HK1173950A - Ophthalmic preparations based on bdnf (brain-derived neurotrophic factor) and their use - Google Patents

Description

Ophthalmic formulations based on BDNF (brain-derived neurotrophic factor) and uses thereof

Technical Field

The present invention relates to an ophthalmic preparation in the form of eye drops containing brain-derived neurotrophic factor (BDNF) and a viscosity-controlling agent, preferably galactoxyloglucan extracted from tamarind seed, also known as TS polysaccharide or TSP.

The formulations are useful in the prevention and treatment of neurodegenerative retinal disorders, particularly retinitis pigmentosa, glaucoma (including congenital glaucoma, infant glaucoma, juvenile glaucoma, adult glaucoma, primary open-angle glaucoma, primary angle-closure glaucoma, secondary glaucoma, iatrogenic glaucoma and acute glaucoma), age-related retinopathies such as age-related macular degeneration, vascular and proliferative retinal disorders, retinal detachment, retinopathy of prematurity (ROP) and diabetic retinopathy, all of which can lead to blindness.

Prior Art

Neurotrophic factors (neurotrophics) are proteins synthesized by nerve cells that control the survival and normal trophism of various cells in the nervous system.

Most well understood is Nerve Growth Factor (NGF), discovered by R.Levi-Montalcini and S.Cohen in the middle of the 20 th century.

Other factors with protein structures similar to NGF were subsequently discovered, and we now consider a class of NGF factors (neurotrophic factors) including BDNF, NT-3, NT-4/5 and NT-6, as well as NGF (the first three are predominantly expressed in the nervous system of mammals, while NT-6 is a new member of the neurotrophic factors found in teleost fish, but not in the brain of mammals).

Neurotrophic factors, including neurotrophic factors, are released by nerve cells that synthesize them and bind to specific receptors on membranes.

Despite their similar structures, various neurotrophic factors act through different receptors and thus have different mechanisms of action.

The binding of neurotrophins to their specific receptors (TrkA binding to NGF; TrkB binding to BDNF and part of NT-4; TrkC binding to NT-3) generates a series of events that induce a specific response in the nerve cells.

Different neurotrophic factor receptors are expressed in different regions, in different cells within the same region, and activate specific intracellular signal transduction pathways. Therefore not all areas or nerve cells are logically responsive to each of the 4 neurotrophic factors described above; the limiting factor is the cellular distribution of specific receptors for a given neurotrophic factor.

The distribution of retinal cells capable of synthesizing and releasing NGF, the primitive form of neurotrophic factors and the distribution of retinal cells expressing NGF receptors (TrkA) is very limited, with practical limits to the subset of ganglion cells and astroglial cells (Garcia et al, 2003).

EP 1161256B1 describes ophthalmic formulations containing 200-500. mu.g/ml NGF for administration to an intact ocular surface for the treatment and/or prevention of conditions affecting the sclera, ciliary body, lens, retina, optic nerve, vitreous humor and/or choroid.

Lambian et al reported that these agents increased retinal levels of NGF; however, it could be demonstrated that NGF is unable to produce neuroprotective effects on the retina.

This is consistent with the results reported recently by Shi et al (2007). NGF binds to receptors in both types of retina, TrkA and P75, which exert opposing effects on the nutrition and survival of nerve cells. When exogenous NGF reaches the retina, it may induce two opposite effects on the retinal cells, thereby cancelling each other.

Furthermore, BDNF is abundantly expressed in the mammalian retina with its receptor TrkB. The retina is composed of multiple types of cells arranged in layers. In particular, BDNF is synthesized by certain ganglion cells and amacrine cells of the inner retinal layer, such as dopaminergic cells (Herzog et al, 1994; Perez and Caminos,1995; Hallbook et al, 1996; Herzog and von Bartheld,1998; Karlsson and Hallbook,1998; Bennett et al, 1999; Pollock and Frost,2003; Seki et al, 2003; Chytrova and Johnson, 2004).

The BDNF receptor, designated TrkB, is expressed in many types of retinal cells, including ganglion cells, amacrine cells, and Muller glial cells (Jelsma et al, 1993; Cellerino and Kohler,1997; Di Polo et al, 2000).

WO 97/45135 relates to stable pharmaceutical compositions of BDNF in the form of aqueous solutions or lyophilisates, and in this document, particularly in the section relating to the prior art, BDNF is believed to be useful in the treatment of a variety of conditions, including retinitis pigmentosa. The only explicitly mentioned administration forms are injection preparations.

JP2003048851 relates to BDNF-based ophthalmic formulations, administered in eye drops over the conjunctiva. The formulations disclosed therein contain a variety of viscosity control agents and are described as being equally effective in delivering BDNF to the retina.

The evidence of activity reported in the literature is not convincing, since the concentration range of BDNF is very wide: 0.001-1% w/v, corresponding to 1X10^-2-a concentration range of 10 μ g/μ l [ claim 3, patent scope; [0006 ] according to the invention]Detailed description of paragraph 3, but in the reported examples, the concentration used =0.004% (wt/vol%), corresponding to 4x10^-2μ g/. mu.l, i.e., well below a concentration effective to increase BDNF retinal levels and prevent retinal changes over time due to light exposure (which is 15 μ g/. mu.l or greater, ranging from 15 to 200 μ g/. mu.l, in accordance with the present invention). It should be noted that the application in JP2003048851 is repeated 3 times a day (10. mu.l/time, 0.004% weight/volume) for 5 days, equal to a dose of 1.2. mu.g/day, for a total dose of 6. mu.g. Even considering the daily dose and the total dose, it is too low for neuroprotective effect; indeed, according to the present invention, in order to obtain neuroprotective effects in a photodamaged retina, at least a minimum total dose of 150 μ g needs to be administered topically. Another testThe new data obtained in the model (glaucoma-developing mice) confirmed that of the 3 BDNF concentrations used (1, 5 and 15. mu.g/. mu.l), only the highest concentration (15. mu.g/. mu.l) was effective.

Furthermore, JP2003048851 relates to the verification of the retinal protective effect by measuring the retinal thickness by histological techniques (hematoxylin eosin staining of retinal sections), but does not additionally demonstrate the recovery of retinal function as measured by electroretinogram recording as described in the present invention. It is well known in the art that to demonstrate the neuroprotective efficacy of treatment at any retinal level, the results obtained with only histological/morphological techniques are inadequate; evidence of recovery of retinal function is also needed. It is therefore believed that BDNF ocular formulations administered topically in concentrations reported in examples in JP2003048851 (more generally in the preferred ranges) cannot pass across the ocular surface to internal tissues in sufficient amounts to produce neuroprotective effects that restore retinal function.

WO 2006/046584 relates to sustained release compositions containing HGF, BDNF or PEDF impregnated with cross-linked gelatin hydrogels, which are useful for treating conditions involving retinopathies, such as retinitis pigmentosa degeneration. In particular embodiments, the composition is in the form of microspheres containing a dose of 0.001-1000 μ g bdnf, and can be administered by intraocular injection or subretinal implantation.

EP0958831 describes ophthalmic compositions containing neurotrophic factors selected from the group consisting of BDNF. The compositions may be administered externally, for example in the form of an ophthalmic ointment or solution, or may be formulated as contact lenses.

The concentration range of neurotrophic factors disclosed in EP0958831 is from 0.0001 to 0.5% (weight/volume), i.e.from 1X10^-3To 5 μ gl. The reported concentration ranges are therefore very broad. EP0958831 is broad in that it involves a variety of neurotrophic factors, including BDNF, which are equally effective over a range of concentrations. Neurotrophic factors are known to differ in their concentration due to the density and distribution of receptors that determine their biological effect in different brain regions and in individual nerve cellsAre not equally effective within the scope.

Furthermore, EP 958831 is very vague and unclear as to the effective BDNF concentration range for topical use. On page 3, line 44 (see paragraphs 0022 and 0033, claims 19 and 20) two concentration ranges are given, which do not match (range A max, between 0.0001-0.5% (W/V), equal to 10)^-3To a concentration in the range of 5. mu.g/. mu.l, range B being between 10^-3To 2x10⁵Mu g/l; these two concentration ranges clearly do not correspond. However, according to the invention, the effective concentration of BDNF is equal to/greater than 15 μ g/μ l (range 15-200 μ g/μ l, i.e. higher than range A reported in EP 958831, i.e. its maximum concentration range). The examples in EP 958831 relate to ophthalmic compositions with BDNF concentrations of 0.02, 0.04 and 10. mu.g/l, i.e.well below (low 1X 10)⁶Fold) the lowest concentration demonstrated according to the invention that was effective in increasing BDNF levels in the retina and preventing photodamage and glaucoma damage, i.e. 15 μ g/μ l.

It can therefore be concluded that ophthalmic compositions of BDNF at concentrations reported in EP 958831 applied topically cannot pass through the ocular surface to the internal tissues in sufficient amounts to increase the retinal levels of BDNF and thereby produce a therapeutic effect.

Another neurotrophic factor, NT-4, which binds TrkB, is expressed at low levels in the retina and acts only on the amacrine cell subset, i.e. those cells that synthesize dopamine (calamus et al, 2007).

A lack of BDNF or its receptor can cause severe alterations in retinal function; for example, mice lacking the TrkB receptor (knockout mice) are characterized by a complete loss of retinal responsiveness to light (complete loss of b-wave in the flashlight retinogram; Rohrer et al, 1999).

The LaVail group has demonstrated that intraocular injection of BDNF, but not NGF, is effective in preventing morphological degeneration of photoreceptors caused by photodamage.

Intraocular injection of BDNF and other neurotrophic factors can reduce damage to retinal ganglion cells caused by optic nerve damage, but it is not clear whether BDNF alone exerts neuroprotective effects, i.e., independent of other neurotrophic factors (Watanabe et al, 2003; Yata et al, 2007).

Other neurotrophic factors such as FGF2 have been shown to be equally effective in preventing morphological changes caused by photodamage, but unlike BDNF, their administration has the undesirable effects of activating factors associated with inflammatory responses (LaVail et al, 1987).

CNTF, another neurotrophic factor, also prevents morphological degeneration caused by photodamage (LaVail et al, 1978); unfortunately, recent trials have shown that CNTF-based therapies alter the response of the retina to light, thus leading to a series of limitations in its potential therapeutic applications (McGill et al, 2007).

These results indicate that merely evaluating the morphological effects of the neuroactive molecules is not sufficient for neuroprotective purposes in a model of retinal disorders; importantly, it is necessary to assess whether these molecules protect retinal function and ensure that they do not damage the response of retinal cells to visual stimuli. There is therefore still a need to identify new BDNF formulations that can deliver BDNF to the retina by non-invasive techniques, which avoid highly invasive administration techniques such as intraocular, subretinal or retrobulbar injection, which are not suitable for long-term chronic treatment, because these techniques risk causing e.g. perforation of the eyeball, infection or bleeding.

The present invention provides topical conjunctival applications of various BDNF-containing formulations, wherein the concentration of BDNF ranges from 15 to 200 μ g/μ l, and the total dose of BDNF per administration ranges from 50 to 4000 μ g depending on the size of the eyeball to be treated, depending on the animal species being treated, including humans.

The formulation preferably contains a viscosity control agent. The viscosity control agent is preferably a galactoxyloglucan (TS polysaccharide or TSP) extracted from tamarind seed, having a molecular weight of 500000 to 800000Da, having the following structural formula:

we demonstrate that the formulation significantly increases the BDNF levels of the retina at a given concentration and dose and prevents i) retinal changes from long term exposure to light, and ii) retinal changes from glaucoma.

It has previously been demonstrated (Uccello-Barretta G et al, 2008; Ghelardi E et al, 2004; Burglassi S et al, 2000; Ghelardi E et al, 2000) that TSP can carry pharmaceutically active molecules for topical treatment of the ocular surface. By increasing the retention time of the formulation on the ocular surface, an increased absorption of the active molecule is observed. This property of TSP has been described in its combination with antibiotics (rufloxacin, gentamicin, ofloxacin), antihistamines (ketotifen) and antihypertensives (timolol), all of which are small molecules.

In pharmaceutical formulations containing recombinant proteins such as BDNF, the active ingredient is a protein, a molecule with a high molecular weight that undergoes post-translational modification and spatial bending adjustments to achieve an active three-dimensional configuration. Due to the interaction with the specific receptors and enzymes that recognize them, the biological activity of proteins is closely dependent on their three-dimensional configuration and therefore their ability to interfere with the biochemical processes of the target cells. Protein configuration can be found to be significantly altered by the environment of the recombinant protein. Formulations containing recombinant proteins must therefore ensure that the protein remains in solution in the active configuration and that its stability is ensured.

The present invention demonstrates that TSP ensures stability of BDNF in the formulation, and increases ocular absorption of the formulation as it remains on the ocular surface for extended periods of time, and that it is important to maintain the biologically active configuration of BDNF.

Summary of The Invention

It has now been found that ophthalmic formulations containing BDNF at a concentration of at least 15 μ g/μ l prevent retinal changes upon prolonged exposure to light, and disorders associated with increased intraocular pressure in glaucoma models.

The present invention therefore relates to an ophthalmic preparation in the form of eye drops, which contains brain-derived neurotrophic factor (BDNF). According to a preferred aspect thereof, the present invention relates to a composition comprising a galactoxyloglucan called TSP extracted from tamarind seed.

The invention also relates to the use of BDNF for the preparation of a medicament in the form of eye drops for the prevention and/or treatment of retinal, optic nerve and lateral geniculate neurodegenerative disorders.

The invention also relates to ophthalmic formulations in the form of eye drops containing BDNF for use in the prevention and/or treatment of retinal, optic nerve and lateral geniculate neurodegenerative disorders.

List of drawings

Figure 1-determination of BDNF levels in retina (a), optic nerve (B) and vitreous humor (C) following topical application of BDNF salt solution. The Y-axis is BDNF levels, measured in BDNF-treated eyes and control eyes treated with saline solution; indicates that the difference is significant.

Figure 2-determination of BDNF levels in retina (a), optic nerve (B) and vitreous humor (C) following topical application of BDNF solution containing sodium carboxymethylcellulose. The legends and labels are shown in FIG. 1.

Figure 3-determination of BDNF levels in retina (a), optic nerve (B) and vitreous humor (C) following topical application of BDNF TSP-containing solutions. The legends and labels are shown in FIG. 1.

Figure 4-compares the effect of TSP, saline solution (NaCl), and sodium carboxymethylcellulose (CMC) in carrying BDNF and increasing its retinal concentration (pg/mg protein, see X-axis). After topical treatment with the TSP-containing solution of BDNF, the retinal levels of BDNF were significantly increased compared to the BDNF levels after topical treatment with the salt solution of BDNF and the sodium carboxymethyl cellulose solution. Data are from column a in figures 1, 2, 3.

Figure 5-kinetics of BDNF levels in retina, optic nerve and vitreous humor following topical application of BDNF TSP-containing solutions. BDNF levels were still significantly high (x) in the first 6 hours after treatment.

Figure 6-topical application of BDNF TSP-containing solutions reduced the photodamage-induced change in retinal flash response (flash ERG). The different brightnesses (cd/m) of the eyes treated with BDNF (black marker) or the control eyes treated with TSP only (photodamage control, white marker) were determined and recorded²See x-axis) amplitude of the excited electroretinogram b wave (μ V, see y-axis); indicates that the difference is significant.

Figure 7-topical treatment with BDNF TSP-containing solutions increased the number of photoreceptors that survived photodamage. Photoreceptors were marked with propidium iodide in transverse sections of the retina. Regardless of the method used (number of photoreceptor cell lines (fig. 7B)) or measurement of Outer Nuclear Layer (ONL) thickness (fig. 7C)), photoreceptors on the central and peripheral retina were significantly more in the BDNF-treated eyes than in the vehicle-treated eyes (control).

Figure 8-local application of salt solution (NaCl) of BDNF reduces photodamage-induced damage to the retina's response to light. The legends and labels are shown in fig. 6.

Figure 9-local treatment with BDNF salt solution increased photoreceptor survival after photodamage. The legends and labels are shown in FIG. 7.

Figure 10-topical treatment of photodamage-induced injury to the photoresponse with BDNF sodium carboxymethyl cellulose-containing solutions. The legends and labels are shown in fig. 6.

Figure 11-effect of topical treatment with BDNF solution containing sodium carboxymethylcellulose on photoreceptor survival following photodamage in BDNF-treated eyes and vehicle (control) -treated eyes, legend and labeling is shown in figure 7.

FIG. 12-increase in intraocular pressure (IOP, mmHg) in DBA/2J mice compared to normal mice (C57bl/6J) in experimental glaucoma mouse model. 6¹/₂The increase in IOP was significant in month-old DBA/2J (×).

Figure 13-record responses of retinas of normal mice (C57bl/6J, black bars) and glaucoma mice (DBA/2J) to visual images (images ERG, P-ERG; stimulation at spatial frequency =0.2C/deg, contrast 90%); the amplitude of the P-ERG response (μ V, see y-axis) was determined by stimulating eyes treated with different concentrations of BDNF (1, 5 and 15 μ g/μ l) and eyes treated with vehicle (control eyes, CTRL); indicates that the difference is significant. P-ERG was recorded at 7 months of age in DBA/2J mice (i.e., after an increase in intraocular pressure (IOP)).

FIG. 14-retinal ganglion cells (whole mount slides) from glaucoma DBA/2J mice (7 months of age) were labeled with fluorescent antibody conjugated transcription factor (Brn3 b). A. The left column shows the effect of two weeks of local treatment of BDNF TSP-containing solutions on the central retina (upper line) and peripheral retina (lower line). After treatment with BDNF (left panel), more cells were labeled than in control eyes (CTRL) treated with vehicle only (right panel). B. Local treatment with BDNF on glaucomatous mice (DBA/2J; eyes treated with BDNF; eyes treated with vehicle, CTRL) and normal mice (C57bl/6J) ganglion cells labeled with Brn3b (density in cells/mm)²Measured, as shown on the y-axis) quantification of the effect.

Detailed Description

It has surprisingly been found that by topical administration of exogenous brain-derived neurotrophic factor (BDNF) to the intact ocular surface, especially the conjunctival sac, the BDNF has a neuroprotective effect on retinal cells at both functional and morphological levels, thereby preventing and/or treating neurodegenerative retinal disorders.

BDNF has been shown to be neuroprotective not only to photoreceptors, but also to ganglion cells, i.e., (i) the innermost retinal cells, which deliver fibers to the visual center, (ii) optic nerve fibers, and (iii) the extraretinal visual center, such as the lateral geniculate body.

The present invention relates to ophthalmic formulations containing BDNF (brain derived neurotrophic factor).

The ophthalmic formulation contains BDNF at a concentration that can range from 15 μ g/μ l to 200 μ g/μ l, preferably from 20 to 100 μ g/μ l, even more preferably from 30 to 50 μ g/μ l. The total bioavailable dose per administration is between 50-4000 μ g, depending on the volume of ocular preparation administered and the species of eye to be treated (including humans).

BDNF can be administered alone or in combination with other active ingredients, such as beta-blockers, prostanoids, and carbonic anhydrase inhibitors.

The formulations are prepared in the form of eye drops and may be solutions, suspensions, gels or ophthalmic ointments containing the active ingredient BDNF or a plurality of active ingredients in an acceptable carrier compatible with the active ingredient and tolerated by the eye.

The pharmaceutically acceptable carrier may be a salt solution, preferably a salt solution containing 0.9% sodium chloride.

It has been found that improved levels of absorption of BDNF can be increased if at least one pharmaceutically acceptable carrier is used in the formulation, preferably galactose xyloglucan (TSP) extracted from tamarind seed, which due to its viscosity allows BDNF to remain on the ocular surface for a longer period of time than when applied in a saline solution and to be washed out of the conjunctiva more quickly.

The TSP concentration is variable, preferably from 0.05 to 2% (weight/volume-w/v), even more preferably from 0.25 to 0.5% (w/v).

TPS is clear, viscoelastic and sterile for corneal protection. TSP also forms a long-lasting film on the ocular surface that lubricates and moisturizes the cornea and conjunctiva.

According to a preferred aspect, the viscous solution contains hyaluronic acid, even more preferably a combination of hyaluronic acid and TSP.

The concentration of hyaluronic acid is variable, preferably between 0.05% and 0.8% (w/v), even more preferably between 0.2 and 0.4% (w/v).

According to a preferred embodiment, the formulation may comprise BDNF at a concentration of 15 μ g/. mu.l in a salt solution with 0.9% NaCl.

According to a further preferred embodiment, the formulation may contain BDNF at a concentration of 15 μ g/μ l in a salt solution containing TSP (preferably 0.25%).

The eye drop formulation can be administered directly to the intact ocular surface topically, i.e., in a non-invasive manner, avoiding the use of invasive methods such as intraocular, subretinal, and retrobulbar injections. In particular, the formulation may be administered to the conjunctival sac. The formulations may also be formulated as eyepatches or in contact lenses.

The retina is a partially independent part of the central nervous system; the presence of different types of barriers, including the blood-retinal barrier, prevents non-specific diffusion of compounds such as macromolecules to the retina. Intraocular penetration of topically applied pharmaceutically active compounds is regulated by barriers in these tissues through systemic absorption and metabolic breakdown of enzymes in the cornea and conjunctiva. After instillation, the pharmaceutically active compound must cross a complex system of blood barriers, including the blood-retinal barrier, to penetrate as far as the underlying tissues of the retina.

In addition, the retina is connected via the optic nerve to the visual center, such as the dorsal lateral geniculate (dLGN), by ganglion cells, which are where optic nerve fibers occur.

As demonstrated in the experimental section, BDNF can be delivered to the retina when administered topically according to the present invention, resulting in an increase in its retinal concentration to a level that can serve a functional and morphological level of neuroprotective effect.

It has also been surprisingly found that, as demonstrated by experimental evidence, ganglion cells allow for the anterograde transport of BDNF, allowing BDNF to prevent and treat degeneration of not only ganglion cells, but also optic nerve fibers, as well as the extension of the condition to the extraretinal visual center, such as the lateral geniculate body.

The present invention also relates to the use of BDNF for the manufacture of an ophthalmic medicament for topical administration to the intact ocular surface in the form of eye drops for the prevention and/or treatment of retinal, optic nerve and lateral geniculate neurodegenerative disorders, especially degenerative retinopathies (e.g. retinitis pigmentosa and glaucoma), age-related retinopathies such as age-related macular degeneration, vascular and proliferative retinal disorders, retinal detachment, retinopathy of prematurity (ROP) and diabetic retinopathy, among other diseases that can lead to blindness. The formulations according to the invention are useful for the prevention and/or treatment of neurodegenerative diseases of the retina, the optic nerve and the lateral geniculate body, such as in particular retinitis pigmentosa and glaucoma (including congenital glaucoma, infantile glaucoma, juvenile glaucoma, adult glaucoma, primary open-angle glaucoma, primary closed-angle glaucoma, acute glaucoma, iatrogenic glaucoma and secondary glaucoma).

Glaucoma is one of a series of progressive conditions affecting the eye that, if not properly treated, can lead to blindness due to loss of ganglion cells and progressive atrophy of optic nerve fibers.

Glaucoma is characterized by increased intraocular pressure (IOP), which can damage ganglion cells and optic nerve fibers either directly (mechanically) or indirectly by inducing ischemia of the retinal blood vessels supplying the medial retina. In the progressive stage, glaucoma can affect the visual center, such as the lateral geniculate body, in addition to the retina, until it eventually affects the visual cortex.

It has been found that treatment with effective concentrations of BDNF not only prevents and reduces degeneration of photoreceptors due to prolonged light exposure (photodamage), but also maintains the response of the retina to light; moreover, the use of the glaucoma test model demonstrates that topical application of BDNF prevents degeneration of retinal ganglion cells in the animal glaucoma model caused by elevated intraocular pressure (IOP); in both animal models, BDNF did not alter retinal responses to visual stimuli.

The following examples further illustrate the invention.

Examples

Examples of formulations

·PreparationSalt solution of 1-BDNF: dissolve 150. mu.g BDNF in 10. mu.l salt solution containing 0.9% NaCl;

·preparation 2-BDNF sodium carboxymethyl cellulose-containing salt solution: mu.g BDNF was dissolved in a solution consisting of 5. mu.l of a salt solution containing 0.9% NaCl and 5. mu.l of a 0.4% sodium carboxymethylcellulose solution.

·Preparation 3-TSP containing salt solution of BDNF: mu.g BDNF was dissolved in 5. mu.l salt solution containing 0.9% NaCl and 5. mu.l 0.5% TSP.

·Preparation 4-hyaluronic acid (0.2%) containing salt solution of BDNF: mu.g BDNF was dissolved in 5. mu.l salt solution containing 0.9% NaCl and 5. mu.l 0.4% hyaluronic acid.

·Preparation 5Hyaluronic acid (0.4%) containing salt solution of BDNF: mu.g BDNF was dissolved in 5. mu.l salt solution containing 0.9% NaCl and 5. mu.l 0.8% hyaluronic acid.

·Preparation 6-hyaluronic acid and TSP containing salt solution of BDNF (I): mu.g BDNF was dissolved in 5. mu.l salt solution containing 0.9% NaCl and 5. mu.l 0.4% hyaluronic acid and 0.4% TSP.

·Preparation 7-hyaluronic acid and TSP containing salt solution of BDNF (II): mu.g BDNF was dissolved in 5. mu.l salt solution containing 0.9% NaCl and 5. mu.l 0.8% hyaluronic acid and 0.4% TSP.

·Preparation 8-hyaluronic acid and TSP containing salt solution of BDNF (II): mu.g BDNF was dissolved in 5. mu.l salt solution containing 0.9% NaCl and 5. mu.l 0.4% hyaluronic acid and 0.6% TSP.

Biological analysis

2.1 example-BDNF levels in the vitreous humor, retina and optic nerve of the eye after 6 hours of topical treatment with BDNF-based formulations.

·Using formulations 1, 2 and 3 above:

the test was performed on white rats (Wistar rats, Harlan, italy); a BDNF sodium carboxymethylcellulose-containing salt solution or a TSP-containing salt solution was topically applied, instilled into the conjunctival sac of one eye, while the other eye was administered as a control with a BDNF-carrying solution (placebo).

·BDNF level determination in retina, vitreous humor and optic nerve

Animals were sacrificed after deep anesthesia 6 hours after intraperitoneal injection of urethane (20%). The eye was then removed and the level of BDNF in vitreous fluid, retinal homogenate and optic nerve homogenate was determined in eyes treated with BDNF and eyes treated with vehicle solution only (control eyes). The assay was performed by immunoassay (ELISA; BDNF Emax immunoassay system, Promega, Madison, Wis., USA). The amount of BDNF in the optic nerve was also determined to determine whether the externally applied BDNF was absorbed and transported by retinal cells, particularly retinal ganglion cells, which form the optic nerve through their fibers.

The results of topical application of BDNF salt solution (0.9% NaCl), expressed as mean BDNF concentration values in retina (a), optic nerve (B) and vitreous humor (C), expressed as pg/mg protein, are shown in fig. 1.

Statistical analysis was performed using student's t-test comparing BDNF treated and control eyes; in all cases, the difference between treated and control eyes was statistically significant (, p < 0.05).

The results of topical application of BDNF to the sodium carboxymethylcellulose-containing solution (0.2%) are shown in fig. 2, and the results of topical application of BDNF to the TSP-containing solution (0.25%) are shown in fig. 3; both were analyzed using student's t-test statistics comparing BDNF treated and control eyes. In all cases, the difference between treated and control eyes was statistically significant (, p < 0.05).

Figure 4 shows comparative levels of retinal BDNF for each type of solution/vehicle used; this assay readily compares the effect of different solutions/carriers at the same BDNF concentration (10 μ l solution, containing 150 μ g BDNF). Topical treatment with the TSP-containing solution of BDNF resulted in significantly higher retinal BDNF levels (student t-test, p <0.05) than the other two formulations used (i.e. BDNF salt solution and BDNF sodium carboxymethyl cellulose-containing solution). Topical treatment with BDNF solutions containing sodium carboxymethylcellulose proved to be the least effective in increasing retinal BDNF levels.

2.2 example-determination of BDNF levels in retina, vitreous humor and optic nerve at various times following topical treatment of eyes with BDNF TSP-containing solutions

It was investigated which content of retina, vitreous humor and optic nerve remained high after a single topical application of BDNF. The study was conducted using TSP-containing carriers, which have proven to be most effective in promoting transscleral entry of BDNF into the retina, optic nerve and vitreous humor. The kinetics of local treatment of BDNF levels in the posterior retina, optic nerve and vitreous humor of the eye were subsequently studied using TSP. Each experimental group treated N =5 eyes. BDNF concentrations in the retina, optic nerve and vitreous humor were determined at different time intervals after administration of 0.25% TSP solution containing BDNF (10 μ l of solution containing 150 μ g BDNF). Control eyes were treated with only the carrier solution containing 0.25% TSP. This test was used to establish the time trend of BDNF levels after a single topical application. Mean BDNF values (y-axis; pg/ml) in the retina, optic nerve and vitreous humor at 6, 12 and 24 hours after application are shown. Figure 5 shows that BDNF levels in the retina remained statistically high, returning to baseline levels within 12-24 hours. Statistical analysis was performed using student's t-test: in a, p <0.01 compared to control eyes. The results of this experiment show that topical application every 12 hours is sufficient to maintain BDNF at high levels in the retina when chronically treated with BDNF carried by artificial tears, particularly based on TSP.

2.3 example-neuroprotective Effect of topical application of BDNF-based formulations

To confirm the neuroprotective effect of BDNF after topical application in the conjunctival sac, an experimental model of retinal degeneration induced by photodamage was used in animal models; this model is widely used to study retinal photoreceptor degeneration caused by long-term exposure to intense light sources (La Vail et al, 1987; Rex et al, 2003). Photoreceptors die by apoptosis, which is caused by excessive absorption of photons by the visual pigment rhodopsin, leading to a change in the pigment regeneration cycle, which ultimately involves pigment epithelial cells. The experimental animal model used was a white rat in which photoreceptors had significant sensitivity to light (Surace et al, 2005). The original experimental protocol proposed by the LaVail et al research group (LaVail et al, 1987) was modified and expanded (Rex et al, 2003).

The following BDNF-based formulations were used:

a) 0.25% TSP solution of BDNF (10. mu.l of solution containing 150. mu.g BDNF).

b) Salt solution of BDNF (0.9% NaCl-10. mu.l solution containing 150. mu.g BDNF).

c) 0.2% sodium carboxymethylcellulose solution of BDNF (10. mu.l of a solution containing 150. mu.g BDNF).

Rats were treated with the formulation listed above for eyes, followed by photodamage treatment. Control eyes were treated with vehicle solution only. Each experimental group treated N =4 eyes. Specifically, after 6 hours of treatment (eyes treated with BDNF, control eyes treated with vehicle solution only), these rats were exposed to light for a long period of 48 hours (animal photodamage model, light source intensity 1000 lux). Prolonged exposure to light results in the degeneration of many photoreceptors on the retina of white rats. The neuroprotective effect exerted by BDNF is validated by morphological methods designed to evaluate photoreceptor survival and functional methods to record the response of the retina to light (iridescent electroretinograms [ ERGs ], widely used to evaluate the functional state of the outer retina in patients with retinal disorders). Considering the small number of cones constituting the basis of ERG response under photoadaptive conditions and the small ERG amplitude under photoadaptive conditions in rats, only the glistened ERG under dark adaptation conditions was recorded, which represents the response of the rod cells as the main component in rat retina. Flash ERG (dark adaptation) was recorded 7 days after the end of the photodamage cycle.

-Preparation a)

Fig. 6 shows the b-wave amplitude of the flash ERG according to the luminance under the dark adaptation condition. As shown in fig. 6, topical application of BDNF TSP-containing solutions significantly reduced the effect of photodamage on retinal glint response (glint ERG). In fact, the amplitude (mean amplitude value in μ V) of the BDNF-treated eyes was significantly higher than the control eyes,. p <0.05 (one-way ANOVA).

-Preparation b)

Fig. 8 shows the b-wave amplitude according to the luminance under the dark adaptation condition. The results indicate that the salt solution of BDNF also reduces the photodamage-induced changes in the retinal photoresponse (iridescent ERG). The amplitude of b-waves in the light-damaged eye of BDNF-treated rats was higher than in the control eye; the amplitude (mean amplitude value in μ V) of the eyes treated with the salt solution of BDNF was significantly higher than the control eyes,. p <0.05 (one-way ANOVA).

-Preparation c)

Fig. 10 shows the b-wave amplitude according to the luminance under the dark adaptation condition. The figure shows that the amplitude (mean amplitude value in μ V) of the eyes treated with BDNF sodium carboxymethylcellulose-containing solution at the highest brightness is significantly higher than the control eye, p <0.05 (one-way ANOVA). In conclusion, sodium carboxymethyl cellulose has proven to be less effective in preventing retinal photoresponsive damage in terms of functional recovery of the retinal response to light than TSP and saline solutions.

The effect of topical treatment with the above BDNF-based formulation on retinal photoreceptor degeneration was subsequently evaluated in the retina of eyes where flashing ERGs were recorded.

The effect of BDNF local treatment on photoreceptor degeneration was quantified by counting the number of rows of photoreceptors surviving photodamage and determining the thickness of the outer nuclear layer of the retina (ONL) containing photoreceptor cell bodies. To perform the assay, photoreceptor nuclei were labeled with iodopropyridine.

-Preparation a)

The results obtained are shown in FIG. 7. Figure 7A shows transverse sections of the retina of eyes treated with BDNF (in 0.25% TSP) and control eyes. To perform the assay, photoreceptor nuclei were labeled with iodopropyridine. Regardless of the method used (photoreceptor cell line count (fig. 7B) or thickness of Outer Nuclear Layer (ONL) (fig. 7C), the photoreceptor number in the central and peripheral retinas in the eyes treated with BDNF was significantly greater than in the eyes treated with vehicle only (control) (student t-test @, p < 0.001).

It was subsequently demonstrated that the BDNF TSP-containing solution protects the retina from light damage when applied topically in the conjunctival sac.

-Preparation b)

Fig. 9(a) shows retinal cross sections of the right eye (saline solution, 0.9% NaCl) and the left eye treated with saline only (control) treated with BDNF. The effect of BDNF local treatment on photoreceptor degeneration was quantified by counting the number of rows of photoreceptors surviving photodamage (fig. 9B) or determining the thickness of the outer nuclear layer of the retina (ONL) containing the photoreceptor cell bodies (fig. 9C). The difference in central and peripheral retina (photoreceptor row number or ONL thickness) between treated and control eyes proved significant (student t-test, p < 0.001).

Local treatment with BDNF salt solution increased the number of photoreceptors surviving photodamage compared to control eyes.

-Preparation c)

Finally, the effect of BDNF topical treatment (sodium carboxymethylcellulose solution) on photoreceptor degeneration was quantified by counting the number of rows of photoreceptors surviving photodamage (fig. 11B) or determining the thickness of the outer nuclear layer of the retina (ONL) containing the photoreceptor cell bodies (fig. 11C).

Considering the results obtained, topical application of TSP of BDNF and salt solutions was considered to have neuroprotective effect on light damage in terms of functional recovery after photodamage and photoreceptor degeneration prevention, whereas treatment with BDNF solutions containing sodium carboxymethylcellulose was less effective at the same BDNF concentration.

It was also demonstrated that treatment with BDNF did not result in altered function of the retina and did not impair its response to visual stimuli.

2.4 example: neuroprotective effects of repeated topical application of BDNF in experimental glaucoma models

Glaucoma is a retinal degenerative disorder of various origins and has various forms (congenital glaucoma, infantile glaucoma, juvenile glaucoma or adult glaucoma depending on age; primary glaucoma, primary open-angle glaucoma or primary closed-angle glaucoma depending on pathogenesis, and secondary glaucoma induced by other disorders, including iatrogenic glaucoma). The most common form of glaucoma, Primary Open Angle Glaucoma (POAG), is characterized by increased intraocular pressure, which causes dysfunction and subsequent degeneration of ganglion cells associated with optic atrophy; symptoms are a gradual loss of vision, eventually blindness. The mechanisms responsible for the dysfunction and degeneration of ganglion cells with optic atrophy are not fully understood at present, but it is generally thought that increased intraocular pressure (IOP) induces mechanical damage to optic nerve fibers in the lamina cribosa and ischemic changes in the optic nerve head and inner retina. In recent years, drug therapy has been aimed at lowering IOP, but a large number of patients are resistant to existing drug therapy and suffer from progressive irreversible loss of visual function. Currently no drug is designed for neuroprotection of retinal ganglion cells and optic nerve fibers to prevent vision loss and return vision to normal. In this patent, we propose local treatment with BDNF in the conjunctival sac in order to increase the BDNF level of the retina through a stable pathway, thereby counteracting the progressive dysfunction and subsequent degeneration and death of ganglion cells. This protocol is based in part on the fact that the BDNF receptor (termed TrkB) is expressed in ganglion cells (Jelsma et al, 1993). To confirm this hypothesis, we used the most common experimental model of spontaneous glaucoma, a double mutant mouse called DBA/2J (John et al, 1998; Chang et al, 1999). DBA/2J mice have homozygous mutations in two independent genes; the first is tyrosine-related protein (Tyrp1-/-) encoding melanoprotein, and the second is membrane glycoprotein (Gpnmb-/-). The mice are characterized by progressive delivery of intraocular pressureIncreasingly, the retina's response to structural visual stimuli is also gradually lost, being dependent on inner retinal/ganglion cells; in humans and animal models, this retinal response is called an image electroretinogram (P-ERG; Domenici et al, 1991; Venturi and Portetti, 2006; Falsini et al, 2008). Following ganglion cell dysfunction, the cells denature and the optic nerve fibers progressively atrophy (Venturi et al, 2006). As shown in fig. 12, in this mouse glaucoma model (DBA/2J), IOP began to increase 5 months after birth; the IOP of DBA/2J mice (N =10) was already significantly higher at 61/2 months (t-test;. p)<0.05) values for normal mice (C57bl/6J; N =5) and 5 months old DBA/2J mice (N = 9). Fig. 13 shows the amplitude of the response of inner retinal/ganglion cells to structural visual stimuli (P-ERG; intensity characteristics of visual image stimuli as spatial frequency =0.2C/deg, contrast 90%), recorded using corneal electrodes connected to an amplifier, connected to a computer for on-line analysis. As shown in FIG. 13, P-ERG of 7-month-old DBA/2J mice (CTRL, N =4) had changed (P-ERG amplitude significantly decreased; student's t-test, P; (P-ERG) was altered<0.05). Starting from 6.5 months (i.e., the time at which IOP is steadily increasing) (fig. 12), BDNF TSP-containing solution is topically applied to one eye for two weeks of repeated treatment (once every 48 hours) and vehicle is applied to the other eye (control eye). Three different BDNF concentrations were used (N =4DBA/2J mice per group): 1. 5 and 15. mu.g/. mu.l. As shown in the histogram, topical application of BDNF at concentrations of 15. mu.g/. mu.l instead of 1 and 5. mu.g/. mu.l (150. mu.g in 10. mu.l of a solution containing 0.25% TSP, ophthalmic preparation a) prevented P-ERG changes in DBA/2J mice (compare data for treated and control eyes; student's t-test, p.<0.05). To confirm whether P-ERG changes correspond to changes in ganglion cells labeled with immunohistochemistry, we used the transcription factor Brn3b expressed in ganglion cells; mice with mutations in this factor (Brn3b-/-) are associated with alterations in ganglion cells (Badea et al, 2009). FIG. 14A shows an enlarged view of a retinal preparation in which ganglion cells were labeled green with a fluorescent antibody and analyzed by confocal microscopy. The number of labeled ganglion cells in the central and peripheral retinas in the eyes of DBA/2J mice decreased significantly. FIG. 14B shows the quantification of labeled cells in terms of density (number of cells/mm)²)。Treatment with the TSP-containing solution of BDNF at a concentration of 15 μ g/μ l for 2 weeks prevented a decrease in Brn3 b-labeled cells compared to vehicle-treated control eyes alone (student t-test, p;)<0.05)。

From the reported data, it can be concluded that in experimental models of glaucoma, repeated local treatment with BDNF prevented the functional changes of the ganglion cells and restored retinal visual function. The minimum effective concentration of BDNF that could exert a protective effect on ganglion cell function was 15 μ g/μ l.

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Claims (12)

1. An ophthalmic formulation in the form of eye drops comprising Brain Derived Neurotrophic Factor (BDNF) at a concentration of at least 15 μ g/μ l.

2. An ophthalmic formulation as claimed in claim 1 wherein the BDNF is in the range 15 to 200 μ g/μ l.

3. The ophthalmic formulation according to claim 1 or 2, further comprising a salt solution as a pharmaceutically acceptable carrier.

4. An ophthalmic formulation as claimed in any preceding claim, wherein the salt solution contains 0.9% sodium chloride.

5. The ophthalmic formulation of any one of the preceding claims, further comprising a viscous solution as another pharmaceutically acceptable carrier.

6. The ophthalmic preparation according to any one of the preceding claims, wherein the viscous solution is a solution comprising polysaccharides (TSP) extracted from tamarind seeds.

7. An ophthalmic formulation as claimed in any preceding claim, wherein the concentration of TSP is in the range 0.05 to 2% w/v.

8. The ophthalmic preparation of any one of the preceding claims, further comprising hyaluronic acid.

9. The ophthalmic formulation of any one of the preceding claims, wherein the viscous solution comprises TSP and hyaluronic acid.

10. Ophthalmic preparation in the form of eye drops comprising BDNF for use in the prevention and/or treatment of neurodegenerative diseases of the retina, optic nerve and lateral geniculate body.

11. The ophthalmic preparation according to claim 10, which is used for the prevention and/or treatment of retinitis pigmentosa.

12. An ophthalmic preparation according to claim 10 for the prevention and/or treatment of primary open angle glaucoma.