Fluorescently labeled intranasal delivery

Abstract

The present invention provides a method of diagnosing a CNS disease comprising administering a fluorescent label of retinal integrity to a subject and generating an image of the eye of the subject wherein delivery of the fluorescent label by intranasal administration and the fluorescent label of retinal integrity of intranasal administration used in this method are also provided. Furthermore, the present invention provides a pharmaceutical composition comprising annexin or a functional fragment or derivative thereof bound to a compound of 2 kDa or less wherein the pharmaceutical composition comprises annexin or a functional fragment or derivative thereof bound at a concentration of at least mg / ml.

Description

[0001] This application is a divisional application of a patent application with an application date of April 23, 2019, application number 201980027922.2, and invention name “Intranasal delivery of fluorescent labels”. Technical Field

[0002] The present invention relates to fluorescent markers for use in diagnosing CNS diseases. Background Art

[0003] Retinal assessment can provide information about the presence and severity of many systemic diseases. For example, the detection of apoptotic retinal cells (DARC) describes a technique for monitoring the rate of retinal cell death and has recently completed a Phase I clinical trial for the diagnosis of glaucoma (Cordeiro et al., 2017) and is currently in Phase II clinical trials for a range of neurodegenerative diseases, including glaucoma, age-related macular degeneration, optic neuritis, and Down's disease (as a model for Alzheimer's disease). The technique currently involves the intravenous administration of a novel fluorescent agent called Anx776, which contains a modified version of the endogenous protein Annexin A5 conjugated to the near-infrared fluorophore Dy-776 (Cordeiro et al., 2017).

[0004] DARC uses the unique optical properties of the eye to directly visualize apoptosis in single nerve cells in patients using a fluorescently labeled derivative of human annexin V. Annexin V is a human protein that has the ability to bind to phosphatidylserine (PS) in the presence of calcium. PS is present in the plasma membrane of every cell, however, apoptotic cells express PS in the outer leaflet of the plasma membrane. Annexin V binds to the exposed PS, thereby identifying apoptotic cells.

[0005] For both the Phase I and Phase II clinical trials of DARC, Anx776 was administered intravenously. Other fluorescent molecules used to detect retinal diseases by confocal scanning laser ophthalmoscopy (cSLO) imaging are also mainly administered intravenously. For example, sodium fluorescein and indocyanine green (ICG) are usually administered intravenously to mark the retinal vasculature to identify abnormal blood vessel growth (angiogenesis) and leakage associated with age-related macular degeneration (AMD) (Yozik et al., 2005).

[0006] The use of intravenous administration requires direct medical supervision, meaning that patients are not routinely evaluated unless suspected pathology is present. Therefore, alternative techniques that can be used to evaluate the retina are needed to allow patients to have more extensive access to such evaluations and potentially diagnose retinal and neurodegenerative diseases earlier. Summary of the invention

[0007] According to a first aspect of the present invention, the present invention provides a fluorescent marker of retinal integrity for diagnosing central nervous system (CNS) diseases, wherein the fluorescent marker is delivered by intranasal administration. Surprisingly, the inventors of the present application found that intranasal administration of the fluorescent marker resulted in rapid accumulation of fluorescence in the retina, and the results of retinal imaging were the same as those obtained after previous intravenous administration of the same fluorescent agent.

[0008] Without being bound by theory, the inventors believe that the intranasally delivered fluorescent marker of retinal integrity is absorbed systemically into the circulation. Due to local administration, the fluorescent marker of retinal integrity may require a reduced dose and have a faster onset of action compared to systemic administration of the same agent.

[0009] As used herein, a fluorescent marker for retinal integrity refers to a fluorescent marker used to query the health of retinal cells and / or the integrity of retinal cells and / or blood vessels. For example, the fluorescent marker can identify and / or distinguish between apoptotic and necrotic cells, or identify areas of vascular leakage or angiogenesis. In response to excitation, the fluorescent marker emits light and may have an emission wavelength of about 400 nm to about 1000 nm, preferably about 500 nm to about 900 nm.

[0010] The fluorescent marker of retinal integrity is preferably provided in a form suitable for topical delivery, especially intranasal delivery. The fluorescent marker of retinal integrity can be provided in the form of a pharmaceutical composition, which can be provided in the form of a solution, suspension or dry powder suitable for inhalation. The pharmaceutical composition comprising the fluorescent marker of retinal integrity can be sterile and can include one or more pharmaceutically acceptable carriers or excipients. Suitable carriers and excipients are familiar to those skilled in the art and can be optimized according to the expected route of intranasal delivery. For example, the composition comprising the fluorescent marker of retinal integrity can also include a buffer, an adhesive, a preservative, a thickener or an antioxidant, such as trehalose.

[0011] In some embodiments of the present invention, the fluorescent marker of retinal integrity can be a fluorescent marker of retinal vascular integrity. Typically, the fluorescent marker will enter and circulate in the retinal blood vessels, making it easy to observe abnormal blood vessel growth (angiogenesis) and / or leakage sites.

[0012] The fluorescent marker of retinal vascular integrity may have a molecular weight of about 2 kDa or less, or 1 kDa or less. In some embodiments of the present invention, the fluorescent marker of retinal vascular integrity may have a molecular weight of about 100 Da, or about 1 kDa, preferably about 300 Da to about 800 Da.

[0013] Suitable fluorescent markers of retinal vascular integrity for use in the present invention include fluorophores, and particularly preferred fluorescent markers of retinal vascular integrity include sodium fluorescein and indocyanine green (ICG).

[0014] As described above, the fluorescent marker of retinal vascular integrity will be administered intranasally, and can be administered at the same or less dosage as the dosage of the same marker administered intravenously. For example, fluorescein sodium can be administered intranasally at a concentration of about 50 mg / mL to about 500 mg / mL, preferably at a concentration of about 50 mg / mL to about 200 mg / mL. In some embodiments of the invention, fluorescein sodium can be administered intranasally at a concentration of about 100 mg / mL. ICG can be administered intranasally at a concentration of about 1 mg / mL to about 100 mg / mL, preferably about 25 mg / mL to about 100 mg / mL. In some embodiments of the invention, ICG can be administered intranasally at about 50 mg / mL.

[0015] In some embodiments of the present invention, the fluorescent marker of retinal integrity can be a marker of retinal cell integrity. Typically, the fluorescent marker of retinal cell integrity includes a fluorescent tag and one or more markers of apoptosis, necrosis, cell activity, cell stress or protein aggregation.

[0016] Fluorescent labels refer to compounds or molecules (such as fluorophores) that emit light in response to excitation, which can be selected for use because the signal-to-noise ratio is improved, thereby improving image resolution and sensitivity while complying with exposure safety standards and avoiding phototoxic effects. Preferably, fluorescent labels cause little or no inflammation when administered. Fluorescent labels can have a wavelength in the infrared or near-infrared range. Fluorescent labels can have an emission wavelength of about 400nm to about 1000nm, preferably about 500nm to about 900nm, and more preferably about 700nm to about 900nm.

[0017] Suitable fluorescent labels include one or more of fluorescein sodium, indocyanine green (ICG), curcumin, IRDye700, IRDye800, Dy-776, Dy-488 and D-781. In a preferred embodiment of the present invention, the fluorescent label is Dy-776.

[0018] Retinal cell integrity fluorescent labels can be prepared using standard techniques for combining fluorescent tags with markers. Such labels can be obtained from well-known sources, such as Dyomics. Suitable techniques for combining labels with markers are known in the art and can be provided by the manufacturer of the label.

[0019] Apoptosis markers refer to markers that distinguish cells undergoing apoptosis from living cells. In addition, the marker should preferably be able to distinguish apoptotic cells from necrotic cells. For example, it can be a compound or molecule that specifically binds to apoptotic cells but does not bind to living cells or necrotic cells. For example, apoptosis markers include annexin family proteins. Annexin is a protein that reversibly binds to the cell membrane in the presence of cations. In particular, annexin can bind to phosphatidylserine (PS) under conditions of calcium presence. PS is present in the plasma membrane of each cell, but apoptotic cells express PS in the outer leaflet of the plasma membrane. Annexin binds to exposed PS, thereby identifying cell apoptosis. Annexins that can be used in the present invention can be natural or recombinant. The protein can be complete or a functional fragment, that is, a fragment or part of the annexin specifically binds to the same molecule as the whole protein. In addition, functional derivatives of such proteins can be used. In particular, functional fragments or derivatives of annexin can include molecules containing "annexin repeats", which are domains of about 70 amino acids that are conserved within a single annexin and between family members. A variety of annexins are available, such as those described in U.S. Patent Application Publication No. 2006 / 0134001A. A preferred annexin is annexin 5, which is well known in the art. Other annexins that can be used include annexins 11, 2, and 6. Other apoptotic markers are known in the art, for example, including the C2A domain of synaptotagmin I, duramycin, non-peptide-based isatin sulfonamide analogs, such as WC-II-89 and ApoSense, such as NST-732, DDC, and ML-10 (St. Hubert et al., 2009).

[0020] In a preferred embodiment of the present invention, the marker of apoptosis is annexin 128 (Tate et al., 2005). Annexin 128 is a variant of annexin 5 that differs from the wild type by two single amino acid mutations. Annexin 128 includes an exposed thiol group at the N-terminus, thereby increasing the binding efficiency of molecular tags (such as fluorescent markers).

[0021] In a preferred embodiment of the present invention, the retinal cell integrity fluorescent marker includes Annexin 128 combined with Dy-776, and Annexin 128 and Dy-776 can be combined at a 1:1 fluorescent Labec labeling ratio.

[0022] Necrotic markers refer to markers that distinguish cells that undergo necrosis from living cells and cells that undergo apoptosis. For example, it can be a compound or molecule that specifically binds to necrotic cells but does not bind to living cells or apoptotic cells. Necrotic markers include propidium iodide (PI), an intercalator that binds to nucleic acids with little or no sequence preference. Other necrosis markers are known in the art, including pyrophosphate, antimyosin, gluconate, hypericin and its derivatives, such as hypericin monocarboxylic acid and palmitic acid, such as bis-hydrazide-bis-DTPA palmitic acid. In particular, 99mTc-pyrophosphate, 111-antimyosin, 99mTc-gluconate and methylene blue are used.

[0023] Other indications of cell activity can also be used to identify apoptotic or necrotic cells. For example, changes in mitochondrial function can be observed, reactive oxygen species (ROS) can be used as a marker, and calcium ions can be used as a marker. Cell activity markers can include one or more of membrane dyes, mitochondrial dyes, autophagy dyes, necrosis dyes, and calcium flow. More particularly, cell activity markers can include Fluo-3, N-(fluorescein-5-thiocarbamoyl)-1,2-hexadecanoyl-sn-glycero-3-phosphoethanolamine, JC-1, dual emission JC-9, reduced rhodamine and romamine, rhodamine 123, and Di-8-ANePPS. Cell stress markers can include one or more markers of lipid peroxidation, glutathione (GSH), or reactive oxygen species (ROS) (such as superoxide, peroxyl radicals, hydrogen peroxide, hydroxyl, and peroxynitrite).

[0024] Protein aggregation in the retina can occur intracellularly or extracellularly within or around retinal neurons, but typically occurs outside the retinal vasculature. The presence of protein aggregates is known to be associated with cells undergoing neurodegeneration. Suitable protein aggregation markers include one or more of Congo red, curcumin or thioflavin S.

[0025] As described above, the present invention provides a fluorescent marker of retinal integrity delivered intranasally for diagnosing CNS disorders in a subject. The subject is preferably a mammal, including a human, and may be a pediatric patient or an elderly patient. CNS disorders can be identified by analyzing retinal integrity, see the methods described in WO2009 / 077750 and WO2011 / 055121. For example, a fluorescent marker of retinal integrity can be used to display the distribution of cell death in the retina by marking apoptotic and / or necrotic cells, and different neurodegenerative diseases with different apoptotic and / or necrotic activity patterns can be distinguished based on the distribution.

[0026] Central nervous system diseases may be inflammatory (such as arthritis or granuloma), infectious (such as viral, encephalitis or bacterial), vascular (such as angiogenesis, occlusion or metabolism), or degenerative (such as glaucoma, age-related macular degeneration (AMD), Alzheimer's disease or Parkinson's disease). In some embodiments of the present invention, the CNS disorder may be a neurodegenerative disease, especially an ocular neurodegenerative disease. The term "ocular neurodegenerative disease" is well known to those skilled in the art and refers to a disease caused by the gradual and progressive loss of ocular neurons, including but not limited to glaucoma, AMD, optic neuritis and diabetic retinopathy. Neurodegenerative diseases include Parkinson's disease, Alzheimer's disease, Huntington's disease and Friedrich's ataxia. In some embodiments of the present invention, CNS disorders may include craniocerebral trauma, stroke, cerebral palsy (such as cerebral palsy caused by neonatal hypoxia), and cancer (including brain tumors).

[0027] The present invention also provides a method for diagnosing a CNS disease, comprising administering to a patient a fluorescent marker of retinal integrity as described herein and generating an image of the patient's retina, wherein the fluorescent marker of retinal integrity is administered intranasally. The image of the patient's retina is preferably generated in vivo and can be obtained using cSLO imaging. The presence or absence of a fluorescent signal in the image can indicate the presence or absence of a CNS disease, thereby enabling a clinician to diagnose the presence or absence of a disease. If a fluorescent signal is present in the image, the location and distribution of the fluorescence can enable a clinician to distinguish the type of central nervous system disease. For example, the fluorescent signal can indicate the site of angiogenesis or vascular leakage. Alternatively, the fluorescent signal can indicate the distribution pattern and / or number of apoptotic and / or necrotic cells.

[0028] The diagnostic method of the present invention can also be used to monitor the progression of a disease, for example, to assess the effectiveness of a treatment or to stage a disease. A first in vivo image of a patient's eye can be compared with a subsequent in vivo image of the same patient's eye, the latter being taken a few days, weeks, or months after the first image. Changes in the fluorescent signal can indicate disease progression or an effective treatment. For example, an increase in the fluorescent signal may indicate an increase in the number of apoptotic cells, which may indicate disease progression. The images can be analyzed as described in WO2011 / 055121.

[0029] In another aspect of the present invention, the present invention provides a pharmaceutical composition comprising the above-mentioned marker of retinal cell integrity, wherein the pharmaceutical composition comprises an annexin or a functional fragment or derivative thereof bound to a compound of 2 kDa or less, wherein the annexin or a functional fragment or derivative thereof is present at a concentration of at least 5 mg / ml. When the annexin is present at a concentration of about 2 mg / ml or more, the composition comprising the annexin encounters precipitation problems. However, the inventors of the present application have been able to solve this problem so as to provide a composition comprising an annexin at a concentration of at least 5 mg / ml. The composition has been shown to be stable for storage for up to 50 days at 25°C under light-proof conditions. Without being bound by theory, the inventors of the present application believe that the binding of a compound of 2 kDa or less to the N-terminus of the annexin can sterically hinder protein aggregation.

[0030] The binding may be electrostatic or covalent, and the compound preferably binds to the N-terminus (i.e., the first 30 amino acids) of annexin or its functional fragment or derivative. In some embodiments of the present invention, annexin or its functional fragment or derivative may be present at a concentration of about 5 mg / ml to about 20 mg / ml, preferably about 5 mg / ml to about 10 mg / ml.

[0031] Compound can be organic compound or inorganic compound, and can have the size of about 100Da to about 2kDa, preferably about 200Da to about 1kDa, more preferably about 300Da to about 800Da.Compound can be fluorescent label as described above, or can be therapeutic agent.Suitable fluorescent label can be selected from one or more of fluorescein sodium, indocyanine green (ICG), curcumin, IRDye700, IRDye800, Dy-776, Dy-488 and D-781.In a preferred embodiment of the present invention, fluorescent label is Dy-776.

[0032] In a preferred embodiment of the invention, the pharmaceutical composition comprises Annexin 128 bound to Dy776. Surprisingly, several methods for concentrating markers of retinal cell integrity have been evaluated and the results demonstrate that Annexin 128 bound to Dy776 is more stable than Annexin 5 at high concentrations.

[0033] The pharmaceutical composition is preferably suitable for intranasal administration (such as a solution, suspension or dry powder suitable for inhalation), and may contain a suitable carrier and / or excipient as described above. The pharmaceutical composition may be in the form of a lyophilized powder and may be administered to a patient in the form of a dry powder suitable for inhalation. Alternatively, the lyophilized powder may be rehydrated to form a suspension prior to administration. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] The present invention will be described in detail below, by way of example only, with reference to the accompanying drawings.

[0035] Figure 1 Intranasal administration of Anx776 rapidly reaches the mouse retina and labels apoptotic retinal cells. cSLO images of the same C57BL / 6J mouse eye at baseline [A], [B], and 3 hours after intranasal administration of Anx776 [C], [D] and intravitreal 4% DMSO [B], [D]. Note that the appearance of apoptosis (white spots) is only seen in the eye in the case of intravitreal injection of 4% DMSO [D]. [E] DARC spots sorted at baseline and 3 hours after IN Anx776 administration. [F] Phosphatidylserine binding assay shows that lyophilized concentrated Anx776 retains good PS binding activity after storage at 25°C for 50 days while protected from light (EC50 ratio <2) compared to fresh Anx776 material.

[0036] Figure 2 cSLO images after administration of 0.025 mL of sodium fluorescein (20% w / v) in C57BL / 6J mice (488 nm excitation).

[0037] Figure 3 . 0.025 mL of indocyanine green (ICG) (795 nm excitation) was administered intranasally in C57BL / 6J mice. DETAILED DESCRIPTION

[0038] Example a) Anx776 rapidly reaches the circulation and retina after intranasal administration

[0039] Annexin 128, in its maleimide form, binds to Dy-776, providing a marker of retinal cell integrity, designated Anx776. The current clinically used Anx776 preparation (0.2 mg / mL) was concentrated 25-fold to 5 mg / mL using a 5 kDa MWCO filter in a buffer containing 20 mM sodium citrate, 280 mM glucose, pH 6.2 water for injection.

[0040] Concentrated Anx776 (hiAnx776) was tested in vivo. Using an established model [6], 4% DMSO (1 μL injection, PBS buffer) was injected into the vitreous cavity to induce retinal cell apoptosis, and hiAnx776 (25 μL) was administered intranasally to C57BL / 6J mice. Figure 1 [A]-[B] show pure representative cSLO images of the retina before treatment and at baseline ( Figure 1 [A]) and DMSO-infection ( Figure 1 [B]) Representative cSLO images of the retina. Figure 1 [D]) and intranasal Anx776 ( Figure 1 [C] and Figure 1 The same eyes were imaged 3 hours after DMSO treatment. DARC spots were clearly visible in the DMSO-treated eyes ( Figure 1 [D]), providing the first evidence that intranasal administration of Anx776 can reach the retina in a concentration sufficient to label apoptotic retinal cells in a manner similar to intravenous or intravitreal administration of DARC. Manual quantification of DARC spots showed that apoptosis was increased in the eye after intravitreal DMSO injury, consistent with previous observations of intravitreal injection of DARC in the model (Yozik et al., 2005).

[0041] We hypothesized that Anx776 would be absorbed systemically after inhalation into the circulation and enter the retina. Our experiments with Anx776 and clinical data confirm that systemic Anx776 can reach the retina after intravenous administration (Cordeiro et al., 2017).

[0042] Anx776 can be easily formulated as a lyophilized powder (with trehalose as cryoprotectant) which retains PS binding activity for up to 50 days when stored at 25°C in the dark ( Figure 1 [F]). Anx776 powders containing up to 10 mg / mL of Anx776 have been prepared, which can be rehydrated prior to IN delivery, or administered directly as a powder formulation.

[0043] b) Small fluorescent molecules currently used to diagnose retinal diseases enter the circulation rapidly enough to be used for diagnosis after intranasal application

[0044] Having observed that the fluorescent protein Anx776 (36 kDa) enters the circulation after intranasal administration, we next sought to determine whether small fluorescent molecules (sodium fluorescein and ICG, 376 Da and 775 Da, respectively) currently administered intravenously to diagnose retinal diseases could also be delivered in this manner. Figure 2 ) or ICG( Figure 3 ) both lead to rapid accumulation of fluorescence in retinal vessels and choroid. Images obtained by cSLO are identical to those obtained after previous intravenous injection of the same fluorescent agent (Kumar et al., 2014). Due to local administration, less dose may be required and the onset of action may be faster compared to formulations of systemically administered agents.

[0045] References

[0046] MF Cordeiro, EM Normando, MJ Cardoso, S. Miodragovic, S. Jeylani, BM Davis, L. Guo, S. Ourselin, R. A'Hern, PA Bloom, Brain 2017, 274, 61.

[0047] JJJorzik,A.Bindewald,S.Dithmar,FGHolz,Retina 2005,25,405.

[0048] S.Kumar,Z.Berriochoa,ADJones,Y.Fu,J.Vis.Exp.2014,e51061.

[0049] De Saint-Hubert M,Prinsen K,Mortelmans L,Verbruggen A,MottaghyFM.Methods.2009Jun;48(2):178-87

[0050] JF.Tait,C.Smith,FG.Blankenberg.J Nucl Med May 1,2005vol.46no.5 807-815

Claims

1. A fluorescent marker for retinal integrity for diagnosis of CNS diseases, wherein: The fluorescent marker was delivered via intranasal administration.

2. The fluorescent marker for retinal integrity for diagnosing CNS diseases according to claim 1, wherein: The fluorescent marker is in the form of powder, suspension or solution.

3. The fluorescent marker for retinal integrity for diagnosing CNS diseases according to claim 1 or 2, wherein: The fluorescent marker is a marker of retinal vascular integrity.

4. The fluorescent marker for retinal integrity for diagnosing CNS diseases according to any one of claims 1 to 3, wherein The molecular weight of the fluorescent label is about 2 kDa or less.

5. The fluorescent marker for retinal integrity for diagnosing CNS diseases according to any one of claims 1 to 4, wherein The fluorescent marker is selected from one or more of sodium fluorescein or indocyanine green (ICG).

6. The fluorescent marker for retinal integrity for diagnosing CNS diseases according to claim 1 or 2, wherein: The fluorescent label is a marker of retinal cell integrity.

7. The fluorescent marker of retinal integrity for diagnosing CNS diseases according to claim 1, 2 or 6, wherein: The fluorescent marker includes a fluorescent tag and a marker of one or more of apoptosis, necrosis, cell activity, cell stress or protein aggregation.

8. The fluorescent marker for retinal integrity for diagnosing CNS diseases according to claim 6 or 7, wherein: The fluorescent tag has an emission wavelength of about 400 nm to about 1000 nm.

9. The fluorescent marker for retinal integrity for diagnosing CNS diseases according to any one of claims 6 to 8, wherein The fluorescent label is selected from one or more of fluorescein sodium, indocyanine green (ICG), curcumin, IRDye700, IRDye800, Dy-776, Dy-488 and D-781.

10. The fluorescent marker of retinal integrity for diagnosing CNS diseases according to any one of claims 6 to 9, wherein The apoptotic marker is selected from one or more of annexin, C2A domain of synapsin I, duramycin, non-peptide based isatin sulfonamide analogs such as WC-II-89 and ApoSense such as NST-732, DDC and ML-10.

11. The fluorescent marker of retinal integrity for diagnosing CNS diseases according to claim 10, wherein: The annexin is selected from one or more of annexin 2, annexin 5, annexin 6, annexin 11 or annexin 128.

12. The fluorescent marker of retinal integrity for diagnosing CNS diseases according to any one of claims 6 to 9, wherein The necrosis marker is selected from propidium iodide (PI), pyrophosphate, antimyosin, oxalate, hypericin, hypericin monocarboxylic acid, palmitic acid, bis-hydrazide-bis-DTPA palmitic acid, 99mTc-pyrophosphate, 111-antimyosin, 99mTc-gluconate and methylene blue.

13. The fluorescent marker of retinal integrity for diagnosing CNS diseases according to any one of claims 6 to 9, wherein The cell activity marker is selected from one or more of membrane dyes, mitochondrial dyes, autophagy dyes, necrosis dyes and calcium flux.

14. The fluorescent marker of retinal integrity for diagnosing CNS diseases according to any one of claims 6 to 9, wherein The marker of cellular stress is selected from one or more of lipid peroxidation, glutathione (GSH) or reactive oxygen species (ROS), such as superoxide, peroxyl radical, hydrogen peroxide, hydroxyl radical, peroxynitrite.

15. The fluorescent marker of retinal integrity for diagnosing CNS diseases according to any one of claims 6 to 9, wherein The marker of protein aggregation is selected from one or more of Congo red, curcumin or thioflavin S.

16. The fluorescent marker of retinal integrity for diagnosing CNS diseases according to any one of claims 1 to 15, wherein CNS diseases are inflammatory (such as arthritis or granuloma), infectious (such as viral, encephalitis, or bacterial), vascular (such as angiogenic, occlusive, or metabolic), or degenerative (such as glaucoma, age-related macular degeneration (AMD), Alzheimer's disease, or Parkinson's disease).

17. A method of diagnosing a CNS disease comprising administering to a subject a fluorescent marker of retinal integrity and generating an image of the subject's eye, wherein: The fluorescent marker was delivered via intranasal administration.

18. The method according to claim 17, wherein: The fluorescent marker is in the form of powder, suspension or solution.

19. The method according to claim 17 or 18, wherein: The fluorescent marker is a marker of retinal vascular integrity.

20. The method according to any one of claims 17 to 19, wherein: The molecular weight of the fluorescent label is about 2 kDa or less.

21. The method according to any one of claims 17 to 20, wherein: The fluorescent marker is selected from one or more of sodium fluorescein or indocyanine green (ICG).

22. The method according to claim 17 or 18, wherein: The fluorescent label is a marker of retinal cell integrity.

23. The method of claim 17, 18 or 22, wherein: The fluorescent marker includes a fluorescent tag and a marker of one or more of apoptosis, necrosis, cell activity, cell stress or protein aggregation.

24. The method according to claim 22 or 23, wherein: The fluorescent tag has an emission wavelength of about 400 nm to about 1000 nm.

25. The method according to any one of claims 22 to 24, wherein: The fluorescent label is selected from one or more of fluorescein sodium, indocyanine green (ICG), curcumin, IRDye700, IRDye800, Dy-776, Dy-488 and D-781.

26. The method according to any one of claims 22 to 25, wherein: The apoptotic marker is selected from one or more of annexin, C2A domain of synapsin I, duramycin, non-peptide-based isatin sulfonamide analogs such as WC-II-89 and ApoSense, such as NST-732, DDC and ML-10.

27. The method according to claim 26, wherein: The annexin is selected from one or more of annexin 2, annexin 5, annexin 6, annexin 11 or annexin 128.

28. The method according to any one of claims 22 to 25, wherein: The necrosis marker is selected from one or more of propidium iodide (PI), pyrophosphate, antimyosin, oxalate, hypericin, hypericin monocarboxylic acid, palmitic acid, bishydrazide-bis-DTPA palmitic acid, 99mTc-pyrophosphate, 111-antimyosin, 99mTc-gluconate and methylene blue.

29. The method according to any one of claims 22 to 25, wherein: The cell activity marker is selected from one or more of membrane dyes, mitochondrial dyes, autophagy dyes, necrosis dyes and calcium flux.

30. The method according to any one of claims 22 to 25, wherein: The marker of cell stress is selected from one or more of lipid peroxidation, glutathione (GSH) or reactive oxygen species (ROS), such as superoxide, peroxyl radical, hydrogen peroxide, hydroxyl group, and peroxynitrite.

31. The method according to any one of claims 22 to 25, wherein: The marker of protein aggregation is selected from one or more of Congo red, curcumin or thioflavin S.

32. A method according to any one of claims 17 to 31, wherein The CNS disorder is inflammatory (such as arthritis or granuloma), infectious (such as viral, encephalitis or bacterial), vascular (such as angiogenic, occlusive or metabolic), or degenerative (such as glaucoma, age-related macular degeneration (AMD), Alzheimer's disease or Parkinson's disease).

33. A pharmaceutical composition comprising an annexin or a functional fragment or derivative thereof bound to a compound of 2 kDa or less, wherein: The pharmaceutical composition comprises the bound annexin or a functional fragment or derivative thereof at a concentration of at least 5 mg / ml.

34. The pharmaceutical composition according to claim 33, wherein The annexin or its functional fragment or derivative is annexin 128, and the compound of 2 kDa or less is Dy776.

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