MXPA98001038A - Treatment of ocular inflammatory conditions with interleucine - Google Patents

Abstract

A method is provided for the treatment of 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 are also provided for the treatment of ocular inflammation, including topical solutions

Description

? TREATMENT OF CONDITIONS WITH EYE INFECTIONS I TFPI EUCINE 10 FIELD OF THE INVENTION The invention relates to the use of mierleu-na-10 (II.-10) "» to treat rondn i OGP;, ocular inflammations, pariularly outside the stromal II induced by the Herpes Simplen Type 1 <HSV-1) BACKGROUND OF THE INVENTION ls inflammatory diseases of the eye can be initiated for viral infection, bacterial, funga! either parasitic or i n by self-invasion. (See, for example, Focal Points, 1992) published by the American Arade and of Ophta lmology). Complications usual ions of ocular inflammation include scars and perforation of the cornea, glaucoma, Neo-iscul ap p a on of the cornea and the resin, scars and detachment of the retup, cataract, damage to the optic nerve and cyclic in the orbital tissues and the eyelids. (See, for example Wakefield, D "and loyd, A., Cyt? Ri i ne 4" l, 199Z1) "E > important n? ni (it?: ar inflammation in tissues For example, the cornea, because the infiltration of leukocytes and blood vessels can cause a significant loss of vision, and repair by corneal transplantation has a very poor prognosis when it occurs. A particularly bad type of condition The inflammatory eye is keratitis est om 3 induced by the simple herpes virus type 1 (HSV-i) or less frequently by the HSV d «-» type 2. The inflammation of the cornea due to HSV is particul evev and can last for years with atami nto. A common sequel is the 5 v -at-yul ar i rae ion and scars in the 3rd cornea. The transplant of the cornea to restore 1 i vision has a high failure rate due to 3 important rates of rejection and re-infection of HSV. (See B .-? Mey N.P. and Foster, CS., Cornea 13: 232, 1 94). Fl diagnosis of stromal keratitis necro i zan is based on clinical findings and can be confirmed by virus isolation and serology while the icognos of stromal medicated immune keratitis is carried out by means of clinical findings, aerology and a study. hi tomorf l gico (See liesegang, T.J. Mayo Clin. Proc. 63 s 1092, 1988; Wi 1 hel niü, K.P., et / - al ", Oph h l mol ogy 101: 1883, 1994.» B n, et al ,, Oph hj Imol ogy 1 < "<!? 1871, 1994), Today, HSV-induced disease is treated by ophthalmology and by applying corticosteroids with anti-viral coverage, usually 2? triflupdma or b i »a iclovir (See, for example, Wilhelmus, K-.R., et al., Ophthal mology., lR83, 1994). However, corticosteroid therapy can prolong and possibly worsen the disease as well as introduce other complications, such as increased viral replication, cataracts, glau-oma, fusion of the c? P \ & ^ infection set swath, and dependence on 1 ov. cor t a osteroídes. (See, I.ieaegang, T.T., Mayo Clin. Proc, 63, 092, 1988). Therefore, there continues to be an important need to develop more effective methods to treat ocular inflammatory conditions such as, for example, stromal keratitis. SUMMARY OF THE INVENTION The invention satisfies the foregoing needs by providing a method for treating inflammatory conditions in a mammal, comprising the administration of a therapeutically effective amount of insulin to a mammal. to within the ocular region of said mammal. It > It also provides pharmaceutical compositions for the treatment of ocular inflammation, including topical solutions. The term "ocular region" refers to the components of or including the cornea, is lerotic, corai e, iliac body, iris, retina, conjunctiva, orbital tissue, eyelids, draining device nasal gr imal, and optic nerve. The nterleucin-1 has the advantage of reducing inflammation without compromising the clearance of the infectious agent of the eye. BRIEF DESCRIPTION OF THE FIGURES This invention can be understood more easily with reference to the appended figures, where! Figures 1A and IB are graphical representations illustrating the effect of local IL-10 on the localized delayed-type hypersensitivity (DTH) response to HSV-1 antigen in rats pre-infected with HSV by inoculation vir l in the .. rnea. In Figure 1, recirculating murine termol-10 (rmIL-10) (28 ng) was inoculated into the ear with the retina antigen (IX). A second inoculum of IL-10 (5 ng) (2X) was given 12 hours later. In FIG. IB, IL-10 was pre-incubated with a monoclonal antibody anti- -TI-10 or control IgS before being inoculated at the DTH piueha site if ul with an antigen and 12 hours later. of the challenge with antigen and was reduced in p ét that of active IL-10, but not neutrized with antibodies. The swelling of the ear was measured 24 hours * - after challenge with antigens. The ear swelling response in native mice was 13 _ • 3 X 1/10, 00 inches. The study was carried out on three to four mice. "By group. Figure 2 is a fi ne reflection that illustrates the effect of rmIL-l? on the development of stromal keratitis caused by herpes and demonstrates a reduced pacification of the corneas according to what was observed by biologics. Ratonen (eight per group) received 1 μl of TL-10 corneal injections (5 ng per injection) 4 hours before and another time. on days 2 and 5 in relation to the time of infection of the cornea by HSV-1 (PE). Ad libically, i.p. 110 ng of IL-10 at the time of viral inoculation and another ve / 3 days later. The controls received a saline solution instead of IL-10, (*) indicates that the treated group was ignitatively (p - 0.0) different from the control group. Figures 3A and 3B are photomicrographs of corneas removed 28 days after inoculation with HSV-1. Cuts were analyzed per group in two independent experiments Cortes of corneas of mice treated with saline (Figure 3A) and mice treated with rmIL-10 (Figure 3B) showed remarkable differences in inflammation, icatrice., and neovasculari? ac? n. Staining H & E. The original amplifier is x200 in the case of the figure 3A,: 100 in the case of Figure 3B. Figures 4A and 4B are graphical ions illustrating the effect of rm? L -10 on systemic DTH sensitization. Patones infected by cornea inoculation with HSV-1 were treated with II. -10 according to what is described in the description in figure 2 above, or they received urine saline solution (control). A DTH examination was performed the day after infection and the swelling of the ear was measured 24 hours later and it was shown that the swelling / t n of the ear in mice treated with 11-10 was not very different > ie the controls that obtained a saline solution, the groups consisted of three to four mice. Figures 4A and 4B illustrate two experiments i dependent. Figure 5 is a graphical representation illustrating the fec or rmll -1 on HSV-1 particles in o o. Patches infected in the cornea with HSV-1 were treated with II-10 in accordance with that described in the description of figure 2 above, or they received a saline solution. The days indicated, four animals in > Each group was killed and the individual children were removed and titled to determine the content of infectious virus proving that it had viral titers in them. > Those treated on II.-10 were progressively reduced compared to the control animals. () treated with 11-10; (o) control animals. Figure 6 is a graphical representation illustrating the effect of rmlt-10 on the synthesis of i oe i na in corneas and fec adas pair HSV-1. Patones infected in the cornea with HSV-1 were treated chron 11-10 according to what is described in the description of figure 2 above or they received a saline solution. Ten days after infection the corneas were removed and individually evaluated by an enzyme-linked immunoabout assay for? L-la3fa, IL-2, and TL-6 and showed selective down-regulation of IL-2 and IL - guest, but not IL ~ -l Ifa. Figures 7A and 7B are graphical ions illustrating the effect of rm? L-10 on the spontaneous synthesis of II. -6 and II -alpha removing mouse cornea buttons, normal corneas were removed from BALB / c mice and sets of three corneas each were incubated in the presence or absence of the indi-vidual amount of 11-10. In Figure 7A, levels of II - in the worker were tested after 12 hours and incubation. In Figure 7B, corneal button lysates were monitored to determine IL-lalfa after 4 hours of incubation. 11-6 of the cornea of the host was significantly inhibited while II-the fa of the cornea of the host was not educated. (*) indicates a specific reduction (p 0.05) in relation to compliance control > on what is evaluated by the Student's t test. Each of the previous figures was originally published in Tumpey et. , J, I munol. 153: 2258 (1994). Additional evidence of inhibitors of HSK IL-10 in humans is illustrated in two additional tests. In "3 nterleule i n - 1 Inhibition of HLA - DR Expression in Human Herpes If it blunts 1 eratitis" by Boorstein, et al., (Ophtha Imology; l? Ts 1529--1 35 (1994), a profound reduction of expression of HLA-DP antigen in stromal keratitis humiiH was observed after incubation of diseased human corylan with recombinant human It-1. íi'hIL-10) in a dose le 100 units per ml. A second study entitled "Interleut i ~ 10 lodulation of inter leuk? N-0 and Monocyte che otac t? C Prote? N-1 Secretion m Human HSK" by Boorstem, et al. (Invest. Ophthalmal Vis S i, 36 (4) sS147, 1995) showed cju * »rhIL-lO is a potent inhibitor of leukocyte leukocytes e? L-8 (80% inhibition) and MCP-- 1 (50 * / * of inhibition) in human corneas affected by stromal keratitis caused by HSV-1 when used in a dsid 1 0 unit des / m1. MADA DESCRIPTION OF THE INVENTION Woven the references mentioned herein are incorporated in their entirety by reference. As used here, "interleukin-10" or "11-10" a chromium is effected by a protein that (a) has a sequence of mature TL-10 inoatides (for example, that does not have a secretory leader sequence) in accordance with what is presented in US Pat. No. 5,231,012 and (ti) has a biological activity as a nabi-IL-10. For the purposes of this invention both 11-10 glycosylation (e.g. produced in eukaryotic cells such as CHO cells), as well as glycosylated iodine (for example, chemically synthesized or bi n produced in E. coli) are equivalent and can be used interchangeably. Muteins and other analgeses are also included, including the BCRF1 prine from the Fpstem-Barr Virus (viral IL-10) which preserves the 1 v i ad iol i i ca of 11 -1.

II-10 suitable for use in the invention can be obtained from culture media conditioned by cells to ivadas that secrete the protein, and purified by standard methods. In addition, the IL-10, or active fragments thereof, can be synthesized chemically using known standard techniques. See Merpfield, t jence 233: 341 (198o >) and Athert n et al, "Sol id Phase Pepticle Synihesiss A Pratemica Aμμroach, 1989. I.P.L. Press, 0: fard. See also U.S. Patent No. 5,231,012. Preferably, the proleary or polypeptide is obtained by methyl from recombinant techniques using < * • - gone nucle? T > r? isolate encoding the IL-IO polypeptide, General methods of molecular biology are described, for example, in Sambol et al., Molecular Cloning, A Labora ory Manual, Cold Spring Habor, New Ya l, 2nd. edii.ión, 1989, and by Ausubel el, al., (eds.) Du- i Protico! s m Molecular Biology, S een / kiley, New York (1987 and periodic supplements). The appropriate sequences can be obtained using standard techniques from > Je genomic libraries or c? DN. Polymerase chain reaction (PCP) techniques can be used. See, for example, PCP Protocola: A Rui of Methods and Applications, 1 90, Inms et al., (Fd.), Acaclemic PGP-JS, New Yorl, New York. Libraries are constructed from the nucleus to the: - 1 ration of > - é1 u 3 ^ »appropriate. See, for example, US Patent No. 5,231,012, which presents rec ominant methods for making IL-10. Genetic sequences can be found, for example, in vain databases, of sequences , for example .. RenBant and BMPl or nucleic acid and PIP and Smiss-Prot for protein c / o Intel li ene í, Mountam View, California, or the Reneti s Computer Group, University of Wisconsin Biotechnology Center, Ma Ison, Wisconsin. Clones that understand sequences and encode human data have been deposited with the American Type Culture CoJlection (ATCC), Ro I-i lie, Maryl nd, ba o Numbers of A ce 8191 and o > 8192. l Identification of other clusters that carry the scripts that code IL-10 e leads to abo either by hybrid nucleic acid ion or by immunological detection. ita e »the encoded μroiein, if a vein of espression is emfilea. Probes of o3 i gonuc le? Tidos drooled in the deposited sequences present in North America No. 5,231,012 are espe cially useful. Sequences of probes and oligonucleotides can also be prepared.1 from conserved regions of related genes in other species »Alternately, degenerate probes based on the sequences of a i or t5 can be used. from II- 10. Standard methods can be used to produce procical cell lines, > : mammals, yeast or transformed insects that are prone to large amounts of lipid. Exemplary strains of E. coli suitable for expression and cloning include 3110 (ATCC Bi, 27325), X1776 (ATCC No. 31244). X2282, and PR1 (ATCC Mp / 31343). Exemplary mammalian cell lines include COS-7 cells, mouse L cells and CHP cells. See Sa brook (1989), supra and Ausubel et al., 1987 complements, supra "Can several f vectors be used? It will be reliable to erect the DNA that has been used for the conventional expression of recombinant proteins in proca r i i t i cells. a-, or euraphoty can be used, the preferred vectors include the pcD viewers described by Ok yanta et al., Mol. Cell. Biol. 3:28 (1983); and Tal be et al., Mol. Cell. B? > -.?3. 8: 4 < W > (1908). Other vecto es, depressure mammals based on SV40 include those presented by r'aufman et al., Mol. Cell. Biol. 2: 1304 (1982) and in U.S. Patent No. 4, < f > 75,285"These SV40-based vectors are particularly useful in C0S-7 monkey cells (ATCC No. CRI.36> 51), neither as in >cells.other mammals such as, for example, cells l > ie mouse. See also, Pouielv, et al., (1989 and supplements) Cloning Vectors? A Laboratory Manu l, Elsev? > .-? r, New York. The J I -J can be produced in soluble form such as, for example, in the form of secreted product of transformed or transfected yeast, insect or mammalian cells. The peptides can then be purified by standards known in the art. For example, purification sites can be pre-injected with ammonium sulfate, ion exchange chromatography, gel ion filtering, electrotransformers, affinity chromatography, and the like. See Methods in Enzyme and Purify ica t ion Principies and Practices (Sμpnger-Ver3 g, New York, 1982). Alternately, it can be produced in an insoluble form, as, for example, in the form of aggregates or inclusion bodies, the IL-10 being purified in this way by means of pt c ed i mi ent standards. in the technique. Examples of purification steps include the separation of the inclusion bodies from cell »hosts decompressed by centrifugation, and then solubilization of 3 bodies of i clu in cort caoirope agent and reducing agent such that the peptide It assumes an ionically active conformation. »Specific details on these procedures, see, for example, Wiukler et al., Biochemistry 25: 4041 (1986), Wmkler et al., Technolgy 3: 9923 (1985). ); Koths et al3, and North American Patent No. 4,569,790, The sequences of numbers used to transfect host cells can be modified using standard techniques, to make IL-10 or fragments thereof, with various properties. leseadas. Such IL-10s can vary from sequences that occur naturally at the level of the primary structure, for example, by insertion, substitution, removal and fusion of amino acids. These mod i f i > They can be used in vain co-processes to produce the fixed chain of Ina modified 1. The vanants of the amino acid sequences can be prepared with several objectives in mind, including the increase of the ia. serum media, easier purification or preparation, 13 improvement of therapeutic efficacy, and decrease of severity or occurrence of side effects during therapeutic use. The vanishing of the amino acid sequences are vanishing, they have predetermined that they do not It is encoded in nature, even though others may be post-translational variants 1, for example, generic variants, or proteins conjugated with pol 1 letg 1 col (PEG), for example, such vanantes can > The invention can be used to the extent that it retains the biological activity of 11-10.Movifications of the sequences encoding the polynucleotides can easily be achieved by various techniques, such as employ utagértesis directed by site (Bi 1 Iman et al. Gene 8.81 (1987)). most of the modified ions are 11 evaluated. 'by routine screening in a suitable test for the desired characteristics. For example, U.S. Patent No. 5,231,012 describes several in vitro tests suitable for the measurement of activity of 11-10. Preferably, human TL-10 was used for human treatment, even though viral IL-10 or 11-10 from other mammalian species could possibly be used. With greater preference, the 11-10 employed is human rectal IL-10. The preparation of human and mouse I L-1 is described in US Pat. No. 5,231,012. cloning and expression of viral II.-10 (BCPF1 protein) from Epstein-Barr virus e1 has been reported by Moore et al., Sience 248.1230 (1990). (See also the application) "International patent W0 91/09127 and US Pat. No. 5,368,854." When referring to II-l, -ei cluyen active fragments, analogs and homologs thereof. Active fragments, analogs and homologs of IL-10 include proteins, polypeptides, peptides that possess one or more of the rare, but ter- rible activities of IL-10. Jl? t osi lada or not gl icosi lada.Examples of the 3> 11-10 include the inhibition or substantial reduction of the level of IL-2, lymphotoin, 11-3, or GM-CSF activity The activity of IL-10 also includes the inhibition of the production of cytosomes by acrylated macrophages, for example IL-1, JL- ?, and TNF-alpha. assays to determine the activity of IL-10, see US Patent No. 5,231,012.This patent also provides proteins having an activity II.-10 and the production of such proteins that includes technical and synthetic techniques. To prepare pharmaceutical compositions including polypeptide 11-10, the polypeptide is either chemically acceptable vehicle or preferably inert excipient. A pharmaceutical vehicle can be any suitable non-toning substance suitable for Provide the patient with the appropriate color. the preparation of such pharmaceutical compositions known in the art; see, for example, Pemington's Pha rm ceu t i cal Se i enees, and U.S. Pharmacupe; National Formulary, Mack Publishmg Company, Ea = »ton, PA (1984); Avis et al. í ds.) (1983) Ph rmaceut i ».. a í Dosage For s: Parenteral Med icat inns: De 'l-er, New York; and Lieber an et al., (ecls.) (1990) Pharmaceutical Dosage Forms: Disperse Systems Dekter, New York. Eye preparations, par- ticularly, may use conventional drops for ointment and ointment, or they may employ the use of preservatives, such as, for example, chloride of chloride, as well as the use of preservatives, or EDTA. ) to increase the penetration; molar adhesive polymers including hyaluronic acid to prolong the contact time of the drug; and icropart 1 / nanoparticles and 1 iposo as co or drug delivery particles. Protein absorption enhancers that make the cornea permeable to proteins and small peptides that include but are not limited to a.one, cetpmida,? Tocalas? I3 B, EDTA, taurocolto and taurodesox i col ato can be roughly apticables. (See 1, for example Aldpch Catalog H rtdbook f Fine Chemicals, 1994-95 Edition, Milwaulee, Wisconsin). For general reference materials on eye preparations, reference may be made, for example to Lee, Pharm. Int., 6: 135 (1985); Hech, et al., In Modern Pharmaceutics (Banker and Rhades, eds.) Marcel DekKar, N.Y. (1979); Maurice, Dphtalmol. Clin. 20:21 (1980); Chiou, et al., Pharmacol. Ther., 17: 269 (1982); Lee, et al., Ocui. Ph r ac 1.,: 7 (198); Camber, et a 1., (1 8), Cu r. Eye R s., 8: 563; M sh, et al., E-p. Eye Res., 11: O (1971); Ra selaat, et al, "Curr. Eyf = Res., 7: 947 (1988); Norn, Acta Oph th imol, 42727 (1964); S ig, et al., J. Pha., 5 »- i. , 66: 122 (1977); Md «en, Int. Ofththalmol. Clin. 20:79 (1980); Higuchi, T. Pharm. S i. , 50: 874 (1961); Saett ne, et al., Int. J. Pharm., 51: 203 (1989); Blaug, et al., Am. J. Of. Hosμ. Pharm., 22: 662 and 1 6); I el ler, et al., E¡ < ? Eye Res., 30: 203 (1980); Grass, et al., Invest Ophthal ol. Vis. S i. , 261-10 (1985); Harris, et al., Bi rna ter i a 1, 11: 652 (1990); Deasy; My Croencapsul a t I on and Related Drug Processes, Mar e] D ck r, Nem Yort (1 84); W? Dd »_ > r, et al. (eds.), Methods m Ert ymology, Vol. 112, Acaclemic Press, Orlando, Florida (1985); Donbro », (ed.), Míe ro * apsules and Na ñopa rt íc les m Medic ine and Pharmat y, CRC Press, Boca Pat n, Florida (1992); Lee, et al., Surv. of Ophth 1 mol. , 29: 335 (1985); Sr ka, et al. , Ann. Rev. Biophy. Hioeng , 9: 467 (1981); Smol iu, et al., Am. J. Ophtha 1 mol, 91: 220 (1981); Ah ed, et al. , Invest., Ophthalmol. vis. S i. 26: 584 (1985); Chiou, et al. , J. Pharm. , It i. , 78: 815 (1989); Yamamoto, et al. , T- Phar. Exp. Ther. , 249: 249 (1989); Bentrel, et al. , Am. T. Phystol. 239: C75 (198?); Mar ínez-Palomo, et al. , T. Cell Biol., 87: 736 (1980); Aldpdge, et al., Chem Commun., 1:26 (1967); Rothweiler, et al. , E pepentia, 22:75 (19¿, 6); Bmder, et al. , Aynew. Chem. Ins. Edi. , 12: 370 (1973). It is desirable to treat ocular inflammations locally. This can take the form of the topical administration of II-10. When topically applied II ~ 10, id IL-10 may be in the form of eye drops, ointment, and other formulations which may be used by the patient. vehicles mentioned above, ls concentrations of 11-10 in these goes. River * vehicles may vary from * 1 micrometre to my ba or conditions (eg inflammation) in which VA penetration may occur up to 2.5 mg / ml, the maximum tolerated dose of topical agents »will < ? mea üc nshnamaorathy and Mitra, 1 93, Ocular Dellvery of Peptides and Protems; m: Ophthal ic Drug Dellvery Systems (A. V. Mi tra, ed.), Marcel Del * ler, New York, pt. 455 et seq.), when the epithelial bap-fra is intact. By c * nns? > in a c.ortcent rae ion that varies within a range of 1 i rogra or by me to 2. 5 mg / ml per drop, preferably 10 mu. rogramo ^ by my .25 mg / ml, a drop can be administered to the eye at a regimen that varies from one drop per hour to one drop every two days, from within-g > A range of one drop a day to four yotas a day. Since the half-life of II .-- 10 may be short in the tear film, it may be necessary to administer the drug more frequently than a 2 to 1 d i 3. This can be determined by the clinician in Liase to the condition > Jel particular patient. The formulation of "yola-" can be preparted in accordance with that indicated in the references p to the vehicles n is mentioned. II .-- 10 can be administered in a ptep manner. A pepocular formulation can be prepared by the use of vehicles already employed in intravenous, subcutaneous, and / or tramuscular injection. the preparation of such formulations is well known in the art. See, for example, Gil an, et al. (e s) (1990) Goodman and Oí Im n 'ss The Pba ma o lica 1 Basev »of Therapen t íe, 9n. edition., Pergamon Press; and Reíiii ng ton's Pharmaceut i »; a 1 Sciences, 17ava. etrl i > ion (1992), Mact Publishing Co., Easton, Fenn, L dosification for administration pepoculur is preseren * - ía of approximately c > Approximately 5 mg per year, preferred maximum * approximately 50? t? it rogramos a aptro; imacally 0. m > j p? > day, according to the biocli sponi bility of the vehicle in ec a and the life me of 11-10 prepared in several vehicles. The TI-10 may also be administered in a sub-uu i v l (se) manner or within the cornea. II-10 is effective in reducing ocular inflammation in the mouse when administered in a dose of, micrograms per day for 7 days with treatment starting 5 days after infection. Formulations raise on jun i vales or on the »óv ^ a could use vehicles use > r) os for the topical administration or the employees ptara 3a a > im? neither rae i or in ravenous, i nt ramust ul or subcutaneous. Therefore, the preferred range of do i for administration subcou unt i v «t 1 in humans is aprot; immedi- ately 5 minutes per week: approximately 5 mg per day, with a higher peak of approximately 50 micrograms per minute, and 0. "In the case of administration to the cornea, the preferred dosage will be approximately 0, my ry to approve: 0.5 mg, with a higher preference of oxygenatedly 5 my rograms to approximately 50 my rograms, using formulations 10 intravenous systems. Since the half-life trie 31-10 is short or ivo (estimated at 2.5 hours in the mouse), it may be necessary to administer the drug more frequently than once a day. This can be determined by the doctor based on the condition of the partici pating patient. IT.- 10 may be »> also useful for the intracameral administration of the drug, including administration of the drug in the anteric chamber and also for the treatment of several patients. ia > The infl m orias int raoc ul ar ar destroras. Formulations can be prepared using vehicles already used for intravenous administration (eg, for example).

Remí gntori 's Pharmaceutical Sciences, I7ava. edition, (1992), Mael < Putil t ^ hing Co., Ea ton, Penn. ) either lipasoma, icroesféra or the supply J ontof or t ico trie 11.-10 (see, for example, Schulman and Peyman, 1993, Intracamer 3, Intraví trea 1, and Retina! Drug Deiivery at: Ophthalmi Drug Delivery Systems (A. Mitra, e »rl.) Marcel Detter, New Ytirl, pages 383 and -following). 11-10 may be useful for the treatment of live allografts of the eye on the ocular kidney, by in- troducing, without limiting itself to them, lobes of the cornea, before its surgical placement in the recipient host. Treatment with II.-10 of aloin ertos. ose »: would require dosages ranging from 0. 01 micrograms per ml to 0.1 g / ml, preferably from 0.1 ml / ml to 10 ml / ml. As used herein, the term "therapeutically effective amount" refers to a suf fi cient amount for improving a symptom > : > sign of inflammation »-, ocular ion. Stromal keratitis caused by the foot, the eyelid, the cornea, clouding of the cornea, neovastulap rar ion, inflammation > The rainbow will begin to appear, and precipitate wills. For reasons, an improvement would be recognized by the use of one or several of these clinical signs. Typical mammals that can be treated include pets such as dogs and cats, and pipmates, including human serejs. Preferably, an IL-3 derived from the target of the target animal of the treatment will be used. The effective amount for a particular patient may vary according to factors such as the condition to be treated, the overall health of the patient. lens, the method, route, and administration of the severity of the collateral examples, the determination of the appropriate dose is made? Part of the clinician uses parameters known in the art. Frt general, the d? Js? S em i za with a bit a bit less than the optimal dosi and is increased by half? slight increases later until reaching the desired or optimal effect. (See generally The Mere I, Manual Article 2¿> 9"Pita r ac ot i net i cs and Drug Administrat ion.").

Under specific circumstances, multiple metrics can be administered in combination. For example, II-10 can be administered in combination with a therapeutically effective dose to one > Therapeutic agents are additionally added. The tonal agent can be a non-infectious agent (for example, tnflundin or 3"-? Clov? R) or an anti-inflammatory agent" -tero "of (for example, acetate). redinsolenta or f luorct et.t3 on) or non-steroidal anti-inflammatory agent (eg, occufen, i nd vi na, or bi nc ic 1 ospor in). 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, II was tested. -10 to determine whether the disease could • suppress the development of et al, romal disease. Two studies give 1 * suppression by II. -10 of the antigen expression HLA-DP (ff u a tein, et al., Ophthalmology 1994; 1 Ol s 1529-1 35) and production of chemo lithia > ie 1 euc ot: i to (Boor- = »tin, et al., Invest. Oμhthal ul Vi-» Si. i. 36 (4): F, 147; 1995) in human corneas affected by stromal keratitis caused by herpes I was praying It-10 human rero bante in 1 units for me, ex vivo. Both of these articles (Boorstein, et al.

Ophthal ology j 101: 1529-1 35, 1994 and Paorstein et al., Invest. Ophthalmol Vis 8c i, 36 (4): S147, 1 95) are hereby expressly incorporated by reference in their entirety. In addition, in a large animal study, rmlL-10 was inoculated into the cornea of mice 4 hours earlier and another time. days +2 and -1-5 in relation to the time of topical inoculation of HSV-1 in the cornea »Ad i c i ona] ly, mice received IL-IO t" p "at the time of admission! st, ra ion of V L? i and again 3 days after the infection. Four weeks after the infection, the incidence of the disease "ie blindness was 95 * / * in the counterparts or they received a saline solution but only 36 * in the treated animals. : > m II. -IO. Histological studies showed extensive cellular infiltrates in the < z of the control animals but not in the CIJOS treated with II -IO, examination of the levels of > - 1 Loi ña piroi nfl lovemaking in the cornea 10 i-jías after the infection revealed that the presence of IL-2 > When I was 10 times smaller and I suffered from II .-- 10 times it was immeasurably smaller than what was found in the control animals, the levels of Il-lalfa were not reduced. The treatment protocol with II -10 empileatki does not rhyme the humoral immune responses or > elular »= s sistém i to autlgenos saw the» 5 »*», iM the regimen of purification of H V-l of the eye differently observed in the * cintróles. In in vitro studies = > e observed »: < that the spontaneous production of ti.- 10 by corneas normally removed was inhibited in more > .! l 5% with a low dose of II. -1 . Mo synthesis of II -alpha was inhibited. > In a collective way, these results indicate that the treatment with II. -JO was 1) to suppress the production of certain cells produced by corneal cells, and 2) to minimize the inflammation of the kidney without interfering with the depletion of the infectious virus. rial is and M tidos Infection p > ? vi vi us - HS5V- 1 d PE, a tcmoci inducer of joke keratitis! dausch, et al., Curr. Eye Res. 8.499, 1989), it was used to initiate the infection. S * = > cul ined the virus and stained it in Vero cells > according to what has been previously described (l usch, et al., Curr. Eye s. 8s499, 1989). BALB / c ratios in four-week-old females (Charle *; Piver Breedmg t abara tor, Wilmington, MA) were measured with 1.0 mg of sodium barbitol in 0.2 ml of a standard phosphate-buffered saline solution. ip The right o was slightly scarified by three twists of a fine tre at the cornea of 2 mm. B * $ then placed in drops a volume of 2 μl which contained d & 1 to 5 x 10,000 units of plaque-forming viruses on the surface of the body and then penetrated by massage using the eyelids, the ÜJUS were examined week Try with a dissecting disc. Be evaluated the corneal opacity in a line from 0 to +5 in accordance with that described elsewhere (Metcal f, et al., Curr. Eye Pes. 6: 173, 1987); a rating of "clear" indicates, while a result of +5 represents a necrotic stromal keratitis. "The eyes were examined in a codi fi ed manner with the uninformed reader of the given treatment. were evaluated, by using the U test of Ma np Whi tney. (Snedecor, GW and Cochran, WG, Stat it? ta 1 Method.5, 6th edition, Itiwa State Uni versi ty Press, Ames, lowa, 19675 pages 130-131.) Rea ives - Obtaining IL-IO rec omnin g of upna írm- TL- 10) t t DIMAX Research T ost i tute, Palo Alto, CA "The content in end» itox ' i U3 e the preparation 'd 11 -10 was -0.1 ng / itg The biological activity of this stage *: ina was 7 H / μl sec not measured in MC / 9 cells of mice (Moore, et al. , Science 748: 1230, 1990), the concentration of IL-10 was determined by the use of a tit for ELISA (Endogen Tn > - "Boston MA.) The hybrid to TERS-2A5.I1 which produces an antic It monf? clortal de It -10 rat anti-rat ion was obtained at American Type Culture Ccillection (ATCCj Roctville, MD). A monoclonal antibody was acquired in a 1 1 neutral 1 11th 11 - 11. mouse anti-mouse moitoc luna 1 in Gen / ryme Corporation (Cambridge, MA). Injections in the cornea - Injections will be carried out on the cornea according to what has been previously described (Hendpcl-s, et al., Invest. Ophtha 1 mol., Vis., I, 37: 366, 1991). Briefly, a disposable needle of no. 30 was used to perforate the epithelial wall of the cornea. A stainless steel needle of 3 cm, no. 32, fixed on a supply Hamil on (Ha i 1 ton, Reno, NV) was then colored in the stroma and injected t μl of rmll ..- 10 or saline in • ?! center of the cornea. Rehearsal »of your persensibi 1? >Delayed type (DTH) - The DTH response in infected mice was determined by the use of the ear swelling test. The Ag de pirueba, HSV-1 (RF), was diluted in a serum-free 1% or 1% PPMI. The virus preparation was then exposed to UV radiation during the minutes. This reduced the infection capacity from 1,000,000 to less than 1.00 plate / 10 μ3 plate units. To P? or the DTH response, 1 μl of viral antigen was inoculated on the dorsal side > I heard right mouse by using a Hami 1 ton 50 μl syringe and a needle of size no. 30 6 days after the infection. The left ear (control) received Iμl trie RPMI 164 with VA of newborn calf serum. Ear swelling was measured 74 hours later by using a Mi tutoyo 7326 icometer (Schlessi nger Tools, New Yort, NY). The results are expressed as ear swelling of the right ear (antigen) minus ear swelling of the left ear (control) in units of 1 / 10,000 inches. To determine if 11-10 suppressed the DTH response, the ears *? ~ 7 Experimental rights received 5 μl of UV-HSV-i (RE) mixed n 5 μl containing 28 ng of IL-10 just before the challenge in the ear, the controls received a single solution instead of 11.-10 . In some experiments IL-10 (110 ng) was incubated with ββ neutralizing 11-10 anti-mouse monoclonal rat (final concentration of 10 μg / l), or bi n IgG control, for 30 minutes on ice before of the inoculation of the ear, the data were evaluated by using f] ^), a. Student t test. Ovarian tissue testing and HSV-1 rtfet ion rs - To test the effect of urine II .-- 10 treatment on the replication of HSV-1 in the cornea, or * .tt »individual integers were removed and placed > . = »N 600 μl of FBS W in DMEM medium with amino acids. The preparations were frozen at a temperature of -70 ° C, declined in the homogenate and homogenized in a ten-year-old hobgenerator (Blic, Vi el and, NH). the homogenates were congel atrios, ascongel attached again, and toned for 15 s with a Sonic 300 Dismembra or (Artel Systems Corporation, Farmingdale, NY). I > The supernatants were then typed for infectious virus in Vero cell monolayers in an AB plate test and hours. Quantification of cytokine - To test the effect of IL-10 on the production of 11-6, IL-2 and II.-lalfa in vivo, corneas were removed from mice infected with control HSV-1 that received a lina solution and other treated with 11-10 IO days after infection. The corneas were cut to 2 mm with the use of a t re ma dt »ierodisection (Roboz Surgical Irtstrument Co., Roctville, MD), and placed» in i idu lly in 600 RPMI of serum-free RPMI 1640 with a solution antifungal Fungi-Bact. The samples were stored at a temperature of -70 ° C until the test. the samples were thawed, zoned for 30 seconds, and clarified by centrifugation at 10 xg for 10 minutes. The 1 cleared cellular isolates were assayed for TI-1, II -2, and IL-6 with the use of ki s The ISA. l s I- its 11-6 (assay sensitivity 15 pg / ml) and II-2 (assay sensitivity 3 pg / ml) were acquired in En > i >;? > rjen, I n »rr. The t-11 of IL-lalfa (sensation 3 test capacity 15 pg / ml) were acquired in Genzyme. The spontaneous synthesis of removed normal corneal tissue was determined according to the third method described (Staats, et al., Imolol 151: 277, 1993). In short, cornea buttons were removed from untreated mice and trimmed to 2mm. The tiulare * preparations were free of limbal vasculature and conformed with that judged by mit splicing. Each sample consisted of three botone *, cornea incubated in the absence or presence of 11-10 in 500 μl of PPMI 1640 (Life Tet hnologi, Inc., Ga i thersburg, MD) with a solution ant, i bi ót i * - a de Fungí -Pac ta a temperature of 37 ° C and 5V < »Ie Co2. After a 12-hour incubation pierce, the supernatants were assayed for II -6. To «detect TL-lalfa, the cornea samples were incubated for 6 hours., Despules l i corneas were soc n} The strips were stripped through 30 seconds with a Sonic 300 dismembran. The lysates were laughed, centrifuged 150 x g for 10 minutes before the test. Hi stol test > rji > o - Eyes and nf ecr tates were enucleated, fixed in the Formal regulated neutral (equivalent to 4% of a solution of neutral regulated pattern), colored n paraf? rt3, and multiple sections of 5 microns were prepared. The sections were stained on hema tox 11? Na ~ eo? na, mounted ean r sma, and placed piara f otrtiti i > "rose op 1 a .. Resul ers 1. Locally provided II.-1 treatment suppresses the DTH response in sensitized hosts Initial experiments were designed to investigate whether a treatment with rmll.-10 could suppress Inflammation mediated by immune cells induced locally., t? They used the choices of these cyclines on the ability of DTH response in mice sensitized to HSV-1. Patons were immunized by topical infection in the scarified cornea with 10,000 units of plaque-forming HSV-1 (RE). Six days later, the animals responded strongly to a viral antigen in DTH tests (Figure 1). However, the injection of rmIL-10 with the test antigen resulted in a reduced ignificative ear swelling (FIG. 1A). The repeated treatment with the cytodite, is to go, at the time of the antigen challenge, Je HSV-í and another one? 1 hour • then (55 n), increased the suppressive effect. This suspension could be reversed by pre-incubation of IL-10 with specialized monoclonal antiviral agents before the 3rd intradermal inoculation of the ear (FIG. IB), speci? Cally the suppression of 31-10 in the response of DTH to the HSV-1 antigen. In Figure 1A, 11-10 (28 ng) was injected into the ear with the rest of antigen i to 3 (IX). 12 hor-J was provided after a second injection of 11-10 (55 n) (2X). E \) Figure IB, II -ÍO was incubated cron mAAB ant? -IL-1 or »on control Ig8 before its i &ula >At the DTH test site, if the patient had a viral antigen and 12 hours later, the urine antigen was detected, the swelling of the ear was measured 24 hours later, the rest with antigen. in native mice was 13 1/3 and 1 / 10,000 inches, the groups were three to 10 mice, 2. The treatment c * rm IL-10 suppresses the development of estuarine keratitis]. by HSV-l (LISIO) The above results indicate that rmIL-10 provided to previously sensitized hosts could down-regulate the inflammatory response mediated by T-cells to HSV-1 antigen, so we raised the possibility that the Also with IL-10 could also suppress the development of stromal keratitis provoked by herpes, a disease that is considered to be mediated at least partially by T cells. , al., Infei t, Im, 26: 1164, 1979) (Russel 1, et al., Invest. Ophth l mol. Vis. S *. i. 25.93R, 1994); (Newell, et al., T. V i ro 1. 63 ü 769, 1 S9); (IMewe11, I., Reg. Immuno., 2; 366, 1909); (Hendr Ics, et al., Invest. Opththa Imol. Vi • = "» Se? "31.1929, 1990) 5 (Doyuw,, et al., Invest. Ophthalmol., Vis S &.t.?: 33: 2165 , 1992); (Ni emi a 1 towsl-i, al., T. Tmmuntl, 149: 3035, 1992). rmTL-10 inoculated directly to the day -1, +1, -4 and +7 in reti i on cti the infection decreased, but did not prevent the development of keratitis and thrombus caused by herpes. When IL-10 was administered if reasonably as t as 1 u > Finally, a remarkable suptresis was achieved, which is the result of a tpuco experiment. The results of the corneal media were negligibly reduced (p> -0.05) in relation to what was observed in the treated control group > in a saline solution at all time points after infection, except in the case At the end of the experiment (day 20), the eyes were removed for inspection by the eye. Figure 3A shows a section of a representative control cornea showing the histological appearance of the stem > ie a stromal keratitis caused by herpes ié mu ina experienced (Wang, et al, Curr. Eye Res. 8:37 (1989).) The cornea was very swollen, and contained a strong infiltrate. Numerous blood vessels were present in the erythromyoma, and an evident epithelial ulceration was observed in the cornea. Figure 3E < Many of the infected corneas protected by treatment with rmll-10 were not swollen, had no epithelial ulcer, had very few infiltrating cells and flamatori s, and had only one neuvasular illoc io tie level. The a > * IL-IO piro reading ion f_ r f i rmad i in the adi * onal experiments. Col * ti it was found that even when the 95 * / (20/21) ci the > > - > Because of the disease and blindness, only 3 * 4 of the animals treated with ci oe ina (8/22) had this disease. The treatment > Orí ÍL-10 did not prevent blepharitis, which is frequently observed afterwards, from an infection of the cornea with HS-1 in mice (Lausch, et al., Inter i rol ogy 31: 159 (1990), in the infectious doses of viruses used (1 to r- "> 10,000 pest-forming units), HSV-l strain RF is sometimes expirterte desce the oo to the nervous system crentr induces a deadly disease The incidence of encephalitis observed in the r-et i bridges of IR-10 (3 / 32.9 * 4) is similar to that observed in the control animals (4 / 32,12.5%). Therefore, the cytokine do ifi used in our experiments It seems that the susceptibility of the host to central nervous system disease has increased the suppressive effector effects on various phases of the immune response, including the inhibition of the immune response. T cells. It was possible that an information redefined from the formula could be the result of a reduction in the generation of ion of T cells sensitized to viral antigen »ie] herpes. To assess the effect that IL-IO has on cell-mediated immunity, a DTH test was carried out in control mice and mice treated with c i to t n is used after infection. The data in Figure 4 are representative of three experiments of this type. We found that treatment with IL-10 protective ep ad the day of infection il did not inhibit the generation of T cells ac iv-is in DTH responses to HSV-l antigens in an assay of ear swelling, neutral titers Before sera collected 4 weeks after infection, part of the 11.10-treated hosts were analogous to those in the control animals (data not shown). Therefore, there is no evidence that the treatment with II-OO suppressed (or increased) the development of cellular immune responses or your 1 1 is aati ene- »HSV - 1. 3. Effect of IL-1 treatment on The replication of the virus in the eye It has been shown that several substances exert anti-viral effects in ocular tissue (Chen, et al., Antiviral Res. 22:15, 1993). Therefore, the possibility arises that treatment with rmIL-10 initiated after infection with HSV-1 could reduce the inflammation in the cornea di ect or in irectamen by inhibiting the viral re l ication in the cornea . To test this hypothesis, the amount of infectious virus recovered from the control mice and mice treated with TL-10 was blasted for a period of time. Figure 5 illustrates that for each period of time examined, the viral titers in the eyes of the treated animals were comparable to the titrations observed in the control animals. These results indicate that the treatment * on II .-- 10 did not accelerate (or retard) the renal clearance of the eye. 4, Effect of IL-10 on the synthesis of IL-lalfa, 11-2 and IL-6 in the cornea It is shown that infections with HSV-1 of the cornea of murirta Z J they are characterized by high levels of IL-6 and IL-lalfa (ñtaats, et al., J "Immunol., 351s277, 1993). Other researchers have reported that II.- 10 pt * edt? suppress the synthesis of prophylactic proteins produced by T cells (De Waal, et al., 7. Immuno., 150: 4754, 1993), leukocytes polymorphs (Cassella, et al. ., J. Exp. Med. 178: 2207, 1993), and onoitos / mar rófgos (Florentino, et al., J. Irtol., 147: 3815 (1991); Bogdam. Et al., T. E pi, Med. 174: 1549 (1991), de Waal, et al., Eip. Med. 174: 1209 (1991)). In order to test whether the traffic in the IL-10 altered the HSV-1-induced keratitis in the keratitis, individual corneas were removed from animals treated with II. -10 and animals > control 10 days after the infi tion and assays were carried out.-, for IL-lalfa, 11.-2, and IL-6 by Fl ISA. The data from the experiments were comptarab 1s and the combined results are illustrated in Figure 6. the levels of II -alpha in the treated animals were not if differently different * »(p - 0.7) from the levels observed trie controls. However, levels of IL-2 and II-6 were very reducible (p? ^ 0.05) in those treated with II. -10. Specifically, only 1 out of 10 corneal samples had a detectable level of TI -2 and only 2 out of 10 had a positive level of 1L - 6. In contrast, 70 * / of the controls were positive for IL-2 and SO * / »had high levels of 11-6. 56 . Effect of II-10 on the spontaneous synthesis of 11-6 and II. -l lfa by mouse cornea buttons removed Because it is known that the origin of IL-10 binding on bone marrow leukocytes results in selective inhibition > ie the synthesis of cytopenia, it was possible that II-10 could inhibit laminen cytokine production by resident corneal cells. It was possible to test this hypothesis because it had previously been shown that normal cornea buttons removed incubated in vi tro spontaneously synthesize IL-lalfa and TI -6 (Staas, et al., T. Immunol. 151: 277, 1993), therefore buttons of cornea removed from uninfected donors were incubated in vitro in the presence or absence of different concentrations of rmIL-10. Membrane supernatants and corneal tissue lysates were subsequently obtained and tested for II. -6 and II -alpha, respectively. the results of three experiments, summarized in figure 7, illustrate that IL-10 in a concentration of 1.5 ug / ml could suppress the synthesis of IL-6 in v 95 * /. Interestingly, the dose of 150 ng / ml was less suppressive. The reason for this situation is not clear, but a pattern of two i -result was observed in all three experiments. In contrast, the synthesis of Illa! Fa was not inhibited by IL-IO in a dose range of 1000 e. Many modifications and variations to this invention will be apparent to those skilled in the art, the specific modalities described herein are offered only by way of example, and the invention is not limited thereto.

Claims (9)

1. B REIVI DICATIONS 1. A method for treating an ocular inflammatory condition in a mammal, comprising the administration of a therapeutically effective amount of mterl euc. ? na-10 to an A > ^ inside > laugh > the ocular region of said mammal.
2. 2. The method of claim 1, wherein the area within the ocular region yields the cornea, sclera, choroid, ciliary body, iris, retina, conjunctiva, orbital tissues, eyelids, nasol agrima drainage apparatus. 1, and optic nerve.
3. 3, The method of claim *, ton 1, where the ocular inflammatory condition is stromal keratitis. .
4. The method of rei indication 3, where stromal keretitis is a stromal keratitis induced pior virus Herpes Si r i 1 «
5. 5. The method of claim 4, wherein the stromal keratitis e * 3 a (judge L 1 is e tromam induced by the i Herpes C i mpt l ex 11 po 1.
6. 6"The method of claim 1, wherein the i nterl eu > . i n-lO is selected within the group consisting ele intern! euc i na- 1 viral and i nter 1 euc i na - 10 human.
7. 7. The re vindication method 1, which further comprises the administration of a therapeutically effective dose of a second, physically active agent,
8. 8, The method of tivi ndi ón 7, where the second agent therapeutically Active is an agent anl i -infere i oso.
9. 9. The method of claim 8, wherein the apti-infant agent is selected within the group consisting of flupdine and i-iclovir, 10. The method of claim 9, wherein the ci-autogenic agent e- =. The method of claim 7, wherein the second active therapeutic agent is an anti-i nf 1 a atop steroid or nu steroid agent. 12. The method of re-indication 7, where the second agent that is therapeutically active is an anti-infi mary people. 13. The method of claim 12, wherein the agent anfci-i flam orio estero.de is predmsol acetate, phosphate ptredm sol ona, dexametarrona or f luorometolone. 14. The method of claim 13, wherein the apty-inflammatory agent is tilde is prefinite acetate at 1 * / .. 15, The method of claim 7, wherein the second therapeutically active agent is a non-steroidal or anti-m-lam-ton agent. 16. The method of claim 15, wherein the anti-inf lamatop et non-estercude agent is ocuferto, i ndoe m, ocic 1 ospi ri, 17. The method of claim 1, wherein the inte rr 10 is administered only. 18. The method of claim 17, wherein the iftlerleue ma-10 is administered in the form of pi ra-lo drops, and the concentration of interleukin-1 per drop is within a range of approximately 1 microgram. by me at approximately 2.5 mg / m. 19. The method of the intermediate concentration of 10 per drop is within a range of approximately 10 micrograms per ml to approximately .25 mg per ml of the rejection method 18, where the Interleukin-10 is administered to the eye in a regimen that is located within a drop of one hour to one drop every two days. 21. The method of the rei indication 20, where the metal-lO < =, e administers to the o or in a regime that is located from a drop pior day up to 4 drops per day. 22. The method of claim 1, where the uterus is administered either as a sub-contract or as a collateral. 23. The method of claim 22, wherein the methyl ester-10 is administered at a dose of approximately 5 fiticrogmos a aproadamente 5 mg pior day. 24. Er 3 method of claim 23, wherein the dosage amount ev, from about 5 micrograms to 0.5 mg per day and 0.5 mg per day. 25. The method of claim t, wherein the ether 3 euc i fta- Al 10 is encapsulated in a liposame. 26. E3 method of rei indication 1, on the uterus 1 euc ina-l -n found encapsulated in a my eracula. 27. A pharmacological composition for the treatment of ocular inflammation, which includes inter-le? e i na -10 »28. A solution f? can? *, e? t ica. Topical for the treatment of ocular inflammation that includes inter- uci-10. 29. the pharmaceutical solution of the re ject ion 28, where the solution is in the form of drops for the children. 7.0. To the pharmaceutical solution of claim 28, give the concentration of leucine-10 per drop is within a range of about 1 microgram per ml to about 2.5 mg / ml. 31. The pharmaceutical composition of rei indi 27, where the composition is to be supplied to a pt? Put region.? lar of the eye. 32, the pharmacological composition of rei indication 27, wherein the composition is for delivery to an intracameral eye region 3/3 The pharmaceutical composition of claim 32, wherein the composition is to add to my previous caima of the eye 34. The pharmaceutical composition of the rei indication 32, where the ir i t ion is to be supplied to a region of the o r.