WO1997005895A1 - Traitement d'inflammations oculaires avec de l'interleukine-10 - Google Patents

Traitement d'inflammations oculaires avec de l'interleukine-10 Download PDF

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WO1997005895A1
WO1997005895A1 PCT/US1996/012459 US9612459W WO9705895A1 WO 1997005895 A1 WO1997005895 A1 WO 1997005895A1 US 9612459 W US9612459 W US 9612459W WO 9705895 A1 WO9705895 A1 WO 9705895A1
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Prior art keywords
interleukin
per
eye
ocular
agent
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PCT/US1996/012459
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English (en)
Inventor
Robert N. Lausch
Victor M. Elner
Terrence M. Tumpey
John E. Oakes
Susan G. Elner
Stephen M. Boorstein
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Schering Corporation
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Priority to EP96928023A priority Critical patent/EP0847277A1/fr
Priority to AU67634/96A priority patent/AU6763496A/en
Priority to JP9508485A priority patent/JPH11510185A/ja
Publication of WO1997005895A1 publication Critical patent/WO1997005895A1/fr
Priority to MXPA/A/1998/001038A priority patent/MXPA98001038A/xx

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/20—Interleukins [IL]
    • A61K38/2066—IL-10
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • 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).
  • IL-10 interleukin- 10
  • HSV-1 Herpes Simplex Virus Type 1
  • Inflammatory diseases of the eye can be initiated by viral, bacterial, fungal, or parasitic infection and by autoimmunity.
  • 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.
  • Wakefield, D. and Lloyd, A., Cytokine 4:1, 1992 See, e.g., Wakefield, D. and Lloyd, A., Cytokine 4:1, 1992).
  • 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.
  • 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.,
  • 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.
  • ocular inflammatory conditions such as herpes stromal keratitis.
  • 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 are also provided.
  • 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.
  • 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 .
  • Fig. 1A recombinant murine interleukin-10 (rmlL-10) (28 ng) was inoculated into the ear with the viral challenge antigen (1X).
  • rmlL-10 murine interleukin-10
  • a second IL-10 inoculum 55 ng was given 12 h later (2X).
  • 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 10TM 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 ⁇ l 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.
  • 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-1 ⁇ , IL-2, and IL-6 and showed selective downregulation of host IL-2 and IL-6, but not IL-1 ⁇ .
  • Figures 7A and 7B are graphical representations showing the effect of rmlL-10 on spontaneous synthesis of IL-6 and IL-1 ⁇ 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.
  • IL-6 levels in the supernatant were assayed after 12 h of incubation.
  • Fig. 7B corneal button lysates were monitored for IL-1 ⁇ after 4 h of incubation.
  • Host corneal IL-6 was significantly inhibited while host corneal IL-1 ⁇ was not reduced.
  • * indicates a significant reduction (p ⁇ 0.05) relative to the control as assessed by Student's Mest.
  • 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.
  • 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.
  • glycosylated e.g. produced in eukaryotic cells such as CHO cells
  • unglycosylated e.g., chemically synthesized or produced in E. coli
  • IL-10 are equivalent and can be used interchangeably.
  • 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.
  • 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.
  • IL-10 IL-10.
  • 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).
  • 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.
  • 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.
  • 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.)
  • 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- ⁇ . 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.
  • 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 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.
  • preservatives such as bezylkonium chloride or ethylenediaminetetraacetic acid (EDTA) to enhance penetration
  • 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.
  • IL-10 it is desirable to treat ocular inflammation locally.
  • This can take the form of topical administration of IL-10.
  • the 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
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.”).
  • 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).
  • 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.
  • mice received IL-10 i.p. at the time of virus administration and again 3 days 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.
  • 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- ⁇ l 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.)
  • Murine recombinant IL-10 (rm-IL-10) was obtained from
  • /6 neutralizing monoclonal antibody was also purchased from Genzyme Corporation (Cambridge, MA).
  • 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 ⁇ l of rmlL-10 or saline was injected into the center of the cornea.
  • Hamilton dispenser Hamilton dispenser
  • 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 ⁇ l.
  • 10 ⁇ l of viral antigen was inoculated into the dorsal side of the mouse's right ear by using a 50 ⁇ l Hamilton syringe and a 30-gauge needle 6 days after infection.
  • the left ear (control) received 10 ⁇ l 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.
  • the right experimental ears received 5 ⁇ l of UV- HSV-1(RE) mixed with 5 ⁇ l containing 28 ng of IL-10 just before ear challenge.
  • the controls received saline in place of IL-10.
  • IL-10 (110 ng) was incubated with monoclonal rat anti-mouse IL-10 neutralizing Ab (10 ⁇ g/ml final concentration), or control IgG, for 30 minutes over ice before ear inoculation. Data were evaluated by using Student's t-test.
  • Cytokine Quantitation To test the effect of IL-10 on IL-6, IL-2 and IL-1 ⁇ 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 ⁇ l 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.
  • trephine Robot Surgical Instrument Co., Rockville, MD
  • 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
  • IL-2 assay sensitivity 3 pg/ml
  • the IL-1 ⁇ kit was purchased from Genzyme.
  • 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 ⁇ l 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.
  • IL-1 ⁇ 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.
  • 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).
  • IL-10 was preincubated with anti-IL-10 mAB or
  • mice 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.
  • IL-10 treatment suppresses the development of HSV-1 induced stromal keratitis (HSK)
  • 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.
  • 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.
  • FIG. 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.
  • IL-10 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.
  • 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
  • I L-10 Effect of I L-10 on synthesis of IL-1 ⁇ .
  • 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-1 ⁇ (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.
  • 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.

Abstract

L'invention concerne un procédé pour traiter des inflammations oculaires, en particulier la kératite du stroma provoquée par le virus de l'herpès du type 1 (VHS-1). Ce procédé consiste à administer dans la région oculaire d'un mammifère une quantité d'interleukine-10 suffisante pour avoir un effet thérapeutique. L'invention concerne également des compositions pharmaceutiques pour le traitement d'inflammations oculaires, en particulier des solutions à usage topique.
PCT/US1996/012459 1995-08-07 1996-08-05 Traitement d'inflammations oculaires avec de l'interleukine-10 WO1997005895A1 (fr)

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EP96928023A EP0847277A1 (fr) 1995-08-07 1996-08-05 Traitement d'inflammations oculaires avec de l'interleukine-10
AU67634/96A AU6763496A (en) 1995-08-07 1996-08-05 Treatment of ocular inflammatory conditions with interleukin-10
JP9508485A JPH11510185A (ja) 1995-08-07 1996-08-05 インターロイキン−１０を用いた眼の炎症性状態の処置
MXPA/A/1998/001038A MXPA98001038A (en) 1995-08-07 1998-02-06 Treatment of ocular inflammatory conditions with interleucine

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US62972196A 1996-04-09 1996-04-09
US08/629,721 1996-04-09
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WO1997041882A1 (fr) * 1996-05-03 1997-11-13 Schering Corporation Methode de traitement de l'inflammation
WO2002078713A1 (fr) * 2001-03-28 2002-10-10 Santen Pharmaceutical Co., Ltd. Medicaments contre les affections retiniennes et choroidiennes contenant des steroides comme principe actif
JP2002534394A (ja) * 1999-01-05 2002-10-15 ザ フリンダーズ ユニバーシティ オブ サウス オーストラリア 眼障害を治療及び診断するための新規な薬物及び方法
WO2009102715A2 (fr) * 2008-02-11 2009-08-20 Board Of Supervisors Of Louisiana State University And Agricultural And Mechanical College Thérapie à base peptidique contre les infections oculaires à virus herpes

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997041882A1 (fr) * 1996-05-03 1997-11-13 Schering Corporation Methode de traitement de l'inflammation
US5753218A (en) * 1996-05-03 1998-05-19 Schering Corporation Method for treating inflammation
JP2002534394A (ja) * 1999-01-05 2002-10-15 ザ フリンダーズ ユニバーシティ オブ サウス オーストラリア 眼障害を治療及び診断するための新規な薬物及び方法
JP2011084578A (ja) * 1999-01-05 2011-04-28 Fliners Univ Of South Australia 眼障害を治療及び診断するための新規な薬物及び方法
WO2002078713A1 (fr) * 2001-03-28 2002-10-10 Santen Pharmaceutical Co., Ltd. Medicaments contre les affections retiniennes et choroidiennes contenant des steroides comme principe actif
WO2009102715A2 (fr) * 2008-02-11 2009-08-20 Board Of Supervisors Of Louisiana State University And Agricultural And Mechanical College Thérapie à base peptidique contre les infections oculaires à virus herpes
WO2009102715A3 (fr) * 2008-02-11 2009-10-08 Board Of Supervisors Of Louisiana State University And Agricultural And Mechanical College Thérapie à base peptidique contre les infections oculaires à virus herpes

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AU6763496A (en) 1997-03-05
JPH11510185A (ja) 1999-09-07

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