CNS delivery of iron-chelators

Abstract

The invention generally concerns a CNS-targeting non-systemic formulation for regulating dopamine and / or serotonin levels in the central nervous system (CNS).

Description

[0001] CNS DELIVERY OF IRON-CHELATORS

[0002] TECHNOLOGICAL FIELD

[0003] The invention generally relates to compositions and methods of regulating dopamine and serotonin levels in a subject.

[0004] BACKGROUND

[0005] The neurotransmitter dopamine is a key factor in the function of the central nervous system (CNS). Apart from regulating processes relating to reward, movement, and cognition, dopamine also regulates critical functions in peripheral organs. Over the years it became apparent that dopamine is also an important immunoregulatory factor that many immune cells take up, produce, store, and / or release. Regulating these pathways could be a promising approach for effective treatments of inflammatory diseases and other dopamine-dependent diseases, as well as regulation of immune signaling in the CNS and peripheral tissues, stressing the role of dopaminergic immunomodulation in diseases such as neuropsychiatric conditions, inflammatory bowel disease, rheumatoid arthritis, and others.

[0006] Serotonin is another neurotransmitter that serves a critical cognitive and behavioural function in humans. Serotonin is involved with mood regulation and is also involved in regulating lactation and involution influencing mechanisms of homeostasis in the mammary epithelium. Deregulation of these epithelial homeostatic systems is in part responsible for breast cancer onset. Regulation of epithelial homeostasis by serotonin is not restricted to the mammary epithelium. In fact, serotonin has also been implicated in epithelial homeostasis of the lung, pancreas, liver and prostate. Therefore, variation in local serotonin signalling may be a common mark of cancer progression in epithelial tumors.

[0007] SUMMARY OF THE INVENTION

[0008] Iron is a critical cofactor for tyrosine and tryptophan-hydroxylases, the ratelimiting enzymes involved in synthesizing dopamine and serotonin, respectively. Maintaining adequate iron levels in the brain is critical, as excess iron can be highly toxic. Iron accumulation can generate oxidative stress and lead to neuronal loss and a variety of neurodegenerative and psychiatric conditions. Noting that systemic exposure of iron- chelating agents used to reduce concentrations of free iron is undesired, the inventors have resorted to developing non-systemic iron-chelator formulations that comprise nonhydrophobic low molecular weight iron-chelators and which exclusively target the central nervous system (CNS).

[0009] In a first of its aspects, the invention concerns a CNS-targeting non-systemic formulation for regulating dopamine and / or serotonin levels in the CNS of a subject, the formulation comprising at least one non-hydrophobic low molecular weight iron-chelator and a pharmaceutically acceptable carrier.

[0010] The invention further concerns a non-hydrophobic low molecular weight iron- chelator for use in a method of regulating dopamine and / or serotonin levels in CNS of a subject, the non-hydrophobic low molecular weight iron-chelator being provided in a formulation suitable for delivery to the CNS of the subject without inducing a systemic effect.

[0011] The “ non-hydrophobic low molecular weight iron-chelator used as an active agent in formulations and methods of the invention is a small molecule having a molecular weight smaller than 300 Da and a water solubility that may be very low or substantial. The active agent is structured to permit interaction with iron ions, e.g., Fe2+and / or Fe3+, through two or more donor atoms (e.g., O, N, S) to form a stable iron complex. Thermodynamically, the iron chelator may be selected to have a stability constant, P (or I< / =[FeL] / (Fe][L])), greater than 106, or greater than IO20The active agent may be any such material, as known in the art, or which is determined to have the aforementioned characteristics.

[0012] In some embodiments, the molecular weight is between 80 and 300 Da. In some embodiments, the molecular weight is between 100 and 300, 120 and 300, 140 and 300, 160 and 300, 180 and 300, 200 and 300, 220 and 300, 240 and 300, 260 and 300, 280 and 300, 100 and 150, 150 and 200, 200 and 250, or between 250 and 300 Da.

[0013] The chelator is said to be non-hydrophobic, namely which is substantially not lipophilic. The chelator may be of any water solubility. The chelator may have water solubility that is between 0.05 mg / ml to 20 g / 1.

[0014] In some embodiments, the iron chelator may be selected from hydroxamates, catecholates, hydroxypyridinones (HOPOs), polyaminocarboxylates and others. Non-limiting examples include enterobactin, seferoxamine, diethylenetriaminepentaacetic acid (DTPA), deferiprone (DFP), deferoxamine, deferasirox, deferitazole, desferrithiocin and others.

[0015] In some embodiments, the active agent is deferiprone (DFP).

[0016] The invention further provides a CNS-targeting non-systemic formulation for regulating dopamine and / or serotonin levels in the CNS of a subject, the formulation comprising a chelator such as DFP and a pharmaceutically acceptable carrier.

[0017] To avoid systemic effects, mainly iron depletion in the blood stream, formulations of the invention are designed and intended for CNS-targeted non-systemic delivery, utilizing administration routes that do not involve wide distribution of the active agent in the body of the subject. These administration routes defining CNS-targeted formulations of the invention may be intranasal or routes of direct administration to the CNS, which are generally regarded non-systemic.

[0018] Excluded from methods of administration are systemic methods such as oral administration and parenteral modes of administration.

[0019] It should be noted that even when a chelator is intentionally delivered to the CNS, systemic exposure and systemic adverse effects may nevertheless occur. Chelators placed into the CSF or brain parenchyma are subject to clearance into meningeal vessels, lymphatics and the general circulation. The rate and extent of this clearance vary with molecule size, lipophilicity and local flow dynamics. With intranasal administration, a fraction of the administered dose follows neuronal routes to the brain, while another portion may be absorbed across the richly perfused nasal mucosa into systemic blood. The systemic effects that can result are predictable and may mainly result from a reduction in the concentration of free iron in the blood.

[0020] Direct delivery into the cerebrospinal fluid (CSF) is a well-established way to place a drug into the CNS while largely bypassing the blood-brain barrier. Intrathecal delivery by a single lumbar puncture or via an implanted intrathecal pump and catheter introduces drug into the spinal subarachnoid space so it mixes with CSF and reaches the spinal cord and, to a variable extent, the brain. This route is commonly used for spinal analgesics, certain antibiotics and chemotherapeutics, and for continuous infusion therapies where steady CNS exposure is desired; implanted pumps can reduce repeated punctures. Intraventricular delivery uses an implanted reservoir / catheter (e.g., an Ommaya reservoir) to deposit the active directly into the brain ventricles, improving CSF distribution for repeated dosing or for agents that poorly reach the brain from the spine. It is commonly used for intrathecal chemotherapy and for situations where serial lumbar punctures are impractical. Both intrathecal and intraventricular approaches greatly reduce required systemic doses and improve local exposure, but they are invasive, require sterile, neuro-tol erant formulations, and still permit gradual clearance from CSF into the systemic circulation.

[0021] When the therapeutic goal is focal brain tissue exposure, direct parenchymal delivery methods may be used. Convection-Enhanced Delivery (CED) uses stereotactically placed microcatheters that generate a local positive pressure gradient to convectively push solution through interstitial spaces, achieving distribution over centimeter-scale volumes of tissue that diffusion alone cannot reach. CED, therefore, allows higher local concentrations with minimal systemic exposure. Other intraparenchymal techniques include stereotactic injections and surgical placement of biodegradable drug-releasing implants (polymeric wafers or depots) in resection cavities may also be used to achieve controlled local release adjacent to target tissues. These direct methods maximize local efficacy and minimize systemic dosing, but they are surgically invasive, require precise targeting.

[0022] In the alternative to directly placing an agent into the CNS, transient blood-brain barrier disruption methods may be used. Such methods transiently increase blood-brain barrier permeability in a controlled region so systemically administered agents can enter the brain tissue. For example, noninvasive focused ultrasound (FUS) combined with systemically injected microbubbles can be used to permeabilize the blood-brain barrier at targeted sites.

[0023] Unlike the aforementioned direct methods of delivery, intranasal delivery exploits the unique anatomy linking the nasal cavity to the CNS, principally the olfactory and trigeminal nerve pathways, to permit movement of molecules from the nasal mucosa into the cribriform plate region and onward into CSF and brain tissue. Because this route can bypass the blood-brain barrier, it offers a non-invasive path to the CNS that is attractive for iron chelator agents, as disclosed herein. However, only a fraction of an intranasal dose typically reaches the brain directly. A substantial portion may still be cleared by mucociliary action. Formulation and carrier design may vary to improve nose-to-brain transfer. Simple spray solutions can be used for agents of the invention. Mucoadhesive polymers (e.g., chitosan and its derivatives) may be used to extend residence time on the olfactory mucosa and can transiently open tight junctions. Nanoparticles, liposomes and nanoemulsions can increase protection of the agents from enzymatic degradation and can be surface-modified to promote neuronal uptake or transcytosis. Thermoresponsive gels, mucoadhesive powders and bioadhesive nanoparticles may also be used to counter rapid mucociliary clearance and increase the chance of transport along neuronal routes.

[0024] The invention further provides an emulsion (such as a nanoemulsion or a microemulsion) comprising at least one chelator, as defined herein, the microemulsion comprising an oil phase and an aqeous phase, wherein the aqeous phase comprising the at least one chelator.

[0025] In some embodiments, the emulsion is a microemulsion.

[0026] In some embodiments, the emulsion comprises a plurality of nanoscale droplets containing the at least one chelator. In some embodiments, the nanoscale droplets having a size or a diameter ranging between 5 and 70 nm, or between 10 and 50 nm, or between 10 and 30 nm.

[0027] In some embodiments, the emulsion comprising the at least one chelator in an amount of at least 180 mg / mL.

[0028] In some embodiments, the emulsion comprises one or more surfactants, e.g., polysorbate 80, polysorbate 20, PEG 400, PG, benzyl alcohol, water and the at least one chelator, e.g., deferiprone.

[0029] In some embodiment’s, the emulsion is referred to herein as Nano IN.

[0030] In some embodiments, the at least one chelator is confined within the nanodroplets. In some embodiments, the relative diffusivity coefficient, as defined herein, of the chelator, e.g., deferiprone, in the emulsion is around 0.2, indicating significant restriction of the chelator mobility within the emulsion and a strong interactions or entrapment at the interface. In some embodiments, the chelator is confined or positioned at an interface between the surfactant-co-surfactant, thereby contributing to the structural stability and efficiency of the emulsion as a drug delivery system.

[0031] In some embodiments, formulations of the invention are configured for intranasal delivery. Intranasal formulations of the invention are tailored to comprise an amount of the iron chelator that is sufficiently high or that is structured (e.g., in a carrier such as a particle) to ensure an effective dose to be delivered to the olfactory / trigeminal region, keeping the agent intact long enough to be absorbed along neuronal or epithelial routes, and avoid local toxicity or rapid mucociliary clearance. The formulations comprise carriers, mucoadhesives, penetration enhancers, stabilizers, and pharmaceutically acceptable excipients that together control residence time, permeability, stability and tolerability. In some cases, formulations of the invention may be aqueous formulations (e.g., as metered sprays, atomizers); dry powders or powder blends suitable for reconstitution in a buffered saline, hydrogel matrices or oil-in-water nanoemulsions or lipid carriers. Mucoadhesive polymers may be included to increase contact with the olfactory mucosa. Biopolymers such as chitosan and its derivatives (e.g., trimethylchitosan), hyaluronic acid, carbomers (Carbopol®), hydroxypropyl methylcellulose (HPMC) and polyvinylpyrrolidone (PVP) may be used. Chitosan is notable because, besides adhesion, it can transiently open tight junctions and improve uptake. Hyaluronic acid and HPMC may also or alternatively used to modulate viscosity and residence time. Permeation enhancers and absorption promoters that increase trans- epithelial or neuronal uptake may also be included. These include bioadhesive / ionic polymers (chitosan), fatty acids (e.g., oleic acid, medium-chain fatty acids), bile salts (e.g., sodium deoxycholate), cyclodextrins (especially hydroxypropyl-P-cyclodextrin) and certain surfactants at low concentrations. Cyclodextrins may be used to improve solubility and to transiently enhance paracellular transport without gross mucosal damage. Stabilizers and protective excipients may also be used for preserving labile chelators. These include protease inhibitors (to reduce enzymatic degradation), antioxidants (glutathione, ascorbate derivatives), tonicity adjusters (sodium chloride, glycerol), vitamins (such as vitamin E, N-acetyl cysteine) and pH buffers to maintain a pH compatible with both the chelator stability and mucosal tolerance (typically near physiologic pH). Colloidal and particulate carriers may also be used to enhanced brain access. Liposomes, solid-lipid nanoparticles, polymeric nanoparticles (PLGA, PEGylated polymers), nanoemulsions and exosome-like vesicles can protect the chelator from degradation, promote uptake by olfactory / trigeminal neurons and enable controlled release. Surface modification (e.g., with targeting ligands, cell -penetrating peptides or mucoadhesive moi eties) may tune biodistribution and neuronal uptake. In another aspect, the invention concerns an intranasal formulation for regulating dopamine and / or serotonin levels in the CNS of a subject, the formulation comprising a chelator as defined herein, e.g., DFP, and a pharmaceutically acceptable carrier.

[0032] Non-limiting examples of agents useful for preparing intranasal or generally nasal formulations include bioadhesive polymers such as mucoadhesive polymers, e.g., chitosan, amylose, amylopectin, carbopol, cellulose, carboxymethylcellulose, sodium alginate, gellan gum, hyaluronan and poloxamer; permeation enhancers such as cyclodextrins, protease inhibitors, cationic polymers and tight junction modulators; surfactants that enhance absorption of the active agent, e.g., phospholipids, bile salts, nonionic surfactants, salts of fatty acids and alkyl glycosides; buffers, such as citrate, TRIS, HEPES, acetate, phosphate, phosphate buffered saline, MOPS and MES; preservatives such as anti-oxidants; viscosity modifiers such as hydroxypropylmethyl cellulose (HPMC), microcrystalline cellulose (MCC), MCC / carboxymethylcellulose mixtures, dextran; tonicity modifiers such as mannitol, dextrose, sodium chloride, sorbitol and maltitol; vitamins and other supplements; and other materials.

[0033] In some embodiments, the intranasal formulation is an aqueous formulation or a formulation comprising any amount of water / saline. The formulation may be in a form of a solution or an emulsion, e.g., a micro or a nanoemulsion.

[0034] In some embodiments, the formulation is an emulsion, e.g., water-in-oil, oil-in- water, or more complex emulsions.

[0035] In some embodiments, the formulation is an oil-in-water microemulsion. The microemulsion may comprise an amount of the active agent, e.g., DFP, present at an interface of oily droplets with dimensions or sizes designed to facilitate transport of the active agent directly to the brain. In some cases, the droplets are of a size between 10 and 50 nm.

[0036] In other configurations of formulations of the invention, the active agent may be encapsulated or carried in liposomes, vesicles, nanocapsules or microcapsules.

[0037] In some embodiments, the invention concerns an intranasal oil-in-water emulsion for regulating dopamine and / or serotonin levels in the CNS of a subject, the formulation comprising at least one iron-chelator, e.g., DFP, and a pharmaceutically acceptable carrier.

[0038] Further embodiments of the invention relate to administration of formulations of the invention to affect regulation of dopamine and / or serotonin levels, to prevent or treat a disease or a condition associated with unregulated levels of the neurotransmitter(s) or increased levels thereof. Regulating the levels of the neurotransmitter(s) is achievable by modulating or regulating levels of iron in the CNS by way of iron chelation, as disclosed herein. The expression regulating dopamine and / or serotonin levels in the CNS” encompasses inducing a change (an increase or a decrease) to the levels of dopamine and / or serotonin in the CNS by regulating iron levels critical for tyrosine and tryptophanhydroxylases involved in synthesizing dopamine and serotonin, respectively. The levels of the neurotransmitter(s) may be decreased by increasing the dosage of the iron-chelator used. Thus, in some embodiments, the regulation encompasses reducing the levels of the neurotransmitter(s). In some instances, regulating the levels in the CNS causes a decrease in the activity of tyrosine and tryptophan-hydroxylases, and therefore a decrease in the levels of serotonin and / or dopamine.

[0039] In some embodiments, the invention concerns an intranasal oil-in-water emulsion for reducing dopamine and / or serotonin levels in the CNS of a subject, the formulation comprising at least one iron-chelator, e.g., DFP, and a pharmaceutically acceptable carrier.

[0040] Within the context of the present application, the dopamine and / or serotonin regulation is for the purpose of achieving prevention or treatment of a psychiatric or a neurodegenerative disease, or any such disease or condition linked to or associated with having too much or too little dopamine or serotonin in the CNS, e.g., brain. In some embodiments, the disease involves having excess serotonin / dopamine.

[0041] It should be noted that while diseases such as schizophrenia are often associated with hyperdopaminergia and hyperserotonergia, the real-life situation is more complex, as some brain regions (i.e. prefrontal cortex) may actually display hypodopaminergia. Moreover, while striatal dopamine (and serotonin) levels are ostensibly decreased following deferiprone administration, there may be other brain regions which could potentially display compensatory (and possibly opposing) changes.

[0042] Therefore, the invention further provides a formulation for CNS delivery for restoring physiologic neurotransmission across various brain disorders (i.e., it is neurotransmitter-focused yet cross-disorder), in a subject suffering from dopamine / serotonin perturbations, the formulation comprising at least one nonhydrophobic low molecular weight iron-chelator and a pharmaceutically acceptable carrier. Further provided is a method for restoring homeostasis in a subject suffering from perturbations in dopamine / serotonin levels, the method comprising non-systemically administering to the subject at least one iron-chelator or a formulation comprising same.

[0043] In another aspect, there is provided a method for preventing or treating a disease or condition associated with or caused by high or irregulated levels of dopamine and / or serotonin in the CNS of a subject, the method comprising non-systemically administering to the subject at least one iron-chelator or a formulation comprising same.

[0044] The iron-chelator and formulation are each as defined herein.

[0045] In some embodiments, the formulation is administered intranasally. In some embodiments, administration of the formulation induces a reduction in dopamine and / or serotonin levels in the subject’s brain.

[0046] The invention further provides a method for preventing or treating a disease or condition associated with high or irregulated levels of dopamine and / or serotonin in the CNS of a subject, the method comprising intranasally administering to the subject at least one iron-chelator or a formulation comprising same.

[0047] The invention further provides a method for preventing or treating a disease or condition associated with high or irregulated levels of dopamine and / or serotonin in the CNS of a subject, the method comprising intranasally administering to the subject DFP or a formulation comprising same.

[0048] The invention further provides a method for preventing or treating a disease or condition associated with high or irregulated levels of dopamine and / or serotonin in the CNS of a subject, the method comprising intranasally administering to the subject an intranasal formulation comprising DFP in an amount effective to reduce levels of dopamine and / or serotonin in the subject brain.

[0049] The disease or condition that can be prevented or treated by administering a formulation of the invention may encompass cognitive deficiencies and emotional disturbances related to aging, neurodegenerative and developmental disorders as well as psychiatric disorders. These include autism spectrum disorders, ADHD, cerebral palsy, Gilles de la Tourette's syndrome, brain injury, Parkinson's disease and related parkinsonian syndromes, Huntington's disease, Alzheimer's disease, dementia disorders, conditions associated with or caused by a serotonin storm, conditions associated with or caused by overdoses of SSRI or SRI therapies, and others. Particularly, formulations and methods of the invention are contemplated for preventing and treating psychiatric and neurological diseases. These include, for example psychosis, schizophrenia, Parkinson's disease, Huntington disease, bipolar disorder and dementia.

[0050] In some embodiments, the disease is schizophrenia or psychosis.

[0051] The invention further provides a method for preventing or treating schizophrenia or psychosis, the method comprising intranasally administering to the subject an intranasal formulation comprising DFP in an amount effective to reduce levels of dopamine and / or serotonin in the subject brain.

[0052] Generally, methods of the invention involve administration of a therapeutic amount of a formulation comprising the iron chelator or a therapeutic effective amount of the chelator itself. The specific chelator and its amount are selected such that they are effective to ameliorate undesired symptoms associated with a disease, to prevent manifestation of such symptoms before they occur, to slow down progression of the disease, slow down deterioration of symptoms, to enhance onset of remission period, slow down irreversible damage caused in the progressive chronic stage of the disease, to delay onset of said progressive stage, to lessen severity or cure the disease, to improve survival rate or more rapid recovery, or to prevent the disease form occurring or a combination of two or more of the above.

[0053] The effective amount for purposes herein may be determined by such considerations as may be known in the art. The amount must be effective to achieve the desired therapeutic effect as described above, depending, inter alia, on the type and severity of the disease to be treated and the treatment regime. The effective amount is typically determined in appropriately designed clinical trials (dose range studies) and the person versed in the art will know how to properly conduct such trials in order to determine the effective amount. As generally known, an effective amount depends on a variety of factors including the affinity of the ligand to the receptor, its distribution profile within the body, a variety of pharmacological parameters such as half-life in the body, on undesired side effects, if any, on factors such as age and gender, etc

[0054] BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1. Schematic representation of DESI-MSI.

[0056] Figure 2. Schematic outline of the developments leading to the invention Figures 3A-D. Characterization and preparation of nano IN microemulsion system. (A) Schematic illustration of the preparation process for nano IN, a deferiprone- loaded nanocarrier system optimized for intranasal delivery. (B) Visual appearance of the final formulation showing a clear and stable microemulsion. (C) Dynamic light scattering (DLS) analysis demonstrating the particle size distribution of nano IN compared to the vehicle control (2X3 replicates). (D) Cryogenic transmission electron microscopy (cryo- TEM) image confirming the nanoscale morphology and uniform structure of nano IN.

[0057] Figures 4A-C. Self-diffusion NMR (SD-NMR) analysis of nano_IN. (A) Diffusion coefficients (D) of individual components — surfactants, co-surfactants, deferiprone, and water — in the nano IN compared to the vehicle, revealing altered molecular mobility upon drug loading. (B) Relative diffusion coefficients (D / Do) of each component, calculated by comparing their diffusivity in the nanocarriers to their unrestricted motion in aqueous solution, indicating increased interfacial association of deferiprone. (C) Schematic illustration of the proposed microemulsion system showing deferiprone localized at the oil-water interface, interacting with both surfactant and cosurfactant layers, consistent with SD-NMR findings.

[0058] Figures 5A-B. Physical assessment of nano_IN using LUMiSizer®. (A) LUMiSizer® transmission profile of nano IN, showing consistent light transmission across the entire cuvette length (x-axis: cuvette position; y-axis: light transmission), indicating no phase separation and excellent physical stability under accelerated conditions. (B) Image of the LUMiSizer® instrument setup, illustrating horizontal placement of the cuvette, which allows light to pass through the full length of the sample for real-time monitoring of destabilization processes.

[0059] Figures 6A-C. Ex vivo permeation of deferiprone across porcine nasal mucosa using Franz diffusion cells. (A) Schematic illustration of the Franz diffusion cell setup, showing the placement of porcine nasal mucosa between the donor and receptor chambers. (B) Quantification of deferiprone extracted from the nasal mucosal tissue over time, demonstrating enhanced mucosal uptake with nano IN compared to aqueous control (lwt% DF in water). (C) The permeation profile of deferiprone collected from the receptor cells at different time points, comparing nano IN to aqueous control (lwt% DF in water).

[0060] Figures 7A-D. In vivo assessment of brain delivery and deferiprone distribution of the nano IN intranasal delivery system using DESI-MSI. (A) Experimental workflow outlining treatment groups, time points, and analytical methods used to evaluate the temporal profile of brain uptake and molecular distribution of deferiprone following intranasal or systemic administration. (B) Representative DESI-MSI ion images of deferiprone detected in coronal brain sections at Bregma +1 mm, comparing spatial distribution across three treatment groups. Signal intensity is represented by a yellow-to- blue color scale, with warmer colors indicating higher deferiprone abundance; corresponding H&E images are shown for anatomical reference. (C) Quantitative analysis of deferiprone intensity from defined brain regions of interest (ROI), presented as fold change across treatment groups. (D) Plasma concentrations of deferiprone were measured via UHPLC-MS, comparing systemic and intranasal delivery approaches across treatment groups.

[0061] Figures 8A-D. Molecular signature analysis of deferiprone effects in the brain following a single dose of nano IN compared to positive and negative controls. (A) Representative DESI-MSI ion images showing spatial distribution of key neurotransmitters across experimental groups: dopamine (top row), serotonin (middle row), and phenylalanine (bottom row). Groups include intranasal nano IN, positive control (i.p. deferiprone), and negative controls (saline, vehicle, and deferiprone in saline). (B) Quantitative analysis of average signal intensity from defined brain regions of interest (ROI), presented as fold change relative to saline control. Bar graphs display fold change for dopamine (left), serotonin (center), and phenylalanine (right), highlighting molecular alterations induced by each treatment. These data illustrate the distinct neurochemical modulation achieved with nano IN compared to control conditions.

[0062] Figures 9A-G. Evaluation of regional brain distribution and molecular effects of deferiprone following multi-dose intranasal nano IN administration. (A) Coronal crosssections of mouse brain spanning from Bregma +3 to -3 mm, representing the anatomical levels used for DESI-MSI analysis. (B) Schematic workflow illustrates the experimental design, including multi-dose intranasal nano IN administration, tissue collection, and subsequent DESI-MSI. (C) Representative DESI-MSI ion images and corresponding hematoxylin and eosin (H&E) stained sections from saline- and nano IN -treated mice across Bregma levels, showing spatial distribution of deferiprone, dopamine, serotonin, and glutamate. (D) Quantification of deferiprone levels across all brain regions, calculated as average signal intensity from DESI-MSI ROIs, showing successful brain delivery by nano IN compared to saline. (E-G) Regional fold changes of dopamine (E), serotonin (F), and glutamate (G) following nano IN administration relative to saline control, highlighting significant molecular alterations in multiple brain regions.

[0063] Figures 10A-D. Evaluation of Nano IN efficacy in an amphetamine-induced psychosis model using DESI-MSI. (A) Representative DESI-MSI images showing brain distribution of deferiprone and its molecular effects on dopamine and serotonin across four experimental groups (n = 3): Group 1 : saline + i.p. saline; Group 2: saline + i.p. amphetamine; Group 3: i.p. deferiprone + i.p. amphetamine; Group 4: Intranasal Nano IN + i.p. amphetamine. The first row displays H&E-stained brain sections following DESI-MSI. Subsequent rows show spatial intensity maps of deferiprone, dopamine, and serotonin, respectively. Color scale represents signal intensity, with yellow indicating highest abundance. (B) Quantification of deferiprone in cortex and striatum across treatment groups (i.p. deferiprone +amphetamine and Nano IN+amphetamine) based on regions of interest (ROI), presented as fold change relative to i.p+amp control at the cortex. (C) Dopamine levels in the striatum presented as fold change relative to the control group saline saline across all experimental groups. (D) Serotonin levels shown as fold change relative to the control group saline saline at the cortex in both cortex and striatum across all groups.

[0064] DETAILED DESCRIPTION OF EMBODIMENTS

[0065] Brain iron dysregulation has emerged as a key pathological feature across a range of neurological disorders. In neuropsychiatric conditions, including major depression, bipolar disorder, autism spectrum disorder, and psychosis. Excessive iron may disrupt neurotransmitter systems and inflammatory signalling, contributing to the emergence and persistence of behavioural and cognitive symptoms. Similarly, growing evidence implicates iron accumulation in neurodegenerative diseases such as Alzheimer’s, Parkinson’s, and Huntington’s diseases, where elevated iron contributes to oxidative stress, mitochondrial dysfunction, and protein aggregation. A striking example of iron- mediated neurotoxicity is found in the rare group of inherited disorders known as Neurodegeneration with Brain Iron Accumulation, which are characterized by abnormal iron deposition primarily in the basal ganglia. These insights have led to increased research efforts exploring the therapeutic potential of brain-targeted iron chelation as a strategy to mitigate neurotoxicity and slow disease progression. Schizophrenia, as an exemplary disease, is a chronic, debilitating condition that fundamentally alters perception, cognition, and social behaviour. Affecting nearly 1% of the global population, schizophrenia typically manifests in early adulthood and ranks among the top global causes of disability due to its persistent impact on functioning and quality of life. While positive symptoms such as hallucinations and delusions are hallmark features, it is the negative symptoms, including avolition, anhedonia, and cognitive deficits, that are often more disabling and poorly addressed by current treatments. Importantly, individuals with schizophrenia experience 20-25 years of reduced life expectancy, largely due to physical comorbidities (e.g., cardiovascular disease, diabetes), lifestyle risk factors, treatment-related side effects, and a strikingly high suicide rate, with up to 10% dying by suicide and nearly 30% attempting it at least once.

[0066] Like most psychotic diseases, schizophrenia is considered to be caused by an imbalance in neurotransmitters (e.g., dopamine, glutamate, and serotonin). Three theories may be involved: 1) The dopamine hypothesis: the original dopamine hypothesis suggests that hyperactive dopamine transmission results in schizophrenic symptoms. This hypothesis evolved to propose regional imbalances, namely, hyperactivity in the mesolimbic system (related to positive symptoms) and hypoactivity in the cortex (linked to negative and cognitive symptoms). 2) The serotonin hypothesis: early studies showed that LSD (d-lysergic acid) could produce hallucinations by antagonizing serotonin in the brain, because of its structural similarity to serotonin and its ability to block serotonin's effects, leading to the initial hypothesis that schizophrenia might involve serotonin deficiency. This hypothesis was later revised to include serotonin excess, as LSD was also found to mimic certain serotonin actions, suggesting that both reduced and heightened serotonergic activity could contribute to psychotic symptoms. 3) The glutamate / NMDA hypothesis: it suggests that dysfunction of NMD A receptors, particularly on inhibitory GABA interneurons, leads to disinhibition of glutamatergic pyramidal neurons, resulting in cortical hyperexcitability and disrupted information processing. This disinhibition contributes to both positive symptoms (e.g., hallucinations) through excessive downstream dopamine activity, and negative / cognitive symptoms through cortical "noise" and impaired signalling.

[0067] Current pharmacological treatments for schizophrenia primarily involve first- and second-generation antipsychotics, such as haloperidol, risperidone, and olanzapine, which exert their effects by blocking dopamine and serotonin receptors. These medications are only effective at reducing positive symptoms of schizophrenia, but they are largely ineffective against negative and cognitive symptoms. Moreover, their overall clinical effectiveness is limited; only about half of the patients experience sufficient symptom control to support independent living, and an estimated 20-50% of individuals have treatment-resistant schizophrenia. Additionally, long-term antipsychotic use is associated with side effects, including extrapy rami dal symptoms, weight gain, metabolic syndrome, sedation, sexual dysfunction, and increased cardiovascular risk. Importantly, these treatments do not address underlying pathophysiological contributors such as iron accumulation, oxidative stress, or neuroinflammation. These limitations underscore an urgent and unmet need not only for alternative medications but also alternative targets and biologically mechanistic therapies that can more effectively address the complex and multifactorial nature of schizophrenia.

[0068] Deferiprone (DF) is a small, hydrophilic molecule that penetrates the blood-brain barrier (BBB) and forms a 3: 1 complex with ferric iron (Fe3+DF3). DF also exhibits iron redistribution properties, transferring excess iron to extracellular transferrin and enabling more physiologic iron homeostasis. These unique characteristics have led to its investigation in brain disorders associated with brain iron overload, including Parkinson’s disease, Alzheimer’s disease, and Friedreich’s ataxia. Despite variability in clinical outcomes, often dependent on disease stage and adjunctive therapy, DF remains one of the few iron chelators with proven central nervous system activity.

[0069] However, systemic administration of DF has side effects. The most significant adverse effects are neutropenia and agranulocytosis, which necessitate frequent monitoring of white blood cell counts. Additional side effects related to peripheral iron depletion include gastrointestinal discomfort, arthralgia, and zinc deficiency. These toxicities are dose-dependent and more likely to occur with chronic systemic exposure. To overcome these challenges and maximize central efficacy while minimizing peripheral toxicity, this invention aims to develop a nasal nanoformulation of DF (Nano IN) designed for direct nose-to-brain delivery. This targeted approach seeks to bypass the systemic circulation, reduce peripheral exposure, and enhances brain bioavailability, ultimately enhancing safety and therapeutic precision for patients with schizophrenia.

[0070] This approach offers a novel disease-modifying therapeutic avenue for brain disorders associated with elevated iron levels, with a primary focus on schizophrenia and psychosis, where modulating iron may restore neurotransmitter balance and improve clinical outcomes. Moreover, this strategy holds promise for broader application in neurodegenerative and neurodevelopmental diseases marked by iron dysregulation.

[0071] DESI-MSI is a particularly valuable technique for in situ imaging of a wide array of molecules, such as fatty acids, lipids, phospholipids, neurotransmitters, and small metabolites, owing to its excellent sensitivity toward these species. This method enables rapid imaging, measurement, and identification of hundreds of molecules at once from an unmodified tissue sample. It is carried out in the open air without the addition of external matrices and thus does not distort the physiological distribution of molecules in tissues. Although it presents a powerful discovery tool, the DESI-MSI workflow is simple: a beam of charged solvent droplets is directed onto a tissue surface to desorb and ionize molecules. The splash of these droplets then carries the resultant ions into a mass spectrometer for analysis (Fig. 1). A two-dimensional (2-D) imaging stage moves the tissue at a controlled speed to record the mass spectra from different spatial coordinates, and the signal is subsequently converted into 2-D images of molecular ion distributions. Each MS spectrum recorded generates a single pixel in the 2-D image, whereas every MS peak distribution can be translated into a separate heat map.

[0072] For this study, a newly established DESI-MS imaging facility was employed. This state-of-the-art facility encompasses two DESI-MSI setups: one coupled to a quadrupole time-of-flight mass spectrometer (QTOF, Xevo G2-XS, Waters Inc.) for high-mass- resolution untargeted imaging, and the other is coupled to a triple quadrupole mass spectrometer (Xevo TQ Absolute, Waters Inc.) for ultra-sensitive imaging. Both setups enable exceptional spatial resolution of approximately 10 pm.

[0073] A summary of the developments leading to the present invention is provided in Fig. 2.

[0074] Design and characterize novel nano-domains (nano IN) to transport DF directly to the brain. Safe and effective intranasal nano-domains have been tailored to encapsulate the DF at the oil-water interface to facilitate its transport directly to the brain. The drug delivery system incorporated only FDA-approved or GRAS (generally regarded as safe) excipients to create a therapy that can be readily translated into the clinic.

[0075] The intranasal microemulsion (ME) system was loaded with DF, with a domain (droplet) size of 10-50 nm. This formulation is optimized to maximize nasal absorption and brain permeation while ensuring stability, biocompatibility, and controlled drug release.

[0076] Evaluation of the therapeutic impact of the novel nano-domain delivery system by characterizing molecular and neurotransmitter alterations using DESI- MSI. The designed nano-domains facilitate efficient nose-to-brain delivery of DF, by passing the BBB and minimizing peripheral exposure and side effects. Iron is a critical cofactor for several enzymatic processes, including the activity of tyrosine and tryptophan hydroxylases, the rate-limiting enzymes in the biosynthesis of dopamine and serotonin, respectively. By chelating excess brain iron, DF modulates the activity of these enzymes, thereby altering neurotransmitter levels and restoring homeostatic balance. This strategy offers a novel, multi-target mechanistic approach to treating disorders associated with iron-induced neurotransmitter dysregulation.

[0077] The nano IN is administered to animal models (ICR mice) and imaging of the brains by DESI-MSI is achieved to map molecular and neurotransmitter changes, with a particular focus on the prefrontal cortex. The data indicates that in an acute amphetamine- induced model of psychosis, intraperitoneally administered deferiprone attenuates molecular perturbations caused in this model by amphetamine administration, downregulating dopamine and serotonin in relevant brain regions.

[0078] Experimental and Design

[0079] Design and characterize novel nano-domains (nano IN) to transport DF directly to the brain.

[0080] Approach: DF is a water-soluble iron chelator with a reported solubility of 130 mg / mL at 25 °C. However, to further enhance its solubility, stability, and brain-targeted efficiency, a microemulsion-based nano-domain system was formed and optimized for intranasal administration. The primary objective was to maximize the solubility and loading capacity of DF within the nanocarriers while enabling effective transport across the nasal mucosa into the brain.

[0081] Microemulsions are ideal for this purpose due to their thermodynamic stability, high solubilization capacity, and ability to form nanoscale droplets (10-50 nm), which facilitate mucosal permeation and enable access to the olfactory and trigeminal nerve pathways. This strategy was designed to bypass the blood-brain barrier, reduce systemic exposure, and ensure precise brain delivery of DF. Results and Methods:

[0082] The microemulsion system was developed with an enhanced deferiprone loading capacity of 180 mg / mL, exceeding its reported aqueous solubility of 130 mg / mL. As depicted in Fig. 3A, the Nano_IN was prepared in a simple three-step steps: First, the “oil phase” was prepared by mixing selected surfactants and co-surfactants. Then, the “aqueous phase” was separately prepared by dissolving DF and additional co-surfactants in water. This aqueous phase was stirred and gently heated at 45 °C for 30 minutes to ensure complete dissolution. Finally, the two phases were combined under continuous stirring for 2 hours to yield a thermodynamically stable and transparent microemulsion (Table 1)

[0083] Table 1. Nano IN Composition

[0084] The nanocarrier was thoroughly characterized using multiple complementary techniques, including visual inspection (clear and transparent appearance), dynamic light scattering (DLS), and cryo-transmission electron microscopy (cryo-TEM) (Fig. 3B-D). DLS analysis revealed that the average droplets size of the deferiprone-loaded nanocarriers was smaller than that of vehicle, suggesting that deferiprone may localize at the oil-water interface, tightening the interfacial packing and reducing droplet diameter.

[0085] To further validate the hypothesis that deferiprone localizes at the droplets interface, a self-diffusion nuclear magnetic resonance (SD-NMR) was used to measure the mobility of each component within the system. The self-diffusion coefficients (D) of the components in both the vehicle and the deferiprone-loaded microemulsion (nano IN) are presented in Fig. 4A. The diffusion coefficients of the surfactants (polysorbate 80 and 20) were nearly identical in both the loaded and unloaded systems, suggesting they codiffuse as micellar aggregates. Accordingly, these surfactants are grouped under a single category in the analysis. Likewise, PEG 400 and PG showed overlapping diffusion behavior and are collectively presented as "co-surfactants."

[0086] SD-NMR provides the diffusion coefficient (D) of each component, which reflects its molecular mobility and interaction with the surrounding environment. The self-diffusion coefficients of the components in nano IN are slightly lower than in the vehicle (Fig. 4A), meaning that DF interacts with components of the ME, thereby hindering their movements. Notably, the diffusivity of DF was closer to that of the surfactant and co-surfactant phases and distinct from that of water, suggesting that the DF interacts more with the cosurfactant and surfactant and less with the water.

[0087] To further understand the spatial behavior of each component, the relative diffusivity coefficients (D / Do) was calculated, where Do represents the diffusion coefficient of each component measured in dilute aqueous solution, reflecting its free, unconstrained motion. A relative diffusivity of 1 indicates that the molecule is freely moving and does not interact with other components in the system, while lower values imply confinement or interaction within the structured environment. As shown in Fig. 4B, the relative diffusivity of deferiprone approached 0.2, indicating significant restriction of its mobility within the microemulsion, indicating a strong interactions or entrapment at the interface.

[0088] Taken together, these findings support the assumption that deferiprone is not freely diffusing in the aqueous phase, but rather locked at the interface between the surfactant-co-surfactant, contributing to the structural stability and efficiency as a drug delivery system (Fig. 4C).

[0089] Initial evidence for the solubilization capacity and physical stability of the nano IN formulation was based on visual observation of the formulation transparency, indicating the formation of a clear and homogeneous microemulsion. To assess long-term physical stability under accelerated conditions, we employed LUMi Sizer®, an advanced analytical centrifugation technology. This instrument applies centrifugal force to rapidly simulate long-term gravitational stress, while simultaneously measuring light transmission across the sample over time. If phase separation or droplet aggregation occurs, changes in light transmittance are detected and converted into instability profiles, providing a predictive measure of shelf life.

[0090] The nano_IN was subjected to centrifugation at 3000 rpm for 13.3 hours at 25 °C, simulating prolonged storage conditions. The nanodomains maintained their physical stability throughout the analysis, almost no changes in light transmission, indicating high colloidal stability under extreme conditions, predicting a shelf life of approximately 2 years (Fig. 5).

[0091] Next, the nasal permeation of deferiprone-loaded nanocarriers (nano IN) was assessed by performing ex vivo permeation studies using Franz diffusion cells equipped with fresh porcine nasal mucosa, obtained from a local slaughterhouse. The mucosal tissues were mounted between the donor and receptor compartments, with the apical side facing the donor chamber and the basolateral side oriented toward the receptor chamber, providing an exposure area of 0.5 cm2(Fig. 6A).

[0092] The receptor chamber was filled with phosphate-buffered saline (PBS, pH 7.4), freshly prepared, and the temperature of the system was set to 32 °C to mimic nasal cavity conditions. Continuous stirring was maintained using a Teflon-coated magnetic stir bar to ensure homogeneity throughout the experiment. A volume of 85 pL of the nano IN formulation was carefully pipetted and applied onto the surface of the mucosa in the donor chamber.

[0093] At predetermined time points (0.5, 1, 2, 4, 6, and 24 hours), the receiver volume samples were collected, and each mucosal membrane was removed for analysis of residual and absorbed drug content. Remaining residues of the formulation were first removed using three sequential strips of Franz cell -compatible adhesive tape, pressed gently onto the mucosa, and discarded after each application. Residual formulation was further cleaned by gently wiping the mucosal surface with Kimwipes®. The mucosal membrane was then immersed in 20 mL of PBS: methanol (1 : 1, v / v) for deferiprone extraction.

[0094] All extraction vials (excluding receptor phase samples) were placed on an orbital shaker at 300 RPM for 2 hours at room temperature, followed by 30 minutes of sonication to enhance drug recovery. The samples were then vortexed for 1 minute, filtered using 0.2 pm pore-size syringe filters, and transferred into fresh glass vials. The extracted samples were stored at 4 °C in the dark until quantification by high-performance liquid chromatography (HPLC) after 24 hours. All experiments were conducted in triplicate (n=3) to ensure reproducibility and statistical reliability. Permeation results for nano IN were compared to a control formulation of 1 wt% deferiprone in water, with control samples collected at 0.5, 1, 2, 6, and 24 hours.

[0095] Nano IN demonstrated enhanced delivery of deferiprone overtime in the mucosal membrane, with a marked increase in permeation observed by 4 hours, followed by a decline in DF levels at 6 and 24 hours (Fig. 6B). This pattern suggests that deferiprone initially accumulates in the superficial layers of the mucosal membrane and subsequently permeates to deeper tissue layers, eventually reaching the receptor compartment. This is evidenced by the substantial increase in deferiprone concentration detected in the receptor compartment at later time points, reaching nearly 90% permeation at 24 hours (Fig. 6C).

[0096] In contrast, the control group (deferiprone solution) showed lower cumulative permeation in the receptor cells (Fig 6C). A direct comparison of the permeability of the control group in the mucosal membrane at the 4-hour time point was not possible due to the absence of corresponding data.

[0097] These findings highlight the superior permeation and retention properties of the nano IN formulation relative to the aqueous deferiprone solution, supporting its potential for efficient nose-to-brain delivery.

[0098] Evaluate the therapeutic impact of the novel nano-domain delivery system by characterizing molecular and neurotransmitter alterations using DESI-MSI.

[0099] Approach: To assess the efficiency of deferiprone delivered via our intranasal nano-domain system (nano IN), we conducted in vivo studies in ICR male mice, employing both healthy and an acute amphetamine-induced model of psychosis.

[0100] Results& Methods:

[0101] To assess the profile of brain delivery and molecular engagement of deferiprone administered via our intranasal nano-domain system (nano IN), this aim was divided into three sub-aims:

[0102] Determine the optimal time point for brain delivery of deferiprone using the intranasal nano-domain system (nano IN).

[0103] Male ICR mice (31-43 g) were divided into three groups, each receiving different treatments and being sacrificed at specific time points to evaluate DF distribution. Group 1: the negative control group received intranasal treatment of either saline, Nano IN vehicle, or DF in saline (1 mL / kg). Group 2: the positive control group received intraperitoneal (i.p.) DF (100 mg / kg) and was sacrificed at 2 and 24 hours post- administration. Group 3: the treatment group received intranasal nano IN (1 mL / kg) and was sacrificed at 0.5, 2, and 24 hours post-administration to evaluate time-dependent brain uptake (Fig. 7A). At each designated time point, mice were anesthetized with isofhirane, and blood was collected via transcardial puncture. The whole brains were rapidly extracted and snap-frozen by immersion for 4 seconds in 2-methylbutane chilled in liquid nitrogen. Frozen brains were stored on dry ice then coronally sectioned using a cryostat at bregma +1. Brain sections were mounted onto glass slides and fixed in a desiccator for 5 minutes before analysis. DESI-MSI was used to map the spatial distribution of deferiprone and associated metabolic changes in the brain. Following DESI-MSI, the same tissue sections were stained with hematoxylin and eosin (H&E) using a previously established protocol for unfixed frozen sections. Optical images of the stained tissues were acquired using a stereo zoom trinocular microscope (Motic Microscopes, USA) to correlate anatomical structures with molecular imaging data.

[0104] To compare brain and peripheral exposure to DF between systemic and intranasal delivery approaches, DF concentrations in the plasma were quantified using liquid chromatography-mass spectrometry (LC-MS).

[0105] The presence of deferiprone was confirmed in the brain following administration using DESI-MSI analysis (Fig. 7B-C). As expected, i.p. administration resulted in higher average deferiprone intensity in brain tissue compared to nano IN, even at its peak time point (30 minutes). This difference is primarily attributable to the substantially higher i.p. dose (approximately six-fold greater than the intranasal dose). Moreover, intranasal administration in mice presents technical challenges due to their small nasal cavities, often leading to partial loss of the administered dose through reflux or external leakage, reducing the effective amount absorbed. Despite these limitations, nano IN successfully delivered deferiprone to the brain, with comparable regional intensity to the deferiprone- in-saline intranasal control (Fig. 7C). The highest signal intensity for nano IN was observed at 30 minutes post-administration, followed by a notable decline at 24 hours, mirroring the temporal profile observed with i.p. administration.

[0106] To assess peripheral exposure, deferiprone concentrations in blood were quantified at multiple time points post-administration using UHPLC-MS. For the i.p. group, only a single replicate was obtained due to technical issues; additional replicates will be included in future studies. As expected, i.p. administration showed higher systemic levels of deferiprone, with blood concentrations approximately 12-fold greater than those observed following intranasal delivery. These findings indicate that nano_IN substantially limits peripheral exposure, potentially reducing the risk of systemic adverse effects such as agranulocytosis. By 24 hours post-dose, both administration routes exhibited minimal residual deferiprone in circulation, consistent with systemic clearance.

[0107] DESI-MSI analysis revealed distinct molecular changes in brain neurotransmitter levels following deferiprone administration (Fig. 8). A reduction in dopamine levels was observed as early as 2 hours post i.p. injection and at both 30 minutes and 2 hours following intranasal nano_IN administration. By 24 hours post-treatment, dopamine levels in both treatment groups returned to near baseline, comparable to those observed in the saline-treated controls. Although deferiprone delivered intranasally in saline resulted in comparable brain concentrations to nano_IN (as confirmed by DESI-MSI), it did not lead to measurable reductions in dopamine levels. This finding suggests that while both nano In and DF in saline reached the brain, only nano IN may facilitate enhanced intracellular or cell-targeted delivery, potentially enabling more effective functional engagement of deferiprone.

[0108] In addition to dopamine, we also observed a reduction in serotonin levels and an increase in phenylalanine following i.p. administration of deferiprone. A similar trend was seen with nano IN, although these changes did not reach statistical significance, likely due to the limited sample size. Nevertheless, qualitative comparisons suggest that nano IN administration also leads to a reduction in serotonin, distinguishable from both saline and deferiprone-in-saline groups. These preliminary findings support the hypothesis that deferiprone delivered via nano IN modulates neurotransmitter pathways beyond dopamine and may offer multi-target neuromodulatory effects, warranting further investigation with larger cohorts.

[0109] Evaluation of the regional distribution gradient of deferiprone in the brain following multiple doses of Nano IN using DESI-MSI

[0110] To further assess the efficacy of nano IN and investigate its distribution across distinct brain regions, a multiple-dose administration study was conducted in male ICR mice. In a previous single-dose study, the maximum intranasal volume allowed (30 pL) limited the deferiprone dose to approximately 0.54 mg, in contrast to the intraperitoneal (i.p.) group, which received 100 mg / kg, equivalent to approximately 3 mg total. Thus, the i.p. dose was approximately 5.5 times higher than the intranasal dose. To address this limitation and evaluate cumulative delivery via the intranasal route, mice were administered three intranasal doses; two intranasal doses of nano IN the day before sacrifice, followed by an additional single intranasal dose (30 pL) administered 2 hours prior to tissue collection. Mice (31-43 g) were randomly divided into three groups (n = 3 / group): Group 1 (Negative Control): Intranasal saline, group 2 (Positive Control): Intraperitoneal deferiprone (100 mg / kg), and group 3 (Treatment): Intranasal nano IN, administered as a multi-dose regimen (Fig. 9B)

[0111] Two hours following the final dose, mice were anesthetized using isoflurane, and whole brains were rapidly extracted. Tissues were snap-frozen by immersion for 4 seconds in 2-methylbutane chilled in liquid nitrogen and stored on dry ice. Brains were coronally sectioned using a cryostat at multiple anatomical levels (Bregma +3, +2, +1, 0, -1, -2, and -3) (Fig. 9A). Sections (10 pm thickness) were mounted onto glass slides and fixed in a desiccator for 5 minutes before analysis.

[0112] Using DESI-MSI we mapped the spatial distribution of deferiprone and associated neurotransmitters alterations across brain regions. Following DESI-MSI analysis, the same tissue sections were stained with hematoxylin and eosin (H&E) using a previously established protocol for unfixed frozen sections. Optical images of the stained tissues were acquired using a stereo zoom trinocular microscope (Motic Microscopes, USA) to correlate anatomical structures with molecular imaging data.

[0113] DESI-MSI successfully detected deferiprone in all analyzed brain regions in the nano IN group, demonstrating broad and uniform distribution across the brain (Fig. 9C). Quantitative analysis confirmed consistent signal intensity across all Bregma levels, indicating effective delivery by the nanocarrier system to both anterior and posterior brain regions (Fig. 9D)

[0114] Molecular signature analysis revealed that nano IN treatment significantly reduced dopamine levels across all analyzed brain regions relative to saline controls (Fig. 9E). Serotonin levels were also significantly decreased at Bregma +2 and +1 (Fig. 9F). Glutamate levels were also significantly reduced in anterior brain regions (Bregma +3 to 0) (Fig. 9G). This reduction in glutamate may reflect decreased excitatory neurotransmission due to iron chelation, which could be beneficial in mitigating glutamate-driven hyperexcitability observed in psychosis.

[0115] Overall, these findings support the efficacy of nano IN in delivering deferiprone to multiple brain regions and modulating key neurotransmitter systems relevant to psychotic disorders. Evaluation of the Therapeutic Efficacy of Nano IN in an Amphetamine -Induced Psychosis Model.

[0116] To assess the therapeutic potential of nano IN in a model of dopaminergic hyperactivity, we employed an amphetamine-induced psychosis model in male ICR mice (31-43 g), a validated system that replicates key molecular features of psychotic disorders. Mice were randomly divided into four experimental groups (n = 3) each receiving specific treatments at two time points: Intranasal or intraperitoneal treatment administered 2 hours prior to sacrifice, and Amphetamine (Amp) or saline injection administered 30 minutes prior to sacrifice. The treatment groups were as follows: Group 1: Intranasal saline (1 mL / kg, at -2 h) + saline (i.p., at -0.5 h). Group 2: Intranasal saline (1 mL / kg, at -2 h) + amp (3 mg / kg, i.p., at -0.5 h). Group 3: i.p. DF (100 mg / kg, at -2 h) + amp (3 mg / kg, i.p., at -0.5 h). Group 4: Intranasal nano_IN (1 mL / kg, at -2 h) + amp (3 mg / kg, i.p., at -0.5 h).

[0117] Thirty minutes following amphetamine or saline injection, mice were anesthetized with isoflurane, and whole brains were rapidly extracted. Brains were snap-frozen by immersion in 2-methylbutane chilled in liquid nitrogen for 4 seconds and stored on dry ice. Coronal brain sections were prepared at Bregma +1 mm using a cryostat. Sections (10 pm thickness) were mounted onto glass slides and placed in a desiccator for 5 minutes before analysis by DESI-MSI. This experimental design enables us to evaluate the ability of nano IN to attenuate amphetamine-induced molecular alterations in the brain.

[0118] Using DESI-MSI, we confirmed successful brain delivery of deferiprone following intranasal administration of Nano IN (Fig. 10B). The signal intensities detected in the striatum were slightly higher than that detected in the cortex at both intranasal and i.p. administration (Figs. 10A-B).

[0119] Amphetamine treatment induced a trend toward elevated dopamine levels in the striatum and increased serotonin levels in the cortex; however, these changes did not reach statistical significance (Figs. 10C-D). Despite this, treatment with Nano_IN significantly attenuated both dopamine and serotonin levels following amphetamine challenge, with effects comparable to those observed following systemic administration of deferiprone in the striatum. These findings underscore the therapeutic potential of Nano IN in modulating key neurotransmitters implicated in neuropsychiatric disorders associated with iron dysregulation, such as psychosis and schizophrenia.

Claims

CLAIMS:

1. A CNS-targeting non-systemic formulation for regulating dopamine and / or serotonin levels in the central nervous system (CNS) of a subject, the formulation comprising at least one non-hydrophobic low molecular weight iron-chelator and a pharmaceutically acceptable carrier.

2. The formulation according to claim 1 , wherein the iron-chelator is a water-soluble material having a molecular weight smaller than 300 Da.

3. The formulation according to claim 1 or 2, wherein the iron-chelator having a stability constant greater than 106.

4. The formulation according to any one of the preceding claims, wherein the iron- chelator having a water solubility between 0.05 mg / ml to 20 g / 1.

5. The formulation according to any one of the preceding claims, wherein the iron- chelator is selected from hydroxamates, catecholates, hydroxypyridinones (HOPOs), polyaminocarboxylates.

6. The formulation according to any one of the preceding claims, wherein the iron- chelator is selected from enterobactin, seferoxamine, diethylenetriaminepentaacetic acid (DTP A), deferiprone (DFP), deferoxamine, deferasirox, deferitazole, desferrithiocin.

7. The formulation according to claim 6, wherein the iron-chelator is selected from deferiprone (DFP), deferoxamine, deferasirox, deferitazole, desferrithiocin.

8. The formulation according to any one of the preceding claims, wherein the iron- chelator is deferiprone (DFP).

9. A CNS-targeting non-systemic formulation for regulating dopamine and / or serotonin levels in the CNS of a subject, the formulation comprising DFP and a pharmaceutically acceptable carrier.

10. The formulation according to any one of the preceding claims, for direct delivery to the CNS or for intranasal delivery.

11. The formulation according to any one of the preceding claims, being an intranasal formulation.

12. An intranasal formulation for regulating dopamine and / or serotonin levels in the CNS of a subject, the formulation comprising at least one non-hydrophobic low molecular weight iron-chelator and a pharmaceutically acceptable carrier.

13. The formulation according to claim 12, wherein the iron-chelator is DFP.

14. The formulation according to any one of the preceding claims, being an aqueous formulation.

15. The formulation according to any one of the preceding claims, being an emulsion.

16. The formulation according to claim 15, wherein the emulsion is a microemulsion or a nanoemulsion.

17. An emulsion comprising at least one non-hydrophobic low molecular weight iron- chelator, the emulsion comprising an oil phase and an aqeous phase, wherein the aqeous phase comprising the at least one iron-chelator.

18. The emulsion according to claim 17, wherein the emulsion is a microemulsion.

19. The emulsion according to claim 17, wherein the emulsion comprises a plurality of nanoscale droplets containing the at least one iron-chelator.

20. The emulsion according to claim 19, wherein the nanoscale droplets having a size or a diameter ranging between 5 and 70 nm, or between 10 and 50 nm, or between 10 and 30 nm.

21. The emulsion according to claim 17, wherein the emulsion comprises the at least one iron-chelator in an amount of at least 180 mg / mL.

22. The emulsion according to any one of claims 17 to 21, the emulsion comprises one or more surfactants.

23. The emulsion according to claim 22, comprising polysorbate 80, polysorbate 20, PEG 400, PG, benzyl alcohol, water and the at least one iron-chelator.

24. The emulsion according to any one of claims 17 to 23, wherein the at least one iron-chelator is confined within the nanodroplets.

25. The emulsion according to any one of claims 17 to 24, wherein a relative diffusivity coefficient of the chelator in the emulsion is below 0.2.

26. The emulsion according to any one of claims 17 to 25, being an intranasal oil-in- water emulsion for regulating dopamine and / or serotonin levels in the CNS of a subject.

27. The emulsion according to claim 26, wherein said regulating of dopamine and / or serotonin levels, prevents or treats a disease or a condition associated with unregulated levels thereof.

28. The emulsion according to claim 27, for decreasing serotonin and / or dopamine levels.

29. A non-hydrophobic low molecular weight iron-chelator for use in a method of regulating dopamine and / or serotonin levels in CNS of a subject, the non-hydrophobic low molecular weight iron-chelator being provided in a formulation suitable for a direct or an indirect delivery to the CNS of the subject without inducing a systemic effect.

30. A method for preventing or treating a disease or condition associated with or caused by high or irregulated levels of dopamine and / or serotonin in the CNS of a subject, the method comprising non-systemically administering to the subject at least one nonhydrophobic low molecular weight iron-chelator or a formulation comprising same.

31. The method according to claim 30, wherein the iron-chelator is selected from enterobactin, seferoxamine, diethylenetriaminepentaacetic acid (DTP A), deferiprone (DFP), deferoxamine, deferasirox, deferitazole, desferrithiocin.

32. The method according to claim 31, wherein the iron-chelator is selected from deferiprone (DFP), deferoxamine, deferasirox, deferitazole, desferrithiocin.

33. The method according to claim 31, wherein the iron-chelator is deferiprone (DFP).

34. The method according to any one of claims 30 to 33, wherein the iron-chelator or formulation comprising same is administered intranasally.

35. The method according to claim 30, wherein the formulation is an emulsion according to any one of claims 17 to 28.

36. A method for preventing or treating a disease or condition associated with high or irregulated levels of dopamine and / or serotonin in the CNS of a subject, the method comprising intranasally administering to the subject at least one iron-chelator or a formulation comprising same.

37. The method according to claim 36, wherein the formulation is an emulsion according to any one of claims 17 to 28.

38. The method according to any one of claims 30 to 37, wherein the iron-chelator is DFP or a formulation comprising same.

39. A method for preventing or treating a disease or condition associated with high or irregulated levels of dopamine and / or serotonin in the CNS of a subject, the method comprising intranasally administering to the subject an intranasal emulsion comprising DFP in an amount effective to reduce levels of dopamine and / or serotonin in the subject brain.

40. The method according to any one of claims 30 to 39, wherein the disease or condition is a cognitive deficiency, an emotional disturbance related to aging, a neurodegenerative and developmental disorder or a psychiatric disorder.

41. The method according to claim 40, wherein the disease or condition is selected from psychosis, autism spectrum disorders, ADHD, Cerebral Palsy, Gilles de la Tourette's syndrome, brain injury, Parkinson's disease and related parkinsonian syndromes, Huntington's disease, Alzheimer's disease, dementia disorders, conditions associated with or caused by a serotonin storm, conditions associated with or caused by overdoses of SSRI or SRI therapies.

42. The method according to claim 40, wherein the disease or condition is a psychiatric or a neurological disease.

43. The method according to claim 41, wherein the disease or condition is psychosis, schizophrenia, Parkinson's disease, Huntington disease, bipolar disorder and dementia.

44. The method according to claim 43, wherein the disease or condition is schizophrenia or psychosis.

45. A method for preventing or treating psychosis, the method comprising intranasally administering to the subject an intranasal formulation comprising DFP in an amount effective to reduce levels of dopamine and / or serotonin in the subject brain.

46. The method according to claim 45, wherein the DFP is provided in a nano or a microemulsion.

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