CA2228620A1 - Treatment of ocular inflammatory conditions with interleukin-10 - Google Patents

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

TRF~TMFI~IT OF OCUI /'R INFLAMMATORY CONDITIONS

FIFI n OF THF INVF~TION

This invention relates to the use of interleukin-10 (IL-10) to treat ocular 15 inflammatory conditions, particularly stromal keratitis induced by Herpes Simplex Virus Type 1 (HSV-1).

R~CKGROUND OF THE INVENTION

Inflammatory ~lise~ses of the eye can be initiated by viral, bacterial, fungal, or parasitic infection and by autoimmunity. (See, e.g., Focal Points 20 (1992) published by the American Academy of Opthalmology). Common complications of ocular inflammation include corneal scarring and perforation, glaucoma, neovasculaIi~ation 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 2~ important to minimize inflammation in tissues such as the clear, avascular cornea bec~use 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 type30 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 v~scul~rization and scarring. Corneal transplantation to W O 97/05895 PCTnJS96/12459 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., Comea 13:232, 1994). Diagnosis of neclo~ g stromal keratitis is made by clinical findings and may be confirmed via virus isolation and serology while that of 5 immune me~lic~te~ 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., ~tal. Ophthalmology 101:1883, 1994; Barron, etal., Ophthalmology 101:1871, 1994). Currently, HSV induced stromal disease is treated by topically applied corticosteroids with an antiviral cover,10 usually trifluridine or acyclovir (See, e.g., Wilhelmus, K.R. et a/., Ophthalmology.101:1883,1994). However,corticosteroidtherapymay prolong and possibly worsen the dice~se as well as introduce other effects such as enhancement of viral repl,c~tion, cataracts, glaucoma, corneal melting, secondary infection, and corticosteroid dependence. (See Liesegang, T.J., Mayo Clin. Proc. 6~.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 20 treating ocular inflammatory conditions in a mammal, comprising administeringa therapeutic~lly 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, 25 choroid, ciliary body, iris, retina, conjunctiva, orbital tissue, eyelids, nasolacrimal drainage apparatus, and optic nerve. Interleukin-10 has the advantage of reducing inflammation without compromising clearance of the infecting virus from the eye.

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i3RIFF DF~CRIPTION OF THE FlGuRFs 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 5 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. lA, 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 10 anti-lL-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 re~uced in the presence of active, but not antibody-neutralized IklO. Ear swelling was measured 24 h after antigen challenge.
The ear swelling response in naive mice was 13 i 3 X 10-4 inches. There 15 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 ~11 intracorneal injections of IL-10 (5 ng per injection) 4 h before and again on 20 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. (~) ind;c~tes the treated group was significantly (p < 0.05) different from the control group.

Figures 3A and 3B are photomicrographs of corneas removed 28 days 25 after HSV-1 innocluation. Four corneas per group were examined in two independent experiments. Cross sections of corneas from saline-treated mice (Fig. 3A) and rmlL-10 treated mice (Fig. 3B) showed marked differences in W O 97/05895 PCT~US96/12459 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 sensit~ ion. Mice infected by corneal innoculation 5 with HSV-1 were treated with IL-10 as described in the description of Figure 2above, or given saline (control). DTH testing was performed on day 6 post-infection and ear swelling was measured 2~ 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 10 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 in~ic~ted four animals in each group were killed and the 15 individual eyes were PYCise~ and titrated for infectious virus conle"l demonstrating that viral titers in eyes treated with IL-10 were progressively redlJced 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 cornea 20 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 exciserl and individually evaluated by enzyme-linked immunosorbent assay for IL-1a, IL-2, and IL-6 and showed selective downregulation of host IL-2 and IL-6, but not IL-1 a.

Figures 7A and 7B are graphical representations showing the effect of rmlL-10 on spontaneous synthesis of IL-6 and IL-la by excised mouse corneal buttons. Normal corneas were removed frorn BALB/c mice, and pools of three W O 97/05895 PCT~US96/12459corneas each were incubated in the presence or absence of the indicated amount of IL-10. In Fig. 7A, IL-6 levels in the supematant were assayed after 12 h of incub~tion. In Fig. 7B, corneal button Iysates were monitored for IL-1Ocafter 4 h of incubation. Host corneal IL-6 was significantly inhibited while host 5 corneal IL-10c was not reduced (~) in~ic~tes a significant reduction (p < 0.05) relative to the control as ~ssessed by Student's t-test.

Each of the above figures was originally published in Tumpey et al., J. Immunol. 153:2258 (1994). Additional evidence for IL-10 inhibitors of HSK
in humans is illustrated by two additional plJblic~t;ons. In Ulnterleukin-10 10 Inhibition of HLA-DR Expression in Human Herpes Stromal Keratitis" by Boorstein, etaL, (Ophthalmology; 101:1529-1535 (1994), profound reduction of HLA-DR antiger- expression in human stromal keratitis was observed following incubation of clice?ced human corneas with recornbinant human IL-10 (rhlL-10)at a dose of 100 units per ml. A second study e.,lillecl 15 ~Interleukin-10 Modulation of lnterleukin-8 and Monocyte Chemotactic Protein-1 Secretion in Human HSK" by Boorstein, et al. (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 20 units/ml.

nFTAII Fn nF.~CRlPTlON OF THF INVFNTION

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 25 (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 coll) IL-10 are equivalent and can be used interchangeably. Also included are muteins and other analogs, including the Epstein-Barr Virus protein BCRF1 5 (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 10 Merrifield, Science 233:341 (1986) and Atherton et al., Solid Phase Peptide Synthesis: A Practical Approach, 1989, I.R.I . 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.
15 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 a/., (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 20 cDNA libraries. Polymerase chain reaction (PCR) techniques can be used.
See, e.g., PCR Protocols: A Guide to Methods and Applications, 1990, Innis et a/., (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 25 methods for making IL-10. Useful gene sequences can be found, e.g., in various sequence d~t~b~ses, e.g., GenBa~k and BMPL or nucleic acid and PIR and Swiss-Prot for protein, c/o Intelligenetics, Mountain View, California, or W O 97/05895 PCTrUS96/12459 the Genetics Computer Group, University of Wisconsin Biotechnology Center, Madison, Wisconsin.

cîones 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 hyb,i~ tiGn 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 us~ful.
Oligonucleotide probes sequences can also be prepared from conserved regions of related genes in other species. Alternatively, degenerate probes based on the amiro 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). ~(2282, and RR1 (ATCC Mp/ 31343). Exemplary mammalian csll lines include COS-7 cells, mouse L cells and CHP cells. See Sambrook (1989), supra and Ausubel et a/., 1987 supplements, supra.

Various expression vectors can be used to express DNA encoding IL-10. Conventional vectors i~sed for expression of recombinant proteins in prokaryotic or eukaryotic cells may be used. Preferred vectors include the pcD
vectors described by Okayama et a/., Mol. Cell. Biol. 3:280 (1983); and Takebe et a/., Mol. Cell. Biol. 8:466 (1988). Other SV40-based mammalian expression vectors include those disclosed in Kaufman et a/., 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 calls (ATCC No. CRL 1651), as well as in W O 97/05895 PCTrUS96/12459 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 5 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 Method~ in Enzyrnology Purification Principles and Pr~ctices (Springer-Verlag, New York, 1982).

Alternatively, 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 agent15 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 et aL, and U.S. Patent No. 4,569,790.

The nucleotide sequences used to transfect the host cells can be 20 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 structura 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 serl~m half-life, facilitating purification or preparation, improving therapeutic efficacy, and lessening the severity or occurrence of sid~ effects during therapeutic use. The amino acid sequence variants are usually predetermined variants not found in nature7 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 5 used in this invention as long as they retain the biological activity of Ik10.
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 modi~icatiGns 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 sui~able 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 etal., Science 248:1230 (1990). (See also international patent application WO 91/09127 and U.S. Patent 5,368,854.) When referring to IL-10, active fragments thereof, analogs and 20 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 canbe glycosylated or unglycosylated. Examples of IL-1 0 activity include inhibition or substantial reduction of the level of IL-2, Iymphotoxin, IL-3, or GM-CSF.
2 5 IL-10 activity also includes inhibition of cytokine production by activated macrophages, e.g., IL-1,IL-6, and TNF-oc.

W O 97/05895 PCTAJS96112459For 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 pharm~ceutic~l compositions including polypeptide Ik10, 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 US. Pharmacopeia:
National Fommulary, Mack Publishing Company, Easton, PA (1984); Avis etal.
(eds.) (1993) Ph~rm~ceuti~l Dot~ge Froms: Parenteral Me,l;~l;GnS: Dekker, New York; and Lieberman et aL (eds.) (1990) Ph~rmaceutir.~l Do~ge Forms:
nisperse 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 penelralion; mucoadhesive polymers including hyaluronlc 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, cytochalasin 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., 2~.21(1980); Chiou, etal., Pharmacol. Ther., 17:269 (1982); Lee, et al., J. Ocul. Pharmacol., 267 (1986); Camber, et al., ~ /0 W O 97/05895 PCT~US96/12459 (1989), Curr. Eye Res., 8:563; Marsh, et al, Exp. Eye Res., 11:43 (1971);
Ramselaar, et al., Curr. Eye Res., 7:947 (1988); Norn, Acta Ophthalmol" 4~727 (1964); Seig, eta/, J. Pharm.; Sci., 6~.122 (1977); McKeen, Int. Ophthalmol.
Clin., 2~.79 (1980); Higuchi, J. Pharm. Sci., 5~.874 (1961); ~~ettone, et al., Int.
J. Pharm., 51:203 (1989); Blaug, et al., Am. J. Of. Hosp. Pharm., 22:662 (1965);Keller, et al., Exp. Eye Res., 3~.203 (1980); Grass, et al., Invest Ophthalmol.
Vis. Sci., 261 10 (1985); Harris, et al., Biomaterials, 11:652 (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., Surv. of Ophthalmol., 29:335 (1985); Szoka, et al., Ann. Rev.
Biophy. Bioeng., g.467 (1981); Smolin, et al., Am. J. Ophthalmol, 91:220 (1981); Ahmed, et al., Invest..Ophthalmol. vis. Sci., 26:584 (1985); Chiou, et al., J. Pharm., Sci., 7~: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., 1:26 (1967);
Rothweiler, et al., Experientia, 22:750 (1966); Binder, et al., Agnew. Chem. Ins.
Edit., 12:370 (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. Concenl,ations 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 concenl,ation 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 5 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 necess~ry 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 indic~te-i in the references for the above-lerer~nced 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: Th~ Pharmacological Bases of Th~rapeutics, 9th ed., Pergamon Press; and Remington's Pharmaceutical Sciences, 17th ed. (1992), Mack Publishing Co., Easton, Penn. The dos~ge 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 various20 vehicles.

The IL-10 can also be administered subconjunctivally (sc) or intracorneally. 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 2~ could employ vehicles used for topical dosin~ or those used for intravenous, intramuscular, or subcutaneous delivery. Thus, the preferred dose range for subconjunctivial adminisl&tiol- in humans is from about 5 micrograms to about 5 mg per day, more preferably from about 50 micrograms to about 0.~ mg. In ~2 _ W O 97/05895 PCTrUS96/12459 the case of intracorneal 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 Ik10 is short in vivo (estimated 2.5 hours in the ~ ~ mouse), it may be necess~ry to give the drug more often than once a day. This can be determine~ by the clinician based on the condition of the particular patie. ~t.

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 dise~-ses. 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 io"lo,vhoretic 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 allGy,drls would require doses ranging from 0.001 micrograms per ml to 0.1 mg/ml, more preferably from 0.1 micrograms/ml to 10 micrograms/ml.

As used herein, the phrase "therapeutically effective amount" means an amount sutriciei-t l:o ameliorate a symptom or sign of ocular inflammation.
Clinical herpes stromal keratitis is characterized by corneal edema, corneal - 25 haze, neovasculali~aLiGn, anterior chamber inflammation and keratic precipitates. Accordingly, amelioration would be recognized by a reduction in one or more of these clinical signs.

W O 97/OS895 PCT~US96/12459 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 5 condition being treated, the overall health of the patient, the method, route, and dose of adminislr~lion 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 10 optimum effect is achieved. (See generally The Merck Manual 269 ~Pharmacokinetics and Drug Administration.n).

In appro~riate circumstances, multiple mecl;cations can be administered in combination. For instance, the IL-10 can be administered in combination with a therapeutically effective dose of one or more ~d~itional therapeutically 15 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 20 the following examples, which are not intended to limit the invention to specific embodiments.
EXAM PLES

In the following examples, IL-10 was tested to determine if this cytokine could suppress the development of stromal dise~se. Two studies 25 demonstrated IL-10 suppression of HLA-DR antigen expression (Boorstein, et a/., Ophthalmology 1994; 101:1529-1535) and leukocyte chemokine production (Boorstein, et al., Invest. Ophthalmol Vis Sci, 36 (4):S147; 1995) in /4 W O 97/05895 PCT~US96tl2459 human corneas affected with herpes stromal keratitis using recombinant human IL-10 at 100 units per ml, ex vivo. Both of these articles (Boorstein, ef al., Ophfhalmology; 101:1529-1535, 1994 and Boorstein, etal., Invest.
Ophthalmol Vis Scl, 36 (4):S147, 1995) are expressly incorporated herein in 5 their entirety by reference.

Further, in an extensive animal study, rmlL-10 was inoculate intracorneally in mice 4 hours before and again on days ~2 and ~5 relative to the time of topical HSV-1 corneal innocul~tion. Additionally, the mice received IL-10 i.p. at the time of virus administration and again 3 days post-infection.
10 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 10-15 fold lower and IL-6 some 50-fold lower than that found in the controls. IL-1a levels were not reduced. The Ik10 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 thecontrols. In vitro studies revealed that spontaneous production of IL-6 by 20 excised normal corneas was inhibited by >95% with low dose IL-10. IL-1a 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 , W O 97/05895 PCTrUS96/12459 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.
5 Virus stocks were grown and titrated on Vero cells as previously described (Lausch, et al., Curr. Eye Res. 8:499,1989). 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 2-111 volume containing 1 to 5 x 104 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. 6:173, 1987); a score of15 0 indicates a clear comea, whereas a +5 score represents severe necroli,il ,gstromal 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 U test. (Snedecor, G.W. and Cochran, W.G., Sl~ lic~l Methods, 6th ed. Iowa State University Press, Ames, lowa, 1967; pp. 130- 131.) Re~ents-- Murine recombinant IL-10 (rm-lL-10) was obtained from DNAX Research Institute, Palo Alto, CA. The endotoxin co,llent of the IL-10 preparation was c0.1 ng/~lg. The biologic activity of this cytokine was 7 U/,ul 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-2A5.11 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 neutralizing monoclonal antibody was also purchased from Gen~yme Corporation (Cambridge, MA~.

Intr~corneal inJections-- Intracorneal injections were performed as previously described (Hendricks, et al. Invest. Ophthalmol. Vis. ScL 3~.366, 1991). Briefly, a 30-gauge disposable needls was used to puncture the corneal epithelial wall. A 30-cm, 32 gauge stainless steel needle attached to a Hamilton dispensel (Hamilton, Reno, NV) was then threaded into the stroma and 1 ,ul of rmlk10 or saline was injected into the center of the cornea.

nel~yed tYDe hyDersensitivity (l~TH) ~s~y-- 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 irr~di~tion for 10 min. This reduced infectivity from 106 to less than 102 plaque forming units/10 ~I. To test for DTH
responsiveness, 10111 of viral antigen was inocul~ted into the dorsal side of the mouse's right ear by using a 50 1ll Hamilton syringe and a 30-gauge needle 6 days after in~ectiGn. The left ear (control) received 10 1ll 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 lef~ (control) ear in units of 10-4 inches. To determine whether IL-10 suppressed the DTH response, the right experimental ears received 5 1ll of UV-HSV-1(RE) mixed with 5 ,ul 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 ,ug/ml final concenlration), or control IgG, for 30 minutes over ice before ear inocul~tiQn. Data were evaluated by using Student's t-test.

W O 97/OS895 PCTnUS96/12459 ACc~y of ocul~r 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 1ll of 2% FBS in DMEM medium with antibiotics. Preparations were frozen at -70~C, thawed, and homogenized in a 5 Ten Broech homogenizer (Bellco, Vineland, INH). The homogenates were frozen, thawed again, and sonicated for 15 s with a Sonic 300 Dismembrator (Artek Systems CGr~,Gralion, Farmingdale, N~). The supernatants were then titrated for infectious virus on Vero cell monolayers in a 48-h plaque assay.

Cytokine t~uan~ on--To test the effect of IL-10 on IL-6, IL-2 and 10 IL-1a production in vivo, corneas were remo~/ed 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 micro~issecting trephine (Roboz Surgical Instrument Co., Rockville, MD), and placed individually in 600 ,ul of serum-freeRPMI 1640 with Fungi-Bact antibiotic solution. Samples were stored at -70~C
15 until assayed. Samples were thawed, sonicated for 30 seconds, and clarified by centrifugation at 150 x 9 for 10 minutes. 1 he clarified cell Iysates 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-1a kit (assay sensitivity 15 pg/ml) was purchased 20 from Genzyme.
Spontaneous cytokine synthesis by excised normal corneas was determined as previously described (Staats, et al., J. Immunol. 151:277, 1993).
In brief, corneal buttons were exciced from untreated mice and trimmed to 2 mm. The tissue preparations were free of limbal vasculature as judged by 25 microscopic inspection. Each sample consisted of three corneal buttons incubated in the absence or presence of murine IL-10 in 500 ~LI 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 1g W O 97/05895 PCTrUS96/12459 assayed for IL-6. For IL-1cc deteclion, cornea samples were inc!Jb~tesl for 6 h.Then the corneas were disrupted by sonication for 30 seconds with a Sonic 300 dismembrator. The tissue Iysates were clarified by centrifugation at 150 x g for 10 min before assay.

HistoloQic FY~min~tion-- Infected eyes were enuc'o~ted 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 hematoxylin-eosin, mounted in resin, and cover-slipped for photomicroscopy.

1 0 Results 1. IL-10 treatment given locally suppresses DTH responsiveness in sensitized hosts Initial experiments were designed to investigate whether rmlL-10 15 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 104 plaque forming units HSV-1 (RE).
Six days later, the animals responded strongly to viral antigen in DTH tests 20 (Fig. 1). However, injection of rmlL-10 with the test antigen resulted in significantly reduced ear swelling (Fig. lA). 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 25 intradermal ear inoculation (Fig. 1 B), specifically the suppresion of IL-10 on the DTH response to HSV-1 antigen. In Fig. lA, IL-10 (28 ng) was injected into the ear with the viral antigen challenge (lX). A second IL-10 injection (55 ng) was given 12 h later (2X). In Fig. lB, IL-10 was preincl~h~ted with anti-lL-10 mAB or W O 97/05895 PCTrUS96/12459 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 ~t 3 X 10~ inches. There were three to four mice per group.

2. IL-10 treatment suppresses the development of HSV-1 induced stromal ker~titis (HSI~

The foregoing results indicated that rmlL-10 given to previously 10 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 ~iise~se believed to be medi~te~ at least in part ~y sensiti~d T cells (Metcalf,et al., Infect. Immun. 26:1164, 1979); (Russell, et al., Invest. Ophthalmol. Vis.
ScL 25:938, 1984); (Newell, et al., J. Virol. 63:769, 1989); (Newell, et al., Reg.
Immunol. 2:366, 1989); (Hendricks, et al., Inv~st. Ophthalmol. Vis. Sci. 31:1929, 1990); (Doymaz, et al. Invest. Ophfhalmol. Vis. Sci. 33:2165, 1992);
(Niemialtowski, et al., J. Immunol. 149:3035, 1992). rmlL-10 inoculated directlyinto the comea on days -1, +1, ~4, and +7 r~lative to infection slowed, but did 20 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 comeal opacity scores were sigi li~icantly reduced (p c 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 de~.icls 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.

W O 97/05895 PCT~US96/12459 Numerous blood vQssels 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 veryfew inrillrtlling inflammatory cells, and only low level neovascularization.

The protective action of IL-10 was coll~i-,-,ed in two ~dditional 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., Int~rl/irology 31:159 (1990). At the virus-in~ecti-,g doses employed (1 to 5 x 104 plaque forming units), HSV-1 strain RE on oc~sion will spread from the eye to the central nervous system and inducs fatal dise~se 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 1~ increase host suscepliLilil~ to central nervous system disease.

IL-10 has multipie suppressive effects on various effector phases of the immune response, including inhibition of T cell proliferation. It was possible that reduced comeal 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-medi~te~ immunity, DTH testing was conducted in cytokine-treate~ 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.
2~ Furthermore, virus neutralizing titers of sera collected 4 wk post-infection from Ik10-treated hosts were analogous to those found in the controls (data not shown). Thus, there was no evidence that IL-10 treatment suppressed (or W O 97/05895 PCT~US96/12459 enhanced) the development of aither the cellular or humoral immune r~sponses to HSV-1 antigens.

3. Fffect of ll -10 trQ~tment on virus repli-!~tit-n in the eye A number of cytokines have been shown to exert antiviral effects in 5 ocular tissue (Chen, et al., AnUviral Res. 2~:15, 1993). Therefore, the possibility was entertained that rmlL-10 treatrnent initiated before HSV-1 Tnfection might directly or indirectly reduce corneal inflammation by inhibitingviral replic~lio" in the corneas. To test this hypothesis, the amount of i"feclious virus recovered from the eyes of IL-10 treated and control mice was monitored 10 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 i".lic~ted that IL-10 treatment did not ~ccelerate (or delay) virus clearance from the eye.

4. Fffect of ll -10 on synthesis of ll -1a.11 -~ and li -6 in the comea It is known that HSV-1 infections of th~ murine cornea are characterized by elevated levels of IL-6 and IL-1a (Staats, et al., J. Immunol . 151:277, 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.
4754, 1993), polymorphonuclear leukocytes (C~-ss~tell~, et al., J. Exp.
20 Med 178:2207, 1993), and monocytes/macrophages (Fiorentino, et al., J.
ImmunoL 147:3815 (1991); Bogdan, et al., J. Exp. Med. 174:1549 (1991); de Waal, et al., J. Exp. Med. 174:1209 (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 25 assayed for IL-1a,1L-2, and IL-6 by ELISA. The data from two experiments were comparable and the pooled results are shown in Figure 6. IL-1a levels in the treated animals were not significantly different (p <0.7) from those seen in ~Z

W O 97/05895 PCTrUS96/12459 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 ~letect~hle 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-la by exciced mouse corne~l buttons Bec~use 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 10 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-1a and IL-6 (St~ts, et al., .1. Immunol. 151:277, 1993). Accordingly, e~cised corneal buttons from uninfected donors were incub~ted in vitro in the presence or 15 absence of different concenlralio"s of rmlL-10. Medium supernatants and Iysates of corneal tissue were subsequently obtained and assayed for IL-6 and IL-1a, respectively. The results of three experiments, summarized in Figure 7, show that IL-10 at a concenllatio" of 1.5 ng/ml could suppress IL-6 synthesis by >95%. Curiously, the 150 ng/ml dose was less suppressive. The reason for 20 this is not clear but a similar dose-response pattern was observed in all three experiments. In contrast, the synthesis of IL-10c 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 25 by way of example only, and the invention is not to be construed as limited thereby.

~3

Claims (34)

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 toan 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.

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 intracorneally 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.

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.