FR2963238A1 - Use of 15-deoxyspergualin derivatives for the treatment and/or prevention of ocular inflammatory diseases, preferably uveitis - Google Patents

Abstract

Use of 15-deoxyspergualin derivatives (I), is claimed for the treatment and/or prevention of ocular inflammatory diseases. An independent claim is included for an aqueous topical formulation comprising (I) as active substance and at least one excipient. ACTIVITY : Antiinflammatory; Ophthalmological. MECHANISM OF ACTION : None given.

Description

FIELD OF THE INVENTION The present invention relates to a new therapeutic use of 15-deoxyspergualine derivatives and their salts. More specifically, the present invention relates to the use of 15-deoxyspergualine derivatives and their pharmaceutically acceptable salts for the treatment and / or prevention of ocular inflammatory diseases, and more particularly of uveitis.

BACKGROUND OF THE INVENTION Inflammatory eye diseases are the leading cause of visual deterioration in the world. More specifically, uveitis refers to the inflammation of the uvea, the vascular middle layer of the eye consisting of the iris, the ciliary body and the choroid. Inflammation in uveitis results from a wide variety of traumatic and immune-mediated aggression.

The main therapeutic choice for patients with uveitis is corticosteroids administered locally or systemically. Nevertheless, corticosteroids have serious systemic side effects and ocular side effects such as cataracts, glaucoma and delayed wound healing.

As an alternative, there are non-steroidal anti-inflammatory agents such as diclofenac or flurbiprofen. However, many subjects do not react or become refractory to steroidal or nonsteroidal therapy. Currently, there are also immunosuppressive drugs such as cyclosporine A, Tacrolimus and Sirolimus administered orally that have systemic side effects, such as risk of viral or bone damage and cancer development. An alternative would be to use immunosuppressive drugs in the oil for topical administration, since their cyclic structures are not soluble in aqueous media. However, the immunosuppressive drugs in the oil have the disadvantage of being irritating, painful and causing blurred vision. The present invention overcomes the disadvantages of the state of the art by providing a novel use of 15-DSG derivatives and their pharmaceutically acceptable salts and, in particular, their use in the treatment and / or prevention of inflammatory eye diseases such as uveitis.

SUMMARY OF THE INVENTION The present invention is based on the unexpected results demonstrating that 15-DSG derivatives and their pharmaceutically acceptable salts are capable of improving clinical signs in uveitis models, particularly with hematoocular barrier and against tissue damage in experimental autoimmune uveoretinitis (UAE) models and without modification of the systemic immune response via ocular pathways. Indeed, the beneficial effects of 15-DSG derivatives and their pharmaceutically acceptable salts, in particular Tresperimus and Anisperimus, on the pathological and molecular changes associated with uveitis have been demonstrated by a regulation of the activation of macrophages and T - mediated response in the eye. The 15-DSG derivatives and their pharmaceutically acceptable salts, with their linear structure, are soluble and stable in aqueous media, so that they can be administered locally in aqueous formulations. According to a first aspect, the present invention thus relates to the use of 15-DSG derivatives and their pharmaceutically acceptable salts, in particular Tresperimus and Anisperimus, for the preparation of a medicament useful for the treatment and / or the prevention of inflammatory eye diseases, in particular uveitis. In a second aspect, the present invention provides a method for treating and / or preventing eye inflammatory diseases, including uveitis, comprising administering to a subject in need thereof a derivative of 15-DSG or a pharmaceutically acceptable salt thereof. In one embodiment, the 15-DSG derivative or its pharmaceutically acceptable salt is Tresperimus or Anisperimus. According to a third aspect, the present invention relates to suitable formulations of a pharmaceutical composition comprising at least as active substance or active compound, a derivative of 15-DSG or a pharmaceutically acceptable salt thereof for local administration to subjects having ocular inflammatory diseases, and more specifically a pharmaceutically acceptable aqueous formulation for local administration. A better understanding of the nature and advantages of the present invention can be gained by reference to the remaining parts of the specification and the claims.

DESCRIPTION OF THE DRAWINGS Figure 1: Effect of an injection of Tresperimus on experimental clinical autoimmune uveoretinitis (UAE) in the rat. Figure 2: Effect of an intravitreal injection (IVT) of Tresperimus on the histopathological scores of UAE (A) and the histopathological changes of UAE in rats treated with Tresperimus (C) compared to rats subjected to an injection of saline solution (B). a, b, d, e = photoreceptor layers; c, f = optical papillae. Figure 3: Effect of Tresperimus in S-antigen-specific delayed-type hypersensitivity (HTR) in rats treated with intravitreal injection. Figure 4: Effect of intravitreal injections of Tresperimus on intraocular proteins (A), cytokines (B) and the expression of nitric oxide synthase-2 (NOS-2) (C). Figure 5: Anatomy of the eye. Figure 6: Eye distribution of Tresperimus after instillation of eye drops of a 1% solution twice daily for 4 days in male New Zealand rabbit. DETAILED DESCRIPTION Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art of this invention. In addition, the following definitions are provided to assist the reader in the practice of the invention. The "subject" is preferably a mammal, more preferably a human. The term "for use in the treatment and / or prevention" as used herein should be understood to cover the direct use of the compound or its derivative or salt for treatment and / or prevention of the specified disease. "Prevention and / or treatment method" shall be understood as covering those processes in which a compound or derivative or salt thereof is administered for the treatment and / or prevention of the specified disease. "Inflammatory eye diseases": is a general term for inflammation affecting any part of the eye or surrounding tissue. Inflammation involving the eye can range from familiar allergic conjunctivitis of hay fever to rare conditions potentially leading to blindness, such as uveitis, scleritis, episcleritis, optic neuritis, keratitis, retrobulbar pseudotumor, Eales syndrome, chronic conjunctivitis or an ocular manifestation of a lesion by systemic disease of ocular tissues, that is to say the retina, which may ultimately lead to blindness. The localization of inflammation governs the diagnostic designation of inflammatory ocular disease. Inflammatory eye diseases can result from several causes. According to the present invention, uveitis is non-infectious and comes from traumatic causes, induced by drugs or malignancies, and from causes consecutive to ophthalmic surgery. The pharmaceutical composition of the present invention may also be used after ophthalmic surgery causing ocular inflammation such as corneal transplantation. "Uveitis" refers to the inflammation of the uvea, the middle vascular layer of the eye consisting of the iris, ciliary body and choroid, and is classified by location, clinical course and laterality. "Anterior" refers to the involvement of the iris, cornea, pupil, aqueous humor or ciliary body. For example, we can mention as anterior uveitis Kawasaki disease. "Intermediate" refers to the vitreous, the posterior zone of the ciliary body, the peripheral retina and the sclera. "Posterior" refers to the choroid or retina by extension, fovea and optic nerve. Noninfectious posterior uveitis includes Behcet's disease, Vogt-Koyanagi-Harada's disease, inflammation of the posterior zone of the ciliary body, sarcoidosis, idiopathic retinal vasculitis and multifocal inflammation of the retina. and the choroid. "Panuheite" is used when two or more segments are affected. The clinical course is defined as "acute", "chronic" or "recurrent". The term "bilateral" is used when both eyes are involved simultaneously. "A derivative of 15-deoxyspergualin or a pharmaceutically acceptable salt thereof" is a compound having a structure related to deoxyspergualine (DSG) or a pharmaceutically acceptable salt thereof. The "salts" can be obtained by the chemical reaction between an inorganic or organic acid and the compounds of formula (I) mentioned below. The preferred inorganic acids for the formation of salts are: hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid. The preferred organic acids for the formation of salts are: fumaric acid, maleic acid, oxalic acid, citric acid, trifluoroacetic acid, tartaric acid and sulphonic acids (acid methanesulfonic acid with dodecane sulfonic acid). In the context of the present invention, therefore, the active substance of the pharmaceutical composition or the drug may be one or more derivatives of 15-deoxyspergualin and a pharmaceutically acceptable salt thereof. Any 15-deoxyspergualine derivative and their pharmaceutical salts known and described in the art can be used for the practice of the use and method described according to the present invention. According to one aspect of the invention, the derivative of 15-deoxyspergualine is a compound of formula (I) NH 0 0 H H 2 NN (CH 2 -N N) Nom / R H 2 (I) in which: n is equal to 6 or 8, A is a bond, a group CH 2, a CH (OH) group, a CHF group, a CH (OCH 3) group, a CH 2 NH group or a CH 2 O group, - R is a hydrogen atom or a CH 3, or a pharmaceutically acceptable salt thereof The compounds of formula (I) are advantageously chosen from: 2 - [[6 - [(aminoiminomethyl) amino] hexyl] amino N- [4 - [(3-aminopropyl) amino] butyl] carbamate 2-oxoethyl; N- [4 - [(3-aminopropyl) amino] butyl] -N '- [6 - [(aminoiminomethyl) amino] hexyl] propanediamide; N- [4 - [(3-aminopropyl); ) amino] butyl] -N '- [6 - [(aminoiminomethyl) amino] -hexyl] -2-hydroxy-propanediamide; N- [4 - [(3-aminopropyl) amino] butyl] -N' - [ 6 - [(aminoiminomethyl) amino] hexyl] -2-fluoro-propanediamide N- [6 - [(aminoiminomethyl) amino] hexyl] -N '- [4 - [(3-aminopropyl) amino] butyl] -2-methoxy-propanediamide; N- [6 - [(aminoiminometh yl) amino] hexyl] -2 - [[[[4 - [(3-aminopropyl) amino] butyl] amino] carbonyl] amino] acetamide; N- [6 - [(aminoiminomethyl) amino] hexyl] -N '- [4 - [(3-aminopropyl) amino] butyl] ethanediamide; N- [8 - [(aminoiminomethyl) amino] octyl] -N '- [4 - [(3-aminopropyl) amino] butyl] ethanediamide; N- [8 - [(aminoiminomethyl) amino] octyl] -N '- [4 - [(3-aminopropyl) amino] butyl] propanediamide; N- [8 - [(aminoiminomethyl) amino] octyl] -N '- [4 - [(3-aminopropyl) amino] butyl] -2-hydroxypropanediamide; N- [8 - [(aminoiminomethyl) amino] octyl] -N '- [4 - [(3-aminopropyl) amino] butyl] -2-fluoro-propanediamide; N- [4 - [(3-aminopropyl) amino] butyl] -2-methoxy-N '- [8 - [(aminoiminomethyl) amino] octyl] propanediamide; N- [8 - [(aminoiminomethyl) amino] octyl] -2 - [[[[4 - [(3-aminopropyl) amino] butyl] amino] carbonyl] amino] acetamide; 2 - [[8 - [(aminoiminomethyl) amino] octyl] amino] -2-oxoethyl N- [4 - [(3-aminopropyl) amino] butyl] carbamate; 2 - [[6- [(aminoiminomethyl) amino] hexyl] amino] -2-oxoethyl N- [4 - [(3-aminobutyl) amino] butyl] carbamate; N- [4 - [(3-aminobutyl) amino] butyl] -N '- [6 - [(aminoiminomethyl) amino] hexyl] ethanediamide; N- [4 - [(3-aminobutyl) amino] butyl] -N '- [6 - [(aminoiminomethyl) amino] hexyl] propanediamide; 2 - [[[[4 - [(3-aminobutyl) amino] butyl] amino] carbonyl] amino] -N- [6 - [(aminoiminomethyl) amino] hexyl] acetamide; N- [4 - [(3-aminobutyl) amino] butyl] -N '- [6 - [(aminoiminomethyl) amino] hexyl] -2-hydroxypropanediamide; N- [4 - [(3-aminobutyl) amino] butyl] -N '- [6 - [(aminoiminomethyl) amino] hexyl] -2-fluoro-propanediamide; N- [4 - [(3-aminobutyl) amino] butyl] -N '- [6 - [(aminoiminomethyl) amino] hexyl] -2-methoxy-propanediamide; N- [4 - [(3-aminobutyl) amino] butyl] -N '- [8 - [(aminoiminomethyl) amino] octyl] ethanediamide; N - [4 - [(3-aminobutyl) amino] butyl] -N '- [8 - [(aminoiminomethyl) amino] octyl] propanediamidecarbamic acid; 2 - [[8 - [(aminoiminomethyl) amino] octyl] amino] -2-oxoethyl N- [4 - [(3-aminobutyl) amino] butyl] -ester; 2 - [[[[4 - [(3-aminobutyl) amino] butyl] amino] carbonyl] amino] -N- [8 - [(aminoiminomethyl) amino] octyl] acetamide; N- [4 - [(3-aminobutyl) amino] butyl] -N '- [8 - [(aminoiminomethyl) amino] octyl] -2-hydroxypropanediamide; N- [4 - [(3-aminobutyl) amino] butyl] -N '- [8 - [(aminoiminomethyl) amino] octyl] -2-fluoro-propanediamide; N- [4 - [(3-aminobutyl) amino] butyl] -N '- [8 - [(aminoiminomethyl) amino] octyl] -2-methoxy-propanediamide; and their pharmaceutically acceptable salts.

The preferred compounds of formula (I) are chosen from 2 - [[6- [(aminoiminomethyl) amino] hexyl] amino] -2-oxoethyl N- [4 - [(3-aminopropyl) amino] butyl] -carbamate or 2 - [[6- [(aminoiminomethyl) amino] hexyl] amino] -2-oxoethyl N- [4 - [(3-aminobutyl) amino] butyl] -carbamate and pharmaceutically acceptable salts thereof. The compounds of formula (I) that are particularly preferred are Tresperimus and Anisperimus. "Tresperimus" is 2 - [[6- [(aminoiminomethyl) amino] hexyl] amino] -2-oxoethyl N- [4 - [(3-aminopropyl) amino] butyl carbamate tris-hydrochloride. "Anisperimus" is 2 - [[6 - [(aminoiminomethyl) amino] hexyl] amino] -2-oxoethyl N- [4 - [(3-aminobutyl) amino] butyl carbamate tetra-hydrochloride. The pharmaceutical compositions of the invention typically comprise at least one derivative of 15-deoxyspergualin or a pharmaceutically acceptable salt thereof, as well as one or more pharmaceutically acceptable carriers or excipients. The pharmaceutically acceptable carriers allow the administration of active compounds, allow and can facilitate or improve the preparation of the composition and can stabilize the composition. In addition, pharmaceutically acceptable carriers can increase the effectiveness of the composition, improve the ocular tolerability of the active compounds and / or modify their release profile. The pharmaceutically acceptable carriers are determined in part by the particular composition that is administered as well as by the particular method that is used to administer the composition.

They must also be pharmaceutically and physiologically acceptable in that they must be compatible with the other ingredients and must be biologically harmless. Such vehicles can take a wide variety of forms depending on the form of preparation desired for administration, for example local administration. "Local administration" should be understood as defining all ocular pathways, ie topical, intraocular (intravitreal, IVT), periocular administration including subconjunctival administration, under Tenon capsule , retrobulbar, intrasclerotic, and the desired form of preparation for administration. The pharmaceutical forms for "topical administration" can be eye drops, for example solutions, suspensions, colloidal systems (for example liposomes, emulsions, nanoemulsions, nanoparticles, microspheres), micelles, complexing systems, for example solutions of cyclodextrins, also sprays, creams, ointments, hydrogels, oleogels, hydrophilic lenses, inserts. "Intravitreal administration" can be performed as injectable or implantable systems. The galenic forms may be solutions, suspensions, colloidal systems (for example liposomes, emulsions, nanoemulsions, nanoparticles, microspheres), micelles, also biodegradable or non-biodegradable implants such as buffers, sticks, disks or pellets. For the two routes of administration, depending on the compound to be delivered, most of the dosage forms mentioned above can potentially increase the residence time of the active ingredient on the surface of the eye or in the vitreous, allow a release slow and prolonged encapsulated compounds and / or avoid toxicity and increase ocular tolerability. According to the present invention, for the treatment and / or prevention of ocular inflammatory diseases, and in particular uveitis, the 15-DSG derivatives or their pharmaceutically acceptable salts are administered via an aqueous acceptable pharmaceutical composition or formulation upon administration. topical or intravitreal. In this case, the pharmaceutically acceptable excipient (s) used should be suitable for topical or intravitreal administration. A major advantage of the present invention is that all ocular tissues (anterior or posterior chambers) are exposed to 15-DSG derivatives after topical administration, for example by instillation of eye drops, for example with an aqueous pharmaceutically acceptable composition. . For example, it was observed that the retina / choroid (posterior chamber) and the ciliary / iris body (anterior chamber) were exposed to Tresperimus levels of 0.5 to 0.7 μM and 0.3 to 0.7 pM for 24 hours following repeated instillation of eye drops twice a day as a simple 1% aqueous solution (see Figure 6). As a result, compared with other topical administration such as intraocular injection or implants, and in addition to better compliance, topical administration of 15-DSG-related compounds may allow for much better control of drug in the eye tissues. In addition, plasma levels were found to be low (<50 ng / mL from 5 min to 2 h after instillation) and for a short time below the lower limit of quantification (Blq) (2 ng / mL 4 hours after the dose) after instillation of eye drops. This helps to limit or avoid unwanted systemic effects. The concentration of the therapeutically active substance in the formulation can range from 0.1 μM to 100 mM with IVT and from 0.001% to 10% with eye drop instillations. Preferably, the concentration of the therapeutically active substance in the formulation can vary from 1 μM to 10 mM with IVT and from 0.1% to 5% with eye drop instillations. These concentrations may be applied for other local routes of administration.

These compositions are prepared by any method well known in the pharmaceutical art. The drug of the present invention may be combined with or used in combination with other therapeutic agents. For example, a subject may be treated with a derivative of 15-deoxyspergualin or a pharmaceutically acceptable salt thereof, particularly Tresperimus or Anisperimus, along with other conventional drugs. In addition, the present invention also relates to a pharmaceutical composition comprising at least one derivative of 15-DSG or a pharmaceutically acceptable salt thereof in combination with one or more drugs used in the treatment of uveitis. The invention will be explained in more detail with reference to Tresperimus, but it will be understood by those skilled in the art that the present invention is not limited to this derivative of 15-DSG.

As we can see from Figure 5, the middle layer of the eye is called the uveal or uveal tract. It consists of the iris, the ciliary body and the choroid. The uvea surrounds the eye like a tunic. The visible part of the uvea, at the front, is the iris. Inflammation of any part of the uveal tract is called uveitis.

In the present invention, we have demonstrated for the first time that local ocular administration of 15-DSG derivatives and their pharmaceutical salts is very beneficial in the treatment of an autoimmune eye disease, UAE, a model of ocular inflammation leading to retinal destruction.

The UAE models help to understand the pathophysiological mechanisms and in particular the involvement of CD4 + lymphocytes (cluster of differentiation 4), macrophages and pro-inflammatory cytokines in the mechanisms of destruction of the retina. UAE is a model of inflammatory disease that shares many clinical and histopathological features with human uveitis such as sympathetic ophthalmia, birdshot retinochoroidopathy, Vogt-Koyanagi-Harada syndrome, Behcet's disease, and sarcoidosis. It is a clinically relevant model for human eye inflammation.

Indeed, biological agents targeting cytokines and various soluble mediators of inflammation have demonstrated their effects for specific ocular inflammatory diseases. The UAE is induced by immunization with the purified retinal autoantigen, S antigen (S-Ag) which is also recognized by subjects with uveitis. The UAE is dependent on the effector cells Thi (interferon-producing cells y) and Th17 (interleukin-17 producing cells) CD4 +, and each effector phenotype can induce a pathological reaction. However, IL-17 (interleukin-17) plays a dominant role in the retinoid-binding KLA between photoreceptors (IRBP), its neutralization prevents or reverse the disease, and Th17 effector cells induce a UAE in the absence of interferon (IFN) -gamma. Then, locally, macrophages and microglial cells amplify the reaction and induce the destruction of photoreceptors and retinal tissue. Monocytes / macrophages as well as neutrophils are important effector cells in UAE whereas T cells act more to initiate and maintain the response. Macrophages cross the blood-retinal barrier and infiltrate the retina, where their release of mediators like NO (nitric oxide) and TNF (tumor necrosis factor) can cause severe retinal lesions and, consequently, loss of vision in humans. We investigated the effect of local administration of Tresperimus on KEP and ocular and systemic immune responses induced by S-Ag immunization.

Interestingly, Tresperimus is a very soluble molecule in saline solutions and its injection into the posterior pole of the rat eye at the posterior zone of the ciliary body allowed diffusion in the anterior and posterior segments of the eye as demonstrated its effectiveness on anterior and posterior ocular inflammation in UAE. In fact, after three intravitreal injections to animals immunized with S-Ag every three days, Tresperimus rapidly spread to the retinal / choroid tissues (- 150 pM one hour after injection) and decreased to reach another 8 days after injection of significant concentrations (-10 μM) that were observed to be effective in transplantation models in rats. In addition, low levels (<90 ng / mL) of Tresperimus were found in the plasma with no effect on the systemic immune response. In fact, the effect of Tresperimus was limited to the eye, confirming that no effective diffusion into the general circulation occurred.

We have shown that three intravitreal injections of Tresperimus performed after immunization with S-Ag during the afferent phase of the disease (days 6, 9, 12) were effective in reducing clinical ocular inflammation and retinal retinal photoreceptors, as shown by Figures 1 and 2. To examine the level of action of Tresperimus, delayed-type hypersensitivity (HTR) to S-Ag, as estimated by an atrial test, was not different in the control rats and the treated rats (Figure 3), suggesting that the treatment did not alter the reactivity of systemic T cells to S-Ag. In addition, we analyzed the systemic and local immune responses. We have shown that ocular treatment has no effect on the systemic immune response. Indeed, in the inguinal lymph nodes draining the immunization site, the level of inflammatory cytokines like TNF-α, and cytokines produced by T lymphocytes like IL-2, IFN-γ (interferon-gamma), IL-17 was not modified by treatment with Tresperimus. In contrast, treatment with Tresperimus was very effective on ocular pathology and local immune response, by inhibiting several cytokines (Figure 4B). The data suggest that treatment with Tresperimus regulates macrophage activation and T-mediated response in the eye. It has been described that macrophages play an important role in tissue destruction. To determine whether the reduction in UAE scores in rats treated with Tresperimus is related to a regulation of macrophage activation, we examined by semi-quantitative RT-PCR the expression of NOS-2 (oxide nitric synthase-2) in infiltrating cells present in the aqueous / vitreous humor from treated rats and control rats. Interestingly, NOS-2 expression was detected by RT-PCR in ocular inflammatory cells collected from control rats while treatment with Tresperimus suppressed NOS-2 expression in infiltrating macrophages (Figure 4C). This result suggests that the reduction in UAE in Tresperimus-treated rats is related to reduced activation of intraocular macrophages or a change in phenotype of macrophages that contribute to reduced tissue injury. This result was confirmed by immunohistochemistry which showed that ED1 + macrophages present in the aqueous humor of rats treated with Tresperimus did not express NOS-2 as compared to control rats. In addition, the treatment reduced the nuclear expression of NF-KBp65 in ocular infiltrating macrophages, suggesting that treatment with Tresperimus has disabled intraocular macrophages.

It has been reported that Tresperimus inhibits the nuclear localization of NF-κB in antigen presenting cells that is required for co-stimulation of T cells. This suggests that local treatment with Tresperimus may be effective on ocular presentation. by S-Ag macrophages to T-cells infiltrating ocular tissue, thus reducing the ocular amplification of the immune response. After antigenic stimuli, different populations of helper T cells developed, which are known as helper T cells of type 1 (Thi: cells producing interferon-γ), Th17 (cells producing IL-17) and Th2 (cells producing IL-4, IL-5 and IL-13). Thi and Th17 cells are involved in autoimmune disorders while Th2 cells play a role in allergies and asthma. We have shown that three intraocular injections of Tresperimus performed after immunization with S-Ag reduced the intraocular concentration of cytokines specific for IL-2, IL-17 T cells (Figure 4B). The concentration of cytokines in pooled aqueous humor and vitreous was determined by multiplex ELISA 21 days after immunization with S-Ag in rats treated with Tresperimus or saline. It shows that reducing the severity of KRA in rats treated with Tresperimus is related to significantly reduced levels of IL-2 and IL-17. It has been reported that IL-2 promotes Th17 cell expansion, which provides explanations for the efficacy of IL-2R antibody therapy in uveitis, and suggests that Th17 cell antagonism to ocular pathology could be used for the treatment of chronic ocular inflammation. Indeed, the UAE in rats can be suppressed by injection of anti-IL-17 antibody with antigen-specific HTR suppression and lymphocyte proliferation assay. This suggests that IL-17 plays a crucial role in the UAE. In addition, we have shown that intraocular administration of Tresperimus reduces the expression of IL-4, IL-6 and VEGF (vascular endothelial growth factor), although not significantly, in the aqueous humor. vitreous body of rats with low UAE. This result is consistent with the observation that administration of recombinant rat IL-4 to Lewis rats exacerbated UAE, with increased production of IFN-γ, TNF-α, and NO. Interestingly, IL-6 has been shown to induce VEGF expression in choroidal neovascularization and tumor angiogenesis. In addition, IL-6 has been shown to be necessary for angiotensin II-induced increases in VEGF expression. Treatment with anti-MR16-1 mouse IL-6 monoclonal antibody suppressed ocular inflammation in mice with UAE and inhibited Th17 cell induction. Anti-IL-6 treatment inhibited not only IRBP-sensitive Th17 cells but also their Th1 equivalents and induced IRBP-responsive regulatory T cells (Tregs) in vitro. Interestingly, the treatment induced a decrease in protein leakage in treated rats (Figure 4A), suggesting that intraocular injection of Tresperimus has protected the blood-eye barriers. The ocular pharmacokinetics of Tresperimus has been performed as a support for pharmacology (see Table 1 below).

Following intravitreal injections of saline at 9 mM Tresperimus, plasma levels of the test sample were low and were quantified only at the first time point 1 h after injection (-40 ng / mL). Ocular tissues were highly exposed to Tresperimus, with significant levels (> 10 μM) in the retina / choroid 8 days after injection. An impact of the pathology induced on the ocular pharmacokinetics of Tresperimus has been observed, with a faster elimination of the ocular tissues of the immunized animals. This observation was related to slightly higher plasma concentrations (range 19-89 ng / mL) and membrane leakage due to immunization of animals with S-Ag.

CONCLUSION Tresperimus, a pharmaceutically acceptable salt of a derivative of 15-DSG, is an active substance useful in the treatment / prevention of ocular inflammatory diseases, after ocular administration in an experimental model of autoimmune uveitis without any modification of the systemic immune response. This is supported by the ocular and plasma pharmacokinetics of Tresperimus showing levels of Tresperimus in ocular tissues and only low systemic exposure, and limiting the immunosuppressive effect to inflammation developed in the eye. The 15-DSG derivatives or their pharmaceutically acceptable salts are promising active compounds for the treatment / prevention of ocular inflammatory diseases because they allow the posterior uveitis to be treated without systemic immunosuppressive effects, without irritation pain and without blurred vision, nor complications caused by corticosteroids or nonsteroidal anti-inflammatory agents. In addition, because of their solubility in aqueous media, 15-DSG derivatives or their pharmaceutically acceptable salts can be injected directly into the vitreous body and can diffuse to eye tissues. Their effect persists at therapeutic levels for at least 8 days in the rat eye. Such an effect results from the reduction of Th1 and Th17 cell responses in the eye and reduced expression of NOS-2.

In addition, the 15-DSG derivatives or their pharmaceutically acceptable salts can be administered in the form of a medicament by topical administration, for example by instillation of eye drops. It is understood that the examples and embodiments described herein are for illustrative purposes only and that different modifications or different changes in light thereof will be suggested to those skilled in the art and should be included in the spirit and the scope of this application and the appended claims. Although methods and materials similar or equivalent to those described herein may be used in the practice or tests of the present invention, preferred methods and materials are described.

MATERIALS AND METHODS I. Induction of KLA in Lewis Rats Female eight week old Lewis rats (R. Janvier, Genest Saint Isle, France) were systemically immunized with 40 μg retinal autoantigen S antigen (S-Ag) purified from bovine retinas and emulsified in complete Freund's adjuvant as previously described (from Kozak Y., Sainte-Laudy J., Benveniste J. and Faure 3.- P., Eur J Immunol 1981, 11: 612-617). The care and use of the animals were in accordance with the ARVO charter for the use of animals in ophthalmic research and vision.

II. Treatment Protocol Administration of Tresperimus was achieved by intravitreal (IVT) injections (5 μL) in both eyes on days 6, 9 and 12 after immunization with S-Ag. At the end of the experiments, that is, 19-20 days after immunization, the rats were anesthetized by intraperitoneal injection of pentobarbital (30 μg / kg) (Sanofi-Aventis, France) prior to blood collection. by intracardiac puncture. The rats were then killed with a lethal dose of pentobarbital and both eyes and samples were collected for analysis. In a first experiment, one group of rats received Tresperimus (9 mM to obtain a final solution at 1 mM in the vitreous) (n = 6 rats), a control group received a vehicle (saline solution) (n = 6 rats). rats) and a control uveitis group of rats was not treated (n = 6 rats). The animals were clinically examined with a slit lamp from day 9 after immunization with S-Ag until the time of euthanasia. Histopathology of the eyes was performed and immunostaining was done on cryostat sections. Inguinal lymph nodes were removed for cytokine analysis by RT-PCR.

In a second experiment, one group of rats received three Tresperimus injections into the vitreous (11 rats) and the controls were injected with saline (12 rats). They were clinically observed and analyzed for delayed-type hypersensitivity (HTR), cytokine / chemokine and NOS-2 analysis in ocular fluids and cell pellets. In a third experiment, Tresperimus levels in plasma and ocular tissue were measured 1h, 3 and 8 days after the third injection (n = 6 at each time point). The blood, aqueous / vitreous humor (right eye) and retina / choroid (left eye) of 6 animals were collected and weighed at each sampling time. All samples were stored at -20 ° C prior to LC-MS / MS analyzes.

III. Evaluation of the severity of UVA 1. Clinical evaluation The animals were examined with a slit lamp on day 7, ie one day after the first IVT injection, to evaluate whether the drug was well tolerated, then every day from day 11 until the time of euthanasia to assess the onset of the disease and the severity of the disease. The intensity of clinical ocular inflammation was scored on a scale of 0 to 7 for each eye as previously described (from Kozak, Eur J. Imm., 2004). 2. Histopathology At the time of euthanasia (days 19 -20 after immunization), the eyes of enucleated rats were fixed, treated, paraffin sections were made in the pupil-optic nerve plane, and were stained with hematoxylin-eosin-saffron for histological evaluation. The sections were examined blindly by a technician who noted the severity of the KIA on a semi-quantitative scale of 0 to 7 as follows (0) no tissue destruction, (1-2) destruction of the outer segments of the rods and cones, (3-4) destruction of the outer nuclear layer, (5-6) destruction of the inner nuclear layer, and (7) destruction of the ganglion cell layer. 3. Immunohistochemistry The eyes (2 eyes / group) were collected, cryostat sections were made (10 μm) and stained for immunochemistry as previously described on day 19-20 after immunization. The following antibodies were used: polyclonal rabbit antibody against NOS-2 (Beckton Dickinson Biosciences, Transduction Laboratories, San Jose, USA), polyclonal rabbit antibodies against NF-0 / p65, and conjugated goat anti-rabbit antibody. to Alexa 488 (Molecular Probes, Eugene, OR), macrosialin anti-CD68 mouse monoclonal antibody (mAb) (clone ED1) (macrophages and dendritic cells) (Serotec, Oxford, UK) and polyclonal goat anti-mouse Alexa antibody 564 (red). The sections were observed by fluorescence photomicroscopy (FXA, Microphot, Nikon, Melville, NY) and digitized micrographs are obtained with a digital camera (Spot, BFI Optilas, Evry, France).

IV. Evaluation of the immune response 1. Delayed type hypersensitivity The HTR was estimated by an atrial test measuring the specific anti-S-Ag response 18 days after immunization. Rats were challenged with 10 μg S-Ag in the right ear and saline in the left ear. Specific atrial swelling was measured 24 and 48 h after challenge and the difference in thickness (mm) between the two ears was calculated. 2. RNA isolation, reverse transcription PCR in lymph nodes and ocular cells Total RNA was isolated from lymph nodes draining the immunization site, 19-20 days post-immunization, and from cells collected after centrifugation of aqueous / vitreous humor from eyes of each group. The inflammatory cytokines TNF-α, IFN-γ, IL-2, IL-17 and the sense and antisense primers of NOS-2 were obtained from Sigma-Genosys (Paris, France), and amplification by PCR was performed according to the manufacturer's instructions. 3. Concentrations of cytokines and chemokines in ocular fluids (pooled aqueous humor and vitreous body) Concentrations of cytokines and chemokines in ocular fluids were measured 19-20 days after immunization with multiplex ELISA (xMAP technology assay, Clinisciences, Montrouge, France) as described in the manufacturer's protocol: MCP-1 / CCL2, MIP1-a / CCL3, RANTES / CCL5, IP10 / CXCL 10 (IFN-inducible protein-10), GRO / KC, IL -113, TNF-α, IL-2, IFN-γ, IL-4, IL-5, IL6, IL-10, IL-13, IL-17 and VEGF. Detection limits for all analytes have been estimated in the neighborhood of 1 to 10 μg / mL.

V. Protein Determination in Aqueous Mood / Vitreous Mood Aqueous humor / vitreous from Tresperimus or saline treated rat eyes were collected from both eyes of each animal and pooled. Total protein concentration was determined using the Bradford test (Biorad, Les Ulis, France).

VI. Statistical Analysis The data are presented as mean ± standard deviation of mean (SEM). The clinical, histological and HTR scores of the UAE are compared using the non-parametric Mann-Whitney U test followed by the Bonferroni multiple comparison test. A p-value adjusted by the multiple comparison tests is calculated in each experiment.

RESULTS I. Pharmacokinetics of Tresperimus in Ocular Tissues and Plasma Following Intravitreal Injections Tresperimus concentrations in plasma, aqueous / vitreous humor, and retina / choroid following intravitreal injections of Tresperimus to Lewis rats are reported in the literature. Table 1: Effect of an intravitreal injection of Tresperimus on the histopathology of the UAE on day 20 after immunization with S-Ag (M ± ETM) Concentrations of Tresperimus 1 h Immune Rats (N = 6) ( 3 injections IVT) Time after injection 3 days 8 days mood Medium 270 2.2 1.8 aqueous / vitreous ETM 61 1 1 (pM) retina / choroid Medium 155 36 11 (pM) ETM 25 4 4 plasma Average 0, 11 blq blq (NM) ETM 0.03 - - Blq: below the lower limit of quantification (6 ng / mL)

After intravitreal injection of a sterile, substantially iso-osmolar and physiological saline solution at 9 mM Tresperimus, plasma levels of the test sample were quantified only at the first time point 1 h after injection, with low mean concentrations close to 0.1 μM (-40 ng / mL). Ocular tissues were highly exposed to Tresperimus, with significant levels (> 10 μM) in the retina / choroid 8 days after injection.

II. Intravitreal injection of Tresperimus is an effective treatment of UAE; Clinical observation Experimental autoimmune uveoretinitis (UAE) was induced in female Lewis rats. At 6, 9 and 12 days post-immunization, the rats received an intravitreal injection of 5 μl / cc of saline, Tresperimus, or no intraocular treatment. Treatment with Tresperimus led to a significant reduction in the clinical severity of UAE from Day 13 after immunization, compared to rats that received saline injections (Day 13: * p <0) , 02, days 14 to 19: *** p <0.0006), or compared with rats that received no intraocular treatment (day 12: * p <0.02; day 19: *** p <0.0006) (Figure 1).

The severity of the disease was significantly reduced by treatment until 19 days after immunization, indicating that intraocular therapy was very effective.

III. Intraocular injection of Tresperimus protects the retina against destruction and modulates macrophage activity. Rats treated with three Tresperimus injections had a very low degree of UAE at the histological level (mean severity of UAE: 1.45 ± 0.26, n = 10, p = 0.007) compared with the lesions observed in control rats subjected to saline injection (mean degree of severity of UAE: 3.25 ± 0.5, n = 10) (FIG. 2A) and by comparison with rats which received no intraocular treatment (mean degree of severity of UAE: 3.15 ± 0.6, n = 10, p = 0.08). The average histopathological score of UAE was based on retinal alterations. Histopathological examination of retinas from control rats subjected to saline injection (Figure 2B) revealed severe posterior uveitis with significant destruction of the photoreceptor cell layer (a, b, white asterisks), infiltration of subretinal space by inflammatory cells (arrow) and exudates of fibrin in the vitreous body (arrowhead). Many inflammatory cells were present in the vitreous body at the level of the optic disc (arrow) (c). In contrast, in rats treated with Tresperimus (Figure 2C), the photoreceptor cell layer was largely unaffected by destruction (e, white asterisks) or exhibited partial loss of the outer segments (d, arrow) with choroidal infiltration by inflammatory cells (d, arrowhead). No inflammation was visible at the level of the optic disc (f, arrow).

As evidenced by immunostaining in salt-treated control rats, many macrophages and lymphocytes positive for ED1 show nuclear and cytoplasmic expression of NF-KBp65, mainly in the vitreous body where many infiltrations by inflammatory cells occur. are visible.

In contrast, in rats treated with Tresperimus, low infiltration by inflammatory cells is visible in ocular tissues, with a reduced number of infiltrating cells in tissues and ocular media and showing only cytoplasmic expression of NF-KBp65, without nuclear expression of NF-KBp65, suggesting that these cells are deactivated and survive. Interestingly, a reduction in NOS-2 expression was detected in ED1 + cells in rats' eyes treated with three Tresperimus injections as compared to saline injected controls.

IV. Intravitreal injection of Tresperimus has no effect on the systemic immune response in vivo 1. Cytokines in the inguinal lymph nodes (RT-PCR) No difference in TNF-α, IL-2, IFN- IL-17 was detected in inguinal lymph nodes from treated rats and control rats, indicating that the treatment has no systemic effect. 2. Delayed type hypersensitivity HTR was estimated by an atrial test measuring the specific anti-S-Ag response. Rats injected intravitreally with Tresperimus 2, 6, 12 days after immunization with S-Ag were tested on day 18 after immunization with S-Ag subcutaneously by subcutaneous injection into one ear. . Tresperimus-treated rats showed no significant reduction in atrial swelling at 24 h and 48 h compared with control rats that received IVT saline injection (p = 0.8, p = 0.4, respectively). ), which shows that the T cell reactivity with S-Ag in vivo is not reduced by treatment with Tresperimus and confirms that the treatment has no systemic effect (Figure 3). ).

V. Effect of an Intraocular Injection of Tresperimus on the Local Ocular Immune Response 1. Protection of the Blood-Eye Barrier Examination of the amount of proteins in the aqueous-vitreous humor from rats injected with Tresperimus in the vitreous shows day 20, sample collection time, a significantly lower amount of protein (p = 0.05) in the samples collected from rats injected with Tresperimus than in rats having an injection of saline solution (Figure 4A), which means that the treatment induces a decrease in protein leakage and protection of the blood-ocular barrier in the treated rats. 2. Cytokines in the aqueous humor / vitreous body (multiplex ELISA test, xMAP technology test) The concentration of cytokines was measured in the aqueous / vitreous humor at the time of sacrifice, i.e. day 20 after immunization. A significant reduction in IL-2 and IL-17 concentrations (p 0.02, ps 0.03 respectively) and no significant effect on IFN-γ were detected in the aqueous / vitreous humor from rats having Tresperimus injection compared to saline-injected rats. Lower, although not significantly different, levels of IL-6, IL-4 and VEGF were found in rats treated with Tresperimus compared to controls (Figure 4B). Quantities of TNF-α, MIP-ia, IL-13, IL-5 and IP10 were undetectable in the treated and control samples. These results suggest that Tresperimus appears to reduce autoimmunity by inhibiting Th1 and Th17 responses. 3. Expression of NOS-2 in infiltrating inflammatory cells present in the aqueous humor / vitreous body (semi-quantitative RTPCR) We evaluated by semi-quantitative RT-PCR the expression of the NOS-2 mRNA in infiltrating ocular cells obtained after centrifugation of ocular fluids of 9 rats injected with saline compared to 11 rats treated with Tresperimus. In agreement with the reduction of NOS-2 expression in ED1 + macrophages present in the eye fluids of rats treated with Tresperimus as detected by immunohistochemistry, we show that the expression of NOS-2 mRNA in the infiltrating intraocular cells contained in the aqueous / vitreous humor from rats treated with Tresperimus was significantly reduced compared to cells from salt-injected rats (Figure 4C). This result suggests that treatment with Tresperimus reduces activation of intraocular macrophages, thereby reducing retinal pathology in rats treated with Tresperimus.

Claims (10)

REVENDICATIONS1. Derived from 15-deoxyspergualine or a pharmaceutically acceptable salt thereof for use in the treatment and / or prevention of ocular inflammatory diseases. 2. Derivative or a pharmaceutically acceptable salt thereof according to claim 1 for use in the treatment and / or prevention of uveitis. 3. Derivative according to claim 1 or 2 which is a compound of formula (I): ## STR1 ## wherein: (I) n is equal to 6 or 8, A is a bond, CH2, CH (OH), CHF, CH (OCH3), CH2NH or CH2O, R is H or CH3, or a pharmaceutically acceptable salt thereof. The compound of claim 3 which is 2 - [[6- [(aminoiminomethyl) amino] hexyl] amino] -2-oxoethyl N- [4 - [(3-aminopropyl) amino] butyl] -carbamate or a pharmaceutically acceptable salt thereof. 5. Derivative according to claim 4 which is the Tresperimus. The compound of claim 3 which is 2 - [[6- [[aminoiminomethyl) amino] hexyl] amino] -2-oxoethyl N- [4 - [(3-aminobutyl) amino] butyl] -carbamate or a pharmaceutically acceptable salt thereof. 10 15 20 25 30 7. Derivative according to claim 6 which is Anisperimus. An aqueous topical formulation which comprises at least one derivative as defined in any one of claims 3 to 7 as the active substance, and at least one pharmaceutically acceptable excipient suitable for topical administration. An aqueous intravitreal formulation which comprises at least one derivative as defined in any one of claims 3 to 7 as the active substance and at least one pharmaceutically acceptable excipient suitable for intravitreal administration. 10. Use of a derivative of 15-deoxyspergualine or a pharmaceutically acceptable salt thereof, in particular Tresperimus or Anisperimus, in the preparation of a medicament useful for the treatment and / or prevention of inflammatory eye diseases, in particular uveitis.