Intranasal Delivery of Fluorescent Marker

Abstract

The present invention provides 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, wherein the fluorescent marker is delivered by intranasal administration is also provided and fluorescent markers of retinal integrity for use in such methods. Also provided is a pharmaceutical composition comprising an annex in or a functional fragment or derivative thereof conjugated to a compound of 2 kDa or less, wherein the composition comprises annex in or a functional fragment or derivative thereof conjugated at a concentration of at least mg/ml.

Claims

1. A fluorescent marker of retinal integrity for use in diagnosing a CNS disorder, wherein the fluorescent marker is to be delivered by intranasal administration.

2. A fluorescent marker of retinal integrity for use according to claim 1, wherein the fluorescent marker is in the form of a powder, a suspension or a solution.

3. A fluorescent marker of retinal integrity for use according to claim 1 or 2, wherein the fluorescent marker is a marker of retinal blood vessel integrity.

4. A fluorescent marker of retinal integrity for use according to any of claims 1 to 3, wherein the fluorescent marker has a molecular weight of about 2 kDa or less.

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

6. A fluorescent marker of retinal integrity for use according to claim 1 or 2, wherein the fluorescent marker is a marker of retinal cell integrity.

7. A fluorescent marker of retinal integrity for use according to claim 1, 2 or 6, wherein the fluorescent marker comprises a fluorescent label and a marker of one or more of apoptosis, necrosis, cell activity, cell stress or protein aggregation.

8. A fluorescent marker for use according to claim 6 or 7, wherein the fluorescent label has an emission wavelength of about 400 nm to about 1000 nm.

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

10. A fluorescent marker for use according to any of claims 6 to 9, wherein the marker of apoptosis is selected from one or more of an annexin, 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.

11. A fluorescent marker for use 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. A fluorescent marker for use according to any of claims 6 to 9, wherein the marker of necrosis is selected from one or more of propidium iodide (PI), pyrophosphate, antimyosin, glucarate, hypericin, hypericin monocarboxylic acid, pamoic acid, bis-hydrazide-bis-DTPA pamoic acid, 99mTc-pyrophosphate, 111In-antimyosin, 99mTc-glucarate and methylene blue.

13. A fluorescent marker for use according to any of claims 6 to 9, wherein the marker of cell activity is selected from one or more of membrane dyes, mitochondrial dyes, autophagy dyes, necrosis dyes and calcium flux.

14. A fluorescent marker for use according to any of claims 6 to 9, wherein the marker of cell stress is selected from one or more of a marker of lipid peroxidation, glutathione (GSH) or reactive oxygen species (ROS), such as superoxide, peroxyl radical, hydrogen peroxide, hydroxyl radical and peroxynitrite

15. A fluorescent marker for use according to any 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. A fluorescent marker of retinal integrity for use according to any of claims to 1 to 15, wherein the CNS disorder is inflammatory (such as arthridides or granulomatous), infective (such as viral, encephalitic, 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 disorder comprising administering a fluorescent marker of retinal integrity to a subject and generating an image of the subject's eye, wherein the fluorescent marker is delivered by intranasal administration.

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

19. A method according to claim 17 or 18, wherein the fluorescent marker is a marker of retinal blood vessel integrity.

20. A method according to any of claims 17 to 19, wherein the fluorescent marker has a molecular weight of about 2 kDa or less.

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

22. A method according to claim 17 or 18, wherein the fluorescent marker is a marker of retinal cell integrity.

23. A method according to claim 17, 18 or 22, wherein the fluorescent marker comprises a fluorescent label and a marker of one or more of apoptosis, necrosis, cell activity, cell stress or protein aggregation.

24. A method according to claim 22 or 23, wherein the fluorescent label has an emission wavelength of about 400 nm to about 1000 nm.

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

26. A method according to any of claims 22 to 25, wherein the marker of apoptosis is selected from one or more of an annexin, 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.

27. A 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. A method according to any of claims 22 to 25, wherein the marker of necrosis is selected from one or more of propidium iodide (PI), pyrophosphate, antimyosin, glucarate, hypericin, hypericin monocarboxylic acid, pamoic acid, bis-hydrazide-bis-DTPA pamoic acid, 99mTc-pyrophosphate, 111In-antimyosin, 99mTc-glucarate and methylene blue.

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

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

31. A method according to any 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 of claims to 17 to 31, wherein the CNS disorder is inflammatory (such as arthridides or granulomatous), infective (such as viral, encephalitic, 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 conjugated to a compound of 2 kDa or less, wherein the composition comprises annexin or a functional fragment or derivative thereof conjugated at a concentration of at least 5 mg/ml.

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

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0033] The invention will now be described in detail, by way of example only, with reference to the figures.

[0034] FIG. 1. Intranasal administration of Anx776 rapidly reaches the retina in mice and labels apoptotic retinal cells. cSLO images of the same C57BL/6J mice eyes at baseline [A,B], and 3 hours after intranasal administration of Anx776 [C, D] and intravitreal 4% DMSO [B,D]. Note the appearance of apoptosis (white spots) is seen only in the eye given intravitreal 4% DMSO [D]. [E] Collated DARC spots at baseline and 3 h after IN Anx776 administration. [F] A Phosphatidylserine binding assay demonstrating that lyophilised concentrated Anx776 retains good PS binding activity compared to fresh Anx776 material after storage for 50 days at 25° C. while protecting from light (EC50 ratio<2).

[0035] FIG. 2. cSLO images after administration of 0.025 mL Sodium fluorescein (20% w/v) in C57BL/6J mice (488 nm excitation).

[0036] FIG. 3. Intranasal administration of 0.025 mL Indocyanine Green (ICG) in C57BL/6J mice (795 nm excitation).

EXAMPLES

[0037] a) Anx776 Rapidly Reaches the Circulation and Retina After Intranasal Administration

[0038] Annexin 128 was conjugated to Dy-776 in its maleimide form, providing a marker of retinal cell integrity identified as Anx776. The formulation of Anx776 presently used in the clinic (0.2 mg/mL) was concentrated 25 times to 5 mg/mL using a 5 kDa MWCO filter, within a buffer containing 20 mM Sodium Citrate, 280 mM Dextrose, pH 6.2 in water for injection.

[0039] Concentrated Anx776 (hiAnx776) was tested in vivo. hiAnx776 was administered intranasally (25 μL) to C57BL/6J mice at the same time as intravitreal 4% DMSO (1 μL injection volume, PBS buffer) was injected to induce retinal cell apoptosis, using a well-established model[6]. FIG. 1[A-B] illustrate representative cSLO images of naïve (FIG. 1A) and DMSO-insulted (FIG. 1B) retina at baseline, before treatment. These same eyes are imaged 3 hours after treatment with 4% DMSO injection (FIG. 1D) and intranasal Anx776 (FIG. 1C and FIG. 1D). Bright DARC spots are clearly visible in the DMSO-treated eyes (FIG. 1D) providing the first evidence to suggest that intranasal administration of Anx776 can reach the retina at sufficient concentrations to label apoptotic retinal cells in a similar manner to intravenously or intravitreally administered DARC. Manual quantification of DARC spots indicates an elevation in apoptosis in eyes subjected to intravitreal DMSO insults, in agreement with previous observations with intravitreally administered DARC in this model (Jorzik et al 2005).

[0040] We hypothesize Anx776 is systemically absorbed after inhalation into the circulation, and gains entry to the retina. Our experimental and clinical data with Anx776 confirms that systemic Anx776 can reach the retina after intravenous administration (Cordeiro et al 2017).

[0041] Anx776 can be readily formulated as a lyophilised powder (with Trehalose as a cryoprotectant) which retains PS binding activity for up to 50 days after storage at 25C (FIG. 1F), protecting from light. Anx776 powders containing up to 10 mg/mL of Anx776 have been prepared which could be rehydrated prior to IN delivery or alternatively administered directly as a powder formulation.

[0042] b) Small fluorescent molecules presently used for the diagnosis of retinal disorders rapidly reach the circulation in sufficient quantities to be of diagnostic use after intranasal application

[0043] Having observed that the fluorescent protein Anx776 (36 kDa) enters the circulation upon intranasal administration, we next sought to determine whether small fluorescent molecules (Sodium fluorescein and ICG, 376 Da and 775 Da respectively) presently administered intravenously for the diagnosis of retinal disorders could also be delivered in this manner. Intranasal administration of Sodium fluorescein (FIG. 2) or ICG (FIG. 3) were both found to result in rapid accumulation of fluorescence in retinal vasculature and choroid. The resulting images acquired by cSLO were identical to those previously obtained after intravenous administration of the same fluorescent agents (Kumar et al 2014) and due to the localised administration, may require reduced doses and have a more rapid onset than systemically administered formulations of these agents.

REFERENCES

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