Intranasal delivery

Abstract

The present invention relates to formulations, devices and delivery of leucine-5-enkephalin (LENK) formulated with N-palmitoyl-N-acetyl-N-monomethyl-N,N-dimethyl-N,N,N-trimethyl-6-O-glycolchitosan (GCPQor Molecular Envelope Technology - MET). In particular, the present invention relates to delivering leucine-5-enkephalin encapsulated in GCPQ. intranasally via a drug or medicine dispersion and delivery system. The dispersion methods employ curved surfaces to impart rotational motions to one or more fluid flows from a pressurized container to create turbulence and improve mixing of medicament with fluid.

Description

[0001] Intranasal Delivery FIELD OF THE INVENTION The present invention relates to formulations, devices and delivery of leucine-5-enkephalin (LENK) formulated with N-palmitoyl-N-monomethyl-N,N-dimethyl-N,N,N-trimethyl-6-O-glycolchitosan or N- palmitoyl-N-acetyl-N-monomethyl-N,N-dimethyl-N,N,N-trimethyl-6-O-glycolchitosan (GCPQ or Molecular Envelope Technology, MET). In particular, the present invention relates to delivering leucine-5-enkephalin encapsulated in GCPQ intranasally via a drug or medicine dispersion and delivery system. The delivery methods employ curved surfaces to impart rotational motions to one or more fluid flows from a pressurized container to create turbulence and improve mixing of medicament with fluid. BACKGROUND TO THE INVENTION The treatment of diseases of the brain is significantly limited by the blood brain barrier. Over the last decade intranasal administration of drugs has gained increasing interest as a non-parenteral therapy. Originally seen as a route of administration for the local treatment of congestion, infection, rhinitis or nasal polyposis, in recent years a variety of products have entered the market for the systemic treatment of a variety of ailments. Nasal delivery of drugs has many advantages, including avoidance of first pass metabolism, avoidance of degradation in the gut, rapid onset with quick diffusion into the systemic system that parallels with intravenous administration and patient compliance due to the non-invasive nature and ease of self-medication. Within the nasal passage there are two main areas of absorption: the respiratory zone, which has the largest surface area and is highly vascularized, where active principles can cross the epithelium via para- or-transcellular routes, and the olfactory epithelium. The latter comprises only 3-5% of the total surface area of the nasal cavity and therefore is not largely involved in systemic absorption, but can allow direct access to the CNS, bypassing the blood brain barrier via the processes of olfactory neurons, through to the synaptic junctions with neurons of the olfactory bulb. There are physical disadvantages associated with administration via the nasal route that must be overcome. These include mucociliary clearance, enzymatic activity of the nasal mucosa, peptidases and drug metabolizing enzymes. In addition, molecular weight and lipophilicity play a part in absorption—low molecular weight molecules having a molecular weight less than 300 Da tend to be rapidly absorbed whereas for molecules between 300-1000 Da, lipid solubility is an important property. Lipophilic molecules diffuse freely, whereas it is thought that hydrophilic molecules must pass through the paracellular route. Molecules with a molecular weight above 1 kDa absorb very slowly and have a low bioavailability. These barriers can be addressed by altering the physiochemical properties of the molecule, increasing permeability by coadministration of an absorption promoter or reducing excretion / degradation by co-administering inhibitors. Absorption promoters currently under development include alkylsaccharides (Intravail®), chitosan (ChiSys™), low methylated pectin (PecSys™) and polyethylene glycol. Chitosan and its derivatives are commonly used as absorption enhancers due to chisosan's well documented ability to facilitate paracellular transport by opening the tight junctions or by interacting with extra-cellular matrix components. Chitosan increases the bioavailability of verapamil when administered nasally to rabbits in comparison to nasal verapamil solution (Abdel Mouez et al; Eur J Pharm Sci 2013, 30, 59-66). In addition, polylactic acid nanoparticles modified with chitosan have been used to encapsulate the analgesic peptide Neurotoxin. Rats intranasally administered with these chitosan modified nanoparticles had an increased concentration of neurotoxin in the periaqueductal gray in comparison to polylactic acid alone loaded nanoparticles (Zhang et al; Drug Development and Industrial Pharmacy 2013, 39, (11), 1618-24). Endogenous opioid peptides Leucine5-enkephalin (LENK) and Methionine5-enkephalin (MENK) are mainly degraded by cleavage of the N-terminal tyrosine. In the presence of polycarbophil-cysteine (0.25%) and glutathione (1%) LENK has shown reduced degradation and enhanced transportation across freshly excised bovine nasal mucosa. The absorption enhancer sodium glycocholate and protease inhibiter puromycin co-administered with LENK reduced degradation in nasal washings. However, this combination of excipients can lead to cell leakage and therefore toxicity. Chitosan formulations can also reduce the degradation of peptides. For instance, a chitosan-EDTA conjugate has been shown to reduce the degradation of LENK (Bernkop-Schnürch et al; 1997, Pharm Res 14, 917-22). LENK has also been nasally administered with trimethyl chitosan nanoparticles and shown enhanced antinociception in two mouse pain models in comparison to LENK alone (Kumar et al; Int J Biol Macromol 2013, 61C, 189-195). WO2004 / 026912 describes polysaccharides which are used to solubilise hydrophobic drugs. The polysaccharides are amphiphilic and are generally selected from any derivatives of the following: chitosans, dextrans, alginic acids, starches, dextran and guar gums. Quaternary ammonium palmitoyl glycol chitosan (GCPQ) and quaternary ammonium hexadecyl glycol chitosan (GCHQ) are used in the Examples of this patent application as solubilising polysaccharides. WO2008 / 017839 describes micellar clusters formed from amphiphilic carbohydrate polymers and their use in formulating hydrophobic drugs. GCPQ is specifically exemplified as an amphiphilic carbohydrate polymer. In U.S. Pat. No.8,278,277 a lipid ester prodrug of LENK is formed and added to a composition comprising GCPQ. The compositions are delivered intravenously or orally. The prodrug was converted to LENK in vivo and shown to result in significant LENK brain levels. US Patent No.10,213,474 describes the delivery of compositions comprising hydrophobic drugs and amphiphilic carbohydrates administered intranasally. There exists a need to deliver enkephalin compositions to the brain intranasally using a delivery device such that the drug is efficacious, stable and safe. SUMMARY OF THE INVENTION The present invention relates to devices for delivering a composition intranasally to a mammal having a therapeutically effective amount of a leucine-5-enkephalin and an amphiphilic quaternary ammonium palmitoyl glycol chitosan (GCPQ) where the amphiphilic GCPQ is capable of self-assembly in aqueous media into particles having a mean particle size between 3-500 nm where the composition is a dried powder having a moisture content of less than 10% and where the composition is contained within a cartridge comprising a pressurized container for a fluid, a chamber for containing the particles, at least one channel running between the container and the chamber to provide fluidic communication between the container and chamber in use, and at least two distinct concave surfaces on or integral with at least part of an internal wall or internal walls of the chamber, the concave surfaces so arranged that once fluidic communication between the chamber and container is established to create a fluid flow from the container to the chamber through the at least one channel and toward the concave surfaces, each concave surface imparts a rotational motion to a fluid flow or portion of fluid flow that impinges upon it, so that within the chamber a rotationally turbulent flow of fluid is produced in order to engage with the particles and to produce a mobile fluid comprising the particulate. The present invention relates to devices where the moisture content of the particles is less than 5% after storage at 25°C and 60% relative humidity for at least 6 months. The present invention relates to devices where the moisture content of the particles is less than 5% after storage at 30°C and 65% relative humidity for at least 6 months. The present invention relates to devices where the change in leucine enkephalin content relative to the powder is less than 10% after storage at 25°C and 65% relative humidity for at least 6 months. The present invention relates to devices where the change in leucine enkephalin content relative to the powder is less than 10% after storage at 30°C and 65% relative humidity for at least 6 months. The present invention relates to devices containing powders with a mean particle diameter of 10 – 50 micrometers. The present invention relates to devices containing powders with a mean particle diameter of about 20 micrometers. The present invention relates to devices where the change in leucine enkephalin content relative to the powder is less than 5% after storage at 30°C and 65% relative humidity for at least 6 months. The present invention relates to devices containing microparticles where the microparticles may be changed on contact with aqueous media to nanoparticles that exhibit a bimodal size distribution in which the particle size is between 10-20 nm for mode 1 and between 80 to 600nm for mode 2. The present invention relates to devices containing microparticles where the microparticles may be changed on contact with aqueous media to nanoparticles with a size distribution of the particles is greater than 60% for mode 2 (80 – 600 nm) and greater than 30% for mode 1 (10 – 20 nm). The present invention relates to devices where the mass of particles delivered by the device is from 0.05mg to 30 mg per kg mammal mass per day. BRIEF DESCRIPTION OF THE FIGURES Figure 1 is a plot of day 1 Plasma LENK Concentration-Time Data Following Intranasal Administration of NES100 to Male Beagle Dogs (Semi-Log). Figure 2 is a plot of Day 1 Plasma LENK Concentration-Time Data Following Intranasal Administration of NES100 to Male Beagle Dogs Without the NES100-Mid Group (Semi-Log). Figure 3 is a plot of Day 1 Plasma LENK Concentration-Time Data Following Intranasal Administration of NES100 to Female Beagle Dogs (Semi-Log). Figure 4 is a plot of Day 14 Plasma LENK Concentration-Time Data Following Repeated Daily Intranasal Administration of NES100 to Male Beagle Dogs (Semi-Log). Figure 5 is a plot of Day 14 Plasma LENK Concentration-Time Data Following Repeated Daily Intranasal Administration of NES100 to Female Beagle Dogs (Semi-Log). Figure 6 is a plot of Day 1 Plasma LENK Concentration-Time Data Following Intranasal Administration of NES100 to Male Beagle Dogs (Semi-Log). Figure 7 is a plot of Day 1 Plasma LENK Concentration-Time Data Following Intranasal Administration of NES100 to Male Beagle Dogs Through 2 Hours (Semi-Log). Figure 8 is a plot of Day 1 Plasma LENK Concentration-Time Data Following Intranasal Administration of NES100 to Female Beagle Dogs (Semi-Log). Figure 9 is a plot of Day 1 Plasma LENK Concentration-Time Data Following Intranasal Administration of NES100 to Female Beagle Dogs Through 2 Hours (Semi-Log). Figure 10 is a plot of Day 14 Plasma LENK Concentration-Time Data Following Repeated Daily Intranasal Administration of NES100 to Male Beagle Dogs (Semi-Log). Figure 11 is a plot of Day 14 Plasma LENK Concentration-Time Data Following Repeated Daily Intranasal Administration of NES100 to Male Beagle Dogs Through 2 Hours (Semi-Log). Figure 12 is a plot of Day 14 Plasma LENK Concentration-Time Data Following Repeated Daily Intranasal Administration of NES100 to Female Beagle Dogs (Semi-Log). Figure 13 is a plot of Day 14 Plasma LENK Concentration-Time Data Following Repeated Daily Intranasal Administration of NES100 to Female Beagle Dogs Through 2 Hours (Semi-Log). DETAILED DESCRIPTION OF THE INVENTION Although chitosan and its derivatives are well documented for the delivery of drugs via the nasal route, nasal delivery with self-assembling amphiphilic carbohydrates such as GCPQ has not been reported with nasal powders. Unlike chitosan, which is soluble at acidic pH, GCPQ is capable of self-assembly at neutral pH, and this confers an advantage over its parent compound for nasal delivery. The addition of the palmitoyl chain to chitosan enables this chitosan derivative to self-assemble and confers greater association with drug compounds and therefore enhanced delivery. Endogenous opioid neuropeptides, preferably neuropentapeptides are particularly preferred drugs for use in this invention. Examples include met-enkephalin (MENK) and leucine-enkephalin LENK. The drug used in this invention may be used to treat brain disorders such as schizophrenia, obesity, pain and sleep disorders, psychiatric diseases, neurodegenerative conditions, brain cancers and infective diseases. Preferred drugs include neuropeptides: enkephalin, neuropeptide S, dalargin, orexin, vasopressin, leptin, cholecystokinin, dynorphin, detorphin I, neurotensin and oxytocin. The amphiphilic carbohydrate compound is typically selected from chitosans, dextrans, alginic acids, starches, guar gums, and their derivatives. Preferably the amphiphilic compound is a chitosan derivative. In a preferred embodiment of the invention, the amphiphilic carbohydrate compound is represented by the formula (I), or a salt thereof, which is reproduced again below for ease of reference: In t s ormua: *is used to represent the continuing polymer chain; the level of unit A (the acetylated unit) is from 0.5% to 30 mole% the level of unit D (the deacetylated unit) is from 1% to 95.5 mole%; the level of unit H (the hydrophobized unit) is from 1% to 95.5 mole%; the level of unit Q (the quaternary amine unit) is from 3% to 97.5 mole%; the level of unit T (the tertiary amine unit) is from 0% to 94.5 mole%. All percentages refer to mole%. It is understood that A + D + H + Q + T will be equal to 100%. It should also be understood that A, D, H, Q and T may form any arrangement in the amphiphilic carbohydrate compound. The arrangement may therefore be entirely random or as a block copolymer form such as ADHQADHQ etc. In one preferred embodiment of the invention, unit T is absent. The following preferred ranges apply whether unit T is present, or not. In a preferred embodiment of the invention, A is in the range 0.5% to 26 mole%, preferably in the range 0.5% to 20 mole%, preferably in the range 0.5% to 15 mole%, more preferably in the range 0.5% to 10 mole%, even more preferably in the range 0.5% to 5 mole% or 0.5 to 4 mole% or 0.5 to 3 mole%. In an alternative preferred embodiment, A is in the range 2 to 20 mole%, preferably 2 to 15 mole%, more preferably in the range 2 to 10 mole%, even more preferably in the range 2 to 5 mole% or 2 to 4 mole%. In an alternative preferred embodiment, A is in the range 1 to 20 mole%, preferably 1 to 15 mole%, more preferably in the range 1 to 10 mole%, even more preferably in the range 1 to 5 mole% or 2 to 5 mole%. In a preferred embodiment of the invention, D is in the range 2% to 94.5 mole%, preferably in the range 10% to 94.5 mole%, more preferably in the range 10% to 90 mole%, typically in the range 20 to 80 mole% or 50% to 75 mole%, more preferably in the range 55% to 75 mole%, even more preferably in the range 65% to 75 mole%. In a preferred embodiment of the invention, H is in the range 2% to 94.5 mole%, preferably in the range 2% to 90 mole%, more preferably in the range 5% to 80 mole%. In a further preferred embodiment, H is in the range 5% to 70 mole%, for instance 5% to 60 mole% or 5% to 50 mole%. In an alternative embodiment, H is in the range 10% to 30 mole%, more preferably in the range 10 to 20 mole% or 20% to 30 mole%. In a preferred embodiment of the invention, Q is in the range 1% to 90 mole%. It is preferably present in the range 2% to 50 mole%, for instance 5% to 30 mole%, 5% to 20 mole%, 5 to 15 mole% or 5 to 10 mole%. In a preferred embodiment of the invention, T is in the range 0% to 20 mole%, more preferably in the range 0% to 10 mole%, even more preferably in the range 0% to 5 mole%. In some embodiments, T is present in the range 0.5% to 20 mole% or 1% to 20 mole%, for instance, 1 to 10 mole% or 1 to 5 mole%. Any of the preferred ranges for A, D, H, Q and T may be combined. In a preferred embodiment the following ranges are present: A is in the range 2 to 30 mole%; H is in the range 14 to 24 mole%; Q is in the range 6 to 14 mole%. In a further preferred embodiment, the following ranges are present: A is in the range 2 to 11 mole%; H is in the range 10 to 24 mole%; Q is in the range 6 to 14 mole%. The amphiphilic carbohydrate may be accompanied by a salt. For instance, the salt can comprise a chloride, iodide, acetate or glucuronide salt. The molecular weight of the amphiphilic carbohydrate compound has a molecular weight in the range 1- 100kDa. Molecular weight is preferably measured using Gel-permeation chromatography – multi-angle light scattering (GPC-MALLS). The amphiphilic carbohydrate compound is capable of self-assembly into nanoparticles in aqueous media. R1, R2, R3, R4and R10are independently hydrogen or any linear, branched or cyclo form of an alkyl, alkenyl, alkynyl, aryl, acyl group, a sugar substituent selected from glucose, galactose, fructose, and muramic acid, or oligo polyoxa C1-C3alkylene units, optionally substituted with amine, amide or alcohol. Preferably these groups are independently selected from hydrogen, a substituted or unsubstituted alkyl group, a substituted or unsubstituted ether group, or a substituted or unsubstituted alkene group. Typically, R1, R2, R3, R4and R10may be C1-C4linear alkyl groups. Conveniently, R1, R2, R3, R4and R10may all be -CH2-CH2-OH. Typically, R1, R2, R3, R4and R10may be C1-C4linear glycol-based groups. Typically, R1, R2, R3, R4and R10are any of the following sugar substituents: glucose, galactose, fructose, and muramic acid. R1, R2, R3, R4and R10may be oligo polyoxa C1-C3alkylene units such as ethylene glycol oligomers. All of R1, R2, R3, R4and R10may be CH2OCH2CH2OH or CH2CH2OH. All of R1, R2, R3, R4and R10may be H or all or one of them may be CH2CH2OH . Typically, R5is a hydrophobic, substituted or unsubstituted, linear, branched or cyclo form of a C4-30alkyl, C4-30alkenyl, C4-30alkynyl, C4-30aryl, amine, C4-30amide, C4-30alcohol or C3-30acyl group. The group R5is from a substituted or unsubstituted group which is an alkyl group such as a C4-30alkyl group, an alkenyl group such as a C4-30alkenyl group, an alkynyl group such as a C4-30alkynyl group, an aryl group such as a C5-20aryl group, a multicyclic hydrophobic group with more than one C4-C8ring structure such as a sterol (e.g. cholesterol), a multicyclic hydrophobic group with more than one C4-C8heteroatom ring structure, a polyoxa C1-C4alkylene group such as polyoxa butylene polymer, or a hydrophobic polymeric substituent such as a poly (lactic acid) group, a poly(lactide-co- glycolide) group or a poly(glycolic acid) group. The R5group may be linear, branched or cyclo groups. Preferred examples of R5groups include those represented by the formulae CH3(CH2)n-CO- or CH3(CH2)n- or the alkeneoic acid CH3(CH2)p-CH=CH-(CH2)q-CO-, where n is between 4 and 30, and more preferably between 6 and 20, and p and q may be the same or different and are between 4 and 16, and more preferably 4 and 14. A particularly preferred class of R5substituents are linked to the chitosan monomer unit via an amide group (including the pendant NH in the formula), for example as represented by the formula CH3(CH2)nCO-, where n is between 2 and 28. Examples of amide groups are produced by the coupling of carboxylic acids to the amine group of chitosan. Preferred examples are fatty acid derivatives CH3(CH2)nCOOH such as those based on capric acid (n = 8), lauric acid (n = 10), myristic acid (n = 12), palmitic acid (n = 14), stearic acid (n = 16) or arachidic acid (n = 18). R6, R7, and R8are independently any linear, branched, or cyclo forms of any alkyl, alkenyl, alkynyl, aryl or acyl group. R6, R7andR8are preferably independently selected from a substituted or unsubstituted alkyl group such as a C1-10alkyl group. R6, R7and / or R8may be linear or branched. Preferably, R6, R7and R8are independently selected from methyl, ethyl or propyl groups. Conveniently, R6, R7and R8form a quaternary ammonium group which is hydrophilic. Hydrophilic groups are groups which are well hydrated by water and associate on a molecular level with water. A further non-ionic hydrophilic group may replace NR6R7R8providing that R6, R7and R8are equal to CH2O-Y where Y is a hydrophilic substituent. In that case both hydrophilic substituents on the carbohydrate polymer may be selected from mono and oligo hydroxy C1-C6alkyl, mono and oligo hydroxy substituted C2-C6acyl, C1-C2alkoxy alkyl optionally having one or more of the hydroxy groups substituted on the alkoxy or alkylene groups, oligo or poly-(oxa C1-C2alkylene), preferably polyethylene glycol comprising up to 120 ethylene oxide units (i.e. a molecular weight of 5000), and C1-C4alkyl (oligo or poly oxa C1-C3alkylene) optionally hydroxy substituted preferably oligo or polyglycerol ether; wherein the replacement group for NR6R7R8is joined via an ether linkage to a saccharide unit of the polysaccharide. The acyl group may contain alkyl, alkenyl or alkynyl groups. R1, R2, R3, R4and R10may also be hydrophilic. The R9group may be present or absent in the general formula. R9may be present or absent and, when present, is a substituted or unsubstituted alkyl group, a substituted or unsubstituted amine group or a substituted or unsubstituted amide group; When absent, it provides a quaternary ammonium functional group that is directly linked to the monomer unit of the chitosan backbone. When the R9group is present it may be a unsubstituted or substituted alkyl group (e.g. a C1-10alkyl group) for example as represented by –(CH2)n- wherein n is preferably 1 to 4. A preferred example of the R9N+R6R7R8substituent is provided by coupling betaine (- OOC-CH2-N+-(CH3)3) to the amine substituent of the b unit providing an amide group such as in: -NH-CO- CH2-N+R6R7R8. R11is a substituted or unsubstituted alkyl group, a substituted or unsubstituted ether group or a substituted or unsubstituted alkene group or hydrogen. Preferably R11is selected from hydrogen and a substituted or unsubstituted alkyl group such as a C1-10alkyl group. R11may be linear or branched. Preferably, R11is selected from methyl, ethyl or propyl groups. Alternatively, it is an OH-substituted alkyl group, preferably of formula CH2CH2OH. R12is a substituted or unsubstituted alkyl group, a substituted or unsubstituted ether group or a substituted or unsubstituted alkene group. Preferably R12is selected from substituted or unsubstituted alkyl group such as a C1-10alkyl group. R12may be linear or branched. Preferably, R12is selected from methyl, ethyl or propyl groups. Alternatively, it is an OH-substituted alkyl group, preferably of formula CH2CH2OH. Typically, R12is a C1-10alkyl group. R12may be linear or branched. Preferably, R12is selected from methyl, ethyl or propyl groups. R13is a substituted or unsubstituted alkyl group, a substituted or unsubstituted ether group or a substituted or unsubstituted alkene group or hydrogen. Preferably R13is selected from hydrogen and a substituted or unsubstituted alkyl group such as a C1-10alkyl group. R13may be linear or branched. Preferably, R13is selected from methyl, ethyl or propyl groups. Alternatively, it is an OH-substituted alkyl group, preferably of formula CH2CH2OH. Most preferably R13is hydrogen. The total number of monomer units of A+D+H+Q+T may be about 10 to 100. Preferably the total number of monomer units of A+D+H+Q+T may be less than about 1000. The amphiphilic carbohydrate compound may also contain additional targeting groups such as peptides, antibodies and other ligands, for example, folate and transferrin ligands which may allow the polymer to target endogenous receptors and thus target its drug payload to such endogenous receptors at the site of pathology. In a preferred embodiment of the invention, the amphiphilic carbohydrate compound is a partially deacetylated form of N-palmitoyl,N-monomethyl,N,N-dimethyl,N,N,N-trimethyl-6-O-glycolchitosan (GCPQ). This is known to be an amorphous compound (Godfrey et al., “Nanoparticulate peptide delivery exclusively to the brain produces tolerance free analgesia”, J. Control Release 2017, 270, 135-144) and so is not bound by the increase in crystallinity observed when acetylated chitosan is converted to deacetylated chitosan. As indicated, some of the substituents described herein may be either unsubstituted or substituted with one or more additional substituents as is well known to those skilled in the art. Examples of common substituents include halo; hydroxyl; ether (e.g., C1-7alkoxy); formyl; acyl (e.g. C1-7alkylacyl, C5-20arylacyl); acylhalide; carboxy; ester; acyloxy; amido; acylamido; thioamido; tetrazolyl; amino; nitro; nitroso; azido; cyano; isocyano; cyanato; isocyanato; thiocyano; isothiocyano; sulfhydryl; thioether (e.g., C1-7alkylthio); sulphonic acid; sulfonate; sulphone; sulfonyloxy; sulfinyloxy; sulfamino; sulfonamino; sulfinamino; sulfamyl; sulfonamido; C1-7alkyl (including, e.g., unsubstituted C1-7alkyl, C1-7haloalkyl, C1-7hydroxyalkyl, C1-7carboxyalkyl, C1-7aminoalkyl, C5-20aryl-C1-7alkyl); C3-20heterocyclyl; and C5-20aryl (including, e.g., C5-20carboaryl, C5-20heteroaryl, C1-7alkyl-C5-20aryl and C5-20haloaryl) groups. The term “ring structure” as used herein, pertains to a closed ring of from 3 to 10 covalently linked atoms, yet more preferably 3 to 8 covalently linked atoms, yet more preferably 5 to 6 covalently linked atoms. A ring may be an alicyclic ring, or aromatic ring. The term “alicyclic ring,” as used herein, pertains to a ring which is not an aromatic ring. The term “carbocyclic ring”, as used herein, pertains to a ring wherein all of the ring atoms are carbon atoms. The term “carboaromatic ring”, as used herein, pertains to an aromatic ring wherein all of the ring atoms are carbon atoms. The term “heterocyclic ring”, as used herein, pertains to a ring wherein at least one of the ring atoms is a multivalent ring heteroatom, for example, nitrogen, phosphorus, silicon, oxygen or sulphur, though more commonly nitrogen, oxygen, or sulphur. Preferably, the heterocyclic ring has from 1 to 4 heteroatoms. The above rings may be part of a “multicyclic group”. A preferred compound of the invention has formula (II): the level of acetylated unit A is from 0.5% to 30 mole%; the level of deacetylated unit D is from 1% to 95.5 mole%; the level of hydrophobized unit H is from 1% to 95.5 mole%; the level of quaternary amine unit Q is from 3% to 97.5 mole%; and the other groups are as defined previously and the preferred percentages defined above apply.

[0002] A further preferred compound of the invention has formula (III): units a and g together correspond to unit D according to claim 1; units b and d together correspond to unit H according to claim 1; unit c corresponds to unit Q according to claim 1; unit e corresponds to unit T according to claim 1; unit f corresponds to unit A according to claim 1; the proportion of units a+b+c+d+e+f+g = 1; and the corresponding levels of A, D, H, Q and T fall within the ranges defined according to claim 1; or salt thereof. In one embodiment of the invention there is provided a method of forming an amphiphilic carbohydrate compound of general formula (I) wherein the method comprises; depolymerising a carbohydrate polymer to form depolymerised carbohydrate; reacting the depolymerised carbohydrate with varying equivalents of a first reactive compound to increase, decrease or maintain the level of acetylation present; reacting the depolymerised carbohydrate with a second reactive compound to form hydrophobic side- groups on the carbohydrate backbone and thus form hydrophobically substituted depolymerised carbohydrate; and adding a third reactive compound to the carbohydrate compound with more, less or the same level of acetylation, to quaternise an amine group and thereby form the amphiphilic carbohydrate compound. The carbohydrate polymer may be selected from a glycol chitosan. The carbohydrate polymer may be depolymerised with any of the following: an acid, a base, or enzyme. The acid used to depolymerise the carbohydrate polymer may be selected from any of the following: HCl, H2SO4, HNO3or HF. The carbohydrate polymer may be depolymerised for a few days, for example, 48 hours, then isolated and subjected to further depolymerisation dependent on the average molecular weight of solubilising carbohydrate polymer required. The average molecular weight of carbohydrate polymer to be depolymerised is about 2 to 100 kDa and is preferably about 5-50kDa or 5-30kDa. The first reactive compound which is used in varying equivalents to increase, decrease or maintain the level of acetylation is typically acetic anhydride. Typically a degraded glycol chitosan is fully acetylated in a first reaction step and then partially deacetylated in a second reaction step to give the desired level of acetylation. The second reactive compound which forms the hydrophobic side-groups on the depolymerised carbohydrate polymer may be selected from any of the following: any type of fatty acid derivative of, for example, stearic acid, oleic acid, palmitic acid; organo halides such as alkyl, alkenyl, alkynyl, cyclic or non-aromatic halides, acyl chlorides, anhydrides, N-hydroxysuccinimide and other activated acyl compounds capable of being attacked on the Cl carbon by a compound capable of nucleophilic attack. By nucleophilic attack is meant compounds which attack atoms with a low electron density. The acyl groups may also contain an alkyl, alkenyl or alkynyl group. Preferably, the second reactive compound which increases, decreases or maintains the level of acetylation on the depolymerised glycol chitosan may be selected form any of the following: hexadecyl bromide, dodecyl bromide, myristic acid N-hydroxysuccinimide. Preferably, the fatty acid derivative may be palmitic acid N-hydroxysuccinimide; palmitic acid benzotriazole carbonate; palmitaldehyde; palmitoyl chloride; and palmitic acid p-nitro phenyl carbonate. The third reactive compound may be an organo halide wherein the organo group may be selected from any linear or branch, substituted or unsubstituted, or cyclo form of any alkyl, alkenyl, alkynyl, aryl, amine, amide, alcohol or acyl group. Typically, the third reactive compound may be any linear or branched, substituted or unsubstituted, or cyclo form of the following alkyl, alkenyl, alkynyl, aryl, amine, amide, alcohol or acyl groups: C1-C30; C1- C12; C1-C6; or C1. Typically, the organo group of the organo halides may be short chain linear alkyl groups. The organo group of the organo halides may be CH3. Preferably, the amphiphilic carbohydrate compound is quaternary ammonium palmitoyl glycol chitosan (GCPQ). The amphiphilic carbohydrate compound is capable of self-assembling into particles in aqueous media without the presence of other agents such as tripolyphosphate. The compositions of the present invention may form particulate aggregates. These may be formed by the aggregation of individual amphiphile molecules and the hydrophilic drug and have a mean particle size of between 10 nm and 20 μm. Preferably the amphiphilic carbohydrate compound forms nanoparticles which can be loaded with hydrophilic, hydrophobic or amphiphilic drug. A dispersion of carbohydrate and drug may be formed which is clear or translucent. Generally, the amphiphilic compound is mixed with drug and a dispersion is prepared by vortexing and probe sonicating the mixture. The mean particle size can readily be determined microscopically or by using photon correlation spectroscopy and is conveniently determined in aqueous dispersions prior to filtration. More preferably, the polymeric micellar aggregates have a minimum mean particle size of at least 10 nm, and more preferably at least 30 nm, and a maximum mean particle size which is preferably 10 μm or less. Typically, the ratio of amphiphilic carbohydrate compound to drug is within the range of from 1:10-20:1; or from 0.5:1 to 10:1 or from 1:5 to 10:1 or from 1:5 to 5:1 or from 1:2 to 10:1 or from 1:2 to 5:1 or from 1:2 to 2:1 or from 0.5:1 to 1:0.5 and preferably from 1:1 by weight. Typically, the ratio of amphiphilic carbohydrate compound to drug to pharmaceutically acceptable carrier may be from about 1:0.05:20 or from 1: 0.1: 20 or from 1: 0.2: 20 or from 1: 0.3:20 or from 1:0.4:20 or from 1:0.05:10 or from 1: 0.1: 10 or from 1: 0.2: 10 or from 1: 0.3:10 or from 1:0.4:10 or from 2:1:20 to 10:1:2; or from 2:1:10 to 10:1:10 or from 2:1:10 to 2:1:1 or from 2:1:10 to 2:1:0.5 or from 2:1:10 to 2:1:0.1 or from 1:1:10 to 1:1:5 or from 1:1:20 to 1:1:5 or from 1:1:1 to 1:1:0.5 or from 1:1:0.1. In one embodment the formulation comprises 1-100 mg of amphiphilic carbohydrate: 50 mg of leucine enkephalin : 1 g of pharmaceutically acceptable carrier. The pharmaceutical composition of this invention may be in a liquid or solid form suitable for intranasal administration. A suitable daily dose can be determined based on age, body weight, administration time, etc. While the daily doses may vary depending on the condition and body weight of the patient, and the nature of the drug, a typical intranasal dose is about 0.1-120 mg / person / day, preferably 0.5-60 mg / person / day. The formulations of the present invention may be delivered intranasally using a variety of intranasal devices. Such devices include the Naltos device (Alchemy Pharmatech), intranasal delimvery devices from Aptar Pharma (Crystal Lake, IL) or Hovione (Loures, Portugal) or any intranasal delivery device which is capable of delivering drugs to the brain via the nose. Such devices are described in WO / 2021 / 005380, WO / 2022 / 208014, WO / 2022 / 123128, WO / 2020 / 11478 and US 7163013. In certain embodiments the formulations of the present invention may be dispensed from a dispenser of a fluid, in particular a gas borne solid or liquid particulate, having: a container for a fluid; a chamber for containing a particulate, in use in fluidic communication with the container; means to cause the fluid to move into the chamber from the container; means as necessary to cause the fluid to engage with particulate accommodated within the chamber to de-aggregate it if aggregated and to agitate it into turbulent flow to produce a mobile fluid comprising the particulate; a discharge outlet, capable of being placed in fluidic communication with the chamber; and a release means for release of the mobile fluid from the dispenser through the discharge outlet. When used herein the term ‘turbulent flow’ includes cyclonic or vortical flow, which are preferred forms of turbulent flow. The dispenser may be used and will operate in any orientation including upright, inverted or laid-down fashion. Another advantage is that it solves the problem of de-aggregating and / or fluidizing particulates using relatively uncomplicated and potentially inexpensive technology. The particulate is generally a solid particulate and is useful for dispensing particulate compositions comprising a medicament in metered doses, especially if the particulate is a solid particulate that tends to clump fairly readily in storage and / or in transit. The properties of any particulate may be any within a wide variety that are compatible with the function of the present device, such as its density, particle size, specific surface area, the desired dose, etc. Preferably the particles have a narrow size distribution and are of similar shape. The fluid in the container may be any that is a sufficiently mobile fluid to agitate the particulate into turbulent flow, and is stable during storage, and is inert to the particulate and the dispensing target. The fluid may be a gas, such as air, or if the particulate is not inert longer-term to air, nitrogen, a conventional optionally fluorinated lower hydrocarbon propellant, such as a hydroflurocarbon (HFC) or carbon dioxide; a liquid, such as water, or if the particulate is not inert longer-term to water, a (usually pressurized) conventional optionally fluorinated lower hydrocarbon propellant, such as butane or an HFC, a hydrofluoro alkane (HFA) propellant or any compatible combination thereof. In some embodiments the fluidic communication between container and chamber often comprises at least one channel, and preferably at least a pair of channels, which runs between the container and the chamber. Any channels that run between the container and the chamber may take the form of conduits, ducts or tubes. The shape and size of any channels for agitating the particulate with the fluid may be any within a wide variety that are compatible with that function. These may be dependent on the particular properties of the particulate, such as its density, particle size, specific surface area, the desired dose, etc, and may be of circular cross section and / or any other regular curved cross-section, e.g. a generally elliptical, semicircular or semielliptical cross-section. However, often each will be of rectilinear cross- section, such as in the form of a slot or triangular, square or oblong cross-sectional duct. For a quantity of a composition comprising a medicament provided in the fully laden present dispenser of 0.5-35 mg, each channel will typically have a cross-sectional area of 0.03 to 3.0 mm2and in particular 1.0 to 1.5 mm2The channels may be of widely varying shape along their length, e.g. curved, but typically are straight. Where there is at least a pair of channels, all the channels will typically have similar and often identical dimensions and configurations. Channels in a pair of channels may of course have opposite handedness where appropriate. In some embodiments the container may be a pressurized fluid container, such that on actuating the release means, the pressurized fluid is urged from the container into the chamber under its own head of pressure. In such cases, the release means may be the same integer as the means to cause the pressurized fluid to move into the chamber to engage with the particulate. A pressurized fluid container has the advantage in the dispenser according to the present invention of potentially providing a more rapid and / or stronger fluid discharge into the chamber. This may be desirable to de-aggregate particulate accommodated within the chamber if aggregated and to agitate it into turbulent flow to produce a mobile fluid comprising the particulate, especially if the particulate tends to clump fairly readily, on storage and / or in transit. The water (moisture) content of the formulations of the present invention upon rapid drying and storage in the delivery device at least 25°C for at least 1 or at least 2 or at least 3 or at least 4 or at least 5 or at least 6 or at least 7 or at least 8 or at least 9 or at least 10 or at least 12 or at least 13 or at least 14 or at least 15 or at least 16 or at least 17 or at least 18 or at least 19 or at least 20 or at least 21 or at least 22 or at least 23 or at least 24 or at least 30 or at least 36 months can be about 0% or less than about 1% or less than about 2% or less than about 3% or less than about 4% or less than about 5% or less than about 6% or less than about 7% or less than about 8% or less than about 9% or less than about 10% or less than about 15% or less than about 20%. The water (moisture) content of the formulations of the present invention upon rapid drying and storage in the delivery device at least 25°C for at least 1 or at least 2 or at least 3 or at least 4 or at least 5 or at least 6 or at least 7 or at least 8 or at least 9 or at least 10 or at least 12 or at least 13 or at least 14 or at least 15 or at least 16 or at least 17 or at least 18 or at least 19 or at least 20 or at least 21 or at least 22 or at least 23 or at least 24 or at least 30 or at least 36 months can be between 0 to 20% or from 0 to 15% or from 0 to 10% or from 0 to 5% or from 1 to 20% or from 1 to 15% or from 1 to 10% or from 1 to 5% or from 2 to 20% or from 2 to 15% or from 2 to 10% or from 2 to 5% or from 3 to 20% or from 3 to 15% or from 3 to 10% or from 3 to 5% or from 4 to 20% or from 4 to 15% or from 4 to 10% or from 4 to 5% or from 5 to 20% or from 5 to 15% or from 5 to 10%. The potency for the formulations of the present invention upon rapid drying and storage in the delivery device at least 25°C for at least 1 or at least 2 or at least 3 or at least 4 or at least 5 or at least 6 or at least 7 or at least 8 or at least 9 or at least 10 or at least 12 or at least 13 or at least 14 or at least 15 or at least 16 or at least 17 or at least 18 or at least 19 or at least 20 or at least 21 or at least 22 or at least 23 or at least 24 or at least 30 or at least 36 months can be about 0% or less than about 5% or less than about 10% or less than about 15% or less than about 20% or less than about 25% or less than about 30% or less than about 35% or less than about 40% or less than about 45% or less than about 50% or less than about 55% or less than about 60% or less than about 65% or less than about 70% or less than about 75% or less than about 80% or less than about 85% or less than about 90% or less than about 95% or less than about 100%. Examples All reagents and chemicals were obtained from Sigma Aldrich Chemical Co., Pool UK, unless otherwise stated. All solvents and acids were obtained from Fisher Scientific, Loughborough, UK. Leucine-5- enkephalin (LENK) was supplied by Ambiopharm Inc. MET was supplied by Nanomerics Ltd. All reagents and chemicals were used without further purification. Milli-Q water was used to prepare the mobile phase and all other aqueous solutions. The terms NM127 and NES100 are used interchangeably and refer to the same composition. Example 1 Production of NM127 (NES100) NM0127 nanoparticles were prepared from GCPQA (N-palmitoyl-N-acetyl-N-monomethyl-N,N-dimethyl- N,N,N-trimethyl-6-O-glycolchitosan, Mw = 18.6 ± 4.6 kDa, Mw / Mn = 1.033 ± 0.027, mole% palmitoylation = 15 ± 1.3, mole% quaternary ammonium groups = 8 ± 0.8) and LENK by vortexing for 5 min in water for injection BP, the pH adjusted to pH = 5.8 with NaOH (1 M) prior to probe sonicating (Qsonica, UK) with the instrument set at 30% of its maximum output for 10 min on ice. In some cases the GCPQ had the following characteristics: Mw = 18.0 ± 3.7 kDa, Mw / Mn = 1.029 ± 0.023, mole% palmitoylation = 16 ± 3.6, mole% quaternary ammonium groups = 10 ± 2.2. NM127 powder was prepared by dispersing MET (12g) in HCl solution (75mM, 240mL). The dispersion was facilitated by mixing in a water bath at 50˚C. Once fully dispersed and cooled, LENK powder (12g) was added to the MET dispersion. Full dissolution of LENK powder was obtained by using a magnetic stirrer. One cycle of high-pressure homogenization was carried out at a pressure of 18,000 psi using the EmulsiFlex TM-C5 (Avestin, Canada). The dispersion was then spray dried using a Buchi Mini Spray-Drier B290 equipped with the Buchi Ultrasonic package at 180°C, pump 5%, nozzle 1.8W, aspirator 85%, air volume 55%. which produced nano-in-micro formulation of NM127. NM127 is present as a microparticulate powder consisting of fused nanoparticles. From the 240mL formulation containing 50mg / mL of LENK and 50mg / mL of MET, a total of 13.95g of NM127 was produced, which corresponds to a yield of 58.1%. The NM127 was aliquoted in 1g batches and stored in tightly sealed 7-mL glass vials. The release characteristics of the NM127 were measured as follows: Drug Content: The LENK content was analysed by dissolving the NM127 powder in methanol, water (50: 50) to a concentration of 1mg / mL and analysed on an Agilent high performance liquid chromatography system (Agilent technologies, UK) consisting of a binary pump (1220 Infinity II LC Gradient system) equipped with a variable wavelength UV detector and an Onyx Monolithic C18 (100 x 4.6 mm) column. The mobile phase used was 0.1% trifluoroacetic acid (TFA), Acetonitrile containing 0.1% TFA. A gradient method (see Table 3) was used to elute the analyte at a flow rate of 1.0mL / min, a column temperature of 40˚C and with UV detection at 214nm. The injection volume was 20µL. Data analysis was done via an Agilent Chemstation. Quantification of LENK was possible using a previously prepared calibration curve Nanoparticle size: The nanoparticle size and polydispersity index (PDI) of the NM127 formulation before spray-drying was analysed by dynamic light scattering using a Zetasizer Nano ZS (Malvern, UK). Samples were diluted 100 times in MilliQ water prior to analyses. Three consecutive measurements were performed at a scattering angle of 173˚ and a temperature of 25˚C. Disposable polystyrene 70μL cuvettes were used to carry out the particle size measurement. Microparticle size: The microparticle size distribution of the spray-dried formulation was determined by laser scattering using a Malvern Mastersizer 3000 (Malvern Instruments Ltd, Worcestershire, UK). Air was used as the dispersion medium for the microparticles from the sample feeding tray. A small quantity of NM127 powder (5-10mg) was loaded into the feeder tray and the microparticle size distribution was characterized by D10, D50, D90 and % particles below 10µm. Moisture content: The moisture content of the NM127 powder was determined by loading about 1mg – 5mg of NM127 into a previously tared aluminum pan and the pan loaded into the TGA Discovery instrument. The sample was heated from 40˚C to 300˚C at a rate of 10˚C / min. The moisture content was determined by calculating the 1st derivative Δ%weight / Δtemperature of the drying process between 60˚C-150˚C and determining the mass loss [%] at the minimum of the derivative. As organic solvents were not used in the manufacture of NM127 powder, an assumption was made that the evaporating solvent was water. The characteristics of the NM127 powder is shown in Table 1 Table 1: Specifications for NM127 Quality Assay Specifications Drug Content 0.5±0.025 per mg of powder Nanoparticle Size Bimodal distribution Peak 1: 50nm-500nm + intensity > 60% Peak 2: 3nm-100nm + intensity < 40% Microparticle Size Not more than 10% of particles < 10µm Moisture Content < 5% The drug content per milligram of powder was determined using Nanomerics’ validated NM127 HPLC SOP for the determination of LENK content. The NM127 powder had an original LENK content of 0.51±0.02mg per milligram of NM127 powder. The LENK content was analyzed via three independent measurements and met the specifications. Nanoparticle size of the formulation after High Pressure Homogenization and before spray drying was determined. The size distribution showed a bimodal size distribution with peak 1 at 362.0±45.8nm (75.6±2.0%) and peak 2 at 15.0 ± 0.7nm (24.4 ± 2.0%). The nanoparticle size distribution was analyzed via three independent measurements and met the specifications set for size distribution (Table 2). The bimodal size distribution is representative of empty polymeric micelles (10–40 nm) and drug filled nanoparticles (>50 nm). The microparticle size of the produced NM127 powder was determined using Nanomerics’ SOP for determination of microparticle size with a Malvern Mastersizer (Malvern, UK). The NM127 powder showed that 9.71±3.34% of particles have a size below 10 µm. The powders’ D10, D50 and D90 were determined to be 10.1±0.76μm, 18.0±1.34μm and 30.9±1.96μm, respectively. The microparticle size was analysed via three independent measurements and met the specifications. The moisture content in the NM127 powder was determined using a Moisture Content Measurement SOP. The NM127 powder showed a moisture content of 3.94±0.04%, which was analyzed via three independent measurements. The NM127 moisture content met the specifications. Example 2 Cartridge Filling NM127 was loaded into a Naltos cartridge by adding a silicon ball in the seating element in the middle of the propellant chamber (large chamber). The powder (9-11mg) was added using the powder dispenser and a spatula, where the powder formulation was filled into the big cavity of the powder dispenser. After pressing slightly, the excess powder was removed with the flat side of the spatula and the content of the powder dispenser was introduced into the Coroflow Chamber (small chamber). Once filled with NM127 powder, the cartridge’s body and lid were ultrasonically welded together according to Alchemy’s Edge weld SOP (SOP-E01). Each cartridge was filled with HFA 134-a propellant at 3.5±0.5 bar according to Alchemy’s Huxley Bertram gassing rig SOP prior to being sealed according to Alchemy’s Swage weld SOP. All cartridges were marked with an individual code and cartridges were weighed before filling and after each step of the process. Example 3 NM127 Stability The stability of the spray dried NM127 formulation was evaluated over 270 days. NM127 was filled into the Naltos cartridges as described in Example 2 and five filled cartridges were loaded into an aluminum blister and sealed. The aluminum pouches also contained a mini desiccant sachet (0.5g silica gel). After insertion of the 5 filled cartridges into the aluminum pouches, the aluminum pouches were immediately heat sealed prior to shipment and storage. Nanoparticle size The stability study was designed to comply with the FDA guidance for Metered Dose Inhaler (MDI) and Dry Powder Inhaler (DPI) products. After preparation and filling of the devices the samples were packaged as would be packaged for a commercial product. There were two storage conditions: 25˚C / 60% relative humidity (RH) and 30˚C / 65% RH. The devices were stored in a horizontal orientation and were tested after pre-defined time points. Additionally, devices were stored in the inverted and upright orientation and were tested at three time points. For each storage condition / orientation, 5 cartridges were analyzed. This study included a total of 11 pre-defined time points over a period of 270 days. The stability study is described in Table 2. Table 2 – Stability study Cartridge Analysis Orientation Time elapsed T1a Microbial limits Horizontal No storage Drug quantitation Nanoparticle size Nanoparticle zeta Microparticle size imaging Moisture content T1b Emitted dose particle size distribution Horizontal No storage Emitted dose Propellant leak rate T2 Propellant leak rate Horizontal 2 days T3 Propellant leak rate Horizontal 4 days T4a Drug quantification Horizontal 7 days Nanoparticle size Nanoparticle zeta T4b Emitted dose particle size distribution Horizontal 4 days Emitted dose Propellant leak rate T5 Drug quantification Horizontal 16 days Nanoparticle size Nanoparticle zeta Microparticle size imaging Propellant leak rate T6 Microbial limits Horizontal 31 days Drug quantification Nanoparticle size Nanoparticle zeta Microparticle size imaging Moisture content Emitted dose particle size distribution Emitted dose Propellant leak rate T7 Drug quantification Horizontal Upright Inverted 60 days Nanoparticle size Nanoparticle zeta Microparticle size imaging Emitted dose particle size distribution Emitted dose Propellant leak rate T8 Drug quantification Horizontal 61 days Nanoparticle size Nanoparticle zeta Microparticle size imaging Emitted dose particle size distribution Emitted dose Propellant leak rate T9 Microbial limits Horizontal Upright Inverted 208 days Drug quantification Nanoparticle size Nanoparticle zeta Microparticle size imaging Moisture content T10 Drug quantification Horizontal 240 days Nanoparticle size Nanoparticle zeta Microparticle size imaging Emitted dose particle size distribution Emitted dose Propellant leak rate T11 Microbial limits Horizontal Upright Inverted 270 days Drug quantification Nanoparticle size Nanoparticle zeta Microparticle size imaging Moisture content Emitted dose particle size distribution Emitted dose Propellant leak rate T1 cartridges were filled with NM127 powder but were not put into storage. The T1 samples were analyzed once the T2-T11 cartridges went into storage. Initially, T1 samples (5 per storage stability condition and assay) were planned to be analyzed; although as the T1 samples were not placed into the stability cabinet and there was no difference between the samples, hence all T1 measurement results were individually carried out and then combined. Further, it was possible to conduct all assays (emitted dose particle size distribution, emitted dose and leak rate) on each intact cartridge and thus up to 15 samples per time point and storage orientation were assayed. However, for T2, T3, and T5, only the leak rate assay was scheduled thus the number of samples analyzed at these time points differ. Drug Quantification and moisture content were determined as described above. Microbial limits, nanoparticle size, zeta potential, Microparticle size imaging, Microparticle size distribution, emitted dose and propellant leak rate were determined as follows. Microbial limits: As per FDA guidance for nasal products, microbial limits were tested for total yeast and mold Count, Total Aerobic Microbial Count, Absence of S. aureus and absence of P. aeruginosa. The assay included a validation of the test product for suitability for microbiological enumeration based on US Pharmacopeia Microbiological Examination of Non-Sterile products: Microbial Enumeration tests, as well as a validation of the test product for suitability for specified microorganism testing based on US Pharmacopeia Microbiological Examination of Non-Sterile products. Nanoparticle size: The nanoparticle size and PDI of the NM127 formulation were analyzed by dynamic light scattering using a Zetasizer Nano ZS (Malvern, UK). Around 1 – 1.5mg of NM127 was weighed out and rehydrated to 2mg / mL in TRIS buffer (pH=7.1; 10mM). Samples were shaken manually, and an aliquot of the dispersion (80 µL) was transferred to BrandTech BRAND UV Cuvettes and covered with parafilm. Cuvettes were kept standing for an hour before measurements were taken, to allow the system to stabilize after rehydration. Three consecutive measurements were performed at a scattering angle of 173˚ and a temperature of 25˚C. Zeta potential: The zeta potential of NM127 was analysed by dynamic light scattering using a Zetasizer Nano ZS (Malvern, UK). An aliquot (100µL) of the 2mg / mL rehydrated sample (see above) was mixed with TRIS buffer (pH = 7.1, 900µL) to obtain a concentration of 0.2mg / mL.900µL of the 0.2mg / mL dispersion was added to Malvern Zetasizer Nano series disposable folded capillary cells (CTS1070) zeta cuvettes. Three consecutive measurements were performed at a temperature of 25˚C. Microparticle size imaging: Microparticles were imaged using a Scanning Electron Microscope (SEM). A self-adhesive carbon disc was placed onto an aluminum stub and with the tip of a spatula the adhesive disc was divided into 5 slices. Each slice was labelled. Using a clean spatula, a small quantity (1-5 mg) of NM127 powder was sprinkled on the self-adhesive carbon disc. An excess of formulation was removed using compressed air. Each sample (activated cartridge) had one slice attributed to itself. The NM127 formulations were gold coated for 60s using a Quorum Q150RS Sputter Coater (Quorum Technologies, UK). The coated samples were then analyzed using the Phenom Benchtop SEM (Phenom-World, Netherlands) where mode = High resolution (10kV), Intensity = Image, Detector = BDS full, Live viewing resolution = 912, quality = medium, acquired image resolution = 2048, quality = high and at an exposure time of 15s. Microparticle size distribution: Particle size distribution was determined according to Alchemy’s Particle size analysis SOP (PRT-NM05) using the Sympatec Helos (Sympatech, Clausthal-Zellerfeld, Germany). Briefly, each cartridge was tapped 3 times, twice on the top of the body and once on the front end before analysis. After verifying that the needle was inside the device casing, the cartridge was introduced into the case. The case was placed and fixed in the support for the actuator. After a reference measurement, the NM127 powder was measured by actuation of the device. A new needle was used for each time point. Results are presented in the form of a distribution diagram of cumulative distribution [%] vs. particle size [µm] and Distribution Density vs Particle size [µm]. Further, a trend diagram representing particle size [µm] vs. time [ms] and Opt. [concentration %] vs. time [ms] were obtained. The report contains D10, D16, D50, D84, D90 and % below 10 µm. From each report the average D10, D50, D90 and % below 10 µm was calculated. For each time point and storage condition the mean and standard deviation was determined. Cartridges that lost pressure before actuation were discarded and not included for this analysis. Further, the Sympatec Helos was not able to record any data if the powder did not physically reach the laser path. This can happen if not enough sample is released from the cartridges, cartridges are not adequately pressurized, the actuator wasn’t correctly pointed at the target (likelihood increased by hand activation) or if there were errors in the software settings. This means that for 60 out of 225 cartridges stored at 25°C / 60% RH and 61 out of 225 cartridges stored at 30°C / 65% RH no particle size distribution data was obtained. Emitted dose: The emitted dose and the residual mass (powder remaining in the device) were determined gravimetrically. Samples are kept for 1 hour on a flat surface in a horizontal orientation after actuation. Each cartridge was weighed according to Alchemy’s Sartorious Micro Balance SOP (SOP-E06). The obtained results correspond to weight of residual mass (WTRESIDUAL MASS). The post filled weight (WTLOADED) was recorded during filling. The emitted dose was calculated using Equation 1 and 2: Equation 1: % residual mass = 100 x WTRESIDUAL MASS / WTLOADEDEquation 2: % emitted dose = 100 - % residual mass Cartridges which did not pierce properly or lost pressure before actuation were discarded and not included for this analysis. This means 57 out of 225 cartridges stored at 25°C / 60% RH and 43 out of 225 cartridges stored at 30°C / 65% RH were discarded. Cartridges should be weighed within 2 hours of actuation in order to avoid the residual powder absorbing moisture. Propellant leak rate: The propellant leak rate was determined according to Alchemy’s Leak rate analysis SOP (PRT-NM04). The leak rate was defined as the percentage of cartridges that lose partially or completely the propellant at any time during the stability study. The leak rate was determined by weighing each cartridge according to Alchemy’s Sartorious Micro Balance SOP (SOP-E06). The % of propellant gas lost was determined by comparing to the pre-analysed WT(g) recorded during filling of the cartridges. Cartridges with a % gas loss higher than 50% are considered to have leaked. The leak rate is calculated as Leak rate = 100 x L / T, where T equals to total analysed samples and L counts samples with a percentage of gas loss above 50%. The LENK content in the NM127 formulation stored in horizontal orientation was assessed over a period of 270 days and results are presented in Table 3. Table 3 Drug Content Cartridge 25°C / 60%RH 30°C / 65%RH T1 0.50±0.01 T4 0.50±0.01 0.50±0.01 T5 0.49±0.01 0.49±0.01 T6 0.50±0.01 0.50±0.01 T7 0.50±0.01 0.48±0.01 T8 0.51±0.01 0.50±0.01 T9 0.48±0.01 0.49±0.01 T10 0.51±0.01 0.50±0.01 T11 0.55±0.01 0.50±0.01 The LENK content in all samples was stable over 270 days and ranged between 0.48 mg per mg powder – 0.51 mg per mg powder, apart from T11 stored at 25°C / 60% RH . All but one of the samples had the required drug content ± 5%. However, no sample is significantly different in drug content to the sample on Day 0 (ANOVA two-way, p>0.05) and no difference between the two storage conditions (p>0.05) was observed. Nanoparticle size distribution and zeta potential of the redispersed NM127 formulation stored in the horizontal orientation was assessed over a period of 270 days. The results for PDI, peak 1, peak 2 and the zeta potential analyses are presented in Table 6, Table 7, Table 8 and Table 9 respectively. Overall, all formulations showed a bimodal size distribution, with peak 1 having a mean size of between 80–150nm and peak 2 having a mean size of between 10–20nm. Size distribution was analysed as intensity vs size and peak 1 had an intensity of >60% of the size distribution for all time points and storage conditions and the intensity of peak 2 remained <30%. Table 4 PDI of the redispersed (2mg / ml) NM127 formulation Cartridge 25°C / 60%RH 30°C / 65%RH T1 0.400±0.046 T4 0.489 ± 0.057 0.483 ± 0.052 T5 0.506 ± 0.058 0.518 ± 0.087 T6 0.510 ± 0.026 0.504 ± 0.027 T7 0.379 ± 0.063 0.597 ± 0.037 T8 0.483 ± 0.028 0.439 ± 0.048 T9 0.248 ± 0.091 0.460 ± 0.097 T10 0.301 ± 0.085 0.262 ± 0.092 T11 0.308 ± 0.073 0.193 ± 0.108 The PDI of the redispersed NM127 formulation is stable over 270 days, apart from T7 and T11 stored at 30°C / 65% RH (Two-way ANOVA, p0.05). With respect to peak 1 and peak 2, there was no significant difference between the timepoints and day 0 nor any significant difference between the peak mean size at the two storage conditions (Two-way ANOVA, p > 0.05). Overall, it can be concluded that the nano-in-micro formulation allows regeneration of nanoparticles on redispersion of the microparticles, when the microparticles are stored for 9 months and thus stability of the nano-in-micro formulation has been demonstrated. For completeness, the numerical values of particle size for peak 1 and peak 2 as well as their respective intensities are presented in Table 5 and Table 6 respectively. Table 5 Particle size and intensity of peak 1 of the redispersed (2mg / ml) NM127 formulation Cartridge 25°C / 60%RH, Size (peak 30°C / 65%RH, Size (peak intensity) intensity) T1 114.00±16.10 (91.69±4.08) T4 152.20±50.10 133.76±21.85 (88.74±7.11) (89.77±6.78) T5 157.21±57.50 152.20±50.10 (86.07±11.82) (86.34±12.26) T6 115.26±57.50 121.67±29.82 (115.26±7.59) (93.48±7.69) T7 127.50±11.93 169.25±25.37 (91.50±2.87) (78.53±10.24) T8 125.15±20.20 122.38±24.74 (89.36±7.92) (92.57±4.83) T9 98.65±5.63 143.31±69.21 (96.97±3.68) (91.45±5.74) T10 98.98±5.26 86.04±5.87 (94.07±3.86) (94.55±5.17) T11 90.91±13.04 80.37±6.19 (95.03±3.59) (97.52±3.47) Table 6 Particle size and intensity of peak 2 of the redispersed (2mg / ml) NM127 formulation Cartridge 25°C / 60%RH, Size (peak 30°C / 65%RH, Size (peak intensity) intensity) T1 13.73±7.28 (7.53±4.27) T4 22.15±10.86 13.92±6.78 (12.16±6.00) (9.59±6.39) T5 16.80±14.39 13.92±7.57 (11.32±10.61) (13.92±10.28) T6 11.28±4.83 11.18±8.38 (10.37±6.40) (7.40±8.16) T7 16.86±2.47 21.43±6.54 (9.21±1.60) (22.16±8.32) T8 13.28±4.71 12.93±2.74 (89.36±7.92) (6.24±2.86) T9 11.16±1.94 14.17±2.86 (6.18±0.71) (7.39±2.47) T10 11.85±1.43 11.24±0.87 (5.77±1.31) (6.20±0.68) T11 11.19±2.73 10.46±1.41 (4.88±3.36) (7.40±1.64) The zeta potential of the redispersed NM127 formulation was analysed over the 9 month storage period and the zeta potential remained positive and, for most time-points, was in excess of +10mV. Statistical analysis demonstrated that the zeta potential was not statistically significantly different on storage for the majority of sampling points, but T4 (Day 7) and T10 (Day 241) are significantly different when compared to Day 0 (Two-way ANOVA, p < 0.05). The sample concentration for T1 (Day 0) and T4 (Day 7) was 0.125 mg / mL. This was initially chosen because of a limitation in powder availability. From T5 (Day 16) all samples were analysed at 0.2mg / mL. Due to an experimental error, samples for T8 (Day 90) were analysed at 2mg / mL. Zeta cuvettes were replaced after T5 (Day 16) and after T10 (Day 241). The buffer was prepared at T0 and replaced at T7 (60 days) and at T9 (209 days). The reduction in positive zeta potential observed at various time points could be partially due to exceeding the lifetime of the zeta cuvette or ageing of the buffer. This could explain why after replacement of buffer / zeta cuvettes the zeta potential once again was close to +20mV. Despite the variations observed, which we attribute to the slight changes in the experimental conditions over the different time points, we judge that that zeta potential of the redispersed powder has been preserved and remained positive with a value close to +20mV. The positive zeta potential of NM127 powder should promote mucoadhesion and thus may enhance the residence time of NM127 in the nares after intranasal administration. Indeed, MET nanoparticles have been shown to adhere and integrate into mucosal surfaces. Table 7 Zeta potential of the redispersed (0.2mg / ml) NM127 formulation. All particles are positively charged. All zeta potential values were positive. Cartridge 25°C / 60%RH 30°C / 65%RH T1 21.9±3.8 T4 11.9±2.9** 14.6±1.9 T5 15.4±4.5 15.2±4.7 T6 13.5±4.2 14.8±5.0 T7 18.0±3.5 18.5±3.5 T8 30.3±2.5 27.4±3.0 T9 19.5±4.9 20.7±3.3 T10 7.8±1.4** 11.4±4.5* T11 19.8±4.1 18.5±3.0 *Significantly different to T1 (p<0.05) **Significantly different to T1 (p<0.01) Microparticle imaging by SEM show microparticles of 5–30 µm in size. The particles are mostly spherical with some appearing deflated and occasionally some in the form of concave discs. Particles appear to be rough rather the being smooth at their surface. This structure is conserved over the 9 months period, suggesting particles are stable in their powder form when stored at the storage conditions tested. The moisture content of the NM127 formulation stored in the horizontal orientation was determined by TGA over a period of 270 days. The result of the analysis is presented in Table 8. Table 8 Moisture content of the redispersed (0.2mg / ml) NM127 formulation Cartridge 25°C / 60%RH 30°C / 65%RH T1 4.02±0.16 T6 3.78±0.19 4.01±0.80 T9 4.19±0.26 3.94±0.28 T11 4.20±0.10 3.97±0.14 The moisture content of the NM127 over the 270 days period was below 5% for all time points and storage conditions and thus NM127 conforms to the specifications for moisture content. Moreover, there was no significant difference in the moisture content at the different time points and the storage conditions. The ability of the formulation to maintain a moisture content below 5% has been shown to be important for its stability and performance. Indeed, it has been observed that with increasing moisture content, the powder agglomerates. Agglomeration of the powder makes handling of the powder more difficult and is likely to negatively affect its pharmaceutical performance as upon actuation of the cartridges the agglomerated powder tends to remain inside the cartridge. Thus, the packaging and storage conditions for NM127 are appropriate to maintain the powder’s pharmaceutical performance in the device. Finally, it was thought that the recovery of the NM127 powder from cartridges could lead to uptake of humidity and thus an increase in moisture content. The results obtained from T11 do not suggest any uptake in humidity as moisture content remained stable. Microbial limit tests of the NM127 formulation stored in the horizontal orientation were carried out according to FDA guidelines, with some modification. The assays determined total yeast and mold count, total aerobic microbial count and tests for presence of S. aureus and P. aeruginosa. After method validation, samples were tested at the different time points. The total yeast and mold count (TYMC) as well as for total aerobic microbial count (TAMC) in NM127 formulations stored at 25°C / 60% RH and 30°C / 65% RH were determined to be < 1000 cfu / g. For T6 stored at 30°C / 65% RH and T9 stored at 25°C / 60% RH it was determined that the specification, 1000cfu / g, was met. These differences are due to limited availability of the NM127 powder. Indeed, tests were carried out using 5 cartridges filled with 10mg of NM127 powder each. Samples stored in the same conditions were pooled for testing. However, as only small amounts of NM127 powder were tested, the limit of detection was <1000cfu / g for microbial enumeration. FDA guidelines for inhalation routes allow for a TAMC of 102 cfu / g (where maximum acceptable count is 200) and a TYMC of 101 cfu / g (where maximum acceptable count is 20). Thus, it is possible that NM127 powder conforms with FDA guidelines, but this could not be demonstrated with this assay due to limit of detection. The microbial limit test for the presence of S. aureus and P. aeruginosa in NM127 powder showed that both microbes were absent in the totality of the powder assayed. FDA guidelines for inhalation routes require absence of both microbes in 1 gram of powder. The particle size distribution of NM127 powder stored at 25°C / 60% RH and activated from Naltos cartridges was measured over time and is presented as D10, D50 and D90 in μm. The percentage below 10 μm was also measured over time. Two-way ANOVA (p<0.05) was applied to compare the two storage conditions and the different time points. For the D10, D50 and the percentage of particles below 10 μm, statistically significant differences were observed over time. However, there was no significant differences in the D90 value over time and the particles did not deviate from the particle size specification given in Table 1. Table 9: NM127 microparticle size diameter (mean ± s.d., µm) for samples stored in Naltos cartridges over 270 days and stored in the horizontal orientation at 25°C / 60% RH Time N 10% diameter 50% diameter 90% diameter % less than point (10% volume (50% volume (90% volume 10µm undersize D10) undersize D50) undersize D90) T1 14 11.1±0.5 20.5±0.9 36.0±2.2 7.3±0.9 T2 3 12.1±1.4 22.1±2.5 36.8±1.9 6.0±1.2 T3 3 11.9±0.8 21.6±1.7 37.2±1.5 5.7±1.2 T4 10 11.4±0.3 20.9±0.7 35.8±0.7 6.7±0.4 T5 5 12.1±0.7 22.3±1.7 37.1±2.4 5.8±0.7 T6 13 12.5±1.1* 23.2±2.0* 37.9±2.2 5.6±0.9* T7 4 13.4±2.4* 24.2±3.6* 38.8±3.0 4.7±1.6* T8 10 10.4±1.0 20.0±1.6 35.0±2.9 9.9±2.4* T9 13 12.4±0.4* 22.4±0.7 37.0±1.2 5.3±0.6* T10 14 12.4±1.4* 21.9±1.7 35.6±1.6 6.0±2.4 T11 15 12.6±0.7* 22.4±1.0 36.5±1.1 5.4±0.8* *= significantly different from Day 0 Table 1: NM127 microparticle size diameter (mean ± s.d., µm) for samples stored in Naltos cartridges over 270 days and stored in the horizontal orientation at 30°C / 65% RH Time N 10% diameter 50% diameter 90% diameter % less than point (10% volume (50% volume (90% volume 10µm undersize D10) undersize D50) undersize D90) T1 14 11.1±0.5 20.5±0.9 36.0±2.2 7.3±0.9 T2 2 11.1±0.4 20.1±0.8 35.1±1.1 7.1±1.1 T3 5 10.8±0.1 19.8±0.3 34.3±0.3 7.7±0.3 T4 15 11.8±0.6 21.6±1.2 37.0±1.9 6.2±0.6 T5 5 12.0±0.5 22.4±1.6 37.7±2.2 5.7±0.6 T6 14 12.3±1.1 22.7±1.9* 37.1±1.9 5.7±1.0* T7 5 12.5±.0.4 22.7±1.0 37.4±1.8 4.7±0.7* T8 14 12.4±0.7* 22.2±1.2 37.3±2.3 5.0±0.8* T9 12 12.2±0.9 21.8±1.8 36.2±2.2 5.6±1.0* T10 10 12.1±1.5 21.5±1.8 35.6±1.3 6.3±1.9 T11 15 12.1±0.7 22.2±1.3 36.4±1.5 5.8±0.9 *= significantly different from Day 0 The emitted weight of NM127 powder contained in the Naltos cartridges was determined gravimetrically over a period of 270 days. All samples retrieved at each time point were analysed for emitted weight. Samples which lost pressure before actuation (>50% pressure) as well as samples not adequately pierced were excluded from the measurement. The emitted weight of NM127 from Naltos cartridges stored in the horizontal orientation was measured using 15 samples per time point apart from T2, T3 and T5 where fewer samples were analyzed. The emitted weight as well as the number of valid samples are presented in Table 11. Table 11 Emitted weight of NM127 powder from samples stored in Naltos cartridges over 270 days and stored in a horizontal orientation. Timepoint Storage Conditions 25°C / 60%RH 30°C / 65%RH n % emitted wt n % emitted wt T1 73.2±9.1 (n=25) T2 3 76.0±2.0 3 68.3±1.7 T3 2 78.8±4.0 5 78.2±4.9 T4 11 77.9±6.3 15 81.5±7.5 T5 5 74.8±9.0 5 75.1±5.1 T6 11 75.8±5.9 13 78.0±6.3 T7 12 79.1±4.9 15 75.6±6.5 T8 9 71.9±9.7 11 79.1±3.1 T9 13 68.4±14.0 13 74.8±10.6 T10 11 74.4±5.8 7 73.0±5.9 T11 13 74.7±7.1 13 68.4±12.8 There was no significant difference in emitted weight between the storage conditions (two-way ANOVA, p<0.05). Moreover, there was no significant difference (Two-way ANOVA p<0.05) between day 0 and all other time points. The average emitted weight from cartridges over 270 days regardless of storage conditions and orientations was 74.9±8.7%. For particle size distribution determination using the Sympatec Helos, cartridges were actuated using an actuator which ensures a reproducible and repeatable actuation force is applied to each device. An electromechanical actuator (EM actuator), actuation by hand using a support and a pneumatic actuator (PA actuator) were employed for this purpose. For most cartridges from T1–T7 the EM actuator was employed, whereas T8 and T9 used hand actuation and T10 and T11 were actuated with a pneumatic actuator.The results are presented in Table 12. Table 12 Comparison of emitted weight (%) obtained by different actuation modes Actuation EM Hand Hand Helios PA n 92 53 116 89 % emitted weight 75.9±9.8 78.0±6.8 74.0±9.7 72.9±10.1 A one-way ANOVA comparison demonstrated that there is a significant difference between actuation by hand and both the Hand Helos (p<0.05) and PA (p<0.01) method of actuation. Indeed, actuation by hand resulted in a significantly higher emitted weight. However, the number of cartridges actuated by hand is limited (53) thus making true comparison difficult. No significant difference was observed when the remaining firing modes were compared to each other. The leak rate of the propellant contained in the Naltos cartridges was determined gravimetrically over a period of 270 days. All samples retrieved at each time point were analysed for loss of propellant. The leak rate of the Naltos cartridges was determined by using 15 samples per time point, apart from T2, T3 and T5, where only 5 samples were analyzed The results of cartridges stored in the horizontal orientation are presented in Table 13. Table 13 Leak rate of Naltos cartridges over 270 days and stored in a horizontal orientation Timepoint 25°C / 60% RH 30°C / 65% RH # samples # lost pressure Leak rate % # samples # lost pressure Leak rate % T1 30 0 0 As 25°C / 60% RH T2 5 0 0 5 2 40 T3 5 0 0 15 0 0 T4 15 3 20 5 0 0 T5 5 0 0 15 0 0 T6 15 2 13.3 15 0 0 T7 15 0 0 15 0 0 T8 15 5 33.3 15 1 6.7 T9 15 2 13.3 15 2 13.3 T10 15 1 6.7 15 2 13.3 T11 15 0 0 15 0 0 Surprisingly, fewer cartridges lost pressure at the higher storage temperature. The most likely explanation is that cartridges stored at 30°C / 65% RH were prepared after the preparation of cartridges stored at 25°C / 60% RH. This led to increasing experience in the cartridge filling procedure and hence likely to fewer cartridges exhibiting propellant leakage. From T6, Nanomerics Ltd. started weighing cartridges to gain a better insight into the leak rate by testing a larger number of cartridges. A comparison of the leak rate over time is limited by the variation in the number of samples tested. However, the leak rate does not increase over time. In actual fact, the leak rate seems to decrease over time. It is noteworthy that improvements in welding and in the ultrasonic settings were implemented before preparing cartridges for the last timepoint (T11) and this is the likely cause in the decrease in the leak rate observed for T11. Of the 415 samples analyzed at 25°C / 60% RH 21 lost pressure (a total of 5.0%). Of the 410 samples analyzed at 30°C / 65% RH 21 lost pressure (a total of 7.6%). Example 4 Orientation Results In accordance with FDA guidelines, different orientations were tested. Prior results were obtained from samples stored in the horizontal orientation. Additional samples were also stored in the upright and inverted orientation and tested at different time points. The results were then compared to the T0 samples and storage in the horizontal orientation to determine if orientation had an impact on the stability and pharmaceutical performance of the formulation. The additional samples were tested for drug content, nanoparticle size and zeta potential, microparticle imaging, moisture content, emitted weight and particle size distribution. The LENK content in all samples was stable over 270 days and was between 0.48–0.52mg per mg of powder, apart from the T11 sample stored in horizontal orientation at 25°C / 60% RH, where drug content was 0.55±0.08mg per mg and for the T11 sample stored in the inverted orientation at 30°C / 65% RH where drug content was 0.54±0.05mg per mg. However, no sample was significantly different from the Day 0 sample and there were no significant differences between all three orientations. The nanoparticle size distribution and zeta potential of the redispersed NM127 formulation stored over a period of 270 days was analyzed to examine the impact of storage orientation on this parameter. Samples were stored in the horizontal, upright and inverted orientations at the two storage conditions and were analyzed at different time points. As was observed for samples stored in the horizontal orientation, all formulations showed a bimodal size distribution, with peak 1 having a mean size of 80–150nm and peak 2 a mean size of 10–20nm. Size distribution was analyzed as intensity vs size and peak 1 had an intensity of >60% of the size distribution for all time points and storage conditions and the intensity of peak 2 remained <30%. The PDI of the redispersed NM127 formulation is stable over 270 days, apart from the T7 sample stored at 30°C / 65% RH. The PDI decreased at the T11 point. Overall, PDI shows some variation over time with a slight decrease in PDI observed on storage. Finally, there was no significant difference in the PDI when the samples were stored at different orientations. In terms of peak 1 and peak 2 there was no significant difference between most timepoints and day 0, or any significant difference between the different orientations (Two-way ANOVA, p< 0.05). Indeed, only peak 1 showed a significant difference to day 0 at T7 when the sample was stored in the horizontal orientation at 30°C / 65% RH. All formulations, independent of storage orientation and the storage conditions showed a bimodal size distribution where the mean particle size was maintained. It can be concluded that the NM127 nano-in-micro formulation allows regeneration of similar sized nanoparticles by redispersion, when the formulation is stored for up to 9 months and thus size stability of the NM127 nano-in-micro formulation has been demonstrated. The zeta potential of the redispersed formulation was analyzed over 9-months when stored at three distinct orientations. The redispersed NM127 formulation had a zeta potential of around +20mV at all time points. Statistical analysis demonstrated that there was no significant difference in the zeta potential when all the samples stored at the various storage conditions were compared to the Day 0 sample. It is noteworthy that the sample concentration used to measure the zeta potential for T1 (Day 0) and T4 (Day 7) was 0.125mg / mL. This was initially chosen because of a limitation in powder availability. All other samples were analyzed at 0.2mg / mL. Furthermore, due to an experimental error only 4 samples were analyzed at T11 for the inverted orientation samples stored at 25°C / 60% RH. Statistical analysis was carried out and these sample size differences were taken into account. NM127 microparticles stored in the horizontal, upright and inverted orientations were imaged by SEM over a period of 270 days. Microparticle imaging by SEM show microparticles with a particle diameter of 5–30µm. As observed for the NM127 samples stored in the horizontal orientation, the particles are mostly spherical with some appearing deflated and occasionally some in form of concave discs. Particles appear to be rough rather the being smooth at their surface. This structure is conserved over the 9-month storage period, suggesting particle morphology is stable when stored as a powder in the afore-mentioned conditions. The moisture content of the NM127 formulation stored in three different orientations was determined by TGA over a period of 270 days. The moisture content of the NM127 over the 270 day period was below 5% for all time points, orientations and storage conditions and thus the samples conform to the specifications. Moreover, there was no significant difference in the moisture content between the samples stored at different orientations. As mentioned above, the ability of the formulation to maintain a moisture content below 5% has been shown to be an important aspect for stability and performance. Overall, there were no significant differences in moisture content between the NM127 samples stored at the different storage orientations. The stability study demonstrated that orientation does not affect moisture content of the powder over a 9- month period. The particle size distribution of NM127 powder stored at 25°C / 60% RH at different storage orientations and activated from Naltos cartridges was measured over time. Two-way ANOVA was applied to compare the different orientations and the different time points. For the D10, D50 and the percentage of particles below 10 μm, statistically significant differences were observed over time. However, there was no significant differences in the D90 value over time. Moreover, no significant differences were observed between the different storage orientations apart from in the % below 10 μm value in the samples stored horizontally or in the inverted position at T7. The particle size distribution of NM127 powder stored at 30°C / 65% RH at different storage orientations and activated from Naltos cartridges was measured over time. The percentage below 10 μm was also measured over time. Two-way ANOVA was applied to compare the different orientations and the different time points. For the D10, D50 and the percentage of particles below 10 μm, statistically significant differences were observed over time. However, there was no significant differences in the D90 value over time. Moreover, no significant differences were observed between the different storage orientations over time. The emitted weight from Naltos cartridges stored in three different orientations was determined gravimetrically over a period of 270 days. The effect of storage orientation on emitted weight has been assessed over 270 days. Results were compared by two-way ANOVA There was no significant difference between samples stored in different orientations over time. The storage orientation does not influence the emitted weight. The stability data of the cartridges stored at different orientations are contained in Tables 14-22 Table 14: Drug content (mean ± s.d.) of the NM127 formulation stored in Naltos cartridges over 270 days and stored at two conditions in the horizontal, upright and inverted orientations (n=5) Timepoint 25°C / 60% RH 30°C / 65% RH [mg] [mg] T1 0.50 ± 0.03 Horizontal 0.50 ± 0.02 0.48 ± 0.02 T7 Upright 0.50 ± 0.01 0.50 ± 0.03 Inverted 0.51 ± 0.03 0.50 ± 0.04 Horizontal 0.48 ± 0.02 0.49 ± 0.00 T9 Upright 0.48 ± 0.01 0.50 ± 0.01 Inverted 0.48 ± 0.01 0.50 ± 0.01 Horizontal 0.55 ± 0.08 0.50 ± 0.02 T11 Upright 0.50 ± 0.08 0.50 ± 0.03 Inverted 0.52 ± 0.03 0.54 ± 0.05 Table15: PDI (mean ± s.d.) of the redispersed NM127 formulation (2mg / mL) stored in Naltos cartridges over 270 days and stored at two conditions in the horizontal, upright and inverted orientations (n=5) Timepoint 25°C / 60% RH 30°C / 65% RH T1 0.400 ± 0.046 Horizontal 0.379 ± 0.063 0.597 ± 0.037* T7 Upright 0.395 ± 0.052 0.398 ± 0.051 Inverted 0.513 ± 0.090 0.373 ± 0.050 Horizontal 0.248 ± 0.091 0.460 ± 0.097 T9 Upright 0.467 ± 0.051 0.480 ± 0.063 Inverted 0.453 ± 0.015 0.359 ± 0.099 Horizontal 0.308 ± 0.073 0.193 ± 0.108 T11 Upright 0.253 ± 0.049 0.275 ± 0.079 Inverted 0.288 ± 0.056 0.252 ± 0.105 * Significantly different from Day 0 Table16: Nanoparticle size of peak 1 (mean ± s.d., nm) and its intensity [%] for the redispersed NM127 formulation (2mg / mL) stored in Naltos cartridges over 270 days and stored at two conditions in the horizontal, upright and inverted orientations (n=5) Timepoint 25°C / 60% RH, Size data (nm), 30°C / 65% RH, Size data (nm), (percentage intensity) (percentage intensity) T1 114.00 ± 16.10 (91.69 ± 4.08) Horizontal 127.50 ± 11.93 169.25 ± 25.37* (91.05 ± 2.87) (78.53 ± 10.24) Upright 114.14 ± 8.27 105.18 ± 7.49 T7 (90.81 ± 4.17) (90.79 ± 4.24) Inverted 147.87 ± 36.70 105.47 ± 8.85 (82.73 ± 13.63) (91.53 ± 3.8) Timepoint 25°C / 60% RH, Size data (nm), 30°C / 65% RH, Size data (nm), (percentage intensity) (percentage intensity) Horizontal 98.65 ± 5.63 143.31 ± 69.21 (91.45 ± 5.74) (96.97 ± 3.68) Upright 137.47 ± 38.15 126.63 ± 28.95 T9 (90.96 ± 9.09) (88.26 ± 12.81) Inverted 107.87 ± 8.25 88.08 ± 12.50 (92.52 ± 4.12) (92.65 ± 5.93) Horizontal 90.91 ± 13.04 80.37 ± 6.19 (95.03 ± 3.59) (97.52 ± 3.47) Upright 88.94 ± 9.39 82.31 ± 4.48 T11 (97.10 ± 3.77) (95.05 ± 5.22) Inverted 85.02 ± 8.31 81.64 ± 10.67 (94.54 ± 3.95) (95.18 ± 4.22) * Significantly different to Day 0 Table 17: Nanoparticle size of peak 2 (mean ± s.d., nm) and its intensity [%] for the redispersed NM127 formulation (2mg / mL) stored in Naltos cartridges over 270 days and stored at two conditions in the horizontal, upright and inverted orientations (n=5) Timepoint 25°C / 60% RH, Size data (nm), 30°C / 65% RH, Size data (nm), (percentage intensity) (percentage intensity) T1 13.73 ± 7.28 (7.53 ± 4.27) Horizontal 16.86 ± 2.47 21.43 ± 6.54 (9.21 ± 1.60) (22.16 ± 8.32) Upright 14.62 ± 4.14 15.02 ± 2.01 T7 (9.69 ± 3.35) (10.25 ± 2.30) Inverted 19.38 ± 6.71 14.12 ± 3.84 (16.15 ± 12.62) (8.41 ± 2.99) T9Horizontal 11.16 ± 1.9414.17 ± 2.86 (6.18 ± 0.71)(7.39 ± 2.47) Timepoint 25°C / 60% RH, Size data (nm), 30°C / 65% RH, Size data (nm), (percentage intensity) (percentage intensity) Upright 20.14 ± 16.52 13.37 ± 7.71 (12.45 ± 13.92) (7.95 ± 6.91) Inverted 11.31 ± 3.95 9.72 ± 2.17 (6.04 ± 3.20) (7.24 ± 3.68) Horizontal 11.19 ± 2.73 10.46 ± 1.41 (4.88 ± 3.36) (7.40 ± 1.64) Upright 10.78 ± 0.31 11.50 ± 3.06 T11 (6.90 ± 1.52) (8.21 ± 3.15) Inverted 11.65 ± 3.15 11.72 ± 3.08 (7.77 ± 2.34) (6.83 ± 2.60) Table18: Zeta potential (mean ± s.d.) of the redispersed NM127 formulation (2mg / mL) stored in Naltos cartridges over 270 days and stored at two conditions and in the horizontal, upright and inverted orientations (n=5). All formulations are positively charged. All zeta potentials were positive values. Timepoint 25°C / 60% RH 30°C / 65% RH T1 21.9 ± 3.8 Horizontal 18.0 ± 3.5 18.5 ± 3.5 T7 Upright 19.3 ± 4.9 19.0 ±3.2 Inverted 18.5 ± 3.5 18.1 ± 2.4 Horizontal 19.5 ± 4.9 20.7 ± 3.3 T9 Upright 21.1 ± 1.8 21.4 ± 3.2 Inverted 21.7 ± 2.4 21.6 ± 2.5 Horizontal 19.8 ± 4.1 18.5 ± 3.0 T11 Upright 18.0 ± 2.3 21.1 ± 1.6 Inverted 19.0 ± 2.2* 19.4 ± 4.5 * n= 4 Table 19:%w / w Moisture content (mean ± s.d.) in NM127 stored in Naltos cartridges over 270 days and stored at two conditions in the horizontal, upright and inverted orientations (n=5) Timepoint 25°C / 60% RH 30°C / 65% RH T1 4.02 ± 0.16 Horizontal 4.20 ± 0.10 3.97 ± 0.14 T11 Upright 3.91 ± 0.24 4.22 ± 0.18 Inverted 4.12 ± 0.28 4.31 ± 0.23 Table 20: NM127 microparticle size diameter (mean ± s.d., µm) for samples stored in Naltos cartridges over 270 days and stored in the horizontal, upright and inverted orientations at 25°C / 60% RH Timepoint n 10% diameter 50% diameter 90% diameter % less than (10% volume (50% volume (90% volume 10µm undersize, undersize, undersize, D10) D50) D90) T1 14 11.1±0.5 20.5±0.9 36.0±2.2 7.3±0.9 Horizontal 4 13.4±2.4* 23.2±3.6 38.8±3.0 4.7±1.6* T7 Upright 9 12.4±2.0 22.7±2.8* 37.6±2.4 5.8±1.4 Inverted 6 11.7±2.0 21.7±3.2 36.1±3.4 7.4±3.9 Horizontal 13 12.4±0.4* 22.4±0.7 37.0±1.2 5.3±0.6* T9 Upright 12 13.4±1.6* 23.9±2.4* 37.9±2.7 4.5±1.2* Inverted 14 12.2±0.6 21.9±1.2 36.0±1.4 5.4±0.8* Horizontal 15 12.6±0.7* 22.4±1.0* 36.5±1.1 5.4±0.8* T11 Upright 15 12.6±0.7* 22.9±1.3 37.2±1.2 5.1±0.8* Inverted 14 12.1±0.4 21.9±0.6 35.7±0.7 5.8±0.5 *= significantly different from Day 0

[0003] Table 21: NM127 microparticle size diameter (mean ± s.d., µm) for samples stored in Naltos cartridges over 270 days and stored in the horizontal, upright and inverted orientations at 30°C / 65% RH Timepoint n 10% diameter 50% diameter 90% diameter % less than (10% volume (50% volume (90% volume 10µm undersize, undersize, undersize, D10) D50) D90) T1 14 11.1±0.5 20.5±0.9 36.0±2.2 7.3±0.9 Horizontal 5 12.5±0.4 22.7±1.0 37.4±1.8 4.7±0.7* T7 Upright 6 12.2±0.3 21.7±0.5 36.0±1.2 5.2±0.4* Inverted 6 12.2±0.6 21.9±1.1 36.5±1.9 5.1±0.7* Horizontal 12 12.2±0.9 21.8±1.8 36.2±2.2 5.6±1.0* T9 Upright 14 12.1±0.9 21.4±1.7 35.3±1.8 5.7±0.7* Inverted 11 11.8±0.8 21.3±1.1 35.2±1.4 6.2±2.0 Horizontal 15 12.1±0.7 22.2±1.3 36.4±1.5 5.8±0.9 T11 Upright 14 12.0±0.7 21.9±1.0 36.4±0.9 5.9±1.1 Inverted 8 12.5±0.7* 22.4±0.9 36.4±1.0 5.2±0.8* *= significantly different from Day 0 Table 22: % Emitted Weight (mean ± s.d.) of NM127 powder from the Naltos cartridges stored at two conditions in the horizontal, upright and inverted orientation over 270 days Timepoint 25°C / 60% RH 30°C / 65% RH, T1 73.2 ± 9.1 Horizontal 79.1 ± 4.9 75.6 ± 6.5 T7 Upright 76.4 ± 5.4 78.1 ± 6.4 Inverted 78.8 ± 7.8 75.7 ± 7.3 Horizontal 68.4 ± 14.0 74.8 ± 10.6 T9 Upright 69.6 ± 11.1 75.2 ± 4.8 Inverted 72.1 ± 6.7 77.2 ± 7.7 T11 Horizontal 74.7 ± 7.1 68.4 ± 12.8 Upright 72.5 ± 8.1 71.0 ± 14.6 Inverted 73.7 ± 12.3 76.1 ± 8.6 Example 5 Dog Study A study was conducted to evaluate the potential toxicity and toxicokinetics of NES100 (NM127), a microparticulate dosage form of LENK (leu-enkephalin) mixed with an excipient polymer MET (Molecular Envelope Technology; Quaternary ammonium palmitoyl glycol chitosan) in a 1:1 ratio, when administered daily for up to 14 consecutive days via intranasal administration as a suspension or dry powder to Beagle dogs. The reversibility of any toxicological effects was evaluated following a 14-day recovery period. Twelve (12) Beagle dogs (6 male and 6 female) were originally assigned to one of six dose groups as presented in the table below. Two additional animals (1 male and 1 female) were subsequently added to Group 5. Vehicle (sterile water), NES100 (formulated in sterile water), NES100 powder, or MET powder was administered intranasally to 1 dog / sex / group. Vehicle or NES100 (12, 60, or 90 mg / mL) was administered over four dosing iterations by instilling 250 µL into each naris for a total of 2000 µL per animal per day. The average actual daily dose of NES100 for the males or females administered the 12 mg / mL formulation was 3.7 or 4.3 mg / kg / day, respectively; for the males or females administered 60 mg / mL was 18 or 21 mg / kg / day, respectively; and for the males or females administered 90 mg / mL was 30 or 31 mg / kg / day, respectively. NES100 powder or MET powder was administered over four dosing iterations via a delivery device (Naltos) into a single alternating naris per iteration. The average actual daily dose of the dogs administered MET powder was 22 mg / kg / day. The average actual daily dose of the male and female dogs administered NES100 powder was 24 and 25 mg / kg / day, respectively. On Day 2, Animal 501 (MET- Powder, male) was administered two doses of NES100 powder and Animal 551 (MET-Powder, female) was administered one dose of NES100 powder, rather than the intended four doses of MET powder. These animals were moved into recovery with no further dose administration and two additional animals, Animals 502 (male) and 552 (female), began 14 consecutive days of four daily dose iterations of MET powder. The formulations were homogeneous suspensions of acceptable concentrations for LENK. The 1.25 mg / mL NES100 and 12.5 mg / mL NES100 formulations were homogeneous formulations of acceptable concentrations for MET. The 90 mg / mL NES100 formulation had a grand RSD of 11.9% and was 15.9% low. The middle sample for the 90 mg / mL NES100 formulation contained foam which likely affected the analysis causing the results to be lower than the top and bottom samples. The foam caused the aliquots of the sample to contain air and lower amounts of MET. For this reason, it is reasonable to discard the results from the middle sample. Without the middle sample, the 90 mg / mL NES100 formulation was within 10% of the target value and a %RSD less than 10%. The 45 mg / mL MET formulation was 42.4% lower than the expected concentration but was homogeneous. The concentrations of the residual dose samples which were analyzed after use for dose administration were high for LENK in the 1.25 mg / mL NES100 formulation (17.5 %RE) and the 12.5 mg / mL NES100 formulation (13.0% RE). The concentrations were low for MET in the 90 mg / mL NES100 formulation (-18.2% RE) and 45 mg / mL MET formulation (- 33.7% RE). All other concentrations were within 10% RE for LENK and MET. In calculating the %REs of the formulations for LENK and MET, the NES100 used to prepare the formulations was assumed to be 50:50 LENK:MET. Table 23: Experimental Design Animal Identification Group Target dose Avg. Ind. Target dose per Target Male Female (mg / kg / day)1Actual Dose animal Formulation Administered (mg / animal / day) Concentration (Male / Female) (mg / mL)1(mg / kg / day) 1-vehicle 0 0 / 0 0 0 101 151 2-NES100 4 3.7 / 34.3 24 12 201 251 low 3-NES100 20 18 / 21 120 60 301 351 mid 4-NES100 30 30 / 31 180 90 401 451 high25-MET 10 22 / 22 60 N / A 501- 551-552 powder350246-NES100 20 24 / 25 120 N / A 601 651 powder1Calculation assumed an average body weight of 6 kg. The actual dosage varied depending on the weight of the animal. The dose delivered per day was a fixed volume / amount. The individual dose administered in mg / kg / day was calculated based on actual individual body weights. Animals were weighed approximately weekly. The calculation of the powder administered per day was determined from the cassette weight prior to and following dose administration.2A target dose of 30 mg / kg / day was the maximum feasible dose based on the dosing volume and concentration of 90 mg / mL.3Provided in ready-to-use cartridges. Beginning on Day 1, the appropriate test, excipient, or Vehicle (control article) was administered intranasally to each animal daily for up to 14 consecutive days. The animals’ body and head were positioned to assure each dose was appropriately administered (vertical and / or vertical recumbency). Animals in Groups 1 through 4 received 8 total dose administrations (4 per naris). Animals in Groups 5 and 6 received 4 total dose administrations (2 per naris). For Groups 1 through 4, on each day of dose administration, the appropriate test material was instilled via calibrated pipette (Rainin L1000) into each naris at a target dose volume of 250 µL per naris. After a 2- minute observation period, a second dose was administered at a target dose volume of 250 µL per naris. Approximately 2 hours after the completion of the second instillation, the dose administration procedure was repeated for a total instillation of 1000 µL per naris (2000 µL per animal). For Groups 5 and 6, on each day of dose administration, the appropriate powdered test material was administered via a delivery device prepackaged with either NES100 or MET, as appropriate. After a target 2-minute observation period, a second dose was administered. Approximately 2 hours after completion of the second administration, the procedure was repeated for a total administration from 2 cartridges per naris (4 cartridges per animal). On Day 2, Animal 501 was administered two doses of NES100 powder and Animal 551 was administered 1 dose of NES100 powder, rather than the intended MET powder. These animals were moved into recovery with no further dose administration and two additional animals, Animals 502 and 552, began 14 consecutive days of dose administration with MET powder. Observations for moribundity and mortality were performed twice daily during the quarantine and study periods, except on the day of study termination when animals were observed once. Cage-side clinical observations were performed on all animals assigned to study at least three times daily during the dose administration phase, starting on Day 1; once prior to the first round of dose administration, once prior to the second round of dose administration, and at 1 to 2 hours following the last dose administration. Cage-side observations were performed at least twice daily during the recovery phase of the study. Detailed clinical observations were performed prior to randomization, once prior to dose administration on Day 1, and at least once weekly thereafter. When the detailed clinical observation coincided with the cage-side clinical observation, only the detailed clinical observation was performed. Blood Specimen Collection Whole blood specimens (target volume 1 mL) were collected from each animal into tubes containing K2EDTA and processed to plasma. Specimens were collected from all groups prior to dose administration and at eight post-dose target time points: 5 min, 10 min, 15 min, 30 min, 45 min, 1.5 hrs, 6 hrs, and 24 hrs following the 4th daily administration on Days 1 (all animals) and 14 (all animals except Animals 501 and 551. Specimens were mixed gently. Protease inhibitor (PI, 10 µL) was immediately added to the 1 mL of blood (scaled up / down as appropriate); the tube was inverted to mix and placed on wet ice until centrifugation (within 30 minutes of collection). Specimens underwent centrifugation for at least 10 minutes in a refrigerated centrifuge (set to maintain 4°C) at a relative centrifugal force (rcf) set to at least 1800 g. Plasma was separated, split into two uniquely labelled polypropylene tubes, and frozen immediately on dry ice and then transferred to a freezer set to maintain -65 to -80°C. The samples were analyzed between 4- and 28-days following collection. The Protease Inhibitor Cocktail (Millipore / Sigma, PN P2714, stored -30 to -15℃, 1 month expiration from the reconstitution date) was prepared in advance by adding 10 mL of water to one bottle, mixing thoroughly, storing, and thawing prior to use. Bioanalysis All plasma specimens (with exceptions described below) were transferred to the designated test site on dry ice and analyzed for the active ingredient, LENK, concentration using a previously qualified bioanalytical LC-MS / MS method. The 5, 10, 30, and 45-minute plasma samples and the 1.5, 6, and 24- hour plasma samples for Group 1 following Day 1 collections and for Groups 1 and 5 following Day 14 collections were retained at the testing facility. Analysis of the blood specimens collected from the Group 1 and Group 5 animals was limited to those specimens collected prior to dose administration and 15 minutes following dose administration. Toxicokinetic Evaluation The toxicokinetic (TK) parameters calculated and evaluated for LENK, the active ingredient of NES100, were Cmax(the maximum observed blood concentration), Tmax(the time to reach the maximum observed blood concentration), elimination half-life (estimated from the terminal linear phase), AUClast(area under the curve from time zero to the last observed time point), AUC0-24hr (area under the curve from 0 to 24 hours), and AUC∞ (area under the curve from time zero extrapolated to infinity). The following acceptance criteria were used to evaluate the concentration-time profiles: (1) the coefficient of determination (r2 ) for the terminal linear phase was greater than or equal to 0.85, (2) the time of the last observed concentration was greater than three times the half-life, and (3) AUC∞ had less than 20% of the area extrapolated. Clinical Pathology Specimens for clinical pathology evaluation were collected prior to group assignment, and on Days 2, 15, and 29 from all groups (via jugular vein). Dogs were fasted overnight prior to blood collection for clinical pathology evaluation according to the following table: Table 24. Clinical Pathology Sample Collection Schedule Assessment Blood Tube Pretest Day 2 Day 15 Day 29 Hematology K3EDTA 2ml 2ml 2ml 2ml Serum Serum 2.5ml 2.5ml 2.5ml 2.5ml Chemistry Separator Tube Coagulation Sodium Citrate 2.7ml 2.7ml 2.7ml 2.7ml Hematology parameters Hematology parameters evaluated included erythrocyte count, hemoglobin, hematocrit, leukocyte count, leukocyte differential, platelet count, reticulocyte count, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration and mean platelet volume. Coagulation Coagulation parameters evaluated were activated partial thromboplastin time, fibrinogen and prothrombin time. Serum Chemistry Serum chemistry parameters evaluated were alanine, aminotransferase, albumin, albumin / globulin ratio, alkaline phosphatase, aspartate aminotransferase, bilirubin, direct bilirubin, total blood urea, nitrogen, calcium, cholesterol, chloride, creatine kinase, creatinine, gamma glutamyl transferase, globulin, glucose, lactate dehydrogenase, phosphorus, potassium, sodium, total bile acids, total protein and triglycerides. Urinalysis Urine was collected overnight (Day 14 to Day 15) via free catch or from the cage pan from all dogs. Urine specimens were submitted for analysis of the following parameters: appearance (clarity and color), bilirubin, glucose, ketones, leukocytes, nitrite, occult blood, pH, protein, specific gravity, total volume, urobilinogen, microscopic examination of sediments. NES100 suspensions (12, 60, and 90 mg / mL) for intranasal instillation were formulated twice. The pH of NES100 ranged from 4.83 to 5.04; the pH of the individual formulations is presented in Table 25. These formulations are referred to NES100-Low, -Mid, or -High, respectively. Table 25. Final pH of each formulation batch Batch Number (Group Designation Target Concentration (mg / mL) Final pH 4-NES100-2 12 4.93 4-NES100-3 60 5.04 4-NES100-4 90 4.96 4-NES100-2 12 4.83 4-NES100-3 60 4.85 4-NES100-4 90 4.92 The group mean nominal and average dose administered of NES100 or MET is presented in Table 26. The dogs administered the suspension formulations received an overall average dosage within 10% of expected and a weekly average dosage within 13% of expected. Based on the pre- and post-dose cassette weights, the dogs within Group 5 (MET powder) received an average of 22 mg / kg / day MET (ranging daily from 19 to 25 mg / kg / day) instead of the targeted 10 mg / kg / day; however, the dogs within Group 6 (NES100 Powder) received an average of 24 (male) or 25 (female) mg / kg / day NES100 (ranging from 16 to 31 mg / kg / day) instead of the targeted 20 mg / kg / day. Table 26. Mean Daily NES100 or MET doses Group ID Sex Target Target per Target Mean Mean Mean Dose Formulation Animal Dose Dose Dose Admin Day (mg / mL) (mg / animal / (mg / kg / Admin Admin 1-14 day) day) Day 1-7 Day 8- (mg / kg / day) (mg / kg / 14 day) (mg / kg / day) 1 101 M 0 0 0 0 0 0.0 Vehicle 151 F 0 0 0 0 0 0.0 2 201 M 12 24 4 3.9 3.5 3.7 (NES100 251 F 12 24 4 4.4 4.1 4.3 low) 3 301 M 60 120 20 19 18 18 (NES100 351 F 60 120 20 21 20 21 mid) 4 401 M 90 180 30 30 29 30 (NES100 451 F 90 180 30 31 30 31 high) 5 (MET 502 M N / A 60 10 23 22 22 powder) 552 F N / A 60 10 23 22 22 6 601 M N / A 120 20 24 25 24 (NES100 651 F N / A 120 20 25 26 25 powder) All animals survived until scheduled removal from the study. Intermittent reduced food consumption, emesis, soft feces, and / or diarrhea were noted during the quarantine period and likely resulted from the transition to facility feed, facility, and staff and / or infection with Giardia, which was identified at quarantine health evaluations as originating at the vendor, and subsequent treatment. Immediately following intranasal administration of NES100-Mid, NES100-High, and NES100 Powder, excess salivation was observed in all dogs. Additionally, NES100-related intermittent emesis was observed on up to 8 of 14 days of dose administration in males administered NES100-Mid and NES100 Powder and up to 7 of 14 days of dose administration in females administered NES100-Low, NES100-High, and NES100 Powder. Though emesis was noted on a single day during the dose administration phase in the male and female dogs administered Vehicle and once in a female dog administered NES100-Mid during the recovery phase, the observation of emesis was considered NES100-related due to the overall frequency and immediacy following dose administration. Though test article-related, the excess salivation and intermittent emesis were deemed not adverse as effects on the body weight, food consumption, or clinical pathology parameters were not noted. Observations noted during the dose administration phase but not related to dose administration of NES100 or MET due to a lack of dose response, presence during the quarantine period and / or in the Vehicle animals, secondary in nature to the dose administration procedure, and / or the intermittent nature of the observation were noted as follows: • reduced food consumption (Vehicle female, Study Day 4) • diarrhea (Vehicle male, Study Day 4, 6, 7; NES100 Mid female, Study Day 9); MET Powder female, Study Days 2 and 4) • soft feces (MET-Powder male, Study Day 8) • sneezing (NES100- Low male, Study Day 12) • nasal discharge (NES100-High male, Study Day 7) • lethargy (NES100-Mid male, Study Day 8; NES100-High male, Study Day 8) During the recovery phase, intermittent occurrences of diarrhea (NES100-Mid male, Study Day 21; NES100 Powder male, Study Days 18 to 19), soft feces (Vehicle female, Study Day 23; NES100-Mid male, Study Day 22), and emesis (NES100-Mid female, Study Day 25) were noted. These observations were deemed either non-adverse or not related to NES100 due to the lack of dose response, presence during the quarantine period and / or observed in the animals administered Vehicle, and / or were intermittent occurrences. Male and female dogs administered NES100 or MET (suspension or powder) exhibited no notable changes in body temperatures following dose administration on Days 1 and 14. Male and female dogs administered NES100 or MET (suspension or powder) gained weight in a manner similar to the animals administered Vehicle. Although sporadic decreases in body weight were observed in all dose groups in both male and female dogs throughout the dose administration and recovery periods, the decreases were usually of small magnitude and not dose related. Male and female dogs administered NES100 or MET (suspension or powder) did not exhibit any notable trends in food consumption. Although sporadic decreases in food consumption were observed in all dose groups in both male and female dogs throughout the dose administration and recovery periods, the decreases were usually of small magnitude, related to periods of protocol-required fasting, and / or not dose related. Bioanalytical and Toxicokinetic Evaluation The plasma samples collected for TK evaluation were successfully analyzed. The LLOQ is defined as the nominal concentration of the lowest calibration standard for the assay. The LLOQ and ULOQ for this method were established in the Sponsor-provided method and were compatible with the instrument response at the testing site. The lower limit of quantification (LLOQ) for the plasma method was 1.01 ng / mL and the upper limit of quantification (ULOQ) was approximately 100 ng / mL extracted from plasma using a 50 µL aliquot of sample. The targe organ for LENK is the brain and not the plasma or any organs distal to the brain. The following acceptance criteria were used to evaluate the concentration-time profiles: (1) the coefficient of determination (r2) for the terminal linear phase was greater than or equal to 0.85, (2) the time of the last observed concentration was greater than three times the half-life, and (3) AUC∞ had less than 20% of the area extrapolated. Only Animal 451 (NES-High) on Day 1 passed all three of the acceptance criteria, and most failed at least two (10 of 15 profiles) with Animal 651 (NES-Powder) on Day 14 unable to estimate the terminal linear phase due to an unexpected increase in LENK concentration at the last time point of 24 hr. The shape of the LENK plasma concentration-time curves typically showed sharp declines over the first 4 to 5 time points (5 min to 30-45 min following the last daily dose administration). The 1.5, 6, and 24 hr time points, when measurable, were typically low in concentration (43 of 48 samples were below 10 ng / mL) which were usually similar in value to the 45-min time point. Some of the Vehicle and MET-Powder samples were above the LLOQ, ranging from 1.22 to 34.5 ng / mL, as well as all Day 1 pre-dose samples for the NES100 groups, ranging from 1.85 to 9.72 ng / mL, suggesting concentration samples nearer to the LLOQ may not be reliable. Additionally, endogenous proteins may also factor into the inconsistent LENK concentrations. Finally, most animals did not have measurable concentrations at all three of the later time points which contributed to the additional variability in the profiles. Together, these factors resulted in variable characterizations of the terminal phase and resulted in the failure of many of the acceptance criteria and poor characterizations of the terminal linear phase. Tmaxtypically occurred at the first sample collection time (target 5 min following the last daily dose administration) in 12 of 16 profiles suggesting rapid absorption of LENK. Elimination half- life estimates were extremely varied. On Day 1, Animal 451 (NES100-High), produced the only profile to pass all three of the acceptance criteria for the characterization of the plasma-time curve, resulting in a half-life estimate of 0.205 hr. Animals 601 (NES100- Powder) (Day 1) and 201 (NES100-Low), 301 (NES100- Mid), and 601 (NES100-Powder) (Day 14), produced profiles which all passed 2 of 3 acceptance criteria and resulted in half-lives ranging from 0.421 to 1.53 hr on those respective study days. All other estimates were longer and highly variable, in particular, Animal 301 on Day 1. This animal had an increase in concentration at 6 hr, followed by a similar concentration at 24 hr. The result was an estimated terminal elimination rate that approached zero, with a corresponding excessively long elimination half-life (333000 hr). Reevaluation of this profile without the later time points (1.5, 6, and 24 hr) resulted in an elimination half-life of 0.0965 hr, which is much more similar to those profiles that passed at least 2 of the acceptance criteria than the original estimate. However, the 0.5 and 0.75 hr samples had concentrations even lower than the 6 and 24 hr samples and closer to the LLOQ suggesting that more than the just the late time points are questionable. In addition, the potential presence of endogenous proteins could affect the measured LENK concentrations all time points. Therefore, all profiles that failed the acceptance criteria, including Animal 301 on Day 1, should be considered to have estimates for the elimination half-life and AUC that may be poorly estimated. When evaluating systemic exposure with Cmax, AUClast, AUC0-24hr, and AUC∞, sex, dose, or formulation effect were not evident. Trends of increasing Cmaxand AUC with increasing dose were also observed. The extremely rapid elimination of LENK over the initial 30 min combined with the extravascular route of dose administration via intranasal administration may explain the variability in both Cmaxand AUC measurements. Additional variability was observed in AUC∞ due to the poor estimation of the terminal elimination phase for many of the animals. Table 27. TK Parameters for LENK Following Intransal Delivery to Dogs Study Sex ID CmaxTmax(hr) Elim. AUClastAUC0-24AUCinfDay (ng / ml) Half life (hr*ng / ml) (hr*ng / ml) (hr*ng / ml) (hr) 1 Male 201 40.4 0.0833 22.0 225 225 359 301 198 0.0833 333000 348 348 5350000 401 60.4 0.0833 12.8 170 170 257 601 188 0.0833 0.421 24.6 25.6 25.4 Female 251 10.9 0.250 30.3 116 116 257 351 53.6 0.0833 86.6 138 138 745 451 62.4 0.0833 0.205 14.1 15.7 15.5 651 427 0.0833 3.07 69.1 76.0 75.9 2 Male 201 70.5 0.0833 1.53 21.4 23.8 23.1 301 96.0 0.167 0.460 22.9 24.4 24.0 401 356 0.0833 12.0 139 139 171 601 335 0.250 1.50 132 134 134 Female 251 28.2 0.0833 4.28 20.0 29.2 29.2 351 271 0.0833 3.76 65.9 82.5 80.9 451 92.2 0.0833 3.76 55.0 78.2 74.9 651 15.2 0.167 ND 203 203 ND Based on the potential impact of unreliable concentrations near the LLOQ, a separate reanalysis of the TK data was performed after exclusion of a subset of samples. Based on the data from the Day 1 pre-dose samples which had a maximum concentration of 9.72 ng / mL, a cutoff value of 10 ng / mL was designated, and all concentrations below this value were excluded from the TK reanalysis. This data censoring resulted in only three profiles having a sufficient number of measurable concentrations in order to estimate the terminal elimination rate constant; Animal 301 (Day 1) which failed all three of the acceptance criteria and Animal 401 (Days 1 and 14) which both passed all of the acceptance criteria. The elimination half-life for Animal 401 was 0.228 and 0.0712 hr on Day 1 and Day 14, respectively. However, Animal 301 (Day 1), which did not pass the acceptance criteria, had an elimination half-life of 9.61 hr. The uncensored TKanalysis showed some trends of increasing Cmax and AUC with increasing dose. This trend remained truefor Cmax, following the reanalysis for all animals since Cmaxand Tmaxvalues remained unchanged. However, for AUClast, this trend was less apparent as the calculated values were more variable than the initial analysis, due to the reduction in number of samples used for the reanalysis. Table 28. TK Parameters for LENK Following Intransal Delivery to Dogs Analyzed without Concentrations below 10ng / ml Study Sex ID Cmax Tmax(hr) Elim. AUClast AUC0-24 AUCinf Day (ng / ml) Half life (hr*ng / ml) (hr*ng / ml) (hr*ng / ml) (hr) 1 Male 201 40.4 0.0833 ND 83.0 ND ND 301 198 0.0833 9.61 503 503 635 401 60.4 0.0833 0.228 28.2 32.8 32.5 601 188 0.0833 ND 17.6 ND ND Female 251 10.9 0.250 ND 6.81 ND ND 351 53.6 0.0833 ND 2.23 ND ND 451 62.4 0.0833 ND 7.58 ND ND 651 427 0.0833 ND 52.2 ND ND 2 Male 201 70.5 0.0833 ND 10.3 ND ND 301 96.0 0.167 ND 16.7 ND ND 401 356 0.0833 0.0712 84.1 85.5 85.5 601 335 0.250 ND 109 ND ND Female 251 28.2 0.0833 ND 1.17 ND ND 351 271 0.0833 ND 43.1 ND ND 451 92.2 0.0833 ND 16.5 ND ND 651 15.2 0.167 ND 336 336 ND NES100- or MET-related changes in survival, body temperature, body weight, food consumption, ophthalmic, hematology, coagulation, and serum chemistry parameters were not observed throughout the study. Intermittent emesis on up to 8 of 14 days of dose administration and / or excess salivation were observed shortly following intranasal dose administration of NES100-Mid, NES100-High, and NES100 Powder. Though emesis was noted on a single day during the dose administration phase in the male and female dogs administered Vehicle and once in a female dog administered NES100-Mid during the recovery phase, the observation of emesis was considered NES100-related due to the overall frequency and immediacy following dose administration. Though test article-related, the excess salivation and intermittent emesis were deemed not adverse as effects on the body weight, food consumption, or clinical pathology parameters were not noted. Urinalysis values for leukocytes, blood, and protein were suggestive of inflammation in both males and females in the Vehicle, NES100 Low, Mid, High, NES100 Powder, and MET Powder groups on Day 15. However, blood parameters compatible with an inflammatory process were not present and urine was free-catch funneled directly into the cage pan and not from a mid-stream free catch; thus, urinary analysis findings were most likely secondary to vulval or preputial skin contamination. Kinetically, Tmaxpost-intranasal administration typically occurred at the first target sample collection time of 5 min, suggesting rapid absorption of LENK. Elimination half-life estimates were extremely varied, ranging from 0.205 to 1.53 hours for profiles that passed at least two of the acceptance criteria. All other elimination half-life estimates were longer and highly variable. When evaluating systemic exposure, sex, dose, or formulation effect were not clearly evident. Trends of increasing Cmaxand AUC with increasing dose were observed. The extremely rapid elimination of LENK, the active ingredient of NES100, over the initial 30 minutes combined with extravascular intranasal dose administration may explain the variability in both Cmaxand AUC measurements. Additional variability was seen in AUC∞ due to the poor estimation of the terminal elimination phase for many of the animals. In conclusion, under the conditions of this study, daily intranasal administration of MET Powder at dosages up to approximately 22 mg / kg / day for 14 consecutive days resulted in no adverse toxicologic findings in the Beagle dog. Additionally, daily intranasal administration of NES100 at dosages up to 180 mg / animal / day (90 mg / mL suspension formulation, translating to approximately 30-31 mg / kg / day) or 120 mg / animal day (powder, translating to approximately 24-25 mg / kg / day) for 14 consecutive days resulted in no adverse toxicologic findings in the Beagle dog. As such, a MET Powder dose of up to 22 mg / kg / day was well tolerated. A NES100 dose of up to 180 mg / animal / day (suspension formulation) or 120 mg / animal / day (powder formulation) when using the administration regimen in this study were deemed the maximum tolerated dose (MTD) for each formulation phase. Kinetically, Tmaxpost-intranasal administration typically occurred at the first target sample collection time of 5 min, suggesting rapid absorption of LENK. Elimination half-life estimates were extremely varied, ranging from 0.205 to 1.53 hours for profiles that passed at least two of the acceptance criteria. All other elimination half-life estimates were longer and highly variable. When evaluating systemic exposure, sex, dose, or formulation effect were not clearly evident. Trends of increasing Cmaxand AUC with increasing dose were observed. The extremely rapid elimination of LENK, the active ingredient of NES100, over the initial 30 minutes combined with extravascular intranasal dose administration may explain the variability in both Cmaxand AUC measurements. Additional variability was seen in AUC∞ due to the poor estimation of the terminal elimination phase for many of the animals. In conclusion, under the conditions of this study, daily intranasal administration of MET Powder at dosages up to approximately 22 mg / kg / day for 14 consecutive days resulted in no adverse toxicologic findings in the Beagle dog. Additionally, daily intranasal administration of NES100 at dosages up to 180 mg / animal / day (90 mg / mL suspension formulation translating to approximately 30 to 31 mg / kg / day) or 120 mg / animal day (powder, translating to approximately 24 to 25 mg / kg / day) for 14 consecutive days resulted in no adverse toxicologic findings in the Beagle dog. As such, a MET Powder dose of up to 22 mg / kg / day was well tolerated. A NES100 dose of up to 180 mg / animal / day (suspension formulation) or 120 mg / animal / day (powder formulation), when using the administration regimen in this study, were deemed the maximum tolerated dose (MTD) for each formulation phase. Example 6 Formulation and Analytic Testing NES100 MET-LENK nano-in-microparticle powder was prepared by spray-drying, using GCP23Q11 MET batch at a ratio of 1:1 (MET : LENK). There were potentially some challenges with the powder rehydration at high concentrations for animal dosing. NES100 powder rehydration was investigated to provide a liquid dispersion of nanoparticles in order to produce a formulation with optimal syringeability, stability, and in vivo dosing compatibility (e.g. pH). Concentrated formulations of NES100 were prepared as follows: For a concentration of 50 mg mL-1LENK, 100 mg powder were dissolve in 0.7 ml water using magnetic stirring at 200 rpm, 37°C (~10 min.). The pH was adjusted while stirring by adding a total of 0.3 ml of NaOH (0.3 M) by adding 0.1 ml at a time (pH = 4.9). For a concentration of 60 mg mL-1LENK, 120 mg powder were dissolve in 0.65 ml water using magnetic stirring at 200 rpm, 37°C (~10 min.). The pH was adjusted while stirring by adding a total of 0.35 ml of NaOH (0.3 M) by adding 0.1 ml at a time (pH = 4.94). For a concentration of 70 mg mL-1LENK, 140 mg powder were dissolve in 0.55 ml water using magnetic stirring at 200 rpm, 37°C (~10 min.). The pH was adjusted while stirring by adding a total of 0.45 ml of NaOH (0.3 M) by adding 0.1 ml at a time (pH = 5.02). Formulation Viscosity The viscosity measurements were performed using m-VROC viscometer (RheoSense). The standard operating procedure included first cleaning the A-05 chip and sample syringe with Aquet, followed by water, before loading the sample into the glass syringe and placing it into the m-VROC system. The measurement protocol was set up as given in Table 29 and all measurements were taken at controlled temperature of 25°C. Table 29 – Parameters of Viscosity Measurements in MET-LENK Formulations Flow rate (µL / min)Measurement Time (s) Waiting Time (s)100 5.3 3.0 200 2.6 3.0 300 1.8 3.0 350 1.5 3.0 400 1.3 3.0 Formulation Syringeability Formulation syringeability was assessed based on its withdrawal using needles of varying gauge (e.g.30G versus 29G) and accuracy of its discharge based on weighing out a sample volume of the formulation (e.g. 50 µl should translate to 50 mg weight). LENK content quantification in non-biological samples by HPLC High-Performance Liquid Chromatography (HPLC) method for LENK detection and quantification in non- biological samples, such as MET-LENK formulations, was set up according to the settings listed in below. An Agilent HPLC-UV system 1220 Infinity LC with a Phenomenex Onyx Monolithic C-18 4.6mm x 100 column was used at 40°C. The mobile phase was water + 0.1% formic acid: acetonitrile + 0.1% TFA (%w / v) with a gradient of 0 min – 80:20, 2 min. – 80:20, 9 min. – 25:75 and 10 min – 80:20. The detection wavelength was 214 nm, flow rate = 1.0 mL / min, injection volume = 10 µL, run time = 14 min and retention time = 4.6-4.8 min. The method was previously validated and its suitability was confirmed by generating a calibration curve (R2 = 0.9999) with the linear range of 15 to 500 µg / mL, based on spiked known concentrations of LENK and yielding well-defined single LENK peaks at matching retention time of ~4.7 min. All samples submitted for HPLC were diluted 200-fold. Data analysis was performed via Agilent Chemstation software. LC-MS bioanalytical method for LENK quantification in biological samples For the LC-MS analysis of LENK, the Agilent Ultivo Triple Quadrupole LC / MS instrument was used. The final quantification method for the target compound, LENK, and its internal standard, LENK-C136,15N in biological samples in MRM (multiple reaction monitoring) scan was set up following identification of a precursor ion for a target compound in the MS2 (tandem mass spectrometry) scan and its respective product ion(s) in the Product Ion scans. The summary of the final LC-MS method used for the LENK detection and quantification, including optimized binary pump, QQQ and source settings is below. An Agilents LC-MS system 1260 Infinity II binary Pump with an AQUITY UPLC BEH C18, 130Å 2.1mm x column was used at 40°C. The mobile phase was water + 0.1% formic acid: acetonitrile + 0.1% formic acid (%v / v) with a gradient of 0 min – 95:5, 2 min. – 95:5, 2.2 min. – 95:5, 10 min – 5:95, 11 min. – 95:5 and 13 min. – 95:5. The flow rate = 0.5ml / min, injection volume = 5µl, run time = 34 min and retention time = 3.5 min. The QQQ parameters were: LENK m / z = 556 -> 397, LENK-13C6,15N (IS) m / z = 562.9 -> 397.2, fragmentor = 150, collision energy LENK =18 and LENK-13C6,15N = 20. Source parameters: flow / sheath gas flow 10L min-1 / 11 L min-1, nebulizer 30psi, capillary 4000V (+) 0V (-) and nozzle voltage 1500 (+) and 0V (- ). LC-MS Analysis of LENK levels in rat brain and plasma matrices Brain and olfactory bulbs (OB) were pre-weighed and homogenates were prepared in LC-MS grade water supplemented with protease inhibitor (0.01 mL per 1 mL; cat. no. P8340-5ML, Sigma-Aldrich), at a dilution of 5:1 (mL : g of tissue). Brains were homogenized in GentleMACS M-tubes (cat. no.130-093-26, Miltenyi Biotec) using GentleMACS instrument (Miltenyi Biotec) and Protein_01_01 program (53s at rpr: 2753), repeated twice (Figure 2A). OB due to low tissue weight, and thus corresponding low homogenate volume, were homogenized manually in 1.5 mL microcentrifuge tube using relevant size disposable pestles (cat. no. 431-0098, VWR) and then being pipetted up and down through tips with decreasing diameter (1 mL and 0.2 mL) until tissue disintegration was complete. Tissues were submerged in protease inhibitor-containing water and homogenized while still not thawed to prevent LENK degradation by proteases. To 0.05 mL of brain or OB homogenate, 0.15 mL of LC-MS grade methanol as a protein precipitation agent (containing 40 ng mL-1of LENK13C6,15N internal standard), was added. The mixtures were vortexed for 15 min., followed by centrifugation (10,000 x g, 20 min, 4°C) to pellet the precipitated proteins and tissue / cell debris, and the supernatants were harvested to amber HPLC vials with insert for LC-MS analysis. 0.05 mL of plasma was mixed with 0.15 ml of LC-MS grade methanol containing 40 ng mL-1of LENK-C136,15N internal standard. The mixture was vortexed for 15 min., followed by centrifugation (4,000 x g, 15 min., 4°C) and the supernatants transferred to amber HPLC vials for the LC-MS analysis (Figure 3B). LENK AUC values of integrated peaks were divided by the LENK-13C6,15N AUC values to achieve LENK: LENK-13C6,15N ratios, normalized by the internal standard. LENK concentrations in samples were computed based on LENK: LENK-13C6,15N AUC ratios of known concentrations in calibration curves generated for the surrogate matrix, brain tissue or plasma. The final LENK concentrations in plasma were expressed as ng mL-1. The concentrations in brain / OB were expressed as ng g-1(per gram of tissue) to normalize for the brain or OB weights of respective animals and this also took into account the homogenate dilution (5-fold) during the LC-MS sample preparation. Example 7 LENK in vivo pharmacokinetics study in rats upon NES100 intranasal dosing Female Sprague-Dawley rats (Charles River, UK) weighing 190 – 230 g and of 8 – 9 weeks of age, were housed five per cage in an air-conditioned unit (20–22 °C, 50–60% humidity) and allowed free access to standard rodent chow and water. Lighting was controlled on a twelve-hour cycle (on at 07.00 h and out at 19.00 h). Animals were habituated for 7 days prior to experimentation and acclimatized to the procedure room for 1 h prior to testing. For the in vivo pharmacokinetics studies NES100 (GCP23Q11 batch) was rehydrated to 50 mg mL-1 of LENK in MilliQ water. The pH of the formulation was adjusted to pH = ~5.5 with addition of NaOH. Rats were stratified into test groups containing n=3-5 animals each (depending on the study and treatment group). Groups were dosed with NES100 and terminated at two timepoints post-dosing (T = 20 min., or T = 1h) or left non-dosed (T = 0 min.). Rehydrated NES100 nanoparticle formulation was dosed intranasally to each nostril, at 25 or 40 mg kg-1of LENK equivalent depending on the study (for 40 mg kg-1two rounds of dosing each nostril was applied as opposed to one round for 25 mg kg-1), using Smiths Medical Portex fine bore polythene tubing of 0.28 / 0.61 mm inner / outer diameter (cat. no.800 / 100 / 100, Smiths Medical International, Kent, UK) of 15 mm length (with ~10 mm length above the tip of the needle) attached to a 0.3 mL insulin syringe with 30G needle (cat. no.324826, BD). Animals were anesthetized by inhalation of isoflurane (~4%) for up to 5 minutes before the dosing, and as needed in between the nares. At timepoints of 20 min. and 1h post-dosing (from completion of second nare) rats were sacrificed by carbon dioxide asphyxiation and deaths were confirmed by cervical dislocation. Blood samples, from which plasma was later sourced, were taken immediately after the termination via cardiac puncture and collected into K2EDTA anti-coagulant vacutainer tubes containing protease inhibitor (0.01 mL per 1 mL blood). Plasma was separated from blood cells by centrifugation (2000 x g, 15 min, 4°C), aspirated into 1.5 mL microcentrifuge tubes and stored frozen at -50°C until LC-MS analysis (storage time ranged from 12 – 16 hours). Brains or olfactory bulbs were extracted and snap-frozen in liquid nitrogen, then stored frozen at -80°C overnight until LC-MS analysis the following day (storage time ranged from 12 – 16 hours). Brain / OB homogenates and plasma then served as matrices for the compound extraction and LC-MS analysis Data were expressed as mean ± standard error of the mean (SEM) unless otherwise stated. Comparisons of more than two groups were performed using ordinary one-way ANOVA followed by Dunnett's multiple comparisons test. The differences between means of two groups were compared by two-tailed unpaired Student’s t test. Statistical significance was reported as p-values, with the following thresholds: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. All statistical analyses were conducted using the GraphPad Prism (version 8.4.2) for Windows (GraphPad Software, San Diego, California, USA). NES100 (GCP23Q11) powder was rehydrated in water at four LENK concentrations (20 mg mL-1, 40 mg mL-1, 60 mg mL-1and 70 mg mL-1) according to the instructions in the Methods section 2.1. to allow optimal reconstitution. Then data were recorded on their viscosity (Table 30) and syringeability (Table 31). Table 30 - Viscosity of MET-LENK formulations rehydrated from NES100 (GCP23Q11) powder at 4 different LENK concentrations: 20, 40, 60, 70 mg mL-1. Data expressed as mean ± SEM (n=3) in mPa / s units. LENK conc, mg mL-1Viscosity, mPa / s ± SEM 20 1.22±0.0 40 2.43±0.0 60 18.92±0.12 70 57.19±0.36 The viscosity (Table 30) was proportional to LENK concentration: lowest at 20 mg mL-1(1.22 mPa / s) and highest at 70 mg mL-1(57.19 mPa / s). Table 31 - Syringeability of MET-LENK formulations rehydrated from NES100 (GCP23Q11) powder at 4 different LENK concentrations: 20, 40, 60, 70 mg mL-1. Values shown are weights (mg) of discharge from insulin syringe with 15 mm tubing attached upon withdrawal of ~50 µL formulation. Data are expressed as mean ± SEM (n=3). Syringes with 30G needles were used in the first instance and if formulation could not be withdrawn or would clog, 29G was applied. “Y” = applicable, “X” = not applicable LENK conc, mg mL-1Weight, mg ± SEM Needle Gauge 30G Needle Gauge 29G 20 52±0.29 Y X 40 51.7±0.34 Y X 60 54.2±1.65 X Y 70 52.2±1.53 X Y The syringeability was measured as a function of ease of withdrawal and discharge depending on needle gauge and accuracy of discharge of a specified volume. In Table 31, we see that while lower concentrations (20 and 40 mg mL-1) could be withdrawn and discharged with 30G needle of smaller diameter, the higher concentrations (60 and 70 mg mL-1) failed and had to be tested with larger needle diameter (29G). Even then the withdrawal was not smooth, especially at the highest concentration of 70 mg mL-1and worked best when proceeded slowly. The discharge of 50 µL was on average accurate (within 5% RSD), but standard error was higher in the higher LENK concentrations of 60 and 70 mg mL-1, indicating higher variability. For animal studies, 50 mg mL-1LENK concentration was decided as most optimal, allowing ease of reconstitution and good syringeability, while still maximizing on concentration. The optimized rehydration protocol increased the pH to ~5.5, which is more compatible with nasal cavity. The optimized protocol allowed reproducible pH values in a repeated experiment and showed by the formulations’ visual appearance that we can achieve clear and stable formulation at pH = 5.5-5.6 (Figure 4). However, above this pH (pH = 5.85), the formulation turned turbid, and was more prone to losing its stability. While the pH=5.33 and pH=5.55-5.6 formulations were found still stable the next day, the pH=5.85 visibly precipitated. Therefore, formulations adjusted to pH=~5.5 were later used for the animal studies. Part of the optimized protocol for NES100 rehydration at higher LENK concentrations constitutes formulation warming. However, in order to rule out LENK degradation due to increased temperature, LENK content was analyzed by HPLC following formulation preparation including stirring on a hotplate for 15 min. at three different temperatures: 25, 37 and 45°C. The LENK recovery in shows minor differences among the temperatures 109% LENK recovery at 25°C, 103% LENK recovery at 37°C and 99% LENK recovery at 45°C. For the optimized protocol, the 37°C was proceeded with, which was closest to the LENK recovery seen at room temperature. In vivo pharmacokinetics of LENK in rat brain tissue and plasma upon intranasal dosing In vivo studies in rats were conducted to determine LENK levels in the brain, olfactory bulb and plasma following intranasal administration of NES100 powder (GCP23Q11 batch) rehydrated to MET-LENK nanoparticles (at 50 mg mL-1) and dosed at 25 mg kg-1(= 1 round of dosing to each nostril) or 40 mg kg-1(= 2 rounds of dosing to each nostril). The formulation was adjusted to pH=~5.5 to make it more compatible with the physiological pH in the nasal cavity (5.5-6.5). Significant dose expulsion via sneezing peri-dosing was not observed. Mild sneezing instances in 2 out of 10 rats dosed at 25 mg kg-1(per 20 dose applications in total) and 3 out of 8 rats at 40 mg kg-1(per 32 dose applications in total) were reported. LENK concentrations were quantified in the following biological samples: olfactory bulb, brain (cerebrum) and plasma by LC-MS bioanalytical method for detection of LENK and its internal standard. The method parameters are listed in Table 32. Table 32 – LC-MS method settings for the detection of LENK. Binary Pump System Agilent 1260 Infinity II Binary Pump Column ACQUITY UPLC BEH C18, 130Å, 1.7 µm, 2.1 mm X 5 mm; 40oC Mobile phase A: Water + 0.1% formic acid; B: Acetonitrile + 0.1% formic acid (%, v / v) Gradient: 0 min. – 95 : 5 2 min. – 95 : 5 2.20 min. – 5 : 95 10 min. – 5 : 95 11 min. – 95 : 5 13 min. – 95 : 5 Flow0.5 mL min-1 Injection volume 5 µL Run time 13 min. Retention time 3.5 min. QQQ LENK m / z = 556 -> 397 LENK-13C6,15N (IS) m / z = 562.9 -> 397.2 Fragmentor 150 Collision energy LENK = 18 LENK-13C6,15N (IS) = 20 Polarity positive Source Gas / Sheath gas temperature 300oC / 350oC Flow / Sheath gas flow 10 L min-1 / 11 L min-1Nebulizer 30 psi Capillary 4000 V (positive), 4000 V (negative) Nozzle voltage 1500 V (positive), 0 V (negative) The above LC-MS method yielded well-defined peaks for LENK or LENK-13C6,15N internal standard eluates at the retention time of ~3.5 min. in non-biological or biological matrices. The calibration curves generated in a non-biological surrogate matrix, as well as brain homogenate and plasma matrices showed good linearity within their respective quantification ranges (surrogate matrix: 0.8-50 ng mL-1, coefficient of correlation R2= 0.9996; brain: 2.5-80 ng mL-1, R2= 0.9988; plasma: 0.8-50 ng mL-1, R2= 0.9953. Clearly defined LENK peak with area under the curve (AUC) of 690.51 (surrogate matrix) or 66.54 (plasma matrix) for the LLOQ (lower limit of quantification) of 0.8 ng mL-1concentration of LENK, or 1394.81 AUC for 2.5 ng mL-1in brain matrix, as well as high signal-to-noise ratios (SNR) of 539.1, 58.6 and 1891.4, respectively (where SNR ≥ 3 is considered acceptable threshold of detection limit), confirmed that our LC-MS quantification method is accurate and highly sensitive. The method was also validated in terms of LENK peak reproducibility based on repeated injections showing similar AUC (RSD = 2.46%) and retention times (RSD = 0.2%), which are within standard 5% RSD, hence satisfying the validity criteria. Representative chromatograms of brain and plasma samples from the rats dosed intranasally with MET- LENK nanoparticles showed well-defined peaks for the LENK or LENK-13C6,15N analytes with flat baselines. The peak integrations indicated fairly high LENK detection signal (AU = 126.79 in brain, 201.52 in OB and 168.72 in plasma) and signal-to-noise ratio (SNR = 98.9, 202, 108.3, respectively) values, confirming high recovery levels within calibration curve range and good sensitivity and detection in the analyzed biological matrices, while no peaks in the blank run in-between samples indicate no analyte cross-over or column retention. Altogether, it validates the developed LC-MS bioanalytical method as suitable for the reliable quantification of LENK levels in the in vivo pharmacokinetics study samples. Table 33 – LENK in vivo pharmacokinetic data summary. CT=0 / CT=20 / CT=60= LENK concentration at T=0 min. / 20 min. / 60 min.; Cmax= maximum LENK concentration; Tmax= time of maximum concentration. Statistical significance relative to baseline is indicated as follows: *P < 0.05, **P < 0.01, ***P < 0.001. 25 mg / kg LENK dose Brain Olfactory Bulb Plasma Baseline: non-dosed,T=0 (ng / g ± SEM) 43.97 ±3.92 22.09 ±3.38 1.77 ±0.04 CT=20(ng / g ± SEM) 78.32 ±7.61 (**) 94.94 ±7.09 (***) 4.86 ±1.02 (**) CT=60(ng / g ± SEM) 63.06 ±2.99 (*) 68.25 ±13.36 (**) 3.42 ±0.13 Cmax(ng / g ± SEM) 78.32 ±7.61 (**) 94.94 ±7.09 (***) 4.86 ±1.02 (**) Tmax(min) 20 20 20 40 mg / kg LENK dose Brain Olfactory Bulb Plasma Baseline: non-dosed, T=0 (ng / g ± SEM) 27.53 ±2.76 43.52 ±9.15 0.6 ±0.08 CT=20(ng / g ± SEM) 44.80 ±4.54 66.67 ±10.06 4.46 ±0.62 CT=60(ng / g ± SEM) 36.64 ±7.25 52.90 ±5.33 5.37 ±0.65 Cmax(ng / g ± SEM) 44.80 ±4.54 66.67 ±10.06 5.37 ±0.65 Tmax(min) 20 20 60 The LC-MS analysis of tissues from rats dosed with NES100 nanoparticles at 25 mg kg-1(Table 33) confirms significantly higher levels of LENK in the brain compared to endogenous baseline of non-dosed rats (43.97 ng g-1, T= 0 min.), with maximum concentration Cmax= 78.32 ng g-1at 20 min. timepoint (= Tmax- time of maximum concentration). At 1h the LENK concentration was still significantly higher relative to non-dosed controls, but lower than at T = 20 min. post-dosing (63.06 ng g-1). Similar pharmacokinetics profile was observed in olfactory bulbs, with Cmax= 94.94 ng g-1and Tmax= 20 min and subsequent decrease to 68.25 ng g-1at T = 1h, both significantly higher compared to non-dosed controls (22.09 ng g-1, T= 0 min.). In the plasma, the LENK Cmaxwas 4.86 ng mL-1(Tmax= 20 min), which is 16-fold lower compared to the brain levels, suggesting fairly low systemic exposure following intranasal administration. Summary of the above data can be found in Table 33. Despite similar pharmacokinetics profile, higher levels of MET-LENK dose when administered two rounds to reach 40 mg kg-1LENK equivalent, did not translate to higher concentrations of LENK reaching the brain (Cmax= 44.80 ng g-1and Tmax= 20 min.) or olfactory bulb (Cmax= 66.67 ng g-1and Tmax= 20 min.) relative to the results of 25 mg kg-1LENK dosing (Table 33). This is likely due to superfluous formulation volumes causing congestion in the nasal cavity and being cleared rather through swallowing to the digestive system instead. Within this disclosure, any indication that a feature is optional is intended provide adequate support (e.g., under 35 U.S.C. 112 or Art. 83 and 84 of EPC) for claims that include closed or exclusive or negative language with reference to the optional feature. Exclusive language specifically excludes the particular recited feature from including any additional subject matter. For example, if it is indicated that A can be drug X, such language is intended to provide support for a claim that explicitly specifies that A consists of X alone, or that A does not include any other drugs besides X. "Negative" language explicitly excludes the optional feature itself from the scope of the claims. For example, if it is indicated that element A can include X, such language is intended to provide support for a claim that explicitly specifies that A does not include X. Non-limiting examples of exclusive or negative terms include "only," "solely," "consisting of," "consisting essentially of," "alone," "without", "in the absence of (e.g., other items of the same type, structure and / or function)" "excluding," "not including", "not", "cannot," or any combination and / or variation of such language. Similarly, referents such as "a," "an," "said," or "the," are intended to support both single and / or plural occurrences unless the context indicates otherwise. For example "a dog" is intended to include support for one dog, no more than one dog, at least one dog, a plurality of dogs, etc. Non-limiting examples of qualifying terms that indicate singularity include "a single", "one," "alone", "only one," "not more than one", etc. Non-limiting examples of qualifying terms that indicate (potential or actual) plurality include "at least one," "one or more," "more than one," "two or more," "a multiplicity," "a plurality," "any combination of," "any permutation of," "any one or more of," etc. Claims or descriptions that include "or" between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. Where ranges are given herein, the endpoints are included. Furthermore, it is to be understood that unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or subrange within the stated ranges in different embodiments of the invention, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise. All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. While this invention has been particularly shown and described with references to example embodiments thereof, it will be understood by those skilled in the art that the various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims. Throughout this specification and claims which follow, unless the context requires otherwise, the word “comprise”, and variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated integer or group of integers or steps but not the exclusion of any other integer or group of integers. As used herein and unless otherwise stated, the term "approximately" means ±20%. It will of course be realised that whilst the above has been given by way of an illustrative example of this invention, all such and other modifications and variations hereto, as would be apparent to persons skilled in the art, are deemed to fall within the broad scope and ambit of this invention as is herein set forth.

Claims

Claims 1. An intranasal nanoparticle suspension comprising leucine enkephalin and quaternary ammonium palmitoyl glycol chitosan (GCPQ), wherein the ratio of the concentration in w / v of GCPQ to leucine enkephalin is from about 1:1 to about 2:1, and wherein GCPQ has a palmitoylation level between 10 mole % and 20 mole % and a quaternization level between 8 mole % and 12 mole %, wherein the formulation has a pH from 5.5 to 6.

5.

2. The intranasal suspension of claim 1 wherein the size of the liposomes comprise a bimodal size distribution of nanoparticles from 10-40nm in diameter and nanoparticles of greater than 50nm.

3. The intranasal nanoparticle suspension of claim 2 wherein the nanoparticles having a diameter of 10- 40nm contain no leucine enkephalin and the nanoparticles having a diameter of greater than 50nm contain leucine enkephalin.

4. A powdered intranasal formulation comprising leucine enkephalin and quaternary ammonium palmitoyl glycol chitosan (GCPQ), wherein the ratio of the concentration in w / v of GCPQ to leucine enkephalin is from about 1:1 to about 2:1, and wherein GCPQ has a palmitoylation level between 10 mole % and 20 mole % and a quaternization level between 8 mole % and 12 mole %, wherein the formulation has a moisture content of below 5%.

5. The powdered intranasal nanoparticle formulation of claim 4 wherein the leucine enkephalin comprises 0.45 to 0.55 mg per mg of the powdered intranasal liposomal formulation.

6. A method for treating pain, comprising intranasally administering to a human or animal in need thereof a composition comprising a therapeutically effective amount of leucine enkephalin and an amphiphilic quaternary ammonium palmitoyl glycol chitosan (GCPQ); wherein the amphiphilic GCPQ is capable of self-assembly in aqueous media into particles having a mean particle size between 20-500 nm; wherein intranasally administering the composition delivers the hydrophilic neuroactive peptide to the brain of the human or animal.

7. The method of claim 6 wherein the particle size of the particles has a bimodal distribution.

8. The method of claim 7 wherein the bimodal distribution has particles having a diameter of 10-40nm which contain no leucine enkephalin and the particles having a diameter of greater than 50nm contain leucine enkephalin. 9.The method of claim 6 wherein the composition is in the form of a suspension. 10.The method of claim 9 wherein the amount of leucine enkephalin administered is from 150 to 180 mg per kg weight of the animal or human per day. 11.The method of claim 6 wherein the composition is in the form of a powder. 12.The method of claim 11 wherein the amount of leucine enkephalin administered is from 100 to 120 mg per kg weight of the animal or human per day.

13. A device for delivering a composition intranasally to a mammal comprising: a therapeutically effective amount of a leucine enkephalin and an amphiphilic compound N-palmitoyl-N- acetyl-N-monomethyl-N,N-dimethyl-N,N,N-trimethyl-6-O-glycolchitosan as dried powder microparticles with a mean particle size greater than 5µm and has a moisture content of less than 5%; wherein the amphiphilic molecule self-assembles in aqueous media into nanoparticles having a mean particle size between 3-500 nm and wherein the composition is contained with a capsule comprising a pressurized container for a fluid, a chamber for containing the particles, at least one channel running between the container and the chamber to provide fluidic communication between the container and chamber in use, and at least two distinct concave surfaces on or integral with at least part of an internal wall or internal walls of the chamber, the concave surfaces so arranged that once fluidic communication between the chamber and container is established to create a fluid flow from the container to the chamber through the at least one channel and toward the concave surfaces, each concave surface imparts a rotational motion to a fluid flow or portion of fluid flow that impinges upon it, so that within the chamber a rotationally turbulent flow of fluid is produced in order to engage with the particles and to produce a mobile fluid comprising the particulate.

14. The device of claim 13 wherein the moisture content of the particles is less than 10% after storage at 25°C and 60% relative humidity for at least 6 months.

15. The device of claim 13 wherein the moisture content of the particles is less than 10% after storage at 30°C and 65% relative humidity for at least 6 months.

16. The device of claim 13 wherein the change in leucine enkephalin content relative to the powder is less than 10% after storage at 25°C and 60% relative humidity for at least 6 months.

17. The device of claim 13 wherein the change in leucine enkephalin content relative to the powder is less than 10% after storage at 30°C and 65% relative humidity for at least 6 months.

18. The device of claim 13 wherein the change in leucine enkephalin content relative to the powder is less than 5% after storage at 30°C and 65% relative humidity for at least 6 months.

19. The device of claim 13 wherein the microparticles have a mean particle diameter of 10 – 50 micrometers.

20. The device of claim 13 wherein the microparticles have a mean particle diameter of about 20 micrometers 22. The device of claim 13 wherein the microparticles when dispersed in aqueous media provide nanoparticles which exhibit a bimodal size distribution in which the particle size of the nanoparticles is between 10-20 nm for the first mode and between 80 to 650 nm for second mode. 23 The device of claim 13 containing microparticles where the microparticles may be changed on contact with aqueous media to nanoparticles with a size distribution of the particles is greater than 60% for mode 2 and greater than 30% for mode 1. 24 The device of claim 13 wherein the mass of particles delivered by the device is from 19 to 25 mg per kg mammal mass per day.

25. A composition comprising leucine enkephalin and an amphiphilic compound N-palmitoyl-N-acetyl-N- monomethyl-N,N-dimethyl-N,N,N-trimethyl-6-O-glycolchitosan as dried powder microparticles with a mean particle size greater than 5µm and has a moisture content of less than 5%; wherein the amphiphilic molecule self-assembles in aqueous media into nanoparticles having a mean particle size between 3-500 nm.

26. The composition of claim 25 wherein the ratio of amphiphilic compound to leucine enkephalin is from 1:10 to 10:1.

27. The composition of claim 25 wherein the ratio of amphiphilic compound to leucine enkephalin is from 1:3 to 3:

1.

28. The composition of claim 25 wherein the ratio of amphiphilic compound to leucine enkephalin is from 1:2 to 2:

1.

29. The composition of claim 25 wherein the ratio of amphiphilic compound to leucine enkephalin is 1:

1.

30. The composition of claim 25 wherein the ratio of amphiphilic compound to leucine enkephalin is 2:

1.

31. The composition of claim 25 further comprising a pharmaceutically acceptable carrier.

32. The composition of claim 31 wherein the ratio of amphiphilic compound to leucine enkephalin to pharmaceutically acceptable carrier is from 1:0.1:10 to 10:10:

1.

33. The composition of claim 31 wherein the ratio of amphiphilic compound to leucine enkephalin to pharmaceutically acceptable carrier is from 2:0.1:10 to 10:5:

1.

34. A method for treating pain, comprising intranasally administering to a human or animal in need thereof a composition comprising a therapeutically effective amount of leucine enkephalin and an amphiphilic quaternary ammonium palmitoyl glycol chitosan (N-palmitoyl-N-acetyl-N-monomethyl-N,N- dimethyl-N,N,N-trimethyl-6-O-glycolchitosan); wherein the amphiphilic GCPQ is capable of self-assembly in aqueous media into particles having a mean particle size between 3-500 nm and wherein the particles are dried and loaded into a capsule which when activated intranasally delivers the particles to the brain of the human or animal.

35. The method of claim 34 wherein the ratio of amphiphilic compound to leucine enkephalin is from 1:10 to 10:

1.

36. The method of claim 34 wherein the ratio of amphiphilic compound to leucine enkephalin is from 1:3 to 3:

1.

37. The method of claim 34 wherein the ratio of amphiphilic compound to leucine enkephalin is from 1:2 to 2:

1.

38. The method of claim 34 wherein the ratio of amphiphilic compound to leucine enkephalin is 1:

1.

39. The method of claim 34 wherein the ratio of amphiphilic compound to leucine enkephalin is 2:

1.

40. The method of claim 34 further comprising a pharmaceutically acceptable carrier.

41. The method of claim 40 wherein the ratio of amphiphilic compound to leucine enkephalin to pharmaceutically acceptable carrier is from 1:1:10 to 10:10:

1.

42. The method of claim 40 wherein the ratio of amphiphilic compound to leucine enkephalin to pharmaceutically acceptable carrier is from 2:1:10 to 10:5:1.

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