Intranasal delivery of fluorescent marker

Abstract

To provide a method of diagnosing a CNS disorder comprising administering a fluorescent marker of retinal integrity to a subject and generating an image of the subject's eye.SOLUTION: The fluorescent marker is delivered by intranasal administration. Also provided are fluorescent markers of retinal integrity for use in such methods. Also provided is a pharmaceutical composition comprising an annexin or a functional fragment or derivative thereof conjugated to a compound of 2 kDa or less. The composition comprises an annexin or a functional fragment or derivative thereof conjugated at a concentration of at least 5 mg / ml.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention relates to a fluorescent marker for diagnosing CNS diseases.

Background Art

[0002] In the evaluation of the retina, it is possible to provide information regarding the presence and severity of many systemic diseases. For example, detection of apoptotic retinal cells (DARC) has just completed a first-phase (clinical) trial for the diagnosis of glaucoma (Cordeiro et al., 2017), and is a technique for monitoring the cell death rate in the retina that is currently undergoing a second-phase (clinical) trial targeting neurodegenerative diseases including glaucoma, age-related macular degeneration, optic neuritis, and Down syndrome (as a model of Alzheimer's disease). The current technique consists of intravenously administering a novel fluorescent agent called Anx776, which contains a modified form of the endogenous protein annexin A5 (fluorescently conjugated to the near-infrared fluorophore Dy-776) (Cordeiro et al., 2017).

[0003] In DARC, specific ocular visual properties are utilized to enable direct observation of single neuronal apoptosis 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 all cells, but apoptotic cells express PS on the outer leaflet of the plasma membrane. Apoptosis is identified by the binding of the exposed PS to annexin V.

[0004] For the first- and second-phase clinical trials of DARC, Anx776 is administered intravenously. Other fluorescent molecules used to detect retinal diseases using confocal scanning laser ophthalmoscopy (cSLO) imaging are also mainly administered by intravenous injection. For example, to identify abnormally proliferated vascular tissue (angiogenesis) and leakage associated with age-related macular degeneration (AMD), generally, fluorescein sodium and indocyanine green (ICG) are administered intravenously to label the retinal vasculature. (Jorzik et al., 2005)

Prior Art Documents

Patent Documents

[0005]

Patent Document 1

Patent Document 2

Patent Document 3

[0006]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

[0007] Intravenous administration requires direct medical supervision. This means that patient evaluation is not usually performed unless a pathological condition is suspected or when it becomes such. Therefore, there is a need for alternative techniques that can be used to evaluate the retina in order for patients to have access to this type of evaluation and potentially increase the chances of early diagnosis of retinal and neurodegenerative diseases. [Means for Solving the Problems]

[0008] In a first aspect, the present invention provides a fluorescent marker for retinal integrity (for confirming integrity) for diagnosing central nervous system (CNS) diseases. This fluorescent marker is delivered by intranasal administration. Surprisingly, when the fluorescent marker was administered intranasally, the retinal image showed the same results as those obtained when the same fluorescent agent was administered intravenously previously. Therefore, the inventors of the present invention discovered that fluorescence accumulates rapidly in the retina with intranasal administration.

[0009] The inventors believe, without being bound by theory, that the fluorescent marker for retinal integrity delivered into the nasal cavity is absorbed into the systemic circulating blood. Due to local administration, the required dose of the fluorescent marker for retinal integrity can be reduced and it can be more immediate-acting than systemic administration formulations of the same drug.

[0010] As used herein, a fluorescence marker for retinal integrity means a fluorescence marker for interrogating the health (soundness) of retinal cells and / or the integrity of retinal cells and / or blood vessels. For example, the marker can identify and / or distinguish apoptotic cells and necrotic cells, or identify regions of vascular leakage or angiogenesis. This fluorescence marker emits light in response to excitation and can have an emission wavelength of about 400 to about 1000 nanometers, preferably about 500 to about 900 nanometers.

[0011] The fluorescence marker for retinal integrity is preferably provided in a form suitable for local delivery, particularly intranasal delivery. The fluorescence marker for retinal integrity is provided as a pharmaceutical composition and can be in the form of a solution, suspension, or dry powder suitable for inhalation. The pharmaceutical composition containing the fluorescence marker for retinal integrity can be made sterile and may contain one or more pharmaceutically acceptable carriers or excipients. Suitable carriers and excipients are those well known to those skilled in the art and can be optimized to correspond to the intended route of intranasal delivery. For example, the composition containing the fluorescence marker for retinal integrity may contain a buffer, binder, preservative, thickening agent, or antioxidant (such as trehalose).

[0012] In an embodiment of the present invention, the fluorescence marker for retinal integrity may be a fluorescence marker for retinal vascular integrity. This type of fluorescence marker typically enters the retinal blood vessels and circulates therein. And sites of abnormally proliferated vascular tissue (angiogenesis) and / or leakage can be easily visualized.

[0013] The molecular weight of the fluorescence marker for retinal vascular integrity can be about 2 kDa or less, or 1 kDa or less. The molecular weight of the fluorescence marker for retinal vascular integrity in the examples of the present invention can be about 100 Da or about 1 kDa, preferably about 300 to about 800 Da.

[0014] Suitable fluorescent markers for retinal vascular integrity used in the present invention contain a fluorophore. Particularly preferred fluorescent markers for retinal vascular integrity include sodium fluorescein and indocyanine green (ICG).

[0015] As described above, since the fluorescent marker for retinal vascular integrity is to be administered intranasally, it can be administered in the same amount or a smaller amount as the amount for intravenous administration of the same marker. For example, sodium fluorescein can be administered intranasally at a concentration of about 50 to about 500 mg / mL (preferably about 50 to about 200 mg / mL). In an example of the present invention, sodium fluorescein can be administered intranasally at about 100 mg / mL. ICG can be administered intranasally at a concentration of about 1 to about 100 mg / mL (preferably about 25 to about 100 mg / mL). In an example of the present invention, ICG can be administered intranasally at about 50 mg / mL.

[0016] In an embodiment of the present invention, the retinal integrity fluorescent marker may be a marker for retinal cell integrity. Usually, the fluorescent marker for retinal cell integrity comprises a fluorescent label and marker for one or more of apoptosis, necrosis, cell activity, cell stress, or protein aggregation.

[0017] A fluorescent label means a compound or molecule (e.g., a fluorophore) that emits light in response to excitation. This substance may be selected and used so as to comply with the light exposure safety criteria to avoid phototoxic effects while improving the signal-to-noise ratio, and thus also improving the image resolution and image sensitivity. The fluorescent label preferably causes little or no inflammation upon administration. The fluorescent label can have a wavelength in the infrared or near-infrared region. The fluorescent label can have an emission wavelength of about 400 to about 1000 nanometers, preferably about 500 to about 900 nanometers, more preferably about 700 to about 900 nanometers.

[0018] Suitable fluorescent labels include one or more of sodium fluorescein, 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.

[0019] A fluorescent marker for retinal cell integrity may be prepared by utilizing standard techniques for conjugating a fluorescent label to a marker compound. Such labels are available from well-known sources (e.g., Dyomics). Suitable techniques for conjugating a label to a marker are well known in the art and may be provided by the label manufacturer.

[0020] A marker for apoptosis means a marker that can distinguish cells undergoing apoptosis from live cells. Additionally and preferably, this marker should be capable of distinguishing cells undergoing apoptosis from necrotic cells. For example, it can be a compound or molecule that specifically binds to apoptotic cells but not to live or necrotic cells. Markers for apoptosis include, for example, the annexin family of proteins. Annexins are proteins that reversibly bind to cell membranes in the presence of cations. Annexins can bind specifically to phosphatidylserine (PS) in the presence of calcium. PS is present in the plasma membrane of all cells, but apoptotic cells expose PS on the outer leaflet of the plasma membrane. Apoptosis is identified by the binding of exposed PS to annexin V. Annexins useful in the present invention may be of natural or recombinant origin. The protein may be a full-length protein or a functional fragment. That is, it may be a fragment or portion of an annexin that specifically binds to the same molecule as the full-length protein. Also, functional derivatives of this type of protein may be used. In particular, functional fragments or derivatives of annexins may include molecules containing an "annexin repeat" (i.e., a domain of about 70 amino acids that is conserved both within individual annexins and between family members). Various annexins are available, for example, annexins such as those described in US Patent Application Publication No. 2006 / 0134001. A preferred annexin is annexin 5, which is well known in the art. Other available annexins include annexin 11, 2, and 6. Other markers of apoptosis are also well known in the art, for example, the C2A domain of synaptotagmin I, duramycin, non-peptidic isatin sulfonamide analogs (e.g., WC-II-89), and ApoSense (e.g., NST-732, DDC, and ML-10) (Saint-Hubert et al., 2009).

[0021] In a particularly preferred embodiment of the present invention, the marker of apoptosis is annexin 128 (Tait et al., 2005). Annexin 128 is a variant of annexin 5 and differs from the wild type by two 1-amino acid mutations. Annexin 128 contains a thiol group exposed at the N-terminal group, which improves the binding efficiency for molecular tags (e.g., fluorescent labels).

[0022] In a particularly preferred embodiment of the present invention, the fluorescent marker of retinal cell integrity comprises annexin 128 conjugated to Dy-776. Annexin 128 and Dy-776 can be conjugated in a 1:1 (fluorescent label:marker ratio).

[0023] The marker of necrosis means a marker that can distinguish cells in which necrosis is occurring from live cells and cells in which apoptosis is occurring. For example, this marker can be a compound or molecule that specifically binds to necrotic cells but not to live cells or apoptotic cells. The marker of necrosis includes an intercalating agent that binds to nucleic acids with little or no sequence preference, such as propidium iodide (PI). Other necrosis markers are also well known in the art, for example, pyrophosphate, anti-myosin, gluconic acid, hypericin, and its derivatives (e.g., hypericin monocarboxylic acid and pamoic acid (e.g., bis-hydrazide-bis-DTPA pamoic acid)). In particular,99m Tc - pyrophosphate, 111 In - antimyosin, 99m Tc - gluconic acid, and methylene blue are used.

[0024] Other signs of cell activity can also be used to identify apoptotic or necrotic cells. For example, by observing changes in mitochondrial function, reactive oxygen species (ROS) can be used as a marker (calcium ions are similar). Markers of cell activity may include one or more of membrane staining, mitochondrial staining, autophagy staining, necrosis staining, and calcium flux. More specifically, markers of cell activity may include one or more of Fluo - 3, N - (fluorescein - 5 - thiocarbamoyl) - 1,2 - dihexadecanoyl - sn - glycerol - 3 - phosphoethanolamine, JC - 1, JC - 9 (with dual emission), diluted rhodamine and roseamine, rhodamine 123, and Di - 8 - ANePPS. Markers of cell stress may include one or more of markers of lipid peroxidation, glutathione (GSH), or reactive oxygen species (ROS) (such as superoxide, peroxyl radical, hydrogen peroxide, hydroxyl radical, and peroxynitrite).

[0025] Protein aggregation in the retina can occur intracellularly, extracellularly, or around retinal neurons, but usually occurs outside the retinal vascular system. The presence of protein aggregates is known to be correlated with cells undergoing neurodegeneration. Suitable markers for protein aggregation include one or more of congo red, curcumin, or thioflavin S.

[0026] As described above, the present invention provides a fluorescent marker of retinal integrity for delivery into the nasal cavity for diagnosing CNS diseases in a subject. The subject is preferably a mammal including a human, and may be a pediatric patient or an elderly patient. CNS diseases can be identified by analyzing retinal integrity (see, for example, the methods described in WO 2009 / 077750 and WO 2011 / 055121). For example, a fluorescent marker of retinal integrity can be used to label apoptotic and / or necrotic cells and to reveal the distribution of cell death in the retina. From this distribution, different neurodegenerative diseases having different patterns of apoptotic activity and / or necrotic activity can be distinguished.

[0027] CNS diseases can be inflammatory (e.g., arthritis or granulomatous), infectious (e.g., viral, encephalitic, or bacterial), vasculovascular (e.g., angiogenesis, obstructive, or metabolic), or degenerative (e.g., glaucoma, age-related macular degeneration (AMD), Alzheimer's disease, or Parkinson's disease). In an embodiment of the present invention, the CNS disease can be a neurodegenerative disease (especially an optic neurodegenerative disease). The term "optic neurodegenerative disease" is well known to those skilled in the art and means a disease caused by a progressive loss of neurons in the eye. This disease includes, but is not limited to, glaucoma, AMD, optic neuritis, and diabetic retinopathy. Neurodegenerative diseases include, for example, Parkinson's disease, Alzheimer's disease, Huntington's disease, and Friedreich's ataxia. CNS diseases in embodiments of the present invention can include traumatic brain injury, stroke, cerebral palsy (e.g., caused by neonatal hypoxia) and cancer (including brain tumors).

[0028] The present invention further provides a method for diagnosing a CNS disease. This method includes the steps of administering to a patient a fluorescent marker of retinal integrity described herein, and generating an image of the retina of a patient to whom the fluorescent marker of retinal integrity has been administered intranasally. Images of the patient's retina are preferably generated in vivo and can be acquired using cSLO imaging. Since the presence or absence of a fluorescence signal in the image can indicate the presence or absence of a CNS disease, a clinician can diagnose the presence or absence of the disease. If a fluorescence signal is present in the image, the clinician can distinguish the type of CNS disease based on the location and distribution of the fluorescence. For example, the fluorescence signal can indicate sites of angiogenesis or vascular leakage. The fluorescence signal can also indicate the number or pattern of the distribution of apoptotic and / or necrotic cells.

[0029] The diagnostic method of the present invention can further be used to monitor the progression of the disease, for example, to evaluate the effectiveness of treatment or the stage of the disease. The first in vivo image of the patient's eye may be compared with an in vivo image of the same patient's eye taken several days, weeks, or months after the first image. Changes in the fluorescence signal can indicate the progression of the disease or effective treatment. For example, an increase in the fluorescence signal can indicate an increase in the number of cells undergoing apoptosis, which is considered to be the progression of the disease. The images can be analyzed as described in WO 2011 / 055121.

[0030] In a further aspect, the present invention provides a pharmaceutical composition comprising a marker of retinal cell integrity as described above. This (pharmaceutical) composition comprises annexin, or a functional fragment or derivative thereof, which binds to a compound of 2 kDa or less, and annexin, or a functional fragment or derivative thereof, is present at a concentration of at least 5 mg / ml. When annexin is present at a concentration of about 2 mg / ml or higher, the composition containing annexin is known to encounter problems with precipitation. However, the inventors of the present invention were able to solve this problem in order to provide a composition containing annexin at a concentration of at least 5 mg / ml. The composition was demonstrated to be stably stored at 25°C for up to 50 days when protected from light. The inventors of the present invention, without being bound by theory, believe that the binding of a compound of 2 kDa or less to the N-terminal group of the annexin protein acts to sterically prevent protein aggregation.

[0031] The binding may be electrostatic or covalent. The compound is preferably bound to the N-terminal group (i.e., the first 30 amino acids) of annexin, or a functional fragment or derivative thereof. In the examples of the present invention, annexin, or a functional fragment or derivative thereof, may be present at a concentration of about 5 to about 20 mg / ml, preferably about 5 to about 1 mg / ml.

[0032] The compound may be an organic compound or an inorganic compound, and the size may be about 100 Da to about 2 kDa, preferably about 200 Da to about 1 kDa, more preferably about 300 Da to about 800 Da. The compound may be a fluorescent label or a therapeutic agent as described above. Suitable fluorescent labels may be selected from one or more of sodium fluorescein, 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.

[0033] In a preferred embodiment of the present invention, the pharmaceutical composition contains annexin 128 bound to Dy776. Surprisingly, multiple methods were evaluated to enrich markers of retinal cell integrity, and annexin 128 bound to Dy776 was demonstrated to be more stable at higher concentrations than annexin 5.

[0034] The pharmaceutical composition is preferably in a form suitable for intranasal administration (e.g., 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 the patient as a dry powder suitable for inhalation. Alternatively, the lyophilized powder may be rehydrated to form a suspension prior to administration.

[0035] As a mere example, the present invention will be described in detail with reference to the drawings.

Brief Description of the Drawings

[0036]

Figure 1

Figure 2

Figure 3

Modes for Carrying Out the Invention

[0037] a) Anx776 reaches the circulating blood and retina rapidly after intranasal administration Annexin 128 was conjugated to maleimide-shaped Dy-776 and provided a marker of retinal cell integrity identified as Anx776. The currently used formulation of Anx776 (0.2 mg / ml) in the clinic was concentrated 25 times up to 5 mg / mL using a 5 kDa MWCO filter in a buffer containing 20 mM sodium citrate, 280 mM glucose, and distilled water for injection at pH 6.2. Tests of concentrated Anx776 (hiAnx776) were performed in vivo. HiAnx776 was administered intranasally (25 μl) to mice (C57BL / 6J). At the same time, using a stable model [6], 4% DMSO (injection volume 1 μl, PBS buffer) was injected into the vitreous body to induce retinal cell apoptosis. Figures 1 [A - B] show representative cSLO images at baseline (before treatment) of an untreated retina (Figure 1A) and a DMSO-insulted retina (Figure 1B). Figures 1 [C - D] are images 3 hours after treatment of the same eyes as in Figures 1A and B. Figure 1C is the result of intranasal Anx776 treatment only, and Figure 1D is the result of 4% DMSO injection and intranasal Anx776 treatment. In the DMSO-treated eyes (Figure 1D), distinct DARC spots were clearly confirmed, which is the first evidence suggesting that when Anx776 is administered intranasally, it can reach the retina at a concentration sufficient to label apoptotic retinal cells, similar to DARC by intravenous or intravitreal administration. Manual quantification of DARC spots showed an increase in apoptosis in eyes receiving intravitreal DMSO insults, which is consistent with previous observations of DARC by intravitreal administration in this model (Jorzik et al., 2005). We hypothesize that after Anx776 is inhaled into the circulating blood, it is systemically absorbed and enters the retina. Our experimental and clinical data using Anx776 confirm that systemic Anx776 can reach the retina after intravenous administration. (Cordeiro et al., 2017) Anx776 can be conveniently formulated as a lyophilized powder (containing trehalose as a cryoprotectant). This lyophilized powder is protected from light and retains PS-binding activity for up to 50 days after storage at 25°C (Figure 1F). Anx776 powder containing up to 10 mg / mL of Anx776 is prepared and can be rehydrated prior to IN (nasal) delivery or administered directly as a powder formulation.

[0038] b) Small fluorescent molecules currently used in the diagnosis of retinal diseases reach the circulating blood rapidly after nasal administration in an amount sufficient for diagnosis. Since we observed that the fluorescent protein Anx776 (36 kDa) enters the circulating blood upon nasal administration, we next sought to determine whether small fluorescent molecules currently administered intravenously for the diagnosis of retinal diseases (fluorescein sodium and ICG, 376 Da and 775 Da respectively) can be delivered in this way. Upon nasal administration of fluorescein sodium (Figure 2) or ICG (Figure 3), it was found that fluorescence rapidly accumulates in both the retinal vasculature and the choroid. The resulting images obtained by cSLO were previously the same as those obtained after intravenous administration of the same fluorescent agent (Kumar et al., 2014). And because of the local administration, the required dose can be reduced and it can be more immediate than the systemic administration formulation of the same drug.

Claims

Claim 1 A composition for use in the diagnosis of CNS diseases, wherein the composition comprises a fluorescence marker for retinal vascular integrity or a fluorescence marker for retinal cell integrity, wherein the fluorescence marker for retinal vascular integrity is a fluorescence marker that enters the retinal blood vessels and circulates within the retinal blood vessels, wherein the fluorescence marker for retinal cell integrity comprises a fluorescent label and one or more markers of apoptosis, necrosis, cell activity, cell stress or protein aggregation, wherein the composition is characterized in that it is delivered by intranasal administration.

Images