WO2009132172A1

WO2009132172A1 - Isoxazoline compounds and their use to suppress inflammation or treat inflammatory disease

Info

Publication number: WO2009132172A1
Application number: PCT/US2009/041514
Authority: WO
Inventors: Ferdinando Nicoletti; Niranjan Sardesai; Jong Joseph Kim
Original assignee: Vgx Pharmaceuticals, Inc.
Priority date: 2008-04-23
Filing date: 2009-04-23
Publication date: 2009-10-29

Abstract

A class of isoxazoline compounds including VGX-1027 and the use of same to reduce inflammation in the eye or to treat inflammatory eye disease, such as uveitis.

Description

ISOXAZOLINE COMPOUNDS AND THEIR USE TO SUPPRESS INFLAMMATION OR TREAT INFLAMMATORY DISEASE

FIELD OF THE INVENTION

[0001] The present invention relates to a class of isoxazoline compounds and the use of same to suppress inflammation in the eye or to treat inflammatory disease therein, such as inflammatory eye diseases like uveitis.

BACKGROUND

[0002] Autoimmune diseases are immunomediated diseases that can be defined according to the organ that is attacked, according the attacking mechanism, whether mediated by autoantibody or T-cells, and the development of chronic inflammation processes often mediated by cytokines. Increasing evidence suggests an important pathogenic contribution of autoimmune phenomena to atherosclerosis, psychiatric disease schizophrenia, epilepsy, baldness, peptic ulcer disease, and others.

[0003] Autoimmune diseases can affect every organ or tissue. There are autoimmune diseases that affect the nervous system, for example, multiple sclerosis, which is a severe disease damages the brain and causes paralysis. Other autoimmune diseases that affect the nervous system are myasthenia gravis and Guillain-Barre syndrome. Myasthenia gravis is a disease characterized by extreme muscle weakness in which receptors that transmit electric impulse from the nerve to the muscle are destroyed. Without the receptors, there is no muscle contraction and therefore muscle weakness develops. Guillain-Barre syndrome may develop after an infection or vaccination.

[0004] There are autoimmune diseases that affect the joints, such as lupus and rheumatoid arthritis that causes deformities to the joints. Finally, there are autoimmune diseases that damage the heart, kidneys, lungs, and eyes.

[0005] A better understanding of immunopathogenic pathways involved in the onset of autoimmune diseases, chronic inflammations or other immunomediated diseases, have allowed identification of what are thought to be key players or modulators of T-cell mediated autoimmune diseases such as cytokines including TNF-α, TNF-beta, IL-I beta, IL- 12, IL- 18 and IFN-gamma. In particular, studies suggesting the key pathogenic role of TNF-α in the pathogenesis of rheumatoid arthritis have translated to the clinical stage in the form of specific inhibitors, such as the neutralizing monoclonal antibody Infliximab (Humana) and the TNF receptor fusion protein Enbrel, which are currently approved for the treatment of RA patients.

[0006] Regarding inflammation in the eye, studies have shown an increased expression of ICAM-I has been observed both in the iris of patients with uveitis (La Heij, 1998) and in vascular endothelium of the rat ciliary body during EIU (Withcup et al., 1993; Withcup et al., 1998). P-selectin is also expressed in the vessels of the iris of rats with endotoxin induced uveitis (EIU) (Suzuma et al., 1997). The involvement of both P- selectin- and ICAM-I -mediated pathways in the pathogenesis of EIU is proven by the ability to block the process by monoclonal antibodies directed against these adhesion molecules (Suzuma et all 997). TNF-α is thought to induce ICAM-I and selectins as well as by contributing to leukocyte adhesion, vascular leakage, and apoptotic cell death in EIU (Kaizumi et al.,2003).

[0007] Furthermore, studies have shown that LPS-induced EIU progresses in part due to activation of NF-κB, which leads to local up-regulated production of TNF-α and subsequent activation of the iNOS-NO pathways. TNF-α is implicated in the NO synthesis pathway in that the induction of iNOS is mediated by the release of endogenous TNF-α It has further been discussed that epithelial cells of the iris ciliary body and cells infiltrating the anterior segment of the eye during EIU are a major source of NO (Goureau et al, 1995; Jacquemin et al, 1996; Mandai et al, 1996; Szabo et al, 1996). It has recently been shown that specific blockade of NF-κB, TNF-α and NO exerts beneficial effects on the course of EIU (Kitamei , 2006; Suzuki et al., 2006; Avunduck et al., 2004; Goureau et al., 1995).

[0008] Double blind clinical studies have proven that the neutralization of TNF can abrogate the early stages of inflammation. Specific inhibitors of TNF-α, in contrast to acetylsalicylic acid which only reduces inflammation, have been presented as a preventive solution to inflammation. Treatment with an anti-TNF alpha drug is now utilized in other autoimmune diseases including Crohn's disease and psoriasis. However, the production of the specific TNF-inhibitors is complex and expensive. Moreover, anti- TNF inhibitors can be only given parenterally and their chronic use may involve a greater risk of developing negative side-effects, including the incidence of tuberculosis.

[0009] Thus, there remains a need for new compounds, and treatments using same, that are effective for treating or eliminating symptoms of inflammatory disease, in particular, uveitis. Further, there is a need for an orally available small compound that inhibits the synthesis and/or the action of endogenous TNF and possibly other cytokines that include IFN-gamma, IL-I, IL- 12,IL- 18 or other cytokines.

SUMMARY OF THE INVENTION

[0010] An aspect of the present invention relates to methods of modulating an immune response in an eye of a mammal by administering to said mammal a pharmaceutical formulation comprising a compound of formula (I):

I.

[0011] Ri _5 represents from one to five substituents independently selected from the group consisting of hydrogen, nitro, cyano, Ci-C₃-alkyl, halogen, carboxy, amino, trifluoromethyl, hydroxy, and Ci-C3-alkoxy, provided when Ri_5 is a hydroxy or alkoxy substituent, said hydroxyl or said alkoxy substituents are ortho or meta in relation to an isoxazole moiety;

[0012] X is a hydrogen, halogen, N₃, SH, =0, =CH₂, an aromatic ring, optionally substituted by substituents selected from the group consisting of hydrogen, nitro, cyano, Ci-C3-alkyl, halogen, carboxy, amino, trifluoromethyl, hydroxy, and Ci-C3-alkoxy, or amino, mono- or disubstituted amino groups, wherein the substituents are selected from C₁-C₄ alkyl, phenyl or benzyl groups optionally substituted by substituents selected from the group consisting of hydrogen, nitro, cyano, Ci-C₃-alkyl, halogen, carboxy, amino, trifluoromethyl, hydroxy, and Ci-C3-alkoxy; and

[0013] Y is hydrogen, C₁-C₄ alkyl, amino, or a group of formula -(CH₂)_0-1A, wherein A is an aromatic ring optionally substituted by substituents selected from the group consisting of hydrogen, nitro, cyano, Ci-C₃-alkyl, halogen, carboxy, amino, trifluoromethyl, hydroxy, and Ci-C3-alkoxy.

[0014] In another aspect of the present invention, there are methods of alleviating symptoms of uveitis in a mammal comprising the steps of identifying a mammal exhibiting uveitis or pathological events leading to uveitis; and administering to said mammal a pharmaceutical formulation comprising a compound of formula (I) as provided herein.

BRIEF DESCRIPTION OF THE DRAWINGS

[0015] The numerous objects and advantages of the present invention may be better understood by those skilled in the art by reference to the accompanying figures, in which:

Fig. 1 displays a bar graph showing the effect of VGX- 1027 on clinical EIU, evaluated 16 hours after LPS injection.

Figs. 2A-2C displays bar graphs showing the effect of VGX- 1027 on the following levels 16 hours after LPS injection: A) protein; B) TNF-α; and C) NO. Fig. 3 displays a bar graph showing the effect of VGX- 1027 on cellular infiltration in the iris ciliary body 16 hours after LPS injection.

Figs. 4 A and 4B display bar graphs showing the effect of VGX- 1027 on A) NFKB nuclear translocation and B) p65 nuclear/cytoplasm ratio on ocular homogenates 16 hours after LPS injection.

Figs. 5A-5C display photographs showing the histological appearance of the iris ciliary body 16 hours after LPS injection: A) vehicle; B) VGX- 1027; and C) sham.

Fig. 6 displays a series of photographs showing immunohistochemical localization of P-selectin, ICAM, and iNOS as a result of one of the following samples: sham, vehicle and VGX- 1027.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

[0016] The pharmaceutical compositions according to the present invention are formulated according to the mode of administration to be used. In cases where pharmaceutical compositions are injectable pharmaceutical compositions, they are sterile, pyrogen free and particulate free. An isotonic formulation is preferably used. Generally, additives for isotonicity can include sodium chloride, dextrose, mannitol, sorbitol and lactose. In some cases, isotonic solutions such as phosphate buffered saline are preferred. Stabilizers include gelatin and albumin. In some embodiments, a vasoconstriction agent is added to the formulation. In some embodiments, a stabilizing agent that allows the formulation to be stable at room or ambient temperature for extended periods of time is added to the formulation. Isoxazoline Compounds

[0017] Compounds of formula I, which are absent a hydroxyl or alkoxy group in the para-position of the phenyl ring:

I. wherein Ri .5 represents from one to five substituents independently selected from the group consisting of hydrogen, nitro, cyano, Ci-C₃-alkyl, halogen, carboxy, amino, trifluoromethyl, hydroxy, and Ci-C3-alkoxy, provided when Ri_5 is a hydroxy or alkoxy substituent, said hydroxyl or said alkoxy substituents are ortho or meta in relation to an isoxazole moiety;

[0018] X is a hydrogen, halogen, N₃, SH, =0, =CH₂, an aromatic ring, optionally substituted by substituents selected from the group consisting of hydrogen, nitro, cyano, Ci-C3-alkyl, halogen, carboxy, amino, trifluoromethyl, hydroxy, and Ci-C3-alkoxy, or amino, mono- or disubstituted amino groups, wherein the substituents are selected from C₁-C₄ alkyl, phenyl or benzyl groups optionally substituted by substituents selected from the group consisting of hydrogen, nitro, cyano, Ci-C₃-alkyl, halogen, carboxy, amino, trifluoromethyl, hydroxy, and Ci-C3-alkoxy; and

[0019] Y is hydrogen, C₁-C₄ alkyl, amino, or a group of formula -(CH₂)_0-1A, wherein A is an aromatic ring optionally substituted by substituents selected from the group consisting of hydrogen, nitro, cyano, Ci-C₃-alkyl, halogen, carboxy, amino, trifluoromethyl, hydroxy, and Ci-C3-alkoxy.

[0020] In another aspect of the present invention, there are methods of alleviating symptoms of uveitis in a mammal comprising the steps of identifying a mammal exhibiting uveitis or pathological events leading to uveitis; and administering to said mammal a pharmaceutical formulation comprising a compound of formula (I) as provided herein, the identifying step comprises: In some embodiments, the identifying step comprises detecting an elevated level of TNF-alpha, NF-kappaB p65, or nitrous oxide (NO), or a combination thereof in aqueous humor in an eye of the mammal.

[0021] A number of the compounds of formula I are known or can be prepared by known methods, as available to one of ordinary skill. For example, for the compounds having the formula according to Formula I wherein Rand R' are both hydrogen and the central ring has a 4,5-dihydro isoxazoline ring system, such as that described in Synth. Comm., 1998,28(13), 2457-2466 and from Synth. Comm., 1997, 27(16), 2733-2742. Preferably, the compounds of formula I are compounds having X selected as hydrogen or Y selected as hydrogen, or both. Preferred compounds of formula I are 3-phenyl-4,5- dihydro-5-isoxazoleacetic acid (hereinafter referred to as VGX- 1027) and 3-phenyl-5- isoxazoleacetic acid, as well as their pharmaceutically acceptable salts, such as sodium, potassium, calcium salts and the like.

[0022] The pharmaceutical compositions presently disclosed are intended for parenteral, topical, oral, intranasal or local administration. In some examples, the pharmaceutical compositions are more effective by administering intravenously, subcutaneously, intradermally, intraperitoneally, intranasally or intramuscularly. In some embodiments, compositions are administered via parenteral administration, which comprise a formulation of the provided compounds described dissolved or suspended in an acceptable carrier, preferably an aqueous carrier. A variety of pharmaceutically acceptable aqueous carriers may be used, e.g., water, buffered water, 0.4% saline, 0.3% glycine hyaluronic acid and known, similar functioning carriers. These compositions may be sterilized by conventional, well known sterilization techniques, or may be sterile filtered. The resulting aqueous solutions may be packaged for use as is, or lyophilized, the lyophilized preparation being combined with a sterile solution prior to administration. The compositions may contain pharmaceutically acceptable carriers, which are substances that help approximate physiological conditions, such as those that are pH adjusting and buffering agents, tonicity adjusting agents, wetting agents and the like, e.g., sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, and etc.

[0023] For solid compositions, conventional nontoxic pharmaceutically acceptable carriers may be used, which include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and known, similar functioning carriers. For oral administration, a pharmaceutically acceptable nontoxic composition is formed by incorporating any of the normally employed excipients, such as those carriers previously listed, and generally 10-95% of active ingredient, or more preferably at a concentration of 25%-75%.

[0024] For aerosol administration, the disclosed compound containing compositions are preferably supplied in finely divided form along with a surfactant and propellant as pharmaceutically acceptable carriers. The surfactant must, of course, be nontoxic, and preferably soluble in the propellant. Representative of such agents are the esters or partial esters of fatty acids containing from 6 to 22 carbon atoms, such as caproic, octanoic, lauric, palmitic, stearic, linoleic, linolenic, olesteric and oleic acids with an aliphatic polyhydric alcohol or its cyclic anhydride. Mixed esters, such as mixed or natural glycerides, may be employed. A carrier can also be included, as desired, as with, e.g., lecithin for intranasal delivery.

[0025] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned hereunder are incorporated herein by reference. Unless mentioned otherwise, the techniques employed or contemplated herein are standard methodologies well known to one of ordinary skill in the art. The materials, methods and examples are illustrative only and not limiting.

[0026] More preferred, a compound useful for the uses and methods described herein is VGX- 1027.

[0027] Preparation of VGX- 1027 (also referred to as GIT-27)

[0028] Benzaldehyde (32.8 mmol) and hydroxylamine hydrochloride (33 mmol) were dissolved in methanol (100 ml) followed by addition of sodium carbonate (65 mmol). Overnight reaction gave the oxime derivative in 95% (30.4 mmol). Chlorination of the oxime using N-chlorosuccinimide (31.6 mmol) in DMF (100 ml) quantitatively furnished chlorooxime. This compound was then dissolved in THF/H2O (*0/20) and treated with 3-butenoate (24.5 mmol) and sodium carbonate (73.6 mmol). After completion (12 h), the product was extracted with ethyl acetate and the organic extracts were washed with brine and dried over magnesium sulfate. VGX- 1027 was crystallized from ethyl acetate/heptane mixture. The structure was confirmed by IH-NMR, 13C-NMR and mass spectroscopy. The daily dosage regimen will presumably vary within wide ranges, for instance from 0.1 to 10 mg/kg body weight.

[0029] VGX- 1027 ((S, R)-3 -phenyl -4,5-dihydro-5-isoxazole acetic acid) (VGX PHARMACEUTICALS, Blue Bell, PA, USA) is an isoxazoline compound that has been previously shown to exert its immunomodulatory affects in vitro by targeting the function of macrophages. VGX-1027 inhibits lipopolysaccharide (LPS)-induced NF-κB, p38 mitogen activated protein (MAP) kinase signalling pathways and TNF-α secretion from purified peritoneal macrophages in vitro (Stojanovic, 2007). We also demonstrated that VGX- 1027 inhibited LPS-induced secretion of IL-I β, TNF-α and IL-IO from peritoneal cells upon in vitro and ex vivo conditions (Stojanovic, 2007). Though not being bound to the following theory, it is believed that the action of VGX- 1027 on macrophages occurs through inhibition of TLR4 and TLR2/6-mediated signalling pathways as the test compound inhibits the release of TNF-α from macrophages stimulated with specific agonists of TLR4 and TLR2/6 such as LPS and zymosan, respectively (Stojanovic, 2007). VGX- 1027 appeared to spare T cell function as it did not modify the proliferation and/or secretion of IL-2, IFN-γ, and IL-4 induced in purified murine CD4+T cells from stimulation with either CD3+CD28 or Concanavalin A (ConA) (Stojanovic, 2007). These effects on macrophages may account for the capacity of VGX- 1027 to markedly ameliorate the course of both acute and chronic immunoinflammatory diseases in rodent models (Stosic-Grujicic, 2007; Stojanovic, 2007).

[0030] VGX- 1027 is effective both when given i.p. and p.o. Acute and sub-acute toxicological studies show that it is not toxic at the doses that exert biological effects in these preclinical models

[0031] The isoxazoline compounds provided herein have been described in earlier filed patent applications, and are hereby incorporated by reference in their entirety: PCT publication WO 06/097273 (Application No. PCT/EP2006/002319), filed March 15, 2006, claiming priority to U.S. Provisional patent application, Serial No. 60/661,500, filed March 15, 2005; and co-pending U.S. patent application, Serial No. 11/908472, filed September 12, 2007 (a National Stage filing off of WO 06/097273). In addition to the methods of use of the compounds disclosed in above-earlier filed applications, including the use of VGX- 1027 to treat TNF mediated immunopatho logical symptoms and diseases, the instant disclosure additionally provides for mode or mechanisms of action of same compounds, and preferably VGX- 1027, in modulating the immune response in the eye and treating inflammation therein, such as uveitis.

EXAMPLES

[0032] The present invention is further illustrated in the following Examples. It should be understood that these Examples, while indicating preferred embodiments of the invention, are given by way of illustration only. From the above discussion and these Examples, one skilled in the art can ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions. Thus, various modifications of the invention in addition to those shown and described herein will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims.

[0033] The data herein show that when VGX- 1027 is administered to mice after LPS challenge, such as preferably i.p. 30 min. after LPS challenge, VGX-1027 counteracts clinical, laboratory and histopatho logical evidence of EIU. Materials and Methods Drugs and Chemicals

[0034] VGX- 1027 was provided by VGX Pharmaceuticals (VGX PHARMACEUTICALS, Blue Bell, USA). Dexamethazone (Soldesan) was purchased from a local pharmacy. Primary anti-P-selectin, anti-ICAM-1, anti-nitrotyrosine, and anti-poly(ADP-ribose) synthetase antibodies were obtained from DBA (Milan, Italy). All other reagents were purchased from Sigma Chemical Co. (St. Louis, MO).

Animal Groups and EIU

[0035] Eight- week-old male Lewis rats (180-220 g) purchased from Harlan- Nossan (S.Pietro al Natisone, Udine) were used for the following experiments. EIU was induced in the rats by injection into the foot with 200 μg LPS from Salmonella typhimurium (Sigma-Aldrich, St. Louis, MO) that had been diluted in 0.2 ml phosphate- buffered saline (PBS, pH 7.4). Different groups of rats were created for the experimental design: The active group was treated with VGX- 1027, a control group with its vehicle, and a positive control group with dexamethazone 1 mg/kg 30 min before, simultaneously with, and 30 min after LPS injection as described (Jin et al., 2006). VGX-1027, dissolved in Na₂ HPO₄ /H₂O was administered i.p. at the dose of 25 mg/kg bd wt. in a final volume of 200 microliters. Only clinical and laboratoristic readouts were considered for the dexamethazone-treated animals. An additional control group received sham-treatment for comparisons. [0036] All animals were treated in accordance with the ARVO Statement for Use of Animals in Ophthalmic and Vision Research.

Clinical grading of uveitis

[0037] Sixteen hours after the induction of uveitis, the eyes were examined by slit lamp by an observer unaware of the treatment. Uveitis in each eye was graded according to a previously reported scoring system (Behar-Cohen, 1998): 0, no inflammatory reaction; 1, discrete dilatation of the iris and conjunctival vessels; 2, moderate dilatation of the iris and conjunctival vessels; 3, intense iridal hyperemia with flare in anterior chamber; and 4, same clinical signs as 3 plus the presence of fibrinous exudate in the pupillary area with intense flare in the anterior chamber. No signs of uveitis were observed in the animals at the beginning of each experiment.

Infiltrating cells, protein concentration, TNF-α and NO levels in aqueous humor

[0038] Immediately after the slit lamp examination, the rats were euthanized and the aqueous humor was collected from both eyes by an anterior chamber puncture (15-20 μl per rat) using a 30-gauge needle under a surgical microscope. The aqueous humor from each eye of the 5 rats from the same group was pooled and diluted 10 times with PBS (pH 7.4).

[0039] For cell counting, the aqueous humor was suspended in an equal amount of Turk stain solution, and the cells were counted with a hemocytometer under a light microscope. The number of cells per field (an equivalent of 0.1 μl) was manually counted, and the number of cells per microliter was obtained by averaging the results of four fields from each sample. The total protein concentration in the aqueous humor samples was measured using a bicinchoninic acid protein assay kit (Pierce, Rockford, IL).

[0040] The levels of TNF-α in the aqueous humor were assessed with a commercially available ELISA kit (R&D Systems, Minneapolis, MN). Intra and inter- assay coefficients of variations were lower than 10%. All aqueous humor samples were stored in ice water until testing on the day of sample collection.

Measurement of intraocular levels of nuclear NF-κB/p65

[0041] NF -KB p65 translocation into the nucleus, as an index of NF-κB activation, was measured using a sandwich ELISA (ActivELISATM; Imgenex, Analitica De Mori, Milano, Italy) according to the manufacturer's protocol. Soon after aqueous humor collection the rats were sacrificed and both eyes enucleated. Ten eyes from 5 animals were pooled together so that each experimental group consisted of 5 samples. The eyes were cut into small pieces, washed with cold PBS, homogenized in hypotonic buffer and centrifuged for 10 min at 10,000 rpm. The supernatants were used as cytoplasmic extract. Nuclear lysis buffer, 500 μl, was added to the pellet and centrifuged at 14,000 rpm for 10 min at 4°C. The supernatant was used as nuclear extract. The anti-NF-κB p65 antibody- coated plate captured the nuclear or cytoplasmic NF -KB p65 of the samples (0.5 to 1 mg/ml of protein) and the amount of bound NF-κB p65 was detected by adding a secondary antibody followed by an alkaline phosphatase-conjugated antibody. The absorbance value for each well was determined at 405 nm using a microplate reader (Bio- Rad, Hercules, CA, USA). The relative ratio of nuclear to cytoplasmic NF-κB p65 was calculated from the respective absorbance values.

Histologic Examination

[0042] A separate set of rats treated in the same way as described above were used for the histologic study. For microscopic histologic evaluation, rat eyes were collected and fixed in a solution of 0.1% glutaraldehyde (25%) and 4% paraformaldehyde for 24 hours at room temperature, dehydrated by graded ethanol, and embedded in paraffin (Paraplast; Sherwood Medical, Mahwah,NJ). Tissue sections (7 μm), cut near the optic nerve, were deparaffmized with xylene, stained with May-Grunwald-Giemsa and studied by light microscopy (Dialux 22; Leitz, Wetzlar, Germany). This was performed in each of the eight eyes (for the four rats in each group).

Localization ICAM-I, P-selectin and iNOS by immunohistochemistry

[0043] Three groups of animals (sham-treated, controls, and VGX-1027-treated ) were used for the immunohistochemical study. Iris-ciliary bodies were fixed in an immuno fixation solution of 0.1% glutaraldehyde (25%) and 4% paraformaldehyde in phosphate buffer. Indirect immunofluorescence staining was performed on 7-μm-thick sections. After deparaffmization, the sections were hydrated (three times for 15 min in PBS) and permeabilized with 0.1% Triton X-100 in PBS for 20 min. Non-specific adsorption was minimized by incubating the section in 2% normal goat serum in PBS for 20 min. Endogenous biotin or avidin binding sites were blocked by sequential incubation for 15 min with avidin and biotin (DBA, Milan, Italy). Sections were incubated overnight with 1) anti-ICAM-1 polyclonal antibody (CD54) (1 :500 in PBS, v/v) (DBA, Milan, Italy), 2) anti-P-selectin polyclonal antibody (1 :100 in PBS, v/v), or 3) anti-iNOS polyclonal antibody (1 :500 in PBS, v/v) (Santa Cruz, DBA, Milan, Italy). Specific labelling was detected with a biotin-conjugated goat anti-rabbit IgG, donkey anti-goat IgG or goat anti-mouse IgG and avidin-biotin peroxidase complex (DBA, Milan, Italy). To verify the binding specificity for ICAM-I, P-selectin, TNF-α and iNOS some sections were also incubated with primary antibody only (no secondary antibody) or with secondary antibody only (no primary antibody). In these situations, no positive staining was found in the sections.

Statistical analysis

[0044] All values are expressed as mean ± SD. The results were analyzed by oneway ANOVA followed by a Bonferroni post hoc test for multiple comparisons. P < 0.05 was considered significant. The figures from histologic and immunohistochemical experiments are representative of at least three experiments (five slides for each eye from different animals) performed on different days.

Results

Inhibition of clinical signs of EIU by VGX-1027

[0045] As shown in Fig. 1, the clinical inflammation score, which was determined 16 hours after injection of LPS, was significantly (P < 0.0001) lower in the group of rats treated with VGX- 1027 than in the negative controls. The effect of VGX- 1027 was comparable to that of the positive control group receiving dexamethazone (Dex) (Fig.l).

VGX-1027 reduces the LPS-induced increase in aqueous humor proteins, TNF-α and NO.

[0046] As previously reported (Bucolo et al., 2003; Jin et al,2006), the aqueous humor of rats challenged with LPS contained significantly larger amounts of proteins TNF-α and NO than those found in the group of sham-treated rats that were not challenged with LPS (Fig.2A-C). However, the LPS-induced elevated levels of both proteins were significantly reduced by treatment with VGX-1027. This effect was comparable to that of Dex (Fig.2A-2C). In addition, the number of inflammatory cells in the aqueous humor was significantly reduced in rats treated with VGX-1027 or Dex (Fig.3).

VGX-1027 reduces intraocular content of NF-κB p65 during EIU

[0047] As shown in Fig. 4, NF-κB p65 were found at increased levels in ocular tissue homogenates from vehicle-treated control mice with consequential increases in the nuclear/cytoplasmic ratios of the transcription factor subunit. In contrast, the nuclear levels and the nuclear/cytoplasmic ratios of NF-κB p65 were significantly reduced both in mice treated with VGX-1027 and in those treated with Dex (Fig. 4 A and B). VGX- 1027 suppresses histological signs of EIU

[0048] As previously reported (Bucolo et al., 2003), the histologic evaluation of iris ciliary body tissues from the LPS-treated rats revealed signs of severe uveitis with massive neutrophil infiltration (Fig. 5A) . Epithelial cells from these rats showed markedly reduced cytoplasm and scarcely visible nuclei (Fig. 5A). On the other hand, rats treated with VGX- 1027 exhibited a significantly milder uveitis than the vehicle-treated rats (Fig. 5B) , and maintenance of the epithelium. The histological appearance of iris ciliary bodies in the rats treated with VGX- 1027 was similar to that observed in the group of sham-treated rats that exhibited no signs of uveitis and intact epithelium (Fig. 5C).

P-selectin, ICAM-I and iNOS immunostaining

[0049] The results of immunohistochemical staining for P-selectin, ICAM-I and iNOS correlated with the histological results and the intensity of the infiltrating cells. Eye tissue sections from the sham-treated rats showed no staining for P-selectin, ICAM-I and iNOS. The tissue sections obtained from rats injected with LPS and treated with the vehicle of VGX- 1027 revealed intense positive staining for P-selectin, ICAM and iNOS (Fig. 6) . In contrast, a much weaker positive staining for these 3 molecules was observed in rats treated with VGX- 1027 (Fig.6). Discussion

[0050] Without being limited to the following theory, it is believed that the inhibitory action of VGX- 1027 on the ocular expression of P-selectin and ICAM-I played a substantial role in its beneficial anti-inflammatory action in this inflammation model. Both P-selectin and ICAM-I are expressed on endothelial cells in response to specific stimuli and both proteins are up-regulated by proinflammatory cytokines such as TNF-α (Ley, 2003; Van de Stolpe and Van der Saag, 1996). These molecules are important for the migration of inflammatory cells into tissues of the eye during EIU development.

[0051] VGX- 1027 is one compound from the described class of isoxazoline compounds that is shown effective in counteracting the uveitogenic effect of LPS, such as when given under a therapeutic regime: 30 min after challenge with LPS, at a time where inflammatory responses, including TNF-α and NO release in the aqueous humor, have already commenced.

References

Avunduk MC, Avunduk AM, Oztekin E, Baltaci AK, Ozyazgan Y, Mogolkoc R (2004). Etanercept treatment in the endotoxin-induced uveitis of rats. Exp Eye Res. 79:357-65.

Behar-Cohen, FF, Savoldelli, M, Parel, JM, et al (1998). Reduction of corneal edema in endotoxin-induced uveitis after application of L-NAME as nitric oxide synthase inhibitor in rats by iontophoresis Invest Ophthalmol Vis Sci 39,897-904

Bora NS, Kaplan HJ (2007). Intraocular diseases - anterior uveitis. Chem Immunol Allergy .92:213-20.

Bucolo C, Cuzzocrea S, Mazzon E, Caputi AP. (2003) Effects of cloricromene, a coumarin derivative, on endotoxin-induced uveitis in Lewis rats. Invest Ophthalmol Vis £ci.44:1178-1184

Goureau, O, Bellot, J, Thillaye, B, Courtois, Y, de Kozak, Y. (1995) Increased nitric oxide production in endotoxin-induced uveitis. Reduction of uveitis by an inhibitor of nitric oxide synthase J Immunol 154,6518-6523

Imrie FR, Dick AD (2007). Biologies in the treatment of uveitis. Curr Opin Ophthalmol.lSAM-6.

Jacquemin, E, de Kozak, Y, Thillaye, B, Courtois, Y, Goureau, O. (1996) Expression of inducible nitric oxide synthase in the eye from endotoxin-induced uveitis rats Invest Ophthalmol Vis Sci 37,1187-1196 Jin XH, Ohgami K, Shiratori K, Suzuki Y, Hirano T, Koyama Y, Yoshida K, Ilieva I, Iseki K, Ohno S. (2006) Inhibitory effects of lutein on endotoxin-induced uveitis in Lewis rats. Invest Ophthalmol Vis Sci. 47:2562-8.

Kitamei H, Iwabuchi K, Namba K, Yoshida K, Yanagawa Y, Kitaichi N, Kitamura M, Ohno S, Onoe K. (2006). Amelioration of experimental autoimmune uveoretinitis (EAU) with an inhibitor of nuclear factor-kappaB (NF-kappaB), pyrrolidine dithiocarbamate. J Leukoc Biol. Jun 79:1193-201.

Koizumi K, Poulaki V, Doehmen S, Welsandt G, Radetzky S, Lappas A, Kociok N, Kirchhof B, Joussen AM (2003). Contribution of TNF-alpha to leukocyte adhesion, vascular leakage, and apoptotic cell death in endotoxin-induced uveitis in vivo. Invest Ophthalmol Vis Sci. 44:2184-91.

La Heij, E, Kuijpers, RW, Baarsma, SG, Kijlstra, A, van der Weiden, CM. (1998) Adhesion molecules in iris biopsy specimens from patients uveitis Br J Ophthalmol 82,432-437

Ley K (2003). The role of selectins in inflammation and disease. Trends MoI Med. 9:263-8.

Mandai, M, Mittag, TW, Kogishi, J, Masayoshi, I, Masanori, H, Yoshimura, N. (1996) Role of nitric oxide synthase isozymes in endotoxin-induced uveitis Invest Ophthalmol Vis Sci 37,826-832

Mangano K, Niranjan S, D'Alcamo M, Libra M, Malaguarnera L, Donia M et al. (2008) In vitro inhibition of enterobacteria-reactive CD4+CD25- T cells and suppression of immunoinflammatory colitis in mice by the novel immunomodulatory agent VGX- 1027 Eur. J. Pharmacol, (in press), doi:10.1016/j.ejphar.2008.02.017

Planck SR, Huang XN, Robertson JE, Rosenbaum JT (1994) Cytokine mRNA levels in rat ocular tissues after systemic endotoxin treatment. Invest Ophthalmol Vis Sci. 35:924-30.

Smith, JR, Hart, PH, Williams, KA. (1998) Basic pathogenic mechanisms operating in experimental models of acute anterior uveitis Immunol Cell Biol 76,497-512

Stojanovic I, Cuzzocrea S, Mangano K, Mazzon E, Miljkovic D, Wang M, Donia M, Al-Abed Y, Kim J, Nicoletti F, Stosic-Grujicic S, Claesson M. (2007). In vitro, ex vivo and in vivo immunopharmaco logical activities of the isoxazoline compound VGX- 1027: modulation of cytokine synthesis and prevention of both organ- specific and systemic autoimmune diseases in murine models. Clin Immunol. 123:311-23.

Stosic-Grujicic S, Cvetkovic I, Mangano K, Fresta M, Maksimovic-Ivanic D, Harhaji L, Popadic D, Momcilovic M, Miljkovic D, Kim J, Al-Abed Y, Nicoletti F. (2007) A potent immunomodulatory compound, (S,R)-3-Phenyl-4,5-dihydro-5-isoxazole acetic acid, prevents spontaneous and accelerated forms of autoimmune diabetes in NOD mice and inhibits the immunoinflammatory diabetes induced by multiple low doses of streptozotocin in CBA/H mice. J Pharmacol Exp Ther.320: 1038-49.

Suzuki Y, Ohgami K, Shiratori K, Jin XH, Ilieva I, Koyama Y, Yazawa K, Yoshida K, Kase S, Ohno S. (2006) Suppressive effects of astaxanthin against rat endotoxin-induced uveitis by inhibiting the NF-kappaB signaling pathway. Exp Eye Res. 82:275-81. Suzuma, K, Mandai, M, Kogishi, J, Shinichiro, JT, Honda, Y, Yoshimura, N. (1997) Role of P-selectin in endotoxin-induced uveitis Invest Ophthalmol Vis Sci 38,1610-1618

Szabό, C, Cuzzocrea, S, Zingarelli, B, O'Connor, M, Salzman, A. (1997) Endothelial dysfunction in a rat model of endotoxic shock: importance of the activation of poly ADP-ribose synthetase by peroxynitrite J Clin Invest 100,723-735

Szabό, C, Zingarelli, B, O'Connor, M, Salzman, A. (1996) DNA strand breakage, activation of poly- ADP-ribose synthetase, and cellular energy depletion are involved in the cytotoxicity in macrophages and smooth muscle cells exposed to peroxynitrite Proc Natl Acad Sci USA 93,1753-1758

Szabό, C. (1996) DNA strand breakage and activation of poly-ADP ribosyltransferase: a cytotoxic pathway triggered by peroxynitrite Free Radic Biol Med 21,855-869

Tellier Z (2007). Human immunoglobulins in intraocular inflammation. Ann N Y Acad Sci.1110:337 ^'-47.

Van de Stolpe A, van der Saag PT (1996). Intercellular adhesion molecule- 1. J MoI Med. 74:13-33.

Whitcup SM, Chan CC, Kozhich AT, Magone MT. (1999) Blocking both E- selectin and P-selectin inhibits endotoxin-induced leukocyte infiltration into the eye. Clin Immunol. 93:107-13. Withcup, SM, DeBarge, LR, Rosen, H, Nussenblatt, RB, Chan, CC. (1993) Monoclonal antibody against CDl lb/CD18 inhibits endotoxin-induced uveitis Invest Ophthalmol Vis Sci 34,673-681

Withcup, SM, Hikita, N, Shirao, M, et al (1995) Monoclonal antibodies against CD 54 (ICAM-I) and CDl Ia (LFA-I) prevent and inhibit endotoxin-induced uveitis Exp Eye Res 60,597-601

Claims

What is claimed are:

1. A method of modulating an immune response in an eye of a mammal, comprising:

administering to said mammal a pharmaceutical formulation comprising a compound of formula (I):

X is a hydrogen, halogen, N₃, SH, =0, =CH₂, an aromatic ring, optionally substituted by substituents selected from the group consisting of hydrogen, nitro, cyano, Ci-C₃-alkyl, halogen, carboxy, amino, trifluoromethyl, hydroxy, and Ci-C₃-alkoxy, or amino, mono- or disubstituted amino groups, wherein the substituents are selected from C₁-C₄ alkyl, phenyl or benzyl groups optionally substituted by substituents selected from the group consisting of hydrogen, nitro, cyano, C₁-C^aIkVl, halogen, carboxy, amino, trifluoromethyl, hydroxy, and Ci-C₃-alkoxy; and

Y is hydrogen, C₁-C₄ alkyl, amino, or a group of formula -(CH₂)_0-1A, wherein A is an aromatic ring optionally substituted by substituents selected from the group consisting of hydrogen, nitro, cyano, Ci-C₃-alkyl, halogen, carboxy, amino, trifluoromethyl, hydroxy, and Ci-C3-alkoxy.

2. The method according to claim 1, wherein the pharmaceutical composition is a compound of formula (I) and X is hydrogen.

3. The method according to any one of claims 1-2, wherein Y is hydrogen.

4. The method according to any one of claims 1-3, wherein the compound comprises 3- phenyl-4,5-dihydro-5-isoxazoleacetic acid or 3-phenyl-5-isoxazoleacetic acid.

5. The method according to any one of claims 1-4, wherein the administering step comprises: oral, intraperitoneal, intravenous, intranasal or intradermal administration.

6. The method according to any one of claims 1-5, wherein the administering step is oral administration.

7. A method of alleviating symptoms of uveitis in a mammal, comprising:

identifying a mammal exhibiting uveitis or pathological events leading to uveitis, administering to said mammal a pharmaceutical formulation comprising a compound of formula (I):

X is a hydrogen, halogen, N₃, SH, =0, =CH₂, an aromatic ring, optionally substituted by substituents selected from the group consisting of hydrogen, nitro, cyano, Ci-C3-alkyl, halogen, carboxy, amino, trifluoromethyl, hydroxy, and Ci-C3-alkoxy, or amino, mono- or disubstituted amino groups, wherein the substituents are selected from C₁-C₄ alkyl, phenyl or benzyl groups optionally substituted by substituents selected from the group consisting of hydrogen, nitro, cyano, Ci-C₃-alkyl, halogen, carboxy, amino, trifluoromethyl, hydroxy, and Ci-C3-alkoxy; and

Y is hydrogen, C₁-C₄ alkyl, amino, or a group of formula -(CH₂)_0-1A, wherein A is an aromatic ring optionally substituted by substituents selected from the group consisting of hydrogen, nitro, cyano, C₁-C^aIkVl, halogen, carboxy, amino, trifluoromethyl, hydroxy, and Ci-C3-alkoxy.

8. The method according to claim 7, wherein the identifying step comprises: detecting an elevated level of TNF-alpha, NF-kappaB p65, or nitrous oxide (NO), or a combination thereof in aqueous humor in an eye of the mammal.

9. The method according to any one of claims 7-8, wherein the pharmaceutical composition is a compound of formula (I) and X is hydrogen.

10. The method according to any one of claims 7-9, wherein Y is hydrogen.

11. The method according to any one of claims 7-10, wherein the compound comprises 3-phenyl-4,5-dihydro-5-isoxazoleacetic acid or 3-phenyl-5-isoxazoleacetic acid.

12. The method according to any one of claims 7-11, wherein the administering step comprises: oral, intraperitoneal, intravenous, intranasal or intradermal administration.

13. The method according to any one of claims 7-12, wherein the administering step is oral administration.

14. A method of alleviating symptoms of uveitis in a mammal, comprising:

identifying a mammal exhibiting uveitis or pathological events leading to uveitis, and administering orally to said mammal a pharmaceutical formulation comprising 3- phenyl-4,5-dihydro-5-isoxazoleacetic acid.