalmol,, 42727 (1964); Seig, et al, J. Pharm.; Sci., 66:122 (1977); McKeen, Int. Ophthalmol. Clin., 20:79 (1980); Higuchi, J. Pharm. Sci., 50:874 (1961); Saettone, et al., Int. J. Pharm., 57:203 (1989); Blaug, et al., Am. J. Of. Hosp. Pharm., 22662 (1965); Keller, et al., Exp. Eye Res., 30:203 (1980); Grass, et al., Invest Ophthalmol. Vis. Sci.. 26110 (1985); Harris, et al., Biomaterials, 77:652 (1990); Deasy, Microencapsulation and Related Drug Processes., Marcel Dekker, New York (1984); Widder, et al, (eds.), Methods in Enzymology, Vol. 112, Academic Press, Orlando, Florida (1985); Donbrow, (ed.), Microcapsules and Nanoparticles in Medicine and Pharmacy, CRC Press, Boca Raton, Florida (1992); Lee, et al., Sυrv. of Ophthalmol., 29:335 (1985); Szoka, et al., Ann. Rev. Biophy. Bioeng., 9:467 (1981); Smolin, et al., Am. J. Ophthalmol, 97:220 (1981); Ahmed, et al., Invest. Ophthalmol. vis. Sci., 26:584 (1985); Chiou, et al., J. Pharm., Sci.. 78:815 (1989); Yamamoto, et al., J. Pharm. Exp. Ther., 249:249 (1989); Bentzel, et al., Am. J. Physiol., 23&.C75 (1980); Martinez-Palomo, et al., J. Cell Biol., 87:736 (1980); Aldridge, et al, Chem Commun., 7:26 (1967); Rothweiler, et al., Experientia, 22.750 (1966); Binder, et al., Agnew. Chem. Ins. Edit. 72:370 (1973). It is desirable to treat ocular inflammation locally. This can take the form of topical administration of IL-10. When the IL-10 is administered topically, the IL-10 can be in the form of eyedrops, ointment, and other formulations which may employ the vehicles listed above. Concentrations of IL-10 in these various vehicles may vary from 1 microgram per ml under conditions (e.g. inflammation) in which 1% penetration may occur to 2.5 mg/ml, the maximal tolerated topical polypeptide dose to the cornea (Krishnamoorathy and Mitra, 1993, Ocular Delivery of Peptides and Proteins; in: Ophthalmic Drug Delivery Systems (A.K. Mitra, ed.), Marcel Dekker, New York, pp. 455 et.seq.), when the // epithelial barrier is intact. Thus, at a concentration that can range from 1 microgram per ml to 2.5 mg/ml per drop, more preferably from 10 micrograms per ml to .25 mg/ml, one drop can be administered to the eye at a rate ranging from one drop per hour to one drop every two days, more preferably in a range of from one drop per day to four drops per day. Since the half life of IL-10 may be short in the tear film, it may be necessary to give the drug more often than once a day. This can be determined by the clinician based on the condition of the particular patient. The eyedrop formulation could be prepared as indicated in the references for the above-referenced vehicles. The IL-10 can be administered periocularly. A periocular formulation could be prepared by using vehicles already employed in intravenous, subcutaneous, and/or intramuscular injection. Preparation of such formulations is well known in the art. See, e.g., Gilman, et al. (eds) (1990) Goodman and Gilman's: The Pharmacological Bases of Therapeutics, 9th ed., Pergamon Press; and Remington's Pharmaceutical Sciences, 17th ed. (1992), Mack Publishing Co., Easton, Penn. The dosage for periocular administration is preferably from about 5 micrograms to about 5 mg per day, more preferably from about 50 micrograms to about 0.5 mg per day, dependent on bioavailability from injected vehicle and half-life of IL-10 prepared in various vehicles. The IL-10 can also be administered subconjunctivally (sc) or intracomealiy. IL-10 is effective at reducing ocular inflammation in the mouse when given sc at a dose of 0.5 microgram daily for 7 days with treatment starting at 5 days postinfection. Subconjunctival or intracorneal formulations could employ vehicles used for topical dosing or those used for intravenous, intramuscular, or subcutaneous delivery. Thus, the preferred dose range for subconjunctivial administation in humans is from about 5 micrograms to about 5 mg per day, more preferably from about 50 micrograms to about 0.5 mg. In the case of intracomeal administration, the preferred dosage would be from about 0.5 micrograms to about 0.5 mg, more preferably from about 5 micrograms to about 50 micrograms, using formulations for intravenous systems. Since the half life of IL-10 is short in vivo (estimated 2.5 hours in the mouse), it may be necessary to give the drug more often than once a day. This can be determined by the clinician based on the condition of the particular patient. IL-10 may also be useful for intracameral drug delivery, including anterior chamber and intra-vitreal drug delivery, for the treatment of a variety of destructive intraocular inflammatory diseases. Intraocular formulations could be prepared by using vehicles already employed for intravenous administration (see, e.g., Remington's Pharmaceutical Sciences, 17th ed. (1992), Mack Publishing Co., Easton, Penn.) or liposome, microsphere, or iontophoretic IL-10 delivery (see, e.g., Schulman and Peyman, 1993, Intracameral, Intravitreal, and Retinal Drug Delivery in: Ophthalmic Drug Delivery Systems (A.K. Mitra, ed.) Marcel Dekker, New York, pp. 383 et seq.). IL-10 may also be useful for treatment of live tissue allografts to the ocular region, including but not limited to corneal allografts, prior to their surgical placement on the recipient host. IL-10 treatment of harvested allografts would require doses ranging from 0.001 micrograms per ml to 0J mg/ml, more preferably from 0J micrograms/ml to 10 micrograms/ml. As used herein, the phrase "therapeutically effective amount" means an amount sufficient to ameliorate a symptom or sign of ocular inflammation. Clinical herpes stromal keratitis is characterized by corneal edema, corneal haze, neovascularization, anterior chamber inflammation and keratic precipitates. Accordingly, amelioration would be recognized by a reduction in one or more of these clinical signs. Typical mammals that can be treated include companion animals such as dogs and cats, and primates, including humans. Preferably, IL-10 derived from the species of the treatment target animal will be used. An effective amount for a particular patient may vary depending on factors such as the condition being treated, the overall health of the patient, the method, route, and dose of administration and the severity of side effects. Determination of the appropriate dose is made by the clinician using parameters known in the art. Generally, the dose begins with an amount somewhat less than the optimum dose and it is increased by small increments thereafter until the desired or optimum effect is achieved. (See generally The Merck Manual § 269 "Pharmacokinetics and Drug Administration."). In appropriate circumstances, multiple medications can be administered in combination. For instance, the IL-10 can be administered in combination with a therapeutically effective dose of one or more additional therapeutically active agents. The additional agent can be an antiinfective agent (e.g'., trifluridine or acyclovir) or a steroidal (e.g., prednisolone acetate or fluorometholone) or nonsteroidal antiinflammatory agent (e.g., ocufen, indovin, or cyclosporine). The broad scope of this invention is best understood with reference to the following examples, which are not intended to limit the invention to specific embodiments. EXAMPLES In the following examples, IL-10 was tested to determine if this cytokine could suppress the development of stromal disease. Two studies demonstrated IL-10 suppression of HLA-DR antigen expression (Boorstein, et al., Ophthalmology 1994; 101:1529-1535) and leukocyte chemokine production (Boorstein, et al., Invest. Ophthalmol Vis Sci, 36 (4):S147; 1995) in human corneas affected with herpes stromal keratitis using recombinant human IL-10 at 100 units per ml, ex vivo. Both of these articles (Boorstein, et al., Oph "ιalmology ; 101:1529-1535, 1994 and Boorstein, et al, Invest. Ophthalmol Vis Sci, 36 (4):S147, 1995) are expressly incorporated herein in their entirety by reference. Further, in an extensive animal study, rmlL-10 was inoculated intracorneally in mice 4 hours before and again on days +2 and +5 relative to the time of topical HSV-1 corneal innoculation. Additionally, the mice received IL-10 i.p. at the time of virus administration and again 3 days post-infection. Four weeks post-infection, the incidence of blinding disease was 95% in the saline-treated controls but only 36% in the IL-10 treated animals. Histologic studies showed extensive cellular infiltrates in control corneas but not in those of the IL-10 treated eyes. Examination of the proinflammatory cytokine levels in the cornea 10 days after infection revealed that the presence of IL-2 was 10- fold Iower and IL-6 some 50-fold Iower than that found in the controls. IL-1α levels were not reduced. The IL-10 treatment protocol employed did not suppress the systemic cellular or humoral immune responses to viral antigens, nor was the rate of HSV-1 clearance from the eye different from that seen in the controls. In vitro studies revealed that spontaneous production of IL-6 by excised normal corneas was inhibited by >95% with low dose IL-10. IL-1α synthesis was not inhibited. Collectively, these results indicate that IL-10 treatment can 1) suppress the production of certain cytokines produced by corneal cells, and 2) minimize ocular inflammation without compromising clearance of the infecting virus from the eye. /5 Materials and Methods Virus Infection — HSV-1 strain RE, a known stromal keratitis inducer (Lausch, et al., Curr. Eye Res. 8:499, 1989), was used to initiate infection. Virus stocks were grown and titrated on Vero cells as previously described (Lausch, et al., Curr. Eye Res. &A99, 1989). Four-week old female BALB/c mice (Charles River Breeding Laboratories, Wilmington, MA) were anesthetized with 1.0 mg of sodium pentobarbital in 0.2 ml of phosphate- buffered saline injected i.p. The right eye was slightly scarified by three twists of a 2-mm corneal trephine. A 2-μl volume containing 1 to 5 x 10 4 plaque forming units of virus was then dropped onto the corneal surface and massaged in using the eyelids. The eyes were examined weekly with a dissecting biomicroscope. Corneal opacity was graded on a scale of 0 to +5 as described elsewhere (Metcalf, et al., Curr. Eye Res. 6J73, 1987); a score of 0 indicates a clear comea, whereas a +5 score represents severe necrotizing stromal keratitis. Eyes were examined in a coded fashion with the reader unaware of the treatment given. The data were evaluated by using the Mann- Whitney l/test. (Snedecor, G.W. and Cochran, W.G., Statistical Methods, 6th ed. Iowa State University Press, Ames, Iowa, 1967; pp. 130- 131.) Reagents — Murine recombinant IL-10 (rm-IL-10) was obtained from DNAX Research Institute, Palo Alto, CA. The endotoxin content of the IL-10 preparation was <0J ng/μg. The biologic activity of this cytokine was 7 U/μl as measured on mouse MC/9 cells (Moore, et al., Science 248:1230 (1990). The concentration of IL-10 was determined by using an ELISA kit (Endogen Inc. Boston, MA). The hybridoma JESS-2A5J 1 that produces a rat anti-mouse IL-10 monoclonal antibody was obtained from American Type Culture Collection (ATCC; Rockville, MD). Monoclonal rat anti-mouse IL-10 /6 neutralizing monoclonal antibody was also purchased from Genzyme Corporation (Cambridge, MA). Intracorneal injections — Intracorneal injections were performed as previously described (Hendricks, et al. Invest. Ophthalmol. Vis. Sci. 32366, 1991). Briefly, a 30-gauge disposable needle was used to puncture the corneal epithelial wall. A 30-cm, 32 gauge stainless steel needle attached to a Hamilton dispenser (Hamilton, Reno, NV) was then threaded into the stroma and 1 μl of rmlL-10 or saline was injected into the center of the cornea. Delayed tvoe hypersensitivity (DTH) assay — DTH responsiveness in ocularly infected mice was determined by using the ear swelling assay. The test Ag, HSV-1 (RE), was diluted in serum-free RPMI 1640 medium. The virus preparation was then exposed to UV irradiation for 10 min. This reduced infectivity from 10 6 to less than 10 2 plaque forming units/10 μl. To test for DTH responsiveness, 10μl of viral antigen was inoculated into the dorsal side of the mouse's right ear by using a 50 μl Hamilton syringe and a 30-gauge needle 6 days after infection. The left ear (control) received 10 μl RPMI 1640 with 1% new born calf serum. Ear swelling was measured 24 hours later by using a Mitutoyo 7326 micrometer (Schlessinger Tools, New York, NY). The results are expressed as ear swelling of the right (antigen) ear minus ear swelling of the left (control) ear in units of 10 -4 inches. To determine whether IL-10 suppressed the DTH response, the right experimental ears received 5 μl of UV- HSV-1(RE) mixed with 5 μl containing 28 ng of IL-10 just before ear challenge. The controls received saline in place of IL-10. In some experiments IL-10 (110 ng) was incubated with monoclonal rat anti-mouse IL-10 neutralizing Ab (10 μg/ml final concentration), or control IgG, for 30 minutes over ice before ear inoculation. Data were evaluated by using Student's t-test. π Assay of ocular tissue for infections HSV-1 — To test the effect of murine IL-10 treatment on HSV-1 replication in the cornea, individual whole eyes were exercised and placed in 600 μl of 2% FBS in DMEM medium with antibiotics. Preparations were frozen at -70°C, thawed, and homogenized in a Ten Broech homogenizer (Belico, Vineland, NH). The homogenates were frozen, thawed again, and sonicated for 15 s with a Sonic 300 Dismembrator (Artek Systems Corporation, Farmingdale, NY). The supernatants were then titrated for infectious virus on Vero cell monolayers in a 48-h plaque assay. Cytokine Quantitation — To test the effect of IL-10 on IL-6, IL-2 and IL-1α production in vivo, corneas were removed from IL-10-treated and saline- control HSV- 1 -infected mice 10 days after infection. The corneas were trimmed to 2 mm with the use of a microdissecting trephine (Roboz Surgical Instrument Co., Rockville, MD), and placed individually in 600 μl of serum-free RPMI 1640 with Fungi-Bact antibiotic solution. Samples were stored at -70°C until assayed. Samples were thawed, sonicated for 30 seconds, and clarified by centrifugation at 150 x g for 10 minutes. The clarified cell lysates were assayed fro IL-1 , IL-2, and IL-6 with the use of ELISA kits. IL-6 (assay sensitivity 15 pg/ml) and IL-2 (assay sensitivity 3 pg/ml) kits were purchased from Endogen, Inc. The IL-1α kit (assay sensitivity 15 pg/ml) was purchased from Genzyme. Spontaneous cytokine synthesis by excised normal corneas was determined as previously described (Staats, et al., J. Immunol. 757:277, 1993). In brief, corneal buttons were excised from untreated mice and trimmed to 2 mm. The tissue preparations were free of limbal vasculature as judged by microscopic inspection. Each sample consisted of three corneal buttons incubated in the absence or presence of murine IL-10 in 500 μl of RPMI 1640 (Life Technologies, Inc., Gaithersburg, MD) with Fungi-Bact antibiotic solution at 37°C and 5% CO2. After a 12-h incubation period, supernatants were assayed for IL-6. For IL-1α detection, cornea samples were incubated for 6 h. Then the corneas were disrupted by sonication for 30 seconds with a Sonic 300 dismembrator. The tissue lysates were clarified by centrifugation at 150 x g for 10 min before assay. Histologic Examination — Infected eyes were enucleated, fixed in 10% neutral buffered Formalin (equivalent to 4% neutral buffered formaldehyde solution), embedded in paraffin, and multiple 5 micron sections were prepared. Sections were stained with hβmatoxylin-eosin, mounted in resin, and cover- slipped for photomicroscopy. Results 1. IL-10 treatment given locally suppresses DTH responsiveness in sensitized hosts Initial experiments were designed to investigate whether rmlL-10 treatment would be able to suppress immune cell-mediated inflammation induced locally. Therefore, the effects of this cytokine on DTH responsiveness in mice sensitized to HSV-1 were studied. Mice were immunized via topical infection on the scarified cornea with 10 4 plaque forming units HSV-1 (RE). Six days later, the animals responded strongly to viral antigen in DTH tests (Fig. 1). However, injection of rmlL-10 with the test antigen resulted in significantly reduced ear swelling (Fig. 1A). Repeated treatment with the cytokine, i.e., at the time of HSV-1 antigen challenge and again 12 h later (55 ng), increased the suppressive effect. This suppression could be reversed by preincubating IL-10 with specific neutralizing monoclonal antibodies before intradermal ear inoculation (Fig. 1B), specifically the suppresion of IL-10 on the DTH response to HSV-1 antigen. In Fig. 1A, IL-10 (28 ng) was injected into the ear with the viral antigen challenge (1X). A second IL-10 injection (55 ng) was given 12 h later (2X). In Fig. 1B, IL-10 was preincubated with anti-IL-10 mAB or i? control IgG before being inoculated at the DTH test site simultaneously with viral antigen and 12 h after antigen challenge. Ear swelling was measured 24 h after antigen challenge. The ear swelling response in naive mice was 13 ± 3 X 10 -4 inches. There were three to four mice per group. 2. IL-10 treatment suppresses the development of HSV-1 induced stromal keratitis (HSK) The foregoing results indicated that rmlL-10 given to previously sensitized hosts could down-regulate the T cell mediated inflammatory response to HSV-1 antigen. Therefore, we postulated that IL-10 treatment might also be able to suppress the development of herpes stromal keratitis, a disease believed to be mediated at least in part by sensitized T cells (Metcalf, et al., Infect. Immun. 261164, 1979); (Russell, et al., Invest. Ophthalmol. Vis. Sci. 25:938, 1984); (Newell, et al., J. Virol. 63:769, 1989); (Newell, et al.. Reg. Immunol. 2:366, 1989); (Hendricks, et al., Invest. Ophthalmol. Vis. Sci. 37:1929, 1990); (Doymaz, et al. Invest. Ophthalmol. Vis. Sci. 33:2165, 1992); (Niemialtowski, et al., J. Immunol. 749:3035, 1992). rmlL-10 inoculated directly into the comea on days -1, +1, +4, and +7 relative to infection slowed, but did not prevent herpes stromal keratitis development. When IL-10 was given systemically as well as locally, marked suppression of stromal keratitis was achieved. Figure 2 shows the result of a typical experiment. Mean corneal opacity scores were significantly reduced (p < 0.05) relative to that seen in the saline-treated control group at all time points post-infection except day 7. At the termination of the experiment (day 28), eyes were removed for microscopic inspection. Figure 3A depicts a section from a representative control cornea displaying the histologic appearance typical of experimental murine herpes stromal keratitis (Wang, et al., Curr. Eye Res. 8:37 (1989). The cornea was greatly swollen, and contained a heavy inflammatory infiltrate. ZO Numerous blood vessels were present in the stroma, and corneal epithelial ulceration was evident. Figure 3B shows that infected corneas protected by rmlL-10 treatment were not swollen, exhibited no epithelial ulceration, had very few infiltrating inflammatory cells, and only low level neovascularization. The protective action of IL-10 was confirmed in two additional experiments. Collectively, it was found that although 95% (20/21) of the controls developed blinding disease, only 36% (8/22) of the cytokine-treated animals did so. IL-10 treatment did not prevent blepharitis, which is commonly seen after HSV-1 corneal infection in mice (Lausch, et al., Intervirology 31: 159 (1990). At the virus-infecting doses employed (1 to 5 x 10 4 plaque forming units), HSV-1 strain RE on occasion will spread from the eye to the central nervous system and induce fatal disease. The incidence of encephalitis seen in the IL-10 recipients (3/32, 9%) was similar to that seen in the controls (4/32, 12.5%). Thus, the cytokine dosage used in our experiments did not appear to increase host susceptibility to central nervous system disease. IL-10 has multiple suppressive effects on various effector phases of the immune response, including inhibition of T cell proliferation. It was possible that reduced corneal inflammation might be a result of a reduction in the generation of sensitized T cells to herpes viral antigen. To evaluate what effect IL-10 has on cell-mediated immunity, DTH testing was conducted in cytokine- treated and control mice 6 days post-infection. The data in Figure 4 are representative of three such experiments. It was found that protective rmlL-10 treatment begun on the day of virus infection did not inhibit the generation of T cells active in DTH responses to HSV-1 antigens in an ear swelling assay. Furthermore, virus neutralizing titers of sera collected 4 wk post-infection from IL-10-treated hosts were analogous to those found in the controls (data not shown). Thus, there was no evidence that IL-10 treatment suppressed (or 2/ enhanced) the development of either the cellular or humoral immune responses to HSV-1 antigens. 3. Effect of IL-10 treatment on virus replication in the eve A number of cytokines have been shown to exert antiviral effects in ocular tissue (Chen, et al., Antiviral Res. 22.15, 1993). Therefore, the possibility was entertained that rmlL-10 treatment initiated before HSV-1 infection might directly or indirectly reduce corneal inflammation by inhibiting viral replication in the comeas. To test this hypothesis, the amount of infectious virus recovered from the eyes of IL-10 treated and control mice was monitored over time. Figure 5 shows that for each time period examined, viral titers in the eyes of treated animals were comparable to that seen in control animals. These results indicated that IL-10 treatment did not accelerate (or delay) virus clearance from the eye. 4. Effect of I L-10 on synthesis of IL-1α. I L-2. and IL-6 in the comea It is known that HSV-1 infections of the murine cornea are characterized by elevated levels of IL-6 and IL-1α (Staats, et al., J. Immunol . 757:277, 1993). Other investigators have reported that IL-10 can suppress the synthesis of proinflammatory cytokines produced by T cells (de Waal, et al., J. Immunol. 750:4754, 1993), polymorphonuclear leukocytes (Cassatella, et al., J. Exp. Med. 178:2207, 1993), and monocytes/macrophages (Fiorentino, et al., J. Immunol. 747:3815 (1991); Bogdan, et al., J. Exp. Med. 774:1549 (1991); de Waal, et al., J. Exp. Med. 774:1209 (1991). To test whether IL-10 treatment altered the cytokine profile of HSV-1 keratitis, individual corneas were removed from IL-10 treated and control animals 10 days after infection and assayed for IL-1α, IL-2, and IL-6 by ELISA. The data from two experiments were comparable and the pooled results are shown in Figure 6. IL-1α levels in the treated animals were not significantly different (p <0.7) from those seen in 2Z the controls. However, IL-2 and IL-6 levels were strikingly reduced (p < 0.05) in the IL-10 treated hosts. Specifically, just 1 of 10 comea samples had a detectable level of IL-2 and only 2 out of 10 were positive for IL-6. In contrast 70% of the controls were positive for IL-2 and 80% had high levels of IL-6. 5. Effect of IL-10 on spontaneous synthesis of IL-6 and IL-1α by excised mouse corneal buttons Because IL-10 binding to leukocytes of bone marrow origin is known to result in selective inhibition of cytokine synthesis, it was possible that IL-10 could also inhibit cytokine production by resident corneal cells. This hypothesis was amenable to testing because it was previously demonstrated that excised normal corneal buttons incubated in vitro spontaneously synthesize IL-1α and IL-6 (Staats, et al., J. Immunol. 757:277, 1993). Accordingly, excised corneal buttons from uninfected donors were incubated in vitro in the presence or absence of different concentrations of rmlL-10. Medium supernatants and lysates of corneal tissue were subsequently obtained and assayed for IL-6 and IL-1α, respectively. The results of three experiments, summarized in Figure 7, show that IL-10 at a concentration of 1.5 ng/ml could suppress IL-6 synthesis by >95%. Curiously, the 150 ng/ml dose was less suppressive. The reason for this is not clear but a similar dose-response pattern was observed in all three experiments. In contrast, the synthesis of IL-1α was not inhibited by IL-10 over a 1000-fold dose range. Many modifications and variations of this invention will be apparent to those skilled in the art. The specific embodiments described herein are offered by way of example only, and the invention is not to be construed as limited thereby. Z3

Claims: WHAT IS CLAIMED IS: 1. A method for treating an ocular inflammatory condition in a mammal, comprising administering a therapeutically effective amount of interleukin- 10 to an area within the ocular region of said mammal. 2. The method of claim 1 , wherein the area within the ocular region comprises the cornea, sclera, choroid, ciliary body, iris, retina, conjunctiva, orbital tissues, eyelids, nasolacrimal drainage apparatus, and optic nerve. 3. The method of claim 1 , wherein the ocular inflammatory conditions in stromal keratitis. 4. The method of claim 3, wherein the stromal keratitis is one that has been induced by Herpes Simplex Virus . 5. The method of claim 4, wherein the stromal keratitis is one that has been induced by Herpes Simplex Virus Type 1. 6. The method of claim 1, wherein the interleukin-10 is selected from the group consisting of viral interleukin-10 and human interleukin-10. 7. The method of claim 1, further comprising administering a therapeutically effective dose of a second therapeutically active agent. 8. The method of claim 7, wherein the second therapeutically active agent is an antiinfective agent. 9. The method of claim 8, wherein the antiinfective agent is selected from the group consisting of trifluridine and acyclovir. 10. The method of claim 9, wherein the antiinfective agent is trifluridine. 11. The method of claim 7, wherein the second therapeutically active agent is a steroidal or nonsteroidal antiinflammatory agent. 12. The method of claim 7, wherein the second therapeutically active agent is a steroidal antiinflammatory agent. 13. The method of claim 12, wherein the steroidal antiinflammatory agent is prednisolone acetate, prednisolone phosphate, dexamethasone, or fluorometholone. 14. The method of claim 13, wherein the steroidal antiinflammatory agent is 1% prednisolone acetate. 15. The method of claim 7, wherein the second therapeutically active agent is a nonsteroidal antiinflammatory agent. 16. The method of claim 15, wherein the nonsteroidal antiinflammatory agent is ocufen, indocin, or cyclosporine. 17. The method of claim 1, wherein the interleukin-10 is administered topically. ?5 18. The method of claim 17, wherein the interleukin-10 is administered in the form of eye drops and the concentration of interleukin-10 per drop is in a range from about 1 microgram per ml to about 2.5 mg/ml. 19. The method of claim 18, wherein the concentration of interleukin- 10 per drop is in a range from about 10 micrograms per ml to about .25 mg per ml. 20. The method of claim 18, wherein the interleukin-10 is administered to the eye at a rate ranging from one drop per hour to one drop every two days. 21. The method of claim 20, wherein the interleukin- 10 is administered to the eye at a rate ranging from one drop per day to four drops per day. 22. The method of claim 1, wherein the interleukin-10 is administered intracomeally or subconjunctivally. 23. The method of claim 22, wherein the interleukin-10 is administered at a dose of from about 5 micrograms to about 5 mg per day. 24. The method of claim 23, wherein the dosage amount is from about 50 micrograms to about 0.5 mg per day. 25. The method of claim 1, wherein the interleukin-10 is encapsulated in a liposome. 26. The method of claim 1, wherein the interleukin- 10 is encapsulated in a microcapsule. 2lβ 27. A pharmaceutical composition for the treatment of ocular inflammation comprising interleukin-10. 28. A topical pharmaceutical solution for the treatment of ocular inflammation comprising interleukin-10. 29. The pharmaceutical solution of claim 28, wherein the solution is in the form of eye drops. 30. The pharmaceutical solution of claim 28, wherein the concentration of interleukin-10 per drop is in a range from about 1 microgram per ml to about 2.5 mg/ml. 31. The pharmaceutical composition of claim 27, wherein the composition is for delivery to a periocular region of an eye. 32. The pharmaceutical composition of claim 27, wherein the composition is for delivery to an eye's intracameral region. 33. The pharmaceutical composition of claim 32, wherein the composition is for delivery to an eye's anterior chamber. 34. The pharmaceutical composition of claim 32, wherein the composition is for delivery to an intravitreal region of an eye.

Inventors: BOORSTEIN STEPHEN M, ELNER SUSAN G, ELNER VICTOR M, LAUSCH ROBERT N, OAKES JOHN E, TUMPEY TERRENCE M, BOORSTEIN, STEPHEN, M., ELNER, SUSAN, G., ELNER, VICTOR, M., LAUSCH, ROBERT, N., OAKES, JOHN, E., TUMPEY, TERRENCE, M.
Applicants: SCHERING CORP, SCHERING CORPORATION
Application year: 1996
Main classification: A61K38
IPC codes: A61K9, A61K31, A61K38, A61K45, A61P27, A61P29, A61P43